Analyzing Electrochemical Sensing Fundamentals for Health Applications

Humans continuously interact with physical, chemical, and biological environments that influence their health, safety, and quality of life. Sensing devices, such as electrochemical sensors that translate environmental qualities into electrical signals, are crucial for detecting biomarker concentrations in various biofluids. However, the understanding of electrochemical sensing is often incomplete, necessitating further study of chemical reactions and sensor‐electrode interactions for healthcare applications. This review analyzes crucial topics in chemical reactions in electrochemical sensing environments. First, the dynamics of chemical energy, the roles of acidic and alkaline fluids, chemical reaction tendencies, thermodynamic equilibria, Gibbs free energy, water dissociation, and the pH scale are discussed. Sensor materials or biomarkers undergo oxidation and reduction reactions in electrochemical sensing. Oxygen‐derived radicals and nonradical reactive species significantly influence biochemical reactions, cellular responses, and clinical outcomes. Then, the review delves into the impact of oxidation reduction reactions on human pathophysiology, redox reactions in hemoglobin, redox environments in human serum albumin and cells/tissues, and thermodynamics of biological redox reactions. Finally, recent advances in electrochemical techniques are presented and research challenges and future perspectives in electrochemical sensing for health applications are addressed.


Introduction
Electrochemical biosensing is a rapidly growing field in global healthcare research.This involves studying the flow of electrical current induced by chemical reactions and measuring the subsequent electrical energy produced. [1]Given that biological systems rely on chemical processes, electrochemical sensing is critical for the real-time detection of target molecules and fast, reliable disease diagnosis.In comparison to lab-based techniques, electrochemical sensing methods are fast, adaptable, portable, costeffective, and do not require skilled professionals. [2]These methods can be conveniently used in point-of-care settings.Electrochemical sensors have been extensively used in detecting biomarkers, analytes, and contaminants in healthcare, environmental monitoring, food packaging, and other applications. [3,4]For instance, low-cost and portable glucose blood sensor strips based on electrochemical sensing generate over $5 billion in revenue annually. [5]mong various reviews, the series titled "Dynamic Electrochemistry: Methodology and Application" in the Analytical Chemistry Journal is particularly notable for its impact on the field of electrochemical sensing.This series includes contributions from Ryan et al. from 1992 [6] and 1994, [7] and Anderson et al. in 1996, [8] 1998, [9] and 2000. [10]Covering a decade, these reviews highlight both fundamental advancements and practical applications in electrochemistry, particularly in the field of electroanalytical chemistry.Topics covered in this series range from ultramicroelectrodes, analytical voltammetry, and electrode kinetics to surface electrode phenomena, modified electrodes, bioelectrochemistry, characterization of various redox couples, spectroelectrochemistry, and instrumentation. [7,10]Although this series offers an extensive overview, our current review is more focused on the application of these techniques in health monitoring, recognizing the growing interest in health assessment through electrochemical methods.Similarly, Bakker et al. in 2002 concentrated only on electrochemical sensors, emphasizing those based on potentiometric and voltammetric techniques, and their applications in areas like immunosensors, deoxyrebonucleic acid (DNA) sensors, electrochemiluminescence sensors, and amperometric gas sensors. [11]In 2003, Stetter et al. reviewed insights on various sensors, including chemical sensors, electrochemical sensors, and the broader purview of the Electrochemical Society (ECS). [12]Given the rapid advancements of electrochemical sensors in health monitoring, our review aims to comprehensively address the bioanalyte environments within the human body, the factors influencing them, and the electrochemical techniques suitable for their detection.
Review articles on electrochemical sensing in health applications cover a wide range of subjects, from pharmaceutical compounds, metals, pathogenic bacteria, and biomolecules to neurotransmitters and glucose. [13,14]One outstanding review delves into modern electroanalytical techniques, highlighting their roles, particularly in the pharmaceutical and metal industries. [13]nother key article emphasizes the capabilities of electrochemical sensors for real-time, in situ chemical assessments, highlighting the significance of microfluidics, immunomagnetic separation, and multiplexing in microbial detection. [15]A unique review casts light on electrochemical biosensors and their ability to convert biological activities directly into electronic signals. [16]urther, a review of electrochemical sensors underlines their adaptability in detecting diverse signals across fields like agriculture, food, and biomedicine. [17]A subsequent review investigates up-to-date electrochemical methodologies, spotlighting the platforms that synergize nanomaterials and biorecognition elements for immediate detection. [18]Another article offers a detailed exploration of glucose biosensors built on nanomaterials, outlining their operational strategies and prospective directions. [14]yclic voltammetry (CV), recognized as a paramount voltammetric technique, has been detailed in a review, [19] granting readers a comprehensive view of the core attributes of electrochemical techniques.However, there is a gap in articles that simplify the basics, strengths, and limitations of each method.8][9][10][11][12][13][14][15][16][17][18][19] Addressing this void and catering to the increasing demand, a timely review could provide clarity on the principles and physics underlying each method.To effectively employ these techniques, an in-depth understanding of reactions at the electrode and the electrical dynamics at the electrode-solution interfaces is essential. [20]Figure 1 provides a pictorial overview of the content of this paper.
In this review, we summarize advanced knowledge of the fundamental principles of various electrochemical techniques for healthcare applications.Section 2 covers crucial topics necessary to understand chemical reactions in an electrochemical cell environment and the concept of reactions on sensing electrodes.In Section 3, we provide a detailed explanation of methods used to understand sensing processes and the formulation and solution of mathematical equations related to the techniques.Sections 4 and 5 present limitations, conclusions, and future research perspectives of electrochemical sensing techniques for health applications.Additionally, we offer a brief description of each technique, including approximations of electrode and electrolyte reactions.

Fundamentals of Analytical Chemistry for Electrochemical Sensing
Electrochemical sensing techniques, a subset of analytical chemistry, focus on analyzing analytes in a solution. [11]They generate signals representing properties of an analyte, such as mass, volume, concentration, or absorbance, facilitating both qualitative and quantitative analyses. [12]Historically, the "classical" technique, which measures signals proportional to the analyte concentration, was widely used. [1]Electrochemical techniques can be categorized into bulk and interfacial methods. [20]The former measures properties related to the total concentration of dissolved ions, while the latter concentrates on potential, current, or charge at the interface. [21]The main concern in electrochemical techniques is the charge transport through the interface and the factors affecting it. [7]The interface comprises an electronic conductor (electrode) and an ionic conductor (electrolyte).An electric potential prompts the current to flow through the interface, representing the rate of change of charges (electrons or holes) flowing through the electrode. [11]The electrode phase includes solid metals (e.g., Pt, Au), liquid metals (Hg, amalgams), carbon (graphite), and semiconductors (indium-tin oxide, Si), while the electrolytes are liquid solutions containing ionic species (H + , Na + , Cl − ) in water or a nonaqueous solvent. [1]Electrochemical cells feature complex environments with multiple interfaces. [20]s illustrated in Figure 1, the relationship between the signal and analyte concentration is a theoretical function that depends on experimental conditions and the measuring instrument used.Now, we will discuss some chemical reactions and thermodynamics, including oxidation-reduction (redox) reactions, which govern the sensing performance, to gain insights into electrochemical sensing techniques.

Equilibrium: Chemical & Thermodynamic
Until the 18th century, chemical reactions were believed to proceed only in one direction, as demonstrated by Equation (1), where substances AB and CD react to produce AC and BD as the products. [22] + CD → AC + BD (1)   These chemical reactions were understood through the lens of chemical affinities.For instance, substances A and B were thought to have affinities for C and D, respectively.Moreover, the idea of a reverse reaction was dismissed.However, after 1798, French chemist Claude Berthollet made a significant discovery while studying the salt water of Natron Lakes during Napoleon's military expedition to Egypt. [23] 2 Co 3 (s) + CaCl 2 (aq) ⇌ 2NaCl (aq) + CaCo 3 (s)  ( This groundbreaking scientific discovery emphasizes the reversible nature of chemical reactions, as indicated by the equilibrium arrow "⇌".Berthollet found that the direction and final composition of a reaction depend on the relative amounts of reactants and products.Initially, the mass of Ca 2+ in the solution decreases, while the mass of CaCO 3 precipitates increases.Once the reaction reaches a steady state with no net changes, it is in equilibrium.This concept plays a vital role in interfacial interactions in electrochemical cells.Rogers et al. 2012 highlighted the importance of chemical equilibria in machine learning for chemical analysis, introducing the term "perturbed isotherms" to describe non-isothermal experimental conditions influenced by temperature and thermal history. [24]hemical equilibria are influenced by individual molecular interactions and thermodynamics, which studies energy changes during chemical reactions. [25]According to Berthollet, the equilibrium chemical reaction represented by aA + bB ⇌cC + dD, where a, b, c, and d are the stoichiometric coefficients of the respective substances, does not guarantee a favorable forward reaction and may depend on initial conditions.Depending on initial conditions, the reaction may shift to the left, to the right, or exist in a state of equilibrium, as seen in Figure 2a,b. [26]The aim of chemical thermodynamics is to understand the conditions affecting a reaction's equilibrium position.The Le Chatelier-Braun principle describes how external factors, such as temperature, pressure, volume, or concentrations of reacting substances, lead to predictable system changes to reach a new equilibrium state. [27,28]igand-protein recognition has potential in assays, [29] inflammation, [30] cancer, [31] and Alzheimer's disease [32] treatments.Zhang et al. [29] developed a label-free electrochemical sensor for the biomarker CD44, detecting human serum and cancer cells. [29]Lerner et al. created a carbon nanotube field effect transistor (NT-FET) for detecting osteopontin (OPN), a prostate cancer biomarker. [33]While traditional methods like the enzymelinked immunosorbent assay (ELISA) are sensitive, they struggle with OPN quantification. [33,34]The NT-FET sensor's response curve was well fitted by a modified Hill-Langmuir equation, shown in Equation ( 3), which describes ligand-receptor binding in thermodynamic equilibrium. [33,35] where ΔI I is the measured responses of devices, c is the OPN concentration, A is the response when all binding sites are occupied, Z is an overall offset to account for the response to pure buffer, K d is the dissociation constant, and n is the Hill coefficient describing cooperativity of binding.However, ligand-protein interactions, such as ligand binding, can impact cell environment thermodynamics, affecting water molecules near binding sites. [36]

Gibb's Free Energy, Water Dissociation Constant, and pH Scale
Thermodynamic changes involve alterations in free energy due to interactions among biomarkers, ligands, solutes, and solvent molecules.These interaction energies depend on fluctuating interatomic distances and orientations, which vary over time in accordance with the conformational dynamics. [25]Under any external conditions, the overall free energy determines the direction of a chemical reaction, be it in the forward or reverse direction.With constant pressure and temperature, Gibbs' free energy (named after J. Willard Gibbs, 1839-1903) is represented by G = H − TS, where H, T, and S represent the enthalpy, the temperature in Kelvin, and the entropy, respectively. [37]nthalpy measures energy flow as heat absorbed or released during a reaction.Endothermic reactions absorb heat and have positive enthalpy, while exothermic reactions release heat with negative enthalpy.Entropy relates to molecular freedom, disorder, and complexity.Change in Gibbs free energy (∆G = ∆H − T∆S) is widely used, with ∆H, T, and ∆S representing the changes in enthalpy, the temperature in Kelvin, and the changes in entropy, respectively.Changes in Gibbs free energy, enthalpy, and entropy are calculated as differences between product and reactant values.
Wallerstein et al. studied the impact of small ligand structure changes on galectin-3C's carbohydrate recognition domain, focusing on binding enthalpy and entropy. [25]They analyzed various interactions in an isothermal environment, finding that minor structural differences affected binding thermodynamics, free ligand chemical potential, and complex structures. [25]Using isothermal titration calorimetry (ITC), they determined binding enthalpy (ΔH), binding-competent protein fraction (n), and dissociation constant (K d ) through a single-site binding model. [38]igure 2. Summary of the changes in Gibbs free energy, chemical equilibrium, and the equilibrium constant.Reproduced by permission.www.cengage.com/permissions, [26] Copyright 2015, Copyright Holder Cengage Learning, https://www.cengage.ca/.For reactions that are a) product-favored and b) reactant-favored when they are at equilibrium.c) The pH and pOH scales indicate the concentrations of [H 3 O + ] and [OH -], respectively.The chart also shows the pH and pOH values of some common substances at standard temperature (25 °C).Reproduced (Adapted) under the terms of the CC BY 4.0 license, https://creativecommons.org/licenses/by/4.0. [57]Copyright 1999-2013, Copyright Holder OpenStax, https://openstax.org/.
The heat released or absorbed during the ith injection is given by the following Equation (4). [39] where V i is the volume of the ith injection, V O is the cell volume, Q off is an offset parameter that accounts for the heat of mixing, and represents the heat following the ith injection, where  = nM i + X i + K d and M i and X i are the total concentrations of protein and ligand, respectively, in the cell at any given point during the titration.Thus, the change in Gibbs free energy can be expressed as Equation (5) for Equation (1).Here, [A] and [A 0 ] represent the concentrations of substance A at any point during the reaction and under standard-state conditions, respectively. [37] R represents the universal gas constant (8.314JK −1 mol −1 ), T denotes the temperature in Kelvin, and Q r signifies the reaction quotient.Standard states are defined as gases with unit partial pressures, solutes with unit concentrations, and a value of 1 for pure solids and pure liquids.The change in Gibbs free energy, ∆G, is zero under standard state conditions or at thermodynamic equilibrium.Hence, the Equation ( 5) can be reduced to under standard state conditions or at thermodynamic equilibrium, where K d is the equilibrium constant or the dissociation constant.The authors found that protein-ligands with similar structures exhibit similar thermodynamic signatures. [25]Wallerstein et al. (2021) calculated the change of entropy (ΔS) of the protein-ligand chemical reaction in a cell using Equation ( 5), ΔG o = − RTlog e K d and − TΔS = ΔG − ΔH.They demonstrated that, for isothermal protein-ligand reactions, the complexes exhibit overall similar thermodynamic signatures, which might be expected due to the similar structures of the ligands. [25]allerstein et al. found several insights on isothermal ligandprotein interactions: 1) chemical potentials in the free state depend on structural differences; 2) binding thermodynamics differences relate to ligand-protein complex variations; 3) binding enthalpy differences can be explained by fewer interactions; 4) total entropy differences come from opposing contributions.More flexibility leads to unfavorable entropic contributions, and galectin-3C's backbone exhibits reduced entropy when bound. [25]sothermal interactions involve entropy-entropy compensation in protein conformation, while enthalpy-entropy compensation is more prominent at higher temperatures. [25,40][42] At 37 °C, red blood cells are oval and flexible, but at 52 °C, they transform into crenated spheres with tiny spicules.[42] Good demonstrated that vesiculation mechanisms in red blood cells are consistent across species and depend on the physical state of water within the cells and cell membranes. [43]Vodyanoy et al. analyzed vesicle loss kinetics in red blood cells during temperature increases due to physical exercise or exposure to external heat. [44,45]Erythrocytes reduce volume and surface area through vesiculation, with the number of vesicles remaining constant under steady-state conditions. [40]der these conditions, an equilibrium exists between vesicles generated by erythrocytes and vesicles destroyed by Kupffer cell mechanisms. [46]he thermodynamic analysis of transformations in living cells is an important tool for the quantitative study of energy transductions that occur during these changes. [40]It allows the definition of the nature and function of the chemical processes underlying these transformations. [47]For two temperature points, T 1 and T 2 measured in Kelvin, the logarithmic form of the Arrhenius equation, k = Ae (− E a RT ) , yields the activation energy, E a , which represents the thermal activation level of transitions from the intact vesicle at T 1 to the destroyed vesicle at T 2 within the context of first-order activation kinetics, as shown in Equation ( 6). [40,48]g where A represents the pre-exponential Arrhenius factor, k stands for the first-order rate constant of the vesicle destruction process, and R denotes the universal gas constant.The value of k depends on the thermodynamic activation parameters of this transition state and can be represented by the Eyring equation, , where ΔG is the standard Gibbs free energy change of activation, h is the Planck's constant, ΔS is the standard entropy change of activation, ΔH is the standard enthalpy change of activation, k b is the Boltzmann constant, R is the gas constant, and T is the absolute temperature in Kelvin. [49]he authors, Vodyanoy et al. (2015), derived a linear relation between the logarithm of the Arrhenius frequency factor and the entropy of activation obtained from the Eyring equation for the transitional state, as shown in Equation (7). [40]g A = log From Equations ( 6) and ( 7), the change of entropy is derived as ΔS = 2.303R{log A − log k b h − log(eT)} and the change of enthalpy can be calculated as ΔH = E a − RT, and the change of Gibbs free energy can be calculated as ΔG = ΔH − TΔS.
Elevated temperatures affect the release of potassium ions (K + ) and acetylcholinesterase (AcChE) from human erythrocytes, as well as methemoglobin formation during erythrocyte transformation and vesiculation. [50]At higher temperatures, erythrocyte vesiculation and hemolysis are endothermic, absorbing heat with positive enthalpy change (∆H > 0). [40]However, entropy changes (∆S) during these processes are negative (∆S < 0), typically associated with decreased molecule's rotational and translational freedom. [40]This negative entropy (ΔS < 0) often involves the dehydration of aqueous solvent moieties and the release of ion, forming the interface between them. [51]The discocyteechinocyte transition also has a negative entropy change due to increased cell membrane curvature. [52]The energy from entropy change, TΔS, correlates with enthalpy change, ΔH, resulting in small Gibbs free energy, ΔG = ΔH − TΔS, compared to both ΔH and TΔS. [40]55][56] A chemical reaction is thermodynamically favorable when its enthalpy decreases and its entropy increases.The change of Gibbs free energy, ΔG = ΔH − TΔS, indicates that a reaction is thermodynamically favorable, unfavorable (where the reverse reaction is favorable), or at equilibrium when ∆G is negative, positive, or zero, respectively, as shown in Figure 2a,b.As a reaction approaches equilibrium through forward or reverse progression, the concentrations of its species experience change.For both Figure 2a,b, the equilibrium mixture points, as described by Equation ( 1), can be represented by Equation (8), where all the species' concentrations are at equilibrium.
A eq B eq + C eq D eq = A eq C eq + B eq D eq (8)   At elevated temperatures, erythrocyte vesiculation and hemolysis processes are endothermic, with a positive change of enthalpy (ΔH > 0) and a negative change of entropy (ΔS < 0).Consequently, the change of Gibbs free energy, ΔG = ΔH − TΔS, is positive (ΔG > 0), making the protein-lipid interaction thermodynamically unfavorable. [40]In contrast, under isothermal conditions, protein-ligand interactions, involving enthalpy-entropy compensation, are thermodynamically favorable, because the change of Gibbs free energy, ΔG = ΔH − TΔS, is negative, while the change in enthalpy is negative (ΔH < 0) and the change in entropy is positive (ΔS > 0). [25]ey chemical reactions include precipitation, acid-base, complexation, and oxidation-reduction (Redox) reactions.Water, as an amphiprotic solvent, can act as a base and an acid by accepting and releasing protons, and it can also interact with itself, as demonstrated by Equation (9).
The equilibrium constant of water, known as the dissociation constant of water, determines its acidic or basic state in acid-base reactions.It can be calculated from Equations (7), (9), and (10) at 25 °C. [57] The value of K w changes with temperature, such as 6.809 × 10 −15 at 20 °C, 1.469×10 −14 at 30 °C, and 1.008×10 −14 at 25 °C.All values are close enough to the value in Equation (10)  that it is used.
Additionally, the p-function of [H 3 O + ] or the pH of pure water at 25 °C can be expressed as Equation (11).
The pH and pOH values of common substances, based on the concentrations of [H 3 O + ] and [OH − ] respectively, are shown in Figure 2c at standard temperature (25 °C).However, there is no pH for vegetable oil or alcohol, because they are not aqueous solutions (solution containing water) at 25 °C. [58]astewater pH is crucial for in-sewer biomarker stability. [59]astewater-based epidemiology (WBE) estimates community chemical usage or exposure by analyzing biomarkers in sewage. [60,61]Li et al. used a first-order kinetics model to simulate biomarker degradation considering pH and other factors in sewers. [59]4][65][66] Ln where C t (μg L −1 ) is the concentration of a biomarker at time t(h), and C 0 is the initial biomarker concentration at t(0) in the reactor.k total (h −1 ) represents the overall transformation rate of a biomarker in sewers, which consists of abiotic transformation (k ww , h -1 ) in the bulk wastewater and biodegradation (k bio , h −1 ) driven by biofilms.It is assumed that suspended solids have negligible biological activity compared to biofilms, and thus, k ww is mainly determined by chemical hydrolysis in the bulk wastewater.Biodegradation is considered a surface process, which is correlated with A V in a sewer section, i.e., k ′ bio × A V .Meanwhile, k′ bio (m/h) includes k′ bioan and k′ bioa , indicating the biodegradation under anaerobic and aerobic conditions, respectively.k ww , k′ bioan and k′ bioa are estimated based on the experimental results obtained from the control reactor, rising main reactor, and gravity sewer reactor, respectively.
In summary, hydrolysis accelerates at higher pH levels, enhancing biomarker removal, while lower pH levels suppress both biodegradation and chemical hydrolysis. [59]The dominant effect of pH on in-sample and in-sewer biomarker stability has been observed.[69][70][71][72] The dissociation of compounds (indicated by pKa) should also be considered, as protonation and deprotonation processes at different pH levels can affect abiotic transformation and biomarker stability. [59]
Chemically, oxidants or oxidizing agents accept electrons, while reductants or reducing agents donate electrons for each compound. [79,82]Reduction involves electron gain, while oxidation involves electron loss. [83]When a reductant donates its electrons, it causes another substance to be reduced, and when an oxidant accepts electrons, it causes another substance to be oxidized. [84]Redox reactions, where reducing agents donate hydrogen or remove oxygen, form the basis for many biochemical pathways, cellular chemistry, biosynthesis, and regulation. [85]nderstanding these reactions is crucial for studying biological oxidation and radical/antioxidant effects.
In biological environments, reductants and oxidants are better termed as antioxidants and pro-oxidants, respectively. [79,84]Prooxidants, including radicals and nonradicals, are called reactive oxygen species (ROS) that can cause damage to biological targets such as lipids, DNA, and proteins, as well as to the cell's defense systems, which are composed of enzymes and reducing equivalents or antioxidants. [83]he transition metals in the first row of the D block in the periodic table contain unpaired electrons and can participate as radicals, except for zinc.They can be converted into relatively stable oxidants. [73]The most abundant transition metals, such as copper and especially iron, are major players in the Fenton reaction, as shown in Equation ( 13), and the metal-mediated Haber-Weiss reaction, as shown in Equation ( 14). [86,87]idation : In 1894, Fenton described the oxidation of ferrous ions (Fe 2 + ) into ferric ions (Fe 3 + ), producing hydroxyl radicals (HO • ), in interaction with H 2 O 2 , explaining oxidative damage in biological environments. [87,88]Loosely bound or removable metals, like these metal ions, participate in redox processes, but metals with one oxidation state hidden in proteins or storage complexes cannot. [89,90]At physiological pH, most iron is oxidized to Fe 3+ and bound to biological chelates, but some cases involve metals in a higher oxidation state. [90,91]Reducing equivalents with suitable oxidation potential and the nature of iron-binding chelates can strongly affect the occurrence of the Fenton reaction in biological surroundings. [92]n contrast, the metal-mediated Haber-Weiss reaction, as shown in Equation ( 14), involves the superoxide radicals, O − 2 , reducing ferric ions into ferrous ions.These ions are more soluble and enable the Fenton reaction, which is responsible for many of the deleterious effects of oxygen radicals. [90,91]The combination of the Fenton and metal-mediated Haber-Weiss reaction is thermodynamically possible, representing the in vivo Haber-Weiss reaction but it is extremely slow. [83]he human body derives energy from glucose, which is primarily stored in plants through the Calvin Cycle, a lightindependent step shown in Figure 3a.This cycle uses ATP (Adenosine Triphosphate), NADPH (Nicotinamide Adenine Dinucleotide Phosphate), and CO 2 to produce carbohydrate molecules like glucose in the stroma.ATP and NADPH are essential energy-rich molecules generated during photosynthesis in plants, algae, and some bacteria.ATP acts as the primary energy currency for cellular processes, while NADPH serves as a coenzyme in anabolic reactions, providing electrons for biosynthesis pathways and maintaining redox balance.Both light-dependent and light-independent reactions can be summarized by Equation ( 16), typically a redox reaction.
Within the plant cell, water is oxidized, losing electrons to oxygen, while carbon dioxide is reduced after gaining electrons, forming glucose.

Redox Environment in Hemoglobin, Human Serum Albumin, Cells, and Tissues
Heme proteins, such as myoglobin (Mb) and hemoglobin (Hb) have various biological functions, including oxygen storage, transport and reduction, electron transfer, and redox catalysis. [94]99][100][101] On the other hand, the free radical nitrite (NO − 2 ), which originates from nitric oxide (NO) metabolism, is responsible for the endothelial dysfunction in humans and is correlated with an increased cardiovascular risk load. [102,103]n the peroxidase cycle of hemoglobin-dependent redox reactions, as shown in Figure 3b, H 2 O 2 can oxidize ferric Mb/Hb (Fe 3 + ) to highly reactive ferryl Mb/Hb (Fe 4 + ), which can cause damage to biological molecules like Mb/Hb through a series of oxidative side reactions. [93,97,98]][105] However, at low concentrations, NO − 2 can promote a pro-oxidant effect on Hb-H 2 O 2 -induced protein oxidation and reduce HepG2 cell viability. [93]In the reduction of ferryl Mb/Hb (Fe 4 + ) to ferric state (Fe 3 + ), NO − 2 is oxidized to a nitrating agent, NO 2 .NO − 2 -triggered tyrosine nitration might make an important contribution to the reduction of enolase inactivation, revealing a potentially protective mechanism in hemoglobin-dependent redox reactions. [93]It is now clear that NO − 2 can act as both an antioxidant and a promising therapeutic agent to protect against myocardial ischemiareperfusion injury through mediating NO homeostasis. [104,105]erum is an oxidizing environment containing 17 disulfide bonds such as cystine, homocystine, cystamine, and glutathione disulfide (GSSG), as well as one unpaired cysteine (Cys 34 ). [106,107]he free cysteine (Cys 34 ) is the most abundant protein in plasma, at levels of 0.6-0.8mm.110] Under pathologic conditions, such as kidney or liver diseases, the level of oxidized albumin increases up to 70%.[113][114][115][116][117] Thus, it is evident that the presence of 70-80% reduced Cys 34 in albumin coexists with 70-80% of oxidized low molecular mass thiols presented as disulfides. [108]Hence, oxidized albumin is considered a short-term biomarker of oxidative stress. [108]xtracellular fluids like HSA are oxidizing environments, while reducing mediums are prevalent inside cells. [108]The movement of electrons from oxidizable organic molecules to oxygen (redox couple) provides the energy required to maintain the ordered state of a living organism, resulting in an overall reducing environment in cells and tissues. [118]Changes in the reducing/oxidizing environment or the redox environment affect the responsiveness of redox couples to electron flow, determining a cell's Redox environment. [118]Bűcher introduced cellular redox biochemistry, which determines the states of various redox couples in cells and estimates the actual cellular reduction potentials (Ist-Potential) for the NAD + /NADH and NADP + /NADPH couples. [119]According to Schafer and Buettner, the redox environment is the summation of the products of the reduction potential and reducing the capacity of all the redox couples found in biological fluids, organelles, cells, or tissues, as shown in Equation (17). [118]dox Environment = n(Couple) where, E i is the half-cell reduction potential for a given redox pair and [Reduced Species] i is the concentration of the reduced species in that redox pair.A representative redox couple, such as the GSSG/2GSH couple, could be used as an indicator of changes in the redox environment since it is the most abundant redox couple in a cell, providing a large pool of reducing equivalents and serving as the cellular redox buffer. [118]However, the ratio of interconvertible oxidized and reduced forms of a specific redox couple is not sufficient for all redox couples, such as GSSG/2GSH couple. [118]To have complete information about a redox couple, the redox state should include the reducing capacity (how large is the pool for the redox buffering system) of a redox couple along with the half-cell reduction potential.Schafer and Buettner proposed a notation for the redox sate/status of a redox pair, such as GSSG/2GSH, which includes half-cell reduction potential, E hc , of the reduced species, in this case GSH, with its concentration, [GSH], shown as follows: {E hc (GSH); [GSH]} = {−180 mV (GSH); 3.5 mM}.

Thermodynamics and Total Reducing Power of Biological Sites
The reduction potential, measured in voltage, can be determined using the Nernst equation developed by Walter H. Nernst in 1889.The reducing capacity, which is the total charge stored or the number of electrons available, can be estimated by determining the concentration of the reduced species in a redox couple. [83]he net transfer of charge, Q in coulombs, for a half-reaction in a redox reaction involving one mole of reactants, can be expressed as Equation (18).
Thus, the change in Gibbs free energy, ∆G in kJ mol −1 , to move the charge, Q, over a potential change, ΔE, is given by Equation (19).
where "-" denotes the favorable direction of a reaction, as shown in Figure 2a,b.According to Equation ( 19), a reaction is thermodynamically favored when ∆G is negative, whereas a redox reaction is favored when E is positive.The Nernst equation, which measures an electrode's potential at 25 °C, can be deduced as Equation ( 20) using Equations ( 5) and (19).
The term ΔE o , which represents the electromotive force under standard conditions, is the difference in the standard reduction potentials of the two half-cells involved in the process.The superscript "°" implies the thermodynamic standard-state conditions.At thermodynamic equilibrium, ∆G = 0, and consequently, ΔE = 0. Equation ( 21) can be derived from Equation (20) under these conditions.
However, Equation ( 21) can be applied in biological sites, such as cells, where the system is reversible and all factors affecting the system are known and can be controlled. [84,85,118]Cells are not in thermodynamic equilibrium because, within them ΔE ≠ 0 and the systems are not fully reversible. [83,118]Therefore, the Nernst equation can be applied for the biological systems described in the set of Equations ( 22), (23), and (24) as demonstrated in Equation (25).
Redox pair 1 : Redox pair 2 : Redox Reaction : where E 2 is the half-cell reduction potential of the electrode associated with the redox pair that is reduced, E Red .Furthermore, E 1 , is the half-cell reduction potential of the electrode associated with the redox pair that is oxidized, E Ox .Consequently, the voltage, ΔE, is the overall potential difference of an electrochemical cell.
Reducing power, unlike redox potential, is not a thermodynamic parameter, but it plays a significant role in conveying information about the overall capability of a cell, biological fluid, or tissue to donate electrons and the concentration of reducing equivalents responsible for this ability. [83]Among many compounds, the major contributors to the reducing power of a resting cell are the low-molecular-weight antioxidants (LMWA), which can be measured to determine the reducing power in cellular and biological environments. [83]Hence, estimating the reducing power may indicate the total LMWA status of a biological system.
There are indirect and direct methods to estimate total LMWA activity. [120]Indirect methods measure consequential factors of redox capacity, such as oxidation products formed or concentrations of major redox couples in the biological environment, using fluorescent or spectrophotometric techniques, or inhibition methods that involve adding a radical species to the sample.Direct methods, both chemical and electrochemical, utilize an electrode and an external probe to measure the current, which is proportional to the concentrations of the LMWA. [83]Chemical methods are primarily colorimetric techniques that measure a redoxactive couple whose reduced and oxidized states have different physical properties, such as the ferric-reducing antioxidant power (FRAP) assay, which is based on the reaction of the ferric/ferrous redox couple with antioxidants in the sample and results in the creation of a blue color. [120]lectrochemical techniques, including voltammetry, are essential and popular measurement methods for various modern cutting-edge research fields in biological sites, such as cells, fluids, and tissues. [121]Voltammetry is applicable for both lipophilic and hydrophilic antioxidants in biological fluids and tissue homogenates, offering several advantages. [83]It can be used without sophisticated extraction and treatment to obtain the reducingpower profile, which provides information about the type and concentration of LMWA. [122]This methodology for quantifying overall LMWA has been used in various clinical situations and pathological disorders, including diabetes, [123][124][125] ulcerative colitis, [126] brain degenerative diseases and head trauma, [127,128] skin status and pathologies, [127][128][129][130] and irradiation therapy, [122] as well as in the study of the aging process [127,128] and stages of embryonic development. [123,131]Biological fluids, such as seminal fluid, cerebrospinal fluid, saliva, sweat, urine, plasma, and gastric juice, possess reducing power derived from their LMWA content. [132]Electrochemical techniques provide necessary knowledge concerning the thermodynamic, kinetic, and analytical features of the tested compounds.We have discussed different electrochemical measuring methods to evaluate the promising aspects, challenges, and prospects of applying them in a biological sample.

Electrochemical Sensing Techniques
Interfacial electrochemical sensing techniques are a broad field that can be classified based on specific techniques, experimental conditions, and output signals, as shown in Figure 4.In this section, we will provide an overview of the sensing performance metrics for each type of sensor by giving some exemplary examples used for detecting neurotransmitters, proteins, and glucose from biofluids for diseases, particularly Schizophrenia, Alzheimer's, and Parkinson's.

Voltammetry
In 1959, Jaroslav Heyrovsky won the Nobel Prize in Chemistry for inventing the first voltammetric method in the early 1920s, commonly known as polarography.Voltammetry measures current as a function of a time-dependent applied potential, which is displayed as a voltammogram.The voltammogram provides information on the quantitative and qualitative characteristics of the analyte in a sample. [133]The faradaic current in a potentiostat arises from the faradaic process, which requires the transfer of electronic charge and involves atoms, ions, or molecules from or to the bulk solution. [134]Besides the faradaic current from the analyte, other types of currents may exist in the cell, such as background currents from electrolysis of impurities, the electrolyte, the electrode material, and capacitive (charging) current.The lat-ter is the only non-faradaic current and depends on the interface that acts as an electrical capacitor under applied voltage.
The electrode surface concentration is used since an equilibrium position is established at the surface.If the initial concentrations of Fe(CN) 3− 6 and Fe(CN) 4− 6 are 1.0 and 0 mM, respectively, the concentrations will not change if a potential of +0.530 V is applied to the working electrode.Hence, there will be no faradaic current flow through the cell.However, if the applied potential is +0.356V, some Fe(CN) 3− 6 will be reduced to Fe(CN) 4− 6 , reaching the equilibrium condition where the concentrations of both ions are equal at the electrode surface but not in the bulk solution (as per Equation ( 28)).This results in a huge amount of faradaic current flowing and eventually reaching zero.The form of the analyte at the electrode surface depends on the applied potential, and its concentration may not be the same at the electrode surface and in the bulk solution. [135]This difference in concentration creates a concentration gradient between the solution at the electrode surface and the bulk solution, which results in a faradaic current flow until even concentrations are reached.The faradaic current is proportional to the rate of charge transfer of the redox reaction and the rate of the chemical reaction. [135]The rate of a cell reaction is determined by two factors: mass and electron transport.The three modes of mass transport are diffusion, migration, and convection.The faradaic current when mass transport occurs alone is given in Equation (29).
where  is the width of the diffusion layer, A is the electrode surface area, D is the diffusion constant, and C is the concentrations.The nature of electron transfer between the electrode and adjacent species also affects the current.If the electrons flow quickly, it means the redox reactions are at equilibrium, ensuring electrochemical reversibility, but if they flow slowly, it means the reactions are irreversible.The Nernst equation can only be applied to reversible redox reactions.In contrast, the short-lived charging current occurs when the electrode's potential suddenly changes from one state to another.The positive or negative surface charge attracts opposite ions at the vicinity of the surface, forming a structured electrode solution interface called the electrical double layer (EDL).

Pulse (Sampled-Current) Voltammetry
The normal linear sweep voltammetry (LSV) is discussed in the CV section, although it is the first voltammetry method.Pulse or sampled-current voltammetry is a popular choice among scientific communities, as shown in Figure 5c-e.To provide a general understanding, Figure 5c-e is discussed briefly below.A redox reaction is observed under stirring when a series of step potentials is applied, as shown in Figure 5c.Initially, the potential doesn't result in any faradaic current flow.As the step potentials increase, some species become electroactive and can be reduced, but not completely to zero surface concentration.The last two step values work in the mass-transfer-limited region, which is diffusion rate dependent and not influenced by potential.The current is sampled at time  for each step and a voltammogram is shown in Figure 5e.
This type of experiment is called sampled-current or normal pulse voltammetry, as shown in Figure 6a, which results in improved sensitivity and lower detection limits.In differential pulse polarography, shown in Figure 6b, the current is the difference between two sampled values taken ≈17 ms before and after the pulse is applied.The benefit of square-wave polarography, seen in Figure 6d, is that the value of  can be as low as 5 ms, reducing the time required for analysis.
For the redox reaction O + ne − ⇌R, if there is no R present at the start, the current after applying the potential can be expressed , as seen in Equation (29).At the limit-ing current, as shown in Figure 6a,b, i , indicating a linear relationship with the bulk concentration of O. Thus, the equation for the Nernst potential and current can be written as From Equations (30-32), the potential equation can be deduced.
where E 1/2 is the known half-wave potential.The half-wave potential equals the standard-state potential if K o and K R are same, which is exactly the case.Differential Pulse Voltametric (DPV) detection of acetylcholine (ACh) has been reported in both enzymatic, [138] and nonenzymatic [137,139] sensors.Depending on the linear range, the nonenzymatic ACh sensors showed good compatibility.Beitollahi et al. 2019 showed that ZnFe 2 O 4 nonenzymatic sensor had a sensitivity of 0.548 μA μm −1 . [139]Nonenzymatic detection of dopamine (DA) by Dursun et al. 2010 using DPV showed better performance based on linear range and limit of detection (LOD). [142]Acute myocardial infarction (AMI), also known as a heart attack, is a common life-threatening cardiovascular disease throughout the world, and cardiac troponin T (cTnT), a cardiac regulatory protein, is its specific biomarker.
AMI results in the rapid release of cTnT (37 kDa) from cardiac muscle cells into the bloodstream, which remains elevated up to 14 days after cardiac ischemia, providing the possibility of prognosis of the disease.DPV-based enzymatic sensing of cTnT was recorded in a couple of scholarly articles. [143,144]The sensor reported by Phonklam et al. 2020 showed a linear range of 0.1-8.0pg mL −1 and LOD 0.04 pg mL −1 .The promising result could be due to the higher surface-to-volume ratio of functionalized multi-walled carbon nanotubes (f-MWCNTs).A hemecontaining protein, cardiac Myo, is one of the markers that increase after AMI onset.Due to its small size of 17.8 kDa, Mb is quickly released into circulation within 1-3 h after symptom onset and serves as a valuable screening molecule with high sensitivity and predictivity for AMI detection. [145]The enzyme-based Myo sensing electrode of Lee et al. showed a linear range of 17.8-1780 ng mL −1 which is a good fit for the serum range of 30-90 ng mL −1 , and LOD of 9.8 ng mL −1 . [145]PV has been used for both enzyme-based [148] and enzymeless [149][150][151][152] detection of glucose in human body.The enzyme-less The current is sampled at the end of each potential pulse for approximately 17 ms before returning the potential to its initial value.Reproduced (Adapted) with permission. [136]Copyright 1999-2023, Copyright Holder David Harvey (https://sites.google.com/depauw.edu/dth/home),and Libretexts (https://LibreTexts.org).Square Wave Voltammetry (SWV) technique has been used to sensing DA in both enzymatic [154] and nonenzymatic [153,156] routes.However, the linear ranges of the above-mentioned DA sensors do not cover the normal physiological range of 50 nm.
Tumor necrosis factor-alfa (TNF-) is naturally produced by activated macrophages and monocytes and has pleiotropic effects on normal and malignant cells.Though it is a key marker of inflammatory diseases, it is involved in a broad range of physiological and pathological responses, such as atherosclerosis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, Alzheimer's disease, and various pulmonary disorders. [157]Moreover, TNF- is rapidly upregulated in the brain after injury and is associated with necrosis or apoptosis. [157]Therefore, measuring TNF- is important for the understanding of inflammation and discovering drugs that alleviate it.Two enzymatic approaches to detect Myo have been reported. [158,159]Both electrodes showed good prospects to sensing the normal physiological range of Myo of 95-500 μg mL −1 .

Cyclic Voltammetry
CV is a powerful and popular electrochemical technique commonly employed to investigate the redox processes of molecular species. [19]CV scans the potential in both directions, estimating the standard-state potential from the average potential between points F and C on the cyclic voltammogram (Figure 7H).When a solution of ferrocenium [Fe(Cp) 2 ] + (Cp = cyclopentadienyl), abbreviated as Fc + , is scanned to negative potentials, Fc + is reduced locally to ferrocene [Fe(Cp) 2 ], abbreviated as Fc, at the electrode, resulting in a measurement of current and depletion of Fc + at the electrode surface, [19] see Figure 7I.Firstly, the negative scan starts from A to D, and during this cathodic scan, [Fc + ] moves away from the surface after being reduced to Fc.The peak current, at point C, is observed due to extra Fc + that has diffused through the interface from the bulk solution.After the peak, the current starts to decrease because of the expansion of the depletion layer that slows down mass transport.The scan is reversed after reaching switching point D, and the anodic or positive scan begins.Due to the more positive potential, the Fc accumulated at the surface during the negative scan is re-oxidized to Fc + .
According to Equation (30), the redox species are equal at points B and E, as per the Nernst equation (E = E 1/2 ).The two peaks are separated due to the diffusion of the analyte to and from the electrode, and the difference between the peaks is 57 mV at 25 °C for a chemically and electrochemically reversible process.The width at half-maximum on the forward scan of the peak is 59 mV.The scan rate is another important parameter of CV, which can control the size of the diffusion layer inversely.A high scan rate means a smaller layer width, which increases the flow of electrons through the interface, resulting in a higher current.
The Randles−Sevcik equation, as shown in Equation (34), relates the peak current to the scan rate v(V s −1 ), the electrode surface area A (cm 2 ), the diffusion coefficient D o (cm 2 s −1 ) of the oxidized analyte, and the bulk concentration C 0 (mol cm −3 ) of the analyte.It is important to note that some analytes undergo oxidation first, in which case the potential would first scan positively. [1]t is evident from Tables 1-3, CV is one of the most popular electrochemical sensing techniques for detecting glucose in the human body.Among so many articles, the scholarly published works with proper details of linear range (sweat 0.05-1 mM; blood 4.9-6.9mM), LOD are presented here in a tabular form.There are enzymatic [162,168,169,[172][173][174] and enzymeless [149,[164][165][166][167]170,171,[175][176][177] electrodes given. Depending on thelinear range and LOD, the glucose sensing electrodes can be categorized into three groups; for blood, [166][167][168][169][170][171][172][173][174][175][176] for sweat, [172] and for both analyte.[164,165,173,177] The glucose sensors fabricated using Au show promising sensing performance of linear range, LOD, and sensitivity, see Table 3. Kurniawan et al. (2006) fabricated an electrode to sense glucose using nano/thin Au, which provided a sensitivity of 160 μA mm −1 cm −2 .[166] On the other hand, Shu et al. reported a dendrite-like gold nanostructures (DGNs) electrode of good sensitivity of 190.70 μA mm −1 cm −2 .[175] Moreover, the electrode of Au nanowires by Cherevko et al. gives the highest sensitivity of glucose sensing of 960 μA mm −1 cm −2 .[149] The better performances of Au nanowires and dendrite-like nanostructures are due to a higher surface-to-volume ratio at the surface of the electrode.However, Au is an expensive material for an electrode to be suitable for mass production.

Stripping Voltammetry
Among anodic, cathodic, and adsorptive stripping, anodic stripping voltammetry is widely used.The applied potential and response current-potential curve are shown in Figure 7J.At the start, the working electrode is held at a cathodic potential sufficient for depositing metal ion Cu 2 + , onto the Hg film electrode, [1] where copper is amalgamated with mercury.During the process, metal is concentrated from the solution to a small volume on the electrode surface and stirring is used to speed up deposition.Before the end, stirring is stopped to eliminate convection mass transport by allowing the solution to become still.In the sweep step, metal is stripped from the electrode to the solution at a sufficient positive voltage.The peak current in the peak-shaped voltammogram represents the metal ion concentration in the solution.The main advantage of this technique is its small detection limits in parts per billion, due to the mass concentration of the analyte.For accurate and precise results, control over experimental conditions is important due to its sensitivity.Key factors include the electrode surface area, deposition time, rest time, stirring rate, and scan rate during the stripping step.The method is mostly useful for metals that form amalgams with mercury. [1]

Amperometry
The amperometry technique produces a current versus time curve after applying a controlled potential, rather than a voltammogram.It is also known as chronoamperometry, single-step amperometry, or forward-step amperometry. [20]Figure 5f shows a step potential function of the technique.When the potential is applied to an electrode submerged in a solution of the electroactive species O (the oxidized form) with a concentration of C o* at the electrode's surface, the species are electro-inactive at E 1 but become rapidly reduced at a more negative potential E 2 .This results in a complete depletion of O at the electrode's surface and drives the experiment into a "mass-transfer-limited" region.Reproduced (Adapted) with permission. [19]Copyright 2017 The American Chemical Society (ACS).Further permissions related to the material excerpted should be directed to the ACS.J) Applied potential and voltammogram for anodic stripping voltammetry (ASV) at a mercury film electrode, with a copper ladder diagram in the upper figure of (J).Typical deposition times are 1-30 minutes, with a lower deposition time for higher analyte concentrations.Reproduced (Adapted) with permission. [136]Copyright 1999-2023, Copyright Holder David Harvey (https://sites.google.com/depauw.edu/dth/home),and Libretexts (https://LibreTexts.org).
At t = 0, O is reduced to the stable anion radical R through the reaction O + ne − → R, causing a large instantaneous current that continues until full reduction is achieved at the surface.The depletion of O creates a concentration gradient that diffuses more O toward the electrode, causing a flux of O and a proportional increase in current.The increasing amount of R thickens the zone of O depletion, causing the slope of the concentration profile and current to decline over time, as shown in Figure 5g,h.Useful Acronym: functionalized single Walled carbon nanotubes (f-SWCNTs); V-phosphomolybdates (PMoV); Graphene flakes (GFs); Indium Tin Oxide (ITO); The current in chronoamperometry is expressed by the wellknown Cottrell equation (see Equation 35), which describes the observed current for a planar electrode at any time following a large forward potential step in a reversible redox reaction as a function of time.The symbols have their usual meaning, which was given previously in ref. [20].
3,4-dihydroxyphenylacetic acid (DOPAC) is a DA metabolite found in the cytoplasm of the brain and neurons, as well as in cerebrospinal fluid (CSF).It is absent or undetectable in the blood of the healthy human body, although a range of 7-13 nm can be found in the CSF of an adult human body during any neural abnormalities. [207,208]An enzymatic amperometric electrode has been fabricated by Liu et al.. [207] Acetaldehyde, also known as ethanal by the international union of pure and applied chemistry (IUPAC), is an organic colorless liquid or gas and one of the most important aldehydes.Although it occurs widely in nature in plants, it is also produced by the partial oxidation of ethanol by the liver enzyme alcohol dehydrogenase and is a contributing cause of hangover after alcohol consumption. [209]The International Agency for Research on Cancer (IARC) has listed acetaldehyde, Me-methyl, (MeCHO, CH 3 CHO) as a Group 1 carcinogen. [210]An enzyme-less sensor has been reported by Blanco et al 2015 which can detect both CH 3 CHO and ACh. [178]An amperometric enzymatic glucose sensor was reported by Li et al. 2020 with excellent sensitivity of 15 000 μA mm −1 cm −2 . [183]The electrode could be used for sensing sweat glucose, since its linear range is 0.008 -1.5 mm.Additionally, a nonenzymatic glucose sensing electrode fabricated by Zhang et al. also suitable for sensing sweat glucose which provides 13 850 μA mm −1 cm −2 . [189]n Table 4, the amperometric glucose sensors that meet the criteria for linear range (sweat 0.05-1 mM; blood 4.9-6.9mM), LOD, sensitivity, reproducibility, stability, and interference analysis are reported.It is evident that the amperometric technique is the most popular electrochemical method for detecting glucose in the human body.This immense popularity is due to the requirement of a single potential, where a range of potentials (mostly −1 to +1 V) is needed to investigate the analyte using CV.The drawbacks (such as required large ranges of sweeping potential from -1 V to +1 V, longer sweeping time, water dissociation after +0.7 V, H 2 evolution at −1 V, etc.) make CV the second most popular electrochemical technique for sensing glucose in the human body.Among the reported scholarly publications, the nonenzymatic [149,177, electrodes show promising prospects over enzymatic [182][183][184][185] ones.
2][193][195][196][197]205,206] Among the enzymatic amperometric glucose sensors, the electrode fabricated by Li et al. using CoS-functionalized MWCNTs with a GOx surface layer exhibits an excellent sensitivity of 15 000 μA mm −1 cm −2 . [183] However, the sensitivit of the electrode may result from two reasons; high surface-to-volume ratio of MWCNTs and electrons transferring capability of CoS in between GOx and MWCNTs.A nickel MOF (Ni-MOF) supported by CNTs yields a sensitivity of 13 850 μA mm −1 cm −2 , which is a non-enzymatic electrode suitable for sweat glucose sensing, fabricated by Zhang et al. [189] Furthermore, electrodes that cover the linear ranges of both sweat and blood are primarily based on nanoparticles of noble  metals, such as Au, Pt, and Ni.This hinders their mass production.[177,193,[195][196][197]206] In contrast, non-precious transition metal oxide (Cu and Co oxides) electrodes mainly provide a linear range for sweat glucose.[201][202][203][204] Thus, non-noble metal oxides are suitable for sweat glucose monitoring and mass production.

Potentiometric Methods
Potentiometric sensors are suitable for determining concentrations higher than 10 −5 m, which is the range required in most cases.For example, the normal blood glucose level in a human body is between 4 and 7 mm.Potentiometry is compatible with multichannel array-type sensory instruments and requires a simple operating circuit.Thus, non-enzymatic potentiometric glucose sensors are attractive for integration with conventional ionselective electrodes, such as pH meters.However, selectivity is a key issue for these sensors.Only a few attempts have been made to create a non-enzymatic potentiometric glucose sensor, as reported by Shoji and Freund. [211,212]They used polymeric membranes with boronic acid units, which have an affinity for diol sac-charides containing two units.The sensor responded to glucose but showed even higher sensitivity to fructose than to glucose. [213]he cell potential is measured under static conditions with little or no current flowing through the electrolyte, so the compositions remain constant and are suitable for quantifying the analyte.In 1889, the first quantitative potentiometric method was invented based on the Nernst equation, which relates a cell's potential to the concentration of electroactive species in it. [1]A potentiometer, shown in Figure 8a, is used to determine the potential.The species are oxidized at the anode and reduced at the cathode electrode.When the switch, T is open, the potential across R cb can be determined by Equation (36).
i cell = i, is zero.Usually, the potential depends on the current flowing through the cell.
The switch T is briefly closed, and the current through the ammeter, which is in the nanoscale, is observed.The slide wire variable resistor is readjusted until the ammeter current is zero to obtain the cell potential.There are two half-cells, shown in Figure 8b, each containing an electrode immersed in a solution of ions whose activities determine the electrode's potential.A salt bridge containing an inert electrolyte, such as NaNO 3 , is used to complete the electron flow path.Porous plugs are used to allow ions to freely move through the bridge.The overall cell reaction and half-reactions of the half-cell reactions are given below.
Oxidation at Anode (Reference) : Cu (s) ⇌ Cu 2+ (aq) + 2e − (39) Reduction at Cathode (Indicator) : 2Ag The cell potential can be calculated by combining Equations ( 21), ( 38), (39), and (40).If both electrodes were placed in the same solution, the reduction of Ag + to Ag would occur on the Cu surface and no electrons would pass through the potentiometer.Equation ( 41) is used to deduce the potentiometric electrochemical cell potential, and Equation ( 42) can be used to determine any unknown value among E cell , a Ag + , and a Cu 2+ if other two are known.(42)   and a Cu 2+ , can be determined if other two are known.

Disadvantages of Potentiometric Methods
The standard state potentials of Equation ( 42) depend on temperature and the medium, as different activities of the same redox couple can vary in different media.For example, the potential for the Fe 3 + /Fe 2 + redox couple is +0.735V in 1 m HClO 4 , +0.70 V in 1 m HCl, and +0.53 V in 10 m HCl. [1]The issue can be addressed by using a matrix-dependent formal potential.Additionally, the junction potential, caused by concentration differences at the interface or bridge between solutions, is a major challenge.A junction potential arises at the interface between two ionic solutions, as shown in Figure 8c if there is a difference in the concentration and mobility of the ions.For example, H + has higher mobility than Cl − .The potential at 0.5 m and 0.1 m NaCl are 10.2 and 18.1 mV, respectively. [214]This potential can be as large as 30-40 mV. [1]To address this issue, a salt with equal cation and anion mobilities, such as KCl, is used with a saturated concentration.However, the junction potential cannot be eliminated, so the cell Equation ( 42) must be rewritten as Equation (43).
First, we will focus on neurotransmitters as summarized in Table 5. Acetylcholine, a neurotransmitter, regulates muscle contraction in the peripheral nervous system (PNS) through acetylcholine receptors (AChR).It also plays a key role in central nervous system (CNS) activities such as behavior, arousal, attention, learning, and memory.It is synthesized from choline by choline acetyltransferase and acetyl coenzyme A in neurons. [215]mbalanced ACh regulation in the brain can cause neuropsychiatric disorders such as schizophrenia, Alzheimer's, Parkinson's, and myasthenia gravis. [215,217]Potentiometric enzymebased detection of ACh has been presented in several literature sources [215,[217][218][219] but the electrode represented in [218] is a better fit with the physiological range but has a high LOD, making it less suitable.The potentiometric ACh sensors listed in Table 5 do not meet the requirements.
DA is another neurotransmitter that conveys messages of pleasure in the brain's reward system.Imbalanced levels of DA are linked to various diseases, including Schizophrenia, Parkinson's, restless legs syndrome, and attention deficit hyperactivity disorder.In 2021, He et al. developed a DA enzyme electrode with a linear range of 0.1 μm to 0.1 m, although the physiological range of dopamine is only 50 nm.
For protein sensors, we will provide a few examples, including CRP, trypsin, CEA, and PSA, due to their roles in inflammation and metastasis.The electrochemical potentiometric technique is primarily used for enzymatic detection of important proteins in the human body, such as CRP, trypsin, CEA, and PSA, as shown in Table 5. CRP, a protein made in the liver, is found in blood plasma and increases in response to an offending agent, activating the first immune responders.In a healthy human, CRP levels are less than 5 mg L −1 . [221]However, surgery, trauma, exercise, heatstroke, and childbirth can result in a drastic increase in CRP, ranging from 1 to 500 mg L −1 .Higher CRP values can indicate cardiovascular diseases, pathogenic diseases, IBD, and colon cancer. [221]An enzymatic anti-CRP layer was immobilized on ZnO nanotubes grown on Au-coated glass to sense CRP.The electrode showed the best response at pH 7, where the optimum pH of the antibody is 7.2.The poor response at pH levels below and above 7 might be due to less activity and detachment of the immobilized antibody. [221]The electrode had the maximum response at 55 °C, but the responses decreased at temperatures below or above 55 °C due to less charge mobility and denaturation of the immobilized antibody.However, the electrode's linear range of 0.01 to 1.0 mg L −1 does not cover the required range of 1-5 mg L −1 .
Trypsin is produced as the inactive zymogen trypsinogen in the pancreas and is activated in the small intestine to break down long chains of proteins into smaller, more easily digestible pieces.A potentiometric polymeric membrane, made using MIP on ISE, is fabricated to sense trypsin in an enzymatic, label-free manner. [222]EA is a large glycoprotein that is typically produced in gastrointestinal tissue and is absent or present in low amounts (less than 2.5 μg L −1 in non-smokers, less than 5.0 μg L −1 in smokers) in the blood of healthy adults.The level of CEA in the blood may be elevated in certain types of cancer, such as colon and rectal, breast, lung, pancreatic, stomach, liver, and ovarian cancer, and in non-cancerous conditions like cirrhosis, hepatitis, diverticulitis, inflammatory bowel disease, peptic ulcer disease, chronic  [ 228] 0.8 -6.obstructive pulmonary disease, cholecystitis, and obstructed bile duct.The CEA test is crucial for the diagnosis of colorectal cancer, as well as for monitoring its response to treatment and the risk of reoccurrence.A CEA-sensing enzymatic layer of hydroxylterminated alkanethiol was created in reference. [223]The thiol molecules of the hydroxyl-terminated alkanethiol layer are chemically bonded to a substrate of a gold-coated silicon chip, and template biomolecules are co-adsorbed and later removed to create footprint cavities.The potential changes upon re-adsorption of the CEA biomolecules are measured potentiometrically. [223]SA is a glycoprotein secreted by the prostate gland and is found in low amounts (<4 μg L −1 ) in the blood. [224]Elevated levels (>10 μg L −1 ) of PSA in the blood can be caused by prostate cancer as well as non-cancerous conditions such as prostatitis (inflammation of the prostate) and benign prostatic hyperplasia (enlargement of the prostate). [224]An enzymatic biosensor with a negative slope of sensitivity, good linear range, and low detection limit has been developed for PSA. [224]otentiometric sensors have been used to detect blood glucose, which is the main source of energy for the human body and is produced from food through digestion.The pancreas produces insulin to help the body convert glucose in the bloodstream into energy.However, uncontrolled blood sugar levels due to ineffective insulin use or deficiency can cause diabetes, which can lead to blindness, kidney failure, heart attack, stroke, and serious dam-age to nerves and blood vessels.Non-enzymatic electrodes made of polyaniline or polyaniline boronic acid could be used for glucose sensing in human blood, but the authors were not given information about their limits of detection or sensitivities. [211,212]ther potentiometric glucose sensors use the GOx enzyme, as shown in Table 5.As shown in Table 5, it is evident that GOx is not an effective enzyme for detecting glucose in blood because its linear range deviates from the desired range of 4.90-6.90[227] However, the linear range of GOx is not suitable for detecting glucose in blood, but it is effective for glucose sensing in urine. [229]

Coulometric Methods
In dynamic methods such as coulometry, current is passed to alter the concentration of species.Coulometry involves complete oxidation or reduction of the analyte at the electrode.The Equation (17) can be represented as Equation ( 44) by substituting N A for the moles of the analyte in the cell.To calculate N A , complete electrolysis must be performed, and 100% current efficiency is required to calculate the charge, Coulometry can be further divided into controlled-potential and controlled-current methods.

Controlled potential (chrono) coulometry
A step potential is applied across the cell.As a result, the current, as shown in Figure 8d, decreases with time as the concentration of the analyte decreases due to oxidation-reduction reactions.The total charge can be found using Equation (45).The method is accurate if no potential-dependent redox reaction is associated, assuming 100% current efficiency.
To select an appropriate potential value, a case study using a platinum working electrode to reduce Cu 2+ as Cu is explained below.The favorable potential is close to 0.342 V, as seen in Figure 8d, but must be greater than 0 V to avoid current from water.The potential required to reduce 99.99% of Cu 2+ can be calculated using Equation (48) if the initial concentration of Cu 2+ is known.A small volume, high surface area, and high stirring rate are suggested to the time needed for the reduction process. [1] 2+ (aq)

Controlled-Current Coulometry
The excitation (red) and charge (blue) are shown in Figure 8f.The method requires less time for both electrolysis time (10 minutes) and processing time, and has a rate constant, but also has two drawbacks.With time, the current decreases due to the rate.
The potential is regulated to keep the current constant, which may result in reduced current efficiency.To ensure 100% current efficiency, a mediator is used.Additionally, the determination of the endpoint is another challenge of the method and requires titrimetry.Please place Table 6 here.
There are limited reports on coulometric sensors for health monitoring.An enzymatic approach to detect alpha-1-fetoprotein (AFP) using H 2 O 2 and a sensing layer of GOx was published in ref. [230].AFP is normally produced by a fetus's liver and yolk sac and decreases significantly within a year after birth.AFP is not generally found (0-20 ng mL −1 ) in healthy adults.Higher values (>20 ng mL −1 ) show the possibility of testicular and ovarian cancer.Adults with cirrhosis and chronic hepatitis also possess higher levels of AFP in their blood.AFP levels of >400 ng mL −1 may indicate liver cancer.Moreover, a higher value of AFP could be a sign of the rare genetic condition ataxia-telangiectasia.In this coulometric sensor, the sensing performance of the device was enhanced by metallizing the electrode and adding microfabricated solution processing channels.After incubation for 120 min, a detection limit of 0.4 ng mL −1 was achieved.However, the main drawback of this approach, as it is based on coulometric sensing, is the time required to obtain the result.

Research Challenges and Future Perspectives
It is clear that electrochemical techniques play a significant role in transitioning health monitoring from expensive, large-scale laboratory apparatus to affordable and easily portable lab-in-a-box solutions. [231]However, some techniques are not as user-friendly, which has led the research community to increasingly focus on voltammetry and amperometry.Junction potential presents a barrier to potentiometric sensing.Furthermore, the requirement for chemical labeling (such as enzyme tags, redox tracers, or nanoparticle labels) poses a significant challenge for specific and direct potentiometric sensing, as the interaction between receptors and bio-analytes typically does not yield a measurable potential signal. [222]Additionally, imprinting bio-analytes is difficult because traditional MIPs are highly cross-linked, making it hard for bio-analytes to access the buried binding sites. [222]On the other hand, coulometry is time-consuming, which renders it unsuitable for rapid sensing.The only coulometric electrochemical sensor listed in Table 6, requires ≈2 h of incubation time to achieve a detection limit of 0.4 ng mL −1 . [230]Although, voltammetry is popular in the academic community, it necessitates scanning the analyte over a range of voltages (mostly -1 V to +1 V), a step that is not required in amperometry, a single potential technique.

Neurotransmitter
][219] In addition, a polymeric structure of PANI was employed as a physical support for the composite of MWCNTs and ANI. [215,217]iO 2 -Np, an insulator, was used to contain the immobilized enzyme AChE. [218]Meanwhile, carboxylated SWCNT-TFserved as an effective pH sensor. [219]A composite of AChE and ChO was used as a sensing surface with a good electronic conducting intermediary layer of molecularly bonded GO and IL of 1-allyl-3-methylimidazoliumbis(trifluoromethylsulfonyl)imide (AMIM TFSI) on a GCE for detecting Ch and ACh, respectively, using DPV. [138]Jahani et al 2020 and Beitollahi et al 2019 have published a few nonenzymatic approaches to detecting ACh using DPV. [137,139]Jahani et al 2020 demonstrated that GQDs can be used as a nonenzymatic ACh electrode surface, [137] while Beitollahi et al. 2019 showed that ZnFe 2 O 4 nanoparticles could serve as an excellent nonenzymatic option for sensing ACh. [139]Notably, Blanco et al 2015 used the traditional direct current (DC) amperometric technique to study a nonenzymatic sensor using a nickel nanowire array electrode, which can detect both CH 3 CHO and ACh. [178]or enzymatic potentiometric DA sensor, He et al. investigated a conducting polymer PEDOT doped with PSS as the solid contact, which was electropolymerized on one end of a gold wire (diameter 0.25 mm). [220]The conducting polymer was covered with a DASM containing 12-crown-4-tetraphenylborate as a neutral carrier, 2-nitrophenyloctyl ether as a plasticizer, and PVC as the membrane matrix.However, for enzymatic DA detection, the DPV technique has been applied on a nanocomposite of POA and CNTs, along with a gel containing MWNTs and room-temperature ionic liquid of OMIMPF( 6). [140,141]Moreover, nonenzymatic detection of DA by Dursun et al. 2010 using DPV showed better performance where the MWCNTs functionalized by electrochemically deposited Pt nanoparticles. [142]he SWV technique has been used to sense DA through both enzymatic [154] and nonenzymatic [153,156] approaches.However, in the enzymatic approach, Fernandes et al. prepared hybrid nanocomposites based on the immobilization of tetrabutylammonium salts of phosphomolybdates (PMo 12 , PMo 11 , PMo 11 V, and PMo 10 V 2 ) on SWCNTss or graphene flakes.The enhancement of the electrochemical properties of polyoxometalates (POMs) was a result of the strong electronic communication between POMs and carbon nanomaterials.Nanocomposites of vanadium-phosphomolybdates exhibited superior V-based electrocatalytic properties for ascorbic acid (AA) oxidation compared to free POMs.Moreover, PMo11V@graphene shows an outstanding sensing performance for the detection of dopamine. [154]n the realm of nonenzymatic detection, both Fernandes et al 2014, and Li et al 2012 applied SWV to N-doped carbon nanotubes functionalized with Fe 3 O 4 nanoparticles (Fe 3 O 4 @CNT-N), and SWCNTs fabricated by sodium dodecyl sulfate (SDS) for detecting DA, respectively. [153,156]

Protein
The electrochemical potentiometric technique is primarily used for the enzymatic detection of important proteins in the human body, such as CRP, trypsin, CEA, and PSA, as shown in Table 5.An enzymatic anti-CRP layer was immobilized on ZnO nanotubes, which were grown on Au-coated glass, to sense CRP. [221]To detect trypsin without chemical labels by potentiometric method, a mussel-inspired surface imprinted polymeric membrane was fabricated on ISE. [222]This biomimetic sensing method is based on a blocking mechanism whereby the recognition reaction between the surface imprinted polymer and a biomarker can block the current-induced ion transfer of an in-dicator ion, thus causing a potential change.A CEA-sensing enzymatic layer of hydroxyl-terminated alkanethiol was created as outlined in ref. [223].The thiol molecules of the hydroxylterminated alkanethiol layer are chemically bonded to a substrate of a gold-coated silicon chip, and template biomolecules are coadsorbed and later removed to create footprint cavities.The potential changes upon re-adsorption of the CEA biomolecules are measured potentiometrically. [223]n the other hand, a potentiometric enzymatic PSA sensor produces protein imprinted materials with charged binding sites (C/PIM) through surface imprinting over graphene layers to which the protein was initially covalently attached. [224]Vinylbenzyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers that labeled the binding site and were allowed to self-organize around the protein.The subsequent polymerization was carried out by the radical polymerization of vinylbenzene.Neutral PIM (N/PIM), prepared without oriented charges, and non-imprinted materials (NIM), obtained without a template, were used as controls.
DPV-based enzymatic sensing of cTnT has been reported in a couple of studies. [143,144]Phonklam et al. 2020 reported that MIP sensor employed an electrodeposited PMB redox probe on MWCNTs modified SPCE, with the electropolymerized polyaniline around the immobilized cTnT templates. [144]However, the biomimetic cavities for targeted cTnT were fabricated by the electro-polymerization of a conductive co-polymer matrix consisting of aniline and carboxylated aniline on GO modified SPE, as described by Karimi et al. 2019. [143]Furthermore, a myoglobin-specific binding peptide peptide (Myo-3R7, CPSTL-GASC, 838 Da) was covalently immobilized on a gold electrode that was functionalized via a DSP SAM to detect myoglobin heme protein. [145]Thiol-terminated PMPC-SH was self-assembled on a gold nanoparticle-modified SPCE for label-free detection of CRP by DPV. [146]In addition, DPA is also used by Zhang et al. for non-invasive monitoring of lactate in the human sweat.This was accomplished by electro-polymerization of 3-APBA with the imprinted lactate molecule on the AgNWs-coated working electrode. [147]ome vacant sites were created on the film of poly(oaminophenol) (PAP) through the electro-polymerization of aminophenol (AP) in the presence of protein Myo, which acted as biomimetic artificial antibodies.Felismina et al. reported the deposition of this enzymatic polymeric membrane on a AuSPE to detect Myo using SWV. [158]The authors also fabricated another SWV-based enzymatic electrode for detecting Myo, in which the SPE modified with a MIP grafted on a graphite support and incorporated in a matrix composed of PVC and the plasticizer o-nitrophenyloctyl ether. [159]The PIM was produced by growing a reticulated polymer around a protein template, followed by radical polymerization of 4-styrenesulfonic acid, 2-aminoethyl methacrylate hydrochloride, and ethylene glycol dimethacrylate.
The polymeric layer was then covalently bound to the graphitic support, and Myo was added during the imprinting stage to act as a template.

Glucose
Potentiometric non-enzymatic electrodes made of polyaniline or polyaniline boronic have been used. [211,212]]229] Both enzyme-based [148] and enzymeless [149][150][151][152] detection of glucose in the human body has been executed using DPV.The enzyme-less electrode delivered superior results compared to the enzymatic one.The sensing material for the enzymatic electrode is a GCE functionalized with ferrocene, two boronic acids, and hexamethylene . [148]Conversely, non-enzymatic electrodes utilize PtAu and PtNPs deposited on MWCNTs and Au disk, respectively.
Table 4 showcases that nonenzymatic amperometric glucose sensors [149,177, show promising prospects over enzymatic [182][183][184][185] sensors. Among th enzymatic amperometric glucose sensors, the electrode fabricated by Li et al. using CoSfunctionalized MWCNTs with a GOx surface layer exhibits an excellent sensitivity of 15 000 μA mm −1 cm −2 [183] . This senitivity may arise from the high surface-to-volume ratio of MWCNTs and the electron transfer capability of CoS between GOx and MWC-NTs.Zhang et al. 2018 fabricated a non-enzymatic electrode suitable for sweat glucose sensing, using a nickel metal-organic framework supported by carbon nanotubes (Ni-MOF/CNTs), yielding a sensitivity of 13 850 μA mm −1 cm −2 .[189] However, electrodes covering the linear ranges of both sweat and blood primarily rely on nanoparticles of noble metals, such as Au, Pt, and Ni, which impedes their mass production.[177,193,[195][196][197]206] In contrast, non-precious transition metal oxide (Cu and Co oxides) electrodes predominantly provide a linear range for sweat glucose, [201][202][203][204] making non-noble metal oxides suitable for sweat glucose monitoring and mass production.

Integrated Sensing Systems and Simultaneous Detection of Biomarkers
Recently, simultaneous analysis of multiple analytes in a single solution using a single electrode or multiple electrodes has been attracting attention from researchers in various fields, from disease diagnosis to food safety and environmental monitoring. [232,233]This approach offers the most economical, rapid, and complete information on the solution being analyzed. [234]In many cases, the detection of multiple analytes is required to determine an individual's health status. [235]Zhao et al. 2016 demonstrated that an electrochemical sensor for simultaneous detection of ascorbic acid (AA), DA, and uric acid (UA) is feasible. [235]Moreover, Fernandes et al. [156] published an article on the simultaneous voltammetric determination of AA, DA, and UA on N-doped carbon nanotubes functionalized by Fe 3 O 4 nanoparticles. [156]Additionally, Dursun et al (2010) presented a simultaneous determination of ascorbic acid, dopamine, and uric acid using pt nanoparticles decorated MWCNTs. [142]owever, designing biosensors capable of simultaneously determining two or more analytes in a single measurement, for instance on a single working electrode in single solution, remains a significant challenge. [233]Yáñez-Sedeño et al. reviewed integrated affinity biosensing platforms that couple multiplexed SPESs, utilizing various electrochemical measurement methods, such as amperometry, electrochemical impedance spectroscopy (EIS), and SWV for simultaneous detection of biomarkers. [236]Kammarchedu et al demonstrated a machine learning (ML)-powered multimodal analytical device based on a single sensing material made of electrodeposited molybdenum polysulfide (eMoS x ) on laser-induced graphene (LIG) for multiplexed detection of tyrosine (TYR) and UA in sweat and saliva. [237]Park et al proposed an all-in-one electroanalytical device (AED), a miniaturized electronic POC device integrated with the most used electroanalytical techniques, such as amperometric, voltammetric, potentiometric, conductometric, and impedimetric techniques. [238]In simultaneous sensing, less solution is needed for analysis, enabling the practical use of matrixes that may be difficult to harvest, such as sweat or breath. [239,240]The potential for sweat or breath to become the matrix of choice for roadside tests and wearable biosensors is increasing, as sensors can detect progressively smaller concentrations of biomarkers. [234]

Access to Biofluids
Heikenfeld et al. categorized the development of bio-analyte instruments into three waves: first, in the 20 th century, the biofluids (Blood, and Urine) were collected and transferred to a separate analytical laboratory.Second, the early 21 st century witnessed the widespread use of point-of-care diagnostics by doctors, nurses, first responders, and patients.The third wave, which is currently ongoing, involves patients carrying wearable devices. [241]However, challenges remain in accessing both invasive (blood, and urine), and non-invasive (saliva, sweat, tears, and breath) bio-fluids.
In modern practices, measuring the presence, concentration, and functionality of analytes in non-invasive biofluids has become increasingly important due to the rapid development of wearable devices.As a result, accessing saliva, sweat, tears, and breath has become highly significant.Saliva is primarily 99% water and is a dilute heterogenous secretion into the oral cavity. [241]he salivary glands receive analyte-rich saliva from capillaries.Due to the difficulties in collecting adequate saliva for conventional tests, there is a scarcity of sensing technologies for saliva.Moreover, sensing in saliva is vital when immediate test feedback is needed for the patient.
Sweat is generated on the skin through eccrine, and apocrine glands and consists of a single tubule. [242,243]The eccrine sweat glands serve the thermal regulation of most of the body surface and provide a good source of analyte-rich bio-fluids.In contrast, sweat from apocrine glands produces oilier and bacteriarich composition that can confound analyte measurement.As the use of sweat as a bio-fluid is very cutting-edge technology, more research is needed in this area. [244]However, an integrated sweat stimulation has been demonstrated to ensure prolonged, continuous access to sweat. [245,246]n the other hand, the use of tears and breaths as bio-fluids is a recent area of investigation.The challenges and prospects of using these bio-fluids are currently being studied.

Standardization of Measured Data by Comparing with Standard Methods like ELISA
[249] ELISA builds upon the work of Avrameas, who used enzyme-linked antibodies in histochemistry. [250,251]This method is a commonly used analytical immunochemistry assay that relies on the specific bond between an antigen and an antibody. [252]It is utilized for detecting and quantifying molecules, such as hormones, peptides, antibodies, proteins, and antigens (both native or foreign).Additionally, it helps to determine the intensity and type of immune response, as well as the innate immunity potential. [252]The test is also applicable for determining antibodies to both native and denatured DNA, [253,254] polysaccharide antigens, [255][256][257] and phospholipids. [258]As ELISA is widely accepted and used, to evaluate the applicability of neurotransmitter, protein, and glucose biosensors, the experimental data of the sensor must be compared with the standardized results of ELISA for the specific bioanalyte.

Analyzing Large Data Using Machine Learning
][261][262][263] These systems can generate a wide range of sensing data using various biofluids, which exceeds the human capacity for processing. [259]his necessitates an analytical technique that can provide easy, fast, and accurate results for disease conditions, as well as predictions of health conditions.As such, cutting-edge deep learning methods, such as artificial neural networks (ANN), can be combined with artificial intelligence (AI) via machine learning (ML).This combination can be used to reduce noise, eliminate redundancy, minimize dimensionality, and make decisions about diseases and their future directions. [259]The deep learning process fundamentally equips ANNs with the ability to mimic the human brain which is the essence of AI, through supervised, unsupervised, and reinforcement training algorithms. [265]Individual diseases can be analyzed using AI by evaluating biomarkers and symptoms. [266,267]

Support Vector Machine (SVM)
The support vector machine (SVM), a supervised ML algorithm, has been used to extract features by reducing data dimensionality for heart failure patients with reduced ejection fraction (HFrEF) in the presence of chronic kidney disease (CKD). [268]ajliwall et al (2019) used SVM to classify cardiovascular disease (CVD) data from a wearable sensor. [269]Additionally, an electronic nose, 'Cyranose 320′, was used to collect breath exhale data, which was then analyzed using SVM to differentiate lung cancer patients. [270]SVM has also been used as a predictive algorithm for the early prediction of asthma, [271] and for analyzing normalized glucose concentration data collected from saliva. [272]urthermore, SVM has been used as a feature selector predictive model for chronic hepatitis-B (HBV) [273] and to classify early liver toxicity based on gene biomarkers. [274]

Random Forest
This is a supervised ML algorithm, widely popular for classification and regression tasks.Rajliwall et al (2019) used a random forest classifier to determine the risk for CVD patients. [269]This model has also been used to extract features from large-scale clinical data for chronic HBV. [273]

Artificial Neural Network (ANN)
Combined mixed potential four-electrode electrochemical sensors have been used to detect methane in natural gas, a potential cause of CVD. [275]The ANN was used to analyze the data with 3layer structure, featuring 3-input nodes and 3-output nodes.ANN has also been used to analyze genetic data of a prostate cancerspecific DNA sequence (PCA3). [276]Furthermore, the ANN algorithm has been successfully used in identifying triple-negative breast cancer (TNBC), a complex molecule to diagnose. [277]ANN was also used to identify the stage of cancer with a remarkable accuracy of 90%. [189]Additionally, the ANN was used to identify lung cancer using data from an electronic nose. [278]Dorner et al. used ANN to classify glucose level data from saliva samples. [272]here have also been articles published on unsupervised learning.One such article used unsupervised learning to analyze dementia among Alzheimer's patients. [279]Data was processed using a non-invasive biosensor to identify heart rate variability (HRV).

Conclusions
In this review, we provide an overview of the fundamental concepts and parameters of analytical chemistry and their applications in various electrochemical sensing methods.The potentiometric technique, while useful, has drawbacks related to temperature and media dependence, the need for a matrix-dependent formal potential, and junction potential.These limitations, along with factors such as specific analyte dependency and agitation, also apply to other techniques like coulometry, stripping voltammetry, and hydrodynamic voltammetry.Redox reactions, which are key in human physiology, can be analyzed with these techniques in various health contexts, including diabetes, brain diseases, aging, and more.Among these techniques, CV and amperometry, which require a single applied potential, are popular in research.Despite drawbacks, recent developments in sensor technology keep the potentiometric technique relevant.However, some techniques like controlled potential coulometry and controlled-current coulometry have significant limitations.Voltametric techniques like CV are widely used for electrode surface characterization despite their limited voltage range.The amperometric technique emerges as the best fit for lab-in-box health monitoring system due to its simplicity and efficiency.Monitoring systems equipped with ANN capabilities can facilitate the diagnosis and classification of chronic diseases, as well as monitor disease status.

Figure 1 .
Figure 1.Pictorial view of electrochemical techniques for health monitoring.

Figure 3 .
Figure 3. a) Plants' photosynthesis process; generation of glucose and oxygen from sunlight, water, and carbon dioxide.b) Dual effects of nitrite on hemoglobin-dependent redox reactions.Reprinted with permission.[93]Copyright 2014 Elsevier Inc.

Figure 4 .
Figure 4.A family tree of several interfacial electrochemical techniques is shown.The specific techniques are in red, the experimental conditions are in blue, the analytical signals are in green, and the red-yellowish octagons show the chronological presentation of the techniques.

Figure 6 .
Figure 6.Potential-excitation signals and voltammograms for a) normal pulse, b) differential pulse, c) staircase, and d) square-wave voltammetry.The black rectangles represent the current sample position.Δi is the current difference between points 2 and 1.The symbols in the diagrams, with typical values for (a), are as follows: (≈1s) is the cycle time; ΔEp(≈2 mV and increase by ≈2 mV) is a fixed or variable pulse potential, ΔEs(≈2 mV) is a fixed change in potential per cycle, and tp(≈50 ms) is the pulse time.The current is sampled at the end of each potential pulse for approximately 17 ms before returning the potential to its initial value.Reproduced (Adapted) with permission.[136]Copyright 1999-2023, Copyright Holder David Harvey (https://sites.google.com/depauw.edu/dth/home),and Libretexts (https://LibreTexts.org).
electrode showed better results compared to the enzymatic one.Both the electrodes of Cherevko et al. 2009 and Lin et al. 2015 provided promising results in terms of liner range, LOD, and sensitivity.

Figure 7 .
Figure 7. A-G): Concentration profiles (mm) for Fc + (blue) and Fc (green) as a function of distance from the electrode (0.5 mm) at various points during the voltammogram.H): Voltammogram of the reversible reduction of a 1 mm Fc + solution to Fc at a scan rate of 100 mV s −1 .I): Applied potential as a function of time for a generic cyclic voltammetry experiment, with the initial, switching, and end potentials represented by (A, D, and G, respectively).Reproduced (Adapted) with permission.[19]Copyright 2017 The American Chemical Society (ACS).Further permissions related to the material excerpted should be directed to the ACS.J) Applied potential and voltammogram for anodic stripping voltammetry (ASV) at a mercury film electrode, with a copper ladder diagram in the upper figure of (J).Typical deposition times are 1-30 minutes, with a lower deposition time for higher analyte concentrations.Reproduced (Adapted) with permission.[136]Copyright 1999-2023, Copyright Holder David Harvey (https://sites.google.com/depauw.edu/dth/home),and Libretexts (https://LibreTexts.org).

Figure 8 .
Figure 8. a) A manual potentiometer with two electrodes (counter electrode and working electrode) is shown schematically.The tapping switch T and the slide-wire variable resistor SW are also indicated.b) A potentiometric electrochemical cell is depicted.Reproduced (Adapted) under the terms of the CC BY 4.0 license, https://creativecommons.org/licenses/by/4.0.[57]Copyright 1999-2013, Copyright Holder OpenStax, https://openstax.org/.c) A junction potential between two ionic solutions with different concentrations (0.5 m NaCl and 0.1 m NaCl) is shown.Reprinted (adapted) with permission.[214]Copyright 2018 American Chemical Society.d) The waveforms of the applied potential and resulting current of a controlled-potential coulometry are shown.e) The reduction steps of different species present in an aqueous solution of Cu 2+ are shown in a ladder diagram.The oxidized species are in blue, and the other species are in red.Reproduced (Adapted) with permission.[136]Copyright 1999-2023, Copyright Holder David Harvey (https://sites.google.com/depauw.edu/dth/home),and Libretexts (https://LibreTexts.org).f) A measured current versus time curve for a controlledcurrent coulometry is shown, with the blue area representing the total charge.

Table 1 .
DPV sensors for health monitoring applications.

Table 2 .
Square wave voltammetric (SWV) sensors for health monitoring applications.

Table 3 .
CV Sensors for Health Monitoring Applications.

Table 4 .
Amperometric sensors for health monitoring applications.

Table 5 .
Potentiometric sensors for health monitoring applications.

Table 6 .
Coulometry sensors for health monitoring applications.