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Ivanov, A.; Shamagsumova, R.; Larina, M.; Evtugyn, G. Electrochemical AChE Biosensor Design. Encyclopedia. Available online: (accessed on 16 April 2024).
Ivanov A, Shamagsumova R, Larina M, Evtugyn G. Electrochemical AChE Biosensor Design. Encyclopedia. Available at: Accessed April 16, 2024.
Ivanov, Alexey, Rezeda Shamagsumova, Marina Larina, Gennady Evtugyn. "Electrochemical AChE Biosensor Design" Encyclopedia, (accessed April 16, 2024).
Ivanov, A., Shamagsumova, R., Larina, M., & Evtugyn, G. (2024, February 20). Electrochemical AChE Biosensor Design. In Encyclopedia.
Ivanov, Alexey, et al. "Electrochemical AChE Biosensor Design." Encyclopedia. Web. 20 February, 2024.
Electrochemical AChE Biosensor Design

Neurodegenerative diseases and Alzheimer’s disease (AD), as one of the most common causes of dementia, result in progressive losses of cholinergic neurons and a reduction in the presynaptic markers of the cholinergic system. These consequences can be compensated by the inhibition of acetylcholinesterase (AChE) followed by a decrease in the rate of acetylcholine hydrolysis. The assessment of cholinesterase inhibitors includes the comparison of the biosensor signals recorded prior to and after the contact of the enzyme with an inhibitor. This makes it possible to calculate the inhibition degree as the relative shift of the enzyme activity and as a measure of the inhibitor content. Even in the case of the direct signal measurement performed in the presence of both the substrate and inhibitor, it is mostly assumed that the initial enzyme activity (for zero inhibitor concentration) is constant and reproducible in the series of experiments required for calibration graph plotting. 

electrochemical biosensor acetylcholinesterase reversible inhibition drug determination

1. Cholinesterase Immobilization

Immobilization of cholinesterase, i.e., its stabilization in an insoluble state on a solid support, preferably on the signal transducer or localized on the transducer interface, is an indispensable part of the biosensor design. The immobilization protocol is intended to establish a long storage and operation period of a biosensor. Meanwhile, its sensitivity toward an inhibitor should be high enough for the analytical applications of the biosensor. This means enzyme manipulations suggested leave the free accessibility of the enzyme active site for the substrate–inhibitor, mostly due to rather low changes in the configuration and flexibility of a protein globule.
The following protocols have been described for AChE/BChE immobilization. Most of them were first described for pesticide detection but have also found application for a reversible inhibitors assay. The immobilization schemes are outlined in Figure 1.
Figure 1. AChE immobilization protocols: (A)—physical adsorption; (B)—implementation in the polymer film; (C)—covalent binding via Au-S bonds; (D)—cross-linking with glutaraldehyde; (E)—carbodiimide binding to the carboxylated carrier; (F)—affinity immobilization via natural receptors.
  • Physical adsorption on solid support (Figure 1A). The bare and modified surface of the electrode as a primary signal transducer or plastic films attached to such an electrode can be used as enzyme solid supports. This method offers the high stability of the enzyme during the storage of the biosensor due to the hydrophilic microenvironment of the enzyme established in the surface layer. Physical adsorption on the solid support including entrapment in the polymer gels (Figure 1B) or in the polyelectrolyte complexes makes it possible to preserve the native structure of an enzyme globule and its affinity toward inhibitors. Polyurethane [1], polyaniline [2][3][4], polypyrrole [5], polysiloxanes [6], sodium alginate [7], poly(vinyl acetate) photocurable polymer (PVA-SbQ) [8] and bovine serum albumin (BSA) [9] were used for this purpose. Possible leaching (desorption) of the enzyme can be suppressed by additional cover films deposited or attached to the enzyme layer. A similar approach has been described for the simultaneous immobilization of AChE with an auxiliary enzyme, choline oxidase (ChO) [10]. Carbon nanomaterials offer many advantages as enzyme supports due to a high surface to volume ratio, electroconductivity and a high adsorption capacity [11][12].
  • The formation of self-assembled monolayers (Figure 1C) is specified because of the high importance of this immobilization protocol for biosensors utilizing golden electrodes or nanoparticles in their assembly [13][14][15][16]. The formation of Au-S bonds offers the site-specific surface immobilization of enzyme molecules. The use of thiolated linkers makes it possible to extend the surface layer and increase its accessibility for inhibitors. Au, as a highly conductive material, improves the conditions of electron transduction and enzyme electric “wiring” and is often combined with other modifiers added to increase the specific surface area (carbon nanomaterials, chitosan films, electropolymerized coatings, etc.).
  • Cross-linking with glutaraldehyde (Figure 1D) increases the average molar mass of the protein so that the enzyme becomes insoluble and deposits on the solid support [9][17]. Glutaraldehyde interacts with amino and thiol functional groups to form Schiff bases. Although the reaction is reversible, the reverse hydrolysis of the product is less probable in the biosensor operation period. The reaction is mostly performed in the presence of the BSA protein protecting the active site of the enzyme from undesired chemical reactions. The reaction is complicated by the partial oligomerization of glutaraldehyde during storage.
  • Covalent carbodiimide binding (Figure 1E) with carboxylated carriers [18][19]. Carbodiimide, specifically, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide chloride (EDC), forms amide bonds between the carboxylic and amino functions and provides the site-specific immobilization of proteins to carboxylated materials, e.g., carbon nanotubes or carbon black. The reaction is performed in mild conditions (at room temperature). The addition of N-hydroxysuccinimide (NHS) prevents the hydrolysis of the unstable intermediate and increases the efficiency of the enzyme binding. Carbodiimide binding is easily combined with the use of Au supports covered with monolayers of mercaptopropionic and mercaptoundecanoic acids.
  • Affine immobilization (Figure 1F) assumes the use of natural receptors like concanavalin A or an avidin–biotin pair [20][21]. This offers mild and oriented immobilization with a high residual enzyme activity and sensitivity toward inhibitors.
Modern approaches elaborated for AChE introduction in electrochemical biosensors for inhibitor determination are summarized in a review [22]. The immobilization protocols include auxiliary reagents introduced with enzymes in the surface layer. They improve the conditions of signal generation, stabilize the enzyme structure or improve the mechanical stability of the surface layer. A variation in the pH and reactant quantities alters the specific enzyme activity and to some extent the sensitivity of the inhibitor determination. It should be noted that the optimization of the immobilization conditions is frequently based on maximizing the signal toward the substrate. This allows for increasing the measurement accuracy but can decrease the inhibition degree of reversible inhibitors showing a maximal influence in different conditions. From a general consideration, the lower enzyme quantities taken for immobilization increase the sensitivity of the signal toward the inhibitor but decrease the accuracy of the inhibition degree determination. Thus, the content of the enzymatic layer is always a compromise between the arguments of the signal value and inhibition detection.

2. Cholinesterase Biosensor Signal Measurement

To date, most cholinesterase biosensors utilize a synthetic substrate, acetylthiocholine, for the monitoring of the enzyme activity and hence for the quantification of the inhibition caused by drugs and other anticholinesterase agents. The reaction is presented in Figure 2.
Figure 2. Amperometric sensing of cholinesterase activity with acetylthiocholine as synthetic enzyme substrate.
Thiocholine can be directly oxidized to disulfide on carbon electrodes at about 0.6 V. Mediated oxidation is preferred to avoid the passivation of the electrode and to decrease the overpotential of the reaction. For this purpose, Ag [19][23][24][25] and Au nanoparticles [13][14][26][27], Prussian blue [28], Co phthalocyanine [29], 7,7,8,8-tetracyanoquinodimethane (TCNQ) [30], macrocycles bearing hydroquinone units [19][31][32] and metal–organic framework (MOF) particles [33][34] are implemented in the surface layer together with enzyme molecules. Mediators are often combined with each other and carbon nanomaterials. The electron transfer path in mediated AchE biosensors is schematically outlined in Figure 3.
Figure 3. Mediated oxidation of thiocholine in AchE biosensor. Ox and Red are oxidized and reduced forms of a mediator system.
The use of the thioanalog of a natural substrate, acetylcholine, complicates the comparison of the inhibition effects with the toxic consequences of poisoning because the nature of the substrate influences the kinetics of reversible inhibition. On the other hand, acetylcholine does not produce an electrochemically active product that could be detected by amperometry. The introduction of a second enzyme, ChO, solves the problem (Figure 4) [10][35].
Figure 4. Cholinesterase activity measurement with acetylcholine and two enzymes (AChE and ChO).
The design of a bi-enzyme sensor is complicated by the difference in the optimum pH of the AChE and ChO activity and in the specific enzyme activity of the preparations used in the immobilization step. In some cases, one of the enzymes was left in the solution while the second one was immobilized on the electrode interface. The accumulative inhibitory effect of the analytes on the two enzymes increased the sensitivity of the inhibitor determination but was less related to the native influence of the anti-AD drugs on the patients’ health. The signal of the AChE–ChO bi-enzyme sensor can be recorded by mediated oxidation or a reduction of the hydrogen peroxide as a final product of reaction. Au-Pt bimetallic nanoparticles [36], MnO2 [37] and phtalocyanines [38] amplify the oxidation of H2O2 while Prussian blue [39] and CdTe quantum dots [40] mediate its reduction. The latter reaction can also be catalyzed by peroxidase, a third enzyme implemented in the biosensor assembly [41].
The potentiometric measurement of the AChE activity assumes the assessment of the quantities of acetic acid or choline released in the enzymatic reaction. The simple design of the measurement equipment applied in potentiometry and the open-circuit mode of the signal measurement are the most mentioned advantages of potentiometric biosensors. In the pH measurements, both acetylcholine and acetylthiocholine can be employed as substrates. It should be mentioned that thiocholine formed in the latter case is ionized in basic media. The acidity constant of thiocholine (pKa = 7.7) is high enough in basic media. Thus, it influences the pH of the solution. This compensates for a lower rate of acetylthiocholine hydrolysis compared to that of acetylcholine. The pH sensitive transducer can be prepared by the deposition of polyaniline, for which the equilibrium potential depends on the pH of the environment. This made it possible to use it as both the enzyme immobilization matrix and signal generation agent [42]. Although the monitoring of the pH changes is one of the oldest approaches in the investigation of enzyme kinetics, it has some obvious drawbacks in the real sample assay and inhibition quantification, i.e., the dependence of the signal on the buffer capacity and pH of the samples tested. The use of choline (thiocholine)-sensitive electrodes based on conventional PVC membranes is an alternative to pH-sensitive biosensors. However, such biosensors were mostly reported for the characterization of the inhibition kinetics [43][44]. The ion-selective membranes consist of the appropriate lipophilic salts of choline and respond on both the substrates (acetylcholine and acetylthiocholine) and the product (choline). The sensitivity of the inhibition measurements was found to be rather modest though the potentiometric sensors were successfully applied for the determination of the inhibition constants. From other potentiometric biosensors, the mediatorless potentiometric detection of H2O2 can be mentioned for a three-enzyme sensor containing BChE, ChO and peroxidase. It showed a rather high sensitivity in irreversible inhibition measurements [45].

3. Anti-AD Drugs Determination

As was mentioned above, the design of the cholinesterase-based biosensors developed for drug determination based on their inhibition are mostly derived from appropriate biosensors previously described for pesticide detection. The difference in their application is mostly related to the consequence of reagent addition and data treatment. Nevertheless, some of the biosensors are used for the quantification of enzyme–drug interactions with particular emphasis on the kinetics or sensitivity of the target reactions. Screen-printed Au electrodes combined with a portable potentiostat [46] is one of the examples of such an approach. In this work, human erythrocyte AChE was mixed with the acetylthiocholine and donepezil standard solution in the well of a microtiter plate, thermostated at 37 °C and then spotted on the working electrode. The thiocholine concentration was assessed by differential pulse voltammetry at 0.16 V. The results obtained were compared with a standard Ellman method of colorimetric enzyme assay with 5,5′-dithiobis(2-nitrobenzoic acid) as the chromogenic AChE substrate. The results were expressed as IC50 values, which were found to be rather similar to both the electrochemical and colorimetric modes of assay. An S-shaped calibration curve complicated the comparison of the concentration ranges of the drug determination. The method was validated using the AChE containing homogenates from the anterior cortex of mice treated with the donepezil.
A similar approach was reported in [47]. Here, the solutions of AChE and ChO were incubated with acetylthiocholine and drugs (donepezil and tacrine) and the decay of the H2O2 concentration was monitored in the chronoamperometry mode on the glassy carbon electrode modified with a coordination polymer formed by L-cysteine and AgNO3. The reaction was first tested for the determination of the cholinesterase activity and H2O2 concentration and then the IC50 of two anti-AD drugs (donepezil and tacrine) was determined in standard solutions.
A microfluidic system based on droplet analysis has been developed for a dose–response inhibition assay [48]. A polydimethylsiloxane chip integrated with microelectrodes has been applied in the gradient generation mode. A whole inhibition assay based on thiocholine oxidation required 6 min. The microfluidic system was tested on carbamate, organophosphate pesticides and tacrine as an anti-AD drug. The latter one exerted inhibition in the concentrations’ range from 0.016 to 87 µM (IC50 37.4 ± 5.8 µM).
In addition to the assessment of relative inhibition abilities, electrochemical biosensors have been described for the determination of the drug concentrations and estimation of possible interferences caused by biological fluid components. It should be noted that the calibration curve of reversible inhibitor determination is not linear and is mostly ascribed by an S-shaped four-parametric logistic function [29][49].
where 𝑐𝐼 is an inhibitor concentration, A1 and A2 are the upper and lower limits of the curve (ideally equal to zero and 100%, respectively), b is a measure of IC50 and p reflects the sensitivity of the enzyme toward the inhibitor, i.e., the slope of the linear approximation in the middle part of the curve. The parameters of the calibration curve are determined using standard statistical software like those of linear regression but require a higher number of experimental points. A similar approach is used in immunoassay, where the signal depends on the equilibrium of the antigen–antibody interaction. In both cases, linear approximation covers a rather narrow middle part of the whole concentration-dependence event though semi-logarithmic plots (inhibition-log𝑐𝐼) are often preferred. The same difference in the metrological assessment of linear and semi-logarithmic plots is the reason for the significant difference in the performance of amperometric and potentiometric sensors used for the assessment of the same inhibitor concentration in similar experimental conditions [42]. Potentiometric sensors are mostly based on the Nernst equation and quantify the pH shift caused by acetic acid released in the substrate hydrolysis. The use of electroactive polymers offers an alternative when similar reactions are monitored by the currents attributed to the shift in the redox equilibrium of the polymeric layer [50].
Flow-through methods of analysis have been gaining increasing attention due to the necessity of screening a large number of samples in similar conditions (biological fluids, drug formulations, waste waters of pharm factories, etc.). In [51], the flow-injection determination of anti-AD drugs has been described with the AChE immobilized on a Au disc. Seven commercially available AChE inhibitors (neostigmine, eserine, tacrine, donepezil, rivastigmine, pyridostigmine and galantamine) were tested and a similarity in the relative inhibition activity was established for various enzyme sources described in the literature. The measurement characteristics were evaluated to obtain both the IC50 values and the enzyme–inhibitor equilibrium constants. The flow conditions also made it possible to estimate the dissociation of enzyme–inhibitor complexes and the recovery of the enzyme activity after its contact with an inhibitor.
A flow-through replaceable thin film cell with the AChE immobilized to the inner walls via carbodiimide binding has been proposed for the determination of donepezil and berberine [52]. All the disposable parts of the devices were produced by 3D printing from poly(lactic acid). Disassembled and assembled flow-through cells with a screen-printed electrode and replaceable reactor are presented in Figure 5.
Figure 5. Schematic outline (a), disassembled (b) and assembled (c) flow-through cell with screen-printed electrode and replaceable reactor for AChE inhibitors detection. Adapted from [52], © mdpi open license.
The products of the substrate hydrolysis migrated to the screen-printed electrode modified with the macrocyclic derivative of hydroquinone, pillar [5]arene, which was involved in the electron transduction path at a low working potential. The flow-through biosensor device was tested on spiked samples of biological fluids and showed a high recovery of the drug determination.
Some other macrocycles on a pillararene platform have also been tested in the assembly of stationary AChE sensors, alone and together with Ag nanoparticles synthesized in situ by the reduction of AgNO3 salts with hydroquinone units of pillararenes [19][25]. Ag accelerates the oxidation of thiocholine and increases the signal of the biosensor toward the substrate due to the accumulation of thiocholine at metal nanoparticles. Meanwhile, the presence of Ag nanoparticles decreases the inhibitor concentrations determined by about one order of magnitude against the same macrocycles taken alone.
Paper-based microfluidic devices are considered as an alternative to flow-through biosensors and intended for the preliminary administration of drugs in neurodegenerative diseases [53]. In those, the enzyme and substrate are located on a different part of the paper support and are combined only on the period of the signal measurement to avoid substrate hydrolysis during the storage period. Such a design, called an origami sensor, assumes the use of screen-printing technologies to obtain conductive pads from carbon containing inks and sample separation parts from commercial membranes that collect the serum and separate red blood cells from their contact with an enzyme. BChE from horse serum was immobilized by spotting on the paper pad and butyrylthiocholine chloride was used as the specific substrate. The thiocholine oxidation was promoted by Prussian blue particles added to the carbon ink. The origami biosensor allowed for assessing the enzyme activity and drugs that inhibit BChE (physostigmine as a model). The measurement period took only 5 min with a deviation of 3.7%.
The idea of the spatial separation of an immobilized enzyme and transducer was also realized in the construction of an electrochemical AChE sensor with aminated magnetic particles used for the immobilization by glutaraldehyde cross-linking [54]. In this work, maghemite superparamagnetic particles were synthesized from the Fe(III) nitrate and modified with the layer containing primary amino groups. After that, the particles were treated with glutaraldehyde and then with the enzyme solution. The reaction resulted in the formation of particles with the active AChE attached to their surface. In the inhibition measurement, the suspension of the particles was first incubated in a microtube with the mixture of an enzyme and inhibitor. After that, the thiocholine formed was separated from the magnetic particles into the microcuvette and the direct oxidation of thiocholine was measured by square-wave voltammetry. The protocol of the biosensor-based measurement is presented in Figure 6.
Figure 6. Determination of the AChE activity and anti-DA drugs with magnetic separation of the enzyme and thiocholine formed in acetylthiocholine hydrolysis. Square-wave voltammetry is used for the signal recording. APTES—γ-aminopropyltriethoxysilane.
Galantamine was chosen as a model inhibitor (limit of detection, LOD, of 1.5 µM). The enzyme activity was found to be quite stable in the presence of some organic solvents (alcohols and dimethylsulfoxide). The measurement results showed good correlation with the colorimetric test of the AChE activity (Ellman test).
The analytical performance of the electrochemical cholinesterase biosensors for the determination of the reversible inhibitors applied as the drugs for the administration of neurodegenerative diseases is summarized in Table 1. It should be noted that some of the articles cited contain data on the determination of other traditional AChE inhibitors, preferably organophosphate and carbamate pesticides.
Table 1. Determination of drugs with electrochemical cholinesterase biosensors 1.
It should be noted that the majority of the sensors presented in the literature are devoted to the determination of appropriate medications in standard solutions. In the case of the inhibition kinetics assessment, the results obtained are compared with the parameters obtained by standard methods used in toxicology and enzymology like the Ellman test of enzyme activity.
Possible interferences present in biological fluids are estimated via the use of spiked samples or of the solutions mimicking the properties of serum plasma or urine (Ringer–Locke solution, artificial urine commercial preparations). On the one hand, they avoid the problems related to the individual diversity of appropriate samples. On another hand, the reliability of such measurements is lower than that based on the reference methods comparison. Indeed, the use of spiked samples with known quantities of drug and artificial biological fluids can be considered as a stepping stone to real sample assays, which is useful and necessary but not fully sufficient for the method validation. Unfortunately, there are no examples of the application of appropriate cholinesterase biosensors for the routine monitoring of drug residues in hospitals.
Regarding point-of-care testing devices, the use of paper-based biosensors in the origami format seems very promising. Its operation is based on intuitively understandable steps that can be performed by unqualified personnel. The immobilization of enzymes provides the stability of their activity within the storage period and a repeatable response toward drugs of interest. It can be expected that such simplified biosensors together with microfluidic devices will find a broader application, especially in developing countries.


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