Recent advances in airborne pathogen detection using optical and electrochemical biosensors

Abstract

The world is currently facing an adverse condition due to the pandemic of airborne pathogen SARS-CoV-2. Prevention is better than cure; thus, the rapid detection of airborne pathogens is necessary because it can reduce outbreaks and save many lives. Considering the immense role of diverse detection techniques for airborne pathogens, proper summarization of these techniques would be beneficial for humans. Hence, this review explores and summarizes emerging techniques, such as optical and electrochemical biosensors used for detecting airborne bacteria (Bacillus anthracis, Mycobacterium tuberculosis, Staphylococcus aureus, and Streptococcus pneumoniae) and viruses (Influenza A, Avian influenza, Norovirus, and SARS-CoV-2). Significantly, the first section briefly focuses on various diagnostic modalities applied toward airborne pathogen detection. Next, the fabricated optical biosensors using various transducer materials involved in colorimetric and fluorescence strategies for infectious pathogen detection are extensively discussed. The third section is well documented based on electrochemical biosensors for airborne pathogen detection by differential pulse voltammetry, cyclic voltammetry, square-wave voltammetry, amperometry, and impedance spectroscopy. The unique pros and cons of these modalities and their future perspectives are addressed in the fourth and fifth sections. Overall, this review inspected 171 research articles published in the last decade and persuaded the importance of optical and electrochemical biosensors for airborne pathogen detection.

Graphical abstract

1. Introduction

Airborne pathogens, such as bacteria (e.g., Mycobacterium tuberculosis), viruses (e.g., Influenza), and fungi (e.g., Aspergillus niger), easily enter living organisms either directly by breathing or indirectly by settling onto surfaces, creating a severe threat to human health and economic growth [[1], [2], [3]]. Currently, the world is facing an adverse condition due to the pandemic of airborne SARS-CoV-2, producing more than 178 million confirmed victims, including 3.8 million deaths, reported to the World Health Organization (WHO) as of June 2021. Thus, a rapid diagnosis of infectious pathogens is necessary to reduce or prevent the pathogen transmission rate within humans [[4], [5], [6]]. The point of care test (POCT) is potentially involved for infectious disease detection, and WHO has set the ASSURED criteria for designing an ideal POCT platform (affordable, sensitive, specific, user-friendly, rapid, equipment-free, and deliverable) [4,7,8].

Several techniques, such as culture, microscopy, immunoassays, and molecular diagnostics, have enormously contributed to infectious disease identification [7,[9], [10], [11]]. Although the culture-based method is one of the best techniques for pathogen detection, the complicated procedure, long time to procure results, and requiring well-trained professionals hinder further applications. Similarly, low magnification microscopy cannot detect pathogens effectively; thus, these methods are not as prevalent as POCT for infectious disease detection [10]. Typically, reverse transcriptase (RT) polymerase chain reaction (PCR) (RT-PCR) and enzyme-linked immunosorbent assay (ELISA)-based methods are widely used for infectious virus detection [12,13]. However, the ELISA technique faces severe issues due to expensive antibodies and more detection time [14]. RT-PCR provides high sensitivity and specificity for detecting pathogens. However, considering the costly analytical equipment and additional purification step, the RT-PCR technique is not robust [12]. Because of user-friendliness, affordability, and rapidity, optical and electrochemical biosensors have recently provided sufficient space for airborne pathogen detection [6,[15], [16], [17]].

In view of selectivity, cost-effectiveness, and detection easily judged by the naked eye, optical biosensors are popular using surface plasmon resonance (SPR), colorimetry, and fluorescence strategies for airborne pathogen detection [[18], [19], [20]]. Only a few nanomaterials (NMs) have an SPR property and exclusively interact with pathogens, developing an instantaneous light signal on their surface that can be seen by the naked eye [5,6]. Noble metals, such as gold (Au) and silver (Ag) nanoparticles (NPs), have unique SPR properties, effectively participating in the detection process [7,21]. Nanozymes are artificial enzymes with unique features, such as easy handling, high stability, and feasible synthetic methodology. Thus, it has been used instead of enzymes in the colorimetric process as a signal transducer for pathogen detection [22]. Currently, metal-organic frameworks (MOFs), Au/Pt NPs, magnetic NPs (MNPs), and carbon nanotubes (CNTs)/Au nanohybrids function as nanozymes for oxidating peroxidase substrates, developing distinguishable color in the presence of airborne bacteria and viruses [19,[23], [24], [25]]. Furthermore, other methods, such as molecular methods, and G-quadruplex DNAzyme, were used in identifying pathogens in the colorimetric platform [[26], [27], [28], [29]]. Recently, the fluorescence method has drawn attention to pathogen detection due to its fast response, high signal-to-volume ratio, simple fabrication, and economical [20,30]. Fluorescent sensor materials only emitted photons when binding with genomic target analytes; thus, it is convenient for identifying pathogens [31]. Furthermore, this technique has higher sensitivity than microscopy or immunoassay-based methods [10,11].

Various features, such as high sensitivity, ease of fabrication, and portability are taken into the account to construct the POCT, and the electrochemical biosensor almost satisfies these criteria. Thus, it is proficiently used in detecting airborne pathogens [2,14]. Electrochemical biosensors read various electrical signals, such as current, resistance, potential, and impedance format, generated through the interaction between the transducer and biomolecules. The transducer is a working electrode modified with proteins or antibodies to detect target analytes [32,33]. The electrical signals produced in the presence of airborne pathogens were measured through various techniques, such as voltammetry (cyclic voltammetry (CV), square-wave voltammetry (SWV), differential pulse voltammetry (DPV)), amperometry, and electrochemical impedance spectroscopy (EIS) [[34], [35], [36], [37], [38]]. Because of the numerous techniques employed in the electrochemical biosensor, it has been efficiently applied in disease monitoring and medical diagnosis [14]. Based on the applications of the optical and electrochemical biosensors, a comparison of these methods with other techniques is shown in Table 1 . Despite numerous articles published in recent times towards airborne pathogen detection, limited review articles are available [4,6]. Even the published review articles did not provide adequate detection strategies for airborne bacteria and viruses. Hence, this review focuses on airborne bacteria and virus detection based on emerging techniques, such as optical and electrochemical biosensors (Fig. 1 ). Specifically, SPR, colorimetry, and fluorescence techniques used in the optical biosensors for infectious pathogen detection are discussed. The electrical signals generated in the presence of airborne pathogens were measured using DPV, CV, SWV, amperometry, and EIS are discussed. Finally, a conclusion and future perspectives are provided for certifying the credibility of this review. In addition, the exponential growth of relative publications on optical and electrochemical biosensors for airborne pathogen detection is shown in Fig. 2 .

Table 1.

Comparison of the optical and electrochemical biosensors with other techniques.

Detection methods	Advantages	Disadvantages
Optical biosensors	●Real-time detection	●Highly sensitive
	●Point of care test	●Low compatibility
	●High sensitivity	●Low reproducibility
	●short detection time
Electrochemical biosensors	●Robustness	●Sensitive to sample matrix
	●Portable	●Low selectivity
	●Excellent detection limits	●Need of trained specialists
Mass-based biosensors	●High sensitivity	●Low sensitivity
	●Rapid	●Interference induces by nonspecific binding
	●Stable output
Culture methods	●Affordable	●Low sensitivity
	●Less equipment usage	●Time-consuming process
	●High selectivity	●Hard to culture some microorganism
		●Biosafety issues
Molecular methods	●High throughput	●Expensive process
	●High selectivity	●Complex procedure
	●High specificity	●Professional equipment needed
Gene Sequencing	●Good stability	●Laborious
	●High selectivity	●Time-consuming process
	●High accuracy	●High measurement cost

Fig. 1.

Schematic diagram showing optical and electrochemical biosensors following various mechanisms for airborne pathogen detection.

Fig. 2.

The charts display the total number of publications per year towards airborne pathogen detection using (A) optical biosensors, (B) electrochemical biosensors, and (C) overall contribution of optical and electrochemical biosensors for airborne pathogen detection. (Data collected from the scifinder-n.cas.org on January 01, 2022, using “colorimetric biosensors”, “electrochemical biosensors”, and “airborne pathogen” as keywords).

2. Optical biosensors for airborne pathogen detection

Pathogens, such as bacteria, viruses, fungi, and protozoa, are prevalent in developing pandemic situations. Because of their severity, bacteria and viruses create more diseases worldwide [1,6]. Significantly, these pathogens can be detected rapidly and on-site using optical biosensors. This section extensively evaluates SPR, colorimetric, and fluorescence-based biosensors. Table 2, Table 3 show a detailed comparison study based on optical biosensors for airborne bacteria and virus detection.

Table 2.

A detailed comparison of the optical biosensor for airborne bacteria detection.

Target bacteria	Detection method	Conjugate materials	Linear range	LOD	Time (min)	Real sample	Ref
B. anthracis	SPR	AgNP	1.0–25 μM	0.31 μM	5	Lake water	43
	Colorimetry	UCNPs−TPP/EBT	2–200 μM	0.9 μM	60	Human serum	44
		Pt/AuNP	–	1 nM	20	–	23
		LAMP primers		500 fg	60	–	47
	Fluorescence	Tb/Eu(MOF)	50–700 nM	4.55 nM	<2	Human serum	48
		Tb/Eu@bio-MOF		34 nM	4	Human serum	50
		R6H/Eu(MOF)	0–80 μM	4.5 μM	–	–	51
		RSPh/Eu(MOF)	–	0.52 μM	<1	–	52
		Si NP/Tb-MOFs	0.025–3 μM	5.3 nM	<1	Bovine serum albumin (BSA)	53
		R6H/EDTA-Eu	10−50 nM	10 nM	–	–	54
		Tb–P/CPs	0−8 μM	5 nM	<1	Urine	40
		Eu/ZnO QD	0−4 μM	3 nM	–	–	39
		Eu/Si QD	0−35 μM	1.02 μM	–	–	56
		Eu/EBT-CD	0.1–12 μM	10.6 nM	10	Urine	57
M. tuberculosis	SPR	AuNP	–	1.25 pM	15	–	59
		AuNP	0–100 ng mL−1	1.56 ng mL−1	90	Urine	60
	Colorimetry	LAMP primers	–	5 pg mL−1	60	–	26
	Fluorescence	MNP	–	102 CFU mL−1	30	Human saliva	62
S. aureus	SPR	AuNP	–	19 CFU mL−1	30	Water	64
		AuNP	5–40 ng μL−1	8.73 ng μL−1	15	–	65
		Labeled AuNP	–	0.02 μM	5	Ocean water	66
		Au/Ag NR	–	25 CFU mL−1	20	Milk	21
	Colorimetry	Au/Fe3O4 NP	10–106 CFU mL−1	10 CFU mL−1	12	Milk	68
		AgNC	10–106 CFU mL−1	4.9 CFU mL−1	5	–	71
		MNP	10–108 CFU mL−1	3 CFU mL−1	<1	Milk	72
	Fluorescence	NBD–Cl	–	1 CFU mL−1	15	Milk	73
		CuNC	10–108 CFU mL−1	80 CFU mL−1	45	Milk	74
S. pneumoniae	Colorimetry	G-quadruplex/hemin	–	156 CFU mL−1	40	–	76
		LAMP primers	–	25 fg μL−1	2	Blood	77
		LAMP primers	2 ng–2 fg μL−1	20 fg μL−1	–	–	8
	Fluorescence	GO		15 CFU mL−1	30	–	30

Table 3.

A detailed comparison of the optical biosensor for airborne virus detection.

Target virus	Detection method	Conjugate materials	Linear range	LOD	Time (min)	Real sample	Ref
Influenza A (H3N2 subtype)	SPR	ConA/GOx/AuNP	0–200 μg mL−1	11.1 μg mL−1	20	–	79
		AuNP	–	7.8 HAU	30	–	18
	Colorimetry	AuNPs/CNTs	–	3.4 PFU mL−1	10	Human serum	25
	Fluorescence	MbO3/g-CN QD	45–250 PFU mL−1	45 PFU mL−1	5	Human serum	85
H1N1 subtype	Colorimetry	AuNP	–	10 pg mL−1	<1	Human serum	81
		PDA vesicles		105 PFU mL−1	–	–	82
		RT-LAMP primers		3 × 10−4 HAU	40	–	84
Avian influenza A (H7N9 subtype)	SPR	AuNP	5–50 pg mL−1	1.25 pg mL−1	10	Human serum	87
	Fluorescence	FMNP	–	0.02 pg mL−1	30	Chicken serum	90
H5N1 subtype	SPR	AuNBP	1–2.5 ng mL−1	1 pg mL−1	35	Human serum	88
	Colorimetry	RCA primers	0.16–1.20 pM	28 fM	–	–	27
H9N2 subtype	Fluorescence	FMNP	300–900 ng mL−1	69.8 ng mL−1	30	Chicken lung	91
		RuSiNP	25 pg–25 ng mL−1	14 fg mL−1	30	Chicken liver	92
Norovirus	SPR	AuNP	10–53 PFU mL−1	10 PFU mL−1	–	BSA	69
	Colorimetry	Graphene/AuNP	–	92.7 pg mL−1	10	Human serum	97
		V2O5	1 pg–100 ng mL−1	0.34 pg mL−1	1	Human serum	98
		Au/AgNP	–	10.8 pg mL−1	–	–	95
	Fluorescence	GO/6-FAM	13 μg–13 ng mL−1	3.3 ng mL−1	5	BSA	99
		AuNPs/CdSeS QDs	1 pg–5 ng mL−1	0.48 pg mL−1	5	Human serum	100
		AgNC	1.8 μM–20 nM	18 nM	–	–	101
SARS-CoV-2	SPR	AuNP	10 pM–100 nM	1 copy	30	Saliva	28
	Colorimetry	ASO/AuNP	0.2–3 ng μL−1	0.18 ng μL−1	10	BSA	106
	Fluorescence	Co-Fe@hemin	0.2–100 ng mL−1	0.1 ng mL−1	16	–	110

2.1. Airborne bacteria detection

Airborne bacteria, specifically Bacillus anthracis, Mycobacterium tuberculosis, Staphylococcus aureus, and Streptococcus pneumoniae widely affect humans, and detecting them using optical biosensors is discussed here.

2.1.1. Bacillus anthracis (B. anthracis) detection

2.1.1.1. Colorimetry strategy

B. anthracis is harmful spore-forming bacteria, that develops a contagious disease called anthrax. Unfortunately, B. anthracis spores endure harsh conditions, such as high temperatures, drought, and ultraviolet (UV) light, and even continuously germinate after the bacteria is dead [39]. Because of the high toxicity and rapid transmission, B. anthracis spores are considered biological weapons and must be monitored through modern techniques to prevent disease outbreaks [40,41]. Dipicolinic acid (DPA), a typical biomarker, presents in dry spores (5%–15%) and maintains the stability of bacterial DNA [42]. Thus, the rapid and facile detection of biomarker DPA identifies bacteria because it constitutes a certain percentage in B. anthracis spores. In general, AgNPs actively participate in detecting pathogens based on their unique SPR property [21]. The yellow color of AgNPs is aggregated while producing a complex with europium ion (Eu³⁺) and the color changes to pink. DPA has excellent coordination ability with Eu³⁺, which helps disperse AgNPs from the Ag–Eu³⁺ complex and retains their original yellow color. Using this SPR phenomenon, the limit of detection (LOD) of B. anthracis in the real sample was measured as 0.31 μM [43]. Significantly, this process has detected the DPA using colorimetry as well as fluorescence methods. These dual-mode strategies can assist to confirm the consistency and accuracy of the detection results. Moreover, the ratiometric colorimetric assay has two wavelengths that could exhibit excellent accuracy toward target analytes detection compared with the measurements achieved at a single wavelength. Hence, this process utilized the ratiometric colorimetric method. Furthermore, in the nano complexes, AgNPs act as a “nanoquencher” which can induce the fluorescence of Eu³⁺, and thus 7.5 μM of DPA was easily perceived by the naked eye. In addition, the Ag–Eu³⁺ complex has an almost similar sensitivity for DPA detection compared with other lanthanide upconversion NPs (UCNPs)-based nanosensors [44].

The anthrax protective antigen (PA₈₃) is another biomarker for B. anthracis, detected using the sandwich immunoassay-based technique. The AuNPs-attached antibodies (Abs) were linked to the PA₈₃ to produce the sandwich immunosensor (Abs–PA₈₃–Abs–AuNP). Then, the platinum (Pt) NPs were stabilized on the surface of the Abs–PA₈₃–Abs–AuNP biosensor, which oxidized the 3,3′,5,5′–tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H₂O₂) and produced blue color. Using this method, up to 1 nM of PA₈₃ was detected within 2 min [23]. This colorimetric assay is more improved than other lateral-flow immunochromatographic assays (LFIA), where various transducers, such as horseradish peroxidase (HRP) and magnetic NPs are used [45,46].

2.1.1.2. Fluorescence strategy

The fluorescence-based strategy has contributed to infectious B. anthracis detection by means of detecting the biomarker DPA [39]. Lanthanide (Ln), such as terbium (Tb) and Eu, was incorporated into the MOF to produce a Tb/Eu(MOF) sensor, sending a ratiometric fluorescent signal for identifying the DPA [48]. Tb/Eu(MOF) sensor exhibited red-orange emissions due to the energy transfer from Tb³⁺ to Eu³⁺ in the system. Substantially, the DPA has more affinity with Tb³⁺, restricting the energy transfer between the lanthanides, showing a consequent yellow-green emission. Notably, 60 mM of B. anthracis spores can be acceptable for humans according to WHO [49]. However, this method has detected DPA up to 4.55 nM, revealing its significance [48]. This method has established a ratiometric self-calibrating MOF sensor by incorporating two lanthanides that did not demand any external instrument for specific calibration. Significantly, the DPA was sensed within 20 s due to the strong interaction between the DPA and Tb, revealing that the lanthanide-MOF sensor will be beneficial for detecting the DPA in biological samples rapidly. More importantly, this process has outstanding specificity toward DPA, providing a precise method for detecting DPA in complex biological samples.

The chitosan-based fluorescent biosensor has been involved in DPA detection due to its simple functionalization and biocompatibility. In the chitosan backbone, the R6H and EDTA were effectively attached, and Eu³⁺ particularly produced a complex with EDTA [54]. The fabricated R6H/EDTA–Eu³⁺ biosensor detected DPA up to 10 nM (Fig. 3 ) which revealed higher sensitivity than the adenine-attached Ln/bio-MOF [50]. Lanthanide phosphonate coordination polymers (Ln–P/CPs) have several unique features, such as large stoke shifts, excellent emissions, and highly stable fluorescence which are induced Ln-P/CPs to perform in the DPA detection [55]. Significantly, the Tb–P/CPs fluorescence intensity gradually increased when increasing DPA from 0 to 8 μM [40]. The LOD was 5 nM and showed higher sensitivity than the NP-based colorimetric method [44]. Due to the high affinity of lanthanides with DPA, various groups have developed the Eu³⁺ functionalized quantum dots (QDs) for B. anthracis detection. Zhou et al. fabricated Eu³⁺/ZnO QDs, where Eu³⁺ was employed as a signal transducer, and ZnO QDs functioned as an internal reference. In this method, the detection process was completed within 8 s due to the large surface area of the ZnO QDs, increasing the contact with DPA [39]. This process was detected as low as 3 nM and exhibited higher sensitivity than other methods [51,52].

Fig. 3.

Schematic illustration of fluorescence-sensing platform for anthrax biomarker (DPA) detection. (a) Fabrication of R6H derivative-based fluorescent probe using chitosan and EDTA–Eu³⁺ complex. (b) The gradual increase of the probe's emission upon the addition of DPA. Reproduced from the published article [54].

2.1.2. Mycobacterium tuberculosis (MTB)

Globally, an estimated 10 million people were affected by MTB in 2019 and 1.2 million were confirmed dead, according to the WHO report. Due to the seriousness of MTB, “early diagnosis and early treatment” is the most effective way to control it and save humans [58].

2.1.2.1. Colorimetry strategy

The SPR-based colorimetric biosensor has been designed for detecting ESAT-6 protein present in MTB. In the process, ESAT-6 was immobilized on specific antibodies, and AuNPs were added to it. The red color of the ESAT-6-contained biosensor changed to blue color due to the salt-induced aggregation, and the LOD was measured up to 1.25 pM [59]. Similarly, another antigen, namely, MTB antigen 85B (Ag85B), was detected using AuNPs in the immunoblotting technique. The recombinant GBP-50B14 fusion antibody was used to produce the immunoassay because it has a high affinity with Ag85B and AuNP. Based on the sustainability of the red color in the biosensor, up to 1.56 ng mL⁻¹ Ag85B could be detected [60]. More importantly, this immunoblotting technique is also capable of detecting another antigen CFB10 of MTB. Furthermore, the sensitivities of Ag85B and CFP10 were 90.5% and 76.2%, respectively, showing higher sensitivity than the ELISA [61]. Hence, detecting these biomarkers using the immunoblotting technique with higher sensitivity can diagnose TB at an early stage.

2.1.2.2. Fluorescence strategy

MTB is pathogenic and has slow growth in the culture medium. Hence, to avoid handling MTB for the experiment, Mycobacterium smegmatis (M. smegmatis) can be used because it has a similar physiological property as MTB and is nonpathogenic. M. smegmatis was functionalized with MNPs for facile magnetic separation, and the progeny mycobacteriophages were used to disrupt the M. smegmatis cells. The fluorescence developed in the system due to the release of intracellular adenosine triphosphate. Using this protocol, the LOD was 3.8 × 10² CFU mL⁻¹ [62].

2.1.3. Staphylococcus aureus (S. aureus)

S. aureus has high pathogenicity and antibiotic resistance causing severe infections; therefore, it is one of the most common human pathogens, inevitably requiring robust analytical detection techniques [63].

2.1.3.1. Colorimetry strategy

The SPR property of AuNPs is significant to identify S. aureus. For instance, a combination of cysteamine (CS), S. aureus-specific pVIII fusion protein (fusion-pVIII), and AuNP have constructed a biosensor (CS/AuNP/fusion-pVIII) for S. aureus detection. In the presence of S. aureus, AuNPs were aggregated on its surface, changing the biosensor color from red to blue. Notably, this method has high specificity toward S. aureus, and the LOD was 19 CFU mL⁻¹ [64]. In this study, the phage display technique was introduced to identify the fusion-pVIII. Indeed, the phage display technique can easily isolate the target protein without losing its activity. Significantly, the fabricated biosensor has shown high specificity due to the strong interaction between the fusion-pVIII and S. aureus. Moreover, the biosensor recovery in the real sample was in the range of 95–110%. Thus, this method could be utilized to monitor S. aureus in environmental samples efficiently [64]. Similarly, another AuNP aggregation-based detection process used inorganic salt for distinguishing positive and negative samples. In the process, the single-stranded DNA (ssDNA) attached to the AuNPs, hybridized with S. aureus DNA, sustaining the red color in it. However, it turned blue when adding sodium chloride (NaCl). Using this method, 8.73 ng μL⁻¹ S. aureus can be detected (Fig. 4 a) [65].

Fig. 4.

(a) Schematic representation for naked-eye detection of S. aureus using AuNPs-probe. Reproduced from the published article [65]. (b) Schematic presentation of a fluorescent immunoassay for S. aureus detection. Reproduced from the published article [73].

Another promising method has proposed an MNP/DNA/alkaline phosphatase (ALP)-based biosensor for monitoring the S. aureus concentration. When adding S. aureus, the ALP was released from the biosensor due to the characteristic behavior of naturally secreted MNase [66]. The released ALP converts the l-ascorbic acid 2-phosphotrisodium salt to ascorbic acid which helps to produce the Ag nanoshell on the Au nanorods (AuNRs). Significantly, this method showed light green to red color, depending on the S. aureus concentration [21]. The detection of S. aureus using the modification of AuNPs with oligonucleotide is making the process more tedious and time-consuming [66]. However, in this method, the unlabelled AuNRs were involved in S. aureus detection, which confirmed its importance in pathogen detection. In addition, the formation of Au–Ag alloy NRs not only has significant benefits in terms of high sensitivity and detection time but also potential for semiquantitative colorimetric pathogen detection due to producing a multicolor based on the S. aureus concentration.

In general, nanozymes have peroxidase-mimicking activities, which were used for S. aureus detection [67]. Zhang et al. synthesized the nanozymes using the composition of Au/Fe₃O₄ NPs, and it was functionalized with specific S. aureus aptamer via Au-sulfur interaction [68]. The aptamer-functionalized nanozymes’ active sites were blocked when adding S. aureus, inhibiting the catalytic oxidation of TMB [19,69]. Thus, this method produced a lighter color in the presence of target bacteria, and the LOD was measured as 10 CFU mL⁻¹ [68]. However, the RCA-based technique developed bright blue color by oxidizing TMB in the presence of S. aureus using an HRP catalyst. Essentially, the RCA method offered extreme sensitivity and selectivity for detecting S. aureus. Under optimum conditions, the RCA method potentially detects the target DNA up to 1.2 pM [70].

In terms of high surface area and more active sites, nanozymes produce a distinguishable color in the presence of bacteria. In fact, MNPs coated on the electrode surface were coupled with specific peptide sequences, displaying black color on the electrode surface. Furthermore, the peptide sequences in the electrode were cleaved when adding S. aureus protease and released MNPs. Consequently, the black color electrode surface changed to golden yellow color. More importantly, this biosensor detected the S. aureus within 1 min and has tremendous specificity against other bacteria [72]. Significantly, this process has performed both colorimetry as well as electrochemical approaches for S. aureus detection. Thus, the pathogen can be determined qualitatively and quantitatively which reveals the impact of this method. Typically, the utilization of expensive antibodies and aptamers is a time-consuming process. Hence, this technique employed nanozymes, and achieved the detection results within 1 min. Moreover, this method has been studied in both colorimetry and electrochemical approaches for S. aureus detection and thus it can be an ideal biosensor for on-site detection of infectious pathogens.

2.1.3.2. Fluorescence strategy

As with SPR and colorimetric-based techniques, the fluorescence method was also extensively employed for target bacteria detection. Deng et al. developed a fluorescent strategy for S. aureus detection using a novel reagent, namely, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD–Cl). In the process, synthesized cysteine (Cys)@liposome immuno-nanocapsules coupled with S. aureus cells and were destructed by Tween-20. Subsequently, Cys was released from the immunosensor, which reacted with NBD–Cl to generate the fluorescence signal. Using this fluorescent strategy, up to1 CFU mL⁻¹ S. aureus was detected (Fig. 4b) [73]. Typically, copper NCs (CuNCs) have unique properties, such as nontoxicity, biocompatibility, and producing efficient fluorescence signals. Thus, CuNCs were attached with aptamer (apt) and antibiotics to produce a biosensor (apt/CuNCs/antibiotic) for quantifying S. aureus [74]. Interestingly, in the presence of S. aureus, CuNCs were aggregated, restricting the free movement of the molecules and producing bright fluorescence in the system. Based on the aggregation strategy, the LOD was 80 CFU mL⁻¹, a lower sensitivity than the AuNP-based aggregation method [64].

2.1.4. Streptococcus pneumoniae (S. pneumoniae)

S. pneumoniae is also known as pneumococcus, which mostly produces pneumonia and meningitis diseases. Furthermore, it is the fourth-largest microbial developing fatal infections, according to a WHO report. Hence, simple, rapid, and affordable analytical techniques are often the ideal choice for S. pneumoniae detection [75].

2.1.4.1. Colorimetry strategy

Recently, combining the catalyzed hairpin assembly (CHA) and signal transducer has been established colorimetric sensor for S. pneumoniae determination. Attaching the target DNA (S. pneumoniae) with hairpin DNA using the CHA process creates a new nucleic acid sequence with a high G-rich DNA content. The G-rich DNA in the presence of hemin produced a G-quadruplex/hemin complex, mimicking HRP activity, producing a distinguishable color. This colorimetric method detected S. pneumoniae at 156 CFU mL⁻¹ [76]. Indeed, signal amplification is a significant process for pathogen detection. CHA can efficiently involve in signal amplification and has the advantage to overcome the drawback of enzymatic amplification. Thus, this technique adopted CHA for S. pneumoniae detection. However, it necessitates intricate engineering for modification with several analytes, which may limit this study to further clinical application.

2.1.4.2. Fluorescence strategy

Significantly, the fluorescence method was also lucrative for S. pneumonia detection. Therefore, a fluorophore-labeled aptamer (FL-apt) attached to target bacteria functions as a biosensor. Indeed, GO interacted with the aptamer via physisorption, reducing the fluorescent intensity of fluorophores through nonradiative excitation energy transfer [78]. Thus, FL-apt/S. pneumoniae-based biosensor gradually decreased their fluorescent intensity while increasing the concentration of GO and LOD was 15 CFU mL⁻¹ [30].

2.2. Airborne virus detection

Viruses spreading through the air can cause serious illness and lead to death. Thus, detecting airborne viruses, such as influenza A (H1N1 and H3N2), avian influenza A (H5N1, H7N9, and H9N2), Norovirus, and SARS-CoV-2, are mandatory and optical biosensors involved in the detection process were discussed in this section [6].

2.2.1. Influenza A (H1N1 and H3N2)

H1N1 and H3N2 viruses have a high transmissibility rate, causing 670,000 deaths annually. Considering the resource-limited settings, identifying these viruses with the naked eye could be a high demand.

2.2.1.1. Colorimetry strategy

A sandwich biosensor (ConA/GOx/AuNP) has been developed using concanavalin A (ConA), glucose oxidase (GOx), and AuNPs for determining the H3N2 virus. The aptamer-functionalized H3N2 was combined with the ConA/GOx/AuNP biosensor, producing red color when the glucose solution was added to it. This method has high selectivity for H3N2 and was detected as low as 11.16 μg mL⁻¹ [79]. In another method, AuNPs combined with CNTs function as nanozymes, oxidizing TMB in the presence of H3N2. This method was detected at 3.4 PFU mL⁻¹, which was 385 times more sensitive than the existing ELISA method (1312 PFU mL⁻¹) (Fig. 5 a) [25]. Substantially, to synthesize the Au-CNT nanohybrid material, an environmentally friendly and inexpensive strategy was introduced in this technique as opposed to the existing methods which utilized harsh conditions for producing the Au-CNT nanohybrid [80]. Moreover, this technique has 500-fold higher sensitivity than commercial immunochromatography kits toward H3N2 detection.

Fig. 5.

(a) Schematic showing the detection of H3N2 virus based on the colorimetric peroxidase-like activity of Au-CNTs. Reproduced from the published article [25]. (b) The AuNPs/MBs-based immunoassay for the colorimetric detection of H7N9. Reproduced from the published article [87].

AuNPs act as nanozymes that can be used instead of HRP to oxidize TMB in the presence of H₂O₂ to develop a colorimetric platform for H1N1 detection. This method simultaneously detects H1N1 and H3N2 with a limit of 10 pg mL⁻¹ and 10 PFU mL⁻¹, respectively [81]. Indeed, although various nanomaterials have exhibited peroxidase-like activity, they are not easy to synthesize. Interestingly, this study utilized AuNP as nanozymes for pathogen detection due to its simple synthetic procedure. Therefore, the AuNP-based immunoassay method could be an useful alternative to other nanozyme-contained techniques for on-site clinical diagnostics [81]. Recently, a polydiacetylene (PDA)-based colorimetric method has been extensively employed for H1N1 detection [5]. The PDA vesicles have unique chromatic properties that were synthesized by photopolymerizing 10,12-pentacosadiynoic acid. Subsequently, the PDA vesicles were functionalized with specific peptide (PEP) sequences to make a PDA-PEP biosensor for capturing target pathogens. The PDA-PEP biosensor efficiently bound the H1N1 due to the high affinity of PEP to the HA protein of the influenza H1 strain. Furthermore, the blue color of the PDA-PEP biosensor changed to red color, and the LOD of this proposed method was 10⁵ PFU mL⁻¹ [82]. Another group followed the same strategy, in which polyvinylidene fluoride was used for immobilizing the PDA vesicles on its surface to produce a paper-like sensor, and smartphone technology was used to quantify the H1N1 [83].

2.2.2. Avian influenza A (AIV)

AIV can function as a gene donor, resulting in a higher death rate than other deadly viruses. Thus, prompt and essential analytical methods for early AIV diagnosis and restrain are needed [86].

2.2.2.1. Colorimetry strategy

The AuNPs/MNPs-based immunoassay was fabricated, in which ALP was attached to enhance the phosphate group's hydrolysis in the H7N9 backbone. Consequently, the AuNPs' color changed from red to blue when increasing the concentration of the target virus. Using this method, the LOD for H7N9 was measured as 1.25 pg mL⁻¹ [87] (Fig. 5b). Another ALP strategy with SPR performance was used for H5N1 determination. The antigen-functionalized Au nanobipyramids (AuNBP) were combined with ALP, generating 4-aminophenol (4-AP) from the cleavage of the 4-aminophenyl phosphate. The produced 4-AP reduced the silver nitrate to AgNPs, which were deposited on the AuNBPs surface. Based on the silver deposition, bright colors were observed, and up to 1 pg mL⁻¹ of H5N1 was detected. Typically, AuNBPs have higher sensitivity than the other nanostructures of the gold due to the high-index faceted on its edges. In addition, the AuNBP appears approximately at 750 nm (near-infrared region) in the UV–vis absorption spectrophotometer, and thus visual detection was easily perceived by the naked eye compared with other colorimetric methods. Owing to the formation of vivid colors, this approach may be employed for the semiquantitative detection of the H5N1 [88].

2.2.2.2. Fluorescence strategy

To perform multiplex detection, the fluorescence method is the easiest because different fluorescent emissions of target pathogens using a single light source can be collected simultaneously [89]. The immunoassay was fabricated using green, yellow, and red fluorescent MNPs (FMNPs) attached with the antibodies of target viruses, such as H9N2, H1N1, and H7N9 [90]. In the UV environment, the LOD of target viruses was 0.02 pg mL⁻¹, exhibiting higher performance than colorimetric methods [86,87]. The formation of hydrogels based on the hybridization between the aptamer and ssDNA was competently used for H5N1 detection. Noteworthy, the aptamer and ssDNA-produced hydrogel remained shrunk in the absence of the target virus. However, when the aptamer in the hydrogel interacts with H5N1, the hydrogel swells. Consequently, the aptamer quencher was released from the hydrogel, producing fluorescence, and this technique could detect 0.4 HAU of H5N1 [20]. In this study, the hydrogel-based aptasensor was introduced which has various advantages such as ease of synthesis, biocompatibility, high chemical stability, simple controllable modification, and low cost. Therefore, the label-free and hydrogel-based fluorescence aptasensor could be an alternative to expensive antibody/antigen-based immunosensors for pathogen detection.

2.2.3. Norovirus (NoV)

NoV is the most ubiquitous pathogen causing diverse diseases, such as diarrhea, food poisoning, and acute gastroenteritis in humans. It can survive any environmental conditions and is transmitted through direct contact, contamination of water, and aerosols [93]. Therefore, earlier diagnosis is preferred to prevent a contagious environment [94,95].

2.2.3.1. Colorimetry strategy

Lactoferrin (LF) is a common recognition molecule, as it attracts any virus in the sample; thus, LF-immobilized cotton swaps have been designed for NoV detection. The NoV was sandwiched between the antibody-conjugated AuNPs and LF-immobilized cotton swaps. The red color was sustained in the cotton swab even after washing with a phosphate-buffered saline solution. This developed immunoassay's LOD for NoV was measured between 10 and 53 PFU mL⁻¹ [96]. Nanozymes, specifically nanohybrid, such as graphene-AuNPs, have intrinsic peroxidase-like properties that were preferentially used for detecting NoV-like particles (NoV-LPs). As using nanozymes for influenza virus detection [25,81], the antibody-attached graphene-AuNPs (nanozymes) adsorbed the NoV-LPs, oxidizing TMB in the presence of H₂O₂ and produced blue color. Remarkably, the LOD of this method was 92.7 pg mL⁻¹, which was 112 times lower sensitivity than the ELISA, and 41 times higher performance than the commercial kit [97]. Recently, vanadium oxide (V₂O₅) NP-encapsulated liposomes produced an immunoassay with NoV [98]. When liposomes break from the sandwich structure, the V₂O₅ NP is released, oxidizing the TMB. The LOD of this colorimetric method was 0.34 pg mL⁻¹ (Fig. 6 a). Interestingly, the released V₂O₅ NPs from captured liposomes not only have peroxidase-like activity but also acted as an electrochemical redox indicator. Therefore, this study has reported colorimetry as well as electrochemical approaches for NoV detection. Furthermore, the 1000-fold higher sensitivity is attained exhibiting the importance of the V₂O₅ NP-encapsulated liposome-based sensor. Also, the fabricated biosensor can be stored for 3 weeks without losing its catalytic activity, showing the remarkable stability of the sensor. Overall, the V₂O₅ NP-encapsulated dual-mode sensor avoids the intricated sample preparation and constraints the use of expensive instruments which makes the detection process facile and rapid [98].

Fig. 6.

(a) Detection of the NoV-based on the effective peroxidase-like activity using the release of V₂O₅ NPs from the captured liposomes. Reproduced from the published article [98]. (b) Schematic showing AuNP/MNP–NC–based sandwich immunoassay for NoV detection using the fluorescence strategy. Reproduced from the published article [100].

2.2.3.2. Fluorescence strategy

To consider developing new techniques, cheap and convenient detection strategies must be developed. Hence, fluorescence-based techniques could fulfill the criteria for target virus detection [89]. The 6-carboxyfluorescein (6-FAM) attached with NoV-specific aptamer and its fluorescence was quenched while adding GO. When introducing the target NoV, the biosensor displayed strong fluorescence due to breakage between the aptamer and GO. Moreover, this fluorescence strategy showed a good linear range between 13 ng mL⁻¹ and 13 μg mL⁻¹ of NoV [99]. The synthesized NCs on the combination of AuNP and MNPs were producing a sandwich immunoassay when attached with a specific antibody, CdSeS QDs, and target NoV. Notably, this immunosensor emitted fluorescence when excited with UV light, and 0.48 pg mL⁻¹ of NoV can be detected (Fig. 6b) [100]. The liposomes encapsulated calcein fluorophore and APTES-functionalized MNPs have been conjugated with the specific antibody. Then, it was attached to target NoV to produce a sandwich-like biosensor (calcein fluorophore/NoV/APTES-MNPs) for NoV detection. This biosensor released fluorescence after the Triton X treatment due to the rupture of the calcein encapsulated liposomes. Using this method, the LOD was as low as 136 copies mL⁻¹ [102]. The specific interaction between the antibody and antigen can significantly reduce the background signal, resulting in a higher detection sensitivity. However, this method has a disadvantage due to the usage of less stable liposomes and antibodies.

2.2.4. SARS-CoV-2

The world is currently challenging an unpleasant situation due to the SARS-CoV-2, producing an infectious disease in humans. Indeed, this virus is environmentally stable and transmitted through direct contact and aerosol. To control SARS-CoV-2, an urgent need exists for simple, rapid, and highly sensitive detection methods [103].

2.2.4.1. Colorimetry strategy

The spherical nucleic acids (SNAs) is a thick shell with highly oriented nucleic acids that were conjugated with AuNPs. Moreover, the PCR-amplified SARS-CoV-2 was incubated with AuNP-functionalized SNAs that sustained red color, even after adding the NaCl solution due to the restriction of single component assembly [104]. The thiol functionalized antisense oligonucleotides (ASOs) capped with AuNPs have a specific interaction with the N-gene of SARS-CoV-2 [105]. Hence, ASO-capped AuNPs sensor was fabricated, which were agglomerated in the presence of the target SARS-CoV-2 N-gene. The RNaseH was introduced to the blue-colored analyte mixture for further signal amplification, producing a precipitate that was visually perceived by the naked eye. This technique enables SARS-CoV-2 detection as low as 0.18 ng μL⁻¹ within 10 min [106]. Significantly, the efficacy of the ASO-capped AuNP biosensor was compared with the FDA-approved diagnostic kit for SARS-CoV-2 detection, confirming a good alternative for the commercially available kits. Due to the rapid mutation of SARS-CoV-2, several diagnostic methods fail to sustain its reliability. But, the ASO-capped AuNP biosensor overcomes this limitation by simultaneously detecting the N-gene of SARS-CoV at various positions in the gene sequences.

The clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease (Cas) (CRISPR/Cas) technique offers to cut the DNA or RNA using the gene-editing technique [107]. Moreover, this method is applied for nucleic acid detection through the colorimetric pathway [108]. Hence, the combination of CRISPR/Cas12a with RT-RPA has been utilized for SARS-CoV-2 detection. In this process, AuNPs were modified with ssDNA, specifically targeting the ORF1ab and N-gene of SARS-CoV-2. After the RT-RPA, Cas12a was activated in the amplified dsDNA. Due to initiating trans-cleavage, the capped DNA in the AuNPs’ surface was gradually hydrolyzed, triggering the aggregation, and the color of the solution turned blue. Because of RT-RPA and Cas12a, this method can detect even one copy of viral RNA [28].

2.2.4.2. Fluorescence strategy

Recently, HRP-labeled anti-human IgA contained with an H₂O₂/luminol-based biosensor was used for SARS-CoV-2 detection through a chemiluminescence platform [109]. Considering the cost and stability of HRP, a Co–Fe@hemin was synthesized and acted as a nanozyme for detecting the target virus. Indeed, the Co–Fe@hemin nanozyme enhanced the luminol intensity in the presence of SARS-CoV-2 [110]. Using this method, the LOD of SARS-CoV-2 was 0.1 ng mL⁻¹, revealing lower sensitivity than the SPR-based method [106].

3. Electrochemical biosensors for airborne pathogen detection

In an electrochemical biosensor, the interaction between immobilized molecules and target analytes (airborne pathogens) produces electrical signals in the system. Based on the detection principle and application, the produced electrical signal can be read using various techniques such as voltammetry (CV, SWV, DPV), amperometry, and EIS [[34], [35], [36], [37], [38]]. Electrochemical biosensors offer various advantages such as sensitivity, portability, easiness, and rapidity for finding airborne bacteria, and this section briefly addresses them. Table 4, Table 5 summarize a detailed comparison study based on electrochemical biosensors for airborne bacteria and virus detection.

Table 4.

A detailed comparison of the electrochemical biosensor for airborne bacteria detection.

Target bacteria	Detection method	Conjugate materials	Linear range	LOD	Time (min)	Real sample	Ref
B. anthracis	Amperometry	ABTS/HRP	103–106 cfu mL−1	102 CFU mL−1	60	–	112
	EIS	aptamer	–	103 CFU mL−1	–	Water	113
	SWV	BiNPs/Nafion-MWCNT	0.1–100 ng mL−1	50 pg mL−1	35	Human serum	114
		Ferrocene/dATP	–	0.8 fM	–	Plasmid pXO1	115
	CV	Au/Pd NP/BNNSs	5 pg–100 ng mL−1	1 pg mL−1	3	BSA	116
MTB	CV	AuNTsA	0.01–100 ng μL−1	0.5 ng μL−1	5	–	117
		Oligomer	1 fM–100 pM	0.2 fM	45	–	119
		AuNPs/C60/PAn	0.02–100 pg mL−1	20 fg mL−1	60	Human serum	122
		NH2–GO/QD	–	8.948 × 10−13M	50	–	118
	DPV	HANPs/PPy/MWCNT	0.25–200 nM	0.141 nM	–	Sputum	120
		C60NP/N–CNT/GO	1 fg–1 ng mL−1	0.33 fg mL−1	60	Human serum	123
		AuNP@C60/N–GO	10 fM–10 nM	3 fM	60	–	124
		AuNC	0.1–1 × 105 fM	0.031 fM	60	BSA	126
		Fe3O4@Ag/GQD	0–500 μg mL−1	0.33 ng mL−1	–	BSA	130
		Si NP/CdSe/ZnS QD	40–100 ng mL−1	1.5 × 10−10 g mL−1	–	BSA	131
S. aureus	DPV	ZrO2/GO	–	3.23 × 10−14 mol L−1	10	–	132
		SWCNT	10–107 CFU mL−1	13 CFU mL−1	30	Milk	134
		BC/MWCNT/PEI	10–107 CFU mL−1	3 CFU mL−1	30	Milk	138
		aptamer	60–6 × 107 CFU mL−1	9 CFU mL−1	–	Water	139
		aptamer	10–108 CFU mL−1	8 CFU mL−1	60	Lake water	140
	CV	zeolite/GO	0.5–100 μM	0.1 nM	–	Fruit juice	133
	EIS	AuNR/PDDA/PSS	103–107 CFU mL−1	2.4 × 102 CFU mL−1	50	Milk	136
S. pneumoniae	CV	oligomer	–	0.5 fM	60	Whole blood	141
	DPV	polyaminophenol	0–200 ng mL−1	54 ng mL−1	–	BSA	143
	SWV	oligomer	0–8 ng mL−1	0.218 ng mL−1	–	BSA	144

Table 5.

A detailed comparison of the electrochemical biosensor for airborne virus detection.

Target virus	Detection method	Conjugate materials	Linear range	LOD	Time (min)	Real sample	Ref
Influenza A (H1N1 subtype)	EIS	aptamer	–	0.9 pg μL−1	30	BSA	17
		BDD	–	1 fg μL−1	–	BSA	147
		TrGO	–	33 PFU mL−1		Saliva	148
	DPV	Cys/oligomer	–	0.002 ng μL−1	30	–	149
		AuNP−MNP−CNT	1 μg–100 fg mL−1	13.66 fg mL−1	–	BSA	150
	Amperometry	antibody	1–104 PFU mL−1	0.5 PFU mL−1	–	–	151
Avian influenza A (H5N1 subtype)	EIS	aptamer	–	0.0128 HAU	30	–	154
		AuNP/aptamer	16–0.125 HAU	0.25 HAU	45	–	155
	CV	pAuNP	–	1 pM	–	Chicken serum	156
	Amperometry	ZnO NR	–	1 pg mL−1	–	BSA	157
Norovirus	EIS	Cys/Nor-1 peptide	–	1.44 μg mL−1	–	Fetal bovine serum (FBS)	158
		Specific binding peptides	0–105 copies mL−1	1.7 copies mL−1	30	FBS	159
		WS2NF/AuNP	0–104 copies mL−1	2.3 copies mL−1	60	FBS	161
	CV	ConA/ALP	102–104 copies mL−1	35 copies mL−1	60	–	162
	Potentiometer	AuNP/MNP/graphene	0.01 pg–1 ng mL−1	1.16 pg mL−1	–	BSA	163
	DPV	AuNP/graphene	100 pM–3.5 nM	100 pM	30	–	36
SARS-CoV-2	Amperometry	Co/TiO2 NT	14–1400 nM	0.7 nM	–	–	167
	EIS	antibody	–	20 μg mL−1	45	BSA	168
	DPV	Au@Pt/MIL–53(Al)	0.025–50 ng mL−1	8.33 pg mL−1	60	Human serum	169
		sulfocalix[8]arene/GO	–	200 copies mL−1	–	Saliva	170
		RPA amplicon/thiol-modified primers	–	3.925 fg μL−1	20	–	171

3.1. Airborne bacteria detection

3.1.1. Bacillus anthracis (B. anthracis)

B. anthracis spores have been mostly detected through its biomarker (DPA) in optical biosensors [43,48]. However, this infectious bacterium is directly detected using electrochemical biosensors [111]. Waller et al. established an amperometric immunoassay for detecting B. anthracis spores. In this immunoassay, the GOx generated H₂O₂ while oxidizing glucose. The produced H₂O₂ develops an electrical signal when oxidizing ABTS using HRB. Notably, this signal was monitored using an amperometric portable instrument and the detection range was 5 × 10³ to 5 × 10⁶ CFU mL⁻¹ [112]. More importantly, even minimal signal differences produced in the presence of environmental interferents including soil and altered pH conditions can be detected, exhibiting the strength of the amperometry method. This technique is cost-effective and rapid, and the sensitivity is either superior or comparable to that of the ELISA and LFDs, thus making it a promising tool for detecting B. anthracis spores on a portable platform.

As with detecting PA using a colorimetric strategy [23], the SWV technique was performed to detect PA at the picogram level. In this process, the immunoassay was fabricated using Nafion-MWCNTs–bismuth NC film modified glassy carbon electrode (GCE) (BiNPs/Nafion-MWCNTs/GCE) and titanium phosphate NP–cadmium ion-contained anti-PA antibodies (TiP–Cd@PA antibodies). Under optimum conditions, the currents increased when increasing the PA concentration, and LOD was measured as 50 pg mL⁻¹ [114]. Typically, the two types of S-layer proteins, such as extractable antigen and surface array protein (Sap), were secreted by B. anthracis in the culture medium. Thus, Sap act as a biomarker, and the fabricated bionanolabel (Au–Pd NPs@BNNSs/Ab₂) converted the 4-AP to 4-quinine imine, resulting in redox peaks that were monitored by CV. Significantly, this proposed immunosensor exhibited a good linear range from 5 pg mL⁻¹ to 100 ng mL⁻¹, and the LOD of B. anthracis Sap was 1 pg mL⁻¹ (Fig. 7 a) [116]. Interestingly, the biomarker Sap is only secreted by B. anthracis and other species of Bacillus will not produce it. Therefore, this method has high selectivity toward B. anthracis detection compared with other techniques. Substantially, the fabricated Au–Pd NPs@BNNSs/Ab₂ immunosensor can quantify the Sap within 1 h due to its high catalytic activity, showing the importance of this electrochemical biosensor.

Fig. 7.

(a) Proposed electrochemical immunosensor for the detection of B. anthracis Sap based on the generation of the redox peaks upon the conversion of the 4-AP to 4-quinine imine. Reproduced from the published article [116]. (b) Target MPT64 sandwiched between the PEI@MOF-modified electrode and the tracer label. TOBA developed the electrical signal in the presence of target bacteria. Reproduced from the published article [123]. (c) Schematic showing a label-free electrochemical immunosensor using PDDA, PSS, and AuNR on the GCE for S. aureus detection. Reproduced from the published article [136].

3.1.2. Mycobacterium tuberculosis (MTB)

The Au nanotubes array (AuNTsA) has a higher electron transfer capacity than the bare Au electrode due to its high surface area; thus, AuNTsA was synthesized using the electrochemical deposition method. To construct the biosensor, the probe DNA was immobilized on the AuNTsA surface, and complementary DNA was hybridized on it. In the process, the electron transfer of redox indicator [Fe(CN)₆]^3−/4− was monitored using CV. The target DNA concentration from 0.01 ng μL⁻¹ to 100 ng μL⁻¹ exhibited a good linear range [117]. In general, the AuNTsA is more delicate and easily damaged. However, this method simply modified the electrodeposition method and produced stable AuNTsA. Moreover, the produced AuNTsA are well separated from each other, which acted as individual electrodes resulting in a substantial improvement in the detection process. Significantly, the AuNTsA electrode is more beneficial compared with the bare Au electrode due to the radial diffusion of ions in constructing efficient electrochemical biosensors. In addition, the fabricated electrode sustained its efficacy toward MTB detection up to three weeks, confirming the stability of the AuNTsA-based electrochemical biosensor [117].

Sypabekova et al. established an aptamer-based technique for MPT64 protein detection, which is an immunogenic polypeptide of MTB. Typically, the aptamer corresponding to the MPT64 protein was designed using the SELEX technique. The designed aptamer was attached to the electrode surface using the thiolated linker. The current fluctuations occurred when introducing the MPT64 protein on the biosensor, monitored using EIS [121]. The fullerene (C60)-incorporated NCs were exclusively involved in MPT64 protein detection due to their electron transfer capacity [122,123]. The MPT64 aptamer was immobilized on the AuNPs@C-60/N-CNT/GO NC surface, developing a sandwich biosensor using polyethyleneimine (PEI)-functionalized MOF. The tetraoctylammonium bromide (TOAB) was used to enhance the redox activity of this sandwich biosensor in the presence of the MPT64 protein (Fig. 7b) [123]. Using this method, the LOD was measured as low as 0.33 fg mL⁻¹, revealing higher sensitivity than the SPR-based method [60].

The IS6110 gene is found at various sites in the MTB genome. Thus, this gene sequence detection was helpful for MTB diagnosis, and the DPV-based electrochemical technique was widely used to monitor the IS6110 gene [[124], [125], [126], [127]]. Arginine contained an active amino group (−NH₂), providing an excellent platform for hybridization between the probe DNA and target DNA. Using MB as the redox signal developer in the DPV technique, up to 4.4 nM of the IS6110 gene was detected [125]. However, the proposed method has lower performance than the target DNA-induced recycling amplification and enzyme-assisted signal amplification-based strategies [126,127]. The culture-filtered protein (CFP-10) is also used as a biomarker to diagnose TB at an earlier stage [[128], [129], [130], [131]]. Tufa et al. devised an immunosensor based on the antigen-antibody interaction. Initially, the CFP-10 antigen was binding with Fe₃O₄@Ag/GQD NCs. The CFP-10 antibody was conjugated with AuNPs which were employed for signal amplification. In this process, the electron transfer and binding ability increased due to the large surface area of the NCs. Thus, the resultant immunosensor revealed high specificity and reasonable LOD against the CFP10 antigen [130]. Significantly, Fe₃O₄ NPs in the nanotriplex (Fe₃O₄@Ag/GQD) sensor enabled facile transportation of reactant and product at the electrode surface. Moreover, AgNPs and GQDs enhanced the electrical conductivity and captured more CFP-10 antigen due to their high surface area. Hence, the nanotriplex (Fe₃O₄@Ag/GQD) sensor achieved good electrochemical performance toward MTB detection, and the properties of these nanomaterials make them a promising candidate to fabricate an efficient electrochemical biosensor for the detection of other pathogenic bacteria in the future.

3.1.3. Staphylococcus aureus (S. aureus)

S. aureus bacteria causes severe health-related issues; thus, electrochemical biosensors were employed for its detection [16,33]. Sun et al. established a novel electrode surface containing ZrO₂/GO for hybridization between the probe ssDNA and target ssDNA. The MB easily attaches to dsDNA and functions as a redox indicator in the electrochemical process. Thus, the S. aureus-contained electrode was dipped in the MB solution and monitored the redox signal variation using the DPV technique. Notably, this fabricated biosensor detected the S. aureus as low as 3.23 × 10⁻¹⁴ mol L⁻¹ [132]. Electrochemical immunosensors were impeccably applied to S. aureus detection [[134], [135], [136], [137]]. AuNRs have good electron conductivity and were functionalized with poly(diallyldimethylammonium chloride) (PDDA) and polystyrene sulfonate (PSS) on the GCE surface for binding the antibodies. The immobilized specific antibodies competently captured the S. aureus cell, and electron transfer on the GCE surface was blocked. Significantly, EIS was used to measure the variation of electron transfer resistance of Fe(CN)₆ ^3−/4− (Fig. 7c). The proposed immunosensor could detect the bacteria as low as 2.4 × 10² CFU mL⁻¹ [136]. In this technique, the simplest self-assembled process was utilized which can control biomolecule density and orientation at a solid-liquid interface resulting in a highly sensitive detection. Most of the S. aureus cells were efficiently captured on the PDDA/PSS/AuNRs/Ab surface without nonspecific adsorption revealing the advantages of the self-assembled immunosensor. Using this method, the whole S. aureus cell has been detected, which eliminated the complex sample preparation processes [136].

In terms of cost and stability of antibodies, devising immunosensors have received increasing challenges recently. Hence, a novel material, such as bacterial cellulose (BC), was modified with MWCNTs for S. aureus detection. The BC/MWCNTs nanohybrid was further treated with PEI to obtain a positive charge on the surface. Finally, the bacterial phage was immobilized on the BC/MWCNT/PEI surface based on the electrostatic interaction. The phage concentration determined the current responses in the biosensor, and up to 3 CFU mL⁻¹ of S. aureus was detected using the DPV method [138]. Significantly, this method was first reported to differentiate live S. aureus cells from the mixture of live/dead cells based on the utilization of the bacteriophages and BC. The BC/MWCNT/PEI film has high lytic activity and thus phages were actively immobilized on its surface, providing excellent sensitivity and selectivity toward live S. aureus cells detection. Moreover, the immobilized phages could be in a humid environment due to the never-dried nature of BC, resulting in bacteriophages with BC-contained electrochemical biosensors that are highly stable and portable.

3.1.4. Streptococcus pneumoniae (S. pneumoniae)

Electrochemical biosensors can detect S. pneumoniae rapidly [141]. Typically, electrode surface modification with polymer components has shown enormous benefits in the electrochemical biosensor field [138,142]. Ferreira et al. modified the graphite electrode surface based on electropolymerization using 4-AP. Due to polymerization, the surface contained –NH₂ and –OH groups, which easily attract oligonucleotides and hybridized with the target ssDNA, producing dsDNA on the electrode surface. Ethidium bromide (EB) functions as an indicator that was efficiently intercalated into the dsDNA. Notably, the current responses generated by EB in the presence of S. pneumoniae were monitored using DPV analysis [143]. Using this process, the LOD was 54 ng mL⁻¹, revealing lower sensitivity than the colorimetric LAMP-based method [8].

3.2. Airborne virus detection

3.2.1. Influenza A (H1N1 and H3N2)

Each year, influenza A affects two billion people, according to the WHO report. Early detection is critical to control the disease, and electrochemical biosensors are increasingly used in identifying these viruses [145]. Matsubara et al. focused on the sialic acid-mimic peptide immobilized on a boron-doped diamond (BDD) electrode using click chemistry for detecting infectious viruses. Significantly, the H1N1 and H3N2 were identified in the limit of 20–500 PFU using EIS analysis [146]. Ravina et al. established a Cys-coated Au-SPE for immobilizing the probe ssDNA. Then, it was hybridized with target ssDNA to produce dsDNA on the electrode surface. Using DPV analysis, 0.002 ng μL⁻¹ of H1N1 was detected (Fig. 8 a) [149]. This study utilized an SPE due to its advantages such as high electrical conductivity, biocompatibility, and stability in various temperatures and pH compared with the other electrodes. Moreover, Cys was easily adsorbed on the SPE surface due to the presence of Au which provided a site to capture the probe DNA on the electrode surface for the electrochemical detection of H1N1. Significantly, this study followed a simple methodology and thus could be employed as a POC diagnosis tool for influenza virus detection.

Fig. 8.

(a) Schematic fabrication of electrochemical biosensor using Cys-linked probe for H1N1 detection. Reproduced from the published article [149]. (b) Schematic representation for Norovirus detection using a WS₂NF/AuNP-peptide electrochemical biosensor. Reproduced from the published article [161]. (c) Process of super sandwich biosensor for the detection of SARS-CoV-2 using a smartphone. Reproduced from the published article [170].

3.2.2. Avian influenza A (AIV)

The H5N1 AIV can rapidly mutate and combine with other subtypes to become a more dangerous pathogen. To prevent its transmission rate, earlier diagnosis is much better, and electrochemical biosensors contributed significantly to this purpose [152]. Lin et al. implemented an impedance immunosensor for H5N1 detection in a real sample. Wet etching microfabrication was employed to modify the electrode and functionalized it with H5N1 antibodies. The impedance magnitude increased when the electrode captured H5N1, and the LOD of this method was 0.2 HAU 50 μL⁻¹ [153]. Compared with the dry-etching process, the improved wet-etching process was utilized in this study which competently reduced the production cost of the interdigitated microelectrode. Moreover, the fabricated electrode can be used at least five times after cleaning with NaOH and HCl solution, however, the recycling process of the electrode may be challenging in the real sample analysis. Significantly, this immunosensor was tested on the chicken swab to identify the H5N1, producing almost comparable accuracy with the RT- PCR method [153]. Indeed, ZnO NR has a high isoelectric point (IEP), stimulating the electrostatic interaction with the lower IEP of AIV antibodies. Using this concept, Han et al. developed a microfluidic electrochemical immunosensor for H5N1 detection [157]. Amperometry analysis measured the oxidation current produced in the process, and LOD was 1 pg mL⁻¹, showing equal sensitivity with the SPR-based method [88].

3.2.3. Norovirus (NoV)

NoV is a lethal human pathogen effectively monitored by electrochemical biosensors due to simplified sample preparation and high sensitivity [6]. Hwang et al. adopted a peptide (Nor-1 peptide)-based evolutionary phage display technique to monitor NoV [158]. In the process, Cys was incorporated into the Nor-1 peptide and immobilized to an Au electrode to produce a biosensor (Au/Cys/Nor-1 peptide). When recombinant noroviral capsid proteins (rP2) were actively attached to the Au/Cys/Nor-1 peptide, a significant increase in impedance was observed due to electrode surface blocking. Using this method, LOD was measured as 1.44 μg mL⁻¹, showing lower performance than the fluorescence-based biosensor [100]. The hydrothermally synthesized 2D-tungsten disulfide (WS₂) nanoflowers (NF) have unique electronic properties. Introducing metal NPs enhanced their charge carrier mobility and thermal conductivity [160]. Hence, Baek et al. synthesized AuNP-decorated WS₂NF and combined it with the bioreceptor attached Cys for capturing the target NoV. Based on EIS analysis, the LOD for NoV was 2.37 copies mL⁻¹ (Fig. 8b) [161]. ConA specifically targeted NoV, not capturing other viruses, such as hepatitis A and hepatitis E. Thus, ConA was attached to a nanostructured Au electrode and immobilized with ALP-contained secondary antibodies. Notably, 4-aminophenyl phosphate was changed to 4-AP due to the presence of ALP in the electrode, and a redox signal was generated when 4-AP was oxidized to 4-quinone imine. CV revealed that the oxidation current increased while increasing the NoV concentration [162]. The detection limit was 35 copies mL⁻¹, disclosing higher efficiency than the fluorescence-based method [102].

3.2.4. SARS-CoV-2

Optical biosensors considerably contributed to SARS-CoV-2 detection, which further demands various analytical techniques due to the high transmittance rate of this infectious virus [[164], [166]]. Hence, electrochemical biosensors were developed with high sensitivity toward SARS-CoV-2 [165]. The receptor-binding domain (RBD) of spike protein is present on the SARS-CoV-2 surface, which can be detected using cobalt-functionalized TiO₂ nanotubes. Amperometry analysis detected the RBD protein in the range of 14–1400 nM within 30 s; thus, this sensor could be used in the rapid diagnostic process [167]. Calixarene is an example of macrocyclic molecules exhibiting excellent enrichment capability to electrochemical indicators, such as MB and toluidine blue (TB). Hence, Zhao et al. fabricated a super sandwich-type electrochemical biosensor based on GO functionalized with p-sulfocalix [8] arene to enrich TB for detecting SARS-CoV-2. Significantly, the GO functionalized p-sulfocalix [8] arene electrochemical biosensor has higher sensitivity (85.5%) than the RT-qPCR (56.5%) when tested with 35 SARS-CoV-2 positive samples. Notably, this super sandwich-type technology does not demand amplification and reverse transcription processes for the detection of SARS-CoV-2. More importantly, this electrochemical biosensor has first established the detection of SARS-CoV-2 based on the smartphone platform (Fig. 8c) [170].

4. Conclusions

Considering the severity and high transmission rate of airborne pathogens, accurate and rapid detection methods are critical. Traditional methods are either expensive and time-consuming or have low sensitivity toward detecting contagious pathogens. Hence, this review focused on emerging techniques, such as optical and electrochemical biosensors to find airborne pathogens, even at low concentrations. Optical biosensors have been inspired by three strategies, such as SPR, colorimetry, and fluorescence used for pathogen detection. Metal NPs, including AuNPs and AgNPs, have unique SPR properties that help to find airborne bacteria and viruses in a short time. In the colorimetry process, a label-free assay is a direct method, that is, the transducer surface generates a distinguishable color in the presence of target analytes due to the biochemical reaction. Significantly, the labeled assay developed an intense color when the target analytes were sandwiched between the capture and fluorophores labeled detector agents. These optical biosensors are easy to fabricate, show prompt results, and have high sensitivity to airborne pathogen detection. Another approachable electrochemical process was unprecedently employed in airborne pathogen detection due to its precision, rapidity, and sensitivity. Moreover, the generated electrical signal based on the interaction between the fabricated electrode and target analytes can easily be measured through various strategies, such as DPV, CV, SWV, amperometry, and EIS. Although there is enormous benefit from the optical and electrochemical biosensors, some critical aspects must be considered to convert them into on-site applications.

5. Challenges and future perspectives

• (i).Devising immunosensors in optical/electrochemical techniques typically used antibodies, antigens, and enzymes (HRP) for producing higher sensitivity toward airborne pathogen detection. However, the stabilities of these biological substances are low, having a shorter shelf life and extra care must be taken to sustain their stability. Hence, immunosensors could not be applied in resource-poor settings.
• (ii).Aptamers provide excellent benefits over antibodies in terms of stability, production cost, and synthesis. Hence, it plays a significant role to develop aptasensors for airborne pathogen detection, where they can function as biorecognition components. However, the shorter-length aptamers do not have high affinities with biomolecules, and longer sequences lead to aggregation or misfolding, affecting the yield of chemical synthesis. Therefore, the perfect length of aptamers must be optimized before fabricating the aptasensor. Furthermore, the immobilization of aptamers is complicated because it relies on adding biotin/streptavidin or a chemical reaction (EDC/NHS coupling), making it an expensive process.
• (iii).Nanozymes have high stability and can be used for pathogen identification based on peroxidase-like activities. However, its synthetic methods are too intricate and offer less reproducibility. Therefore, the advancement of easy-to-prepare methods and fabrication of nanozyme-based highly sensitive biosensors for airborne pathogen detection are needed.
• (iv).The inorganic material containing fluorescence probes (optical biosensor) has higher stability and improved sensitivity against infectious pathogens. Furthermore, the ratiometric fluorescence probes measured the variation of the fluorescence intensity ratio developed in the presence of the pathogen and obtained an accurate result. However, each fluorophore has individual fluorescence intensities and many experiments must be conducted using a combination of different fluorophores to obtain perfect ratiometric fluorescence sensors. Therefore, the fluorescence technique is considered expensive and laborious.
• (v).Due to technology improvements, the nucleic acid amplification-based method is widely used for airborne pathogen detection, offering improved sensitivity and fast responses over other techniques. However, this method requires expensive reagents, sophisticated equipment, and a modernized laboratory to perform the biochemical reaction. To simplify this technique, the device should be modified as a portable platform to identify the pathogen competently.
• (vi).PDA-fabricated biosensors have received much attention due to their characteristic chromatic properties and are employed in colorimetric infectious disease monitoring, but only limited experiments have been performed so far. Thus, elaborate research based on PDA biosensors is necessary to apply for on-site applications.
• (vii).In the electrochemical biosensor, various electrodes are available, and choosing a suitable electrode for devising the potential sensor is critical. Furthermore, recently developed electrochemical biosensors have many advantages in terms of miniaturization and portability. Nevertheless, the device fabrication process is time-consuming and costly, which is a demerit and should be considered before designing effective electrochemical biosensors.
• (viii).

  Colorimetric biosensors using the LFIA technique produced a successful kit that is commercially available for HIV and pregnancy tests. Similarly, airborne bacteria (B. anthracis, MTB, and S. pneumoniae) were monitored using the LFIA-based colorimetric biosensor and other infectious pathogens have to be detected using a similar platform due to its uniqueness. Despite the LFIA being a qualitative assay, they are desirable for using POCT due to their simplicity, portability, and ease to see the obtained result with the naked eye. Overall, each technique has its advantages and limitations, but further research is needed for each technique to become an attractive POCT diagnostic kit. Finally, this review provided an in-depth knowledge of optical and electrochemical biosensors based on consolidated recently published articles; thus, it will benefit those working in the field of airborne pathogen detection.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Biographies

Doctor Rajamanickam Sivakumar received a Ph.D from Annamalai University, India and conducted postdoctoral research at Inha University, South Korea. In 2019, he joined as an Assistant Professor at Gachon University, South Korea. His major research direction lies in biosensors fabrication, colorimetric nucleic acid detection, and microfluidics.

Professor Nae Yoon Lee received a Ph.D from the University of Tokyo in 2004 and worked as a postdoctoral fellow in Korea Advanced Institute of Science and Technology (KAIST) and as a research professor at Ewha Womans University in South Korea from 2004 to 2007. She is currently a Professor in the Department of BioNano Technology at Gachon University since 2007. Her main research field is concerned with the development of Lab-on-a-Chip system particularly focusing on microdevice fabrication and sealing, surface modification, and developing portable diagnostic devices for point-of-care testing (POCT).

Abbreviations

WHO

World Health Organization

POCT

Point of Care Test

PCR

Polymerase Chain Reaction

LAMP

Loop-mediated isothermal amplification

RCA

Rolling Circle Amplification

RPA

Recombinase Polymerase Amplification

ELISA

Enzyme-Linked Immunosorbent Assay

SPR

Surface Plasmon Resonance

NPs

Nanoparticles

MOFs

Metal-Organic Frameworks

MNPs

Magnetic Nanoparticles

CNTs

Carbon Nanotubes

CV

Cyclic Voltammetry

SWV

Square-Wave Voltammetry

DPV

Differential Pulse Voltammetry

EIS

Electrochemical Impedance Spectroscopy

B. anthracis

Bacillus anthracis

MTB

Mycobacterium tuberculosis

S. aureus

Staphylococcus aureus

S. pneumoniae

Streptococcus pneumoniae

DPA

Dipicolinic acid

LOD

Limit of Detection

EBT

Eriochrome Black T

UCNPs

Upconversion Nanoparticles

PA₈₃

Anthrax Protective Antigen

TMB

3,3′,5,5′–tetramethylbenzidine

H₂O₂

Hydrogen peroxide

LFIA

Lateral-Flow Immunochromatographic Assay

HRP

Horseradish Peroxidase

CDs

Carbon Dots

Ag85B

MTB antigen 85B

MNase

Micrococcal Nuclease

ALP

Alkaline Phosphatase

NCs

Nanocomposites

ConA

Concanavalin A

AIV

Avian Influenza A

4-AP

4-aminophenol

NoV

Norovirus

LF

Lactoferrin

6-FAM

6-carboxyfluorescein

SNAs

Spherical Nucleic Acids

ASOs

Antisense Oligonucleotides

SPE

Screen-Printed Electrode

Sap

Surface array protein

MB

Methylene Blue

C60

Fullerene

TOAB

Tetraoctylammonium bromide

SWCNTs

Single-Walled Carbon Nanotubes

GCE

Glassy Carbon Electrode

PDDA

Poly(diallyldimethylammonium chloride)

PSS

Polystyrene sulfonate

BC

Bacterial Cellulose

EB

Ethidium Bromide

CFP10

Culture-filtered protein

PspA

Pneumococcal surface protein A

BDD

Boron-Doped Diamond

Cys

Cysteine

RBD

Receptor-Binding Domain

Data availability

No data was used for the research described in the article.