Analytical Biochemistry 421 (2012) 208–212

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Analytical Biochemistry

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Assay of acetylcholinesterase activity by potentiometric monitoring of acetylcholine

M. Cuartero a, J.A. Ortuño a,⇑, M.S. García a, F. García-Cánovas b

a Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, Murcia E-30100, Spainb Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Murcia E-30100, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 1 August 2011Received in revised form 26 September2011Accepted 3 October 2011Available online 12 October 2011

Keywords:Acetylcholine-selective electrodeEnzymatic hydrolysisAcetylcholinesterasePotentiometric monitoringActivity assay

0003-2697/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.ab.2011.10.008

⇑ Corresponding author. Fax: +34 868 887409.E-mail address: jortuno@um.es (J.A. Ortuño).

1 Abbreviations used: ACh, acetylcholine; AChE, acetDOS, dioctyl sebacate; ISEs, ion-selective electrodes;bis-(trifluoromethyl)phenyl]borate; NPOE, 2-nitrophenphosphate; THF, tetrahydrofuran.

An acetylcholine-selective electrode based on a plasticized polymeric membrane has been developed. Theelectrode exhibited good selectivity for acetylcholine (ACh) over choline and some common ions, low drift,and a fast response to ACh. The response was linear over an ACh concentration range of 1 � 10�6 to1 � 10�3 M with a slope of 59.1 ± 0.1 and a detection limit of 1.5� 10�7 ± 1.2� 10�8 M. The electrode wasused to monitor enzymatic ACh hydrolysis catalyzed by acetylcholinesterase (AChE) at different substrateand enzyme concentrations. A kinetic data analysis permitted the determination of the Michaelis–Mentenconstant of the enzymatic hydrolysis and AChE activity in the range of 2 � 10�5 to 3.8� 10�1 U ml�1.

� 2011 Elsevier Inc. All rights reserved.

Acetylcholinesterase (AChE)1 terminates the transmission of im- application to a potentiometric AChE assay based on the monitor-

pulses in the cholinergic synapses through the rapid hydrolysis ofacetylcholine (ACh) to choline (Ch) [1]. ACh is an important cationicneurotransmitter associated with attention, learning, memory, con-sciousness, sleep, and control of voluntary movements [2].

The history of recent developments in assays for AChE activityhas recently been reviewed [3]. Such assays are spectrometric, col-orimetric, radiometric, calorimetric, and biosensor based. The vastmajority of the assays reported in the literature made use of non-natural substrates of AChE, such as acetylthiocholine and butyryl-thiocholine. However, the use of the natural substrate ACh hasseveral advantages. For example, the value of the Michaelis–Menten constant is much higher than for nonnatural substrates,increasing the sensitivity of the assays for AChE activity determina-tion. Moreover, the constant value for ACh is more biochemicallyrelevant. One reason for using nonnatural substrates is that AChis not easily monitored since it does not absorb in the UV band,does not present fluorescence, is not redox electroactive, and is dif-ficult to derivatize. Fortunately, acetylcholine cation ACh+ is morelipophilic than its hydrolysis product choline cation Ch+, as shownby their respective values of ion standard transfer potential fromwater to nitrobenzene [4]. In this work, we take advantage of thislipophilicity difference to develop an ACh-selective electrode for its

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ylcholinesterase; Ch, choline;KTPB, potassium tetrakis[3,5-yl octyl ether; TCP, tricresyl

ing of the natural substrate (ACh) monitoring.The methods reported to date that use ACh as substrate are

based on the potentiometric measurement of the pH variationdue to the appearance of the hydrolysis product, H+ [3]. However,this approach has a series of disadvantages related with the need touse unbuffered or weakly buffered media [3,5]. These disadvan-tages are overcome with the use of the ACh-selective electrodehere developed, since it works properly in buffered media.

The calculation of reaction rates and the quantitative determi-nation of enzymes, substrates, and products of enzymatic reactionsare among the applications of ion-selective electrodes (ISEs) [6]. Tobe able to monitor the kinetics of a hydrolysis reaction, an ISE mustsatisfy a set of general requirements common to all detection sys-tems, i.e., sufficient sensitivity and stability, low background noise,low drift, and rapid response time [7]. Furthermore, when the ISEmonitors the substrate concentration of a reaction, it must havesufficient selectivity for the substrate over the product of the reac-tion. To our knowledge, only one ACh-selective electrode has beenapplied to the monitoring of the ACh hydrolysis [8,9]. The methodin question consists of a primitive type liquid membrane electrodewhich has nowadays been totally replaced by the plasticized poly-meric version that presents several advantages, among themmechanical stability and durability. In this paper an ACh-selectiveelectrode based on a plasticized polymeric membrane that exhibitsthe general requirements noted above.

The main goal of the present work was to develop a robustpotentiometric assay for AChE activity using the ACh-selectiveelectrode to monitor the enzymatic hydrolysis of ACh. This assayis used here to determine the Michaelis–Menten constant and

Potentiometric assay of AChE / M. Cuartero et al. / Anal. Biochem. 421 (2012) 208–212 209

the enzyme activity. However, it has a wide scope of further appli-cations in the determination of AChE inhibitors.

Materials and methods

Materials

All chemicals were of analytical reagent grade and Milli-Q waterwas used throughout. Polyvinyl chloride (PVC) of high molecularweight, dioctyl sebacate (DOS), 2-nitrophenyl octyl ether (NPOE),tricresyl phosphate (TCP), potassium tetrakis [3,5-bis-(trifluoro-methyl)phenyl]borate (KTPB), acetylcholine chloride, and tetrahy-drofuran (THF) were purchased from Fluka (Munich, Germany).Acetylcholinesterase (type VI-S) from Electrophorus electricus (elec-tric eel, EC 3.1.1.7, 288 U/mg solid) and choline chloride were ob-tained from Sigma-Aldrich (Munich, Germany).

Phosphate buffer of different pH values between 5.8 and 8.9was prepared by accurately mixing appropriate volumes of0.01 M KH2PO4 and 0.01 M NaOH solutions. An AChE stock solutionwas prepared by dissolving 0.46 mg of AChE in 2.5 ml of phosphatebuffer and diluting with water to 5.0 ml in a calibrated flask, result-ing in a solution of 25.8 U ml�1. A 0.1 M ACh stock solution wasprepared by dissolving 0.1817 g of acetylcholine chloride in10.0 ml of water. Working ACh solutions were prepared by dilutingthis with water. A 0.1 M Ch solution was prepared by dissolvingcholine chloride in water. All these solutions were stored in arefrigerator at 4 �C.

Potentiometric measurements were recorded using a home-made high-impedance data acquisition 16-channel box connectedto a personal computer by USB (Universal Serial Bus). A Fluka(Munich, Germany) electrode body ISE and an Orion Ag/AgCl dou-ble-junction reference electrode (Orion 90-02), containing a10�4 M KCl solution in the outer compartment, were used.

Membranes and electrodes preparation

The compositions of the membranes evaluated in the presentwork are shown in Table 1. The selected membrane was preparedby dissolving 100 mg of PVC, 200 mg of the plasticizer NPOE, and1.5 mg of the ionic additive KTPB in 3 ml of THF. This solutionwas poured into a Fluka glass ring (inner diameter 28 mm, height30 mm) on a Fluka glass plate, and allowed to settle overnight untilall the THF had evaporated, to obtain a thin plastic membrane. A 6-mm-diameter piece was cut out with a punch and incorporatedinto a Fluka electrode body ISE containing 1 � 10�4 M KCl as inter-nal filling solution. The electrodes were conditioned in water untilthey reached a constant potential. When not in use, the electrodeswere kept immersed in water.

Potentiometric measurements

All potentiometric measurements were carried out with contin-uous magnetic stirring in a thermostated vessel at 28 ± 0.1 �C.

Table 1Percentage of PVC, plasticizer (DOS, NPOE, or TCP) and ionic additive (KTPB) of thedifferent membranes evaluated.

Membrane Percentage (w/w) of components in membranes

PVC DOS NPOE TCP KTPB

A 33.2 66.3 0.5B 33.2 66.3 0.5C 33.2 66.3 0.5D 33 66 1E 33.5 66.2 0.3

Serial calibration graphs of the electrodes for ACh, Ch, H+, Na+,and K+ were made by adding with micropipettes consecutive smallvolumes of the corresponding solutions in 50.0 ml of water to cov-er the concentration interval 10�8–10�3 M for ACh, 2 � 10�6–10�3 M for Ch, and 10�5–10�3 M for the rest. The steady-statepotentials recorded were then plotted versus logarithmic valuesof the corresponding concentrations.

The data within the linear portion of the ACh calibrations werefitted to E = E0 + S logCACh to obtain the calibration parameters, E0

and S, standard potential, and slope of the electrode, respectively.The selectivity coefficients KACh;j were calculated by using the

separate solution method [10] by comparing the concentrationsof the primary (ACh) and interfering ion (j) that generate the samepotential.

Kinetic monitoring of the acetylcholine hydrolysis

A volume of 50.0 ml of the pH 7.5 phosphate buffer solution wastransferred into the thermostated vessel, the ACh-selective elec-trode and the reference electrode were immersed and the dataacquisition was started. Once the potential stabilized, an appropri-ate volume of ACh working solutions was injected. When the po-tential stabilized again, an appropriate volume of AChE stocksolution was injected. The potential kinetic curve was left to devel-op. The resulting curve was transformed into a concentration ki-netic curve using a calibration graph for ACh made in pH 7.5phosphate buffer solution following the calibration procedure de-scribed above. The corresponding initial slope, equivalent to theinitial rate of the enzymatic hydrolysis, was calculated from theconcentration kinetic curve.

Results and discussion

Development of an acetylcholine-selective electrode

The effect of the membrane composition on the potential re-sponse of the electrode to ACh was studied by varying the plasti-cizer and the concentration of the ionic additive used in themembrane construction.

The potential responses of PVC membranes plasticized withDOS, NPOE, or TCP (membranes A, B, and C of Table 1, respectively),all of them containing 0.5% of KTPB as ionic additive, for increasingconcentrations of ACh in water are shown in Fig. 1. As can be seen,the potential increased with the ACh concentration over the wholeconcentration range assayed. The membrane plasticized withNPOE displayed the highest slope and linear range, 58.6 mV/decand 10�6–10�3 M, respectively. In the case of the other mem-branes, containing DOS and TCP, the slopes were sub-Nernstian(46.4 and 42.3 mV/dec, respectively).

The responses of the electrodes built with the three membranesfor the ACh hydrolysis product (Ch) for the alkaline cations com-monly present in buffers (Na+ and K+) and for H+ are included inFig. 1. As can be seen, for any of the three membranes the responsetoward Ch was less intense than toward ACh. The selectivity coef-ficients, KACh;Ch, calculated by using the separate solution method[10], were �0.9, �1.8, and �0.9 for the membranes constructedwith DOS, NPOE, and TCP, respectively. These values indicated thatthe best selectivity corresponded to the membrane plasticizedwith NPOE, whose response toward ACh was about 60 times stron-ger than toward Ch, permitting the ACh concentration during theinitial state of its hydrolysis reaction to be monitored withoutinterference from the reaction product, Ch.

As regards Na+, K+, and H+, it can be seen that while the mem-branes plasticized with DOS and TCP showed a degree of potentialresponse toward them, the membrane plasticized with NPOE

Fig.1. Calibration graphs obtained for acetylcholine (ACh), choline (Ch), Na+, K+, and H+ using membranes containing 33.2% of PVC, 0.5% of KTPB, and 66.3% of the plasticizerTCP, NPOE, or DOS, membranes A, B, and C, respectively.

210 Potentiometric assay of AChE / M. Cuartero et al. / Anal. Biochem. 421 (2012) 208–212

showed practically no response. This means that the membraneplasticized with NPOE can be used for ACh monitoring in manypH buffers. Following the recommendations given by Bakkeret al. [10] to avoid biased values, no selectivity coefficients weredetermined for Na+, K+, and H+ since no Nernstian portion was ob-tained. Instead, the entire set of calibrations graphs obtained isshown in Fig. 1.

Taking all this into account, the membrane containing NPOE asplasticizer, membrane B, was selected for further experiments be-cause it showed the best sensitivity and selectivity.

The influence of the amount of ionic additive (KTPB) in theNPOE plasticized membrane on the potential response to AChwas also studied using membranes B, D, and E (Table 1). Mem-branes B and E, containing 0.5% and 0.3% of KTPB, respectively, pro-vided similar calibration graphs, while the response of membraneD, containing the highest KTPB content (1%), was the weakest. Ofthe two membranes B and E, we selected membrane B becauseof its longer lifetime, more than 2 months in continuous use.

Fig.2. Comparison of the acetylcholine concentration versus time profiles achievedby continuous dilution of 50.0 ml of 4 � 10�4 M acetylcholine solution with buffersolution (—) at different flow rates, 10, 5, and 2.5 ml min�1, and the correspondingprofiles obtained by monitoring the dilution with the ACh-selective electrode (s).

Taking all this into account, membrane B, containing 33.2% PVC,66.3% NPOE. and 0.5% KTPB, was selected.

The influence of pH on the potentiometric response of the se-lected ISE to ACh was studied by making calibrations at differentpH values within the range 5.8–8.9 by using phosphate buffers ofdifferent pH. No significant differences in the corresponding cali-bration parameters (E0 and S) were obtained.

Response characteristics of the electrode

The response characteristics of the electrode were obtained bymaking out calibration graphs for ACh in a 7.5 pH phosphate buffersolution since this is a convenient medium for carrying out theenzymatic hydrolysis of ACh. The calibration graph parameterswere calculated as described under Materials and methods. A lin-ear response was obtained over a wide concentration range,1 � 10�6–1 � 10�3 M, with a slope value of 59.2 mV/dec. The elec-trode displayed a small segment with a super-Nernstian responseat the lowest concentration. The detection limit (LD) was obtainedfollowing a criterion recommended by IUPAC to calculate thedetection limit for this type of response [11,12] from the cross sec-tion of the two linear parts of the response function, i.e., theextrapolated lines of the Nernstian and nonresponsive segmentsof the ISE response curve. A value of 1.4 � 10�7 M was obtained.

The reproducibility of these parameters was studied by makingsuccessive calibration graphs with the same membrane on thesame day (n = 3), with the same membrane on different days(n = 10) during a period of 40 days, and with two different mem-branes. The results obtained for the slopes were 59.1 ± 0.1,57.7 ± 1.5, and 58.0 ± 1.2 mV/dec, respectively, for E0 were382.4 ± 0.8, 373.0 ± 12.3, and 354.7 ± 31.3 mV, respectively, andfor LD were 1.5 � 10�7 ± 1.2 � 10�8, 3.3 � 10�7 ± 2.2 � 10�8, and1.9 � 10�7 ± 1.4 � 10�8 M, respectively. These results showed thatthe ISE presented excellent repeatability; however, the slight vari-ation in the reproducibility between days makes it convenient tocarry out a diary calibration of the ACh-selective electrode.

The drift of the electrode potential was studied at the lowestand the highest ACh concentrations of the linear range,(1 � 10�6–1 � 10�3 M) over a period of 10 min. The values ob-tained were 0.05 and 1.1 mV/h, respectively, low values which al-low the kinetic monitoring of the enzymatic ACh hydrolysis underthe conditions used in the present work without the need for drift

Fig.3. (A) Plot of potential versus time obtained by making successive injections of (1) 5 ll of 0.1 M ACh solution and (2) 10 ll of 25.8 U ml�1 AChE solution, into 50.0 ml ofphosphate buffer. (B) Corresponding concentration versus time plot calculated from the previous plot.

Fig.4. Plot of the initial reaction rates obtained with the ACh-selective electrode atseveral ACh initial concentrations fitted to the Michaelis–Menten equation.

Potentiometric assay of AChE / M. Cuartero et al. / Anal. Biochem. 421 (2012) 208–212 211

correction. This is because reaction rates registered in the presentwork were equal to or higher than 84.6 mV/h.

To check the suitability of the ACh-selective electrode for fol-lowing a continuous decrease in the ACh concentration, the elec-trode was immersed in a 4 � 10�4 M ACh, 0.01 M buffer solutionof pH 7.5. Once the potential was stabilized, a continuous dilutionwas made by adding buffer solution at a constant flow rate using adouble piston pump. The potential was monitored and the corre-sponding values were transformed into ACh concentration, as de-scribed under Materials and methods. The concentration vs timeplot obtained at three different flow rates is shown in Fig. 2, to-gether with the theoretical values calculated from the dilution pro-gram. As can be seen, the corresponding plots at each flow rate aresuperimposed, indicating that the ISE is able to follow the contin-uous decrease in ACh concentration.

Potentiometric monitoring of acetylcholinesterase-catalyzedhydrolysis of acetylcholine

Since the optimal pH and temperature for AChE are about 7.5and 28 �C [13,14], the enzymatic hydrolysis of ACh was carriedout using a pH 7.5 phosphate buffer solution and at a constanttemperature of 28 �C.

A typical kinetic-potentiometric plot of the AChE-catalyzedhydrolysis of ACh obtained as described under Materials and meth-ods is shown in Fig. 3A. Arrow 1 corresponds to the injection of analiquot of the ACh stock solution in the phosphate buffer solution,which was followed by a fast increase of the potential signal toreach a constant value, corresponding to the electrode response to-ward ACh. Arrow 2 corresponds to the injection of an aliquot of theAChE stock solution, after which the potential decreased due to adecrease in the concentration of ACh in the medium as a resultof the hydrolysis. Fig. 3B shows the corresponding kinetic curveobtained by transforming the potential data into ACh concentra-tion using the calibration graph.

Kinetic data analysis

The influence of initial substrate concentration, [ACh]0, on thehydrolysis kinetic was studied using a fixed concentration of AChE(5.4 � 10�3 U ml�1) and varying the substrate concentration in therange 8 � 10�7–5 � 10�4 M. The potential was monitored and con-verted into ACh concentration as described above. The initial reac-tion rates obtained were plotted vs [ACh]0 and fitted to the

Michaelis–Menten equation (Fig. 4). As can be seen, a good fit(r = 0.9993) was found. This fit provided values of4.7 � 10�5 ± 0.3 � 10�5 M and 8.3 � 10�8 ± 0.2 � 10�8 M s�1 forthe characteristic parameters of the enzymatic reaction, Km andVmax, respectively.

The effect of AChE concentration on the initial rate of the enzy-matic reaction was studied using an ACh concentration of4 � 10�4 M, which is in the plateau of the graph in Fig. 4, and vary-ing the concentration of enzyme in the range of 2 � 10�5 to3.8 � 10�1 U ml�1. A linear relationship (r = 0.9994) was obtainedin a concentration range of almost four decades. This linear regres-sion might be used for the determination of AChE in problemsamples.

Conclusions

The selectivity for acetylcholine over choline, the fast response,and low drift of the plasticized polymeric membrane ACh-selectiveelectrode developed permit the hydrolysis of ACh to be monitored.The electrode can be used for AChE activity assay as a convenientalternative to other techniques.

212 Potentiometric assay of AChE / M. Cuartero et al. / Anal. Biochem. 421 (2012) 208–212

Acknowledgments

The authors gratefully acknowledge the Ministerio de Ciencia yTecnología, Spain (project CTQ2008-04806/BQU). M.C. thanks Uni-versity of Murcia for a grant.

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