Sensing the Future—Frontiers in Biosensors: Exploring Classifications, Principles, and Recent Advances

Abstract

Biosensors are transforming healthcare by delivering swift, precise, and economical diagnostic solutions. These analytical instruments combine biological indicators with physical transducers to identify and quantify biomarkers, thereby improving illness detection, management, and patient surveillance. Biosensors are widely utilized in healthcare for the diagnosis of chronic and infectious diseases, tailored treatment, and real-time health monitoring. This thorough overview examines several categories of biosensors and their uses in the detection of numerous biomarkers, including glucose, proteins, nucleic acids, and infections. Biosensors are commonly classified based on the type of transducer employed or the specific biorecognition element utilized. This review introduces a novel classification based on substrate morphology, offering a comprehensive perspective on biosensor categorization. Considerable emphasis is placed on the advancement of point-of-care biosensors, facilitating decentralized diagnostics and alleviating the strain on centralized healthcare systems. Recent advancements in nanotechnology have significantly improved the sensitivity, selectivity, and downsizing of biosensors, rendering them more efficient and accessible. The study examines problems such as stability, reproducibility, and regulatory approval that must be addressed to enable the widespread implementation of biosensors in clinical environments. The study examines the amalgamation of biosensors with wearable devices and smartphones, emphasizing the prospects for ongoing health surveillance and individualized medical care. This viewpoint clarifies the distinct types of biosensors and their particular roles, together with recent developments in the “smart biosensor” sector, facilitated by artificial intelligence and the Internet of Medical Things (IoMT). This novel approach seeks to deliver a comprehensive evaluation of the present condition of biosensor technology in healthcare, recent developments, and prospective paths, emphasizing their significance in influencing the future of medical diagnostics and patient care.

1. Introduction

Biosensors have emerged as transformative tools in healthcare, uniting biology, chemistry, physics, and engineering to address various diagnostic and monitoring needs.¹ A biosensor is an analytical device that combines a biological sensing element, such as enzymes, antibodies, or nucleic acids, with a physicochemical detector to convert biological interactions into measurable signals.^2,3 The healthcare sector has widely embraced biosensors for their ability to detect and monitor biomarkers associated with diseases, including infectious agents and chronic conditions such as Parkinson’s disease and cancer.⁴⁻⁷ The glucose biosensor represents a notable progression in diabetes therapy by enabling continuous, noninvasive monitoring of blood glucose levels.^8,9 Biosensors offer several advantages over traditional diagnostic methods, including rapid reaction times, increased sensitivity, portability, and the capacity for real-time, point-of-care (POC) applications, hence facilitating prompt clinical decision-making by healthcare professionals.^10,11

There is a current need in the medical and analytical sensing industries for telemedicine, healthcare monitoring, and mobile e-health services, and technological advancements have hastened their expansion. Improvements in the design of biosensing devices have spurred large areas of study, leading to the creation of new sensing technologies. Innovative biosensors utilizing nanoparticles are evolving as analytical tools for diverse biomolecules, highlighting the crucial role of nanomaterials in this context.¹² The incorporation of nanotechnology into biosensor development has significantly enhanced their functionality. Nanomaterials, such as nanoparticles, and graphene are employed to increase the surface area of sensors, thereby improving their sensitivity and detection limits.¹³ This has been particularly important in the detection of low-abundance biomarkers, such as certain cancer markers, enabling earlier diagnosis and more effective treatment plans.¹⁴ Significant progress in biosensor technology may be achievable through the thorough exploration and systematic development of metal oxide-based nanomaterials, such as nanowires (NWs), nanorods (NRs), carbon nanotubes (CNTs), quantum dots (QDs), and nanocomposite dendrimers. An analysis of the design and fabrication of these nanoparticles reveals new opportunities for enhancing biosensor detection efficacy. The exceptional platform offered by these nanomaterials for manipulation and customization allows researchers to accurately alter their characteristics to satisfy the requirements of diverse biosensing applications.¹⁵ This precise engineering improves the sensitivity of biosensors while enabling the adjustment of their selectivity and overall effectiveness.

A biosensor can detect an enzyme or nucleic acid by utilizing a biological interaction to ascertain the concentration of the substance.¹⁶ The integration of mechanical contact with the unique adhesion, affinity, and mobility of biological components facilitates the assessment of molecular or subcellular variations in mass, displacement, and other parameters.¹⁷ The primary mechanism via which biosensors operate is signal transduction. Heat sterilization is not feasible for these devices due to the denatured characteristics of biological components.¹⁸ Effective early detection and diagnosis can diminish future healthcare expenditures. Diagnostic tools are proteins located on the surfaces of bacterial and viral cells. Among the various biosensors employed in medical diagnostics, a glucose sensor is particularly notable. The enzyme glucose oxidase on the sampling strip biochemically interacts with the glucose in the blood sample, enabling the calculation of glucose concentration.¹⁹ Molecular interactions among biomolecules or faulty genes have been linked to numerous diseases, including cancer, Alzheimer’s, Parkinson’s, autoimmune disorders, and AIDS, among others.²⁰⁻²⁴ Currently, biosensors employing electrochemical, optical, and piezoelectric principles can identify a diverse range of analytes, such as lactate, glucose, cholesterol, and specific DNA sequences. Moreover, miniaturization and progress in microfabrication have facilitated the creation of portable biosensors that may be seamlessly incorporated into wearable devices. This facilitates ongoing surveillance of patients’ physiological metrics, enhancing personalized therapy and improving outcomes, particularly for individuals with chronic conditions necessitating prolonged monitoring.

This comprehensive overview analyses the many categories of biosensors and their main uses in identifying biomarkers, including glucose, proteins, nucleic acids, and infections. Biosensors are traditionally grouped based on the transducer type (electrochemical, optical, piezoelectric, or thermal) or the biorecognition element (enzymes, antibodies, aptamers, nucleic acids, etc.) employed for object detection. Optical biosensors are employed for the detection of proteins and nucleic acids in cancer diagnostics, while electrochemical biosensors are predominantly utilized for glucose monitoring in diabetes management. Furthermore, due to their capacity to identify subtle variations in mass, piezoelectric sensors are advantageous for illness detection. This overview presents a novel framework for categorizing biosensors based on the morphology of the substrate, enhancing prior classifications. The structural characteristics of diverse substrates, including paper and electrospun fiber mats (or thin films), are examined concerning their influence on the sensitivity, stability, and selectivity of biosensors. This substrate-based classification elucidates biosensor technologies, highlighting the significance of material morphology in augmenting detection efficacy. This comprehensive method facilitates a deeper understanding of biosensor functionality and enhances the development of next-generation medical biosensors with more efficiency.

2. Biosensors

Biosensors can quantify analytes or evaluate the degree of biochemical interactions by integrating a biochemical transducer with a biological recognition element. The preliminary phase of the operational principle requires the biological component—such as an antibody, nucleic acid, aptamer, or enzyme—to selectively attach to the target analyte. A biological change, such as the production of electrons, a shift in optical properties, or a shift in mass, can be induced by this contact. After this change occurs in the body, the physicochemical transducer turns it into a measurable signal like a voltage, light intensity, or sound wave.²⁵⁻²⁷ Electrochemical biosensors work by detecting currents generated by oxidation or reduction reactions induced by analytes and enzymes, respectively. Optical biosensors detect changes in light intensity due to changes in the refractive index or fluorescence caused by analyte binding. To accurately identify the connection and transform it into a measurable signal, the bioreceptor, transducer, and signal processor all work together as part of a biosensor.^28,29 Blood glucose concentration or pathogen detection are examples of understandable outputs that can be produced by a signal processor after it has been amplified and processed by the transducer, which ensures high specificity by identifying only the target analyte. Biosensors deliver sensitive, quick, and precise measurements across various medical applications due to the seamless integration of biological and physicochemical elements. Figure 1 depicts the different components of a biosensor as an illustration, while Figure 2 provides a simplified schematic diagram of (a) a screen-printed electrode, (b) the detection mechanism of a bioanalyte, and (c) an electrochemical cell.

Figure 1.

Illustration of the different components of a biosensor.

Figure 2.

Schematic representation of (a) the main components of a screen-printed electrode, (b) the detection mechanism of the bioanalyte, and (c) an electrochemical cell.

2.1. Principle and Working of a Biosensor

Traditional approaches, such as membrane encapsulation, physical adsorption, covalent or noncovalent binding, etc., are used to immobilize a bioreceptor on a suitable substrate, making it the sensor’s main component.³⁰ When the sample’s analyte binds to the sensing platform’s bioreceptor, a biomolecular interaction occurs, eliciting an optical, electrical, or chemical response. Sometimes, a new product will be formed during the interaction, and byproducts like ions, gases, heat, light, etc. will be released. These surface responses from the transducer are then transformed into electrical or optical signals, which may be read with the help of an amplifier. The differential gap between the sample signal and the reference signal is amplified typically in the signal processing stage. The microprocessor unit receives the digital signal and processes, calibrates, and displays the data in the chosen units.

Essential to a biosensor’s performance are its sensitivity, selectivity, and stability. The biosensor has what is called a “working range”, or a range of concentrations of the bioanalyte in which it is effective. Precancer diagnostic biomarker detection requires a biosensor sensitive enough to pick up on even trace concentrations of the analyte. Graphene was described by M. Pumera et al. to be a promising material for detecting the target particle at concentrations as low as ppm or ppb.³¹ Graphene’s low noise and strong electrical conductivity allow for its extraordinary sensitivity.³² Researchers have also shown that graphene and reduced graphene oxide’s sensitivity and specific affinity can be greatly enhanced through functionalization³³⁻³⁵

Having the biosensor properly identify the biomarker molecule is a crucial step in the process. Different proteins or antigens may be present in the target blood sample. However, even in this case, the biosensor should react solely to the target biomolecule and no extraneous substances. Higher and improved sensitivity can be achieved thanks to the lock-and-key mechanism disclosed by antigen–antibody interaction.³⁶ To detect the cancer biomarkers carcinoembryonic antigen (CEA), CA 125, and Her-2/Neu, Jokerst and his colleagues created a semiconductor QD-based biosensor that can do all three at once. They found that it was far more sensitive than the standard ELISA (Enzyme-based Immunosorbent assay) test.³⁷ Researchers have used MWCNTs on the electrodes of the immunosensor’s scaffolds to detect several antibodies, including TGF-beta 1 cytokine, thanks to the material’s high conductivity and electro-catalytic characteristics.^38,39

The accuracy of the measurement is crucial for any machine performing quantitative analysis. The signal strength was more affected by the concentration of the analyte. Maintaining a consistent signal is essential for accurate readings. It is also important for a biosensor to have a quick response time, as this is essential for real-time monitoring. It has been stated that a submicron-sized glucose sensor implanted under the skin can provide results in under a millisecond.⁴⁰ The biosensor’s durability is determined by several elements, including the accuracy with which the transducer electronics are tuned, the bioreceptor’s affinity, and the bioreceptor’s degradation time or rate, among others.⁴¹

2.2. Types of Biosensors

The biorecognition elements, sensor substrate, and sensing methods used in biosensors all contribute to the devices’ unique classifications. Figure 3 is a high-level overview diagram of the several kinds of biosensors now in use in the field of biomedicine. The classification of biosensors in healthcare, as shown in diagram 3, is structured into several categories based on their transduction techniques, biorecognition elements, microelectromechanical systems (MEMS), and the nature of substrates. Under the transduction techniques category, biosensors are divided into mass-based, electrochemical, and optical biosensors. Mass-based biosensors include types like magnetoelastic, piezoelectric, and quartz crystal microbalance (QCM) biosensors, which measure changes in mass or mechanical properties. Electrochemical biosensors are further classified into amperometric, potentiometric, conductometric, and impedimetric types, all of which convert biochemical interactions into electrical signals. Optical biosensors utilize light-based phenomena and include surface plasmon resonance (SPR), optical fiber-based, and surface-enhanced Raman spectroscopy (SERS) biosensors.

Figure 3.

Classification of biosensors based on the transducer element, biorecognition element, MEMS, and nature of the substrate.

In terms of biorecognition elements, biosensors can be classified based on the biological material used, such as antibodies, DNA, enzymes, or phages. These bioreceptors selectively bind to their target analytes, enabling the detection of specific biomolecules. MEMS-based biosensors, leverage advanced miniaturization technology to improve precision and sensitivity.

Finally, the classification also includes biosensors based on the nature of the substrate, such as those using electrospun materials, and paper. These various biosensors offer various benefits, including enhanced surface area for improved sensitivity, biocompatibility, and the ability to fabricate flexible, wearable biosensors. This diverse classification framework reflects the wide range of biosensor technologies available for healthcare applications, each designed to meet specific diagnostic or monitoring needs. Biomolecules can take many forms, including enzymes, cells, antibodies, DNA, and many others. Physicochemical interactions between the biorecognition component and the sample’s target molecule are crucial to the signal transduction process and various types are discussed below.

2.2.1. Transduction Techniques

It is possible to carry out transduction in several methods. Mass, electrochemical interactions, and optical phenomena serve as the primary dividing lines. There are countless potential transductions because each class contains several subclasses.⁴² The following sections examine the major biosensor classifications based on the transducing techniques used.

2.2.1.1. Mass-Based Biosensors

2.2.1.1.1. Magnetoelastic Biosensors. Many different types of sensors, such as biosensors used to detect Bacillus anthracis, Salmonella typhimurium, etc., are built using the magnetoelastic phenomena.⁴³⁻⁵² It is based on magnetostriction that magnetoelastic sensors function. Some ferromagnetic materials undergo morphological transformations when their magnetization is altered. We all know that when a system is operating at its resonant frequency, the amplitude of its vibrations is maximized. Mechanical resonance in magnetostrictive materials can be explained as having a magnetic source. External mechanical strain is caused by a vibrating magnetic field. Any change in magnetic field strength is translated into a change in strain by the ferromagnetic material. As a result of the strain reaching its maximum value at resonance, the magnetic values similarly peak at their highest level.⁵³ Therefore, the magnetoelastic biosensor is a product of both magnetic and mechanical resonance, as suggested by the name.

Various detecting methods, such as optical, acoustic, magnetic, etc., are available for the operation of a magnetoelastic sensor strip.⁵⁴ Magnetostrictive materials exhibit a linear strain when subjected to a magnetic field. Magnetoelastic sensors can detect in both the frequency and time domains.⁵⁵⁻⁵⁷ Frequency mode enables effective wireless real-time monitoring by continuously exciting the sensor over a frequency range to determine its resonance frequency. The resonance frequency was determined by Zeng et al.,^58,59 who employed multiple cycles at a specified frequency. Sensors with a larger collecting area benefit from time-domain detection, whereas those with a smaller footprint should use the frequency mode. Due to background noise, the output detection signal from tiny sensors operating in liquid media may not be particularly reliable. Better detection was achieved by Wen et al. time’s domain approach using a pulse-initiated magnetic field rather than a biased magnetic field since the sensitivity is DC-dependent.⁶⁰

Vibrations at the resonant frequencies will occur when a magnetic field is applied in an alternating fashion. For a material with length L, Elasticity E, and density, the frequency-density relation is:⁶¹

1

which is also valid for the vibrational frequency of a ribbon-like thick film sensor with these same parameters. When a biorecognition element, such as a bacterium or an enzyme, is captured on the sensor platform, its mass will increase relative to the reference, causing the resonant frequency to drop.^60,62 The operation of a magnetoelastic biosensor is depicted in a simplified diagrammatic form in Figure 4.

Figure 4.

Schematic working of a magnetoelastic biosensor.

The equation⁶¹

2

describes the change in resonant frequency when an additional mass Δm is attached to a magnetoelastic sensor with an initial resonant frequency f₀ and initial mass m_s. To detect the Bacillus anthracis bacteriophage, Johnson et al. reported fabricating an effective gold-coated magnetoelastic sensor platform utilizing photolithography and sputtering.⁶³ To detect swine fever virus E2 antibody at a cheap cost and with high throughput, Guo and colleagues created a magnetoelastic biosensor.⁶⁴

2.2.1.1.2. Piezoelectric Biosensor. By applying mechanical stress, piezoelectric materials can produce electrical current. Some substances also display the opposite of the piezoelectric effect, known as the inverse piezoelectric effect, in which mechanical strain is generated in response to an electrical current. Quartz, Rochelle salt, zinc oxide, aluminum phosphate and nitrides, topaz crystal, lead titanate, polyvinylidene fluoride, etc. are examples of anisotropic materials that are more likely to exhibit piezoelectricity.⁶⁴⁻⁷¹ A mass attached to the sensor surface causes an oscillation shift, which is the basis for the piezoelectric biosensor’s operation. Figure 5 is a simplified schematic depiction of this concept.

Figure 5.

Simplified schematic illustration of the working of a piezoelectric sensor.

The following equation defines the frequency shift Δf, brought about by the bound mass, Δm on the piezoelectric crystal:

3

where f₀ means the fundamental mode of the crystal oscillation in hertz and A is the piezoelectrically active area in centimeters.⁷² Environmental conditions, as well as the equation related to viscosity reported by Kanazawa et al., are taken into account, even though equation 3 is employed for thin film adhered to the transducer surface.⁷³ There are several types of piezoelectric transducers on the market, but they may be broken down into two main categories: bulk acoustic wave (BAW) and surface acoustic wave (SAW).⁷⁴ Quartz crystal microbalances (QCM) are the most common type of BAW device, and they are made up of a large piezoelectric crystal (often quartz) and two conducting electrodes.⁷⁵

2.2.1.1.2.1. Quartz Crystal Microbalance (QCM). In 1959, Sauerbrey established a connection between variations in resonant frequency and the mass density of the sensor surface. QCM sensors have subsequently been utilized for various applications. Figure 6 depicts a QCM.

Figure 6.

Schematic diagram of a QCM.

A QCM sensor has a quartz oscillator disc and circular electrodes. AC in these electrodes causes the quartz to vibrate. When an analyte molecule attaches to quartz, the mass changes, and the vibration frequency changes. Quartz signal transducer translates a nanogram shift in mass (67 Hz cm ^–2 ng^–1 sensitivity at 170 MHz) generated by biorecognition element and bioreceptor hybridization.⁷⁶ Using diverse bioreceptor surfaces increases QCM’s adaptability.

There are piezoelectric QCM immunobiosensors.^77,78 Viruses and bacteria have unique cell surface proteins. Hemagglutinin and neuraminidase are H1N1 virus coat proteins. If these proteins are present, the sample contains the H1N1 Virus. Li et al. developed a QCM-based biosensor to detect H5N1.⁷⁹ Alzheimer’s illness requires molecular peptide detection. M. K. Mustafa and his team utilized QCM and spectroscopic ellipsometry to detect -amyloid peptide (1–16) and amyloid precursor protein (APP770), enabling Alzheimer’s disease early diagnosis.⁸⁰ Mannelli et al. reported direct DNA probe attachment for affinity biosensors.⁸¹ Setyawan P Sakti and colleagues produced milk adulteration detection equipment with a 1 ppm detection limits.⁸²

2.2.1.1.2.2. Surface Acoustic Wave (SAW) Transducer-Based Biosensors. Electroacoustic devices use piezoelectric crystals based on sound vibrations. BAW and SAW propagation transducers are common. Any change in the transducer’s mass, viscoelasticity, or dielectric characteristics will affect the output. Bioaffinity membrane on piezoelectric substrate affects SAW phase velocity and propagation loss. SAW devices can detect very tiny vapor concentrations, making them electronic noses. Analyte changes in acoustic wave characteristics: due to their high sensitivity, they’re commonly utilized in defense to detect TNT, RDX, and other explosives.^83,84 Several cancer cells release certain gas vapors which are specific to the type of cancer and can be treated as a biomarker for cancer. Hence, the gas/vapor detecting ‘electronic bio nose’ has significant importance in the present era. IDT (micrometallization patterns made of aluminum, gold, platinum (and its alloys), titanium, conducting ceramics, etc.),⁷⁴ piezoelectric substrates (quartz, Gallium Arsenide, etc.), and chemically active thin surfaces with affinity are the basic components of a SAW device. SAW biosensors can detect even a little change in mass or viscosity, which affects the acoustic wave’s frequency response.⁸³ SAW devices have better sensitivity than QCM devices because their operating frequency range is a few MHz to GHz.⁸⁵Figures 7 and 8 show the SAW sensor and two-port delay line with the sensing screen and analyte gas/vapor molecules.

Figure 7.

Schematic diagram of a SAW sensor.⁸⁶ [Reprinted with permission from Liu, J.; Lu, Y. Response Mechanism for Surface Acoustic Wave Gas Sensors Based on Surface-Adsorption. Sensors 2014, Vol. 14, Pages 6844–6853 2014, 14 (4), 6844–6853. 10.3390/S140406844]. Copyright [2014] [Liu et al. Licensee MDPI, Basel, Switzerland].

Figure 8.

(a, b) Two-port delay line with analyte on the sensing film.⁸⁴ [Reprinted with permission from Devkota, J.; Ohodnicki, P. R.; Greve, D. W.; Luo, J.; Xuan, W.; Fu, R. Y. Q. SAW Sensors for Chemical Vapors and Gases. Sensors 2017, Vol. 17, Page 801 2017, 17 (4), 801. 10.3390/S17040801]. Copyright [2017] [Devkota et al. Licensee MDPI, Basel, Switzerland].

A brief consolidation of piezoelectric biosensor research over the years is shown in Table 1.

Table 1. Brief Survey of Piezoelectric Biosensor Research.

Piezoelectric technique/ device used	Analyte	Bioreceptor	Sensitivity	Ref
8 μm thick lead magnesium niobite-lead titanate piezoelectric plates	DNA	DNA probe	10–19 mol/L in urine samples	(87)
SAW biochip	Escherichia coli l-asparaginase	Chip immobilized polyclonal antibody (pAb) as the bioreceptor	Method quantified E. coli l-ASNase.	(88)
SAW biochip	HIV	Functionalized (ink jet printing) dual-channel biochips were with capture coatings to detect either anti-p24 or anti-gp41 antibodies,	100% sensitivity (for anti-gp41) and 64.5% (for anti-p24)	(89)
ZnO based FBAR	Tumor marker Mucin (MUC1)	Streptavidin self-assembled gold electrode	4642.6 Hz/nM	(90)
Flexural Plate Wave (FPW) gravimetry	Mc5	Gold particles attached to a secondary Ab (Goat Anti-Mouse IgG) and NP5	3-fold higher at 5V, with 180 ° out of phase acoustic streaming IDTs, compared to no mixing.	(91)
SH-SAW (love mode)	Interleukin-6 (IL-6)	Immobilized monoclonal IL-6 antibody	Could detect even in the fg range	(92)
Lead zirconate-lead titanate glass piezoelectric microcantilever sensor	Marker of cancer Her2	Antibody	0.06 nmol/L	(93)
QCM	Drug melphalan	Molecularly Imprinted Polymer from electropolymerized 3-thiophene acetic acid	5.40 ng/mL	(94)
QCM	Taurine	Electrochemically polymerized I-methionine with molecularly imprinted taurine	0.12 μmol/L	(95)
Piezoelectric PVDF platform	COVID-19 virus	polyvinylidene fluoride (PVDF) unsmooth nanofibers-based MEMS	Fempto molar sensitivity, introduced a mathematical model	(96)
Piezoelectric biosensor	immunoglobulin Y	carboxymethyl chitosan (CMCS)+ antibody matrix	270 ng/mL	(97)
Piezoelectric biosensor	Identifying anomalies related to a thrombus in cardiovascular grafts	NaNbO3 fibers) in elastomeric matrix	130 mV m N1–	(98)

2.2.1.2. Electrochemical Biosensors

More than half a century has passed since the introduction of the first electrochemical sensor on the market.⁹⁹ The electrochemical biosensor takes advantage of electrical conductivity-dependent electrochemical interactions between electrodes and electrolytes.¹⁰⁰ Scopus data on biosensor publications from 2018 to 2024 February are shown in Figure 9 indicating a rise during the period of COVID-19.

Figure 9.

Scopus data from 2018 to 2024 February on biosensor publications.

In this recent pandemic scenario, the values are still rocketing. This popularity of electrochemical biosensors can be attributed to their high-sensitivity transducer qualities, low cost, and capacity to integrate with the next generation of downsizing technologies in the nano regime.⁴² The electrochemical biosensor has multiple modes of operation, which are depicted in Figure 10.¹⁰¹

Figure 10.

Schematic diagram of different working modalities of an electrochemical biosensor.¹⁰¹ [Reprinted with permission from Sumitha, M. S.; Xavier, T. S. Recent Advances in Electrochemical Biosensors – A Brief Review. Hybrid Advances 2023, 2, 100023. 10.1016/j.hybadv.2023.100023]. Copyright [2023] [Sumitha M S, Xavier T S, Elsevier B.V.].

MXenes, a novel class of two-dimensional transition metal carbides, nitrides, and carbonitrides, exhibit exceptional electrochemical, mechanical, and catalytic properties, making them suitable for electrochemical sensor and biosensor applications. Mohanapriya et al. provided a comprehensive analysis of the synthesis, design, and engineering techniques utilized to enhance the unique properties of MXenes for the detection of environmentally and biologically significant analytes, including nitro and phenolic compounds, metal ions, biomolecules, and small molecules. Considerable focus is placed on the role of MXenes in point-of-care electrochemical detection systems, highlighting their potential for innovative and effective identification of pollutants and advantageous chemicals. This analysis reviews recent advancements and methodologies in MXene-based electrochemical sensing technologies to offer perspectives on future progress.¹⁰² Wang et al. described the creation of a very sensitive nanonanosensor for detecting microRNA-21 (miR-21), a biomarker linked to colorectal cancer, utilizing electric field force to align multiwalled carbon nanotubes (MWCNTs) on the electrode surface. The independent MWCNT electrode, with enhanced active sites, demonstrates a 150-fold increase in peak current responsiveness compared to a bare electrode. The sensor has remarkable sensitivity (48.24 μA μm^–1), a broad detection range (0.01 × 10^–15 to 100 × 10^–12 m), and a low detection limit (1.2 × 10^–18 m), making it an essential tool for the early diagnosis of colorectal cancer through miR-21 detection in human serum.¹⁰³

Li et al. fabricated an ultrasensitive CNT-Pt composite electrochemical sensor with a printed wrinkled carbon nanotube (CNT) nanostructure for the concurrent detection of dopamine (DA) and uric acid (UA). The sensor, designed to emulate the architecture of taste buds, features a biomimetic wrinkled CNT layer with nanoscale Pt electrodes, enhancing sensitivity by around 40% relative to traditional Pt sensors. The distinctive wrinkled structure and hydrophilicity of the CNT sheet improve detection precision, significantly minimizing interference from ascorbic acid (AA). The sensor exhibits significant promise for electrochemical analysis, especially in the diagnosis of metabolic illnesses by the measurement of DA and UA.¹⁰⁴

The generation or consumption of charged particles, which can be detected by a voltage change or a current change, is a defining feature of the electrolyte solution upon the interaction between the immobilized biomolecules and the analyte molecules in the sample.^105,106 A variety of electrochemical biosensor approaches for detecting a wide range of analyte molecules are reviewed here.

2.2.1.2.1. Amperometric Biosensor. In amperometry, the oxidation or reduction that occurs at the electrode as a result of the interaction between the analyte and the biorecognition element is measured at a constant or variable voltage. It is possible to use the electric current produced to determine the concentration of the analyte.¹⁰⁶ The current in Clark’s amperometric glucose detector is first determined by the oxygen concentration at the oxygen electrodes. Figure 11 shows a schematic representation of a first-generation glucose sensor.

Figure 11.

Schematic picture of the first-generation glucose sensor.

The KCl solution is placed between a Pt (Platinum) cathode and an Ag (silver) anode electrode. Gluconic acid and hydrogen peroxide are byproducts of the oxidation of glucose, which enters the cell through the bottom membrane. H₂O₂ and gluconic acid are produced when glucose is exposed to oxygen. The result is less oxygen at the cathode and a smaller amount of electricity flowing through the circuit. It is also feasible to use a relative potential of + 0.68 V to cause the following reactions without having to measure current in terms of oxygen concentration:

4

5

All commercially available glucometers have a disposable test strip where a small amount of blood is measured. Figure 12 depicts a typical glucometer and each strip includes electrodes, enzymes, and separators.

Figure 12.

Photograph of a commercially available glucometer.

A blood-sugar level of 80–120 mg/dL, while fasting is considered normal. The ideal glucose sensor would be able to accurately detect both hypo and hyperglycemia.¹⁰⁷ The electrochemical biosensors have developed from this to artificial intelligence-assisted real-time monitoring nano biosensors and due to the recent developments in the biosensor business, we now have implantable, continuously monitoring sensors that can quantify the transfer of enzyme to electrode.¹⁰⁸ Numerous diseases, including cancer, Alzheimer’s, Parkinson’s, heart problems, premature aging, and others, have been linked to free radicals, also known as Reactive Oxygen Species (ROS).¹⁰⁹ In 2015, Mauro Tomassetti and colleagues created an amperometric sensor with the enzyme superoxide dismutase (SOD) and used it to detect the antioxidant capabilities of various mixed berries, comparing the results to those obtained using spectrophotometric and spectrofluorimetric methods.¹¹⁰ Here, Pt serves as the anode, and Ag/AgCl/Cl serves as the cathode. Interest enzyme is immobilized in a Kappa-carrageenan gel sandwiched between a cellulose acetate and commercially available dialysis membrane (A-9777).¹¹⁰ Changes in current due to hydrogen peroxide release were recorded by adding xanthine oxidase and xanthine solutions to the sensor’s phosphate buffer solution at regular intervals and in optimized proportions. The xanthine concentration in both the control and test samples was plotted against the slope of the current fluctuation. The slope was found to be steeper for the one that lacked antioxidant properties.

Electrospun Cu/CNF electrodes are more sensitive than undoped electrospun CNF, suggesting that they may be a promising amperometric biosensing candidate for catechol, as reported by Jiapeng Fu et al. Cu/CNFs/Lac/Nafion/GCE was found to have a sensitivity of 33.1 A/mM.¹¹¹ Glass carbon electrode. GCE was utilized as the working electrode, Pt wire was employed as the counter electrode, and Ag/AgCl (saturated KCl) was used as the reference electrode in the amperometric experiments. At an operating potential of 0.5 V, the current–time curve of Cu/CNFs/Lac/Nafion/GCE is depicted in Figure 13. Calibrating the plot is included here as an insert (current Vs concentration).

Figure 13.

Amperometric reading for Cu/CNFs/Lac/Nafion/GCE electrode¹¹¹ [Reprinted with permission from [Fu, J.; Qiao, H.; Li, D.; Luo, L.; Chen, K.; Wei, Q. Laccase Biosensor Based on Electrospun Copper/Carbon Composite Nanofibers for Catechol Detection. Sensors 2014, Vol. 14, Pages 3543–3556 2014, 14 (2), 3543–3556. 10.3390/S140203543]. Copyright [2014] [Fu et al, Licensee MDPI, Basel, Switzerland].

Polymer films of the thiophenes (2,2′-BT) and (4,4′-bBT) were electrochemically polymerized by Maria Pilo and colleagues to entrap glucose oxidase. Real samples, such as pears, apricots, peaches, etc., had their glucose levels measured, and it was discovered that a thinner polymer layer generated at the electrode resulted in a lower detection threshold, which could have an impact on how well the enzyme attached to the electrode.¹¹² Some cationic metal complexes will bind to double-stranded DNA electrostatically in a process called Indirect DNA hybridization if they are present.¹¹³ As a result of using a DNA intercalator called PIND-Ru, Gao and colleagues were able to speed up the oxidation of amines and detect DNA at a lower limit (1.5 pmol/L) at 0.65V (Ag/AgCl) in their studies.¹¹⁴ An amperometric sensor electrode containing a biotin-streptavidin horseradish peroxide complex was utilized by Wang et al. to detect promyelocytic leukemia/retinoic acid receptor alpha fusion gene in a concentration range of 0.05 to 5.0 nmol/L.¹¹⁵ To identify West Nile Virus (WNV), Lonescu, et al. created a femtogram (fg) sensitive DNA sensor.¹¹⁶

2.2.1.2.2. Potentiometric Biosensor. Variations in voltage are measured to deduce analyte concentrations, and hence voltammetry and potentiometry are likewise included among the electroanalytical techniques. The concentration of the biomolecule to be detected is proportional to the potential of the working electrode, which is determined by the difference between the reference and working electrodes when no current is flowing. The logarithmic format is commonly used to record the concentration vs potential difference data. Because of their sensitivity, portability, moderate cost, and real-time fast measurements¹¹⁷ electrochemical sensors, particularly those using potentiometric characterization tools, are well established, and can be used to detect a wide range of organic or inorganic materials, including urea, sugar, neurotransmitters, biological entities, etc.¹¹⁸ Several commercial potentiometric glucose sensors exist; these employ standard glass pH electrodes for their measurement. The pH will drop as a result of the electrochemical reaction with the glucose oxidase occurring at the electrode, and this will be measured. Between 2004 and 2007, Pusoschi et al. immobilized the reactive enzyme in several glucose sensors using membranes such as Nylon, cellophane, nitrocellulose, etc.^119−122 Enzyme entrapment on a platinum electrode has also been achieved with a polypyrrole sheet.¹²³ There are several commercially available methods for determining cell viability or toxicity, including dye exclusion (Trypan blue, eosin, Congo red, erythrosine B assays), colorimetric (MTT assay, MTS assay, XTT assay, WST-1 assay, WST-8 assay, LDH assay, SRB assay, NRU assay, and crystal violet assay), and fluorometric (Alamar Blue assay).¹²⁴ These traditional approaches typically rely on cellular metabolic activity, which does not lend itself to a rapid and precise measurement.¹²⁵ Potentiometric cell viability testing has the advantage of providing fast, real-time, continuous data for a variety of analytes, regardless of their potential concentration.^125,126 A negative potential is generated by the adherence of cells to the electrodes; this potential is then measured with a standard.^{125,127−129} Tuberculosis (TB) can be detected in many ways, including the Tuberculin Skin Test (TST; DTH measurement),¹³⁰ IGRAs (interferon-gamma release assays; ELISA (QuantiFERON V R-TB), ELISpot (T-SPOTV -TB test),^130,131 Antigen Detection Assay (IDA; Immunochromatography),¹³² PCR based nucleic acid amplification tests,^133−135 etc. Voltammetry can be carried out in several different ways; however, the most common and well-established methods are cyclic voltammetry (CV), square wave voltammetry (SWV), and differential pulse voltammetry (DPV), all of which show the current response as peaks that are proportional to the concentrations of the analyte being measured. The different sensor advancements for the detection of Mycobacterium tuberculosis and tuberculosis biomarkers are discussed in a 2019 review by Behrouz Golichenari et al.¹³⁶ The voltammetric biosensor created by Chi Hung Tzang et al. is highly sensitive to the detection of formate and glucose 6-phosphate.¹³⁷ Voltammetric responses at 100 mV/s for a 3,4-DHB modified GC electrode in 0.1 M phosphate buffer solution (pH 7.0) and different concentrations of NADH and NADPH and the lower detection limits for these compounds, respectively, are 2.5 × 10^–5 M and 1.0 × 10^–4 M. The toxicity to cells can be understood using potentiometric measures. Using a potentiometric assay, Ramya Kolli and colleagues determined how different electrodes affected cell survival.¹²⁵ The work of Yashas etal showcases the creation of a polyphenol oxidase (PPO) biosensor to detect catechol, utilizing a matrix of strontium copper oxide (SrCuO₂) and polypyrrole nanotubes (PPyNT). The manufactured sensor has a longer shelf life and great reproducibility. The wide linear range of 1–50 μM is where the suggested biosensor finds its use. Using the devised sensor, a real sample analysis of tap water, mineral water, and home wastewater revealed adequate recovery. Thus, the biosensor aims to be used in environmental protection and monitoring.¹³⁸

Formaldehyde is produced in the human body and is found in food, animal products, and bodily fluids. To detect the presence of formaldehyde, Y.H. I. Korpan et al. developed an ion-selective field effective transistor. Using cells of the methylotrophic yeast Hansenula polymorpha, they were able to produce alcohol oxidase (AOX) with an enzyme-based sensor response time of one minute and a cell-based biosensor configuration response time of two minutes.¹³⁹ To detect urea in blood serum with a long shelf life of nearly five months, S. V. Marchenko and colleagues developed two pH-sensitive FETs, one with BSA (Bovine Serum Albumin) in PVA/SbQ photopolymer as the reference and the other with immobilized recombinant urease in the same photopolymer platform. Rapid responses of 60–120 s were achieved throughout a sensitive detection range of 0.5–40 mM.¹⁴⁰ To detect uric acid in real time, Juliana de Fa’tima Giarola et al. developed a platinum electrode and polypyrrole/uricase/graphene composite, (Pt-PPy/UOx/Grap) and measured a sensitivity of 0.541 nmol/L.¹¹⁷

2.2.1.2.3. Conductometric Biosensors. Like amperometric and potentiometric biosensors, conductometric biosensors benefit from a lack of a reference electrode, allowing them to function more effectively at lower alternating voltages by preventing Faraday processes at the electrodes. These biosensors also allow us to create compact, portable conductometric biosensors for use in point-of-care diagnostics.¹⁴¹

There was research into conductometric glucose sensors in the 1990s; Shul’ga et al. used thin film interdigitated metal electrodes, and the detection limit was in the 0.01 mM range.^142,143Equations 6 and 7 outline the fundamental enzymatic processes:¹⁴¹

6

7

Renal impairment may be indicated by a decreased urine urea level and an increased blood urea level.¹⁴⁴ An increase in positive and negative ions produced by the initial enzymatic reaction will cause a shift in the biosensors’ membrane conductivity. In 2012, Saiapina and colleagues created a conductometric l-arginine detection biosensor based on an arginase-urease system.¹⁴⁵

Conductometric biosensors have been used by Hnaien and others to detect Escherichia coli (E. coli),¹⁴⁶ protein denaturation,¹⁴⁷ and other phenomena. Compared to the original protein, the conductivity was found to be drastically different after protein denaturation (nearly a 3-fold difference).¹⁴⁷ Hnaien et al. also compared conductometric bienzymatic sensor action for the detection of myoglobin release with the UV/vis spectroscopy characterization technique.¹⁴⁸ It is possible to detect substances such as lactose,¹⁴⁹ maltose,¹⁵⁰ insecticides, and more with the use of a variety of conductometric biosensors. In 2007, Phuong and colleagues created a biosensor to identify GMO soybeans. DNA sensor sensitivity was measured to be 4.28 V /pM, and the reaction time was measured to be less than 60 s. Detection is achieved through a change in conductance brought about by the hybridization of matching DNA sequences.¹⁵¹ Johne’s illness is caused by Mycobacterium avium subsp. Paratuberculosis (MAP), and its source were identified. A conductometric biosensor developed by Okafor et al. has a rapid reaction time of 120 s for the IgG antibody.¹⁵² The choice of electrodes for a conductometric sensor with an interdigitated transducer is crucial for optimizing its performance. Commonly used sensor electrode materials include platinum,^153,154 gold,^155−157 nickel/cobalt,¹⁵⁸ copper,¹⁵⁹ silver,¹⁶⁰ carbon,¹⁶¹ titanium,¹⁶² aluminum,¹⁶² etc.

The relationship between membrane thickness and the active area of electrodes is crucial to the miniaturization of the conductometric transducer, which is easier to build than other electrochemical transducers. Photolithography, thick film printing technology, etc., are just a few of the main methods utilized to create conductometric transducers. Conductometric methods can also be used to determine the catalytic activity of enzymes. In the case of amidases, a change in conductivity results from the creation of ion groups, whereas in the cases of dehydrogenases and decarboxylases, it results from the separation of various charges. Researchers are working on different experimental approaches to address the potential drawbacks of conductometric sensors, such as a high signal-to-noise ratio, low specificity, and electrode polarization during enzyme activities. Glucose oxidase silicalite modified glutaraldehyde (GOxSME-GA) stainless steel electrode was developed by Oleksandr Ye Dudchenko et al., who also compared it to a nonmodified one (GOx-GA) and found that the silicalite modification improved enzyme entrapment on the electrodes, which improved the biosensor’s signal reproducibility and storage stability.¹⁶³Figure 14 shows the SEM image of an electrode from a conductometric transducer.

Figure 14.

SEM picture of a (a) single set of electrodes and the (b) entire bare conductometric transducer zoomed portion¹⁶³ [Reprinted with permission from Dudchenko, O. Y.; Pyeshkova, V. M.; Soldatkin, O. O.; Akata, B.; Kasap, B. O.; Soldatkin, A. P.; Dzyadevych, S. V. Development of Silicalite/Glucose Oxidase-Based Biosensor and Its Application for Glucose Determination in Juices and Nectars. Nanoscale Res Lett 2016, 11 (1), 1–7. 10.1186/S11671-016-1275-2]. Copyright [2016] [Dudchenko et al. 2016, PMCID: PMC 4740475 PMID: 26842792].

2.2.1.2.4. Impedimetric Biosensors. Electrochemical impedance spectroscopy (EIS) is a technique for the sensitive detection of biomolecules that takes advantage of electrical changes at the modified electrode surface caused by biomolecular events such as the binding of receptors or proteins or cells, enzymatic activities, etc. Many types of immunosensors and aptasensors rely on impedimetric measurements to function. The key and basic mechanisms between antigen and antibody are utilized in immunosensors. The presence of an antigen can be understood through an increase in electron transfer resistance caused by the creation of an antigen–antibody complex. The impedance change serves as the basis for impedimetric aptasensors, which detect DNA damage, conformational changes, or the binding of a specific sequence.¹⁶⁴ In this way, the EIS can be thought of as a molecular fingerprinting technology. By connecting capacitors, resistors, and other electrical components in series or parallel, we can create electrical circuits that are equivalent to the various electrochemical reactions. The electrolyte resistance R_s; which depends upon solution conductivity and cell geometry, Charge-transfer resistance (R_ct) at the electrode/electrolyte interface, which can be thought of as the ratio of over potential to current in the absence of mass transfer limitation; double-layer capacitance (C_dl), which is a function of electrode area, nature, the electrolyte ionic strength, and permittivity and mass transfer resistance (R_mt) and Warburg impedance (W) are the characteristic features of impeimetric measurements.¹⁶⁴ Nyquist or Bode plots are used to show the frequency-related impedance spectra, where the straight line region in the low-frequency range represents the diffusion-limited process and the semicircle region in the high-frequency range accounts for the electron-transfer-limited process. For highly rapid electron transfer processes, only the straight line segments will be present, but for slower processes, a larger semicircular segment will be present. The diameter of the semicircle is a measure of electron transfer resistance.¹⁶⁵

Using mercaptobenzoic acid (MBA) and cysteine-coated magnetic Fe₃O₄ NPs on gold electrodes as the DNA immobilization probe, Maurilia and co-workers have developed an impedimetric sensor for the detection of tuberculosis DNA. The use of electrochemical impedimetric sensors has benefits and drawbacks. The EIS is a nondestructive method that addresses disturbances of low amplitude from a steady state. Impedance measurements can be performed with or without the redox couple, making them either faradaic or nonfaradaic.¹⁶⁴ Important parameters that are vital in the selection of a redox probe include charge, hydrophilicity/hydrophobicity, redox couple size, electrode properties, etc.¹⁶⁹ The precision of the measuring instruments and the methods used are crucial in determining the accuracy of the impedance measurement, as stated by Yuan et al.¹⁶⁵

Immunosensors, which employ antigen–antibody complex production with impedimetry as the detecting approach, have been the subject of increased study. Specific antigen binding to an antibody inhibits the Faradaic reaction of the redox pair, leading to a significant increase in electron transfer resistance relative to the initial state.¹⁶⁶ Epidermal growth factor receptor (EGFR) was identified as a cancer marker by Elshafey and colleagues. Anti-EGFR antibodies are more likely to bind to the electrode since it has been modified with G proteins.¹⁶⁷ There have been multiple reports of impedimetric immunosensors for human epidermal growth factor receptor-3 (HER-3),^168−170 PSA,¹⁷¹ etc. Aptamers, which are merely synthetic DNA or RNA strands, outperform antibodies in terms of specificity and stability. Impedimetric techniques allow for three different kinds of detections to be made: by measuring changes in impedance, resistance, or current. Once the aptamer has bound to the target nucleic acid sequence, we can either detect it directly without a label or use a labeled redox active particle or nanoparticle to do so. When using signal probes, for example, we can also employ indirect labels.¹⁷²

Researchers have reported using a wide variety of materials, including ZnO,¹⁷³ the core/shell structure of FePt/ZnS,¹⁷⁴ Thiol-modified DNA,¹⁷⁵ Streptavidin-modified DNA,¹⁷⁶ graphene oxide-modified Gold NPs,¹⁷⁷ etc., to boost the sensitivity of DNA hybridization and amplify changes in the charge transfer resistance (Rct) for improved impedimetric detections. The development of impedimetric biosensors based on enzymes is also actively being researched. Affinity-based attachments or self-assembled monolayer-based immobilization are being exploited for more selective linking of the target molecule.¹⁷⁸ A biosensor based on impedimetric measurements was developed by Reddy and colleagues to detect Creatinine. As little as 20 ng/mL was detected.¹⁷⁹ Using glucose oxidase, Shervedani et al. successfully commercialized a glucose impedimetric sensor with a detection limit of 15.6 μM.¹⁸⁰ Using the breakdown of glutamate oxidase as a catalyst, Maalouf et al. created a glutamate biosensor with a detection limit of 20 μM.¹⁸¹ Detecting cells is vital in the medical field for identifying dangerous pathogens and cancerous cells. The phospholipid bilayer that makes up cell membranes provides the membrane with an insulating value of about 10^–7 S/m¹⁸² and keeps ions out until ion channels are opened.¹⁸³ In this analogy, each ion channel corresponds to a parallel resistor,¹⁸² with the total resistance ranging from 105 to 1 M Ω μm.^2,184,185 An increase in impedance is measured and used for sensing when the desired cells adhere to a working electrode, thereby decreasing the available area. To detect ssDNAs, several research teams have reported using impedimetric biosensors that immobilize materials such as gold nanoparticles, graphene,¹⁸⁶ etc. Droplet evaporation on an electrode has been reported by Alam et al. to trigger osmoregulatory pathways, allowing for the detection of 104 living cells/mL and the discrimination between naturally occurring and genetically engineered S. typhimurium, as well as between the living and dead E. coli and S. epidermidis.¹⁸⁷

Understanding how mammalian cells interact with artificial surfaces in real-time is made possible by EIS technology.¹⁸⁸ Also, impedimetric sensing is a breeze with the lab-on-a-chip method.¹⁸⁹ A microorganism-detecting impedimetric lab-on-a-chip with a total fluidic path volume of 30 nL was fabricated by Gomez and colleagues. By injecting a viable cell suspension of Listeria innocua, the impedance properties were altered according to the metabolic activities of the bacterium.¹⁹⁰ With the use of closed dielectrophoretic (DEP) cages, Medoro et al. created a parallel lab-on-a-chip impedimetric system. The prototype was based on commercially available printed circuit board technology, and particle presence was confirmed by a change in the electric field above the electrode location.¹⁹¹

In light of the current COVID-19 situation, it would be highly beneficial to increase the number of tests utilizing EIS, which can be used to analyze pulmonary edema, respiration rate, body temperature, and lung composition.¹⁹² Results from a study using AD5933 to take bioimpedance measurements at a frequency range of 10 kHz to 100 kHz with a four-electrode interface indicated some encouraging trends.^193,194 Recent research by Minsoo Kim et al. produced an impedimetric biosensor for detecting vascular endothelial-derived growth factor (VEGF) with a detection limit of 0.5 pg/mL, making it a potentially useful tool in the clinical diagnosis of VEGF-related ocular illnesses.¹⁹⁵ A consolidated analysis of electrochemical biosensor performance is tabulated in Table 2.

Table 2. A Brief Consolidated Electrochemical Biosensor Performance.

Electrochemical detection technique	Disease or infectious agent	Sample/analyte/enzyme used for detection	Electrode/immobilization	Limit of detection	Ref
Amperometric	Glucose	Glucose oxidase	Polyimide-laser-engraved porous graphene (LEPG)/Pt NPs/Glucose oxidase	0.3 μM	(196)
Amperometric	Sarcosine (prostate cancer biomarker)	Sarcosine oxidase	GCE/Chitosan+Ti based MXene/SO	18 nM	(197)
Amperometric	Cancer marker L-fucose	The recombinant dehydrogenase domain (PQQ domain: residues 240–649) of Coprinopsis cinerea pyranose dehydrogenase (CcPDH)	The isolated PQQ domain from CcPDH /(AuNP)/SAM	13.6 μM	(198)
Amperometric	Uric acid	Uricase	Uricase+Boron nitride nanosheets/ Nafion	0.14 μM	(199)
Enzymatic Amperometric	Nitrite	Salivary nitrite	Cyanocobalamin (VB12) bionic enzyme-assisted system	1 nM	(200)
Amperometric	miRNAs	(His-Tag-Zinc Finger Protein/ dsRNA)+ (biotin/ synthetic complementary RNA detector probe)+ streptavidin–horseradish peroxidase conjugate	SPE	0.91 nM	(201)
Amperometric	Catecholamine	Tyr (tyrosinase)/EDC(1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide)-NHS(N-hydroxysuccinimide)/ChitNPs nanocomposite	The screen-printed graphene electrode with ChitNPS+ Tyr enzyme	0.17 μM	(202)
Enzymatic Amperometric	Uric acid	Uricase	(AuNPs/TiS2 + Uricase/Nafion) SPE	0.18 μM	(203)
Amperometric	Glucose	Nonenzymatic	Nickel NPs + graphene	0.0152 μM	(204)
Cyclic voltammetry	Alicyclobacillus acidoterrestris	DNA	Reduced graphene oxide(rGO)/ a polymer derived from 3-hydroxybenzoic acid/a specific DNA probe sequence complementary with the genomic DNA of A. acidoterrestris	12 ng/mL	(205)
SWV	Saxitoxin (STX)	STX aptamer	Porous Pt NP’s Round-type microgap electrode (RMGE)/aptamer	4.669 pg/mL	(206)
Potentiometric	Urea in sweat	Polyaniline ink, urease bioink, and a polyvinyl chloride membrane	Screen-printed carbon electrodes (SPCEs)	5 to 200 mM at pH 7	(207)
DPV	Mtb	DNA	AuNP/probe/Target DNA/probe/MP/SPCE	0.01 ng/mL	(208)
Label free voltametric aptasensor	Inflammatory response checker- Lipopolysaccharides (LPS)/endotoxins	Au NP-thiol functionalized LPS binding Aptamer	The PEI-rGO-MoS2 nanocomposite	3.01 × 10–5 ng/mL	(209)
Conductometric	Salmonella Enteritidis	monoclonal anti-Salmonella Enteritidis antibodies	polyaniline/zinc oxide (PANI/ZnO) nanocomposite film on a gold electrode	6.44 CFU/mL in 0.1% peptone water	(210)
Impedimetric	SARS-CoV-2	Spike (S) protein	secondary antibody-gold nanoparticle (AuNP) conjugates and target Abs/IDEs (dielectrophoresis (DEP))	200 ng/mL (Au NP) and 2 μg/mL (DEP method)	(211)
Impedimetric	L-hydroxyproline (L-hyp)	3-aminophenylboronic acid (3-APBA)	3-APBA + o-phenylenediamine (OPD)/L-hyp/screen-printed electrode	0.13 μg/mL	(212)
Impedimetric	Escherichia coli O157:H7	Bacterial phage	AuNPS/multiwalled carbon nanotubes (MWCNTs)/tungsten oxide nanostructures (WO3)	3.0 CFU/mL	(213)
Impedimetric	SARS-CoV-2	angiotensin-converting enzyme 2 (ACE-2) transmembrane receptor	ACE-2/indium tin oxide electrode (ITO)	10.0 pg/mL (ITO-APTES-mVero) and 7.25 pg/mL(ITO-APTES-mCalu)	(214)
Impedimetric	-	Thrombin	Histidine-labeled thrombin-binding aptamers (TBA)/Cu2+/poly(pyrrole-nitrilotriacetic acid film	4.7 × 10–12–5 × 10–10 mol/L	(215)
Impedimetric	caspase-9	-	zinc oxide and copper oxide at the electrode–solution interface	0.07 U/mL (0.032 μM)	(216)

2.2.1.3. Infrared and Raman Spectroscopy Facilitated Biosensors

Numerous health issues and early illness diagnostics rely on the detection of biofluids containing lipids, proteins, nucleic acids, etc. Due to the complexity of biomolecules, chemometrics, and other enhanced statistical approaches are being proposed as necessary for characterization studies of biomolecules using FTIR and Raman Spectroscopy.²¹⁷ The discipline of bio analyte detection relies heavily on vibrational spectroscopy, which consists of Infrared and Raman spectral series that complement each other and encompass a broad range of fingerprint molecule vibrational wavenumbers from 14,000 to 50 cm^–1.^218−220 The primary benefits of utilizing vibrational spectrometry as the biosensor detecting equipment are its blameless sensitivity with extremely low detection limit and its nondestructive susceptibility, which allows the samples to be reused for subsequent analytical procedures.²²¹ Compared to more traditional detection methods, vibrational spectroscopy excels due to its ability to precisely determine the molecular structure composition of an analyte. In addition to these benefits, it is considered a “green” analytical method because only a small amount of potentially polluting chemical reagents and solvents are used.²²² The FTIR spectrum is a plot of light intensity vs wavenumber that can be used to detect transmission or absorption.²¹⁷ Spectroscopy methods like Fourier transform infrared (FTIR) and Fourier transform infrared (FT-Raman) are currently widely utilized for the detection of a wide range of lipids, proteins, nucleic acids, etc.^223,224

In recent years, the study of biomolecular spectroscopy has become increasingly interested in FTIR/Raman studies of the biological activities of specific chemical compounds and its halogen substitutions, such as 2-amino-5-flurobenzoic acid with Cl/Br substitution.²²⁵ As the disease progresses, a noticeable shift in the lipid profile of RBCs will be present.²²⁶ Lack of phospholipids and increased LDL cholesterol and triglyceride levels are described as depression indicators by researchers.^222,227 Additionally, elevated levels of immunoglobulin G (IgG) may be indicative of an infection or inflammatory response in the body, and corresponding alterations in nucleic acids may be indicative of malignancy.^228,229 When combined with chemometrics analysis, spectroscopic techniques like Fourier transform infrared (FTIR) and Raman spectroscopy can be utilized as a potent tool for detecting a wide range of biological components. Principal Component Analysis (PCA), Linear Discriminant Analysis (LDA), Partial Least Squares Discriminant Analysis (PLS-DA), Orthogonal Projection to Latent Structures Discriminant Analysis (OPLS-DA), Stepwise Multiple Linear Regression (SMLR), and Multiplicative Scattering Correction (MSC) are all examples of spectral processing techniques.^219,230,231 Because the amount of light absorbed is governed by the Beer–Lambert equation, FTIR and the attenuated total reflectance approach can be used to estimate biomolecular concentrations.²³² The studies conducted in the field of biosensors employing FTIR and Raman spectroscopy are summarized in Table 3.

Table 3. Recent Biosensing Research Works Using FTIR and Raman Spectroscopy.

Sample	Analyte	Experimental conditions	Tool used	LOD/Y and R2 values/ key features	Ref
Serum	Glucose	Wavenumber scanning in the 1800–400 cm–1	PLSR	R = 0.86	(293)
		Range, 25mW 830nm diode laser
Gaseous Sample	Formaldehyde	SiO2-shelled AuAg alloy nanoparticles/agarose gel	3-methyl-2-benzothiazolinone hydrazine (MBTH) patch (known as M-hydrogel patch)	1.46 × 10–8 mg/mL	(294)
Urine	Platelet-derived growth Factor (PDGF)-BB dimer, biomarker for Chronic Heart Disease and atherosclerosis	Ag Np colloids	SERS+PCA-LDA (Principal component analysis (PCA) combined with linear discriminant analysis (LDA))	87.9% sensitivity	(295)
Serum	MCF-7 tumor marker	Gold nanowires	SERS	LOD 0.2 cells mL–1	(296)
Liquid sample	Sunitinib malate (SM)	filter paper substrates/silver nanoparticles (AgNPs)	SERS	10–10 M	(297)
Liquid sample	carbendazim (CBZ) detection on apple peel	electrospun PVDF/CQDs film/Au-Ag NPs	SERS	1.20 ng/cm2	(298)
Serum	Nucleoside diphosphate kinase NM23-H2 (NDKB)	Au@Ag NPs	MIP-SERS	0.82 pg/mL	(299)
Liquid sample	Zika Virus	silver nano islands	SERS	0.11 ng/mL	(300)

Xie and colleagues used FTIR with PLSR and ATR techniques within a wavenumber range of 1600–900 cm^–1 to efficiently detect glucose in the blood serum.²³³ Albumin and immunoglobulin G (IgG) from serum can be detected by FTIR and PLSR in distinct wavenumber domains, as demonstrated by the work of Perez Guaita et al.,²³⁴ and Spalding et al.,²³⁵ respectively. Using the principal component analysis (PCA) technique over a scan range of 4000–400 cm^–1, Gok and coresearchers distinguished between cancer cells and normal cells.²³⁶ Similarly, Lima et al. used FTIR, PCA-LDA, and GA-LDA to identify ovarian cancer markers in plasma at a wavenumber range of 1800–900 cm^–1.²³⁷

Figure 15 shows the FTIR spectra of biofluids²³⁸ (representing different lipids, nucleic acids, and amide bonds).

Figure 15.

FTIR spectra of biomolecules²³⁸ [Reprinted with permission from Wang, R.; Wang, Y. Fourier Transform Infrared Spectroscopy in Oral Cancer Diagnosis. Int J Mol Sci 2021, 22 (3). 10.3390/ijms22031206]. Copyright [2021] [Wang et al. Licensee MDPI, Basel, Switzerland].

Based on the reviews of the literature, the absorption peaks of amide I, amide II, and amide III are located at 1648 cm^–1, 1544 cm^–1, and 1314 cm^–1, respectively.^224,239 Additionally, peaks at 1644 cm^–1, 1650–1658 cm^–1, 1640–1620 cm^–1, and 1695–1670 cm^–1 are unmistakable vibrational identifiers of the random coil structure, -helic structure, and -sheet structures, respectively.²³⁹ Absorption peaks at 2963 cm^–1, 2922 cm^–1, and 2850 cm^–1 verify the existence of lipids in the biological sample by their corresponding asymmetric stretching vibrations of CH₃, CH₂, and symmetric vibrations of CH₂.²²⁴ Additional evidence that phospholipids are present in the sample may be found at 1057 and 1249 cm^–1.^240,241 There was a significant difference between the serum samples of depressed and healthy individuals regarding the severity of absorption peaks in the lipid profile.²⁴² Proteins and immunoglobulins, which can serve as biomarkers for a wide range of disorders, can be analyzed quantitatively and qualitatively using ATR-FTIR spectroscopy.²⁴³

The molecular interactions between staphylococcal protein A (SpA) and anti-Escherichia coli immunoglobulin G (IgG) were investigated by Kengne et al. using Raman spectroscopy. Since they discovered that the tyrosine amino acid residue in the -helix structure of SpA is crucial for its interaction with IgG, this discovery paved the way for the rapid and simple application of Raman spectroscopy to the detection of disease-causing pathogens.²⁴⁰ The vibrational analysis with Density functional theory calculations of stanozolol and oxandrolone molecules at the B3LYP/6–31G (d, p) basis set was performed by TibebeLemma and colleagues, and it will aid in the detection of a wide variety of steroids.²⁴¹ In Figure 16, the spectra of both living and dead cells are shown.²⁴²

Figure 16.

Comparison Raman spectrum of dead and viable cells²⁴² [Reprinted with permission from Notingher, I.; Verrier, S.; Haque, S. A.; Polak, J. M.; Hench, L. L. Spectroscopic Study of Human Lung Epithelial Cells (A549) in Culture: Living Cells versus Dead Cells. Biopolymers 2003, 72 4, 230–240]. Copyright [2003] [Wiley Periodicals, Inc.].

I. Notingher and colleagues studied and compared dead cells and healthy cells, derived from the A549 lung cell line, and understood the difference from viable cells through a decline in the various peak intensities, such as protein peaks at 1320 cm^–1 and 1342 cm^–1 and phenylalanine at 1005 cm^–1 and also by the sharp dip at 788 cm^–1 and 782 cm^–1 indicating the breakdown of phospho diester bonds and DNA pairs, etc.; which can be used to get information.²⁴² As in Owen et al experimental work, which combined Raman with least-squares fitting for the quantitative detection of topoisomerase II inhibitor, several studies employ Raman spectroscopy to learn more about how medications interact with living cells.²⁴³

2.2.1.3.1. Surface Enhanced Raman Spectroscopy (SERS). Despite its many benefits, Raman spectroscopy has been spurred to find ways to boost the Raman signal, leading to the development of Resonance Raman and Surface Enhanced Raman Spectroscopy.²⁴⁴ Field studies have shown that a boost of roughly 10¹⁶ in the Raman signal can be obtained by combining the two approaches.²⁴⁵ We have known about the SERS mechanism ever since Fleischmann first noticed it in 1974.²⁴⁶ Because SERS may boost a signal by a factor of several, it has the potential to be used for single-molecule detection.^247−250 Using metal nanoparticles like gold, silver, or copper, SERS compensates for Raman’s low cross-section by controlling the local electromagnetic field generated by the analyte biomolecule’s adsorption to the nanoparticle.

SERS can be explained in terms of chemical enhancement due to polarizability or electromagnetic (EM) enhancement that can be attributed to an applied electric field.²⁵¹ The molecular orbital concepts of HOMO (Highest Occupied Molecular Orbit) and LUMO (Lowest Utilized Molecular Orbit) can be used to express chemical enhancement in three distinct ways.^252,253 The simplest and initial method involving noncovalent binding of the analyte with no charge transfer is ground-state chemical enhancement. Metallic contact alters the electrical charge distribution of the analyte, which in turn affects its polarizability. The second method involves the analyte molecule’s covalent binding to the nanoparticle surface, either directly or indirectly, and results in an increase in Raman resonance. Because of these interactions, a new electronic state will be created at the surface, which could be in resonance with the laser, leading to an amplified Raman signal.²⁵¹ Charge transfer resonance, which is related to the transfer of charge between the analyte and the metal nanoparticle, is the third chemical enhancement mechanism. In this case, there is a resonance between Charge transfer transitions and excitation caused by the laser energy and the difference in energy level between the Fermi level of the metal and the HOMO or LUMO energy levels of the molecule. Chemical improvement has a smaller all-around effect than electromagnetic improvement²⁵¹

Surface plasmon resonance (SPR), a unique phenomenon of metallic nanoparticles, has been linked to electromagnetic enhancement; this will be covered briefly here, while a comprehensive description will be given in the optical sensor section. The great sensitivity of SPR is due to the resonant response of the metallic nanoparticles’ free electron charges to the wavelength of an external laser. Nanoparticles made of metal have a highly variable refractive index or dielectric constant. The diameter of the metallic nanoparticle, d, and the wavelength of the incident laser energy, determine the wavelength at which Localized Surface Plasmon Resonance (LSPR) occurs.²⁵⁴ Field studies have revealed that the nature, size, and geometry of the metallic nanoparticle are the determining elements in SPR amplification. Different metal nanoparticles showed varying degrees of enhancement in various parts of the electromagnetic spectrum; for example, silver nanoparticles showed stronger enhancement than gold nanoparticles in the infrared area.²⁵⁴ Because there will be a greater concentration of excited atoms in a bigger volume as a result of quantum confinement, the intensity of the electromagnetic field will be amplified, leading to an increase in the SERS effect. In the nano domain, for example, the SERS effect was amplified over one hundred times when the particle geometry changed from a sphere to a nanorod.²⁵⁵ Enhancement was also observed to be greater for nanoparticles with sharper edges.²⁵⁶ Surface roughness of metallic nanoparticles was found to affect the resonance frequency, and it was shown that an increase in roughness led to a boost in SERS because of increased scattering.^257,258

Researchers Deepak et al. looked into how LPSR affected solar cells by fine-tuning metallic nanoparticles, and they discovered that the addition of plasmonic materials improved scattering cross sections and light absorption.^259,260 Tuan Vo-Dinh looked at silver-coated substrates in the 1990s to see if they may improve the Raman signal used to detect compounds in liquid or gaseous samples. Using SERS technology, a fiber optic sensor, and vapor dosimeter were used to detect and identify the chemicals.²⁶¹ SERS technology is commonly used for the nanomolar detection of neurotransmitters utilizing nanometals. Tiwari et al. used AgNPs for the detection of glutamate (10^–7 M) and γ-amino butyric acid (GABA) (10^–4 M), two amino acid neurotransmitters with a strong link to epilepsy.²⁶² The detection limits of 2 μM for choline, 4 μM for acetylcholine, 10 μM for dopamine, and 0.7 μM for epinephrine were achieved by Siek et al., who created a SERS substrate containing Ag NPs by electrodeposition.²⁶³ Combining SERS with Spatially Offset Raman Spectroscopy (SESORS) allows for more accurate in vivo measurements at greater depths than is possible with either technique alone.²⁶⁴ Here, the light needs to travel laterally when it is gathered from an off-center point before it can be scattered by the surface, making it an ideal candidate for below-ground surveillance.²⁶⁵ Moody et al., used Surface-enhanced Spatially Offset Raman Spectroscopy (SESORS) with the use of Principal Component Analysis (PCA) and multivariate analysis to gain a detection limit of 100 μM for melatonin, serotonin, and epinephrine traces in a cat’s skull.²⁶⁶ Tian et al. employed SERS probes with biocompatible gold nanostars and Raman/FTIR imaging to track the in vivo transport of Mitoxantrone, a cancer medication that can be used in anti-inflammatory therapy for persons with cardiovascular illnesses.²⁶⁷ SERS has been used in numerous studies to identify potential indicators of cardiovascular disease.²⁴⁴ Pucetaite et al. used FT-Raman and SERS to detect uric acid, and their substrate of choice was colloidal Ag NPs.²⁶⁸ Another promising measure for detecting chronic cardiovascular disease is C-reactive protein (CRP). Leucomalachite green (LMG) was reduced to malachite green (MG) by Guo and colleagues, and this transformation was used to detect CRP. Au-SERS substrates with antibody (Ab) adhesions corresponding to CRP, Horse Radish Peroxidase (HRP), and LMG were employed in the experiment.²⁶⁹ Important cardiac marker N-terminal pro-brain natriuretic peptide (NT-proBNP) was detected by He et al. with a limit of detection (LOD) in the subfg mL^–1 range using a SERS sandwich assay tool consisting of Au NPs, Toluidine, Blue reporter, and primary and secondary antibodies in conjunction with the magnetic compound of cobalt (CoFe₂O₄).²⁷⁰

To enhance the SERS sensitivity in molecule detection, Chahinez DAB et al. developed a plasmonic platform based on a cubic pattern of gold spheres. The COSMOL Multiphysics software was used for the simulation, which employed finite element methods. Using both 20 nm Large Plasmonic Nanostars (LPN) and 2 nm sized Small Plasmonic Nanostars (SPN), an 85% SERS boost in the intensity was reported, opening the door to potential future models for detecting biomolecules with a variety of structures.²⁷¹ Tip-Enhanced Raman Spectroscopy (TERS) is a newer SERS instrument that takes advantage of the imaging prowess of Scanning Probe Microscope (SPM) technology by using a metal-coated SPM tip that is placed on the substrate during mounting to produce a strong LSPR when irradiated with a laser.^272−274 This clever method, which may be used for single-molecule detection, allows for high-resolution SERS imaging with a short acquisition time because the tip radius is lower than the diffraction-limited spot size. Another perk of TERS is that the analyte does not have to be in physical touch with the SERS substrate to generate a detectable surface-enhanced Raman signal.²⁷⁵ Functionalizing the substrate with the right nanomaterials was found to increase its stability and affinity for analytes. By altering the AgFON (silver film over nanosphere) substrate with atomic layer deposition (ALD) of subnano level Alumina, Zhang et al. demonstrated an improved anthrax spore detection limit of 1400 from 2600.²⁷⁶

Since adenine has a larger Raman scattering cross section than any other DNA base pair, it can be used for single-molecule identification.^277,278 For DNA or aptamer detection, the employment of intrinsic SERS and TERS has become commonplace. In their SERS experiment, Halas and colleagues attributed the peak shift in vibrational bands at 729 cm^–1 to the DNA base pair adenine, which they detected using biocompatible gold nanoshells encased in a glass substrate.²⁷⁹ In the realm of cancer diagnosis and treatment, they also used the SERS tool’s detection and imaging capabilities to track and analyze dsDNA’s spectral shifts in response to chemotherapeutic agents like cisplatin and transplatin.²⁸⁰ Multiple investigations have shown that TERS is an effective method for direct label-free detection of DNA and RNA, including viral detection.²⁸¹ Tobacco mosaic virus (TMV) was detected in a work conducted by Deckert and colleagues by taking advantage of the interaction between an Ag-coated AFM tip and the envelope protein.²⁸² When compared to semiconductor nanoparticles emitting in the NIR range, SERS imaging tags are superior for tumor recognition even in vivo. In their study, Qian et al. employed PEG/AuNP SERS substrates to successfully measure tumor locations at a depth of 2 cm below the skin.²⁸³

Numerous studies aimed at detecting cancer cells rely heavily on SERS. Twenty MCF-7 breast cancers were found by Zhang et al. Core–shell nanorod SERS substrates for tracking circulating tumor cells in a 1 mL blood serum-like liquid including a mixture of HeLa cells and Human Embryonic Kidney cells.²⁸⁴ SERS (Scintillation Enhanced Recovery after Surgery) is the foundation for REMI (Raman Encoded Molecular Imaging), a technique that accurately pinpoints surgical margins during cancer therapies.²⁴⁴ Epidermal growth factor receptor (EGFR) and programmed death ligand 1 (PGL1) were revealed to be part of a multiple tumor marker breast cancer screening tool discovered by Webb and colleagues.²⁸⁵ It is possible to use photothermal therapy in conjunction with REMI to provide a more targeted surgical approach to cancer treatment.²⁴⁴ Using Au nanoshell SERS substrates and the cancer cell lines H1299 and H522, Park et al. created a biosensor with a sensitivity of 97.3% for exosomes produced by malignant cells.²⁸⁶ The detection of aldehydes using an electronic nose may be useful in the early detection of lung cancer. Qiao and colleagues’ SERS active substrate including Au NPs formed into Au superparticles functionalized with 4-ATP and ZIF-8 metal–organic framework enabled the detection of aldehyde at a 10 ppb level.²⁸⁷ Using Prostate Specific Antigen (PSA), Silica cores, and Raman-labeled Ag NPs, Chang and colleagues created a SERS sensor for prostate cancer with a 0.11 pg mL^–1 detection limit.²⁸² Using an aptamer-based SERS sensor, Yang et al. were able to detect PSA at a level as low as 5 pg mL^–1.²⁸⁸ These Au NPs were functionalized with 4,40-dipyridyl (DP) and a PSA complementary DNA strand, and they were attached to a magnetic NP in a new way.²⁸⁸

In this scenario involving the spread of COVID-19, the requirement for quick and accurate identification of the viral genome is of the utmost importance. For the identification of nucleoproteins like H1N1, Maneeprakorn et al. developed a SERS-based lateral Flow Immunochromatographic Assay. Detection was found to be possible down to 6.7 ng mL^–1.²⁸⁹ A schematic illustration of the quantitative detection of human IgM using an SERS-based lateral flow immune assay is shown in Figure 17.²⁹⁰

Figure 17.

A schematic illustration of the quantitative detection of human IgM using an SERS-based lateral flow immune assay²⁹⁰ [Reprinted with permission from Jia, X.; Wang, C.; Rong, Z.; Li, J.; Wang, K.; Qie, Z.; Xiao, R.; Wang, S. Dual Dye-Loaded Au@Ag Coupled to a Lateral Flow Immunoassay for the Accurate and Sensitive Detection of Mycoplasma pneumoniae Infection. RSC Adv 2018, 8, 21243–21251. 10.1039/C8RA03323D]. Copyright [2018] [The Royal Society of Chemistry].

Utilizing a head-flocked nanopillar SERS substrate, Woo Hyun Kim et al. created a label-free, ultrasensitive cancer diagnostic senor for the marker miRNA.²⁹¹ Reactive ion etching (RIE) with a 10.67 mW excitation at 785 nm was employed to create the nanopillars. Multiple tumor markers miR-10b, miR-21, and miR-373 all had LODs of 3.53 fM, 2.17 fM, and 2.16 fM, respectively, in this experiment.²⁹² Additionally, SERS has the potential to be employed for the detection of a wide range of bacteria.²⁹² Assigning distinctive peaks in bacterial SERS is difficult because of the potential for overlapping peaks of different lipids, proteins, carbohydrates, etc. The outer bacterial cell membrane will separate from the sample bacteria when it comes into touch with the capped plasmonic nanoparticles.²⁹² Consequently, SERS can prove why it is the gold standard in biosensing by offering a large range of analyte possibilities at an unrivaled sensitivity. A consolidated research work exploiting FTIR and Raman spectroscopy is shown in Table 3.

2.2.1.4. Optical Biosensors

As mentioned before, the physicochemical changes that take place upon engagement between the target molecule and the biorecognition element are crucial to the signal transduction process. Because optical signals are more sensitive, insensitive to noise, and stable than other physical signals,³⁰¹ biosensors that use the optical transduction technique have promising applications in the performance arena.³⁰² Corona virus pandemics are currently unfolding over the world, making timely diagnoses and ongoing monitoring of the virus’s genome crucial for containing the outbreak.³⁰³ There are several direct and indirect ways of detecting viruses, but they all have drawbacks, such as being time-consuming, having low sensitivity, and requiring access to specialized equipment.³⁰³ We are reminded of the necessity for a commercially available truly quick sensitive and specific biosensor by the emergence of new viral strains around the world such as Nipah, Zika, COVID-19, etc., which pose a grave threat to human survival. In this case, an optical biosensor would be ideal because it would solve the problem and provide the required data within no time. The next sections will go over the many categories of optical biosensors.

2.2.1.4.1. Surface Plasmon Resonance (SPR). Biosensors based on surface plasmon resonance (SPR) have been available in the industry since the early 1980s²⁵⁴ and have reached remarkable progress by overcoming the limitations in detecting low biomolecular concentrations through evolving innovative techniques and configurations.³⁰⁴

2.2.1.4.1.1. Principle of SPR. Delocalized electron oscillations at a metal-dielectric interface called surface plasmons are in resonance with incident radiation.³⁰⁵ SPR, which is sensitive to the refractive index at the nanometallic border, amplifies the localized electromagnetic fields.^306,307 Thin film optical refractometers detect changes in refractive index brought on by biomolecular interactions.³⁰⁸ The SERS section explains how electromagnetic or chemical enhancement can account for the functioning mechanisms. The localized surface plasmons are responsible for the broad frequency enhancement seen in electromagnetic enhancement, while in chemical enhancement we rely on the amplification of the Raman signals of biomolecules linked to SERS substrates.³⁰⁸Figure 18 is a schematic depiction of the SPR’s operational mechanism.

Figure 18.

Schematic diagram of the SPR sensor operational mechanism³⁰⁹ [Reprinted with permission from Murali, S.; Rustandi, R.; Zheng, X.; Payne, A.; Shang, L. Applications of Surface Plasmon Resonance and Biolayer Interferometry for Virus–Ligand Binding. Viruses 2022, 14, 717. 10.3390/v14040717]. Copyright [2022] [Murali et al. Licensee MDPI, Basel, Switzerland].

By undergoing total internal reflection at small reflecting angles, incident light generates an evanescent field at interface.³⁰⁸ When p-polarized light shines on a metallic interface, it causes the creation of surface polaritons, leading to a dramatic decrease in the reflected light intensity at the angle where SPR waves are induced. This perpendicular electric field component is responsible for the near-surface amplification. Distance (d) over which the intensity decreases to 1/e of the original value is roughly estimated to be λ/2 in the sample buffer above the metallic surface.³¹⁰ When a bioanalyte binds to a functionalized metallic surface, it shifts the SPR angle, which in turn changes the refractive index of the interface; by calculating these shifts, we may determine the concentration of biomolecules at the surface. The biosensor has trouble detecting receptor–ligand interactions at distances greater than 600nm from the sensor chip, which is a key limitation of the SPR sensor. To our relief, however, the sensor’s sensitivity may be adjusted by varying the affinity coatings on the metal surface and the buffer solutions.³⁰⁸ Depending on the electromagnetic wave’s propagation constant, the surface plasmon frequency can be calculated using the dispersion relation shown below:³⁰⁸

8

where β_sp is the propagation constant of the surface plasmon on the interface, λ is the wavelength of incident light, and ε_m and ε_d are the permittivities of metal and dielectric, respectively.³¹¹ Collectively oscillating surface plasmons in the excited state raise the field adjacent to the analyte–ligand interaction at resonance, resulting in a localized electromagnetic enhancement as these surface plasmons transform their energy into electromagnetic waves.³¹² Localized surface plasmon resonance (LSPR) and propagating surface plasmon polariton (PSPP) sensors are the two most common varieties of SPR biosensors.²⁵⁴Figure 19 depicts the LSPR sensor mechanism.³¹³

Figure 19.

Schematic diagram of LSPR mechanism³¹³ [Reprinted with permissions from Unser, S.; Bruzas, I.; He, J.; Sagle, L. Localized Surface Plasmon Resonance Biosensing: Current Challenges and Approaches. Sensors 2015, 15 (7), 15684–15716. 10.3390/s150715684]. Copyright [2015] [Unser et al., licensee MDPI, Basel, Switzerland].

Quantum confinement effects of metallic nanoparticles are relied on to generate strong electromagnetic fields for use in the LSPR sensor.³⁰⁸ Charge density oscillations are generated by incident light striking the nano metallic substrate surface, and LSPs are confined to the surface, giving rise to vertically attenuated electronic oscillations.³¹⁴ The resonant frequency is determined by extinction peaks, which are in turn determined by the refractive index, and the richer electromagnetic fields increase interactions at the interface.³⁰⁸ Using PSPP sensors, in which excitation occurs via the coupling between SPs and photons at the interface, negates the drawbacks of interference and the damage created by direct irradiation of laser in sample.³¹⁵ Here the surface plasmon polaritons waves can travel along the interface.^308,315 Thin-film refractometer spectral extinction measurements of LSPR peak shifts are used for SPR sensor detection.³¹⁶ To properly interpret the findings, we need a firm grasp of how scattering spectra differ from ensemble extinction spectra of the metal nanoparticle.^317,318 In the lab, the shift and refractive index can be determined after locating the LPSR peak.³⁰⁸ Direct, label-free biomolecular detection can be achieved via refractive index fluctuations, as seen by a shift in the sensogram proportionate to the amount of mass adsorbed on the SPR chip surface.³¹⁹

SPR biosensor test for Epstein–Barr virus detection was developed by Riedel et al., and validated by detecting three antigens of the virus at the same time.³²⁰ The SPR biosensor developed by Pimkova and colleagues detects VEGFR-1, the protein marker for myelodysplastic syndrome, with a sensitivity of 25 μg L ^–1.³²¹ Arsenic (III) detector with a 1nM detection level was the contribution of Choi and colleagues while study activities of Pennacchio et al. provided us with an immune-chemical SPR biosensor with a limit of detection of 0.1 nM for the mycotoxin; -patulin.^322,323 The SPR cancer detector developed by Altintas et al. involves immobilizing antibodies against a carcinoembryonic antigen on a gold nanoparticle-built sensor chip with the use of 11-mercaptoundecanoic acid functionalization.³²⁴ When the refractive index of the environment changes, LPSR biosensors use this shift to their advantage, allowing them to detect changes in the spectral composition of the environment.³²⁵ Computational studies reveal that bioligand binding affinities are characterized by association/dissociation features; for example, molecules with relatively low binding affinities or K_i values can be ascribed to strong target recognition.³²⁶ About the HIV protease inhibitor molecule, Ha ma la inen et al. characterized almost 300 structurally distinct compounds.³²⁷ A pharmaceutical firm in Sweden developed a biosensor design called BIAcore TM that uses surface plasmon resonance technology to detect atrazine in real time without using a label and with a detection limit of 0.05 μgL^–1.³²⁸

Recently, Kim et al. have made use of the harmless and selective sticking properties of M13 bacteriophage to fabricate a highly sensitive SPR sensor.³²⁹ Recent years have seen the development of plasmon assisted microscopy of nano-objects (PAMONO) sensors for bioimaging, which are particularly useful for real-time viral quantifications, detection of extracellular vesicles, etc.³³⁰ There are commercially accessible aptamer-based affinity-based SPR sensor platforms. Researchers from several institutions have created SPR-based aptamer sensors specific to diverse avian influenza virus strains and subtypes.^331,332 Despite the current worldwide viral pandemic, there is hope for the future of biomedicine with the development of SPR-aided vaccine designs (VSV-Ebola immunization).³³³

2.2.1.4.2. Optical Waveguide. An optical waveguide, as the name indicates is a structure that guides light energy; usually constructed on a thin substrate using glass or polymer.^334,335 The configuration in which light is confined in the transverse direction comes under the slab waveguide category and that in which light can travel only in the longitudinal direction comes under the stripe waveguide category. Figure 20 gives the schematic representation of the classifications of optical waveguide.³³⁶

Figure 20.

Schematic representation of a classification of the optical waveguide.

Dimensions and nature of material intensely affect the decreasing electric field and its penetration depth. A good sensor candidate is one with a small optical waveguide having a significant difference in refractive index values between the core and cladding.³⁰¹ Two types of biosensors are discussed below whose sensor arm is generally a rib waveguide.

2.2.1.4.2.1. Slot Waveguide Biosensor. To create a slot biosensor, a low refractive index medium is sandwiched between two high refractive index slab waveguides.³³⁷ The polarization charge generated by the dielectric discontinuity interacts with the plasma oscillations existing at the interface. Because of the break in the electric field (the normal component’s), light is restricted in the low refractive index area.³³⁸ Light energy can be stored in the slots with less propagation loss and an assumed optical capacitance in this waveguide design, which is compatible with Complementary Metal-Oxide-Semiconductor (CMOS) technology.³³⁸ Resonance redshift calculations for the biosensor built by Barrios et al. using a slot waveguide with a microring resonator showed a sensitivity of 212 nm/RIU and a limit of detection of 2.3 × 10^–4 RIU.³³⁹

2.2.1.4.2.2. Interferometric Waveguide Biosensors. The optical navigation property of waveguides is utilized in these interferometric waveguide biosensors, making them a type of cutting-edge biosensor. As explained in the earlier section, the analyte adsorption at the interface results in a phase shift in the guided mode due to a change in the refractive index. When the reference signal is combined with the phase-shifted signal, an interference pattern is produced that provides a quantitative reading of the bioanalyte.³⁰¹ The experimental interferometric sensing setup is depicted in Figure 21 with mechanical and electrical noise generators. Figure 22 provides a broad category for interferometric waveguide biosensors, which is briefly explored below.

Figure 21.

Experimental setup of the interferometric sensing system, with mechanical and electrical noise sources³⁴⁰ [Reprinted with permission from Leuermann, J.; Fernández-Gavela, A.; Torres-Cubillo, A.; Postigo, S.; Sánchez-Postigo, A.; Lechuga, L. M.; Halir, R.; Molina-Fernández, Í. Optimizing the Limit of Detection of Waveguide-Based Interferometric Biosensor Devices. Sensors 2019, 19 (17). 10.3390/s19173671]. Copyright [2019] [Leuermann et al, Licensee MDPI, Basel, Switzerland].

Figure 22.

Schematic representation of interferometric waveguide biosensor classification.

2.2.1.4.2.2.1. Mach–Zehnder Interferometer (MZI) Biosensor. The Mach–Zehnder interferometer (MZI) is a type of interferometer that uses two arms, one of which serves as a reference and the other as a sensor. To create the two beams, a single source of monochromatic light is split in half and sent down each of the arms.³⁰¹ Light entering the sensor arm reacts with the analyte, causing a phase shift, while light entering the reference arm remains unaffected. These are put back together, and the result is transformed into fluctuations in field intensity, from which analyte concentrations can be calculated.³⁴¹ Short, schematic explanation of MZI categorization is provided in Figure 23.

Figure 23.

Classification of MZIs based on coupling manners of light.

Misiakos et al. fabricated a broad-band MZI sensor having a detection limit of 10^–6–10^–7 RIU.³⁴¹ In the 90s alone, several hybrid MZI interferometer sensors were developed, and Heideman and co-workers were successful in getting a sensitivity of 4 × 10^–6 RIU.³⁴² The integration of broadband MZI (silicon nitride waveguide) on a lab-on-chip arrangement enhanced the multibiomolecular analyte detection to a sensitive pM level.^343,344

2.2.1.4.2.2.2. Hartman Interferometric Biosensor. Hartman interferometric biosensors came as a solution to reduce the sensor surface complexity of a fully integrated MZI, which led to heavy costs and complicated fabrication techniques.³⁴⁵ The components of the Hartman interferometer include an input grating, waveguiding films, and an optical element. These interferometers were used to detect pathogens, nucleic acid, proteins, etc.³⁴⁶ This category of biosensor is not appreciated and promoted much, as its design was not successful in reducing the complexity needed.³⁰¹

2.2.1.4.2.2.3. Young Interferometric Biosensor. The sensitivity decline issue of biosensors, related to field variations addressed in the Young interferometer sensor, through the well-known configuration known as dual polarization interferometry (DPI); which uses a slab waveguide system where only one transverse direction confines the guided modes (lateral modes-infinite).³⁴⁷ This configuration allows the instantaneous switching of polarization which is the key to immediate analyte detection.³⁰¹Figure 24 represents the working principle of DPI.³⁴⁸ In DPI, the sensor waveguide is in contact with the sample and it is vertically above the reference waveguide. The output from both the waveguide interferes to produce the pattern in the far field. The fringe positions get shifted instantaneously with the modifications or variations in the sample layer.³⁰¹

Figure 24.

Pictorial representation of DPI.

Many researchers have developed an interesting taste in the field of Integration of DPI with other detecting techniques recently, due to the enhancement in the sensing capability. DPI + quartz crystal microbalance (QCM) with dissipation was implemented by Ouberai et al., which studied the effect of different parameters at the interface.³⁴⁹ DPI+ellipsometric studies done by Liang and coresearchers revealed that lower NaCl concentration leads to the uniform distribution of ssDNA (single-stranded DNA) initiators whereas, accumulation on the surface was the outcome of higher concentration values.³⁵⁰ When a sensor chip is Integrated with a Young interferometer, then the interference patterns obtained are recorded by the CCD camera, and fast Fourier transform (FFT) is the technique used for measurements.³⁵¹ Schmitt et al. succeeded in enhancing the detection limit to 9 × 10^–9 RIU for chip-assisted interferometric sensing.³⁵² Even DPI provides real-time quantitative data with high sensitivity; the trouble in the compilation of all the interference fringes and the relatively high cost of the equipment set its limitation.³⁴⁷

2.2.1.4.3. Optical Fiber. Optical fiber is a common type of dielectric waveguide with a circular cross-section, consisting of a core and cladding with different refractive indices. Total internal reflection occurs when light rays traveling from a dense to a less dense medium encounter an angle of incidence larger than the critical angle, resulting in an evanescent field at the interface and a net flow of energy across the reflecting surface as the light rays interact with molecules within the penetration depth.³⁵³ Attenuated total reflection (ATR) sensors take advantage of the fact that reflectance decreases at the contact.³⁵³

The fluorescence emitted by the fluorophore is focused on detectors in TIRF (Total Internal Reflection of Fluorescence) sensors. Optical fiber biosensors can operate based on spectroscopic techniques such as Raman, SPR, fluorescence, phosphorescence, absorption, etc., or a combination of these.³⁵³ The optical properties of silica-based optic fiber were improved through the doping of GeO₂, Al₂O₃, B₂O₃, and F, and the fiber optic biosensor may be divided into intrinsic and extrinsic types.³⁵⁴ Researchers Ferreira and co. were able to successfully use an optical fiber biosensor to detect E. coli 0157:H7 in the early 2000s.³⁵⁵ The results showed that the detection limit of E.Coli varied from 6.0 × 10³ CFU/mL ± 1 log for polystyrene to 6.0 × 10³ CFU/ mL ± 1 log for silica.³⁵⁶ Geng and co-workers designed an optic fiber immunosensor that could detect Listeria monocytogenes in food with a sensitivity of 4.3 × 10³ CFU/mL.³⁵⁷ The biotinylated selective antibodies were immobilized on polystyrene fibers and used to detect Bacillus anthracis spores in less than 60 min with a limit of detection of 3.2 × 10⁵ spores/mL, as demonstrated by the work of Tims and Lim et al.³⁵⁸ Polystyrene fibers sped up the reaction time for Salmonella typhimurium detection from 40 to 20 min, and the limit of detection was increased to about 5 × 10⁵ CFU/mL.³⁵⁹

M. Yunianto and colleagues created a fiber optic sensor for measuring serum cholesterol levels by transmitting light via an optical fiber that had been damaged.³⁶⁰ Serum concentrations between 140 and 250 mg per deciliter (mg/dL) were shown to be optimal. A UV–vis spectrometer test and a light spectrometer test were conducted. The UV–vis spectrometer showed excellent linearity (0.96), while the light spectrometer test showed 0.94 linearity in white light LED. When imaging fiber optic microarrays, Epstein and coworkers employed a modified epi-fluorescence microscope with a xenon arc lamp for excitation.³⁶¹ Fibers were placed into a big piece and positioned using an X-Y stage; imaging was performed using a charge-coupled device (CCD) camera with a resolution of up to 3 m diameter sensor.³⁶¹

Literature from 2016 discusses several techniques for fabricating polymer optical fiber sensors,³⁶² various optical fiber designs,³⁶³ and the imbuing of molecular imprinting technology (MIT) with surface plasmon resonance (SPR) and long-range surface plasmon resonance (LSPR).³⁶⁴ The work of Anuj K. Sharma and colleagues describes the several types of optic fibers and LPSR methods that have been developed over the past decade to improve the area of fiber optic sensors. Fiber optic SPR sensor developments in terms of detecting capabilities are discussed in this review.³⁶⁵ Single-mode fibers (SMFs) are optical fibers that have a core diameter between 1 and 10 m and support only the fundamental mode (Gaussian-shaped field distribution across core and cladding) in two orthogonal polarizations.³⁶⁵ The light interaction of SMF sensors occurs in the fiber itself, allowing for minor phase shifts in the modulated light, which contributes to their renown for great sensitivity. Multimode fiber (MMF), in which light can propagate along many channels, was developed because of the limitations imposed on SMF sensor performance by Polarization instability as a result of temperature fluctuations and mechanical deformations. In this case, the diameter of the core is between 75 and 200 m.³⁶⁵

Graphene, as discovered by Fu et al., increased the sensitivity of a biosensor based on fiber optic surface plasmon resonance.³⁶⁶ Zhang et al.‘s unique, promising design provides the answer to many real-world problems in the field of optical fiber biosensors. Two tandem SPR sections are integrated into a single optical fiber in this design, which eliminates the impact of temperature fluctuations and noise-related issues.³⁶⁷ Kanso et al. used analyte modeling in their simulation research to look at how the roughness of metal layers affected the performance of SPR sensors.³⁶⁸ The performance of an optic fiber sensor is critically dependent on the interaction of surface plasmons with the analyte at resonance, which is related to the dip in the SPR curve.³⁶⁵

DNA–protein interaction can be detected with the use of Pollet et al.’s fiber optic SPR sensor, which is made possible by a self-assembled monolayer (SAM) of polyethylene glycol (PEG) functionalized with Streptavidin that coats the gold-layer-sputtered optic fiber.³⁶⁹ The SPR characteristics of Siver nanolayers were used by Sharma et al. for the accurate detection of A, B, and O blood types.³⁷⁰ According to theoretical studies, ITO-coated optic fiber sensors are more sensitive than gold-coated ones, and the SPR wavelength of conducting metal oxides like Indium tin oxide (ITO) is in the Infrared range.³⁶⁵ Although optical fiber sensors can be integrated into several ways for many different analyte detection purposes, their availability in a user-friendly approach is slightly limited by difficulties for miniaturization and its relatively higher cost, both of which can be eliminated through future research works.³⁷¹

2.2.1.4.4. Optical Resonator. When light is restricted to move within a cavity, self-interference occurs, resulting in an electrical oscillation with specific resonant frequencies.³⁷¹ Here, any modification in the resonant parameters will be unveiled as a change in the light intensity, making the microcavity an optical signal transducer.³⁰¹ The optical cavities are divided into the following categories whose Q factor is the parameter that defines its efficiency:³⁷²

• Fabry–Perot microcavity

• Whispering Gallery Mode (WGM) resonator

• Asymmetric Cavity

• Photonic Crystal Cavity

2.2.1.4.4.1. Biosensors Based on WGM Resonator. In the WGM, the resonant modes got confined in the cavity obeying Snell’s Law and undergoing the total internal reflection.³⁷³ The mathematical representations of the relation between resonant frequencies and cavity radius are given in Equation 9(373) and that between transmitted and input power is given in Equation 10:³⁷⁴

9

where the resonance wavelength is represented by λ, RWGM is the radius of the cavity, n_eff is the effective mode index of the WGM mode, and m is the polar order of the WGM modes;

10

where the symbols represent the following parameters. P_out (ω) – transmitted power; P₀ – input power; K – coupling coefficient (determines how much energy is coupled into the cavity); ω₀ – resonance frequency; fwhm – full-width at the half-maximum of the resonance spectrum profile.

Jiangang Zhu et al. developed a novel group of nano couplers based on nano scatterer’s Rayleigh Scattering and plasmonic effects and successfully demonstrated the whispering gallery microlaser optical effects.³⁷⁵ The coupling of free space light into whispering gallery modes is represented in Figure 25.

Figure 25.

(a) Due to Purcell enhancement, almost all the light is collected into the cavity mode when the nanoscatterer is near the cavity. (b) Schematic representation of the experimental setup for characterization of nano coupler. PC-polarization controller, WDM-wavelength division multiplexer, NPDS nanoparticle delivery system. (c) Finite element simulation showing that free space light cannot couple into a WGM resonator by direct illumination. (d) Magnified view of the WGM area shown in c. (e) Shows how effectively the nanoparticle on the surface of resonator helps for the excitation from free space. (White dots are nanoparticles)³⁷⁵ [Reprinted with permission from Zhu, J.; Özdemir, S. K.; Yilmaz, H.; Peng, B.; Dong, M.; Tomes, M.; Carmon, T.; Yang, L. Interfacing Whispering-Gallery Microresonators and Free Space Light with Cavity Enhanced Rayleigh Scattering. Sci Rep 2014, 4 (1), 6396. 10.1038/srep06396]. Copyright [2014] [Zhu et al. Nature/scientific reports].

WGM resonators are available in different configurations, such as microspheres, microring, microdisk, etc.³⁰¹ In the spherical cavity, only certain modes are allowed due to self-interference of the recirculating rays; which is very different from the “open-loop” evanescent field sensor principle of optical fibers or waveguides.³⁷⁶ Both the resonance condition and the dielectric properties within the evanescent field will get changed due to the size increase, which happened because of the adsorption, and it will be perceived as a redshift, obeying the relation as shown in equation 11:³⁷⁶

11

This relation becomes crucial in the biomolecular detection scenario, as the biomolecular layer thickness becomes significant compared to sphere size at regime³⁷⁶

Research shows high Q factor values for the microsphere resonators in the near IR-VIS regions, making it suitable even for single molecule detection.³⁰¹ Liquid-core optical ring resonator (LCORR) where the light is confined along the perimeter can be combined with microfluidics, which will promote the sensing ability and such a design put forward by Andres et al. for glucose detection exhibited a limit of detection value less than 10^–6.^377,378 Silicon-on-insulator (SOI) wafers where silicon substrate and waveguides are separated by a silica buffer can be combined with MEMS (Micro Electro Mechanical Systems or with recent NEMS (Nano Electro Mechanical Systems) for multianalyte detection.³⁴⁹ Nadgaran and coresearchers’ design using triangular nanoprisms of gold coupled with hybrid WGM microtoroids delivered a Q factor value of 3.6 × 10⁴ and a red shift of 26.45 fm while detecting a single BSA protein.³⁷⁹

2.2.1.4.5. Others. 2.2.1.4.5.1. Photonic Crystal-Based Biosensors. Photonic crystals (PCs) are characterized by a photonic band gap (PBG), which is the frequency range over which propagation of light inside the crystal is not possible. The presence of defects whose shape, size, etc., can be tuned, will introduce localized photonic energy states in the gap, resulting in higher field confinement, small mode volume, and low extinction loss and thereby making the PC a better candidate in the detection field.³⁸⁰ A sensor for virus detection having a limit of detection of 1.4 nM was designed by Miller and coresearchers, in which they made a channel for the propagation of modes within the band gap of a 2D PC silicon slab with a triangular lattice of air holes, by removing the central array of air holes.³⁸¹Figure 26 schematically represents the working of an IgG detector using a photonic crystal developed by Park et al.³⁸² A color change from green to orange (observed using an optical microscope with a CCD camera) occurred on adding 10 mg/mL of IgG solution, which was the result of a large band gap shift of the order of 50 nm (measured using spectrum analyzer)

Figure 26.

Schematic representation of IgG detector using photonic crystal³⁸² [Reprinted with permission from Choi, E.; Choi, Y.; Nejad, Y. H. P.; Shin, K.; Park, J. Label-Free Specific Detection of Immunoglobulin G Antibody Using Nanoporous Hydrogel Photonic Crystals. Sens Actuators B Chem 2013, 180, 107–113. 10.1016/j.snb.2012.03.053]. Copyright [2012] [Elsevier B.V.].

An avidin detection of the order of 15nM was executed through an integrated system of PC sensors and a PDMS microfluidic system designed by Scullion et al.,.³⁸³ A portable, fast, user-friendly biosensor for detecting the influenza virus (H1N1) with a detection limit of 138 pg/mL was designed by Park and coresearchers, in which optical signals from quantum dot aptamer beacons were amplified by the three-dimensional PC and were captured using an ordinary smartphone camera.³⁸⁴ The working principle is shown in Figure 27.

Figure 27.

Principle of the “OFF–ON” detection utilizing quantum dots conjugates and nanoporous PC. (a) “OFF” state, i.e., loading the prepared quenched quantum dots conjugates. (b) “ON” state, i.e., capturing the H1N1 virus and recovering quantum dots signal. (c) Quantum dots signal was enhanced by the emission light propagating in the PC structure to make the virus detection visualized³⁸⁴ [Reprinted with permission from Lee, N.; Wang, C.; Park, J. User-Friendly Point-of-Care Detection of Influenza A (H1N1) Virus Using Light Guide in Three-Dimensional Photonic Crystal. RSC Adv 2018, 8 (41), 22991–22997. 10.1039/C8RA02596G]. Copyright [2018] [The Royal Society of Chemistry].

Figure 28 is the photo and SEM image of a sensor made with a flexible PC structure designed by Xu et al.³⁸⁵ Zhao and coresearchers gave out the successful integration of microneedle arrays with PC barcodes.³⁸⁶ These multianalyte detecting bar code patches which can be put on just like a sticker, in the body, open up a way toward the painless, noninvasive, fast, multifunctional futuristic sensor platforms (Figure 29).

Figure 28.

(a) Photo of the proposed microcavity sensor which was made up of a flexible monocrystalline PC structure. (b) SEM image of the PC microcavity³⁸⁵ [Reprinted with permission from Xu, X.; Subbaraman, H.; Chakravarty, S.; Hosseini, A.; Covey, J.; Yu, Y.; Kwong, D.; Zhang, Y.; Lai, W.-C.; Zou, Y.; Lu, N.; Chen, R. T. Flexible Single-Crystal Silicon Nanomembrane Photonic Crystal Cavity. ACS Nano 2014, 8 (12), 12265–12271. 10.1021/nn504393j]. Copyright [2014] [American Chemical Society].

Figure 29.

(a) Sketch of application of the proposed microneedle arrays which were integrated with PC barcodes in the detection of biomarkers in skin interstitial fluid. (b) Optical images of the microneedle arrays with one barcode loaded in a single tip.³⁸⁶ [Reprinted with permission from Zhang, X.; Chen, G.; Bian, F.; Cai, L.; Zhao, Y. Encoded Microneedle Arrays for Detection of Skin Interstitial Fluid Biomarkers. Advanced Materials 2019, 31 (37), 1902825. 10.1002/adma.201902825]. Copyright [2019] [John Wiley and Sons].

2.2.1.4.5.2. Optical Biosensors with Nanomaterials. The properties like large surface-to-volume ratio, quantum confinement, etc. of nanomaterials make them a good candidate in the biosensor field.³⁸⁷ The major advantage of using nanomaterials in the detection field is that physio-chemical reaction outputs are tunable in the case of NPs.³⁸⁷ A brief description of the classification of Nanomaterial optic biosensors is explained in the following sections.

2.2.1.4.5.2.1. Quantum Dots. Band gap tunability to the desired energy value is the major advantage of semiconductor nanoparticles or Quantum Dots (QD). The smaller the size, the larger the band gap, and vice-versa. In addition to this, properties like photostability, high quantum yield, broad absorption, narrow emission, etc. further help the QD to be the first choice for making a sensitive fluorescent bioanalyte detector.^388,389 QD does not need multiple excitation sources as its excitation wavelength will be distant from emission wavelengths, enabling it to be used in multiplex arrays. QDs are excellent fluorescent energy transfer (FRET) sensor candidates as there is no significant overlapping between the excitation and emission spectra.^388,389Figure 30 represents the schematic diagram of the size dependency of QDs.³⁹⁰

Figure 30.

A schematic representation of the band structure in solids: (a) quantum confinement effect on changing quantum dot size; (b) surface trap sites with their electronic energy states localized within the QDs bandgap³⁹⁰ [Reprinted with permission from Mansur, H. S. Quantum Dots and Nanocomposites. WIREs Nanomedicine and Nanobiotechnology 2010, 2 (2), 113–129. 10.1002/wnan.78]. Copyright [2010] [John Wiley and Sons].

Several QD fluorescent pathogen detectors are available such as detectors for E. coli O157:H7,³⁹¹ Bacillus thurinigensis,³⁹² respiratory syncytial virus, etc.; which exploit the QD-FRET principle.³⁹³ Several issues need to be addressed while considering the QD–antibody conjugation. They are

• 1).The number of antibodies per QD control is very difficult.
• 2).The proper functionalization needs proper orientation of the antibody or aptamer, control which is also difficult.
• 3).Room temperature denaturing may happen to antibodies, hence they need to be cryopreserved. But at the same time, QD cryopreservation is not advisable.³⁸⁷

Long and the research group developed carrier-protein haptens and conjugated with the QDs to overcome the practical difficulties while dealing with QD/antibody group.³⁹⁴ Carboxyl QDs were conjugated with fluorescently labeled antibodies in the experiment to detect 2,4-dichlorophenoxyacetic acid (2,4-D) (pesticide). 2,4-D/BSA conjugates were allowed to interact with the optical immunosensor probe. Increasing concentrations of the analyte lessened the fluorescence, and the detection limit was 0.5 nM.³⁹³Figure 31 represents the Schematic QD-based FRET genosensor.³⁹⁵ Here the dye-sensitized complementary DNA conjugates with the QD-DNA hybrid and that results in the QD-sensitized dye FRET signals indicating the presence of labeled DNA.³⁹⁶

Figure 31.

Schematic QD-based FRET genosensor³⁹⁵ [Adapted with permission from [Zhang, H.; Feng, G.; Guo, Y.; Zhou, D. Electronic Supporting Information (ESI) Robust, Specific Ratiometric Biosensing Using a Copper-Free Clicked Quantum Dot-DNA Aptamer Sensor Experimental Section; 2013]. Copyright [2013] [The Royal Society of Chemistry].

CdTe QD functionalized with Citrus tristeza virus (CTV) coat protein derived antibody performs as a specific analyte detecting helper in CTV detecting sensors.^397,398 Shojaei et al. developed a sensor using the antibody derived from CTV coat protein attached to Au NP and CdTe.³⁹⁹ Coat protein on QD and recombinant coat protein on rhodamine 123 were designed as the donor–acceptor pair in a sensor that used FRET detection,³⁹⁷ and they are schematically represented in Figure 32.

Figure 32.

Schematic presentation of specific CVT nano biosensor³⁹⁷ [Reprinted (Adapted with permission from Reza, M.; Samiee, F.; Tabatabie, M.; Mohsenifar, A. Sensors and Transducers Development of Quantum Dot-Based Nanobiosensors against Citrus Tristeza Virus (CTV); 2017; Vol. 213. http://www.sensorsportal.com].Copyright [2017] [IFSA Publishing, S. L, http://www.sensorsportal.com/HTML/DIGEST/P_RP_0227.htm].

Table 4 illustrates different QD configurations used for various biomolecular detection.

Table 4. Tabular Representation of Different QD Configurations for Biomolecular Detection.

^a(Adapted with permissions - References (400−406)) (400) Yeh, H.-C.; Ho, Y.-P.; Wang, T.-H. Quantum Dot–Mediated Biosensing Assays for Specific Nucleic Acid Detection. Nanomedicine 2005, 1 (2), 115–121. 10.1016/j.nano.2005.03.004. Copyright [2005] [Elsevier Inc.]. (401) Singh, S.; Chakraborty, A.; Singh, V.; Molla, A.; Hussain, S.; Singh, M. K.; Das, P. DNA Mediated Assembly of Quantum Dot–Protoporphyrin IX FRET Probes and the Effect of FRET Efficiency on ROS Generation. Phys. Chem. Chem. Phys. 2015, 17 (8), 5973– 5981. 10.1039/C4CP05306K. Copyright [2015] [The Royal Society of Chemistry]. (402) Gill, R.; Willner, I.; Shweky, I.; Banin, U. Fluorescence Resonance Energy Transfer in CdSe/ZnS-DNA Conjugates: Probing Hybridization and DNA Cleavage. J Phys Chem B 2005, 109 (49), 23715–23719. 10.1021/jp054874p. Copyright [2005] [American Chemical Society]. (403) Abu-Salah, K. M.; Zourob, M. M.; Mouffouk, F.; Alrokayan, S. A.; Alaamery, M. A.; Ansari, A. A. DNA-Based Nanobiosensors as an Emerging Platform for Detection of Disease. Sensors 2015, 15 (6), 14539–14568. 10.3390/s150614539 [www.mdpi.com/journal/sensors ]. (404) Bae, P. K.; Chung, B. H. Multiplexed Detection of Various Breast Cancer Cells by Perfluorocarbon/Quantum Dot Nanoemulsions Conjugated with Antibodies. Nano Converg 2014, 1 (1), 23. 10.1186/s40580-014-0023-5. Copyright [2014] [Bae and Chung; licensee Springer]. (405) Zhang, C.; Johnson, L. W. Quantum-Dot-Based Nanosensor for RRE IIB RNA-Rev Peptide Interaction Assay. J Am Chem Soc 2006, 128 (16), 5324–5325. 10.1021/ja060537y. Copyright [2006] [American Chemical Society]. (406) DAI, Z.; ZHANG, J.; DONG, Q.; GUO, N.; XU, S.; SUN, B.; BU, Y. Adaption of Au Nanoparticles and CdTe Quantum Dots in DNA Detection* *Supported by the Natural Science Foundation of Tianjin (Nos. 06TXTJJC14400, 07JCYBJC15900) and Young Teacher Foundation of Tianjin Polytechnic University (No. 029624). Chin J Chem Eng 2007, 15 (6), 791–794. 10.1016/S1004-9541(08)60004-X. Copyright [2007] [Chinese Journal of Chemical Engineering]. (Reprinted with permissions - Reference (407)) (407) Ai, X.; Niu, L.; Li, Y.; Yang, F.; Su, X. A Novel ß-Cyclodextrin-QDs Optical Biosensor for the Determination of Amantadine and Its Application in Cell Imaging. Talanta 2012, 99, 409–414. 10.1016/J.TALANTA.2012.05.072. Copyright [2012] [Elsevier B.V.].

2.2.1.4.5.2.2. Gold Nanoparticles. In the earlier sections, details of several gold nanoparticle-based SPR biosensors -were discussed. Generally, size-dependent color change or FRET is the method of detection used in gold-based biosensors.³⁸⁷ Several Gold NP-based sensors are available for the detection of heavy metal ion presence. 5ng/ml detection limit was obtained for the Hg2+ ion detection through the sensor developed by Darbha and co-workers.⁴⁰⁸ Cysteine conjugated Gold NP sensor made Cu2+ ion detection rapid (sensitivity 10^–5 M) and gave for visual approval with a color change.⁴⁰⁹ Long et al in their review on optical biosensors mentioned the detector developed by Cheng et al. using a rhodamine 6G conjugated Au NP fluorescent sensor for Hg²⁺ detection with a 0.012 ppb detection limit.³⁸⁷ In 2012, Fang Luo and coresearchers developed a novel fluorescent assay detection method using gold nanoparticle.⁴¹⁰ Only the presence of the target molecule revealed the fluorescence, as there were enhancements of fluorescent dyes and size changes to the ssDNA-attached gold NP. The model was able to conduct in vitro-specific detection of adenosine, thrombin, and cocaine. Figure 33 illustrates the schematic representation of aptamer-based fluorescence biosensors using unmodified AuNPs.⁴¹¹

Figure 33.

Schematic representation of gold nanoparticle-based colorimetric detection assay format.⁴¹¹ [Reprinted with permission from Mondal, B.; Ramlal, S.; Lavu, P. S.; N, B.; Kingston, J. Highly Sensitive Colorimetric Biosensor for Staphylococcal Enterotoxin B by a Label-Free Aptamer and Gold Nanoparticles. Front Microbiol 2018, 9. 10.3389/fmicb.2018.00179]. Copyright [2018] [Mondal, Ramlal, Lavu, N and Kingston, Frontiers in Microbiology]

Zeng et al. coupled horse radish peroxide (HRP) with gold NP to find out the glucose level.⁴¹² Han et al. developed a colorimetric ELISA with gold NP for the detection of ractopamine.⁴¹³ The H₂O₂ will oxidize gold (III) ions and will result in a red color. In the absence of ractopamine, NPs will aggregate and will result in a purple color. The limit of detection was 0.35 ng mL^–1 in urine. The design for H₂O₂ detection using gold nanoparticles is schematically represented in Figure 34.⁴¹⁴

Figure 34.

Schematic representation of fabricated hemoglobin/gold nanoparticle heterolayer immobilized on the microgap for H₂O₂ detection⁴¹⁴ [Reproduced with permission from Lee, T.; Kim, T.-H.; Yoon, J.; Chung, Y.-H.; Lee, J. Y.; Choi, J.-W. Investigation of Hemoglobin/Gold Nanoparticle Heterolayer on Micro-Gap for Electrochemical Biosensor Application. Sensors 2016, 16 (5). 10.3390/s16050660]. Copyright [2016] [Lee et al. Licensee MDPI, Basel, Switzerland].

The various steps involved in a sandwich-type gold nanoparticle-based biosensor for E. granulosus are described below and is schematically represented in Figure 35.⁴¹⁵

• Step I: Colloid gold nanoparticles were synthesized using chitosan.

• Step II: Chi–GNPs surface was activated by GA and conjugated with protein A.

• Step III: Hydatid cyst antigen (Ag B) was immobilized on the NC membrane, membranes were blocked with BSA, and then, treated with serum sample, and finally, each sample was dipped into Chi–GNPs–GA–P.A conjugate

Figure 35.

Schematic illustration of sandwich type E. granulosus biosensor⁴¹⁵ [Reprinted from Safarpour, H.; Majdi, H.; Masjedi, A.; Pagheh, A. S.; Pereira, M. de L.; Rodrigues Oliveira, S. M.; Ahmadpour, E. Development of Optical Biosensor Using Protein A-Conjugated Chitosan–Gold Nanoparticles for Diagnosis of Cystic Echinococcosis. Biosensors (Basel) 2021, 11 (5). 10.3390/bios11050134]. Copyright [2021] [Safarpur et al. Licensee MDPI, Basel, Switzerland].

2.2.1.4.5.2.3. Graphene and Carbon Nanotubes. Optical biosensors based on QDs are excellent in terms of their sensitivity and specificity in the case of in vitro detection of biomolecules. What makes biosensors with Graphene a little upper hand, is its biocompatibility in addition to other properties such as excellent mechanical strength, electrical and thermal conductivity, fast electron transportation, and large active surface area available. Electrochemical biosensors with graphene are known for their excellent sensitivity, but graphene’s optical properties are not that much exploited in the sensor fabrication field.³⁸⁷ Wen and coresearchers fabricated a DNA detector working based on surface-enhanced Raman spectroscopy with a graphene-Au NP substrate, which showed a detection limit of 10 pM.⁴¹⁶

CRET (chemiluminescence resonance energy transfer) in which nonradiative energy is transferred from donor to acceptor without any external excitation.³⁸⁷ A 1.6 ng mL^–1 limit of detection was found in a graphene CRET platform sensor developed by He et al., for C-reactive protein (CRP).⁴¹⁷ The advantage of using Graphene Oxide (GO) is that it can undergo easy conjugation with the biomolecules through its functional end groups. Rotavirus detection was executed by using a GO immunosensor in which the virus was attached to the ant-rotavirus functionalized GO substrate when it was introduced; which resulted in the fluorescent quenching of GO.⁴¹⁸ A detailed review of graphene-based biosensors is done by Phitsini Suvarnaphaet and Suejit Pechprasarn in which the biosensor works related to pristine and modified (graphene oxide, graphene quantum dot, reduced graphene oxide) are discussed. Various configurations for optical sensing are schematically categorized in the review.⁴¹⁹Figure 36 schematically represents the graphene, GO, and rGO.⁴²⁰

Figure 36.

Schematic diagram of the formation of graphene, GO, and rGO from graphite⁴²⁰ [Reprinted with permission from Filip, J.; Tkac, J. Is Graphene Worth Using in Biofuel Cells? Electrochim Acta 2014, 136, 340–354. 10.1016/j.electacta.2014.05.119]. Copyright [2014] [Elsevier Ltd. All rights reserved].

2.2.1.4.5.2.4. Magnetic Nanoparticles. Superparamagnetic nanomaterials can be used for specific attachment of the desired analyte molecule and the analyte separation can be easily done. Biosensors for use in a wide variety of applications, including medical diagnosis, food safety, environmental monitoring, etc., have made extensive use of magnetic nanobeads (MNB). There have been numerous reports of the use of magnetic beads ranging in size from nanometers to micrometers for the separation and detection of a wide range of biological and chemical targets, such as Escherichia coli O157:H7,^421,422Salmonella typhimurium,⁴²³Listeria monocytogenes,⁴²⁴ Avian Influenza A Virus,⁴²⁵ cancer cells,^426,427 and so on. Because of their many advantages over larger magnetic microbeads (>500 nm in diameter), such as greater specific area, faster reaction kinetics, less steric hindrance, and more homogeneous distribution, 20- to 200-nanometer-diameter magnetic nanobeads (MNBs) have attracted a lot of attention in recent years. In many of their uses, MNBs must do double duty:⁴²⁸

• as a magnetic carrier, allowing for the separation of targets from complicated samples to reduce background interferences; and

• as a magnetic label, amplifying signals to improve detection sensitivity.

When produced, magnetic nanobeads (MNBs) typically have two parts: a magnetic core that provides MNBs with their superparamagnetic capabilities and a polymer shell that confers additional functional groups on MNBs.⁴²⁸ In the presence of an external magnetic field, MNBs are driven along the field lines by their superparamagnetic capabilities, before dispersing once the field is no longer present. By virtue of their functional groups, MNBs can be further modified with a wide range of biochemical components to facilitate selective binding to biological and chemical targets. The magnetic force on the MNBs is proportional to

• 1).the magnetization and
• 2).physical size of the MNBs; as well as
• 3).the magnetic intensity and
• 4).gradient of the magnetic field

making the core material and physical size of the MNBs two important characteristics that play a key role in the behavior of the MNBs under a magnetic field. Out of the many magnetic core materials, many commercial items and scientific studies rely on iron oxides because of their high magnetization, low cost, and outstanding biocompatibility.⁴²⁸

For magnetic nanobeads to couple with specific biological recognition elements for selective binding with targets to accomplish separation, detection, or both, the polymer shell is critical. Coating the cores with polymeric stabilizers/surfactants, depositing really thin layers of inorganic metals onto the cores, or enveloping the cores into liposomes are all common ways to generate polymer shells, which can increase the stability of MNBs by preventing irreversible agglomeration and allowing for more even distribution.⁴²⁹ After proper functionalization with hydroxyl groups and amino groups, further activation with 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) and n-hydroxy-succinimide sodium salt (NHSS) allows the functionalized groups especially the carboxyl or amino groups on the MNBs to bind with proteins such streptavidin, antibodies, protein A, etc.⁴²⁸ Different separation techniques are discussed in Table 5 and illustrated in Figure 37.

Table 5. Comparison of Different Magnetic Separation Techniques.

Conventional magnetic separation	High-gradient magnetic separation	Magnetophoretic separation	Magnetic continuous flow separation	Magnetic grid separation
First, in a tiny centrifuge tube, the MNBs with antibodies are combined with the sample containing targets to generate the MNB–analyte complexes.	After being magnetized by an externally applied magnetic field, the soft magnetic materials in the form of balls or wires in a separation column are then employed to trap the immune MNBs as they stream through the column.	First, enough MNBs are coupled with the sample targets to create the magnetic targets that will serve as the sample flow	Before being evenly dispersed in a fluidic channel, the immune MNBs are caught by an external magnetic field.	The antibody-modified MNCs were initially utilized to create an MNC virtual net in a tiny rectangle glass tube that was wrapped in copper tape.
The sample solution background can then be pipetted away, and the magnetic targets can be caught against the tube wall using an external magnetic field.	The column is constantly fed with a large sampling volume containing the targets, and the targets are separated from the background as the antibodies on the MNBs catch only the targets	A gradient magnetic field is given to a magnetophoretic channel into which the sample and carrier flows are injected.	The sample holding the targets is then injected into the channel, where it is bound specifically to the MNBs.	Then, the tube was injected with the E. coli O157:H7 specimen, which the immune MNCs then captured to create the magnetic bacterium.
The magnetic targets are then resuspended using a reduced volume of buffer solution.	Once the magnetic field has been removed, the magnetic targets can be collected from the column and sent on to the detection step further down the line.	As the magnetic field pulls the magnetic targets in the direction of a greater gradient, the targets are continuously separated from the rest of the sample while the nonmagnetic background stays put in the flow.	Finally, after the magnetic field has been removed, the channel is purged, and the magnetic target complexes are concentrated in a tiny volume of buffer solution.	After the external magnetic field was removed, the magnetic pathogens were finally gathered.
		At last, the magnetic targets are gathered at the allotted exit, where they can be enriched by yet another magnetic field.		With this magnetic grid separation, 85% of the E. coli O157:H7 bacteria could be removed from a 10 mL sample in just 10 min.428
				The creation of the MNC net based on Lenz’s Law was the key achievement because it made it possible for high MNC chains to develop in the separation channel at a high flow rate.
ADVANTAGES (over centrifugation and filtration)	ADVANTAGES	ADVANTAGES (over traditional methods)	ADVANTAGES (over magnetophoretic separation)	ADVANTAGES
(1) High concentration since significantly less buffer solution is needed to resuspend the magnetic targets, and (2) High specificity because of the antibodies’ ability to preferentially react with the targets.	(1) A sizable volume of sample may be separated	(1) Automatic and continuous flow helps to separate the analyte from a large volume of material.	(1). A smaller quantity of the MNBs is needed to achieve a high separation efficiency	With dot-array high gradient magnetic fields, the dispersed MNBs can create MNB chains in the fluid separation channel
(2) Just a minimal quantity of the MNBs is required.		DISADVANTAGES	DISADVANTAGES	The improved control of the MNBs to form chains and distribute uniformly in the channel can be done by using an iron hoop to change the direction of magnetic field lines resulting in a 70%, fast, 1-h separation efficiency of pathogen (Salmonella) from a 50 mL food sample
		(1). A large number of conjugated MNBs are required to raise the cost-effective	(1) The MNBs often clumped together when they were ensnared against the canal wall, with only the top layer of MNBs being able to successfully catch their prey.
		(2). Complete elimination of contaminants is not always possible	(3). In addition, the efficiency of the interaction seen between targets and the MNBs was typically poor because it was based largely on the diffusion of the targets from the liquid phase to the solid phase, where the MNBs were located

Figure 37.

Schematic diagrams of various magnetic separations: (a) Conventional magnetic separation, (b) magnetophoretic separation, (c) magnetic continuous flow separation⁴²⁸ [Adapted with permission from Wang, L.; Lin, J. Recent Advances on Magnetic Nanobead Based Biosensors: From Separation to Detection. TrAC Trends in Analytical Chemistry 2020, 128, 115915. 10.1016/j.trac.2020.115915]. Copyright [2020] [Elsevier B.V. All rights reserved].

Magnetic nanobeads’ electromagnetic responses at a single or dual mixing frequency are used in electromagnetic biosensors because they are proportional to the concentration of magnetic targets. Nonlinear magnetization properties of magnetic nanobeads form the basis for the electromagnetic responses.⁴³⁰ Since MNBs are frequently employed to isolate targets from mixed samples, they provide for a great detection signal in and of themselves. The electromagnetic detector or reader is at the heart of this biosensor and consists primarily of three coils:

• (1)an outer driving coil (onto which a sinusoidal magnetic driving field at low frequency is applied to drive the MNBs into saturation);
• (2)a middle excitation coil, (onto which a magnetic exciting field with high frequency is generated to magnetize the MNBs); and
• (3)an inner detection coil, (onto which the induced signal is picked up to calculate the concentration of MNBs).

This method, known as magnetic frequency mixing, determines the nature of the induced signal. Because the exciting field magnetizes the MNBs, the induced signal is robust when the driving field is shut off and as the MNBs are magnetically saturated when the driving field is turned on to its maximum, the induced signal is faint. The aggregate frequency at which the amplitude of these two signals is greatest is used to measure the concentration of MNBs. At concentrations as low as 2.5 ng/mL,⁴³¹Yersinia pestis may be detected by this electromagnetic biosensor. Also, by coupling this magnetic nanobead detector with a nitrocellulose membrane, a comparable electromagnetic biosensor was described for the rapid detection of two influenza A viruses with a low detection limit of pictograms of virus per well.⁴³²

Electromagnetic biosensors are rapid, easy, and cheap. However, the inherent background noise from heterogeneous MNBs and the poor detection signal from trace amounts of magnetic nanobeads impair electromagnetic biosensor sensitivity and make practical applications difficult. Superparamagnetic MNBs and repeated magnetic induction-based MNB measurements may solve this.

DNA/microRNA,⁴³³ bacteria/viruses,^434−436 and protein biomarkers^437,438 have all been detected using magnetic relaxation switching (MRS) biosensors, a novel form of magnetic nanobead-based biosensor. Depending on whether they assess longitudinal (T1) or transverse (T2) relaxation, MRS biosensors can be classified into one of two categories.⁴²⁹ When MNBs are in a scattered or aggregated condition due to the presence of targets, the T2 of the biosensor might shift, and this shift is typically measured by T2-mediated MRS biosensors. As a result of their interaction with the targets, MNBs in solution can undergo a transition from a dispersed to an aggregated form, which can lead to a drop in T2.⁴²⁹ T2 is larger when no target is present in the sample because MNBs stay scattered; however, T2 is lower when targets are present because MNBs aggregate after being conjugated with them. Salmonella enterica was detected in milk samples down to 103 CFU/mL using this MRS biosensor.⁴³⁹ Poly vinyl alcohol (PVA) functionalized magnetic nanobeads were used to create a T2-mediated MRS biosensor for the detection of boric acid or borate ester (BA/BE) relying on the covalent interaction of the hydroxyl groups of PVA and BA/BE, which leads to their aggregation.⁴⁴⁰

Developed magnetic immunoassay techniques directly detect the magnetic field generated by the magnetically labeled targets using a sensitive magnetometer.⁴⁴¹ Weitschies et al. propose a novel magnetic relaxation/remanence immunoassay (MARIA) employing a superconducting quantum interference device (SQUID) as a magnetic field sensor.⁴⁴² This technique involves immersing an immobilized target in a suspension of superparamagnetic nanoparticles bound to antibodies specific to the target. To align the dipole moments of the particles, a periodic external magnetic field is used. The SQUID detects the magnetic field generated by the target-bound particles. Magnetic marker monitoring (MMM) can provide insight into the digestive tract transit of a solid oral dose form. Only the bandwidth of the data-collecting technology restricts the passage’s temporal resolution. The resulting spatial resolution was on the order of a few millimeters. This is significantly further than what is possible with standard techniques like γ -scintigraphy. MMM’s key benefits are:

• 1).It may be used in any setting without worrying about radiation exposure.
• 2).It provides an opportunity to learn even more about the behavior and fate of solid pharmaceutical formulations as they travel through the digestive tract.
• 3).Furthermore, MMM provides data on how the digestive tract reacts as it passes the magnetically tagged object. This quality may prove especially helpful in identifying digestive system problems.
• 4).The utilization of a minimal quantity of magnetic material to label the pellet, as well as the compact size of the pellet contained within the capsule, indicate that the implementation of MMM is not limited solely to items of considerable dimensions, such as a capsule.
• 5).Moreover, the method of multiple unit pellet system (MMPS) can be readily employed for the encapsulation of any orally delivered, monolithic, pharmacological dosage form.

However, magnetic marker monitoring (MMM) has a restriction of the localization to only one magnetically marked object.⁴⁴¹

Xiao et al. describe in detail the optomagnetic biosensors which employ alternating magnetic fields to generate periodic movements of magnetic labels.⁴⁴³ The strategies discussed are consolidated in the flowchart in Figure 38. As stated earlier in this section optomagnetic sensing strategy requires that the magnetic particles (or aggregates) exhibit some type of optical anisotropy, caused either by (magnetically or binding-induced) clustering/alignment or by the intrinsic shape anisotropy of the particles.⁴⁴⁴ The magnetic field-enhanced agglutination assays (MFEAs) employ an external alternating magnetic field to induce the magnetic particles’ dipole–dipole attraction to enhance interparticle binding kinetics. Both homogeneous and gradient magnetic fields can be used for MFEAs, inducing the formation of magnetic particle chains and clusters in the suspension, respectively. The colliding frequency is significantly increased in MFEAs due to the magnetic force-controlled particle encounter, stirring, and increased local particle concentration (due to the magnetic field gradient), all of which help to overcome the limitations governed by Brownian translational diffusion.

Figure 38.

Volumetric sensing strategies of optomagnetic sensors.

In addition, because of the magnetic interaction between their dipoles, MNPs can self-organize into various shapes when subjected to a magnetic field.⁴⁴³ Magnetic objects, like MNP dimers/multimers or shape anisotropic magnetic structures (like magnetic nanorods), can experience rotational motion in relation to the field when subjected to a rotating magnetic field. Using an oscillating magnetic field for magnetic actuation resulted in less phase lag in magnetic nanoparticles (MNP) than a rotating magnetic field, according to the reports. As a result, bioassays are more sensitive:

• When subjected to a magnetic field, magnetic nanoparticles (MNPs) aggregate due to dipole attraction.

• Distance between MNPs considerably impacts the strength of this attraction.

• Individual magnetic nanoparticles (MNP) can undergo Brownian relaxation without clustering by adjusting their concentration and oscillating field strength/ frequency.

• MNPs are randomly oriented near zero magnetic fields but partially align their magnetic moments under nonzero magnetic fields.

• If MNPs have optical anisotropy, particle rotation changes light intensity. To measure the increase in the hydrodynamic size of magnetic nanoparticles, measure the change in transmitted light’s second harmonic to an oscillating magnetic field.

• This method gave optomagnetic biosensor’s performance comparable to pricey magnetic susceptometers.⁴⁴⁵

Advantages

• Low cost

• Ease of use

Disadvantages

• The imprecise and low limit of detection (LOD) is caused by the increased

• interparticle binding kinetics, which in turn amplify the nonspecific reactions and result in a noisy background.

• The technique of integrating multistep assays onto a chip is intricate.

• Multiplex sensing requires labels or analytes to exhibit distinct signals, whereas

• volumetric optomagnetic sensing lacks the ability to differentiate signals.

A brief consolidation of optical biosensor performance is given in Table 6.

Table 6. A Brief Tabulation of Optical Biosensor Performance.

Transducer	Analyte	Limit of Detection	Ref
SPR	As(III)	1 nM	(323)
LSPR multiarray	Immunoglobulins, C-reactive proteins, fibrinogen	100 ng/L	(446)
LSPR nanochip	Ovarian cancer marker HE4	4 pM	(447)
LSPR (gold nanorods)	Ochratoxin (Mycotoxin)	<1 nM	(448)
MachZehnder Interferometer (MZI) with double-slot hybrid plasmonic (DSHP) waveguide	2-propanol	Sensitivity 1061 nm/RIU	(449)
Broad-band MZI	-	10–6–10–7 RIU	(450)
Hybrid MZI interferometer	-	Sensitivity 4 × 10–6 RIU	(451)
ZnO films	GVA	1 pg/mL–10 ng/mL	(452)
CdSe/ZnS	Methylated DNA	1 aM	(453)
CdSe/ZnS	PSA	0.05 nM	(453)
Fe3O4/CdSe	Cancer cell	98 cells/mL	(454)
Magnetic NP	atrazine	3 pg/L	(455)

2.2.2. Biorecognition Elements Based

The overall well-being of the population relies heavily on the prompt identification of diseases and the subsequent administration of suitable therapy. The successful design of a biosensor depends on a comprehensive understanding of the properties of the biorecognition element and its effects on the bioanalytes. The sensor’s performance requires a specific affinity toward the analyte.⁴⁵⁶ The sensor field employs a wide range of biorecognition elements, which can be classified into many categories as shown in the graphic chart (Figure 39).

Figure 39.

Graphic classification chart showing the different categories of biorecognition elements.

Noticeable elements from each category are discussed further.

2.2.2.1. Antibody-Related Biosensor/Immunosensor

Antibodies are natural protein structures having a three-dimensionality. The structure of antibodies is shown in Figure 40.

Figure 40.

Monoclonal antibody (mAb) structure⁴⁵⁷ [ADCP: antibody-dependent cell phagocytosis; CDC: complement-dependent cytotoxicity; ADCC: antibody-dependent cell cytotoxicity; C: constant; V: variable; L: light chain; H: heavy chain][Reprinted with permission from Liguori, L.; Polcaro, G.; Nigro, A.; Conti, V.; Sellitto, C.; Perri, F.; Ottaiano, A.; Cascella, M.; Zeppa, P.; Caputo, A.; Pepe, S.; Sabbatino, F. Bispecific Antibodies: A Novel Approach for the Treatment of Solid Tumors. Pharmaceutics 2022, 14 (11). 10.3390/pharmaceutics14112442]. Copyright [2022] [Liguori et al. Licensee MDPI, Basel, Switzerland].

Only the specific antigen corresponding to the respective antibody site will come and bind, forming an antigen–antibody immunocomplex whose interaction profiles can be measured through various transducing methods.⁴⁵⁶ Immunosensors are one of the fastest growing sensing technologies because of their fast and specific detection.⁴⁵⁸ Antibody technology used generally is monoclonal antibody (mAb) technology or hybridoma technology⁴⁵⁹ immobilization of Abs on sensor substrates also depends on the sensitivity and limit of detection of the sensor. If the antibody binds through the constant region Fc, then the antigen binding sites will be available to the maximum extent and the sensitivity will be higher. If the binding occurs through the antigen binding sites surrounding the VH and CH regions, the antigen binding possibility will decrease reducing the sensitivity.⁴⁶⁰ In addition to the orientation of attachment of Abs, the extent of linkage to the substrate is also an important factor considering the binding of cognate antigen to the specific antigen binding sites of antibodies.⁴⁵⁶ Getting a specific substrate surface with proper antibody immobilization can be done with functionalization using specific groups such as glutaraldehyde, cabodiimide, succinimide ester, etc.⁴⁶¹Figure 41 and Table 7 represent and compare the different antibody immobilization techniques.

Figure 41.

Antibody immobilizations: (a) noncovalent, (b) covalent, (c) affinity.

Table 7. Comparison of Different Antibody Immobilization Techniques.

Immobilization Approaches (459)
Non-Covalent	Covalent	Affinity
Forces – electrostatic or ionic bonds, hydrophobic interactions, and van der Waals forces	Involves surface modification through the groups such as hydroxy, thiol, carboxy, or amino groups	Covalent immobilization’s disadvantages– partial denaturation and the lack of definite orientation
Adsorption–physisorption or chemisorption	Surface modification techniques–chemical modification, photochemical grafting, plasma gas discharge, ionizing radiation graft copolymerization, etc.	Affinity immobilization involves
Ab entrapment into conducting polymer films is another Ab immobilization		The use of biotin–avidin or immobilizing intermediate binding proteins, such as Protein A or G, first onto the sensor surface onto which Abs are attached.

Graphene-based biosensors are well-known for their excellent sensitivity. Figure 42 schematically represents the immobilization of different biorecognition elements on the surface.⁴⁶² As previously mentioned, the many functional groups on Graphene Oxide (GO) allow for the attachment of numerous types of antibodies. The most versatile strategy for GO Ab functionalization among the three options, namely EDC/NHS chemical conjugation, electrostatic bonding, and bonding through PASE linker, is the first one. This is because it involves a direct attachment between the carboxyl and amine groups. In this method, EDC refers to 1-ethyl-3-(3-dimethylamino propyl) carbodiimide, NHS stands for N-hydroxy succinimide, and PASE represents 1-pyrene butanoic acid succinimidyl ester.⁴⁶³

Figure 42.

Immobilization schemes of different biorecognition elements on graphene surface⁴⁶² [Reprinted with permission from [Peña-Bahamonde, J.; Nguyen, H. N.; Fanourakis, S. K.; Rodrigues, D. F. Recent Advances in Graphene-Based Biosensor Technology with Applications in Life Sciences. J Nanobiotechnology 2018, 16 (1), 75. 10.1186/s12951-018-0400-z]. Copyright [2018] [Janire Peña-Bahamonde et al, Springer Nature].

Various virus and bacteria detectors were developed for using Graphene – Ab platform for clinical diagnostics; for instance, detectors for Zika virus Afsahi,⁴⁶⁴ dengue,⁴⁶⁵ rotavirus,⁴¹⁸ avian flu virus H7(Au NP modified Graphene),⁴⁶⁶ Hepatitis C Virus, HCV (surface modification by AgNP⁴⁶⁷), and E.Coli,⁴⁶⁸Salmonella typhimurium,⁴⁶⁹ etc. Lee et al.,⁴⁷⁰ in 2010 developed a carcinoembryonic antigen (CEA) detector using Aluminum Nitride FBAR (Film Bulk Acoustic Resonator). AIN film deposition was done using the RF Sputtering method. On the substrate, 0.2–10 mg/mL of anti-CEA protein was functionalized and the measuring mode worked at the resonant frequency of 2.477 GHz. After the specific adhesion of CEA on the corresponding Ab, the resonant frequency was shifted drastically, which ensured the CEA presence.

2.2.2.2. DNA Sensor

The pursuit of comprehending the human genome has sparked a revolution in genome analysis, resulting in the creation of DNA-based biosensors capable of detecting a wide range of bioanalytes.⁴⁷¹ The synthesis and reproduction of nucleic acid recognition layers offer a distinct advantage over antibody or enzyme-modified layers.⁴⁷¹ The immobilization of DNA should ensure both accessibility to the target analyte and secure attachment to the substrate.^472,473 DNA molecules can be affixed to the substrate using many methods, similar to the ones mentioned for antibody attachment. For example, thiolated DNA can be connected to a gold nanoparticle substrate, biotinylated DNA can be linked to an avidin-streptavidin complex, or DNA can be simply adsorbed onto electrodes.⁴⁷¹ The sensor’s sensitivity can be enhanced through two methods: utilizing dendrimers, which possess several branch-like functional groups suitable for DNA hybridization,⁴⁷⁴ or employing Peptide Nucleic Acid (PNA) that features a pseudo peptide bond instead of a sugar–phosphate backbone.⁴⁷⁵

Graphene-based DNA sensors utilize π–π interactions for immobilizing DNA and primarily employ electrochemical and fluorescence transducers. These sensors assess fluctuations in physical quantities resulting from DNA hybridization or nucleobase oxidation.⁴⁶² The EDC/NHS immobilization technique is the most versatile method, as previously discussed. Akhavan et al. utilized the presence of sharp and active edges of reduced graphene nanowalls (RGNW) to promote the uniform electron transport between the DNA and the electrode in the DPV mode.⁴⁷⁶ The detection sensitivity for dsDNA ranged from 0.1 fM to 10 mM. The fluorescent DNA biosensor utilizes a fluorescently labeled single-stranded DNA (ssDNA) that is functionalized to the substrate. This ssDNA is designed to be complementary to the target DNA. The operating principle of this biosensor is based on the quenching effect of graphene.⁴⁶² Qaddare et al. created a very sensitive DNA sensor for detecting the HIV-1 gene. The sensor utilizes luminous carbon dots combined with AuNPs/GO as a sensing platform. The sensor is capable of detecting the gene at a femtomolar level of sensitivity.⁴⁷⁷ The chosen measurement technology was FRET (fluorescence resonance energy transfer). The fluorescence was emitted upon hybridization with the target DNA sequence, and the minimum detectable concentration was 15 femtomolar (fM). Systemic lupus erythematosus is an autoimmune illness. Buhl et al. have created a DNA sensor that utilizes SPR technology to identify the presence of disease-causing dsDNA autoantibodies.⁴⁷⁸ In the detection system, human transferrin was modified with biotin, and a synthetic oligonucleotide was linked to its corresponding sequence that matches the specific antigenic structure to be identified.

To functionalize ssDNA tagged with 6-mercapto-1-hexane and Au NPs, Xu et al. created a CuS-Graphene composite platform.⁴⁷⁹ To characterize the material, impedance spectroscopy and cyclic voltammetry were employed. The target DNA was detected as low as 0.1 pM. Schematic illustration of the electrochemical DNA biosensor fabrication process is shown in Figure 43.

Figure 43.

Schematic illustration of the electrochemical DNA biosensor fabrication process⁴⁸⁰ [Reprinted with permission from Wang, T.; Zhang, Z.; Li, Y.; Xie, G. Amplified Electrochemical Detection of MecA Gene in Methicillin-Resistant Staphylococcus aureus Based on Target Recycling Amplification and Isothermal Strand-Displacement Polymerization Reaction. Sens Actuators B Chem 2015, 221, 148–154. 10.1016/j.snb.2015.06.057]. Copyright [2015] [Elsevier B.V. All rights reserved].

Riyadh Abdulmalek Hassan et al. have recently created an innovative electrochemical biosensor to identify carrageenan. They achieved this by immobilizing calf thymus dsDNA on a carbon-based solid-phase extraction (SPE) and employing the redox indicator methylene blue.⁴⁸¹ The measurement technique employed was differential pulse voltammetry (DPV), and the determined limit of detection was 0.08 mg/L. Figure 44 depicts the competitive electrostatic interaction between dsDNA and carrageenan in their conjugation with methylene blue. It also shows the electrical setup of the DNA device utilized in this research study.⁴⁸¹ Despite the high specificity, quick analysis, and multianalyte detection ability of DNA sensors, their significant drawback is in the deteriorating nature of DNA, which necessitates particle analysis and specialized storage settings.

Figure 44.

Schematic illustration of competitive electrostatic interaction of dsDNA and carrageenan toward binding with methylene blue. (a) High DPV signal obtained after accumulation of MB in the dsDNA. (b) Lower DPV signal generated after immersion of the dsDNA electrode in the carrageenan solution. (c) The experimental setup of the three-electrode system consists of a carbon SPE working electrode, an Ag/AgCl reference electrode, and a glassy carbon counter electrode⁴⁸¹ [Reprinted with permission from [Hassan, R. A.; Heng, L. Y.; Tan, L. L. Novel DNA Biosensor for Direct Determination of Carrageenan. Sci Rep 2019, 9 (1), 6379. 10.1038/s41598-019-42757-y]. Copyright [2019] [www.nature.com/scientificreports/].

2.2.2.3. Enzymes-Based Biosensor

Enzyme-based biosensors are readily available and more stable than DNA biosensors.⁴⁶² Developing an enzyme-based biosensor offers advantages such as the production of biocompatible and reusable byproducts, as well as the ability to catalyze specific reactions.⁴⁸² However, high temperatures can negatively impact stability by causing enzyme denaturation, resulting in poor performance.⁴⁶² Various physical factors, including pH, presence of chemical inhibitors, solvent polarity, ionic strength, and temperature, can affect the performance of an enzyme sensor.⁴⁶² Technological advancements such as site-directed mutagenesis, chemical modifications, and recombinant DNA technology can enhance enzyme stability.⁴⁸²Figure 45 illustrates the timeline classification of enzyme-based biosensors,¹⁰¹ while Figure 46 depicts the enzyme-labeled indirect detection of antigen and antibody.⁴⁸³

Figure 45.

Chronological classification of enzyme-based biosensors¹⁰¹ [Reprinted with permission from Sumitha, M. S.; Xavier, T. S. Recent Advances in Electrochemical Biosensors – A Brief Review. Hybrid Advances 2023, 2, 100023. 10.1016/j.hybadv.2023.100023]. Copyright [2023] [Sumitha M S, Xavier T S. Elsevier B.V.].

Figure 46.

Schematic representation of the configuration of enzyme-labeled biosensors for the detection of (a) antigen and (b) antibody.

The preceding segments of the classification within the transducer category encompassed various biosensors that are dependent on enzymes. Hence, the subsequent article offers a succinct summary of enzyme biosensors that rely on graphene. Using graphene as a substrate for enzyme-based biosensors has numerous advantages, including

• higher stability,

• improved enzymatic activity,

• superior immobilization of enzymes, and direct electron transmission between electrodes and enzymes.⁴⁸⁴

Out of the immobilization methods stated before, direct physical adsorption without the use of chemicals is considered the best option for attaching enzymes to the surface of graphene.⁴⁶²

Mechanisms of electrochemical enzyme biosensor⁴⁸⁵

• 1)Based on the catalytic properties of enzymes, i.e., undetectable form to detectable form of analyte.
• 2)Based on enzyme activity, i.e., inhibition or moderation.

The laccase and horse radish peroxidase (HRP) enzymes are widely utilized in the biosensor business because of their versatility and cost-effectiveness.⁴⁶² Enzymes can be immobilized using a combination of reduced graphene oxide (rGO) and a magnetic Fe₃O₄ substrate, resulting in improved sensitivity.⁴⁸⁶ In a study by Filip et al., they immobilized bilirubin oxidase on a graphene oxide (GO) substrate to identify aberrations in liver function.⁴⁸⁷ Glucose sensors utilizing glucose oxidase (GOx) are widely prevalent in the industry. Feng and his colleagues developed a detector using a composite substrate of graphene and polyaniline. This detector achieved a detection range of 10 μM–1.48 Mm.⁴⁸⁸

2.2.2.4. Phage Sensor

A “bacteriophage” is a type of virus that specifically infects harmful bacteria cells and inhibits their reproduction, ultimately causing their destruction through lysis. Figure 47 depicts the mechanism of action of a bacteriophage.

Figure 47.

Influence of phage deficiency in acne, atopic dermatitis, and psoriasis⁴⁸⁹ [Reprinted with permission from Natarelli, N.; Gahoonia, N.; Sivamani, R. K. Bacteriophages and the Microbiome in Dermatology: The Role of the Phageome and a Potential Therapeutic Strategy. Int J Mol Sci 2023, 24 (3). 10.3390/ijms24032695]. Copyright [2023] [Natarelli et al. Licensee MDPI, Basel, Switzerland].

Bacteriophage-based biosensors are rapidly emerging as a promising sensor option for the particular detection of pathogens.^490,491 According to Lu et al., the combination of phage cocktails or collections of phage-derived recognition proteins can be utilized to precisely target pathogenic bacteria.⁴⁹² Richter confirmed the effectiveness of targeting a specific strain of pathogen using a specific bacteriophage.⁴⁹³ Bioprobes can range from whole phage probes to phage display peptide (PDP) probes to the more recently developed receptor binding proteins (RBP) from phage probes.⁴⁹⁴ Srivastava and colleagues created a sensor capable of detecting bacteria. The sensor has a limit of detection (LOD) of 100 CFU/mL. This was achieved by immobilizing the T4 phage on a modified Si/Ag film/glutaraldehyde/4-aminothiophenol substrate.⁴⁹⁵ The transduction approach they employed was SERS technology. Horikawa et al. conducted research using the E2 phage to detect strains of Salmonella enterica ser and Salmonella typhimurium. They employed magnetoelastic transduction techniques for this purpose.⁴⁹¹

Ullah et al. found that the presence of functional groups like carboxyl, hydroxyl, and aldehyde on the surface of bacteriophages allows them to interact with various protein groups on the surface of the analyte cell. These functional groups also assist in immobilizing the bacteriophages on the substrate surface.⁴⁹⁶ Anany et al. stated that to immobilize the phage systemically, the substrate surface should be functionalized with positively charged groups. This is because the head of the phage carries a net negative charge, allowing the positively charged tail portion to bind to the receptors on the target bacteria.⁴⁹⁷ Different methods, including laser printing, adsorption, and inkjet, for the immobilization of phages on paper strips are also available. Fernandes and his colleagues devised a magneto-resistive phage sensor by fixing phage onto gold nanoparticles.⁴⁹⁸Figure 48 illustrates a schematic representation of how an impedimetric phage sensor operates.⁴⁹⁹

Figure 48.

Schematic illustration of impedimetric biosensor based on T4-phage. (A) Cysteamine is assembled on a gold electrode. (B) Stimulation by cross-linker, i.e., 1,4-dithiocyanate (PDICT). (C) T4 phage immobilization and blocking with the addition of ethanolamine. (D) E. coli cells capturing. (E) E. coli cell detection, based on impedimetric /LAMP dual-response⁴⁹⁹ [Reprinted with permission from [Tlili, C.; Sokullu, E.; Safavieh, M.; Tolba, M.; Ahmed, M. U.; Zourob, M. Bacteria Screening, Viability, And Confirmation Assays Using Bacteriophage-Impedimetric/Loop-Mediated Isothermal Amplification Dual-Response Biosensors. Anal. Chem 2013, 85 (10), 4893–4901. 10.1021/ac302699x]. Copyright [2013] [American Chemical Society].

Rajnovic et al. have recently created a detection system that has a limit of detection (LOD) of 108 pfu/mL. This technique relies on the significant reduction in optical density resulting from cell lysis. The researchers tested over 90 distinct combinations of bacteria and phage concentrations.⁵⁰⁰ Merwe et al. provide a comprehensive overview of bacterial detection methods utilizing phages in their brief review.⁵⁰¹ Gizem Ertürk et al. have created a new type of biosensor called a molecularly imprinted capacitive biosensor for detecting phages. This biosensor is capable of quickly and accurately detecting phages in river water, allowing for real-time monitoring of contamination levels.⁵⁰²

2.2.2.5. Cell-Based Biosensors

Biosensors utilizing antibodies, DNA, and similar components are indeed costly, as previously mentioned. An economical approach to biosensor development involves utilizing the cell itself as the biorecognition element. The reasons for this are as follows:⁵⁰³

• 1)Cells can reproduce themselves and produce elements like antibodies without the need for additional processes to purify the antibodies.
• 2)They can provide insights into the feasibility of the analyte.
• 3)Optimal cell biorecognition elements, such as E.Coli, possess the ability to be modified through genetic engineering and can be cultivated with ease.

Cell detectors have been developed to identify the presence of metabolites⁵⁰⁴ and hazardous compounds in urine,⁵⁰⁵ as well as nitrogen oxides in serum and urine.⁵⁰⁶ Cell-based biosensors are limited by their reliance on intracellular interaction with analytes.⁵⁰⁷ The utilization of both surface display technology and antibody engineering will facilitate the identification of extracellular analytes.⁵⁰³ The study conducted by Kylilis et al. involved the creation of a biological alternative to the Latex Agglutination Test (LAT). They achieved this by utilizing E. coli with a surface display of single domain antibodies (nanobodies) as the component that performs biorecognition.⁵⁰³ The nanobodies were chemically bonded to a paired dimeric GFP analyte, and the limit of detection (LOD) was mainly influenced by the overall quantity of nanobodies available. The device was created as a comprehensive cell biosensor designed to detect the human fibrinogen (hFIb) factor, a well-established biomarker for cardiovascular disease. This sensor device, which detects extracellular proteins, is cost-effective and does not necessitate the transcription or translation of the reporter protein.⁵⁰³ Wong et al. have created a cell-based biosensor using Anabaena torulosa to detect heavy metals.⁵⁰⁸ The bacteria adhered to cellulose by the process of filtering. The measurement was conducted using optical fiber technology. The lower limit of detection (LLD) for heavy metals and pesticides ranged from 0.025 to 1.195 μg/L.

Electrical cell-substrate impedance sensing (ECIS) and light addressable potentiometric sensors, along with fluorescent imaging, are commonly used methods for transduction and detection in the fabrication of mammalian cell biosensors.⁵⁵⁹ Electrochemical impedance sensing (EIS) platforms are highly regarded in the advancement of whole-cell biosensors. This is because they

• can monitor cell-substrate interaction without the need for labels;

• can assess various aspects [attachment, spreading, motility, growth, proliferation, cell-to-cell interactions, the metabolic and electrophysiological status].

In addition, various techniques for measuring impedance at specific locations have been created and utilized to study specific properties of samples in complex heterogeneous structures or mixtures, as well as in cell and particle research. These techniques include electrical impedance tomography, scanning electrochemical microscopy, and microelectrode arrays.⁵⁰⁹ The development of mammalian-cell-based devices for monitoring and modulating physiological processes in humans is a promising and rising area in therapeutic synthetic biology.

2.2.2.6. Biomimetic Biosensor

Aptamers are short chains of nucleotides or peptides, typically consisting of around 40 bases, that have a high degree of specificity for particular target molecules. They are chosen from a sizable pool of randomly generated sequences. Riboswitches contain endogenous aptamers. An electrochemical biosensor that generates an electrochemical signal in response to the interaction between an analyte and an aptamer is referred to as an E-AB (electrochemical aptamer based) biosensor. These biosensors offer advantages over other aptamer-based biosensors, such as fluorescence aptamer biosensors, as they enable real-time in vivo measurements. The efficiency is contingent upon the aforementioned criteria

• 1)Density packing of DNA/RNA
• 2)Nature of Self-Assembled Monolayer
• 3)Potentiostat frequency

Figure 49 schematically illustrates two types of aptamer biosensor.⁵¹⁰

Figure 49.

(a) VEGF detecting aptamer E-AB biosensor. On target attachment, the partially unfolded aptamer probe folds completely resulting in a closed structure and the variation in the electron transfer kinetics leads to an elevated MB current (b) An aptamer cocaine detector. Here before the target binding, the aptamer probe is fully or partially extended and once the target binds with the aptamer it kind of forms a “three-way junction”, which will alter the electron transfer kinetics and the output current⁵¹⁰ [Reprinted with permission from [Rebecca Lai, Research Area 1: Biosensor Design | The Lai Lab. http://chemweb.unl.edu/lai/research-area-one-biosensor-design/ (accessed 2024–10–18)] [Department of Chemistry | Hamilton Hall 651| University of Nebraska-Lincoln | Lincoln, NE 68588 | 402–472–5340].

A molecularly imprinted substrate is a synthetic polymer material with chemically active and particular recognition sites that mimic the natural biorecognition elements. It is utilized for the detection of different bioanalytes.^511,512 Molecular imprinting is a process where a monomer is polymerized along with an analyte within a template.⁵¹³ The elimination of the template results in the presence of distinct binding sites in the product, leading to the formation of molecularly imprinted polymers (MIPs).⁵¹³ The adaptability of MIPs allows for their development with nearly all biomolecules, making them an ideal choice in the field of sensors. Vera L. V. Granado et al. provided a comprehensive analysis of the fabrication techniques for potentiometric and amperometric MIP-based biosensors. Electropolymerization was used to deposit a conducting polymer on the electrode, which included spherical MIP particles with a diameter of 1 μm.⁵¹³Table 8 gives a brief consolidated comparison of various transduction techniques used for various bioanalytes.

Table 8. Transduction Techniques Used for Various Biological Elements.

Electrode/biorecognition element	Analyte	Transduction technique	Ref
Antibody	HIV-1 virion infectivity factor	Piezoelectric	(514)
Antibody	Listeria monocytogenes	Electrochemical	(515)
Antibody	Entamoeba histolytica antigens	Amperometric	(516)
Antiadenovirus, Group II (HEV) polyclonal antibody	Adenovirus	Optoelectronic	(517)
Anti-CT	Cholera toxin	SPR	(518)
Anti-HIV-1 gp120 antibody	HIV	Fluorescence	(519)
Cy3 Ab	Alzheimer disease	SERS	(520)
Horse polyclonal anti-SARS-CoV [C]	SARS-associated coronavirus (SARS-CoV)	piezoelectric	(521)
GO probe functionalized with DNA	ssDNA	FRET	(522)
GO-DNA	Staphylococcus aureus DNA	Fluorescence	(523)
GO/pencil graphite electrode (GO/PGE)+DNA (HBV)	Hepatitis B virus	Electrochemical	(524)
Graphene/Au+DNA	BRCA1 DNA	Electrochemical	(525)
Graphene–Nafon composite film+DNA	ssDNA of HIV-1 gene	Impedimetric	(526)
Laccase	Caffeic acid	electrochemical	(527)
HRP	H2O2	Electrochemical	(528)
S. aureus phage St.au9IVS5 (displaying peptide RVRSAPSSS)	S. aureus	Fluorescent Microscopy	(529)
Phage for P. aeruginosa	P. aeruginosa	Raman	(530)
S. arlettae specific phages	S. arlettae	EIS	(531)
B1–7064 Phage	Bacillus cereus	Amperometric	(532)
AT20	E. coli G2–2	Bioluminescence	(533)

2.2.3. MEMS Based

Li et al. investigated the development of advanced electrical connections for two-dimensional semiconductors, focusing on monolayer molybdenum disulfide (MoS₂). The authors achieve a notable improvement by hybridizing MoS₂ with semimetallic antimony, reducing contact resistance to 42 ohm-micrometers while preserving stability at 125 °C. This approach provides significant improvements in transistor performance, surpassing current silicon-based technologies, and has possibilities for scalable next-generation electronics.⁵³⁴

A category of devices called microelectromechanical systems (MEMS) has emerged as a result of the remarkable progress made in the miniaturization of electronics.⁵³⁵ MEMS devices are extensively employed for many purposes and are succinctly classified in Figure 50. Transducers, specifically sensors or actuators, are the most prevalent form of MEMS. They are capable of converting one signal type into another signal type. Alternatively, they can be fabricated as cantilever or string configurations, which correspond to structures resembling single- or double-clamped beams, respectively. The operation of MEMS devices relies on energy transduction, which facilitates the passage of information from the sensing unit to the controller. The controller then uses a control algorithm to make decisions and then sends commands to the actuation unit. The advantages and challenges faced by MEMS devices are listed below in Table 9.

Figure 50.

MEMS classifications and uses.

Table 9. Advantages and Disadvantages or Challenges of MEMS Devices.

Advantages	Disadvantages/challenges
Low power consumption	Due to the small size significant power transfer is not possible
Lightweight	The base material cannot be loaded with a large load
Ease of integration	Design standards are not well-developed
High performance in wearable electronics	Complex thermo mechanical behaviors
High resonant frequency	Complex motion controls
Low cost (due to bulk production)	Expensive materials
High sensitivity	Some reliability issues
High accuracy	Temperature-dependent effects
New career opportunities	Low operational speed, contamination, break/crack possibility, and poor fatigue properties

In the field of medicine and healthcare, MEMS devices also referred to as bio micro electromechanical systems (BioMEMS), micro total analysis systems (μTAS). Lab-on-chips (LoCs), or biochips, have the potential to be used in various applications such as drug synthesis, drug delivery, microsurgery, micro therapy, diagnostics and prevention, artificial organs, genome synthesis and sequencing, and cell manipulation and characterization too.⁵³⁵ This is schematically represented in Figure 51.

Figure 51.

Schematic diagram of MEMS in biology.

MEMS devices are made mostly of silicon and silicon compounds, like integrated circuits.⁵³⁶ The field considers doped single crystalline silicon and polycrystalline silicon (polysilicon) as representative materials due to their unique properties, such as strength, conductivity, resilience, robustness, reliability, easy processing, process reproducibility, and low unit costs.⁵³⁷ Silicon dioxide can be utilized for insulation, passivation, and sacrificial purposes, while silicon nitride can be used for insulation.⁵³⁸ Even though silicon is abundant in nature, its expanding functionality and complexity have made it insufficient for MEMS and demand the incorporation of new materials such as single-crystal silicon carbide, germanium-based materials, metals, metal nanocomposites, and polymers. Photolithography, oxidation, sputtering, thin layer deposition, and other well-known micromachining processes utilized in the integrated circuit domain have been employed to produce MEMS/Bio-MEMS devices. Both surface micromachining and bulk micromachining are extensively utilized in microfabrication.

2.2.3.1. Bulk Micromachining

Bulk micromachining is a process that includes selectively etching or removing material from the bulk substrate to create MEMS devices. By selectively removing excess material, it is possible to create microelectromechanical systems (MEMS) components such as beams, cantilevers, and membranes. The elimination of the majority of the material can be accomplished by wet/dry chemical techniques or physical techniques. A frequently employed technique is chemical wet/dry etching, in which a reactive chemical, either in a wet or dry state, selectively removes the exposed areas of the bulk material. Chemical wet etching is advantageous when a rapid etch rate is necessary. Dry etching is advantageous for creating structures with high aspect ratios and complex geometries that cannot be achieved with wet etching procedures.⁵³⁹ The drawbacks of bulk micromachining include difficulties in controlling the etch depth over time throughout the substrate due to variations in the diffusion of etchant species, etchant aging, loading effects, and variations in substrate thickness. Furthermore, devices that do not require electronics to be connected with MEMS devices will find bulk micromachining to be economically inefficient.⁵³⁹

2.2.3.2. Surface Micromachining

The technique of surface micromachining entails depositing and creating a mechanically supportive layer, known as the “sacrificial layer”, on the substrate. This is followed by depositing and creating thin films, referred to as the “structural layers”, on top of the sacrificial layer. Upon finishing the fabrication process, the sacrificial layer is specifically removed, resulting in the liberation of the movable micromechanical devices from the substrate.⁵³⁹Figure 52 depicts the fabrication processes in surface micromachining.

Figure 52.

Surface micromachining processes.

The primary benefit of surface micromachining is that it allows for the creation of structures with much lower dimensions compared to bulk micromachining. Additionally, surface micromachined devices can be easily integrated with electronic components. Polymeric devices offer distinct advantages over silicon devices considering bio-MEMS. These advantages include their biocompatibility, the ability to tune the surface groups for different applications, the feasibility of creating complex 3-D structures, and cost-effective fabrication processes. Figure 53 schematically shows the various classifications of polymeric device fabrication techniques.

Figure 53.

Schematic representation of various polymeric device fabrication techniques.

Biosensing and biomolecule immobilization on nanogold substrates were covered earlier. Silicon oxide and nitride, CMOS-compatible materials, are commonly employed in MEMS/Bio-MEMS structures. In cantilever structures and BIOFETs, silicon nitride/oxide serves as structural layers or dielectrics. Immobilization techniques for binding biomolecules on these layers are highly relevant. Silicon nitride sheets are nonporous and uncharged, preventing biomolecule attachment via electrostatic or physical adsorption. Silanization with head groups such as NH₂, COOH, and CHO is a common approach for modifying silicon oxide and nitride surfaces Plasma and oxygen create oxynitride, followed by anhydrous silanization. Nonplasma processes are preferred to avoid damage to suspended objects and substrate surfaces from high-energy particles. Acrylic acid vapor treatment at radio frequency plasma leads to the formation of carboxylic groups on silicon nitride surfaces. Silicon oxide surface absorbs water, forming hydroxylated surfaces that can be changed by silane molecules, analogous to silicon nitride. Surface modification is important for biomolecule covalent attachment.⁵³⁹ A wide range of noninvasive Bio-MEMS technologies and devices have been documented and created. Capacitive or impedimetric sensors are a type of sensor that can be easily integrated into microchannels in a miniaturized configuration. They are also sensitive enough to detect molecules in a sample volume of nano- or microliters.

The creation of microphysiological systems has advanced through the combined progress in microsystems engineering, tissue engineering, and stem cell biology. Consequently, numerous designs for microscale culture systems have been created to recreate tissue and organ functioning at unprecedented levels. There are two main approaches to developing micro physiological systems to better mimic biological processes: (1) creating 3D culture systems with more complex structures, and (2) engineering microfluidic 3D culture devices that include dynamic fluid flow, known as organ chips (organ-on-a-chip).⁵⁴⁰

Through micropump perfusion, microfluidics allows for the dynamic growth of cells and the timely discharge of waste and nutrition. The cellular milieu in which cells reside is more akin to in vivo settings than static culture. Furthermore, the application of fluid shear stress results in the development of organ polarity.⁵⁴¹ Crucially, the chip applies essential physical force to the regular biological processes of endothelial cells by triggering cell surface chemicals and related signaling cascades. Furthermore, the inclusion of fluid in the organs on a chip device enables biological evaluations to be conducted at the individual organ level.⁵⁴² The chip system may summarize fluid flow using either a simple “rocker” motion on a chip or a more complex programmable “pulsatile” format.⁵⁴³ The fluid predominantly functions as a laminar flow at the microscale level, creating a steady gradient of biological molecules that are controlled in both space and time. Biological events, such as angiogenesis, invasion, and migration, involve different biochemical signals that are influenced by concentration gradients.⁵⁴³ By adjusting flow velocity and channel shape with the use of microvalves and micropumps, microfluidics mimics intricate physiological processes seen in the human body and creates stable, 3D gradients of biochemical concentration. Typical physiological pressures experienced in daily life include arterial pressure, pulmonary pressure, and skeletal pressure. The pressures mentioned are crucial for the maintenance of mechanically strained tissues, including skeletal muscle, bone, cartilage, and blood vessels.⁵⁴⁴ Microfluidics utilize elastic porous membranes to generate periodic mechanical stresses. Mechanical stimulation is recognized as a crucial factor in determining differentiation throughout physiological processes.⁵⁴⁵ A complex and well-organized arrangement of several cells is necessary for the human body’s organization to generate functional whole-body connections. Cell patterning is controlled using microfluidics to create intricately geometrized in vitro physiological models. Surface alterations, templates, and 3D printing aid in the process of arranging cells on the chip. The utilization of the 3D printing technique allows for the creation of hydrogel scaffolds that possess intricate pathways, hence facilitating the patterning of cells at several scales. 3D printing offers the benefit of enabling user-defined digital masks, which allows for versatility in cell patterns. This versatility is crucial for reconstructing the cellular milieu in vitro.⁵⁴³ Li et al. devised techniques to achieve the fast arrangement of different types of cells on glass chips through controlled manipulation of their spatial arrangement.⁵⁴⁶ This process involves continuously seeding cells on a glass chip while also covering it with polyvinyl acetate and ablation with a carbon dioxide laser. This technique facilitates regulated interactions between epithelial and mesenchymal cells. Furthermore, it is possible to arrange mesenchymal cells with comparable characteristics on glass chips. This technique is beneficial for doing extensive research and pharmaceutical experiments on the interaction between skin epithelial and mesenchymal cells. It can also be used to create patterns in other types of cells. Thus, the critical elements influencing in vitro organ culture are cell patterning, gradient concentration, fluid shear force, dynamic mechanical stress, etc.

A continual circulation of media and interactions between tissues is necessary for an assortment of physiological pathways. Organ chips, when used individually, do not accurately capture the intricate nature, functional alterations, and overall integrity of organ function. The “multiorgan-on-a-chip”, also known as the “human-on-a-chip”,⁵⁴⁷ is a device that may create many organs at the same time, which has garnered significant research interest. From two-dimensional cell cultures to animal models and three-dimensional organoids to OOC, technology has evolved to allow more options for applications such as disease models and drug screening. Figure 54 gives the schematic representation of the evolution of the models and the application of OOC.

Figure 54.

Schematic representation of the evolution of the models and applications of OOC⁵⁴⁸ [Reprinted the image with permission fromJiang, L.; Li, Q.; Liang, W.; Du, X.; Yang, Y.; Zhang, Z.; Xu, L.; Zhang, J.; Li, J.; Chen, Z.; Gu, Z. Organ-On-A-Chip Database Revealed—Achieving the Human Avatar in Silicon. Bioengineering 2022, 9 (11). 10.3390/bioengineering9110685]. Copyright [2022] [Jiang etal, Licensee MDPI, Basel, Switzerland].

2.2.4. Based on the Nature of the Substrate Platform

The preceding sections discussed the categorization of biosensors according to the methods of transduction, the components responsible for biorecognition, and the use of microfluidics in the sensor platform. A thorough grasp of the biosensor, including its types, principles, recent research outcomes, etc., can be obtained by performing an additional categorization based on the substrate’s nature. Biosensors can be classified into two categories based on the type of substrate: electrospun and paper-based biosensors. These two will discussed briefly in the following sections.

2.2.4.1. Electrospun Biosensors

Electrospinning is a method for manufacturing polymeric nanofibers, which was first developed and patented in the 1930s specifically for textile fiber production.⁵⁴⁹ Electrospinning enables the production of nanofibers with diameters ranging from nanometers to micrometers. These nanofibers can be created by using either an electrostatically driven polymer solution or a molten form.⁵⁵⁰Figure 55 illustrates the experimental configuration of the electrospinning device and displays a scanning electron microscope (SEM) image of a nanofiber produced through electrospinning.

Figure 55.

(a) Schematic representation of electrospinning experimental setup (b) electrospun Nylon 6 nanofibers in the form of nonwoven fabric⁵⁵¹ [Reprinted with permission from Fong, H. Electrospun Nylon 6 Nanofiber Reinforced BIS-GMA/TEGDMA Dental Restorative Composite Resins. Polymer (Guildf) 2004, 45 (7), 2427–2432. 10.1016/j.polymer.2004.01.067]. Copyright [2004] [Elsevier Ltd.].

The following parameters affect the nature and dimensions of polymeric nanofibers:⁵⁵²

• 1.Polymer and solution parameters (Molecular weight, conductivity, surface tension, viscosity)
• 2.Process variables (applied voltage, flow rate, collector distance)
• 3.Environmental conditions (temperature, humidity, auxiliary gas)

Numerous fields, including tissue engineering, biosensing, energy storage, catalysis, and others, benefit from the use of electrospun fibers due to their well-known large active surface area, porosity, easy functionalizations, and tunability of fiber and pore dimensions.^553,554 Electrospun biosensors outperform other types of sensors in terms of sensitivity, selectivity, and response time due to their distinctive quantum features. The utilization of electrospun nanofibers as biosensors can be categorized into two methods.

1. Direct Use of Electrospun Fibers⁵⁵⁵

• Here functional polymers like PAN, PANI, etc. are electrospun and are directly used as the inducing agents of the respective biosensors

• Simple, fast, multianalyte detection possibility, better biocompatibility, etc.

Indirect Use of Electrospun Fibers (556)

• Here, electrospun nanofibers are template-like, on which further functionalizations are done and are then used as biosensing electrodes.

In 2019, Jeong et al. created a detector for cortisol utilizing N-doped carbon nanofibers, which is based on a field-effect transistor (FET).⁵⁵⁷ The substrate transducer layer of the Field-Effect Transistor (FET) consisted of electrospun Polyvinyl Alcohol (PVA) nanofibers embedded with polypyrrole. Carbon nanofibers are renowned for their exceptional electrical conductivity and expansive active surface area.⁵⁵³ Wang and his team utilized this characteristic and created a detector for levodopa and uric acid. The detector consisted of MoS₂ NSA/CNFs nanofibers as the electrode, which were produced using electrospinning and hydrothermal synthesis.⁵⁵⁸ Demiroglu and his colleagues have confirmed that the conductivity of an electrode is increased when it is treated with carbon nanofibers in an acetaminophen-DPV sensor.⁵⁵⁹ Wang et al. developed a biosensor using a Burkholderia cepacia lipase (BCL)@MOF nanofibrous material (MOF-Metal Organic Nanofiber) to detect the presence of methyl parathion in vegetables. The BCL@MOF nanofibers were attached to the glassy carbon electrode using a chitosan cross-linker. The limit of detection (LOD) for this conjugation was 0.067 μM.⁵⁶⁰ Salahandish et al. discovered that by enhancing the original electrode with multiple layers of PANI nanofibers, the detection limit and response time for the SK-BR3 breast cancer biomarker were significantly enhanced.⁵⁶¹ Moreover, the QCM biosensor exhibited a significant increase in binding efficiency, reaching a 10-fold improvement, when the gold electrode surface was altered using electrospun bovine serum albumin nanofiber.⁵⁶²

Dawei et al. devised an innovative polyphenol biosensor by integrating CNFs, laccase, and Nafion to detect catechol. This biosensor exhibits a detection limit of 0.63 μM with a response time of 2 s.⁵⁶³Figure 56 displays the SEM pictures of electrospun carbon nano fiber (ECNFs) and the surface of laccase-Nafion-electrospun carbon nano fibers/glassy carbon electrode. The first photo includes an inset showing the distribution of fiber diameters.

Figure 56.

SEM images of the (a) ECNFs and (b) the surface of laccase–Nafion–ECNFs/GCE⁵⁶³ (Inset: the diameter distribution diagram of the ECNFs) [Reprinted with permission from Li, D.; Pang, Z.; Chen, X.; Luo, L.; Cai, Y.; Wei, Q. A Catechol Biosensor Based on Electrospun Carbon Nanofibers. Beilstein Journal of Nanotechnology 2014, 5, 346–354. 10.3762/bjnano.5.39.]. Copyright [2014] [Li et al; licensee Beilstein-Institut.].

The benefits of electrospun nanofibers for cancer diagnosis and detection are covered by Sahar et al. in their review.⁵⁶⁴Figure 57 depicts a schematic depiction of an electrochemical nanofiber-based biosensor designed for the detection of the p53 gene.⁵⁶⁵

Figure 57.

Schematic representation of the preparation of the electrochemical biosensor (MB – methylene blue)⁵⁶⁵ [Reprinted with permission from [Wang, X.; Wang, X.; Wang, X.; Chen, F.; Zhu, K.; Xu, Q.; Tang, M. Novel Electrochemical Biosensor Based on Functional Composite Nanofibers for Sensitive Detection of P53 Tumor Suppressor Gene. Anal. Chim Acta 2013, 765, 63–69. 10.1016/j.aca.2012.12.037]. Copyright [2012] [Elsevier B.V.].

The detection of cancer antigen-CA125 was performed using a unique electrospun nanofiber platform composed of PAN/MWCNT/ZnAc.⁵⁶⁶ The antiCa125 antibody was attached to the nanofiber surface utilizing EDC/NHS conjugation. LOD was 1.13 ×10^–3 units per milliliter (U/mL). The process is depicted in Figure 58.

Figure 58.

Schematic representation of the fabrication of carcinoma antigen-125 immunosensor⁵⁶⁶ [Reprinted with permission from [Paul, K. B.; Singh, V.; Vanjari, S. R. K.; Singh, S. G. One Step Biofunctionalized Electrospun Multiwalled Carbon Nanotubes Embedded Zinc Oxide Nanowire Interface for Highly Sensitive Detection of Carcinoma Antigen-125. Biosens Bioelectron 2017, 88, 144–152. 10.1016/j.bios.2016.07.114]. Copyright [2016] [Elsevier B.V.].

Juliana and her colleagues created an immunosensor to detect the pancreatic cancer antigen CA19–9.⁵⁶⁷ The immobilization of anti-CA19–9 antibodies occurs within a 3D structure, which is achieved by applying a coating of either multiwalled carbon nanotubes (MWCNTs) or gold nanoparticles (AuNPs) onto electrospun nanofibrous mats composed of polyamide 6 and poly (allylamine hydrochloride). The experiment was conducted utilizing impedance spectroscopy. The limit of detection achieved for MWCNT coating was 1.84 U mL^–1, while for Au NP it was 1.57 U mL^–1.⁵⁶⁷Table 10 presents the LODs of four distinct glucose detectors that are based on electrospun fibers.

Table 10. Electrospun Fiber Sensors for Glucose Detection.

Analyte	Transduction technique	Nanofibers/electrode specifications/transducer	LOD (in μM)	Ref
Glucose	Amperometric	L-Cys-GOx/PVA/ZnO NFs/Au electrode	1	(568)
Glucose	Amperometric	GOx/Mesoporous ECNFs/SPE	-	(569)
Glucose	Amperometric	MWCNTs/Au NPs/PAN NFs/Au electrode	4	(570)
Glucose	EIS	Au NPs/GOx-PVA-PEI NFs/Au electrode	0.9	(571)

2.2.4.2. Paper Based

Point-of-care (POC) devices are what spurred the biosensor industry’s extraordinary expansion. These devices are renowned for their user-friendly interface, simplicity in sample manipulation, portability, requirement of minimal sample volume, and cost-effective manufacturing.⁵⁷² A paper-based analytical device (PAD) is a POC (point-of-care) device that utilizes cellulose fibers as its primary substrate. It has become increasingly popular in diverse industries.⁵⁷³ The choice of appropriate paper material is crucial for the optimal functioning of the paper sensor. Table 8 displays the transduction specifics of different paper-based sensors. Whatman brand chromatography paper exhibits favorable flow speed, retention time, and other characteristics, rendering it the highest-rated paper substrate for biosensors.⁵⁷⁴ Nitrocellulose possesses functional groups that enable it to covalently bond with the analyte molecule, allowing for its entrapment. These molecules’ membranes are extensively utilized in ELISA and AuNP-based sensing platforms.⁵⁷⁵ Another often employed type of paper is a biomimetic substrate platform known as bioactive paper. In this case, the biomolecules are attached to the cellulose fiber scaffold either by aldehyde or amide bonding, inkjet printing,⁵⁷⁶ or graft copolymerization.⁵⁷⁷ Alternative cost-effective solutions for paper-based sensor substrates include glossy paper⁵⁷⁸ and paper towel.⁵⁷⁹ The new review work by Amrita et al. provides a thorough comparison and discussion of the merits and disadvantages of several paper sensor fabrication processes, including photolithography, inkjet printing, laser cutting, hydrophobic silanization, and origami-kirigami approaches.⁵⁷⁴

Ratajczak and Magdalena Stobiecka provide comprehensive descriptions of diverse paper-based biosensors utilized for the early detection of cancer in their recently accepted review article.⁵⁸⁰ The addition of graphene sheets and carbon nanotubes will improve the biosensor’s electrical conductivity and catalytic capabilities. In 2018, Ji et al. developed a paper-based immunosensor with a LOD of 1–18 ng/mL for the detection of prostate cancer.⁵⁸¹ On the micropore filter paper, multiwalled carbon nanotubes were applied, followed by functionalization of the paper with PSA antibodies using EDC/NHS conjugation. Subsequently, measurements of relative electrical resistance were conducted. Figure 59 illustrates the operational mechanism of the PSA paper sensor in a diagrammatic manner.

Figure 59.

(a) Functionalization of PSA antibody on MWCNT. (b) Steps leading to an assembled biosensor substrate⁵⁸¹ [Reprinted with permission from [Ji, S.; Lee, M.; Kim, D. Detection of Early Stage Prostate Cancer by Using a Simple Carbon Nanotube@paper Biosensor. Biosens Bioelectron 2018, 102, 345–350. 10.1016/j.bios.2017.11.035]. Copyright [2017] [Elsevier B.V.].

Given that cell-labeled sensors have a short shelf life, paper-based sensors without any cell functionalization are becoming more and more popular. Gräwe A and colleagues have created a Fluorescent cell-free paper sensor capable of detecting mercury ions and gamma hydroxybutyrate, a substance commonly used as a date-rape medication. The detection methods employed are straightforward, allowing anyone with a smartphone to access and analyze the results.⁵⁸²Figure 60 illustrates the optical detection of a paper sensor using cell-free protein synthesis (CFPS) technology. The sensor is enclosed in a 3D container and measurements are taken using a smartphone.⁵⁸²

Figure 60.

(a) Optical readout with different filter combinations and (b) 3D case and smartphone for bioanalysis⁵⁸² [Reprinted with permission from Gräwe, A.; Dreyer, A.; Vornholt, T.; Barteczko, U.; Buchholz, L.; Drews, G.; Ho, U. L.; Jackowski, M. E.; Kracht, M.; Lüders, J.; Bleckwehl, T.; Rositzka, L.; Ruwe, M.; Wittchen, M.; Lutter, P.; Müller, K.; Kalinowski, J. A Paper-Based, Cell-Free Biosensor System for the Detection of Heavy Metals and Date Rape Drugs. PLoS One 2019, 14 (3), e0210940, 10.1371/journal.pone.0210940]. Copyright [2019] [Gräwe et al PLoS One].

Scala-Benuzzi et al. developed a paper-based fluorescent immunosensor to detect ethinylestradiol.⁵⁸³ The sensor has a limit of detection (LOD) of 0.05 ng L^–1.Figure 61 illustrates the configuration and operation of a strip sensor.⁵⁸⁴ Mahato and Chandra developed a naked-eye detector for detecting adulterations in milk samples.⁵⁸⁵ The detector utilizes the catalytic activity of alkaline phosphatase (ALP) and integrates digital image colorimetry (DIC) with a smartphone for optical readout. ALP antibodies were immobilized on a paper substrate, and the binding of ALP molecules from the sample caused a change in ALP-BCIP (5-bromo-4-chloro 3-indolyl phosphate) activity. The LOD was determined to be 0.87 ± 0.07 U/mL.⁵⁸⁵

Figure 61.

Working configuration of aptamer-based paper strip sensor⁵⁸⁴ [Reprinted with permission from [Xu, H.; Mao, X.; Zeng, Q.; Wang, S.; Kawde, A.-N.; Liu, G. Aptamer-Functionalized Gold Nanoparticles as Probes in a Dry-Reagent Strip Biosensor for Protein Analysis. Anal. Chem 2009, 81 (2), 669–675. 10.1021/ac8020592]. Copyright [2008] [American Chemical Society].

Fan and colleagues have created a cellulose paper sensor that is made of pure cellulose and has a screen-printed electrode. This sensor has been modified to include reduced graphene oxide (rGO) to detect the cancer antigen CA125. Figure 62 illustrates the operational setups of a lateral flow nucleic acid biosensor (LFNAB) in a diagrammatic manner.⁵⁸⁶Table 11 presents a summary of various types of paper used in the biosensor field.

Figure 62.

(A) working configuration of lateral flow nucleic acid biosensors (LFNAB); (B) LFNAB action with and without the presence of target microRNA; (C) multiple analyte detection⁵⁸⁶ [Reprinted with permission from [Zheng, W.; Yao, L.; Teng, J.; Yan, C.; Qin, P.; Liu, G.; Chen, W. Lateral Flow Test for Visual Detection of Multiple MicroRNAs. Sens Actuators B Chem 2018, 264, 320–326. 10.1016/j.snb.2018.02.159]. Copyright [2018] [Elsevier B.V.].

Table 11. Summary of Various Types of Paper Used in the Biosensor Field.

Type of Paper	Analyte	Transduction Techniques	Ref
Whatman RC60 regenerated membrane filter	Bromide, chloride, iodide	CV	(587)
Whatman No. 1 filter paper	CEA and CA125	DPV	(588)
Whatman Chromatographic Paper 3 mm	Staphylococcus aureus	DPV	(589)
180 gsm office paper	Hemoglobin	EIS	(590)
PVDF filter membrane	WBC	CV and DPV	(591)
Filter paper (102 mm, 15 mm)	Glucose	CV and chrono-amperometric	(592)
Cellulose acetate filter paper	Small molecules	CV and amperometric	(593)
Whatman grade 1 chromatic filter paper	Protein	Colorimetric	(594)
Whatman grade 1 chromatic filter paper	K-562 cell	Electrochemical	(595)

3. Recent Research Works in Biosensors

The growing field of biosensor technologies for disease detection has garnered significant academic interest, particularly in response to the COVID-19 pandemic. Xi et al. discuss a G-quadruplex-based biosensor, which utilizes the unique G-quadruplex structure identified in the SARS-CoV-2 genome, offering a novel approach for detecting disease-causing viruses.⁵⁹⁶ Similarly, Cui et al. analyze various COVID-19 detection methods, emphasizing the use of AI-related, SPR, and SERS-based sensors.⁵⁹⁷ Another important development is described in Graphene field-effect transistor biosensor for detection of biotin with ultrahigh sensitivity and specificity by Wang et al., where a graphene-based FET with a detection limit of 90 fg/mL is engineered, exploiting biotin–avidin conjugation chemistry with Cr/Au electrodes.⁵⁹⁸ Furthermore, a new probe for DNA sensing and drug delivery is introduced for employing p-nitrophenyl carbonate (NPC), which is highly reactive to amines and offers enhanced optical stability.⁵⁹⁹ Neurotransmitter detection is also advanced by Banerjee et al. where various neurotransmitter sensors using CV/FSCV and DPV measurement techniques are reviewed.⁶⁰⁰ Additionally, Heredia et al., report a novel method for gold nanoparticle adhesion using starch hydrogels, achieving a detection limit of 25 ng/mL.⁶⁰¹ Ozbeck et al. present a potentiometric PVC membrane-based biosensor that measures concentrations of valproic acid and other ions in blood samples, with a detection limit of 9.75 × 10^–7 mol/L for valproic acid.⁶⁰² A mathematical modeling of a pH-based potentiometric biosensor for detecting organophosphorus pesticides is explored by Shanti et al., using the Akbari-Ganji approach. They derived analytical expressions for substrate concentrations, with MATLAB employed for simulations.⁶⁰³ These studies collectively contribute to the expanding research on biosensors for diagnostic and therapeutic applications.

Öndes et al. developed a novel and exceptionally stable urea biosensor utilizing poly(2-hydroxyethyl methacrylate-glycidyl methacrylate) nanoparticles as a nanocarrier for the urease enzyme. An ammonium-selective electrode was used to create the potentiometric urea biosensor, which incorporated urease-immobilized nanoparticles. LOD was 0.77 μM and the response time was 30 s.⁶⁰⁴ The study done by Ribeiro et al., introduces a potentiometric biosensor designed for point-of-care analysis that can detect amyloid β-42; which relies on the molecular imprint polymer (MIP) process, where covalently immobilized Aβ-42 is utilized to generate distinct detecting cavities on the surface of SWCNTs. The target peptide was extracted from the polymer matrix through the enzymatic degradation catalyzed by proteinase K. The capacity of the sensing material to reattach to Aβ-42 was exhibited by integrating the MIP material as an electroactive compound in a PVC/plasticizer combination, which was then applied to a solid conductive substrate made of graphite. LOD was found to be 0.72 μg/mL.⁶⁰⁵ In the research work done by Bouri et al., a filter paper is coated with Pt and then covered with a Nafion membrane to create a working electrode. The Pt electrode’s versatile capability enables the identification of H₂O₂ produced through the oxidation of galactose in the presence of the galactose oxidase enzyme.⁶⁰⁶ A bioselective membrane has been developed by Lyudmyla et al., as a potentiometric sensor. The membrane is built on butyrylcholinesterase and pH-sensitive field-effect transistors (FETs), which have been enhanced with gold nanoparticles (GNPs). The laboratory prototype of the biosensor has a linear range of 0.025–0.2 mM, the lowest limit of detection of 0.018 mM, and a response time of 1.4 min.⁶⁰⁷ It is crucial to promptly recognize and monitor topiramate (TOP) medication, which is among the most frequently used anticonvulsant and antiepileptic medications in humans. In this work, Khalifa et al., new molecularly imprinted polymer (MIP) based potentiometric (MIP/PVC/GCE) and voltammetric (MIP/GO/GCE) sensors were created for the sensitive and selective measurement of TOP.⁶⁰⁸ An experimental paradigm was utilized, by Soldatkin and colleagues, employing the bioselective components of acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and glucose oxidase (GOD) in biosensors. The immobilization of enzymes on conductometric transducers’ surfaces was achieved through the cross-linking of the respective enzymes using glutaraldehyde.⁶⁰⁹

The biosensor fabricated by Berketa et al. is based on a recombinant arginine deiminase (ADI) enzyme from Mycoplasma hominis, which was synthesized in Escherichia coli to detect the presence of l-arginine. The biosensor was created by immobilizing ADI on the sensitive surface of the conductometric transducer’s interdigitated electrodes through cross-linking with glutaraldehyde.⁶¹⁰ Zouaoui et al., present the development of a mathematical model to accurately describe and optimize the configuration of the urea biosensor. The biosensor utilizes interdigitated gold microelectrodes that have been modified with a urease enzyme membrane. The presented model specifically emphasizes the enzymatic reaction and diffusion phenomena that take place within the enzyme membrane and the diffusion layer.⁶¹¹ The conductometric biosensor can specifically identify and measure the levels of two model proteins and samples of the SARS-CoV-2 RBD spike protein variant in real time. It is highly sensitive and capable of detecting concentrations as low as 7 pg/ml within 10 min of sample incubation. The biosensor is equipped with a battery-free, wireless near-field communication (NFC) interface to enable rapid and contactless detection of different strains of SARS-CoV-2. The smartphone’s capability to promptly detect and display real-time results for different strains of SARS-CoV-2 can effectively mitigate the spread of the outbreak by promptly notifying users of their infection status.⁶¹² In the work done by Razmshoar et al., the suggested limit for the discharge of Methanol vapor of the sensor is 200 ppm (ppm); which utilizes alcohol oxidase (AOX) attached to electrospun nanofibers made from a blend of polystyrene and poly(amidoamine) dendritic polymer (PS-PAMAM-ESNFs) on interdigitated electrodes (IDEs). The conductometric sensor exhibits a sensitivity of 150.53 μS.cm^–1.⁶¹³

Using glucose as a common analyte, Lui et al., present an amperometric biosensor on a toothbrush. The toothbrush is coated with carbon graphite ink and Ag/AgCl ink, which serve as the sensing electrodes. Subsequently, the enzyme is immobilized. The sensor exhibits exceptional detection capabilities for glucose within a concentration range of 0.18 mM to 5.22 mM and achieves a rapid detection time of under 5 min.⁶¹⁴l-Lactic acid (LA) is a noteworthy biomarker for the detection of several illnesses, including cancer. Tvorynska et al., fabricated a readily replaceable lactate oxidase (LOx)-based minireactor, positioned in front of a screen printed electrode with silver amalgam and it serves as a transducer with covalently immobilized LOx and filled with mesoporous silica powder (SBA-15). Amperometric detection has excellent operational (93.8% of the initial signal after 350 measurements) and storage (96.9% of the initial biosensor response after 7 months) stability.⁶¹⁵l-Dopa, a naturally occurring bioactive molecule found in many Leguminosae plants, is the most efficacious pharmacological treatment for alleviating symptoms of Parkinson’s disease. Motor difficulties arise due to variations in plasma levels of L-DOPA with the development of the disease. Cembalo et al., present a new amperometric biosensor for detecting L-Dopa. The biosensor utilizes tyrosinase that is cocrosslinked onto a graphene oxide layer created by electrodeposition and the LOD is found to be 0.84 μM.⁶¹⁶

Jalalvand et al. created an innovative biosensor that utilizes multivariate calibration techniques to accurately measure the levels of triglycerides (TGs) in lyophilized serum samples.⁶¹⁷ (Electrode used: modified glassy carbon electrode). The investigation by Pedersen et al., revealed that the suggested approach for functionalization was appropriate for both the thin films and the majority of the solid-phase extractions (SPEs). The thin-film electrodes provided a precise surface chemistry that was suitable for thiol-based self-assembled monolayer (SAM) formations. Additionally, high-quality SAMs were effectively produced on most of the solid-phase electrodes, allowing for the effective immobilization of antibodies. LOD was determined to be 1.0 ng/mL.⁶¹⁸ Wang et al. present the development of a highly effective, enzyme-free, and uncomplicated biosensor that prevents fouling. The biosensor is based on a self-screened peptide aptamer. The surface of a glassy carbon electrode (GCE) was modified using a polyaniline (PANI) polymer to provide antifouling interfaces. The peptide aptamer, consisting of the amino acid sequence Cys-Pro-Pro-Pro-Pro-Arg-Glu-Asn-Ile-Gln-Arg-Leu-Thr, was attached to the electrode using a cysteine linker that can conduct electricity. The electrode was then used to measure the presence of L-Lys.⁶¹⁹ Paziewska-nowak et al., present a novel impedimetric biosensor that employs a distinctive H2N-(CH2)6–5′[TAGAGGATCAAA-AAA]4TAGAGGATCAAA3′ dsDNA oligonucleotide as a biorecognition element to specifically detect lactoferrin in human saliva. LOD was found to be 1.25 nM.⁶²⁰ Procalcitonin (PCT) shows great promise as a highly sensitive biomarker for severe inflammation and infection in the early diagnosis of sepsis. The study done by Selimoglu et al. present a biosensor for PCT (procalcitonin) detection that utilizes graphene doped with silver nanoparticles (Ag Np). The LOD (limit of detection) value of the PCT/Ab/AgNp/SLG@ITO impedimetric biosensor that was created was found to be 0.55 ng mL^–1.⁶²¹ An impedance biosensor was created to identify miRNA-10b by utilizing Cu²⁺ modified NH2-metal organic frameworks (NMOF@Cu²⁺) in combination with a three-dimensional (3D) DNA walker signal amplification technique by Tang et al., The plentiful Cu²⁺ can attach to the MOF via a coordination reaction with NH2, acting as a scaffold to rapidly form CuFe Prussian blue analogue@NMOF (CuFe PBA@NMOF). The carboxyl group in the organic ligands of the NMOF can construct DNA strands (complementary strand, CS) for biorecognition reactions. The target activated a 3D DNA walker, leading to the removal of substantial amounts of assistant strands (AS) which were then connected to the surface of GCE. CS-NMOF@Cu²⁺ can be formed on the GCE by combining it with AS. LOD of 0.5 pM under optimal conditions was achieved.⁶²²

Calabrese and researchers created an electrochemical biosensor that performs impedimetric readings on both liquid and air samples to detect VOC compounds in food. The biosensor utilizes the pig odorant-binding protein (pOBP) as the molecular recognition component. We assessed the binding strength of pOBP to three distinct volatile organic molecules (1-octen-3-ol, trans-2-hexen-1-ol, and hexanal) associated with food rotting. LOD was found to be 0.1 μM.⁶²³ Swiftly detecting and quantifying fentanyl in bodily fluids is essential for law enforcement to combat fentanyl abuse and for medical professionals to promptly care for victims. An innovative freestanding surface-enhanced Raman spectroscopy (SERS) biosensor named FrEnSERS biosensor is presented by Su et al., for accurate label-free identification of trace quantities of fentanyl in biological fluids, demonstrating outstanding enrichment capabilities. The biosensor comprises a reduced graphene oxide membrane coated with many hydrophobic gold nanostars. The FrEnSERS biosensor has good sensitivity and accuracy in detecting fentanyl in serum and urine over a wide range, with a detection limit of 0.47 ng/mL for serum and 0.73 ng/mL for urine.⁶²⁴ Fiore et al., describe an initial paper-based microfluidic system that uses filter paper to regulate flow and introduce reagents for a reagent-free competitive magnetic-bead-based immunosensor. This device is specifically built for measuring cortisol in sweat. The paper-based microfluidic design was fabricated using wax printing and laser-cutting techniques to enable capillary-driven microfluidics. Monoclonal antibodies bound to magnetic beads aid in identifying cortisol in the reaction zone, allowing for accurate detection of the target substance. The interaction between the target cortisol and labeled cortisol with the acetylcholinesterase enzyme results in a response that is inversely proportional to the concentration of the target cortisol between 10 to 140 ng/mL by folding the pad holding the enzymatic substrate. A paper-based microfluidic device was connected to a Near-Field Communication wireless module to create a versatile integrated wearable gadget for detecting cortisol in sweat.⁶²⁵ Verma et al., describe the development of an affordable real-time sensing module designed to detect uric acid on a simple, disposable paper surface. The detecting method uses a capacitive measurement apparatus with ZnO hexagonal rods on Cu interdigitated electrodes (IDEs) on hydrophobic A4 paper. The Arduino IDE software is used to code the Arduino Mega board to detect variations in capacitance and display the corresponding uric acid levels on an LCD screen. The experiment showed a direct correlation between uric acid levels ranging from 0.1 mM to 1 mM, with a sensitivity of 9.00 μF mM^–1 cm^–2 at 0.1 mM.⁶²⁶ Cheng et al. developed a cost-effective, compact optical biosensor for detecting blood urea nitrogen (BUN) levels in blood obtained from the fingertip. The biosensor features a simple, stackable, and layered paper-based strip design. It offers a linear optical reaction to BUN concentration and reduces the complexity of the optical scanner. The approach demonstrated a wide detection range of 2.46 to 38.14 mM and a low detection limit of 0.03 mM. The device may connect to a cloud-enabled smartphone via Bluetooth to transmit test data (IoMT).⁶²⁷

Pornprom et al., introduce a cost-effective paper-based electrochemical biosensor that uses a gold particle-decorated carboxyl graphene (AuPs/GCOOH)-modified electrode to detect heat shock protein (Hsp16.3), a biomarker indicating the onset of TB infection. The device pattern was created to assist in detecting operations and printed on low-cost filter paper to form hydrophobic and hydrophilic regions using a wax printing technique. Immunoassays were performed in a half-sandwich format as it is a reagent-free technique that avoids the necessity for a labeling step. The immunosensor was developed by drop casting GCOOH, electrochemically coating AuPs, and creating a biorecognition layer against Hsp16.3 utilizing EDC/NHS-sulfo standard chemistry. Hsp16.3 led to a notable decrease in the electrochemical signal of the redox probe [Fe (CN)6]3–/4– due to the insulating properties of the immunocomplexes produced. GCOOH aids in the direct immobilization of antibodies, whereas AuPs enhance the electrochemical properties of the sensor. The suggested immunosensor, requiring only a small sample amount of 5 μL, showed outstanding performance with a detection limit of 0.01 ng/mL. This method is highly specialized for Hsp16.3 and can rapidly identify TB-infected sera in just 20 min without the need for pre-enrichment. This makes it a feasible choice for early tuberculosis detection in regions with restricted resources.⁶²⁸ Feng et al. created an advanced electrochemiluminescence (ECL) biosensor by merging a flexible paper-based sensing tool with a disposable three-electrode detecting setup. An aptamer was utilized as the recognition element in creating a cellulose paper that detects Staphylococcus aureus (S. aureus). The paper also contained probe DNA-GOD, which was linked to glucose oxidase (GOD) to amplify signaling. The aptamer-S. aureus pair enabled the precise liberation of probe DNA-GOD in a quantifiable manner. The remaining probe DNA-GOD on the paper-based aptasensor was activated by glucose, causing a significant decrease in the ECL signal. Ru(bpy)32+ molecules were added to porous zinc-based metal–organic frameworks (MOFs) to improve the electrochemiluminescence capabilities of the biosensor, leading to the creation of Ru(bpy)32+ functionalized MOF nanoflowers (Ru-MOF-5 NFs).⁶²⁹ The work of Khorshed et al. introduces a portable biosensor that uses lectin array technology to identify glycans in IgG based on impedance. It utilized biotinylated Griffonia simplicifolia (GSL II) and Ricinus communis agglutinin I (RCA I) lectins in our biosensor setup to measure the N-acetyl glucosamine (GlcNAc) to galactose (Gal) ratio, known as the agalactosylation factor (AF). Streptavidin gold nanoparticles were linked to biotinylated lectin attached to the carbohydrate in the glycoprotein to amplify the variation in impedance signal and improve detection sensitivity. Furthermore, it demonstrates the feasibility of using the biosensor to distinguish between COVID-19-positive patient samples and negative patients.⁶³⁰

4. Artificial Intelligence, Machine Learning, Internet of Things (IoT), and Biosensors

The advancement of artificial intelligence (AI) has significantly impacted biosensors and changed the approach to bioanalytical applications. Biosensors, which integrate biological elements with transducers, usually provide exceptional sensitivity and accuracy in detection. However, the incorporation of AI technology has greatly improved their performance to unmatched levels. Machine learning algorithms, particularly those used in deep learning, have demonstrated remarkable capability in extracting important patterns and insights from complex biological data generated by biosensors. This combination allows biosensors to precisely detect analytes and enhance their responses gradually. Artificial intelligence (AI) and biosensors have been extensively used in various fields, including medical diagnostics, environmental monitoring, and food quality management. AI-powered biosensors in medical contexts have accelerated illness detection by studying intricate biomolecular interactions, hence enhancing customized and proactive healthcare. AI-driven environmental biosensors offer instant identification and tracking of contaminants, facilitating efficient resource management and environmental conservation. Artificial intelligence (AI) plays a vital role in improving the speed and accuracy of biosensor data analysis, facilitating quick decision-making in critical scenarios. Despite significant advancements, there are still issues with ethical considerations, data security, and standards. The integration of artificial intelligence (AI) with biosensors has significant promise as it progresses, influencing the advancement of analytical technologies and fostering chances for scientific exploration and creativity. Data science and machine learning have proven effective in several computer vision applications such as autonomous vehicles, motion detection, image categorization, and smart robots. The job descriptions are properly defined, making the problem transparent and allowing for easy verification of the answer. Healthcare responsibilities have inherent safety and security hazards, leading to privacy concerns. The absence of precise definitions for these challenges hinders the ability to conclusively identify their solutions. Assessing the likelihood of severe and even deadly illness in individuals infected with pathogens like the SARS-CoV-2 virus is an urgent and essential concern. Data science has been used to suggest prediction measures using a variety of genetic and physiological markers, as well as symptoms.⁶³¹

Integrating machine learning into conventional biosensors has the potential to enhance measurement accuracy and data interpretation reliability.⁶³² Measurements are affected by variability, uncertainty, drifts, and noise, which impede precision and might pose challenges and incur costs. Advanced physics simulations that could help create detailed and comprehensive data sets have not been produced or widely adopted yet. Moreover, numerous biosensing systems suffer from a lack of substantial parallelism, which hampers the collection of comprehensive information for multiple analytes. Machine learning can be categorized in multiple ways depending on different criteria. Machine learning can be categorized into four divisions according to the learning system: unsupervised learning, supervised learning, semisupervised learning, and reinforcement learning. Unsupervised learning involves working with training samples when the labeled information is not provided. The goal is to uncover the intrinsic characteristics and patterns of the data by using unlabeled training samples. Unsupervised learning has been widely applied in the organization of computing clusters, social network analysis, market segmentation, and astronomical data analysis.⁶³³ The study done by Taha et al., evaluate the most efficient strategies for managing the pandemic, considering developments in optical sensor technology for detecting COVID-19.⁶³⁴ The review also involves examining alternative medical service delivery methods utilized during the pandemic, including telehealth/telemonitoring, tele-management, tele-screening, and teleconsultation. The optical detection system presented uses teleservices and artificial intelligence to identify and categorize COVID-19 patients in healthcare settings. Examining the categorization of nanophotonic biosensors with tele-modules might help understand the methods and technology utilized for detecting SARS-CoV-2 biomarkers.⁶³⁴

The Internet of Medical Things is an expanding network of interconnected medical devices and systems that create an ideal setting for the advancement of biosensors. The study done by Bhupinder Singh delineates the structure of the Internet of Medical Things (IoMT), encompassing intelligent medical equipment, encrypted data transfer, and sophisticated data analysis.⁶³⁵ Biosensors are described as crucial components in the IoMT system, transitioning from passive devices to active participants in patient care. An e study examines ethical issues with patient data privacy, security vulnerabilities in IoMT ecosystems, and the importance of robust regulatory frameworks to avoid misuse. It delves into the challenges faced by healthcare firms in adopting technologies, including issues like interoperability, scalability, and integration problems. The study explores emerging topics such as implanted biosensors, applications of nanotechnology, and the utilization of artificial intelligence for data processing despite encountering obstacles. The study commences by elucidating the fundamental principles of biosensors and their diverse applications in healthcare. The article delves into various biosensors, including glucose monitoring for diabetics and wearable electrocardiogram (ECG) devices, showcasing their significance in early disease detection, ongoing monitoring, and personalized medication.⁶³⁵

Alam et al. collected COVID-19 data over a year by utilizing various data sets obtained from advanced biosensor technologies.⁶³⁶ These data sets comprise patient symptoms, bodily condition, COVID-19 sorts (Alpha, Beta, Gamma, And Delta), and recovered cases. The data preparation function aims to eliminate unwanted noise and data. Optimal selection and feature extraction are performed using the principle component of African buffalo optimization (PCABO). A shuffle shepherd optimization-based generalized deep convolutional fuzzy network (SSO-GDCFN) is proposed for diagnosing COVID-19 illness states, types, and recovery categories. The proposed generalized deep convolutional fuzzy network integrates the deep convolutional approach with generalized approximation reasoning-based intelligent control. The SSO algorithm is utilized to enhance the parameters of the approach. The research’s implementation is conducted using the MATLAB program. The efficacy of the recommended strategy has been validated through a comparison of the simulation outcomes generated by the proposed technique with those of several conventional methods. Figure 63 represents the typical block diagram of a NFC sensor and Figure 64 represents the applications of Bluetooth low energy (BLE) devices.

Figure 63.

Schematic illustration of a typical block diagram of a batteryless NFC sensor⁶³⁷ [Reprinted with permission from Lazaro, A.; Villarino, R.; Lazaro, M.; Canellas, N.; Prieto-Simon, B.; Girbau, D. Recent Advances in Batteryless NFC Sensors for Chemical Sensing and Biosensing. Biosensors (Basel) 2023, 13 (8). 10.3390/bios13080775]. Copyright [2023] [Lazaro et al. Licensee MDPI, Basel, Switzerland].

Figure 64.

Applications of BLE in wireless devices. (a) The wearable mouthguard salivary uric acid biosensor design integrated a wireless amperometric circuit board.⁶³⁸ (b) Real-time monitoring of salivary uric acid level using the integrated mouthguard biosensor with BLE 4.0 graphic interface on a laptop⁶³⁸ [Reprinted with permission from [Kim, J.; Imani, S.; de Araujo, W. R.; Warchall, J.; Valdés-Ramírez, G.; Paixão, T. R. L. C.; Mercier, P. P.; Wang, J. Wearable Salivary Uric Acid Mouthguard Biosensor with Integrated Wireless Electronics. Biosens Bioelectron 2015, 74, 1061–1068. 10.1016/j.bios.2015.07.039]. Copyright [2015] [Elsevier B.V.].

5. Challenges and Future Trends

Substantial progress has been made; yet, many obstacles persist concerning the extensive adoption of biosensors in healthcare. In clinical settings, the dependability and longevity of biosensors are essential due to their extended application. The regulatory framework is a significant challenge, as biosensors are required to adhere to strict testing and approval procedures to confirm their safety and efficacy in clinical settings. This process can be laborious and expensive, presenting challenges for smaller businesses or startups. Furthermore, biosensors that can identify biomarkers and deliver quantitative data are crucial for efficient clinical decision-making. Achieving improved sensitivity and selectivity for specific biomarkers or analytes remains a technical challenge. Inaccurate or ambiguous readings may lead to misdiagnoses or erroneous data, which is particularly critical in medical diagnostics.

Many biosensors, particularly enzymatic and biological varieties, face limitations concerning shelf life and stability. Maintaining operational efficiency over extended periods, especially in varying climatic circumstances, is difficult. The incorporation of biosensors into wearable technology for continuous, real-time monitoring, such as glucose or heart rate sensors, has challenges concerning power consumption, miniaturization, and data accuracy. Mass-producing biosensors while maintaining uniformity, quality, and cost-effectiveness is a substantial challenge. Augmenting output may also result in fluctuations in sensor performance. Biosensors generate significant volumes of data, raising issues related to data storage, management, and security. In healthcare, the confidentiality of patient information is a critical issue, and compliance with regulations such as GDPR (The General Data Protection Regulation, or GDPR, 2018) and HIPAA (The Health Insurance Portability and Accountability Act of 1996 (HIPAA)) is imperative. Alternative diagnostic techniques, such as laboratory assays and imaging modalities, contend with biosensors. Biosensors must offer a clear benefit in terms of speed, cost, or convenience to distinguish themselves. For biosensors to succeed in the consumer market, there must be assurance in the accuracy and reliability of the devices. Issues with prior biosensor devices, like imprecise measurements, could negatively impact the reputation of promising technologies in the marketplace.

Biologists, chemists, physicists, electronics engineers, and data analytics specialists are all needed for biosensor development. Logistical challenges and a lack of inspiration could result from coordinating interdisciplinary teams. Integrating biosensors with digital health platforms, such as cloud-based data systems and mobile health applications, is the main emphasis of the research. Healthcare providers can get vital information through these technologies’ real-time data analysis and remote patient monitoring. Traditional healthcare is likely to include biosensor technology more and more in the coming years. This technology has the potential to revolutionize diagnosis, patient management, and tailored medication. Recent developments show that these technologies have a lot of potential in the future, even though there are still problems with stability, repeatability, and regulatory hurdles. As biosensors continue to advance in capability, they will play an increasingly important role in healthcare delivery, patient monitoring, and illness identification.

The future of medical biosensors is set for substantial transformation through the incorporation of artificial intelligence (AI), providing considerable advancements in healthcare diagnosis and monitoring. AI-driven biosensors provide the capability to transform health data detection and analysis through real-time, continuous monitoring and offering predictive analytics for early illness identification. Through the integration of machine learning algorithms, these biosensors can evaluate extensive volumes of intricate biological data, identify trends, and discern small anomalies associated with the early onset of diseases such as cancer, diabetes, and cardiovascular disorders. This improves diagnostic precision and facilitates individualized treatment strategies. Moreover, AI can enhance biosensor efficacy by optimizing calibration procedures, reducing noise, and augmenting the specificity and sensitivity of the devices. AI-powered biosensors, when integrated with wearable or implantable technology, can provide remote patient monitoring, notifying healthcare personnel of significant alterations in a patient’s state and allowing for prompt actions. As AI advances, it will facilitate the development of self-learning biosensors that adjust to unique physiological patterns, paving the way for a future characterized by highly personalized, proactive, and predictive healthcare. The collaboration of AI and biosensor technology will improve patient care quality and expand global access to healthcare solutions, especially via telemedicine and remote diagnostics.

6. Conclusion

The significance of biosensors has increased throughout the global pandemic due to their critical role in the rapid and precise detection of infectious illnesses. Biosensors are crucial instruments that provide the swift identification of specific biological markers, such as antigens or antibodies, associated with infections like viruses. In light of the persistent health challenges, biosensors are essential for facilitating accurate and comprehensive testing. They enable the rapid detection of illnesses and the execution of prompt public health measures. Their ability to provide timely results, often with remarkable accuracy and precision, is essential in controlling and preventing the spread of infectious illnesses. Biosensors alleviate pressure on healthcare systems by improving testing protocols and enabling swift responses. This enhances our ability to efficiently manage and mitigate the impacts of pandemics. The integration of biosensors into diagnostic techniques is becoming increasingly essential for an effective and resilient public health system, as technological improvements continue. The review offers a thorough catalog of nearly all biosensor categories, clarifying fundamental concepts, subclassifications, new developments, benefits of different approaches, output efficiencies, and supplementary details. This comprehensive review publication categorizes biosensors according to substrate characteristics, thereby offering a unique contribution to the field. The selection of an appropriate transduction mechanism has a substantial influence on the performance and applications of biosensors. Biosensors utilize several approaches to transform biological processes into detectable signals. Optical transduction techniques, including surface plasmon resonance, fluorescence, and absorbance, allow for highly sensitive and real-time monitoring. They excel in the realm of biomolecular interactions and the alteration of biological material. In contrast, electrochemical transduction is characterized by its ease of miniaturization and high sensitivity to electrical signals produced during metabolic processes. This method is extensively employed by glucose and DNA biosensors. The piezoelectric transduction method utilizes the frequency shift in quartz crystal microbalances to accurately detect even the slightest changes in mass, without the requirement of using labels. The advantages and disadvantages of each transduction method impact aspects like as cost, sensitivity, and specificity. The effectiveness of a method relies on the particular needs of the biosensing application.

The incorporation of AI has markedly enhanced biosensors’ capacity to analyze data, identify patterns, and render conclusions. Artificial intelligence algorithms improve the sensitivity and specificity of biosensors by facilitating rapid and accurate processing of complex biological data. Machine learning is an artificial intelligence approach that allows biosensors to integrate new data and adapt to their environment, hence improving their effectiveness. The integration of artificial intelligence (AI) into biosensors enables concurrent real-time monitoring and predictive analysis. This advancement facilitates the rapid detection of illnesses and the delivery of customized healthcare.

The Internet of Things (IoT) has markedly accelerated the progress of biosensor technology by enabling seamless network access, hence promoting efficient data transmission and remote monitoring. The integration of IoT-enabled biosensors enables rapid data collection, storage, and analysis, hence improving the efficiency of healthcare systems. This linkage facilitates continuous monitoring of health indicators, swift identification of anomalies, and timely intervention. The use of IoT-enabled biosensors in healthcare promotes the development of novel systems that prioritize patient-centered methodologies and improve healthcare results.

Wearable biosensors in healthcare technology are transformative as they provide continuous patient monitoring without intrusive procedures. Wearable real-time biosensors offer multiple benefits, including the continuous assessment of diverse physiological indicators, thereby improving our understanding of an individual’s health status. Furthermore, these biosensors can continuously monitor essential physiological parameters, enabling the early detection of health problems and the implementation of preventive measures. Users and healthcare professionals may easily access and assess the comprehensive health data offered by wearable biosensors, hence improving decision-making. Moreover, it enables consumers to engage actively in their healthcare by providing essential information about their health metrics. This fosters a sense of personal responsibility for their well-being and enhances patient engagement. Wearable biosensors, connected to the Internet of Things (IoT), provide remote patient monitoring by healthcare practitioners, enhancing the efficiency of healthcare delivery and reducing the necessity for frequent hospital visits. This is especially beneficial for persons with chronic diseases.

In summary, biosensors play a crucial role in healthcare and medical diagnostics. Nanobiosensors, engineered to identify perilous infections like COVID-19 and Zika, exemplify only a few applications of these versatile devices. Biosensors have become indispensable tools owing to their capacity to swiftly, sensitively, and precisely identify biological markers linked to various illnesses. The domain of cancer nano biosensors possesses the capacity to markedly improve patient outcomes through its remarkable accuracy and early detection abilities, facilitating timely intervention. The advancement of biosensors for infectious diseases has greatly enhanced global initiatives in monitoring, controlling, and avoiding the spread of infectious agents. This was especially apparent in their vital function during the COVID-19 pandemic. These advanced approaches improve diagnostic accuracy and allow for continuous monitoring, enabling swift responses and interventions in public health. The ongoing technology breakthroughs have increased the importance of biosensors in the pursuit of enhanced, more efficient, and widely accessible healthcare solutions for various medical conditions. The continuous advancement and incorporation of these technologies into global healthcare systems underscore their vital importance in transforming disease identification and treatment. The biomedical research community is pivotal to the future of human health and survival within this global epidemic, particularly concerning the early diagnosis and screening of diverse infectious diseases. The paper outlines the tactics implemented for detecting diverse bioanalytes and highlights areas where the scientific community may unite as one entity.

Glossary

Full form Abbreviation

AChE

acetylcholinesterase

AF

agalactosylation factor

AOX

alcohol oxidase

AOX

alcohol oxidase

ACE-2

angiotensin-converting enzyme 2

Ab

antibody

ADI

arginine deiminase

AI

artificial intelligence

ALD

atomic layer deposition

BioMEMS

bio micro electromechanical systems

BUN

blood urea nitrogen

BLE

Bluetooth low energy

BSA

bovine serum albumin

BAW

bulk acoustic wave

BCL

Burkholderia cepacia lipase

BuChE

butyrylcholinesterase

CD

carbon dots

CNTs

carbon nanotubes

CMCS

carboxymethyl chitosan

CEA

carcinoembryonic antigen

CFPS

cell-free protein synthesis

CCD

charge-coupled device

R_ct

charge-transfer resistance

CRET

chemiluminescence resonance energy transfer

CTV

citrus tristeza virus

CMOS

complementary metal-oxide-semiconductor

CcPDH

Coprinopsis cinerea pyranose dehydrogenase

CRP

C-reactive protein

CV

cyclic voltammetry

DEP

dielectrophoretic

DPV

differential pulse voltammetry

C_dl

double-layer capacitance

DSHP

double-slot hybrid plasmonic

DPI

dual polarization interferometry

ECIS

electrical cell-substrate impedance sensing

ECG

electrocardiogram

E-AB

electrochemical aptamer based

EIS

Electrochemical impedance spectroscopy

ECL

Electrochemiluminescence

R_s

electrolyte resistance

ECNFs

electrospun carbon nano fibers

ELISA

enzyme-based immunosorbent assay

EGFR

epidermal growth factor receptor

E.coli

Escherichia coli

FFT

fast Fourier transform

FET

field-effect transistor

FBAR

film bulk acoustic resonator

FPW

flexural plate wave

FRET

fluorescent energy transfer

FTIR

Fourier transform infrared

GOx

glucose oxidase

GO

graphene oxide

Hsp

heat shock protein

HCV

Hepatitis C virus

HOMO

highest occupied molecular orbit

LUMO

lowest utilized molecular orbit

HRP

horse radish peroxide

HER-3

human epidermal growth factor receptor-3

hFIb

human fibrinogen

IgG

immunoglobulin G

ITO

Indium tin oxide

IDEs

interdigitated electrodes

IGRAs

interferon-gamma release assays

IoMT

Internet of Medical Things

LoCs

lab-on-chips

LFNAB

lateral flow nucleic acid biosensor

LAT

latex agglutination test

LMG

leucomalachite green

LOD

limit of detection

LDA

linear discriminant analysis

LCORR

liquid-core optical ring resonator

LSPR

localized surface plasmon resonance

LLD

lower limit of detection

MZI

MachZehnder interferometer

MFEAs

magnetic field-enhanced agglutination assays

MMM

magnetic marker monitoring

MNB

magnetic nanobeads

MRS

magnetic relaxation switching

MARIA

magnetic relaxation/remanence immunoassay

MG

malachite green

R _mt

mass transfer resistance

MB

methylene blue

μTAS

micro total analysis systems

MEMS

microelectromechanical systems

MIT

molecular imprinting technology

MIPs

molecularly imprinted polymers

mAb

monoclonal antibody

MMF

multimode fiber

MMPS

multiple unit pellet system

MSC

multiplicative scattering correction

MWCNT

multiwalled carbon nanotube

MAP

Mycobacterium avium subsp. paratuberculosis

NEMS

nano electro mechanical systems

NRs

nanorods

NWs

nanowires

NFC

near-field communications

NHS

N-hydroxy succinimide

NHSS

N-hydroxy-succinimide sodium salt

NT-proBNP

N-terminal pro-brain natriuretic peptide

OPLS-DA

orthogonal projection to latent structures discriminant analysis

PAD

paper-based analytical device

PLS-DA

partial least squares discriminant analysis

PNA

peptide nucleic acid

PDP

phage display peptide

PBG

photonic band gap

PC

photonic crystals

pOBP

pig odorant-binding protein

PAMONO

plasmon assisted microscopy of nano-objects

NPC

p-nitrophenyl carbonate

POC

point-of-care

PVA

poly vinyl alcohol

PEG

polyethylene glycol

PPO

polyphenol oxidase

PPyNT

polypyrrole nanotubes

Pt-PPy/UOx/Grap

polypyrrole/uricase/graphene composite

PS-PAMAM-ESNFs

polystyrene and poly(amidoamine) dendritic polymer

PVDF

polyvinylidene fluoride

PCA

principal component analysis

PCABO

principle component of African buffalo optimization

PSPP

propagating surface plasmon polariton

QDs

quantum dots

QCM

quartz crystal microbalance

REMI

Raman encoded molecular imaging

ROS

reactive oxygen species

RBP

receptor binding proteins

RGNW

reduced graphene nanowalls

rGO

reduced graphene oxide

RCA I

Ricinus communis agglutinin I

RMGE

round-type microgap electrode

SARS-CoV

SARS-associated coronavirus

SEM

scanning electron microscope

SPM

scanning probe microscope

SPE

screen printed electrode

SAM

self-assembled monolayers

SSO-GDCFN

shuffle shepherd optimization-based generalized deep convolutional fuzzy network

SOI

silicon-on-insulator

SMFs

single-mode fibers

ssDNA

single-stranded DNA

SWCNTs

single-walled carbon nanotubes

SPN

small plasmonic nanostars

SPE

solid-phase extraction

SWV

square wave voltammetry

SpA

staphylococcal protein A

S. aureus

Staphylococcus aureus

SMLR

stepwise multiple linear regression

SrCuO2

strontium copper oxide

SQUID

superconducting quantum interference device

SOD

superoxide dismutase

SPR

surface plasmon resonance

SERS

surface-enhanced Raman spectroscopy

SENSORS

surface-enhanced spatially offset Raman spectroscopy

TBA

thrombin-binding aptamers

TERS

tip-enhanced Raman spectroscopy

TMV

tobacco mosaic virus

TOP

Topiramate

TST

tuberculin skin test

TB

tuberculosis

VEGF

vascular endothelial-derived growth factor

W

Warburg impedance

WNV

West Nile Virus

WGM

whispering gallery mode

EDC

1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride

PASE

1-pyrene butanoic acid succinimidyl ester

MBTH

3-methyl-2-benzothiazolinone hydrazine

The authors declare no competing financial interest.