Recent advances in electrochemical sensors for vitamin sensing: toward point-of-care micronutrient assessment

Abstract

Micronutrients, including vitamins and minerals, are essential for maintaining normal health. Micronutrient deficiency can lead to various health complications. Assessing micronutrient levels is crucial, as early and routine micronutrient assessment and supplementation can help prevent deficiencies. Current assessment methods, such as Immunoassays, high-performance liquid chromatography (HPLC)-ultra-violet (UV) spectroscopy/fluorescence detection (FLD), liquid chromatography coupled with mass spectrometry (LC–MS), and similar techniques, are sophisticated, expensive, time-consuming, and require trained professionals. These limitations have prompted the development of point-of-care (POC) micronutrient screening devices that are simple, quick, reliable, and cost-effective. Electrochemical biosensors are one of the most promising analytical platforms for healthcare and other applications. This review focuses on the recent advances in electrochemical biosensors for vitamin sensing. It covers various types of electrochemical biosensors, including amperometric, potentiometric, and impedimetric biosensors, and discusses challenges associated with biosensors for potential use in healthcare as a routine vitamin assessment method.

Introduction

Micronutrients comprise minerals and vitamins are one of the major groups of nutrients that the human body needs to sustain normal health through several physiological functions [1, 2]. Minerals are the elements in food that are essential to building support strong bones and muscles and supporting the maintenance of various body functions [3] while Vitamins are required organic compounds that must be consumed in trace amounts for to carry out multiple biological functions necessary to develop and maintain overall human health [1, 4]. Vitamins are categorised into fat-soluble (A, D, E, and K) and water-soluble (vitamins B and C) based on solubility (Fig. 1). The water-soluble B-complex vitamins include thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), biotin (vitamin B8), folic acid (vitamin B9), and cyanocobalamin (vitamin B12) [5, 6].

Fig. 1.

Classification of Vitamins based on solubility

As these vitamins are not produced in our body; therefore, they must be obtained through either diet or vitamin supplementation. Vitamins through diet include meat, fish, poultry, legumes, nuts, eggs, dairy products, and leafy greens and vegetables, which are rich in these vitamins (refer Table 1) [7, 8]. WHO has given general recommendations on dietary intake to satisfy the nutritional need for vitamins for a human [9]. Also, countries often provide guidelines for"dietary reference intakes"to promote proper nutrition among the population [1]. These are known as the RDA, or ‘recommended dietary allowance’ based on age/sex [10, 11].

Table 1.

The functions and dietary sources of water-soluble vitamins [3, 10, 12–29]

Vitamins	Co-enzymes Coenzymes	Dietary sources	Biochemical functions	Deficiency symptoms	Recommended dietary allowance (RDA)
Vitamin A	cis-retinal	Meat, eggs, fortified margarine, butter, cream, cheese, fortified milk, vegetables, fruits, and dark green leafy vegetables	Development of cells Vision	Night blindness Vision disturbances Joint and bone pain Poor appetite Nausea and vomiting Sunlight sensitivity	Men: 900 mcg/day Women: 700 mcg/day
Vitamin E	α-Tocopherol	Nuts, seeds, egg yolks, liver, whole grain products, Potato, carrot, broccoli, cereals, olive oil, and orange	Antioxidant	Anaemia, Neurological problems	22.4 IU/day (15 mg/day)
Vitamin D	1,25-Dihydroxy vitamin D3	Fatty fish, meat, egg yolk, dairy products	Calcium homeostasis and bone metabolism	Rickets, osteomalacia, cardiovascular cancer	Age 1–70: 15 mcg/day Age 70 and older: 20 mcg/day
Vitamin K	γ-carboxylases	Cabbage, broccoli, sprouts, spinach, apple, and banana	Blood clotting	Bleeding diathesis, abnormal blood coagulation diseases	Men: 120 mcg Women:90 mcg
Vitamin C	Hydroxylation reactions	Orange, grapes, potatoes, tomatoes, and peppers	Collagen synthesis	Scurvy, heart disease, and cancer	Men: 90 mg/day Women: 75 mg/day
Thiamine (B1)	Thiamine pyrophosphate (TPP)	Animal sources: Pork, liver, heart, kidney, milk sunflower seeds, and wheat	Nutrients into energy	Irritability in infants Diabetes and Beriberi, Chronic neurological and Wernicke-Korsakoff syndrome	Adults: 1–1.5 mg/day Children: 0.7–1.2 mg/day Pregnancy/old age:2 mg/day
Riboflavin (B2)	Flavin mononucleotide (FMN) Flavin adenine dinucleotide (FAD)	Organ meats, eggs, beef, and mushrooms Milk & Milk-products	Conversion of food into energy an antioxidant	Burning sensation on the skin Digestive disorders Cheilosis Dermatitis	Adults: 1.2–1.7 mg/day Pregnancy/lactating women:0.2–0.5 mg/day
Niacin (B3)	Nicotinamide Adenine Dinucleotide (NAD+) Nicotinamide adenine dinucleotide phosphate (NADP+)	Chicken, tuna, yeast, whole grains, beans, peanut milk, egg, fish, and lentils	Signal to cell production and repair of DNA	Pellagra: affects the skin, gastrointestinal tract & central nervous system Dermatitis, Diarrhea, Dementia	Adults: 15–20 mg/day Children: 10–15 mg/day
Pantothenic acid (B5)	Coenzyme A or CoA(A-acetylation)	Liver, fish, yoghurt, milk, egg, and avocado	Hormone and cholesterol production	Insomnia Depression, irritability Upper respiratory infections	Adults: 5–10 mg
Pyridoxine (B6)	Pyridoxal-5′-phosphate (PLP) Pyridoxamine-5′-phosphate (PMP)	Egg yolk, fish, milk, wheat, corn, cabbage	Metabolism of Amino acid Production RBC Neurotransmitters creation	Alzheimer’s convulsions Peripheral neuropathy	Adults: 2–2.2 mg/day Pregnancy/lactation/old age:2.5 mg/day
Biotin (B7)	Biotin	Salmon, Yeast, cheese, eggs, and liver	Metabolism of carbohydrate and fat Regulating gene expression	Red rash on the face and the genital area Hallucination	100–300 mcg per day
Folate (B9)	Tetrahydrofolate Methyl tetrahydrofolate	Leafy greens, Liver and beans or in supplements such as folic acid	Cellular growth Metabolism of Amino acid Formation of red and white blood cells Cell division	Neurological problems Anaemia Rheumatoid arthritis A higher risk of lower bone density	Adults: 100 mcg per day Pregnancy:300 mcg per day Lactation:15 mcg per day
Cobalamins (B12)	Methyl cobalamin adenosyl cobalamin	Animal sources like meats, eggs, seafood, and dairy	Neurological function Production of DNA Development of RBC	Parkinson’s Alzheimer’s Pernicious anaemia Megaloblastic anaemia	Adults: 2.4 mcg per day Pregnancy: 2.6 mcg per day Lactation: 2.8 mcg per day Child:0.5–1.5 mcg per day

Vitamins are widely recognized for their various physiological functions in the metabolism of energy as well as in the maintenance of healthy liver, muscles, skin, eyes, and other body tissues. They serve as coenzymes and enzyme cofactor precursors in various metabolic processes that occur inside the body, including lipid, carbohydrate, and protein metabolism. Vitamin deficiency can lead to various health complications (Refer Table 1) [3, 12, 13].

As aforementioned, vitamins are a class of nutrients that play an important role in a variety of physiological processes, and both insufficiency and/or deficiency can be harmful to human health. As a result, it is important to have a screening technique for tracking vitamin concentrations in various matrices. Techniques such as ELISA, Spectrophotometry, capillary electrophoresis (CE), immunoassay, high-performance liquid chromatography (HPLC)-ultra-violet (UV) spectroscopy/fluorescence detection (FLD), liquid chromatography–mass spectrometry (LC–MS), for determining the presence of vitamins in biological fluids [30–42] have been compiled in Table 2.

Table 2.

Various analytical methods for the detection of vitamins in biological fluids targeting the recent articles

Vitamins	Detection range in blood	Assay/methods available	Reference
Vitamin A	15 −60 µg/dL	HPLC*, LC–MS** Immunoassay	[43–50]
Vitamin E	3–18.4 µg/mL	HPLC*, LC–MS** Spectrophotometric Immunoassay	[30, 43–46, 48–50]
Vitamin D	30—50 ng/mL	Immunoassay HPLC*, LC–MS**	[44–48, 50–54]
Vitamin K	0.2–3.2 ng/mL	HPLC–MS*, LC–MS** Immunoassay	[46, 48, 55–59]
Vitamin C	0.6–2 mg/dL	Spectrophotometric HPLC*, LC–MS** Capillary electrophoresis Immunoassay	[60–67]
Thiamine (B1)	2.5–7.5 μg/dL,	HPLC*, LC–MS** Capillary electrophoresis Immunoassay	[34, 68–76]
Riboflavin (B2)	4–24 µg/dL	Spectrofluorimetry HPLC*, LC–MS** Capillary electrophoresis Immunoassay	[34, 68, 70, 72–76]
Niacin (B3)	0.5–8.45 µg/mL	HPLC*, LC–MS** Capillary electrophoresis Immunoassay	[34, 70, 72–76]
Pantothenic acid (B5)	1.6–2.7 µmol/L	HPLC*, LC–MS** Immunoassay	[34, 70, 72–77]
Pyridoxine (B6)	5–50 µg/L	Spectrofluorimetry LC–MS**, HPLC* Capillary electrophoresis Immunoassay	[34, 68–76]
Biotin (B7)	33–329 pmol/L	HPLC*, LC–MS** Microbiological Assay	[34, 70, 72–76, 78]
Folate (B9)	2.7–17.0 ng/mL	LC–MS**, HPLC* Competitive immunoassay	[34, 70, 72–76]
Cobalamins (B12)	160–950 pg/mL	LC–MS**, HPLC* Chemiluminescence Capillary electrophoresis Competitive immunoassay	[34, 70, 72–76, 79–81]

*HPLC high-performance liquid chromatography, **LC–MS liquid chromatography–mass spectrometry

Studies of the literature reveal that these methods have many extraordinary advantage (Table 3), like high sensitivity and selectivity [48]. However, they have a few drawbacks (Table 3), like taking a long time to analyse, requiring complex sample preparation, having a large sample size, cross-reactivity, and expensive instruments, and needing highly trained people to operate [46, 82]. Routine screening may not be feasible with current screening methods available. Therefore, the above-mentioned issues have elevated the demand for assessment techniques/devices that meet the criteria of cost-effectiveness, rapid detection, high throughput, and portability.

Table 3.

Comparison of different detection techniques for the determination of micronutrients

Method	Advantages	Disadvantages	Refs.
Spectrophotometric	Simple and low-cost High throughput	Low sensitivity	[83]
Capillary electrophoresis	High throughput High separation efficiency Short analysis time Low sample and electrolyte consumption	Due to the small dimension of capillary, heat dissipated cases diffusion which results in the resolution	[84–86]
Chemiluminescence	Rapid analysis time High sensitivity and specificity Reduced incubation time Low consumption of reagents	The high price of Chemiluminescence reader Limited antigen detection	[87–90]
HPLC*	Determine, quantify, and sensitively separate the mixture’s components	Expensive, Columns clogging	[32, 38, 91, 92]
ELISA**	Sensitive and rapid, High throughput, Time-saving, Specific, strong affinity	Sample preparation, Antibody variability, cross-reactivity, and false positives, Produce radioactive waste	[41, 79, 93–95]
LC–MS***	High sensitivity, Selectivity, and specificity	Costly equipment and a high level of technical expertise are needed	[45, 96–98]

*HPLC high-performance liquid chromatography, **ELISA enzyme-linked immunosorbent assay, ***LC–MS liquid chromatography–mass spectrometry

In recent years, biosensors have become an ideal option to overcome the shortcomings mentioned above. This shift allows for real-time monitoring, on-site testing, and early intervention in healthcare, nutrition, and food quality, making them highly advantageous for modern diagnostic needs [99]. The creation of biosensors can be accomplished through a wide variety of methods. We provide a general overview of biosensors, including brief explanations of the variety of biosensors and approaches. Biosensor is an analytical tool that transforms biological processes into quantifiable physiochemical signals that measure the presence of specific analyte/concentration [100–102]. Conventional biosensors consist of two main components: a transducer for monitoring analyte concentration and a biological sensing or recognition element [103]. The molecule that specifically recognizes the analyte is referred to as a bioreceptor. Examples of bioreceptors include enzymes, cells, aptamers, deoxyribonucleic acid (DNA), and antibodies [104]. Signal generation occurs when the analyte interacts with the bioreceptor, resulting in changes such as pH, mass, heat, light, or charge [105–107]. Biosensors can be further categorized based on the type of bioreceptor employed such as enzymatic, immunological, or nucleic acid-based as well as the type of transducer used, which includes electrochemical, optical, piezoelectric, or thermal transducers, each tailored to specific applications and detection methods. These have various advantages over Traditional Methods such as (i) High Sensitivity: Detect very low concentrations of analytes, allowing for the identification of analytes present in trace amounts. (ii) Specificity: Selectively interact with target analytes, reducing interference from other substances and enhancing measurement accuracy. (iii) Fast Response Time: Provide rapid results, often within minutes, making them crucial for time-sensitive applications such as medical diagnostics and environmental monitoring. (iv)Miniaturization: Designed to be compact and portable, making them ideal for real-time and point-of-care diagnostics. (v) User-Friendly Operation: Designed for ease of use, often requiring minimal training, facilitating widespread adoption in clinical and field settings. (vi) Cost-Effectiveness: The potential for low-cost manufacturing and the ability to perform multiple tests simultaneously can make biosensors more economical compared to traditional laboratory methods. (vii) Real-Time Monitoring: Biosensors enable continuous monitoring of analyte levels, providing dynamic information that aids in timely decision-making and treatment adjustments. (viii) Integration with Technology: Integrated with mobile devices and data analytics tools, enhancing data collection, analysis, and sharing, which is valuable for personalized medicine and public health initiatives.

Electrochemical biosensor research has seen a significant increase since 2005, both in general and healthcare applications. While the first electrochemical biosensor was published in 1979, its use in healthcare wasn’t reported until 1987. As of 2025, over 12,793 publications in chemical analysis and over 8639 publications on electrochemical biosensors, with nearly 6,000 specifically focused on healthcare (Fig. 2) [108, 109].

Fig. 2.

Annual trends in electrochemical biosensor research: a comparative analysis of general and healthcare-focused publications.

Copyright © Sumeyra Savas Feb 2025

The increase in publication counts linked to electrochemical biosensors began in 2005 and has continued to rise. This increase represents not just increased research activity, but also a rising recognition of the importance of electrochemical biosensors in a variety of applications, particularly in healthcare settings. The growing corpus of literature reflects technological improvements, novel materials, and improved techniques, all of which help to produce more sensitive, selective, and flexible biosensors [110, 111]. As these devices evolve, their ability to address crucial difficulties in diagnostics, environmental monitoring, and food safety becomes more widely recognized, generating additional interest and investment in the healthcare industry.

To address various challenges, this review explores various electrochemical biosensing techniques, including amperometric, potentiometric, Electrochemiluminescence biosensing and bioimaging and impedimetric approaches, followed by an analysis of current developments in electrochemical biosensors for vitamin detection, with a particular focus on their potential for point-of-care applications. Finally, it presents future perspectives and potential research directions for integrating electrochemical biosensors into routine healthcare for vitamin assessment.

Electrochemical biosensors

Electrochemical biosensors operate by converting a biochemical reaction into an electrical signal, which can be quantitatively analysed. These sensors consist of a biorecognition element (such as enzymes or antibodies) that interacts specifically with the target molecule and an electrode that transduces this interaction into a measurable electrical response [112–114].

Key components of an electrochemical biosensor:

The performance of an electrochemical biosensor is influenced by several key components, each of which plays a crucial role in transforming the biochemical interaction into a detectable and quantifiable electrical signal:

Biorecognition element

This is the biological component that selectively binds to the target analyte. Examples include enzymes, antibodies, aptamers, and nucleic acids. The selection of the biorecognition element is based on the characteristics of the target analyte and the level of specificity needed for accurate detection [115].

• Classification based on the biorecognition element

1. Aptamers (DNA or RNA)-based biosensors

DNA biosensors are biological receptor-based biosensors. Their high selectivity for their target analytes in a matrix of chemical or biological components is perhaps the most attractive aspect of biosensors [116, 117]. Nucleic acids serve as the biological receptors for DNA biosensors, which are used to identify non-macromolecular and protein substances that interact with specific DNA segments called DNA probes/primers [118, 119]. The interaction that has been seen is the result of stable hydrogen bonds forming between the strands of the DNA. The immobilization of the probe becomes the most important stage in the development of the sensor [120]. Because of the strong coupling of lined-up nucleotide strands between bases in their complementary sections, biosensors based on DNA, RNA, and peptide nucleotide acids are among the most sensitive [105, 121, 122].

• (b)Enzyme-based biosensors

An enzyme-based biosensor’s working principle is determined by the target analytes binding capabilities and catalytic reaction [123, 124]. The process of recognizing an analyte may involve several different potential mechanisms: (i) Analyte metabolized by enzyme, by monitoring the enzyme’s catalytic transformation of the analyte, the concentration of the enzyme can be calculated.; (ii) An enzyme that is activated or inhibited by the analyte means less enzymatic product formation (iii) Tracking the change in the characteristics of the enzyme [105, 125, 126]. Different kinds of biosensors can be made based on enzyme specificity because enzyme-based biosensors have been around for a long time [123]. However, enhancing the enzyme’s sensitivity, stability, and adaptability is costly and difficult due to the enzyme’s extremely sensitive structure [127]. Enzyme-based biosensors make the best use of electrochemical transducers [128]. Biosensors based on glucose and urea are the most widely used enzyme-based biosensors [127].

• (c)Antibody-based biosensors

Due to the potent antigen–antibody interactions and a wide variety of applications of antibodies, they have been used as affinity bio-recognition components for over two decades [129]. Immunosensors are biosensors that either function on antibody-antigen interaction or have an embedded antibody as a ligand. There are two types of immune sensors as Non-labelled and labelled. Non-labelled immune sensors are designed to precisely identify the antigen–antibody combination by calculating the physical changes brought about by the emergence of the complex. A sensitive, detectable label is used in the case of a labelled immunosensor [105, 130, 131]. Through label measurement, the antigen–antibody complex is evaluated with greater sensitivity.

Electrodes

Electrodes function as conductors, enabling the flow of electrical current produced by the biochemical reaction. The material composition and surface characteristics of the electrodes are key factors in enhancing the sensor’s sensitivity and ensuring the accuracy of the signal detected [113, 132, 133].

Three kinds of electrodes:

1. Working electrode: the heart of electrochemical biosensors

1. Directly interacts with the analyte and bioreceptor.

2. The site where the electrochemical reaction occurs [134].

3. Often made of materials like gold, platinum, carbon, or glassy carbon [135, 136].

• (b)Reference electrode: a stable anchor for electrochemical measurements

1. Maintains a stable potential, providing a reference point for measuring the potential at the working electrode.

2. Common reference electrodes include the silver/silver chloride (Ag/AgCl) electrode and the calomel electrode/or Glass Electrode/Ion-Selective Electrodes [137, 138].

• (c)Counter electrode: completing the electrochemical circuit

1. Completes the electrical circuit and allows for the passage of current.

2. Often made of the same material as the working electrode or a less reactive material like platinum.

Transducer

It converts the biological interaction into an electrochemical signal. Common transducers include amperometric [139], potentiometric [140], impedimetric [105], and electrogenerated chemiluminescence [141]. The transducers role is to detect and amplify changes in the system such as voltage, current, impedance, conductivity, capacitance and light [141–144] that occur due to the biochemical reaction [107].

• Classification based on the transducing element

The transducing element, which transforms the biological recognition event into a quantifiable electrical signal, enables electrochemical biosensors to be categorized. Depending on the target analyte and application, these biosensors have different operating principles and each have special benefits and drawbacks (Table 4). The main categories consist of

Table 4.

Comparison of different biosensor types: principles, advantages, applications, and limitations

Biosensor type	Principle	Advantages	Applications	Disadvantages
Amperometric-based biosensors	Measures current generated by redox reactions at a constant voltage	High sensitivity, real-time monitoring, widely used in medical diagnostics	Glucose monitoring, vitamin analysis, neurotransmitter detection	Susceptible to electrode fouling, requires precise control of applied voltage, interference from non-target species
Potentiometric-based biosensors	Measures potential differences developed at the sensor surface due to sensor-analyte interactions	Low power consumption, stable response, selective for specific ions or molecules	pH sensing, ion-selective electrodes, and environmental monitoring	Slow response time compared to amperometric sensors, sensitive to temperature and ionic strength variations
Conductometric-based biosensors	Measures changes in electrical conductivity or impedance caused by biological interactions	No reference electrode is required, cost-effective, simple fabrication, and real-time detection	Cell fluid analysis, microbial detection, enzyme-based biosensors	Lower sensitivity compared to other electrochemical methods, affected by solution composition changes
Electrochemiluminescence (ECL) biosensors	Utilizes light emission generated by electrochemical reactions for analyte detection	High specificity, suitable for imaging applications, provides strong signal output	Bioimaging, immunoassays, pharmaceutical analysis	Requires complex instrumentation, potential instability of luminescent species, and expensive setup

1. Amperometric-based biosensors

Amperometric-based biosensors use redox processes to measure the difference in current potentials when antigen/antibody pairing occurs [40]. Amperometric sensors monitor the current generated by electrochemical oxidation or reduction of electroactive species at the working electrode when a constant voltage is given to the working electrode with the reference electrode [144]. The concentration of the analyte in the solution is inversely proportional to the current that is generated on the surface of the working electrode. Amperometric sensors apply a voltage to initiate electrochemical oxidation or reduction, measuring the resulting current using the Cottrell equation [132]:

$$i=(\text{nFAC}\surd D)/\surd (\pi t)$$

where, i: Current (in amperes) measured at the working electrode. n: The number of electrons transferred in the electrochemical reaction. F: The Faraday constant (96,485 C/mol), a proportionality constant between the amount of electricity and the amount of matter involved in an electrochemical reaction. A: The area of the planar electrode (in cm²) where the electrochemical reaction occurs. C: The initial concentration of the analyte (in mol/cm³) before the experiment begins. D: The diffusion coefficient of the analyte (in cm²/s), which represents how quickly the analyte diffuses through the solution. t: Time (in seconds) elapsed since the start of the experiment.

• (b)Potentiometric-based biosensors

For potentiometric sensors [140], a specific equilibrium is established at the sensor surface due to sensor-analyte interactions, providing information about the analyte concentration. The relationship between the potential and the analyte concentration is typically described by the Nernst equation [145]:

$$\text{E}={\text{E}}^{\circ }+(\text{RT}/\text{nF})\text{ln}([\text{A}]/[{\text{A}}_0])$$

where: E: Measured potential (in volts); E°: Standard electrode potential (in volts); R: Gas constant (8.314 J/mol K); T: Temperature (in Kelvin); n: Number of electrons transferred in the redox reaction; F: Faraday constant (96,485 C/mol); [A]: Concentration of the analyte; [A₀]: Reference concentration of the analyte.

For it to function, electrochemical reactions must result in a build-up of charge potential at the working electrode relative to the reference electrode [40, 144]. Potentiometric Ion-selective electrodes are used in biosensors to convert biological reactions into electrical responses.

• (c)Conductometric-based sensors

Conductometric sensors, also the same as impedimetric sensors, measure changes in surface conductance to detect and quantify analytes. The change in the electrical conductivity of cell fluids can be measured using conductometric biosensors. The majority of reactions result in a change in the solution composition [144]. Therefore, conductometric biosensors are capable of identifying any reactive change that takes place in a solution [40]. Conductometric biosensors have several important advantages, such as the fact that they don’t require the use of a reference electrode, operate at low-amplitude alternating voltage to prevent Faraday processes on electrodes, are light-insensitive, and are straightforward to integrate using a cost-effective thin-film standard technology. There is a growing trend toward the creation of Impedimetric biosensors right now [144]. Impedimetric techniques have been used to analyse the design of biosensors as well as the catalysed reactions of enzymes, lectins, nucleic acids, receptors, whole cells, and antibodies [40, 143, 146].

• (d)Electrochemiluminescence biosensing and bioimaging

ECL biosensors can be considered a subset of electrochemical biosensors that specifically harness the electrochemiluminescence phenomenon for signal generation.

Electrochemiluminescence (ECL) is a phenomenon where light is emitted as a result of electrochemical reactions at or near the surface of an electrode.

It involves the generation of excited states through electrochemical processes, followed by their relaxation with the emission of light [147]. Biological recognition events lead to changes in the electrochemical reactions, which, in turn, affect the intensity of the emitted light. This change in light emission is then utilized to quantify the presence or concentration of a specific analyte [141, 148].

Immobilization matrix

The immobilization matrix acts as a connecting link between the biorecognition element and the transducer surface, ensuring the biorecognition elements stability and function. Structure/material utilized to firmly attach the biorecognition element to the transducer surface of the sensor [149]. Various materials are utilized to create immobilization matrices, and the choice depends on factors such as compatibility with the biorecognition element, desired sensitivity, and application requirements.

For example, some compounds, such as glucose, cholesterol, etc., are not electroactive. When target analytes interact with the biorecognition elements, they induce changes in the electrochemical properties of the transducer producing measurable voltage, current, impedance, conductivity, capacitance and light [141, 143, 144].

Materials for immobilization matrices

A variety of materials have been employed in developing immobilization matrices, each delivering unique benefits tailored to targeted applications. Materials for immobilization matrices can be classified into polymers such as (e.g., chitosan, polyaniline, alginate) (including natural and synthetic types), nanomaterials (e.g., graphene, carbon nanotubes), metal nanoparticle (e.g., gold, silver, zinc oxide), biomaterials and sol–gel materials. Researchers can enhance the performance of electrochemical biosensors, improving their sensitivity, specificity, selectivity, reusability longevity and overall effectiveness in detecting target analytes [150–152]. Since some target analytes (e.g., glucose, cholesterol) are not inherently electroactive, their interaction with the immobilized biorecognition element leads to measurable changes in the sensor’s electrochemical properties, such as voltage, current, impedance, conductivity, capacitance, or optical responses [152].

The widespread success of electrochemical sensor research has made it difficult to cover all achievements in this review. Therefore, we aimed to demonstrate the diversity of the field rather than focusing on a specific type of electrochemical sensor [153, 154].

Electrochemical sensors for vitamin

Numerous electrochemical sensors have recently been suggested for the detection of vitamins. In this review, current advances in electrochemical sensors for vitamin detection during the past few years are reviewed. Electrochemical sensors have tremendous success in the detection of vitamins [155–157]. Electrochemical sensors are a widely used technique to evaluate the redox characteristics of vitamins since each one contains distinct components that can undergo the transfer of electrons in solutions. Based on the electrochemical oxidation or reduction of the analyte in the electrolyte, electrochemical sensors can measure the current on the working electrode to determine the concentration of the analyte [105, 144, 157]. Currently, the most appealing electrochemical methods such as LSV (Linear Sweep Voltammetry), SWV (Square wave Voltammetry), CV (Cyclic Voltammetry), DPV (Differential Pulse Voltammetry), and Amperometry have shown substantial increases in vitamin detection with improved sensitivity [158].

But even while conventional biosensors that use these electrochemical techniques operate dependably, their sensitivity and detection limits are still limited. In order to improve the sensitivity and selectivity of electrochemical applications, electrode modification and functionalization have become a major area of effort. To improve sensor performance and functionality, a variety of nanomaterials, including metallic nanoparticles, carbon nanotubes, nanospheres, Quantum dots and nanorods, have been added to electrode modifications [155, 159, 160].

Impact of nanomaterials on vitamin detection

Nanomaterials have revolutionized the field of biosensors, enhancing sensitivity and selectivity [105, 161, 162]. Because of their distinct physicochemical (high surface area, excellent conductivity, and biocompatibility) characteristics, nanomaterials have significantly advanced electrochemical biosensors [160]. A critical characteristic that enables increased biomolecule immobilization and enhances the interaction between the sensing element and the target analyte is high surface area. Considering an example of a nanosphere of radius “r”. The surface area of the sphere = 4πr²; Volume of the sphere = 4/3πr³, Therefore the surface area to the volume ratio will be 4πr²/{4/3(πr³)} = 3/r, which means the given volume is divided into smaller piece, the surface area increases. So, we can say that the surface area to volume ratio is very large for Nano-particles [105, 163]. Since nanoparticles have a very small size, r is small, so the ratio of surface area to volume is very large. Therefore, it increases the chemical reactivity of nanoparticles and also helps in tailoring the nanomaterials for various sizes, shapes etc. A given mass of nanomaterial will be substantially more reactive than a similar quantity of material made up of large particles because growth and catalytic chemical reactions occur at surfaces [164]. Therefore, this characteristic allows for the immobilization of a larger number of bio-receptors, such as enzymes or antibodies, resulting in a higher number of binding events with target analytes. Some of the key nanomaterials used in vitamin sensors include Carbon-based nanomaterials, such as graphene, graphene oxide (GO), and carbon nanotubes (CNTs), metal and metal oxide nanoparticles, including gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), and metal oxides (ZnO, TiO₂, Fe₃O₄), play a crucial role in improving conductivity and enhancing electrochemical vitamin detection. Additional contributions are made by polymer-based nanocomposites and hybrid nanomaterials like as metal–organic frameworks (MOFs), which have catalytic qualities that improve conductivity, sensor response, enzyme stability, and selective vitamin detection by facilitating electrochemical oxidation and reduction [144, 165–172].

Nanomaterials increase biosensor stability, biocompatibility, and enzymatic activity, hence prolonging sensor longevity. They also facilitate miniaturization and portability, enabling real-time vitamin monitoring via point-of-care (POC) devices [40, 104, 169].

To further enhance biosensor performance, Electrodes like ITO-coated glass, GCE, SPCE, Glassy Carbon Electrode, CPE etc. have been modified with nanomaterials like ZrO₂NPs, NPs, CNTs, Gd₂O₃ NRs etc. by various research groups(Table 5) across the globe [173, 174]. Accurate vitamin quantification has been achieved using a variety of techniques(Table 5). To properly diagnose and treat metabolic illnesses, vitamin levels in human plasma, urine, diet, and medications must be determined.

Table 5.

A summary of sensors for the detection of vitamins

Vitamins	Sample	Electrode	Technique	Linear range	LOD	Refs.
Vitamin A	Tablet and food	Nanoalloy (Pt: Co) Carbon Paste Electrode	SWV	0.1 μM to 100 μM	0.04 μM	[189]
	Dietary supplements	SPGNE	SWV	0.1 µg mL−1 to 5 µg mL−1	0.018 µg mL−1	[175]
Vitamin E	Standard samples	CPE/MWCNTs/Tyrosinase/NafioN	CV		9 × 10−7 mol L−1	[190]
	Standard samples	GCE	SWADSV	12 to 30 µmol L−1		[191]
	Standard samples		SWASV* CV	0.5–4.0 µg L−1 40.0–160.0 µg L−1	0.056 µg L−1	[192]
	Dietary supplements	SPGNE	SWV	0.08 µg mL−1 to 5 µg mL−1	0.012 µg mL−1	[175]
Vitamin D	Pharmaceutical samples	Boron Doped Diamond Electrode	SPGE	2 to 200 μmol dm−1	0.17 μmol dm−3	[193]
	Standard samples	Gd2O3 NRs	DPV	10–100 ng mL−1	0.10 ng mL−1	[173]
	Dietary supplements	SPGNE	SWV	0.08 µg mL−1 to 5 µg mL−1	0.013 µg mL−1	[175]
Vitamin K	Green leafy vegetables	screen-printed graphene electrode (SPGE)	SWASV		0.099 μg mL−1	[194]
	Blood serum	2-Amino-5-Chloro Benzophenone On Pencil Graphite Electrode	SWV	50–700 nmol L−1	16.58 nmol L−1	[195]
	Standard samples	GCE	SWADSV	0.4 to 7.1 µmol L−1		[191]
	Dietary supplements	SPGNE	SWV	0.2 µg mL−1 to 1.6 µg mL−1	0.004 µg mL−1	[175]
Vitamin C	Orange Juice sample	PEDOT/Fe(CN)64−/GCE	CV	0.04–200 Mm 0.7–1500 mM 1–2000 mM	0.02 mM 0.3 mM 0.5 mM	[176]
	Multivitamin tablets	Silver-doped poly(L-arginine)- GCE	CV	5.0 × 10–6–4.0 × 10–3 M	3 × 10–6 M	[178]
	Honey samples	PEDOT/ZrO2NPs/GCE	DPV	1–1500 μM	0.45 μM	[174]
	Human plasma samples	p-AMTa	DPV	30 μM–270 μM	7.14 × 10−7 M	[177]
	Food samples	ZrO2/NPs/IL/CPE	SWV	0.07–850 M	0.009 M	[185]
	Real Samples	LIG-based Closed BPE electrode	ECL	1–1000 μM	0.96 μM	[188]
Thiamine (B1)	Standard	CPE-MIPpy	CV	–	6.9 × 10–5 M	[196]
	–	Glassy Carbon Electrode	DPV	0.6 mM–1.6 mM	5.14 × 10−4 M	[197]
	Multi-vitamin tablets, urine, and human blood serum	f-MWCNTs	DPASV	0.60–19.43 ng mL−1	0.17–0.2 ng mL−4	[180]
	Multivitamin drinks, multi-vitamin tablet	CoPC-SPCE	CV	0.1–20 µg mL−1	6.3 ng mL−1	[198]
	Serum, plasma, and urine sample	DNA-modified MWCNPTE and PMWCNTPE	SWADSV DPV	0.0025–0.80 μg mL−1 1.0–80 μg mL−1	1.1 ng mL−1 0.44 μg mL−1	[182]
Riboflavin (B2)	Orange Juice sample	PEDOT/ClO4−/GCE	CV	0.15–300 mM 1–1500 mM 2–2000 Mm	0.08 mM 0.5 mM 0.9 mM	[176]
	Multivitamin tablets	Silver-doped poly(L-arginine)- GCE	CV	1.0 × 10–7 to 2.3 × 10–5 M	8 × 10–8 M	[178]
	Oral, syrup, and tablet samples	BiFE	SWADSV	0.3–0.8 μmol L−1 and 1.0–9.0 μmol L−1	100 nmol L−1	[199]
	Tablets	MnO2/CPE	DPV	0.02 to 9 M	15 nM	[200]
	Food and Pharmaceutical Samples	Shewanella oneidensis MR-1	CV	10–120 nM	0.85 ± 0.09 nM	[201]
	Food and Pharmaceutical Samples	MnTPP/CPE	DPV	1.0 × 10−8 to 1.0 × 10−5 M	8.0 × 10−9 M	[202]
	Honey samples	PEDOT/ZrO2NPs/GCE	DPV	0.05–300 μM	0.012 μM	[174]
	Optineuron injection and Beplex Forte	GC/MWCNTs-MnIIIsalen	DPV	1.0–400 μM	0.73 μM	[179]
	Human plasma samples	p-AMTa	DPV	10 μM–90 Μm	4.54 × 10−8 M	[177]
Niacin (B3)	Pharmaceutical samples	polycrystalline gold electrode	CV	2.4 mM to 2.7 M 2.4 mM to 3.3 M	0.27& 0.33 µM	[183]
Pantothenic acid (B5)	Human urine samples	Glassy carbon electrode	SWV	1.0–0.008 μg/mL	0.5 μg/mL	[203]
Pyridoxine (B6)	Orange Juice sample	PEDOT/Fc−/GCE	CV	0.1–300 mM 0.5–1500 mM 1.5–2000 mM	0.05 mM 0.1 mM 0.7 mM	[176]
	Multivitamin tablets	Silver-doped poly(L-arginine)- GCE	CV	1.0 × 10–5–3.0 × 10–3 M	5 × 10–6 M	[178]
	Honey samples	PEDOT/ZrO2NPs/GCE	DPV	0.5–1000 μM	0.20 μM	[174]
	Optineuron injection and Beplex Forte	GC/MWCNTs-MnIIIsalen	DPV	1.0–300 μM	0.42 μM	[179]
	Blood serum and multivitamins	Au-CuO/MWCNTs/GC-modified electrode	CV	0.79 mM–18.4 mM	0.15 mM	[204]
	Food samples	ZrO2/NPs/IL/CPE	SWV	0.8–550 M	0.1 M	[185]
	Soft drink and pharmaceutical formulation samples	F-MWCNTs) modified GCE	CV EIS	0.5 to 20 µM 20 to 200 µM	0.038 µM 0.125 µM	[205]
Biotin (B7)	Plasma	Nafion-modified BDD electrode	DPV		5 nM	[206]
		Gold electrode modified with 3-mercapto propanoic acid (MPA)	CV EIS		1.5 ng L−1	[207]
Folate (B9)	Human, Mouse, and Rabbit serum	Fe2O3/NiO/Mn2O3 (NPs) on a glassy carbon electrode	CV	0.1 nM–0.01 mM	96.89 ± 4.85 pM	[208]
	Human plasma samples	p-AMTa	DPV	20 μM to 180 μM	2.5 × 10−7 M	[177]
		ZnFe2O4MNPs/SPE	CV	1.0–100.0 μM	0.3 μM	[209]
	blood plasma	GC/oligo DAT electrode	DPV	5–25 μM	3.5 × 10−11 M	[210]
	Std,serum	graphene nanosheets (GrNs) decorated indium tin oxide (ITO)		5–100 nM	0.52 nM	[211]
	Wheat flour	DNA/PGE	DPV	0.1–10.0 μmol L−1	1.06 × 10−8 μmol L−1	[212]
Cobalamins (B12)	Pharmaceutical samples	MAA/SAM/Au	CV	4.0 × 10−9 to 4.0 × 10−5 mol L−1	1.0 × 10−9 mol L−1	[213]
	Real samples	PGE	SWV	200–9500 nmol/L	0.093 µM	[214]
	Food samples	Au/PPy/FMNPs@TD	CV EIS DPV	2.50 nM–0.5 µM	0.91 nM	[215]
	Pharmaceuticals and Supplements	Carbon Paste Electrode (CPE) Modified by a Manganese(II) Polymeric Film	SWV	13.86 ng L−1 to 1500 ng L−1	4.34 ngL−1	[216]
	Pharmaceutical samples	SWCNT–chitosan modified PGE	SWV	5 nM–100 nM at pH 2.0 5 nM–80 nM at pH 5.0	0.89 nM 2.1 nM	[217]
	Pharmaceutical samples	D-phenylalanine Nanotubes	CV EIS DPV	–	1.6 µM	[218]
	Milk, pharmaceutical samples	poly(PBHQ)/MWCNTs/GCE	CV	0.1–10 M	0.01 M	[219]
	Real samples	LIG-based Closed BPE electrode	ECL	0.5–1000 nM	0.109 nM	[188]
	Standard	LIG-based BPE and SE electrodes	ECL	0.5–700 nM (BPE) 0.5–1000 nM (SE)	107 and 94 pM	[187]

DPASV differential pulse anodic stripping voltammetry, VB-1 vitamin B1 (thiamine), VB-2 vitamin B2 (riboflavin), CV cyclic voltammetry, DPASV differential pulse anodic stripping voltammetry, BiFE Bismuth-film electrode, SWAdSV square-wave adsorptive stripping voltammetry, SWV square wave voltammetry, DPV differential pulse voltammetry (DPV),3-amino-5-mercapto-1,2,4-triazole modified glassy carbon, MnTPP/CPE Manganese(III) tetraphenyl porphyrine, VB2-/PoAP/GCE VB2 molecularly imprinted polymers modified electrode, FFV Fourier transform continuous cyclic voltammetry, poly(PBHQ)/MWCNTs/GCE Poly(2,2′-(1,4-phenylenedivinylene) bis-8-hydroxyquinoline)/multi-walled carbon nanotube-modified glassy carbon electrode, PGE pencil graphite electrodes, Au/PPy/FMNPs@TD Gold/polypyrrole/ferromagnetic nanoparticles/triazine dendrimer electrode, EIS electrochemical impedance spectroscopy

Using a screen-printed graphene/Nafion electrode (SPGNE), a novel electrochemical sensor was presented by Jeerakit, et al., for the simultaneous measurement of fat-soluble vitamins (A, D, E, and K). Square-wave voltammetry (SWV) has been used to investigate the electrochemical behaviours of fat-soluble vitamins in an ethanol and sodium perchlorate monohydrate solution. According to the study, each fat-soluble vitamin’s peak of oxidation emerged at a separate potential, which allowed for simultaneous detection [175]. Researchers also used a carbon paste electrode (CPE) that was modified using a nanoalloy (Pt: Co) room-temperature ionic liquid (RTIL) for the detection of Vitamin A in food samples.

Nie et al., showed adding two electroactive species, ferricyanide (Fe(CN)₆⁴) and ferrocene carboxylic acid ((C₅H ₅)Fe(C ₅H ₄CO ₂H)), as doping anions during the electro polymerization of PEDOT at glassy carbon electrodes (GCEs), functionalized PEDOT films were created. Such nanostructured films were used as electrochemical sensors for the simultaneous measurement of vitamins B2, B6, and C [176]. The same group demonstrated the first electrochemical sensor of the simultaneous determination of riboflavin (RB), ascorbic acid (AsA), and folic acid (FA) using a poly(3,4-ethylene-dioxythiophene)/Zirconia nanoparticles (PEDOT/ZrO₂NPs) composite film on a glassy carbon electrode (GCE)to enhance the sensitivity. The PEDOT/ZrO₂NPs/GCE electrode, on the other hand, displays stable voltammetric signals for RB, AsA, and FA in a mixture. Furthermore, Nie et al., showed that in the presence of significant amounts of the other two vitamins, the PEDOT/ZrO₂NPs/GCE electrode was able to specifically detect each vitamin separately [177]. Similarly, several other researchers showed the simultaneous measurement of B2, B6, and C in Optineuron injection and Beplex Forte, multivitamins, and human plasma samples [174, 178, 179].

Prasad et al., In their study, demonstrate the use of hexamine ruthenium (II) chloride as a probe molecule for an electrochemical sensor based on MIP for the indirect determination of thiamine (Vitamin B-1). The limit of detection was 0.17 ng mL⁻¹ for a range from 0.60 to 19.43 ng mL⁻¹ (S/N = 3), with no cross-reactivity or false-positive results [180]. Wahyuni et al. put forth a technique that uses a glassy carbon electrode as the working electrode via in situ pH modulation to both quantify the analytical response and locally adjust the pH value. Thiamine (vitamin B1) is detected in aqueous KCl as a model system, and the pH is modulated by applying negative potentials to the working electrode (scan rate 100 mV s⁻¹) [181].

A new electrochemical biosensor was developed by Brahman et al., using a ds-DNA-modified MWCNPTE (multiwalled carbon nanotube paste electrode) and PMWCNTPE (pretreated multiwalled carbon nanotube paste electrode), it was possible to study the electrochemical interaction of vitamin B1 with DNA based on the reduction of the oxidation signal of guanine and adenine bases using differential pulse voltammetry. After interacting with vitamin B1, the strength of the guanine and adenine oxidation signals decreased, and this signal was employed as an indication for the sensitive measurement of vitamin B1 [182].

On a polycrystalline gold electrode, the oxidation of nicotinic acid and nicotinamide happened at almost the same potentials, while their reduction happened at distinct peak potentials. By using cyclic voltammetry and bulk electrolysis, it was possible to explain the redox reaction processes of nicotinic acid and nicotinamide by the development and disappearance of new nitrogen–oxygen bonds in the pyridine rings. With detection limits of 0.27 and 0.33 M for nicotinic acid and nicotinamide, respectively, the cathodic peak currents at roughly 0.20 V were linear with their concentrations in the range of 2.4 mM to 2.7 M and 2.4 mM to 3.3 M.Voltammetry was subsequently used for the selective monitoring of medicines nicotinic acid and nicotinamide concentrations [183].

Conductive carbon black (CB) and nanoporous natural zeolite were exchanged with Ni²⁺ ions (NiZ) to create a unique and selective voltammetric sensor for vitamin B6 (VB6). Ni-zeolite/carbon black modified glassy carbon electrode (NiZCB-GCE) modified by Porada and his coworkers, demonstrated that Under ideal circumstances, the oxidation peak current was linear to the VB6 concentration in the range of 0.050 to 1.0 mg L⁻¹ at the potential + 0.72 V vs. Ag | AgCl |3 M KCl reference electrode. Comparing the calculated limit of detection to chemically modified electrodes made of other carbon-based materials, it was found to be substantially better at 15 gL⁻¹ (0.09 mol L⁻¹) [184]. For the detection of vitamin C, (ascorbic acid (AsA)) in the presence of vitamin B6, a quick and easy voltammetric approach based on the usage of ZrO₂ nanoparticles and ionic liquids carbon paste electrodes (ZrO₂/NPs/IL/CPE) was given by Amin et al. They synthesized carbon paste electrodes modified with 1-butyl-3-methylimidazolium tetrafluoroborate ([B_mim] BF₄) as a binder for the voltammetric analysis of AsA and vitamin B6 in food samples, the application of synthesized nanoparticles was used. The two peaks for AsA and vitamin B6 are separated by approximately 0.44 and 0.82 V at an ideal pH of 7.0, which indicates that AsA can be determined in the presence of B6. In the ranges of 0.07–850 M for AsA and 0.8–550 M for vitamin B6, the peak current of square wave voltammograms (SWV) of AsA and B6 increased linearly with their concentrations. The AsA and vitamin B6 detection limits were 0.009 and 0.1 M, respectively [185]. Recently, a group, Hossein Sadeghi et al., detected vitamin B-6 with NiO-CNTs/MOHFPE/CPE, as an analytical tool [186].

They constructed an electroanalytical food sensor using a carbon paste electrode (CPE), a nanocomposite made of NiO and CNTs, and a material called 1-methyl-3-octylimidazolium hexafluorophosphate (MOHFPE). In addition to demonstrating catalytic activity for the measurement of vitamin B6, the NiO-CNTs/MOHFPE/CPE also increased the oxidation current of vitamin B6 by up to 2.5 times. The effectiveness of the modification of CPE utilizing NiO-CNTs and MOHFPE was further demonstrated by the alteration of the oxidation potential of vitamin B6 to a negative value of about 100 mV in comparison to CPE. A linear relationship between the current and vitamin B6 concentrations in the range of 4.0 nM to 500 M with a detection limit of 0.9 nM was found by the results of differential pulse voltammetric (DPV) experiments [186].

Here Karastogianni et al., the fabrication of a newly modified carbon paste electrode with a manganese complex film including thiophene-2-carboxylic acid and Triethanolamine as ligands, along with its electrochemical characterization. For the electrochemical analysis of cyanocobalamin, this electrode was utilized (vitamin B12). Square wave voltammetry was used to determine the amount of cyanocobalamin in tablets and nutritional supplements [209].

Bhaiyya et al. developed a 3D-printed miniaturized portable system for the detection and monitoring of ECL signals (Fig. 3i). In the current study, the detection of vitamin B12 was successfully achieved using two Electrochemiluminescence (ECL) platforms: one employing a Bipolar Electrode (BPE), and the other utilizing a Single Electrode (SE). The electrodes were manufactured on a polyimide (PI) substrate through the creation of optimized Laser-Induced Graphene (LIG). By employing a CO₂ laser with optimized speed and power, the non-conducting portion of the PI substrate was transformed into a conducting zone, forming the electrodes essential for ECL imaging. An Android smartphone played a dual role in driving the DC to DC buck-boost converter and capturing the ECL images. The sensing of vitamin B12 demonstrated linear ranges of 0.5–700 nM and 0.5–1000 nM, with corresponding limits of detection (LOD) of 0.107 nM (R² = 0.98, n = 3) and 0.094 nM (R² = 0.977, n = 3) for the BPE and SE-based ECL platforms, respectively [187]. The same group developed an in-house miniaturized portable 3D printed system which accommodates a smartphone, creating a standalone ECL sensing platform. The smartphone not only captures the ECL signal but also powers the ECL device through a DC-to-DC buck-boost converter. The performance of the two-channel LIG-C-BPE-ECL device was validated by individually sensing Hydrogen peroxide (H₂O₂), Vitamin B12, and Vitamin C [188].

Fig. 3.

(i) Simultaneous detection of Vitamin B12 and Vitamin C, a 3 channel LIG-C-BPE-ECL system having Vitamin B12 = 0.5 nM, Luminol = 4 mM, Vitamin C = 1 μM and PBS = 0.1 M, applied voltage = 7 V, b Vitamin B12 = 100 nM, Vitamin C = 100 M, c Vitamin B12 = 300 nM, Vitamin C = 300uM, d Vitamin B12 = 500 nM, Vitamin C = 500 μM, e Vitamin B12 = 1000 nM, Vitamin C = 1000 M, f Bar graph for vitamin B12 Vs ECL intensity (RLU), g Bar graph for vitamin C vs. ECL intensity (RLU), Error bar represents standard deviation for three experiment Copyright

© Elsevier (ii) DPV curves at GC/MWCNTs-Mn^IIIsalen with consecutive additions of vitamin B2 (A) or vitamin B6 (B). C DPV curves at GC/MWCNTs -Mn^IIIsalen with consecutive additions of vitamin B2 in presence of 20.0 μM of vitamin B6. D DPV curves at GC/MWCNTs-Mn^IIIsalen with consecutive additions of vitamin B6 in presence of 20.0 μM of vitamin B2. Inset of A and B represents the calibration plot for the determination of vitamin B2 and B6, respectively (in 0.1 M pH 7.0 phosphate buffer).Copyright © Elsevier (iii) ZnO thin film based sensing device-1′ × 1′ substrate size can produce 35 devices along with I–V Plot for various concentration of VB6 (Pyridoxine)

Piyuesh et al. created an multiwalled carbon nanotubes (MWCNTs) and Mn(salen)Cl (Mn³⁺salen) nanocomposite is combine the catalytic qualities of Mn³⁺salen with the high conductivity of MWCNTs. Utilizing a variety of methodologies, such as electrochemical ones, the MWCNTs-Mn³⁺salen nanostructure is used to detect vitamin B2 (riboflavin) and B6 (pyridoxine) simultaneously utilizing differential pulse voltammetry (DPV). The sensitivity, detection limit, and repeatability of a glassy carbon electrode (GC) modified with MWCNTs-Mn³⁺salen (GC/MWCNTs-Mn³⁺salen) are high (R.S.D. = 3.7% for B2, 3.4% for B6). The electrode additionally exhibits long-term stability (>10 days at room temperature). The technique makes electrochemical analysis of vitamins B2 and B6 in pharmaceutical formulations easy and efficient (Fig. 3ii).

In our current work, we developed a ZnO thin film-based device for bio-sensing applications using two probe measurement set-up (Fig. 3iii). ZnO thin film was developed using a sol–gel spin coating (Zinc acetate dihydrate (Zn (CH₃COO)₂·2H₂O),₂-methoxy ethanol (CH₃–O–C₂H₅) and Monoethanolamine (MEA) as metal alkoxide precursor, solvent and sol stabilizer respectively) and followed by pre & post heat treatment for better crystallinity. Structural, morphological and optical characterizations confirmed the crystallinity, orientation, surface morphology, size distribution and transparency of ZnO thin film. Metal (Silver-Ag) electrodes were deposited on ZnO thin film and tested using two probe measurement set-ups for various concentrations of vitamin B6 molecules [220].

The current response was measured between the electrodes for various concentrations of VB-6 which showed a linear response upon the addition of vitamin B6. The developed biosensor response time of a few seconds (~2 s), less amount of analyte (0.5 µL), low power consumption and mass-produced potency (1′ × 1′ substrate size can produce 35 devices), have been expected for use in practical applications. Nevertheless, some challenges still need to be overcome regarding the sensitivity, specificity, and influence of interference ions in body fluids.

Advantages of electrochemical biosensors for vitamin assessment

1. Cost-effectiveness

Less expensive than conventional techniques. Miniaturization of these and the use of easily accessible materials contribute to reduced production and operational costs.

• (b)High sensitivity and selectivity

High sensitivity and selectivity provided by electrochemical biosensors enable precise vitamin detection even at low concentrations. By employing certain biorecognition components, like enzymes or antibodies, these sensors can target particular vitamins while minimizing interference from other chemicals.

• (c)Rapid and convenient analysis

Rapid analysis times, often within minutes. This enables speedier action and results, which could lead to better patient outcomes. These sensors are portable and ease of use makes them appropriate for point-of-care (POC) testing, which eliminates the need for specialized lab equipment and skilled workers.

Challenges and future directions for vitamin sensing

Despite the various advantages of electrochemical biosensors vitamin evaluation nevertheless have a number of issues that need to be resolved before they can be widely used. Real-world applications face challenges because to factors such as sensor reliability, the capacity to detect many vitamins at once, and large-scale manufacturing. Furthermore, even though these sensors have a high sensitivity, their selectivity, repeatability, and integration with contemporary digital health systems frequently need to be improved [221, 222]. These issues are examined in the section that follows, along with possible future paths to improve the efficiency of electrochemical vitamin biosensing systems.

Challenges

1. Simultaneous detection of multiple vitamins

Several electrochemical sensors have been developed for the detection of various vitamins. Jeerakit et al. introduced a sensor for fat-soluble vitamins (A, D, E, K) using a screen-printed graphene/Nafion electrode where square-wave voltammetry was employed to analyze the vitamins’ electrochemical behaviour [175]. Nie et al. created nanostructured films for the simultaneous measurement of vitamins B2, B6, and C. Additionally, they developed a sensor to detect riboflavin, ascorbic acid, and folic acid using a poly(3,4-ethylene-dioxythiophene)/Zirconia nanoparticles composite film to enhance the sensitivity [176, 177]. These could simultaneously measure more than one vitamin with good sensitivity. But these can have some drawbacks such as sensors may demonstrate cross-sensitivity, resulting in overlapping responses because of the analogous chemical structures of vitamins, complicating their differentiation. Each vitamin necessitates distinct calibration and optimization, hence challenging the development of a sensor capable of concurrently detecting numerous vitamins.

• (b)Choice of electrochemical electrode/detection method

The preparation of the modified electrode involves multiple steps, which can be time-consuming and require specialized equipment.

Amperometric-based biosensors are better suited for mass production because they enable a sensitive, quick, precise, and linear response in comparison to potentiometric biosensors [162]. However, these sensors’ disadvantages include poor selectivity and interference from other electroactive substances [144]. In biosensors, this may be the most widely used electrochemical detection method. Biotest samples containing electroactive species can be detected by this high-sensitivity biosensor.

While Electrochemiluminescence (ECL) biosensors offer several advantages, they also come with certain disadvantages: Compared to some other biosensor types, ECL biosensors have limitations in multiplexing capabilities, i.e., the simultaneous detection of multiple analytes. Developing ECL biosensors for multiple targets can be challenging and may require additional technological advancements. Luminescent materials used in ECL biosensors may be susceptible to degradation over time, leading to reduced stability and long-term reliability of the sensor. This can affect the sensor’s performance and lifespan. ECL measurements often require specialized instrumentation, such as a photomultiplier tube (PMT) or a charge-coupled device (CCD), which may limit the accessibility of the technology in certain settings. ECL biosensors may not be well-suited for certain analytes or biological molecules due to the specific requirements of the electrochemical and luminescent reactions involved. Despite these disadvantages, it’s essential to note that ongoing research and technological advancements continue to address and mitigate some of these challenges, contributing to the improvement and broader adoption of ECL biosensors in various applications.

• (c)Interference in body fluids

In regard with the influence of interference i in body fluids, one has to be careful in choosing recognition elements, such as enzymes or antibodies, that specifically bind to the target vitamin while minimizing cross-reactivity with interference ions and significantly enhancing the biosensor’s specificity. Sample preparation and treatment such as filtration, centrifugation, or chemical treatment can help in removing or minimizing interference ions from the sample before analysis. Biosensor surfaces can be functionalized with coatings or molecular imprints that are specific to the target vitamin. Advanced electrochemical techniques, such as differential pulse voltammetry or amperometry or Cyclic Voltammetry, can be employed to distinguish the electrochemical responses of the target vitamin from interference ions, based on their distinct oxidation/reduction potentials [223]. Electrochemical biosensors need to work well in complex biological environments such as blood, urine, and food matrices, where signal interference might be introduced by non-target biomolecules. The following strategies are used to improve selectivity and accuracy: functionalized electrode surfaces (e.g., nanomaterials and self-assembled monolayers); specific biorecognition elements (e.g., enzymes, aptamers, and antibodies); differential measurement approaches (e.g., dual-electrode systems and background subtraction); and optimized electrochemical techniques (e.g., differential pulse voltammetry and electrochemical impedance spectroscopy). High specificity in real-world biological samples may be achieved by biosensors through the integration of advanced materials, selective coatings, and precise electrochemical methods, allowing for reliable and interference-free vitamin detection for clinical and food safety applications [150–152].

• (d)Miniaturization and portability

Miniaturization considerably reduces biosensor detection limits by increasing surface area-to-volume ratio, improving analyte mass transport, and decreasing sample diffusion distances. As electrode size reduces, more analyte molecules interact with the sensing surface, resulting in increased signal intensity and sensitivity. Furthermore, nanoelectrodes/or materials coated with nanomaterials minimize background noise, enabling the detection of extremely low analyte concentrations. However, downsizing brings new obstacles, such as greater susceptibility to electrical noise, fabrication complications, and potential signal drift. Optimizing nanomaterials, microfluidics, and advanced signal processing is critical for getting the most out of tiny biosensors while retaining accuracy and reproducibility. To create compact, user-friendly, and completely integrated point-of-care devices, advancements are needed. Real-time data transfer, smooth wearable and mobile technology integration, and effective battery management for ongoing monitoring are among the difficulties.

Future directions

Future developments in electrochemical biosensors for vitamin detection can delve deep into hybrid nanomaterials to improve sensitivity, selectivity, stability and multi-analyte detection for increased effectiveness [159]. Also, Embedding AI and machine learning will optimize data interpretation, allowing individualized nutrition monitoring [111]. While microfluidic lab-on-a-chip systems will enable compact, high-throughput analysis, wearable and implantable biosensors will enable non-invasive, continuous tracking, and wireless and IoT-enabled sensors will enable real-time monitoring and remote healthcare applications, thereby increasing accessibility to vitamin assessment [224]. In addition to establishing standardization and regulatory approvals for broad usage in clinical and commercial settings, future activities will promote cost-effective fabrication methods.

Summary

Vitamins are essential nutrients for an everyday healthy life. A daily healthy diet or supplements are required by the body for various physiological functions to maintain normal health. Regular monitoring of vitamin levels is important to reduce the risk associated with deficiency-related disorders. Also, taking vitamin supplements beyond the recommended levels causeshealth side effects. So, routine screening tools may be required for determining vitamin levels and follow-up.

It is crucial to have a routine screening method for monitoring vitamin levels in body fluids. Conventional methods while reliable for vitamin assessment, are often limited by their complexity, cost, and time-consuming nature (Table 3). There is a need for alternative tools for quantitative or qualitative assessment of vitamin levels in a range of matrices to overcome the limitations associated with the present tools. Owing to the advancement in the nanoscience field especially in sensing, the electrochemical biosensor can be a promising candidate for vitamin sensing being easy to use, portable, sensitive, selective, cost-effective and can use small analyte quantities [105]. The speed at which the molecular interaction between the receptor and the analyte may be converted into detectable signals, affinity-based biosensors, such as electrochemical biosensors, have an advantage over conventional analytical procedures in terms of High throughput rate [105, 158].

Detecting only a single analyte can be limiting, especially when that analyte may be associated with various conditions. Therefore, for vitamins, which often involve multiple complexities, employing a multi-analyte sensing platform becomes crucial for accurate diagnosis. In this review, we summarize various types of electrochemical sensors related to vitamins and explore non-invasively accessible biofluids suitable for sensing applications.

Despite of above advantages, currently, there are associated difficulties in implementing an ideal biosensor as a routine screening tool for assessment of vitamins is challenging. For a large number of important biomedical applications, these have semi-quantitative or qualitative results with poor sensitivity. Despite many recent efforts, signal amplification to raise the required detection limits, stability, suitability, shelf life, optimum driving voltage, current response, potential leakage (weak acid electrolyte interactions), and inability to be recycled are persisting challenges that need to be addressed. Additionally, analytes interact with electrochemical sensors to produce electrical signals that are inversely proportional to the concentration of the analyte. Due to these constraints, the scope of electrochemical sensor technology is not able to satisfy the pre-requisite as an ideal biosensor. However, in recent years a variety of inorganic and organic contaminants have been monitored using electrochemical sensors. Through the use of microfluidics and multiplexing, which have been made possible by the fast breakthroughs in nanotechnology over the past few years, the present scarcity in the lack of automation and the time lag between the sensing measurements should be reduced for real-time monitoring of the events. Sensing technology has not been used to its full potential and has a lot of room to improve its sensing capabilities.

Despite several studies on sensing biomolecules, vitamins have not been explored much and still a lot needs to be done to improve the vitamin assessment capabilities to work in the desired range of matrices of interest. Even though some studies have reported encouraging results, electrochemical sensors for the simultaneous detection of vitamins remain an open challenge. It is also essential for the quantification of vitamins along with other important biomarkers in complex sample environments for success in clinical applications [157, 225]. POCT (point-of-care testing) and implantable vitamin detection devices are the need for future development avenues [156].

In conclusion, we are certain that vitamin assessment may become a new norm and may be used routinely in clinical diagnostics in the future. Electrochemical sensors may play a major role in these assessments by finding applications from bench side to bedside. Current research is concentrated on finding solutions to the current issues and realizing the full potential of portable electrochemical sensors. Therefore, electrochemical sensors have a great chance to surpass conventional techniques in the near future [144, 156].

The aforementioned conversation highlights the potential advantages of integrating various sensing methodologies and employing multiplexing, contingent upon the specific application and analytical requirements. By strategically combining different sensing modalities, it becomes feasible to enhance sensitivity, and selectivity, and expand the dynamic detection range, surpassing the capabilities of individual sensing mechanisms [226].

Their ability to provide rapid, real-time analysis makes them suitable for point-of-care applications, especially in resource-limited settings where traditional methods are impractical. However, to realize their full potential in healthcare, challenges related to selectivity, stability, and data interpretation must be overcome. Further research and development, guided by WHO guidelines on micronutrient deficiencies, can help ensure that these technologies become widely accessible, improving global health outcomes through better monitoring and management of micronutrient deficiencies.

Author contributions

B A: Conceptualization, Writing – original draft. Saritha U K: Supervision. G M R: Supervision. Shobha U K: Supervision. A R P: Supervision. S D K: Supervision. S D: Supervision. A P: Supervision, Writing- Reviewing and Editing.

Funding

Ms. Bhavana Anchan acknowledges financial support from the Manipal Academy of Higher Education (MAHE) through Dr. TMA Pai PhD. Scholarship. Ajeetkumar Patil and Saritha Kamath U. are grateful to MAHE for supporting this research through the MAHE-Intramural Fund.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Ethics and Consent to Participate declarations: Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Clinical trial registration

Not applicable.