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Application of NAD<sup>+</sup>-Dependent Electrochemical Dehydrogenase Biosensors in Human Physiological Fluids: Opportunities and Challenges

Application of NAD+-Dependent Electrochemical Dehydrogenase Biosensors in Human... Hindawi Journal of Analytical Methods in Chemistry Volume 2023, Article ID 3401001, 13 pages https://doi.org/10.1155/2023/3401001 Review Article Application of NAD -Dependent Electrochemical Dehydrogenase Biosensors in Human Physiological Fluids: Opportunities and Challenges 1 1 1 1 2 2 Xinrui Jin , Min Zhong , Zixin Zhu , Jingling Xie , Jinkang Feng , Yusen Liu , 1 1 1 Jinglan Guo , Baolin Li , and Jinbo Liu Department of Laboratory Medicine, Afliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, China Southwest Medical University, Luzhou, Sichuan 646000, China Correspondence should be addressed to Jinbo Liu; liulab202204@163.com Received 25 May 2022; Revised 13 October 2022; Accepted 10 January 2023; Published 11 February 2023 Academic Editor: Ricardo Jorgensen Cassella Copyright © 2023 Xinrui Jin et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Electrochemicalenzymaticbiosensorsrepresentapromising,low-costtechnologyforpoint-of-care(POC)diagnosticsthatallows fast response and simple sample processing procedures. In this review, we summarize up-to-date literature on NAD /NADH (β-nicotinamide adenine dinucleotide)-dependent electrochemical dehydrogenase biosensors and highlight their applications in human physiological fuids. A brief comparison of various enzyme immobilization procedures is frst presented, discussing preparation processes and principal analytical performance characteristics. In the following section, we briefy discuss classes of biosensors based on redox mediators-mediated electron transfer systems (METs). Finally, the conclusion section summarizes the ongoing challenges in the fabrication of NAD -dependent electrochemical dehydrogenase biosensors and gives an outlook on future research studies. electrons and protons are transferred [9]. Amperometric 1. Introduction techniques and cofactor regeneration approaches bring new Te history of sensor design and fabrication began in 1962, opportunities for the fabrication of biosensors and the de- whenClarkandLyonsfrstproposedtheconceptofenzyme- velopment of new electrochemical devices. Nevertheless, the based electrode [1]. Inspired by the seminal work of Clark existing sensing applications are limited and hampered by and Lyons, various types of enzymatic electrochemical drawbacks such as sophisticated immobilization and sta- biosensors have been developed successively for the de- bilization protocol for enzymes, selectivity and stability in tection of diverse targets (lactate, ethanol, bile acid, etc.), clinical complex samples, and the need for cofactor re- which enabled high-throughput and onsite analysis of bi- generation [10]. To expand the analytical possibilities of ological samples [2–4]. All such procedures need the ad- NAD /NADH-dependent electrochemical dehydrogenase ditionofsensingelementstotheelectrodestructurethrough biosensors,studiesareunderwaytoimproveimmobilization multiple strategies, including physical adsorption, covalent methods, miniaturization of sensor components, and im- bonding techniques, and mediators [5–7]. provement of enzyme stability [11, 12]. Diverse nano- In recent years, enzymatic electrochemical biosensors materials derived from carbon materials, metal materials, havegainedpopularityowingtothehighbiocatalyticactivity polymer composites (e.g., conducting polymers or molec- and specifcity of enzymes, along with the fnancial acces- ularly imprinted polymers), and hybrid materials (e.g., sibility to purifed enzymes [8]. β-nicotinamide adenine hydrogel) have also been attempted. + + dinucleotide (NAD /NADH) is an important coenzyme Tis article reviews the recent developments in NAD / coupleinvolvedindiverseenzymaticreactionsduringwhich NADH-dependent electrochemical dehydrogenase 2 Journal of Analytical Methods in Chemistry biosensors, which include diferent strategies for biosensor construction, various immobilization methods, analysis performance, and application of sensors in actual sample analysis. Along with this, the merits and challenges of Electroactive current NAD -dependent electrochemical dehydrogenase product biosensorsarehighlightedanddiscussed.Asfarasweknow, this is the frst review on NAD -dependent electrochemical Analyte Transducer dehydrogenase biosensors in human physiological fuids. Biological (Electrode) Element 2. Basic Principle of Electrochemical Biosensors Electrochemical biosensors pose an attractive solution for point-of-care (POC) diagnostics because they are readily integrated with microelectronics and they require minimal instrumentation. Enzyme-coupled biosensing electrodes are Transducing Electrical Signal Detector designed to immobilize biocatalysts near the electrode surfacewherethebiocatalystsinvolvedmustcatalyzespecifc Figure 1: Basic analytical scheme of an enzyme-modifed elec- electrochemical reactions. Typically, the biometric element trochemical biosensor. is attached to the surface of the transducer, which provides substrate specifcity. Te consumption or production pro- cedure is then detected by a transducer which generates shuttle electrons between the enzyme and electrode. Te a measurable signal (most often an electric current) pro- general equation of electrochemical dehydrogenase bio- portional to the concentration of analytes as shown in sensors is given as follows: Figure1.Electrochemicaldehydrogenasebiosensorsoperate in the presence of NAD /NADH, acting as a medium to +EnzymeCatalysis + (1) Substrate + NAD ⟶ Product + NADH +H . Instead, the physical adsorption of CNTs onto aromatic 3. Enzyme Immobilization Technology residues is found to be mainly hydrophobic interactions. To As powerful biocatalysts, enzymes have unique substrate test 3-hydroxybutyrate (3-HB), Khorsand et al. [14] utilized specifcity and high catalytic activity. For the preparation of single-walled carbon nanotubes (SWCNT) as a binder to electrochemical sensors, the immobilization of enzymes is attach 3-hydroxybutyrate dehydrogenase (HBDH) to the a very complicated process and has a great impact on the screen-printed carbon electrode (SPCE) surface. After the performanceof thesensors[13].Immobilization of enzymes addition of CNTs, the oxidation potential of NADH de- commonlyisperformedbyfourmethods,includingphysical creased to −0.05V. When the biosensor was used to analyze adsorption on a support material, covalent binding to 3-HBinserumsamples,thelinearitywasupto2.00mMwith a surface (that provides stronger, more stable, and irre- the detection limit of 80.00 μM and a good storage stability versible linkages compared to other methods), entrapment (180days) at 4 C. In a similar research study by Khorsand within polymers, and crosslinking between molecules. De- et al. [15], the 3-HB biosensor was established by using tailed results are presented in Table 1. SWCNT to fx the cofactor NAD on the surface of SPCE, and then HBDH was deposited on the modifed electrode. Tis biosensor presented the linear range of 0.01–0.10mM 3.1. Physical Adsorption. Physicaladsorption isasimple and and the detection limit of 9.00 μM. Te established assay fast way for attaching enzymes to the biosensor surface. Te correlated well with the standard β-hydroxybutyrate assay application of nanomaterials can enhance the performance kit available on the market. It is worth mentioning that only of biosensors in several aspects, for instance, it can increase + the frst drop of NAD would be enough, instead of the transducer stability and lifetime, improve sensitivity, and addition of NAD for each test. Lately, a simple and rapid can achieve a better time of response [31]. procedure was reported for the construction of the an- drosterone biosensor, in which the strong pi-stacking in- teraction between cofactors and CNTs provided excellent 3.1.1. Carbon Nanotubes. Amongavarietyofnanomaterials, stability [16]. Te use of the Nafon flm enabled the ac- carbon nanotubes (CNTs) with superior electrical conduc- curate detection of androsterone in the presence of inter- tivity, high surface area, and good chemical properties make ferents (uric acid and ascorbic acid). As mentioned above, them a promising material for enzyme immobilization. such composite electrodes integrate the capacity of CNTs to However, their hydrophobic aromatic structure is not facilitate electron transport with the desirable benefts of suitable for electrostatic bonding (unless functionalized). Journal of Analytical Methods in Chemistry 3 Table 1: Enzyme immobilization schemes and analytical performance of electrochemical dehydrogenase sensors in human physiological fuids. Storage Sensing Limit Linear Response Target stability Sample Immobilization Ref scheme of detection range time (days) HBDH/SWCNT/SPCE; 3-HB 0.009mM 0.10–2.00mM 40s 180 Serum Physical adsorption [14] HBDH/NAD /SWCNT/SPCE; 3-HB 0.009mM 0.01–0.10mM 40s 180 Serum Physical adsorption [15] 3a-HSD/MWCNTs/OPPF /NAD ; Androsterone 0.15 μM 0.50–10.00 μM — 6 Serum Physical adsorption [16] PheDH/PAD/rGO/SPCE; Phenylalanine 0.20 μM 1.00–600.00 μM 60s 60 Blood Physical adsorption [17] GLUD/rGO-Au /GCE; α-KG 9.20 μM 66.70–494.50 μM — — Serum Physical adsorption [13] nano NADH/LDH/Nano-CeO /GCE; Lactate 50.00 μM 0.20–2.00mM <4s 12 Blood Physical adsorption [18] 0.55–5.50 μM/ LDH/Au/EVIMC/TiNTs/PANI; Lactate 0.165 μM 8s 30 Serum Physical adsorption [19] 5.50–3330.00 μM PRODH/Fe O /MCM-41/nPrNH /GCE; L-proline 0.006 μM 0.01–0.15 μM — — Blood Physical adsorption [11] 3 4 2 LDH/NAD /pTTCA/MWNTCOOH/gold Lactate 1.00 μM 5.00–90.00 μM 10s 30 Serum Covalent bonding [20] electrode; Fe O /MWNTCOOH/LDH/NAD /GCE; Lactate 5.00 μM 50.00–500.00 μM — — Serum Covalent bonding [21] 3 4 FDH/MWNT-COOH/PBA/AuSPE; HCHO 6.00ppb 10.00 ppb–10.00ppm — 1.25 Urine Covalent bonding [12] SHL/HBDH/PCS/tefon membrane; 3-HB 3.90 μM 8.00–800.00 μM 2s 20 Serum Entrapment [22] LDH/rGO-AuNPs/SPCE; Lactate 0.13 μM 0.01–5.00mM 8s 25 Serum Entrapment [23] ADH/NAD /MDB/GMCs/CS/SPCE; Ethanol 80.00 μM 0.50–15.00mM 5s 40 Blood Entrapment [24] ADH/MADQUAT/SWCNT-rGO/GCE; Ethanol 0.16 μM 5.00–400.00 μM — — Blood Entrapment [25] URS/GLDH/N-DNW; Urea 3.87mg/dL 10.00–100.00mg/dL 10s 30 Urine Cross-linking(EDC/NHS) [26] GDH/GDI/CS/CNT/GCE; Glucose 3.00 μM 5.00–300.00 μM 5s 8 Urine Cross-linking (GA) [27] BSS/β-HSD/NHO/Nation/Pt; Bile acid — 2.00–100.00 μM 5min 28 Urine Cross-linking (GA) [28] ADH/NAD /MDB/Nation/SPCE; Ethanol 11.00 μM −5.00mM 30s 30 Serum Cross-linking (GA) [29] 2+ ADH/Ru(bpy) /rGO/BSA/GCE; Ethanol 0.10 μM 1.00–2000.00 μM — 30 Serum Cross-linking (GA) [30] 1-ethyl-3-vinylimidazolium chloride, EVIMC; glutaric dialdehyde, GDI; mobile crystalline material 41, MCM-41; n-propylamine, nPrNH ; octylpyridinium hexafuorophosphate, OPPF6; polyaniline, PANI; and titania nanotubes, TiNTs. 4 Journal of Analytical Methods in Chemistry paste electrode materials. Enzymes or other substances can inertness. Te immunity to heat can hamper proteins from bephysicallymixedbynoncovalentapproaches.Meanwhile, experiencing abundant conformational transforms inside the prepared electrodes preserve the features of traditional the pores of solid supports [11]. Hasanzadeh et al. [11] fxed carbon paste electrodes such as the feasibility of achieving proline dehydrogenase (PRODH) onto a novel carbon paste background current, easy renewal, and recombination electrode modifed with mesoporous silica nanomaterials 2 −1 properties. which have a large surface area (362m ·g ). Te catalytic activity of PRODH-entrapped magnetic mesoporous silica nanomaterials remained stable at 70 C. Te engineered 3.1.2. Reduced Graphene Oxide. Reduced graphene oxide biosensor has a linear range of 0.01–0.15 μM and a detection (rGO) with large specifc surface areas and abundant limitof0.006 μM,whichcanbeusedtomeasureL-prolinein functional groups is an ideal substrate for enzyme immo- whole blood, normal, and malignant cell lines. Te immo- bilization [32]. Moreira et al. [17] immobilized the phe- bilized PRODH exhibited greater activity over wider ranges nylalanine dehydrogenase enzyme (PheDH) onto the paper of pH values and temperatures than the free form. microzone by physical adsorption for the phenylketonuria (PKU) screening in neonatal samples (Figure 2). Te elec- trochemical oxidation was investigated by diferential pulse 3.2. Covalent Bonding. Covalent bonding ofers stronger voltammetry (DPV) at 0.6V. Te response was linear from interactions than physical adsorption because it can ofer an 1.00 μMto600 μMwiththedetectionlimitof0.20 μM.Ithas exceptionally thin, uniform, and stable surface. Chemical been reported that the fxation of metals and metal oxide conjugation via the coupling of carboxylic acid group nanoparticles on the surface of RGO prevents the aggre- (COOH), amino group (NH ), alcohol group (OH), or gation of graphene sheets and promotes ion transfer [33]. azide-alkyne cycloaddition, and sulfhydryl-maleimide cou- More recently, metal and metal oxide nanoparticles have pling are usually used to covalently attach hydrophilic been integrated on GO through a single-step synthesis in functional groups to the surface of the nanomaterial [35]. whichmetalsaltswerecoreducedsimultaneouslyalongwith Accordingly, most chemical covalent modifcations in GO [13, 33]. A selective biosensor for α-ketoglutarate electrochemical dehydrogenase biosensor studies were (α-KG) analysis was developed through the attachment of formed using an amide bond between amine-modifed ol- glutamate dehydrogenase (GLUD) onto the surface of the igonucleotides and the carboxylic acid groups of the rGO-Au composite[13].Itexhibitedalinearbehaviorin nano nanotube [12, 20, 21]. Rahman et al. [20] constructed an the66.70–494.50 μMand thedetectionlimitof 9.20 μM.Te electrochemical method for lactic acid detection, in which precision of the spiked serum (n �3) was in the range of + LDH and NAD were successively fxed on poly-5,2′-5′,2″- 3.8%–4.5%, with recoveries of 97.9%–102.4%. terthiophene-3′-carboxylic acid (pTTCA)/multiwalled car- bon nanotubes (MWCNTs) membrane, followed by the activation step of N-(3-dimethylamino-propyl)-N′- 3.1.3. Metal Oxide. Among nanostructured metal oxides, it ethylcarbondiimide hydrochloride (EDC). Te biosensor is noted that CeO nanoparticles (isoelectric point 9.2) can responsewaslinearfrom5.00 μMto90.00 μMwithalimitof immobilize biomolecules with low isoelectric points via detection of 1.00 μM. In another study, Teymourian et al. electrostatic interactions, which helps to preserve their bi- [21] used a simple coprecipitate procedure to in-situ load ological activity [34]. Nesakumar et al. [18] coated a glassy magneticFe O nanoparticlesontothesurfaceofMWCNTs. 3 4 carbon electrode (GCE) with a thin layer of carbon paste in LDH and NAD were immobilized through a similar pro- which CeO nanoparticles with a face-centered cubic cedure where the -COOH groups present on the Fe O / 3 4 structurewereembedded,andthenimmobilizedNADHand MWCNTs flm form a covalent bond with the amino group lactate dehydrogenase (LDH) at the interface. Te amper- of the enzyme. DPV detection of the biosensor to lactate ometric response to the standard concentrations of lactate displayed linear responses over the concentration range of was found to be linear from 0.20mM to 2.00mM with the −1 50.00–500.00 μM with a detection limit of 5.00 μM and sensitivity of 571.19 μA·mM and a response time of ≤4s. −1 a sensitivity of 7.67 μA·mM . In addition to this, a solid ionic lactate biosensor was + Te determination of targets in complex substrates designed to immobilize LDH-containing NAD onto employing traditional electrodes remain a major challenge a doped graphene-like membrane [19]. Te biosensor under the infuence of interferences. In view of this, Pre- showed two linear responses in the concentration range of maratne and his colleagues [12] constructed a biosensor 0.55–5.55 μM and 5.50–3330.00 μM, respectively, with employing a pyrenyl carbon nanostructure complex, with a detection limit of 0.165 μM. Te recoveries ranged from the capability of eliminating interferences. Tis system was 96.7% to 105.8%, with a relative standard deviation of conjugated with a fow injection analysis (FIA) system to (RSD) ≤3.16%,indicatingthatthebiosensorwassuitablefor determine formaldehyde (HCHO) in urine (Figure 3). For the analysis of lactate in real samples. this purpose, the gold screen printed electrodes (AuSPEs) were modifed with polymer flms via strong π-π in- 3.1.4. Inorganic Mesoporous Materials. Compared to poly- teractions between MWNTs and 1-pyrenebutyric acid mers, inorganic mesoporous materials have drawn signif- (PBA). Tereafter, the polymer-modifed AuSPEs were cant attention as support materials for molecular catalysts coated with a freshly prepared mixture of 0.35M 1-ethyl-3- owing to their excellent thermal stability as well as chemical [3-dimethylaminopropyl] carbodiimide hydrochloride Journal of Analytical Methods in Chemistry 5 NAD L-Phe PheDH Electrochemicalreduction Phenylpiruvate NADH (-1.2 V;800 s) Figure 2: Diagram of the electrochemical paper-based analytical device for Phe detection in neonatal samples (reprinted from reference [17]). (EDC) and 0.1M N-hydroxysuccinimide (NHS) followed by conditions, while enzyme embedding in appropriate polymer aliquots of formaldehyde dehydrogenase (FDH) solution. matrix lattices ofers a relatively better enzyme retention. Te constructed electrodes were connected to an internal fow cell that was concatenated to an injection pump and 3.3.1. Sol-Gel. Sol-gel materials ofer efcient means for a sample injector. Te fow injection method for the fxing enzymes via inorganic oxo (M-O-M) or hydroxo (M- designed bioelectrode signifcantly reduced the LOD to OH-M) bridges to formulate a continuous network con- 6ppb, which was 12-fold less than the agitation-solution taining liquid phases which can then be dried out to form method. Te sensor showed improved selectivity to HCHO solid matrices. with a moderate cross-reactivity for acetaldehyde Adual-enzymeClarkelectrodeforthedetectionof3-HB (CH CHO) and negligible cross-reactivity for propio- was established by specifc dehydrogenation of HBDH and naldehyde, acetone, methanol, and ethanol. Te response of salicylate hydroxylase (SHL) coated with a poly(carbamoyl) the bioelectrode to HCHO in 10-fold diluted urine was sulfonate (PCS) hydrogel on a Tefon membrane [22]. Te found to be linear from 10ppb to 10ppm with the detection operation of the biosensor was based on the specifc de- limit of 6ppb. hydrogenation of 3-HB consuming NAD catalyzed by HBDH,whichleadstotheproductionofNADHasshownin 3.3. Entrapment. Physical adsorption techniques lead to the following equation: problems with protein desorption due to changes in external + HBDH 3 − hydroxybutyrate + NAD ⟶ acetoacetate +NADH. (2) Ten,SHLcatalyzestheirreversibledecarboxylationand Employing the similar procedure, the researchers con- thehydroxylationofsalicylateinthepresenceofoxygenand structed the L-lactate detecting device by combining en- NADH as shown in the following equation: zymes with rGO-AuNPs in a sol-gel matrix derived from tetramethoxysilane and methyltrimethoxysilane [23]. Te SHL + (3) salicylate + NADH + O ⟶ catechol + NAD + CO . 2 2 determination of L-lactic acid could be almost free from the interferences of urate, paracetamol, and L-ascorbate. It Clearly, the presence of interference in human body provided a sensitivity of 154 μA/mM·cm , a linearity of fuids was found to be minimal due to the combination of 0.01–5.00mM, and a coefcient of variation of 2.5%. Te HBDH and Tefon membranes. Te total reaction time was constructedbiosensorcouldbestoredinadesiccatorat4 C·s less than 5min with the linear range of 8.00–800.00 μM and for over 25days. the detection limit of 3.90 μM. 6 Journal of Analytical Methods in Chemistry HCHO HCOOH HCOO Formaldehyde dehydrogenase NADH NAD Reference electrode - + Q + 2e + 2H QH (At electrode) Counter electrode Fabricated PBA MWCNT Working electrode (a) (b) Figure3:(a)Schematicofthemicrofuidicssystemand(b)surfacemodifcationofAuSPEsandtheprocedurefordetectingHCHObyfow injection or stirring solution amperometry (redrawn from [12]). 3.3.2. Composite Membrane. Apart from immobilizing When the applied voltage is +0.5V, the electrochemical enzymes, the membrane can also insulate the electroactive reoxidation of NADH to NAD results in the analytical substances and reduce signal interference. Nevertheless, response as expressed by the following equation: they also have some key weaknesses including leakage and + + − NADH ⟶ NAD + H + 2e . (6) conductivity. To solve these difculties, Hua et al. [24] established a novel simple and valid enzyme embedding Te principle of ethanol oxidation was catalyzed by device employing a nanobiocomposite synthesized by se- ADH, which consists of four crystallographically distinct, quentially adding graphitized mesoporous carbons but structurally similar, subunits arranged as two dimers. (GMCs), Meldola’s blue (MDB), alcohol dehydrogenase Using the device, the reduction of NADH produced by the (ADH), and NAD into chitosan (CS) solution. In this enzyme was accomplished at a relatively lower potential device, CS possessed cationic properties, good membrane (+0.5V vs. Ag/AgCl) and the limit of detection for ethanol −1 forming ability and adhesiveness, and great bio- was 0.16 μM, with the sensitivity of 1.84 μA·mM ; these compatibility,whichcannotonlybeusedasadispersantof results showed that ADH catalyzing ethanol with MAD- GMCs but also as a medium for fxing ADH. Te con- QUAT as a redox mediator was successful. Te accurate structed disposable biosensor presented a fast ampero- determination of ethanol in complex specimens is of great −1 metric response (5s), good sensitivity (67.28nA·mM ), signifcanceinclinicalandforensicmedicine.Tebiosensors widelinear range(0.50–15.00mM),and low detectionlimit for ethanol detection are based on either alcohol oxidase (80.00 μM) towards ethanol. Te recoveries ranged from (AOX) or ADH. Te ADH-based biosensor was superior to 97.2% to 106.0% and the coefcient of variation within and the AOX-based on comparisons of stability and between batches was less than 5%. specifcity [37]. Te binding of aldehyde to NAD resulted in the for- mation of NADH and aldehyde being bound to the zinc activesite.ADHusuallycatalyzesethanoloxidationthrough 3.4. Crosslinking. Typical crosslinking occurs through the the following bi-bi mechanism: [36]: application of a chemical agent called a cross-linker, most commonly a lysine linker, to covalently concatenate two E + A + B↔EA + B↔EAB↔EPQ↔EQ + P↔E + P + Q, (4) residues that are close in space within or between proteins. Te most commonly used crosslinking additives are EDC/ where E represents the enzyme, A represents NAD , B standsforethanol, Pisacetaldehyde,and Q denotesNADH. NHS [26] and glutaraldehyde (GA) [27–30] for crosslinking tissue scafolds and enhancing structural stability. Similarly, Adhikari and coworkers [25] reported a new enzyme embedding device coated with a special cationic polymer, poly(2-(dimethylamino)ethyl methacrylate) 3.4.1. EDC/NHS. An amperometric urea biosensor was (MADQUAT) on a SWCNT-rGO nanohybrid thin flm, developed by immobilizing urease (Urs) and glutamate which catalyzed the oxidation and reduction of electroactive dehydrogenase (GLDH) on a silicon substrate precoated substances. Te chemical oxidation of NAD by the ethanol with N -incorporated diamond nanowire (N-DNW) thin occurred as shown in the following equation: 2 flms which were synthesized by a microwave plasma en- + ADH + CH CH OH + NAD ⟶ CH CHO + NADH + H . (5) 3 2 3 hanced chemical vapor deposition (MPECVD) technique [26].TeEDC–NHS-initiated-COOHgroupwascovalently Journal of Analytical Methods in Chemistry 7 connected to the NH terminal of Urs and GLDH to form the surface of the working electrode (Figure 4). A suitable a covalent amide bond (CO-NH). Te biosensor exhibits mediator in the course of electrocatalytic reaction should be good performance in sensitivity (6.18 μA/mg·dL/cm ), lin- (i) long-term stability, (ii) antifouling efect, (iii) higher 0’ earity range (10.00–100.00mg/dL), lower detection limit electrontransferrateconstant(k ),(iv)formalpotential(E ) (3.87mg/dL), and fast response time (>10s). Te biosensor of the redox mediator should be less than the oxidation maintained 80.0% of its original activity towards urea after potential of NADH, and (v) reduction of considerable being stored in the refrigerator at 4−6 C for 1month. overpotential. Mediators used for electrochemical de- hydrogenase biosensors in human physiological fuids can be divided into four types as quinine groups, metals and 3.4.2. GA. A model enzyme glucose dehydrogenase (GDH) metal complexes, aromatic diamines, and organic dyes was attached to the CNT-CS membrane and used for (Table 2). detecting glucose in the urine matrix without interference [27].GDHcouldbecovalentlyfxedbythereactionofamino groups in highly biocompatible and hydrophilic CS with the 4.1. Quinine Groups. Luoetal.[38]constructedadisposable bifunctional crosslinking agent GA. Te device permitted amperometric biosensor fabricated by immobilizing ADH relatively rapid (∼60s) determination of glucose at low andNAD coatedwithNafon combinedwithAuNPsonto potentials (0.40V) without the presence of redox mediators. the surface of SPCE modifed with MDB. As an electronic Te biosensor displayed a fast response time (<5s), a wide medium, MDB could facilitate the conversion of NADH to measuring range (5.00–300.00 μM), a high sensitivity NAD atapotentialof0.0V(vs.saturatedcalomelreference −1 −2 (80 ±4mA·M ·cm ), and a low detection limit (3 μM). electrode, SCE), thereby, eliminating interferences of oxi- Koide et al. [28] reported a similar procedure for the de- dizable substances present in real blood samples. Te bio- tection of bile acids in urine by employing an electro- sensor was linear from 2.00mM to 8.00mM with chemical biosensor coated with Nafon , which both a correlation coefcient of 0.996. In addition to this, reducedthepresenceofinterferingsubstancesandenhanced a reagentless biosensor has been successfully constructed to thelong-termstabilityofthereferenceelectrode.Ten,three detect glutamate in food and clinical samples by mixing enzymes (bile acid sulfate sulfatase: BSS, 3α-hydroxysteroid unpurifed MWCNTs with CS to immobilizing GLDH and dehydrogenase: 3α-HSD, and NADH oxidase: NHO) were NAD and coating them layer by layer on the surface of fxed with GA onto the Nafon coating sensor chip. Te MDB-modifed SPCE [6]. Te glutamate content in the response time was 5min with a linearity of 2.00–100.00 μM. sample was calculated by employing the standard addition It also exhibited high reproducibility (CV values: ≤10%) and method where the currents produced by each addition were continuous repeatability of measurement (CV value: 5%– plotted and the resulting line was extrapolated. Te elec- 11%). trocatalysis occurred at very low potentials (from approxi- Furthermore, several devices for the detection of serum mately +0.1V vs. Ag/AgCl). However, analytical indexes ethanol were successfully manufactured by using a chem- suchassensitivity,linearrange,anddetectionlimitwerenot ical crosslinking method with GA as the crosslinker. Luo explained. It has also been proposed that MDB hindered the et al. [29] produced a disposable serum ethanol biosensor development of biosensor electrodes by inhibiting the that fxed ADH and NAD on the Nafon -MDB-modifed NAD -dependent enzyme. Tese mediators were covalently SPE based on the cross-linking method. When the voltage bound to important thiol groups in the enzyme. However, isappliedtotheAg/AgClreferenceelectrodeat −0.17V,the the inhibitory efect can be eliminated by employing a 1,10- response time of the biosensor is less than 30s, and the phenanthroline quinone (1,10-PQ) medium, which can linearrangeis5.00mM.Gaoetal.[30]alsoproducedanew hinder 1,4-nucleophilic addition with enzyme amino acid electrochemiluminescence (ECL) ethanol biosensor based residues [39]. Tese mediators incorporated the reactive 2+ on Ru(bpy) and ADH fxed by the rGO/bovine serum 3 quinone double bonds into heteroaromatic rings. When the albumin (BSA) composite membrane. Te ECL response of SPCE bioelectrodes incorporating 1,10-PQ, NAD , and 2+ the ADH/Ru(bpy) /rGO/BSA electrode to standard 3 HBDH were used to analyze 3-HB in blood, it exhibited concentrations of ethanol was found to be linear in the a linear response range of 0.00mM–6.00mM, using a small rangeof1.00–2000.00 μMwithadetectionlimitof0.10 μM. volume of blood (5 μl) and it was stable for up to 18months Te ECL strength maintained about 90% of the original ° at30 C,andshortenedthetotalassaytimeto30s.Inanother value after being kept in the refrigerator at 4 C for research, Wang et al. [40] constructed a “pop-up” electro- one month. chemical paper-basedanalyticaldevice(pop-up-EPAD)that allowedanenzymaticassayfor3-HBinbloodtobereadwith a commercial glucometer. NAD and HBDH reagents were 4. Mediated Electron Transfer stored on the devices. Te sample and 1,10-PD were added To help to overcome the limitations of accessibility and to the reaction area on the top layer of the paper device and proximity and to reduce the susceptibility to interfering thenenteredintothedetectionarea.Oncethesamplepassed species by lowering electrode potentials, redox mediators throughtheelectrodes,theglucosemeterbegantestingatthe have been used in electrochemical dehydrogenase bio- potential of +0.2V and displayed the fnal values. Te pop- sensors. Te medium is attached to the redox enzyme and up-EPADs exhibited the linear range of 0.10–6.00mM and mediateselectrontransferfromtheenzyme’sredoxcenterto the detection limit of 0.30mM. 8 Journal of Analytical Methods in Chemistry Figure 4: Electrochemical reactions involved in NAD /NADH-dependent electrochemical dehydrogenases biosensors operating with mediator. 4.2. Metal and Metal Complexes. Liao et al. [41] built an utilized it for triglyceride (TG) in human serum. Te cat- amperometric biosensor by employing an iridium nano- alytic and electrochemical procedures of TG can be de- particle as a redox mediator doped into a carbon paste and scribed by the following reactions: lipase + + TG ⟶ fatty acid + glycerol + NAD Glycerol  dehydrogenase  dihydroxyacetone + NADH +H , (7) 4+ + 3+ + NADH + 2Ir ↔NAD +2Ir +H . (8) 3+ −1 Followed by the electroregeneration of the Ir at an L) . At storage conditions of around 23 C, the design in- applied potential of +0.15V vs. Ag/AgCl which is expressed creased shelf-life to more than 60days. as Moreover, Zhang et al. [43] developed a 3α-HSD-based indirect electrochemical method to accurately determinate 3+ 4+ − 2Ir ↔2Ir + 2e . (9) bile acids concentrations in serum with unmodifed SPCE. Te amperometric response of the optimized bile acids Te linearity was found to be in the range of −1 detectoronexposuretostandardconcentrationsofbileacids 0.00–10.00mM with a sensitivity of 7.50nA·mM . Te was linear from 5.00 μM to 400.00 μM. Known concentra- method for the detection of TG can overcome the in- tions of bile acids were spiked into serum samples and the terference of UA and AA. + recovery values ranged from 75.10% to 113.1%. Using the Moreover, Li et al. [42] fxed HBDH, NAD , and the similar biosensor design, Tian et al. [4] utilized the electron electron mediator of potassium ferricyanide (K [Fe(CN) ]) 3 6 mediator of tris(2,2′-bipyridine)ruthenium(III) (Ru(bpy) onto the SPCE precoated with a layer of hydrophilic gel 3+ ) to react with NADH, thereby indirectly detecting bile sodium carboxymethyl cellulose (CMC). A comprehensive + 3− acids. It exhibited a linear behavior from 5.00pmol/L to evaluation of the immobilized HBDH/NAD /Fe(CN) / 150.00pmol/L with a detection limit of 0.40pmol/L (based CMC/SPCE system showed that the procedure was suitable on 3× the baseline noise) in a 10 -fold dilution serum. Te for thedetection of 3-HB in whole blood or serum with very 3+ use of Ru(bpy) greatly enhanced the electrical conduc- small sample volumes (2.0 μL) and a response time of 50s. tivity and the sensitivity of the device (Figure 5). Te amperometric response was found to be linear from 1.50mg/L to 500.00mg/L with a sensitivity of 0.011 μA·(mg/ Journal of Analytical Methods in Chemistry 9 Table 2: Electrochemical dehydrogenase sensors comparison: detection mechanism and performance. Storage Sensing Limit Linear Response Electron Target stability Sample Ref scheme/target of detection range time mediators (days) ADH/NAD /Nafon/AuNP/MDB/SPCE; Ethanol 16 μM 2.00–8.00mM 40s 30 Serum MDB [38] GLDH/NAD /MWCNTs/CS/MDB/ Glutamate — — — — Serum MDB [6] SPCE; 1,10-PQ/NAD /HBDH/SPCE; 3-HB — 0.00–6.00mM <30s 540 Blood 1,10-PQ [39] 1,10-PD/NAD /HBDH/EPAD; 3-HB 0.30mM 0.10–6.00mM — — Blood 1,10-PQ [40] 4+ 4+ Ir /SPCE; TG — 0.00–10.00mM — — Serum Ir [41] + 3− 3− HBDH/NAD /Fe(CN) /CMC/SPCE; 3-HB — 1.50–550.00mg/L 50s 60 Serum Fe(CN) [42] 6 6 SPCE; Bile acid — 5.0–400.0 μM — Serum — [43] Ru(bpy) SPCE; Bile acid 0.40pM 5.00–150.00pmol/L — — Serum [4] 2+ SH/Tyrosinase/PheDH/CPE; L-Phenylalanine 5.00 μM 20.00–150.00 μM 45–60s 11 Serum and blood Catechol [44] rGO/PhNHOH/GCE; Lactate 2.50 μM −90.00 μM 23s — Serum PhNHOH [45] HBDH/THI/rGO/SPCE; 3-HB 1.00 μM 0.010–0.40mM 7 s 20 Serum THI [46] 50.00–400.00mg/ LIP/GDH/TB/ERGO/ITO; TG 0.18mM 12s 112 Serum TB [47] dL 10 Journal of Analytical Methods in Chemistry NAD 3α-HSD 2+ Ru (bpy) t (s) t (s) PBS solution Diluted serum R R COCR COCR 3 3α-HSD R R 1 + NAD NADH HO R R 3+ + 2+ NADH + Ru (bpy) NAD + Ru (bpy) 3 3 2+ 3+ Ru (bpy) - e Ru (bpy) 3 3 Figure 5: Principle of enzymatic coupling double oxidization electrochemical detection of bile acids (reprinted from [4]). 4.3. Aromatic Diamines. An enzyme carbon paste electrode L-phenylalaninedetection[44].WiththecatalysisofPADH, (CPE) based on tyrosinase, salicylate hydroxylase (SH), and L-phenylalanine acids reacted specifcally with NAD to L-phenylalaninedehydrogenase(PADH)wasestablishedfor produce NADH as shown in the following equation: L − Phenylalanine +NAD PADHPhenylpyruvate +NADH. (10) Ten, the irreversible decarboxylation and the hydrox- couple (-NHOH/-NO) covalently functionalized with rGO. ylation of salicylate catalyzed by SH were described by the Te sensor can determinate lactate as low as 2.5 μM without −1 −2 following equation: interference, with a sensitivity of 10.57 ±0.38nA·μM ·cm , a linear range up to 90.00 μM, and a response time of 23s. salicylate + NADH + 2H + O SH catechol (11) +NAD +H O + CO . 2 2 ´ ´ 4.4. Organic Dyes. Martınez-Garcıa et al. [46] established an Te oxidation of catechol to o-quinone by tyrosinase enzymatic electrochemical device for the detection of 3-HB could be described by equation (12). Also, the reaction in serum by fxing HBDH onto a rGO and thionine (THI)- current was measured by the chronoamperometry assay at modifed SPCE. Te THI’s redox mediator combined with E � −50mV vs. Ag/AgCl. appl an rGO-modifed electrode could reduce the detection potential to 0.0V. Te optimized device showed a linear catechol +2H + O tyrosinase o − quinone + 2H O. (12) 2 2 response range of 0.010–0.40mM, a limit of detection of Tis relies on the circulation of catechol and o-quinone 1.00 μM, and a response time of 7s. betweentheelectrodeandthetyrosinase,therebyamplifying Furthermore,theTGdetectiondevicewasestablishedby the electrocatalytic signal. Te reaction time for L- fxing lipase (LIP) and GDH on the indium-tin-oxide (ITO) phenylalanine was <60s with a linearity range from glasselectrodecoatedwithanelectrochemicallyreducedGO 20.00 μM to 150.00 μM. Te biosensor also lost >50% of its (ERGO) membrane doped by toluidine blue (TB) [47]. original response to galactose within 11days. Glycerol was oxidized in the presence of GDH, and TB then In addition, Manna and Retna Raj [45] described an extracted the H ions released from the glycerol, leaving the electrochemical device for the detection of lactic acid in hydrogen to ERGO for performing the subsequent redox humanserumemployingrGO-PhNO compositescovalently reaction. At a potential of +0.34V, ERGO was reoxi- 2 (Red) functionalized with a p-nitrophenyl moiety of p-nitroaniline. dized to ERGO , resulting in an electrochemical re- (Oxid) Te rGO-PhNHOH was formed by the potential cycling of sponse. With a rapid response within 12seconds, the device the rGO-PhNO -modifed GC electrode. Te current signal is linear in the range of 50.00mg/dl to 400.00mg/dl and has −1 was detected by the oxidation of NADH mediated by a redox a sensitivity of 29Pa·mg ·dl. I (μA) I (μA) Journal of Analytical Methods in Chemistry 11 density. Te immobilization procedure of redox-active en- 5. Challenges and Critical Issues toward zymeontheelectrodehasagreatinfuenceontheindexesof Clinical Applications the detection device. It is therefore a key issue, especially if Although considerable resources have been invested in large-scale applications are envisaged, to ensure that the attenuation of the catalytic activity of the immobilized building electrochemical biosensing devices based on de- hydrogenase over the past two decades, there has been little biocatalystisasreducedaspossible.Anotherdirectionbeing explored is the use of mediators such as metal and metal apparent success in the productization of these devices for use in human physiological fuids such as serum and urine. complexes, aromatic diamines, and organic dyes as the electron shuttles between the enzyme and the transducer. Tecriticalproblemofsuchdevicesistheefectiveexchange of electrons between the active center and the supporting Biosensors, which use electron mediators, often show high composite or electrode. Direct electron transfer (DET) current densities and a good sensitivity. However, they are through the bare electrode is extremely hard because the usually less stable due to the immobilization of the medium redox-active sites of NAD /NADH-dependent de- and the mediators. Tus, there still are challenges yet to be hydrogenases are deeply embedded in the well-electrically overcome to achieve better sensitivity, selectivity, stability, insulated shells [13]. Te most common is that distances and lifetime, before NAD -dependent electrochemical de- greaterthan20apreventDETbetweentheactivesiteandthe hydrogenase can be used in practical applications. electrode.Tisproblemisoftenaddressedthroughtheuseof mediators,whichcanreachtheactivesiteoftheenzymeand Conflicts of Interest act as electron shuttles by mediating the exchange of elec- Te authors declare that they have no conficts of interest. trons between the enzyme and the electrode. Due to its excellent electrical conductivity and high sensitivity, METis proven to be promising in the construction of various Authors’ Contributions biosensors and biofuel cells. It is also important to address Xinrui Jin, Jinglan Guo, Baolin Li, and Jinbo Liu con- the issues of mediator-based biosensors, such as leakage of ceptualised and designed the study. Zixin Zhu, Yusen Liu, mediators, thehigh costof complexes, or theharmfulness of and Min Zhong constructed fgures and tables. Xinrui Jin, shuttling molecules must be considered in future studies. Jingling Xie, and Jinkang Feng drafted the manuscript. All Other challenges associated with electrochemical de- authors contributed to writing the fnal drafts of the hydrogenase biosensors are that NADH biosensing on bare manuscript. electrodes typically leads to the accumulation of the oxi- dation products on the electrode surface which causes high overpotential and unstable detection signals [48]. Te sur- Acknowledgments face characteristics of the sensor and its selectivity to targets Te work was supported by the grants from Southwest must be improved, otherwise many components in bi- Medical University (Grant nos. 2021ZKQN063, 2020500, ologicalfuidssuchasbloodandurinesamplescouldquickly and S202210632152) and the Science and Technology De- contaminate the electrode. Furthermore, the bulk of the partment of Sichuan Province (Grant nos. 2021YFS0171and research efort has been on small initial sample sizes, por- 2021YFH001). table devices, greater sensitivity or selectivity, and wide linear ranges with low detection limits. In contrast, the number of publications dealing with thermal stability, References storage stability, shelf life, and the reuse of electrochemical [1] L. C. Clark and C. Lyons, “Electrode systems for continuous dehydrogenase biosensors is very small. 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Application of NAD<sup>+</sup>-Dependent Electrochemical Dehydrogenase Biosensors in Human Physiological Fluids: Opportunities and Challenges

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2090-8865
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10.1155/2023/3401001
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Abstract

Hindawi Journal of Analytical Methods in Chemistry Volume 2023, Article ID 3401001, 13 pages https://doi.org/10.1155/2023/3401001 Review Article Application of NAD -Dependent Electrochemical Dehydrogenase Biosensors in Human Physiological Fluids: Opportunities and Challenges 1 1 1 1 2 2 Xinrui Jin , Min Zhong , Zixin Zhu , Jingling Xie , Jinkang Feng , Yusen Liu , 1 1 1 Jinglan Guo , Baolin Li , and Jinbo Liu Department of Laboratory Medicine, Afliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, China Southwest Medical University, Luzhou, Sichuan 646000, China Correspondence should be addressed to Jinbo Liu; liulab202204@163.com Received 25 May 2022; Revised 13 October 2022; Accepted 10 January 2023; Published 11 February 2023 Academic Editor: Ricardo Jorgensen Cassella Copyright © 2023 Xinrui Jin et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Electrochemicalenzymaticbiosensorsrepresentapromising,low-costtechnologyforpoint-of-care(POC)diagnosticsthatallows fast response and simple sample processing procedures. In this review, we summarize up-to-date literature on NAD /NADH (β-nicotinamide adenine dinucleotide)-dependent electrochemical dehydrogenase biosensors and highlight their applications in human physiological fuids. A brief comparison of various enzyme immobilization procedures is frst presented, discussing preparation processes and principal analytical performance characteristics. In the following section, we briefy discuss classes of biosensors based on redox mediators-mediated electron transfer systems (METs). Finally, the conclusion section summarizes the ongoing challenges in the fabrication of NAD -dependent electrochemical dehydrogenase biosensors and gives an outlook on future research studies. electrons and protons are transferred [9]. Amperometric 1. Introduction techniques and cofactor regeneration approaches bring new Te history of sensor design and fabrication began in 1962, opportunities for the fabrication of biosensors and the de- whenClarkandLyonsfrstproposedtheconceptofenzyme- velopment of new electrochemical devices. Nevertheless, the based electrode [1]. Inspired by the seminal work of Clark existing sensing applications are limited and hampered by and Lyons, various types of enzymatic electrochemical drawbacks such as sophisticated immobilization and sta- biosensors have been developed successively for the de- bilization protocol for enzymes, selectivity and stability in tection of diverse targets (lactate, ethanol, bile acid, etc.), clinical complex samples, and the need for cofactor re- which enabled high-throughput and onsite analysis of bi- generation [10]. To expand the analytical possibilities of ological samples [2–4]. All such procedures need the ad- NAD /NADH-dependent electrochemical dehydrogenase ditionofsensingelementstotheelectrodestructurethrough biosensors,studiesareunderwaytoimproveimmobilization multiple strategies, including physical adsorption, covalent methods, miniaturization of sensor components, and im- bonding techniques, and mediators [5–7]. provement of enzyme stability [11, 12]. Diverse nano- In recent years, enzymatic electrochemical biosensors materials derived from carbon materials, metal materials, havegainedpopularityowingtothehighbiocatalyticactivity polymer composites (e.g., conducting polymers or molec- and specifcity of enzymes, along with the fnancial acces- ularly imprinted polymers), and hybrid materials (e.g., sibility to purifed enzymes [8]. β-nicotinamide adenine hydrogel) have also been attempted. + + dinucleotide (NAD /NADH) is an important coenzyme Tis article reviews the recent developments in NAD / coupleinvolvedindiverseenzymaticreactionsduringwhich NADH-dependent electrochemical dehydrogenase 2 Journal of Analytical Methods in Chemistry biosensors, which include diferent strategies for biosensor construction, various immobilization methods, analysis performance, and application of sensors in actual sample analysis. Along with this, the merits and challenges of Electroactive current NAD -dependent electrochemical dehydrogenase product biosensorsarehighlightedanddiscussed.Asfarasweknow, this is the frst review on NAD -dependent electrochemical Analyte Transducer dehydrogenase biosensors in human physiological fuids. Biological (Electrode) Element 2. Basic Principle of Electrochemical Biosensors Electrochemical biosensors pose an attractive solution for point-of-care (POC) diagnostics because they are readily integrated with microelectronics and they require minimal instrumentation. Enzyme-coupled biosensing electrodes are Transducing Electrical Signal Detector designed to immobilize biocatalysts near the electrode surfacewherethebiocatalystsinvolvedmustcatalyzespecifc Figure 1: Basic analytical scheme of an enzyme-modifed elec- electrochemical reactions. Typically, the biometric element trochemical biosensor. is attached to the surface of the transducer, which provides substrate specifcity. Te consumption or production pro- cedure is then detected by a transducer which generates shuttle electrons between the enzyme and electrode. Te a measurable signal (most often an electric current) pro- general equation of electrochemical dehydrogenase bio- portional to the concentration of analytes as shown in sensors is given as follows: Figure1.Electrochemicaldehydrogenasebiosensorsoperate in the presence of NAD /NADH, acting as a medium to +EnzymeCatalysis + (1) Substrate + NAD ⟶ Product + NADH +H . Instead, the physical adsorption of CNTs onto aromatic 3. Enzyme Immobilization Technology residues is found to be mainly hydrophobic interactions. To As powerful biocatalysts, enzymes have unique substrate test 3-hydroxybutyrate (3-HB), Khorsand et al. [14] utilized specifcity and high catalytic activity. For the preparation of single-walled carbon nanotubes (SWCNT) as a binder to electrochemical sensors, the immobilization of enzymes is attach 3-hydroxybutyrate dehydrogenase (HBDH) to the a very complicated process and has a great impact on the screen-printed carbon electrode (SPCE) surface. After the performanceof thesensors[13].Immobilization of enzymes addition of CNTs, the oxidation potential of NADH de- commonlyisperformedbyfourmethods,includingphysical creased to −0.05V. When the biosensor was used to analyze adsorption on a support material, covalent binding to 3-HBinserumsamples,thelinearitywasupto2.00mMwith a surface (that provides stronger, more stable, and irre- the detection limit of 80.00 μM and a good storage stability versible linkages compared to other methods), entrapment (180days) at 4 C. In a similar research study by Khorsand within polymers, and crosslinking between molecules. De- et al. [15], the 3-HB biosensor was established by using tailed results are presented in Table 1. SWCNT to fx the cofactor NAD on the surface of SPCE, and then HBDH was deposited on the modifed electrode. Tis biosensor presented the linear range of 0.01–0.10mM 3.1. Physical Adsorption. Physicaladsorption isasimple and and the detection limit of 9.00 μM. Te established assay fast way for attaching enzymes to the biosensor surface. Te correlated well with the standard β-hydroxybutyrate assay application of nanomaterials can enhance the performance kit available on the market. It is worth mentioning that only of biosensors in several aspects, for instance, it can increase + the frst drop of NAD would be enough, instead of the transducer stability and lifetime, improve sensitivity, and addition of NAD for each test. Lately, a simple and rapid can achieve a better time of response [31]. procedure was reported for the construction of the an- drosterone biosensor, in which the strong pi-stacking in- teraction between cofactors and CNTs provided excellent 3.1.1. Carbon Nanotubes. Amongavarietyofnanomaterials, stability [16]. Te use of the Nafon flm enabled the ac- carbon nanotubes (CNTs) with superior electrical conduc- curate detection of androsterone in the presence of inter- tivity, high surface area, and good chemical properties make ferents (uric acid and ascorbic acid). As mentioned above, them a promising material for enzyme immobilization. such composite electrodes integrate the capacity of CNTs to However, their hydrophobic aromatic structure is not facilitate electron transport with the desirable benefts of suitable for electrostatic bonding (unless functionalized). Journal of Analytical Methods in Chemistry 3 Table 1: Enzyme immobilization schemes and analytical performance of electrochemical dehydrogenase sensors in human physiological fuids. Storage Sensing Limit Linear Response Target stability Sample Immobilization Ref scheme of detection range time (days) HBDH/SWCNT/SPCE; 3-HB 0.009mM 0.10–2.00mM 40s 180 Serum Physical adsorption [14] HBDH/NAD /SWCNT/SPCE; 3-HB 0.009mM 0.01–0.10mM 40s 180 Serum Physical adsorption [15] 3a-HSD/MWCNTs/OPPF /NAD ; Androsterone 0.15 μM 0.50–10.00 μM — 6 Serum Physical adsorption [16] PheDH/PAD/rGO/SPCE; Phenylalanine 0.20 μM 1.00–600.00 μM 60s 60 Blood Physical adsorption [17] GLUD/rGO-Au /GCE; α-KG 9.20 μM 66.70–494.50 μM — — Serum Physical adsorption [13] nano NADH/LDH/Nano-CeO /GCE; Lactate 50.00 μM 0.20–2.00mM <4s 12 Blood Physical adsorption [18] 0.55–5.50 μM/ LDH/Au/EVIMC/TiNTs/PANI; Lactate 0.165 μM 8s 30 Serum Physical adsorption [19] 5.50–3330.00 μM PRODH/Fe O /MCM-41/nPrNH /GCE; L-proline 0.006 μM 0.01–0.15 μM — — Blood Physical adsorption [11] 3 4 2 LDH/NAD /pTTCA/MWNTCOOH/gold Lactate 1.00 μM 5.00–90.00 μM 10s 30 Serum Covalent bonding [20] electrode; Fe O /MWNTCOOH/LDH/NAD /GCE; Lactate 5.00 μM 50.00–500.00 μM — — Serum Covalent bonding [21] 3 4 FDH/MWNT-COOH/PBA/AuSPE; HCHO 6.00ppb 10.00 ppb–10.00ppm — 1.25 Urine Covalent bonding [12] SHL/HBDH/PCS/tefon membrane; 3-HB 3.90 μM 8.00–800.00 μM 2s 20 Serum Entrapment [22] LDH/rGO-AuNPs/SPCE; Lactate 0.13 μM 0.01–5.00mM 8s 25 Serum Entrapment [23] ADH/NAD /MDB/GMCs/CS/SPCE; Ethanol 80.00 μM 0.50–15.00mM 5s 40 Blood Entrapment [24] ADH/MADQUAT/SWCNT-rGO/GCE; Ethanol 0.16 μM 5.00–400.00 μM — — Blood Entrapment [25] URS/GLDH/N-DNW; Urea 3.87mg/dL 10.00–100.00mg/dL 10s 30 Urine Cross-linking(EDC/NHS) [26] GDH/GDI/CS/CNT/GCE; Glucose 3.00 μM 5.00–300.00 μM 5s 8 Urine Cross-linking (GA) [27] BSS/β-HSD/NHO/Nation/Pt; Bile acid — 2.00–100.00 μM 5min 28 Urine Cross-linking (GA) [28] ADH/NAD /MDB/Nation/SPCE; Ethanol 11.00 μM −5.00mM 30s 30 Serum Cross-linking (GA) [29] 2+ ADH/Ru(bpy) /rGO/BSA/GCE; Ethanol 0.10 μM 1.00–2000.00 μM — 30 Serum Cross-linking (GA) [30] 1-ethyl-3-vinylimidazolium chloride, EVIMC; glutaric dialdehyde, GDI; mobile crystalline material 41, MCM-41; n-propylamine, nPrNH ; octylpyridinium hexafuorophosphate, OPPF6; polyaniline, PANI; and titania nanotubes, TiNTs. 4 Journal of Analytical Methods in Chemistry paste electrode materials. Enzymes or other substances can inertness. Te immunity to heat can hamper proteins from bephysicallymixedbynoncovalentapproaches.Meanwhile, experiencing abundant conformational transforms inside the prepared electrodes preserve the features of traditional the pores of solid supports [11]. Hasanzadeh et al. [11] fxed carbon paste electrodes such as the feasibility of achieving proline dehydrogenase (PRODH) onto a novel carbon paste background current, easy renewal, and recombination electrode modifed with mesoporous silica nanomaterials 2 −1 properties. which have a large surface area (362m ·g ). Te catalytic activity of PRODH-entrapped magnetic mesoporous silica nanomaterials remained stable at 70 C. Te engineered 3.1.2. Reduced Graphene Oxide. Reduced graphene oxide biosensor has a linear range of 0.01–0.15 μM and a detection (rGO) with large specifc surface areas and abundant limitof0.006 μM,whichcanbeusedtomeasureL-prolinein functional groups is an ideal substrate for enzyme immo- whole blood, normal, and malignant cell lines. Te immo- bilization [32]. Moreira et al. [17] immobilized the phe- bilized PRODH exhibited greater activity over wider ranges nylalanine dehydrogenase enzyme (PheDH) onto the paper of pH values and temperatures than the free form. microzone by physical adsorption for the phenylketonuria (PKU) screening in neonatal samples (Figure 2). Te elec- trochemical oxidation was investigated by diferential pulse 3.2. Covalent Bonding. Covalent bonding ofers stronger voltammetry (DPV) at 0.6V. Te response was linear from interactions than physical adsorption because it can ofer an 1.00 μMto600 μMwiththedetectionlimitof0.20 μM.Ithas exceptionally thin, uniform, and stable surface. Chemical been reported that the fxation of metals and metal oxide conjugation via the coupling of carboxylic acid group nanoparticles on the surface of RGO prevents the aggre- (COOH), amino group (NH ), alcohol group (OH), or gation of graphene sheets and promotes ion transfer [33]. azide-alkyne cycloaddition, and sulfhydryl-maleimide cou- More recently, metal and metal oxide nanoparticles have pling are usually used to covalently attach hydrophilic been integrated on GO through a single-step synthesis in functional groups to the surface of the nanomaterial [35]. whichmetalsaltswerecoreducedsimultaneouslyalongwith Accordingly, most chemical covalent modifcations in GO [13, 33]. A selective biosensor for α-ketoglutarate electrochemical dehydrogenase biosensor studies were (α-KG) analysis was developed through the attachment of formed using an amide bond between amine-modifed ol- glutamate dehydrogenase (GLUD) onto the surface of the igonucleotides and the carboxylic acid groups of the rGO-Au composite[13].Itexhibitedalinearbehaviorin nano nanotube [12, 20, 21]. Rahman et al. [20] constructed an the66.70–494.50 μMand thedetectionlimitof 9.20 μM.Te electrochemical method for lactic acid detection, in which precision of the spiked serum (n �3) was in the range of + LDH and NAD were successively fxed on poly-5,2′-5′,2″- 3.8%–4.5%, with recoveries of 97.9%–102.4%. terthiophene-3′-carboxylic acid (pTTCA)/multiwalled car- bon nanotubes (MWCNTs) membrane, followed by the activation step of N-(3-dimethylamino-propyl)-N′- 3.1.3. Metal Oxide. Among nanostructured metal oxides, it ethylcarbondiimide hydrochloride (EDC). Te biosensor is noted that CeO nanoparticles (isoelectric point 9.2) can responsewaslinearfrom5.00 μMto90.00 μMwithalimitof immobilize biomolecules with low isoelectric points via detection of 1.00 μM. In another study, Teymourian et al. electrostatic interactions, which helps to preserve their bi- [21] used a simple coprecipitate procedure to in-situ load ological activity [34]. Nesakumar et al. [18] coated a glassy magneticFe O nanoparticlesontothesurfaceofMWCNTs. 3 4 carbon electrode (GCE) with a thin layer of carbon paste in LDH and NAD were immobilized through a similar pro- which CeO nanoparticles with a face-centered cubic cedure where the -COOH groups present on the Fe O / 3 4 structurewereembedded,andthenimmobilizedNADHand MWCNTs flm form a covalent bond with the amino group lactate dehydrogenase (LDH) at the interface. Te amper- of the enzyme. DPV detection of the biosensor to lactate ometric response to the standard concentrations of lactate displayed linear responses over the concentration range of was found to be linear from 0.20mM to 2.00mM with the −1 50.00–500.00 μM with a detection limit of 5.00 μM and sensitivity of 571.19 μA·mM and a response time of ≤4s. −1 a sensitivity of 7.67 μA·mM . In addition to this, a solid ionic lactate biosensor was + Te determination of targets in complex substrates designed to immobilize LDH-containing NAD onto employing traditional electrodes remain a major challenge a doped graphene-like membrane [19]. Te biosensor under the infuence of interferences. In view of this, Pre- showed two linear responses in the concentration range of maratne and his colleagues [12] constructed a biosensor 0.55–5.55 μM and 5.50–3330.00 μM, respectively, with employing a pyrenyl carbon nanostructure complex, with a detection limit of 0.165 μM. Te recoveries ranged from the capability of eliminating interferences. Tis system was 96.7% to 105.8%, with a relative standard deviation of conjugated with a fow injection analysis (FIA) system to (RSD) ≤3.16%,indicatingthatthebiosensorwassuitablefor determine formaldehyde (HCHO) in urine (Figure 3). For the analysis of lactate in real samples. this purpose, the gold screen printed electrodes (AuSPEs) were modifed with polymer flms via strong π-π in- 3.1.4. Inorganic Mesoporous Materials. Compared to poly- teractions between MWNTs and 1-pyrenebutyric acid mers, inorganic mesoporous materials have drawn signif- (PBA). Tereafter, the polymer-modifed AuSPEs were cant attention as support materials for molecular catalysts coated with a freshly prepared mixture of 0.35M 1-ethyl-3- owing to their excellent thermal stability as well as chemical [3-dimethylaminopropyl] carbodiimide hydrochloride Journal of Analytical Methods in Chemistry 5 NAD L-Phe PheDH Electrochemicalreduction Phenylpiruvate NADH (-1.2 V;800 s) Figure 2: Diagram of the electrochemical paper-based analytical device for Phe detection in neonatal samples (reprinted from reference [17]). (EDC) and 0.1M N-hydroxysuccinimide (NHS) followed by conditions, while enzyme embedding in appropriate polymer aliquots of formaldehyde dehydrogenase (FDH) solution. matrix lattices ofers a relatively better enzyme retention. Te constructed electrodes were connected to an internal fow cell that was concatenated to an injection pump and 3.3.1. Sol-Gel. Sol-gel materials ofer efcient means for a sample injector. Te fow injection method for the fxing enzymes via inorganic oxo (M-O-M) or hydroxo (M- designed bioelectrode signifcantly reduced the LOD to OH-M) bridges to formulate a continuous network con- 6ppb, which was 12-fold less than the agitation-solution taining liquid phases which can then be dried out to form method. Te sensor showed improved selectivity to HCHO solid matrices. with a moderate cross-reactivity for acetaldehyde Adual-enzymeClarkelectrodeforthedetectionof3-HB (CH CHO) and negligible cross-reactivity for propio- was established by specifc dehydrogenation of HBDH and naldehyde, acetone, methanol, and ethanol. Te response of salicylate hydroxylase (SHL) coated with a poly(carbamoyl) the bioelectrode to HCHO in 10-fold diluted urine was sulfonate (PCS) hydrogel on a Tefon membrane [22]. Te found to be linear from 10ppb to 10ppm with the detection operation of the biosensor was based on the specifc de- limit of 6ppb. hydrogenation of 3-HB consuming NAD catalyzed by HBDH,whichleadstotheproductionofNADHasshownin 3.3. Entrapment. Physical adsorption techniques lead to the following equation: problems with protein desorption due to changes in external + HBDH 3 − hydroxybutyrate + NAD ⟶ acetoacetate +NADH. (2) Ten,SHLcatalyzestheirreversibledecarboxylationand Employing the similar procedure, the researchers con- thehydroxylationofsalicylateinthepresenceofoxygenand structed the L-lactate detecting device by combining en- NADH as shown in the following equation: zymes with rGO-AuNPs in a sol-gel matrix derived from tetramethoxysilane and methyltrimethoxysilane [23]. Te SHL + (3) salicylate + NADH + O ⟶ catechol + NAD + CO . 2 2 determination of L-lactic acid could be almost free from the interferences of urate, paracetamol, and L-ascorbate. It Clearly, the presence of interference in human body provided a sensitivity of 154 μA/mM·cm , a linearity of fuids was found to be minimal due to the combination of 0.01–5.00mM, and a coefcient of variation of 2.5%. Te HBDH and Tefon membranes. Te total reaction time was constructedbiosensorcouldbestoredinadesiccatorat4 C·s less than 5min with the linear range of 8.00–800.00 μM and for over 25days. the detection limit of 3.90 μM. 6 Journal of Analytical Methods in Chemistry HCHO HCOOH HCOO Formaldehyde dehydrogenase NADH NAD Reference electrode - + Q + 2e + 2H QH (At electrode) Counter electrode Fabricated PBA MWCNT Working electrode (a) (b) Figure3:(a)Schematicofthemicrofuidicssystemand(b)surfacemodifcationofAuSPEsandtheprocedurefordetectingHCHObyfow injection or stirring solution amperometry (redrawn from [12]). 3.3.2. Composite Membrane. Apart from immobilizing When the applied voltage is +0.5V, the electrochemical enzymes, the membrane can also insulate the electroactive reoxidation of NADH to NAD results in the analytical substances and reduce signal interference. Nevertheless, response as expressed by the following equation: they also have some key weaknesses including leakage and + + − NADH ⟶ NAD + H + 2e . (6) conductivity. To solve these difculties, Hua et al. [24] established a novel simple and valid enzyme embedding Te principle of ethanol oxidation was catalyzed by device employing a nanobiocomposite synthesized by se- ADH, which consists of four crystallographically distinct, quentially adding graphitized mesoporous carbons but structurally similar, subunits arranged as two dimers. (GMCs), Meldola’s blue (MDB), alcohol dehydrogenase Using the device, the reduction of NADH produced by the (ADH), and NAD into chitosan (CS) solution. In this enzyme was accomplished at a relatively lower potential device, CS possessed cationic properties, good membrane (+0.5V vs. Ag/AgCl) and the limit of detection for ethanol −1 forming ability and adhesiveness, and great bio- was 0.16 μM, with the sensitivity of 1.84 μA·mM ; these compatibility,whichcannotonlybeusedasadispersantof results showed that ADH catalyzing ethanol with MAD- GMCs but also as a medium for fxing ADH. Te con- QUAT as a redox mediator was successful. Te accurate structed disposable biosensor presented a fast ampero- determination of ethanol in complex specimens is of great −1 metric response (5s), good sensitivity (67.28nA·mM ), signifcanceinclinicalandforensicmedicine.Tebiosensors widelinear range(0.50–15.00mM),and low detectionlimit for ethanol detection are based on either alcohol oxidase (80.00 μM) towards ethanol. Te recoveries ranged from (AOX) or ADH. Te ADH-based biosensor was superior to 97.2% to 106.0% and the coefcient of variation within and the AOX-based on comparisons of stability and between batches was less than 5%. specifcity [37]. Te binding of aldehyde to NAD resulted in the for- mation of NADH and aldehyde being bound to the zinc activesite.ADHusuallycatalyzesethanoloxidationthrough 3.4. Crosslinking. Typical crosslinking occurs through the the following bi-bi mechanism: [36]: application of a chemical agent called a cross-linker, most commonly a lysine linker, to covalently concatenate two E + A + B↔EA + B↔EAB↔EPQ↔EQ + P↔E + P + Q, (4) residues that are close in space within or between proteins. Te most commonly used crosslinking additives are EDC/ where E represents the enzyme, A represents NAD , B standsforethanol, Pisacetaldehyde,and Q denotesNADH. NHS [26] and glutaraldehyde (GA) [27–30] for crosslinking tissue scafolds and enhancing structural stability. Similarly, Adhikari and coworkers [25] reported a new enzyme embedding device coated with a special cationic polymer, poly(2-(dimethylamino)ethyl methacrylate) 3.4.1. EDC/NHS. An amperometric urea biosensor was (MADQUAT) on a SWCNT-rGO nanohybrid thin flm, developed by immobilizing urease (Urs) and glutamate which catalyzed the oxidation and reduction of electroactive dehydrogenase (GLDH) on a silicon substrate precoated substances. Te chemical oxidation of NAD by the ethanol with N -incorporated diamond nanowire (N-DNW) thin occurred as shown in the following equation: 2 flms which were synthesized by a microwave plasma en- + ADH + CH CH OH + NAD ⟶ CH CHO + NADH + H . (5) 3 2 3 hanced chemical vapor deposition (MPECVD) technique [26].TeEDC–NHS-initiated-COOHgroupwascovalently Journal of Analytical Methods in Chemistry 7 connected to the NH terminal of Urs and GLDH to form the surface of the working electrode (Figure 4). A suitable a covalent amide bond (CO-NH). Te biosensor exhibits mediator in the course of electrocatalytic reaction should be good performance in sensitivity (6.18 μA/mg·dL/cm ), lin- (i) long-term stability, (ii) antifouling efect, (iii) higher 0’ earity range (10.00–100.00mg/dL), lower detection limit electrontransferrateconstant(k ),(iv)formalpotential(E ) (3.87mg/dL), and fast response time (>10s). Te biosensor of the redox mediator should be less than the oxidation maintained 80.0% of its original activity towards urea after potential of NADH, and (v) reduction of considerable being stored in the refrigerator at 4−6 C for 1month. overpotential. Mediators used for electrochemical de- hydrogenase biosensors in human physiological fuids can be divided into four types as quinine groups, metals and 3.4.2. GA. A model enzyme glucose dehydrogenase (GDH) metal complexes, aromatic diamines, and organic dyes was attached to the CNT-CS membrane and used for (Table 2). detecting glucose in the urine matrix without interference [27].GDHcouldbecovalentlyfxedbythereactionofamino groups in highly biocompatible and hydrophilic CS with the 4.1. Quinine Groups. Luoetal.[38]constructedadisposable bifunctional crosslinking agent GA. Te device permitted amperometric biosensor fabricated by immobilizing ADH relatively rapid (∼60s) determination of glucose at low andNAD coatedwithNafon combinedwithAuNPsonto potentials (0.40V) without the presence of redox mediators. the surface of SPCE modifed with MDB. As an electronic Te biosensor displayed a fast response time (<5s), a wide medium, MDB could facilitate the conversion of NADH to measuring range (5.00–300.00 μM), a high sensitivity NAD atapotentialof0.0V(vs.saturatedcalomelreference −1 −2 (80 ±4mA·M ·cm ), and a low detection limit (3 μM). electrode, SCE), thereby, eliminating interferences of oxi- Koide et al. [28] reported a similar procedure for the de- dizable substances present in real blood samples. Te bio- tection of bile acids in urine by employing an electro- sensor was linear from 2.00mM to 8.00mM with chemical biosensor coated with Nafon , which both a correlation coefcient of 0.996. In addition to this, reducedthepresenceofinterferingsubstancesandenhanced a reagentless biosensor has been successfully constructed to thelong-termstabilityofthereferenceelectrode.Ten,three detect glutamate in food and clinical samples by mixing enzymes (bile acid sulfate sulfatase: BSS, 3α-hydroxysteroid unpurifed MWCNTs with CS to immobilizing GLDH and dehydrogenase: 3α-HSD, and NADH oxidase: NHO) were NAD and coating them layer by layer on the surface of fxed with GA onto the Nafon coating sensor chip. Te MDB-modifed SPCE [6]. Te glutamate content in the response time was 5min with a linearity of 2.00–100.00 μM. sample was calculated by employing the standard addition It also exhibited high reproducibility (CV values: ≤10%) and method where the currents produced by each addition were continuous repeatability of measurement (CV value: 5%– plotted and the resulting line was extrapolated. Te elec- 11%). trocatalysis occurred at very low potentials (from approxi- Furthermore, several devices for the detection of serum mately +0.1V vs. Ag/AgCl). However, analytical indexes ethanol were successfully manufactured by using a chem- suchassensitivity,linearrange,anddetectionlimitwerenot ical crosslinking method with GA as the crosslinker. Luo explained. It has also been proposed that MDB hindered the et al. [29] produced a disposable serum ethanol biosensor development of biosensor electrodes by inhibiting the that fxed ADH and NAD on the Nafon -MDB-modifed NAD -dependent enzyme. Tese mediators were covalently SPE based on the cross-linking method. When the voltage bound to important thiol groups in the enzyme. However, isappliedtotheAg/AgClreferenceelectrodeat −0.17V,the the inhibitory efect can be eliminated by employing a 1,10- response time of the biosensor is less than 30s, and the phenanthroline quinone (1,10-PQ) medium, which can linearrangeis5.00mM.Gaoetal.[30]alsoproducedanew hinder 1,4-nucleophilic addition with enzyme amino acid electrochemiluminescence (ECL) ethanol biosensor based residues [39]. Tese mediators incorporated the reactive 2+ on Ru(bpy) and ADH fxed by the rGO/bovine serum 3 quinone double bonds into heteroaromatic rings. When the albumin (BSA) composite membrane. Te ECL response of SPCE bioelectrodes incorporating 1,10-PQ, NAD , and 2+ the ADH/Ru(bpy) /rGO/BSA electrode to standard 3 HBDH were used to analyze 3-HB in blood, it exhibited concentrations of ethanol was found to be linear in the a linear response range of 0.00mM–6.00mM, using a small rangeof1.00–2000.00 μMwithadetectionlimitof0.10 μM. volume of blood (5 μl) and it was stable for up to 18months Te ECL strength maintained about 90% of the original ° at30 C,andshortenedthetotalassaytimeto30s.Inanother value after being kept in the refrigerator at 4 C for research, Wang et al. [40] constructed a “pop-up” electro- one month. chemical paper-basedanalyticaldevice(pop-up-EPAD)that allowedanenzymaticassayfor3-HBinbloodtobereadwith a commercial glucometer. NAD and HBDH reagents were 4. Mediated Electron Transfer stored on the devices. Te sample and 1,10-PD were added To help to overcome the limitations of accessibility and to the reaction area on the top layer of the paper device and proximity and to reduce the susceptibility to interfering thenenteredintothedetectionarea.Oncethesamplepassed species by lowering electrode potentials, redox mediators throughtheelectrodes,theglucosemeterbegantestingatthe have been used in electrochemical dehydrogenase bio- potential of +0.2V and displayed the fnal values. Te pop- sensors. Te medium is attached to the redox enzyme and up-EPADs exhibited the linear range of 0.10–6.00mM and mediateselectrontransferfromtheenzyme’sredoxcenterto the detection limit of 0.30mM. 8 Journal of Analytical Methods in Chemistry Figure 4: Electrochemical reactions involved in NAD /NADH-dependent electrochemical dehydrogenases biosensors operating with mediator. 4.2. Metal and Metal Complexes. Liao et al. [41] built an utilized it for triglyceride (TG) in human serum. Te cat- amperometric biosensor by employing an iridium nano- alytic and electrochemical procedures of TG can be de- particle as a redox mediator doped into a carbon paste and scribed by the following reactions: lipase + + TG ⟶ fatty acid + glycerol + NAD Glycerol  dehydrogenase  dihydroxyacetone + NADH +H , (7) 4+ + 3+ + NADH + 2Ir ↔NAD +2Ir +H . (8) 3+ −1 Followed by the electroregeneration of the Ir at an L) . At storage conditions of around 23 C, the design in- applied potential of +0.15V vs. Ag/AgCl which is expressed creased shelf-life to more than 60days. as Moreover, Zhang et al. [43] developed a 3α-HSD-based indirect electrochemical method to accurately determinate 3+ 4+ − 2Ir ↔2Ir + 2e . (9) bile acids concentrations in serum with unmodifed SPCE. Te amperometric response of the optimized bile acids Te linearity was found to be in the range of −1 detectoronexposuretostandardconcentrationsofbileacids 0.00–10.00mM with a sensitivity of 7.50nA·mM . Te was linear from 5.00 μM to 400.00 μM. Known concentra- method for the detection of TG can overcome the in- tions of bile acids were spiked into serum samples and the terference of UA and AA. + recovery values ranged from 75.10% to 113.1%. Using the Moreover, Li et al. [42] fxed HBDH, NAD , and the similar biosensor design, Tian et al. [4] utilized the electron electron mediator of potassium ferricyanide (K [Fe(CN) ]) 3 6 mediator of tris(2,2′-bipyridine)ruthenium(III) (Ru(bpy) onto the SPCE precoated with a layer of hydrophilic gel 3+ ) to react with NADH, thereby indirectly detecting bile sodium carboxymethyl cellulose (CMC). A comprehensive + 3− acids. It exhibited a linear behavior from 5.00pmol/L to evaluation of the immobilized HBDH/NAD /Fe(CN) / 150.00pmol/L with a detection limit of 0.40pmol/L (based CMC/SPCE system showed that the procedure was suitable on 3× the baseline noise) in a 10 -fold dilution serum. Te for thedetection of 3-HB in whole blood or serum with very 3+ use of Ru(bpy) greatly enhanced the electrical conduc- small sample volumes (2.0 μL) and a response time of 50s. tivity and the sensitivity of the device (Figure 5). Te amperometric response was found to be linear from 1.50mg/L to 500.00mg/L with a sensitivity of 0.011 μA·(mg/ Journal of Analytical Methods in Chemistry 9 Table 2: Electrochemical dehydrogenase sensors comparison: detection mechanism and performance. Storage Sensing Limit Linear Response Electron Target stability Sample Ref scheme/target of detection range time mediators (days) ADH/NAD /Nafon/AuNP/MDB/SPCE; Ethanol 16 μM 2.00–8.00mM 40s 30 Serum MDB [38] GLDH/NAD /MWCNTs/CS/MDB/ Glutamate — — — — Serum MDB [6] SPCE; 1,10-PQ/NAD /HBDH/SPCE; 3-HB — 0.00–6.00mM <30s 540 Blood 1,10-PQ [39] 1,10-PD/NAD /HBDH/EPAD; 3-HB 0.30mM 0.10–6.00mM — — Blood 1,10-PQ [40] 4+ 4+ Ir /SPCE; TG — 0.00–10.00mM — — Serum Ir [41] + 3− 3− HBDH/NAD /Fe(CN) /CMC/SPCE; 3-HB — 1.50–550.00mg/L 50s 60 Serum Fe(CN) [42] 6 6 SPCE; Bile acid — 5.0–400.0 μM — Serum — [43] Ru(bpy) SPCE; Bile acid 0.40pM 5.00–150.00pmol/L — — Serum [4] 2+ SH/Tyrosinase/PheDH/CPE; L-Phenylalanine 5.00 μM 20.00–150.00 μM 45–60s 11 Serum and blood Catechol [44] rGO/PhNHOH/GCE; Lactate 2.50 μM −90.00 μM 23s — Serum PhNHOH [45] HBDH/THI/rGO/SPCE; 3-HB 1.00 μM 0.010–0.40mM 7 s 20 Serum THI [46] 50.00–400.00mg/ LIP/GDH/TB/ERGO/ITO; TG 0.18mM 12s 112 Serum TB [47] dL 10 Journal of Analytical Methods in Chemistry NAD 3α-HSD 2+ Ru (bpy) t (s) t (s) PBS solution Diluted serum R R COCR COCR 3 3α-HSD R R 1 + NAD NADH HO R R 3+ + 2+ NADH + Ru (bpy) NAD + Ru (bpy) 3 3 2+ 3+ Ru (bpy) - e Ru (bpy) 3 3 Figure 5: Principle of enzymatic coupling double oxidization electrochemical detection of bile acids (reprinted from [4]). 4.3. Aromatic Diamines. An enzyme carbon paste electrode L-phenylalaninedetection[44].WiththecatalysisofPADH, (CPE) based on tyrosinase, salicylate hydroxylase (SH), and L-phenylalanine acids reacted specifcally with NAD to L-phenylalaninedehydrogenase(PADH)wasestablishedfor produce NADH as shown in the following equation: L − Phenylalanine +NAD PADHPhenylpyruvate +NADH. (10) Ten, the irreversible decarboxylation and the hydrox- couple (-NHOH/-NO) covalently functionalized with rGO. ylation of salicylate catalyzed by SH were described by the Te sensor can determinate lactate as low as 2.5 μM without −1 −2 following equation: interference, with a sensitivity of 10.57 ±0.38nA·μM ·cm , a linear range up to 90.00 μM, and a response time of 23s. salicylate + NADH + 2H + O SH catechol (11) +NAD +H O + CO . 2 2 ´ ´ 4.4. Organic Dyes. Martınez-Garcıa et al. [46] established an Te oxidation of catechol to o-quinone by tyrosinase enzymatic electrochemical device for the detection of 3-HB could be described by equation (12). Also, the reaction in serum by fxing HBDH onto a rGO and thionine (THI)- current was measured by the chronoamperometry assay at modifed SPCE. Te THI’s redox mediator combined with E � −50mV vs. Ag/AgCl. appl an rGO-modifed electrode could reduce the detection potential to 0.0V. Te optimized device showed a linear catechol +2H + O tyrosinase o − quinone + 2H O. (12) 2 2 response range of 0.010–0.40mM, a limit of detection of Tis relies on the circulation of catechol and o-quinone 1.00 μM, and a response time of 7s. betweentheelectrodeandthetyrosinase,therebyamplifying Furthermore,theTGdetectiondevicewasestablishedby the electrocatalytic signal. Te reaction time for L- fxing lipase (LIP) and GDH on the indium-tin-oxide (ITO) phenylalanine was <60s with a linearity range from glasselectrodecoatedwithanelectrochemicallyreducedGO 20.00 μM to 150.00 μM. Te biosensor also lost >50% of its (ERGO) membrane doped by toluidine blue (TB) [47]. original response to galactose within 11days. Glycerol was oxidized in the presence of GDH, and TB then In addition, Manna and Retna Raj [45] described an extracted the H ions released from the glycerol, leaving the electrochemical device for the detection of lactic acid in hydrogen to ERGO for performing the subsequent redox humanserumemployingrGO-PhNO compositescovalently reaction. At a potential of +0.34V, ERGO was reoxi- 2 (Red) functionalized with a p-nitrophenyl moiety of p-nitroaniline. dized to ERGO , resulting in an electrochemical re- (Oxid) Te rGO-PhNHOH was formed by the potential cycling of sponse. With a rapid response within 12seconds, the device the rGO-PhNO -modifed GC electrode. Te current signal is linear in the range of 50.00mg/dl to 400.00mg/dl and has −1 was detected by the oxidation of NADH mediated by a redox a sensitivity of 29Pa·mg ·dl. I (μA) I (μA) Journal of Analytical Methods in Chemistry 11 density. Te immobilization procedure of redox-active en- 5. Challenges and Critical Issues toward zymeontheelectrodehasagreatinfuenceontheindexesof Clinical Applications the detection device. It is therefore a key issue, especially if Although considerable resources have been invested in large-scale applications are envisaged, to ensure that the attenuation of the catalytic activity of the immobilized building electrochemical biosensing devices based on de- hydrogenase over the past two decades, there has been little biocatalystisasreducedaspossible.Anotherdirectionbeing explored is the use of mediators such as metal and metal apparent success in the productization of these devices for use in human physiological fuids such as serum and urine. complexes, aromatic diamines, and organic dyes as the electron shuttles between the enzyme and the transducer. Tecriticalproblemofsuchdevicesistheefectiveexchange of electrons between the active center and the supporting Biosensors, which use electron mediators, often show high composite or electrode. Direct electron transfer (DET) current densities and a good sensitivity. However, they are through the bare electrode is extremely hard because the usually less stable due to the immobilization of the medium redox-active sites of NAD /NADH-dependent de- and the mediators. Tus, there still are challenges yet to be hydrogenases are deeply embedded in the well-electrically overcome to achieve better sensitivity, selectivity, stability, insulated shells [13]. Te most common is that distances and lifetime, before NAD -dependent electrochemical de- greaterthan20apreventDETbetweentheactivesiteandthe hydrogenase can be used in practical applications. electrode.Tisproblemisoftenaddressedthroughtheuseof mediators,whichcanreachtheactivesiteoftheenzymeand Conflicts of Interest act as electron shuttles by mediating the exchange of elec- Te authors declare that they have no conficts of interest. trons between the enzyme and the electrode. Due to its excellent electrical conductivity and high sensitivity, METis proven to be promising in the construction of various Authors’ Contributions biosensors and biofuel cells. It is also important to address Xinrui Jin, Jinglan Guo, Baolin Li, and Jinbo Liu con- the issues of mediator-based biosensors, such as leakage of ceptualised and designed the study. Zixin Zhu, Yusen Liu, mediators, thehigh costof complexes, or theharmfulness of and Min Zhong constructed fgures and tables. Xinrui Jin, shuttling molecules must be considered in future studies. Jingling Xie, and Jinkang Feng drafted the manuscript. All Other challenges associated with electrochemical de- authors contributed to writing the fnal drafts of the hydrogenase biosensors are that NADH biosensing on bare manuscript. electrodes typically leads to the accumulation of the oxi- dation products on the electrode surface which causes high overpotential and unstable detection signals [48]. Te sur- Acknowledgments face characteristics of the sensor and its selectivity to targets Te work was supported by the grants from Southwest must be improved, otherwise many components in bi- Medical University (Grant nos. 2021ZKQN063, 2020500, ologicalfuidssuchasbloodandurinesamplescouldquickly and S202210632152) and the Science and Technology De- contaminate the electrode. Furthermore, the bulk of the partment of Sichuan Province (Grant nos. 2021YFS0171and research efort has been on small initial sample sizes, por- 2021YFH001). table devices, greater sensitivity or selectivity, and wide linear ranges with low detection limits. In contrast, the number of publications dealing with thermal stability, References storage stability, shelf life, and the reuse of electrochemical [1] L. C. Clark and C. Lyons, “Electrode systems for continuous dehydrogenase biosensors is very small. 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