development of a disposable organophosphate biosensor

Development of a Disposable Organophosphate Biosensor

Melissa Espinosa, Plamen Atanasov, and Ebtisam Wilkins*

Department of Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, New Mexico 87131, USA

Received: February 18, 1999

Final version: April 29, 1999

Abstract

A screen-printed enzyme electrode has been developed for the determination of organophosphorus pesticides based on the detection ofcholinesterase inhibition by analytes. A cholinesterase enzyme is co-immobilized with choline oxidase peroxidase on the surface of a screenprinted carbon electrode. The determination of cholinesterase activity is based on a sequence of enzymatic reactions: butyryl-cholinehydrolysis catalyzed by cholinesterase; choline oxidation catalyzed by choline oxidase and the formation of hydrogen peroxide; detection ofhydrogen peroxide via peroxidase catalyzed electron transfer. Electroreduction of hydrogen peroxide causes an electrode potential shift dueto a decrease of the reaction overvoltage. The rate of electrode potential increase is proportional to the activity of cholinesterase. Theelectrodes allow rapid quantitative assay of cholinesterase inhibitors, such as organophosphate pesticides, with a low detection limit in sub-micromolar concentration ranges with an overall assay time of 20 minutes. The technique employs low cost disposable sensing elements.Reproducibility of the enzyme sensor technology and the shelf lifetime of the disposable biosensors are evaluated. Applicability of the assayis illustrated by detection of common of organophosphorus pesticides.

Keywords: Organophosphorus pesticide determination, Environmental biosensor, Direct electron transfer

1. Introduction

Organophosphorus compounds (OPCs) are signi®cant envir-onmental and food chain pollutants [1, 2] due to their intensiveuse as pesticides in agriculture. Chromatographic techniques aregenerally the most widely used methods for determination ofOPCs. These techniques allow selective and quantitative deter-mination. However, they have a number of disadvantages limitingtheir applications primarily to laboratory settings and prohibittheir use for rapid analyses under ®eld conditions. Currently, theavailable chromatographic equipment is complex and expensiveand can only be operated by highly trained technicians. Pre-treatment and assay procedures are lengthy, hence fast analysesare impossible in most cases. Environmental issues require sen-sitive, selective and quantitative methods capable of fast detec-tion of pollutants in ®eld conditions: streams, ground, wastewaters, in the soils and plants and in food as well [1].

1.1 Electrochemical Biosensors for OPCs

The most general approach for determination of OPCs is basedon their inhibition of the activity of cholinesterase enzymes[3, 4]. Cholinesterases are hydrolases catalyzing the hydrolysisreaction of a particular choline ester (butyrylcholine, acetyl-choline, etc) to the corresponding carboxylic acid with therelease of choline:

R-choline� H2O ÿ!Cholinesterase

�R�acetyl-;butyryl-; etc:�R-COOH� choline

�1�The presence of low concentrations of inhibitors strongly and

speci®cally affect enzyme activity. Therefore, by measuring theenzyme activity the concentration of the organophosphoruscompounds can be assayed.

Electrochemical methods for cholinesterase activity assay thatare based on pH-shift potentiometry have been described [5±11].Conventional pH electrodes [7±11] and pH sensitive ®eld effecttransistors [5, 6] were employed as transducers coupled with

cholinesterase enzymes. The main disadvantage of the pH-shiftbased method is a strong requirement for low buffer capacity ofthe sample. In addition, the sensitivity of pH based analyticaltechniques, in general, is less than that based on amperometricassay. The theoretical threshold of pH based assay methods is aslow as 58 mV per decade of analyte concentration. Ion-selectivemembranes [12] and mediator-assisted potentiometry [13] havealso been proposed for assays of cholinesterase inhibitors.

A number of articles describe techniques for determination ofcholinesterase activity based on amperometric measurement ofproducts formed as a result of enzymatic hydrolysis (Reaction 1).In this case, arti®cial (butyryl- or acetylthiocholine) cholin-esterase substrates are used. Thiocholine, formed as a result ofcholinesterase-catalyzed hydrolysis can be measured amper-ometrically on a platinum electrode [14, 15] or mercury electrode[16]. Analyses based on thiocholine determination employing anelectrode modi®ed by cobalt phthalocyanine [17±22] or cobalttetraphenylporphyrin [23] have been described. Enzymatichydrolysis of aminophenyl acetate leads to formation of amino-phenol. The technique of determination of cholinesterase activitybased on sensitive amperometric detection of aminophenol hasbeen described elsewhere [24, 25].

A popular method of determination of cholinesterase activityis based on coupling a cholinesterase enzyme with a cholineelectrode [26±39]. This coupling results in two consecutiveenzyme reactions, ®rst catalyzed by cholinesterase (Reaction 1),and second, catalyzed by choline oxidase:

choline� O2 ÿ!Choline oxidasebetaine� H2O2 �2�

The choline electrode usually consists of an amperometrictransducer and immobilized choline oxidase. The most frequentlyused electrochemical transducers are hydrogen peroxide elec-trodes [26±28, 33±36]. The amperometric signal in this case isdue to electrooxidation of hydrogen peroxide, which is thecoproduct of the enzymatic choline oxidation (Reaction 2).Oxygen amperometric sensors (Clark-type electrodes) have beenalso used as basic transducers for choline electrode construction[29, 32, 37]. The signal in this case is based on the reduction ofmolecular oxygen which is the coreactant in Reaction 2. Redox

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mediators: hexacyanoferrate [26], ferrocene derivatives [38] andtetracyanoquinodimethane [39] have also been used in the con-struction of choline electrodes.

In order to facilitate hydrogen peroxide detection, a thirdenzyme, horseradish peroxidase has been employed as a catalystfor hydrogen peroxide reduction with the enzymatic oxidation ofan electrode donor substance. Redox mediators have been used inthis case for cathodic detection. An amperometric sensor forcholine based on electron transfer between horseradish perox-idase and a redox polymer has been described [19, 20]. The useof redox mediators facilitate electron exchange but lead to systemcomplications.

1.2. OPC Assay Based on Direct Electron Transfer

The coupling of horseradish peroxidase acting as a hydrogenperoxide detector, with enzymes forming hydrogen peroxide as aco-product of analyte oxidation, has been used in earlier designsof potentiometric sensors based on direct mediatorless bio-elec-trocatalysis for determination of glucose [40] and lactate [41].

In such systems peroxidase catalyzes the reaction of hydrogenperoxide electro-reduction. The mechanism of this reaction ismediatorless, based on direct electron transfer from electrode tosubstrate molecule via the enzyme active site [42]:

H2O2 � 2eÿ � 2H� ÿ!Horseradish peroxidase on electrode2H2O �3�

Therefore, the enzyme peroxidase acts as an electrocatalyst(bio-electrocatalyst) lowering the overvoltage for hydrogen per-oxide reduction at the electrode. As a result a signi®cant increaseof the electrode potential (DE), anodic shift towards the equili-brium potential of the redox couple H2O2=H2O, occurs. The rateof this increase, (DE=Dt), is proportional to the hydrogen per-oxide production rate. Dynamic potentiometric signal (dE=dt)provides an extremely sensitive mean of monitoring hydrogenperoxide production rates.

A potentiometric electrode based on direct mediatorless bio-electrocatalysis for determination of choline and butyrylcholinehas been described in our earlier work [43]. Three enzymes:peroxidase, choline oxidase, and butyrylcholinesterase are co-immobilized on the electrode surface. Choline oxidase catalyzesthe reaction of choline oxidation accompanied with hydrogenperoxide formation (Reaction 2). Hydrogen peroxide acts as asubstrate of the enzyme peroxidase. Using the two enzymesperoxidase and choline oxidase in a coupled system allowsdetermination of choline concentration as a result of the con-secutive enzyme Reactions 2 and 3. In the system choline con-centration assay is based on the measurement of the rate ofelectrode potential increase (DE=Dt). The ability of the electrodeto detect choline over a wide concentration range allows its usefor measurement of butyrylcholine concentration in assay sys-tems employing coupled butyrylcholinesterase. The butyryl-choline esterase enzyme causes choline production due toenzymatic hydrolysis of butyrylcholine (Reaction 1). This elec-trode was applied as a sensing element for detection of organo-phosphate inhibitors of butyrylcholinesterase [43, 44].

The advantages of this technology over other disposableelectrochemical systems [22, 23] are associated ®rst with thepotentiometric method of signal generation which providesindependence on the electrode surface area and resulting in sig-ni®cant improvement in reproducibility of the individual sensor'sresponse parameters. In addition, the bioelectrocatalytic signaltransduction provides extreme sensitivity [43, 44] towards

changes in cholinesterase activity in much lower limits ofdetection. The derivative potentiometric protocol adopted for thestudy (measurements of DE=Dt) contributes to the sensitivity ofanalysis and provides a mean to obtain results before reaching thesteadystate, thus expediting the assay. Present work describes thedevelopment of this sensing element as a biosensor prototype forenvironmental analysis of organophosphorus pesticides. It bringsthe concept idea (discussed in [43]) to a technological solutionaddressing the issues of sensor design, optimization, technolo-gical and analytical characterization of the sensing elements, theirstorage lifetime and application in the assay of several commonpesticides.

2. Experimental

The biosensor employs a multi-enzyme system arranged in theform of a disposable miniature electrode. Figure 1 presents a topview of the sensor prototype. The electrode is manufactured by ascreen-printing technology which allows mass fabrication of thesensing element with good reproducibility. The three enzymes:peroxidase, choline oxidase and cholinesterase are co-immobi-lized on the screen-printed carbon electrode surface. This tri-enzyme system provides a mean for determination of cholin-esterase activity as a result of the consecutive enzyme reactions[1±3]. Figure 2 presents a schematic of the consequence of theenzymatic reactions at the electrode surface: the sequence ofsubstrate-to-product transformations concluded by the electro-reduction of hydrogen peroxide, which is the signal generatingreaction.

2.1. Reagents and Electrodes Preparation

Peroxidase (EC 1.11.1.7, 1100 U=mg, from horseradish),choline oxidase (EC 1.1.3.17, 10 U=mg, from Arthobacter

globiformis), butyrylcholinesterase (EC 3.1.1.8, 300 U=mg, from

Fig. 1. Schematic view of the screen-printed sensor prototype.

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Electroanalysis 1999, 11, No. 14

horse serum), choline, butyrylcholine, trichlorfon (2,2,2-thri-chloro-1-hydroxyethyl phosphonic acid) and glutaraldehyde wereproducts of Sigma Chem. Co. (St. Louis, MO). Pesticides:dichlorvos (2,2-dichlorovinyl dimethylphosphate), parathion(0,0-diethyl-0-p-nitrophenyl phosporothioate) and diazinon (0,0-diethyl-0-[2-isopropyl-4-methyl-6-pyridimy]phosporothioate)were obtained from Supelco Corp. (Bellefonte, PA). All otherreagents used were of analytical purity grade.

The screen-printed carbon electrodes used in this study werefabricated at Dr. Joseph Wang's laboratory, New Mexico StateUniversity (Las Cruces, NM). Ercon carbon ink (referred in thestudy as ink Type A) or Acheson ink (Type B carbon ink) wereused to fabricate the screen-printed carbon electrodes. A group often electrodes were printed onto an alumina ceramic plate(33.36100 mm) through a patterned stencil. The printed elec-trode area was 1.5630 mm. The electrodes were subsequentlydried for 30 minutes at 100 �C and allowed to cool to the roomtemperature. A layer of an insulator (Ercon) was then printedusing the same procedure as above and allowed to dry for 30minutes at 100 �C (see Fig. 1).

The electrodes were cleaned with alcohol and distilled waterand then depolarized in phosphate buffer solution (PBS) atÿ0.8 V (vs. Ag=AgCl) until the establishment of a constantresidual current (ca. 30 minutes). The screen-printed carbonelectrode was then doped with 10mL of a solution containingperoxidase, choline oxidase and butyrylcholinesterase (1 mg ofeach in 10mL of PBS buffer solution). The electrode was thensoaked for 30 minutes in 5 % (v=v) solution of glutaraldehyde inPBS at room temperature and stored overnight at 4 �C. Thisresulted in the preparation of a trienzyme electrode with the threeenzymes co-immobilized on the electrode surface by glutar-aldehyde cross-linking. The electrode was then allowed to dry atroom temperature for 3 hours. The electrodes were stored dry at4 �C.

2.2. Measurements Procedure and Instrumentation

Measurements were performed in a cell containing 1 mL ofPBS. The reaction was started by adding a 20mL aliquot ofsubstrate (butyryl choline) into the cell. Potential changes weremeasured by means of a high impedance voltmeter (DM 2010,

Keithley Instruments, Inc., Cleveland, OH). As a reference, aAg=AgCl electrode was used. The potentiometric output (elec-trode response) was obtained as the rate of potential change(mV=min) and registered on a X �t� ÿ Y recorder (Omnigraphic-2000, Houston Instruments Inc, TX). The electrode response fora particular substrate concentration characterizes the electrodeactivity.

Trichlorfon (a low-toxic laboratory model OPC) was used asa model analyte for the optimization of the inhibitor assay pro-cedure. The procedure consists of three steps:

i) incubation stage: precalibration of the sensing element byobtaining its response to a standard concentration of butyryl-choline as dE=dtjin at certain pH level and temperature(procedure optimization parameters);

ii) inhibition stage: incubation of the sensing element in asolution containing inhibitor at certain pH level and tem-perature (procedure optimization parameters);

iii) detection stage: obtaining the resulting sensor response afterinhibition as @E=@tjres and normalizing it to the initialresponse to obtain the sensor response (SR) which is afunction of the inhibitor concentration (Cinh) as:

SR�Cinh� � f�@E=@tjin� ÿ �@E=@tjres�g=�@E=@tjin� �4�Following the initial measurement during the incubation stageand after the inhibition stage, the electrode was washed by dis-tilled water, immersed into the measuring cell containing freshPBS and depolarized for 30 seconds by forced polarization inorder to return the electrode potential to the background value.The potential of depolarization of 0.0 mV (vs. Ag=AgCl refer-ence electrode) was used. A potentiostat (CV-1B, BioanalyticalSystems, Inc., West Lafayette, IN) was used for electrode depo-larization connected to the trienzyme working electrode,Ag=AgCl reference electrode and an auxiliary Pt electrode. Afterdepolarization the electrode was ready to perform next mea-surement: detection stage.

3. Results and Discussion

3.1. Electrode Materials and Immobilization of the

Enzymes

Screen-printed carbon electrodes were made using two typesof carbon inks as basic electrode material. The surface propertiesof these materials were studied and modi®ed in order to allowsimple immobilization procedure of the enzymes, preferably byphysical sorption. Cyclic voltamperometry was used as a mainmethod for electrode surface investigations. The cyclic voltam-mograms of the Ercon carbon ink electrodes (Type A) andAcheson ink printed electrodes (Type B) are shown in Figure 3. Itcan be seen that the Type A carbon expresses some quasi-reversible redox transformations (manifested as a pair of cathodicand anodic peaks) probably corresponding to some iron-con-taining substances in the carbon matrices. This is a typicalresponse for some activated carbons used as electrocatalysts.Carbon Type B does not demonstrate any redox transformationsand is considered a graphite-type material. Immobilization con-ditions on these two types of carbon surfaces were found to bedifferent. Type A carbon (activated carbon) demonstrated thecapability to immobilize all three enzymes by simple physicalsorption without any pretreatment.

Fig. 2. Consequence of the enzymatic reactions at the electrode surface.

Disposable Organosphosphate Biosensor 1057


In order to increase the immobilization capacity of carbonType B, an electrochemical pretreatment (activation) has beenundertaken. Electrodes were polarized at � 2.0 V for 5 to 10minutes (at pH 7.4 phosphate buffer solution) which lead tooxidation of the electrode surface resulting in formation of qui-none-type oxygen-containing surface groups and increased in thewet surface area. This result was con®rmed by the cyclic vol-tammogram of the Type B electrode after activation (see Fig. 3)which demonstrates both the increasing capacitance current andthe wide maxima of surface groups redox transformations. Aftersuch a procedure, Type B carbon (graphite) signi®cantlyincreased its capability to immobilize the three enzymes bysimple physical sorption similarly to Type A carbon. Electrodesof both types were cathodically treated (at 7 0.8 V for 5 min-utes) for surface cleaning before enzyme immobilization.

Immobilization of the enzymes on the electrode surface is acritical step in biosensor development. In order to simplify thefabrication procedure of the sensing element, a mixture of thethree enzymes was co-immobilized directly on the surface in apresumable excess of enzyme loading. Effectively only perox-idase has to be in intimate contact with the carbon electrodesurface in order to achieve the optimum conditions for directelectron transfer (Reaction 3). Other enzymes (choline oxidaseand butyrylcholinesterase) are immobilized on the electrode ason an inert carrier. Co-immobilization of the enzymes from amixture provides also conditions for close proximity of theenzymes in the chemically coupled assay scheme (Reactions 1±3). Simple physical sorption (the technologically preferred

method) and adsorption followed by additional glutaraldehydebinding were tested on both screen printed electrodes Type A andType B. Both techniques provided satisfactory reproducibility ofthe results and comparable shelf life times for the sensing ele-ments. The investigations con®rmed that the trienzyme electrodesremain active when stored (at � 4 �C) for more than one month(see below). Some preference could be given to the methodologybased on physical sorption followed by glutaraldehyde treatmentin order to ensure better shelf life times. Carbon Type A isadvantageous because it does not need the electrochemical pre-activation procedure for enhancement of its adsorption proper-ties.

3.2. Assay Reproducibility

Figure 4 presents typical recordings of the consecutive mea-surements of @E=@tjin and @E=@jres as described in the inhibitorassay procedure. The assay allows determination of OPCs insubmicromolar concentration ranges. The overall assay timeincluding all operation stages is 20 minutes. Technology forelectrode fabrication was evaluated in terms of its reproducibility.

Figure 5 demonstrates the responses of 8 individual sensors,from the same manufacturing batch, to a given standard con-centration of trichlorfon (model OPC). The error bars associatedwith the individual measurements represent the measurementerror. It can be seen that the distribution of the individualresponses is well grouped and the relative uncertainty of the

Fig. 4. Recordings of the electrode potential shift a) before inhibition[@E=@tjin] and b) after inhibition [@E=@tjres] during exposure to increasingconcentrations of OPC. Results obtained with 3 individual sensors arepresented.

Fig. 3. Cyclic voltammograms of the electrodes screen-printed with twotypes of carbon ink: a) Type A and b) Type B.



assay does not exceed 15 % (the 15 % of the average signal valueinterval is shown as shaded zone in the graph which correspondsto the SD for the 8 individual measurements).

Series of sensors (manufacturing batches) were fabricatedwhile varying the number of the electrodes in the series (from 3to 15) and the number of electrodes which undergo simulta-neously the electrochemical pretreatment (depolarization steps inthe fabrication process). The limitations of the manufacturingprocess were assessed demonstrating the advantages of largerbatch production. The overall reproducibility of the sensorsanalytical characteristics was in the acceptable level of 15 %deviation from the expected sensitivity within a batch. Batch-to-batch reproducibility was found to be within 25 % of theexpected value.

3.3. Optimization of the Assay

Electrodes were incubated in solutions during the incubationstage and during the inhibition stage at different pH values (andconstant temperature) and the sensor response was tested. Resultsof this investigation are presented in Figure 6. Figure 6a illus-trates the effect of pH variation during the incubation stage on thesensor response. A plateau of high sensor response is observedbetween pH 5.8 and pH 10 followed by a decrease in response athigher pH values. It is probable that the protonized form of tri-chlorfon inhibits butyrylcholinesterase faster than the deproto-nized one. Figure 6b represents the data obtained when the pHwas varied during the inhibition stage. In the low pH range,practically no inhibition is observed even for high concentrationsof the inhibitor. The increase in the pH of the inhibitor solutionresults in a signi®cant increase in inhibition. This is apparently,due to a deprotonized form of butyrylcholinesterase which is lessresistant to inhibition than the protonized form. Increasing pHbeyond 8.4 leads to a rapid decrease in inhibition. Figure 6cpresents a subtraction of the data from Figure 6a and b illus-trating the optimal pH conditions for the assay performance.Maximum inhibition is observed for pH 10. As a result of theinvestigation of the dependence of pH of the inhibitor solution onthe inhibition effect of trichlorfon pH 10 was selected for theOPC assay optimization at different temperatures.

An increase in temperature of the inhibitor solution enhancedthe inhibition effect. However, a temperature above 45 �C leads tothermal inactivation of the enzymes. Electrodes were incubated atdifferent temperatures (in thermostated cell) during the incuba-tion stage and during the inhibition stage and the sensor responsewas tested at pH 10. Results are presented at Figure 5. Figure 7aillustrates the effect of temperature variation during the incuba-tion stage on the sensor response. It was found that temperaturechange during the incubation stage from ambient (23 �C) toelevated (43 �C) does not signi®cantly affect the sensor response.Figure 7b represents the data obtained when the temperature wasvaried during the inhibition stage. The sensor response decreaseswith increase in the temperature from 23 �C to 37 �C anddemonstrates a local maximum at 40 �C. Figure 7c presents asubtraction of the data from Figure 7a and Figure 7b indicatingthe optimal temperature range for the assay. It should be notedfrom the practical point of view that the temperature dependenceis expressed as a plateau of high sensor response from 32 �C to40 �C which provides an advantage or relative insensitivity of theassay performance to temperature variations. When incubatingthe sensor at 40 �C for 10 min., a detection limit of 5 nM(1.3 ppb) concentration of trichlorfon was achieved.

3.4. Disposable Sensor Shelf Lifetime

The sensor shelf lifetime has been investigated in a series ofexperiments. Batches with uniformly fabricated sensors werekept in de®ned conditions: in refrigerator at 4 �C, at room tem-

Fig. 5. Intrabatch reproducibility: responses of 8 individual sensors,from the same manufacturing batch, to a given standard concentration oftrichlorfon.

Fig. 6. Effect of pH during incubation (a) and inhibition (b) stages of theassay; overall effect (c).



perature of 20 �C and in an incubating box at 40 �C. At particulartime intervals sensors from those batches were tested with amodel OPC (trichlorfon) to determine the sensitivity of the sensorresponse to the inhibitor. Having in mind that the sensors aredisposable, the shelf lifetime of the electrodes made by thistechnology was determined as the period of time within whichthe sensor sensitivity obtained from such a test does not falloutside the deviation range of the intra-batch reproducibility(15 %). Dropping the sensitivity below 15 % from the averagewas considered as a manifestation of the decline in the sensorperformance. The experimental results of this study are presentedin Figure 8. This ®gure shows the relative value of the initialsensor response (compared to the ®rst measurement made with asensor from a given batch) as a function of the storage time. Itcan be seen from Figure 8 that shelf lifetime of the sensors, whenstored at elevated temperature (40 �C) were shorter then two days.During the ®rst measurement, the sensor results showed a lowersignal than the level of expectation. While stored in ambienttemperature (20 �C) the sensors demonstrated a shelf lifetime ofone week. Sensitivity of the sensors remained practically constantfor more that 56 days when kept in refrigerated conditions (4 �C).Experiments with sensor batches stored at 4 �C are still in pro-gress, showing more than two months of shelf lifetime. Theseresults can be considered acceptable from the practical point ofview as pertinent to a system where no special packaging wasused for sensor preservation. Introducing packaging technologiesas a part of the development of this assay system could sig-ni®cantly improve shelf lifetime of the sensors.

3.5. Organophosphate Pesticides Assay

Figure 9 demonstrates the calibration plots for OPCs studied inthis work: the model low-toxic OPC trichlorfon (Fig. 9a) andsome common organophosphate pesticides: parathion (Fig. 9a),dichlorvos (Fig. 9a) and diazinon (Fig. 9b). These calibration

Fig. 8. Shelf life-time of the sensors: dependence of the relative value ofthe initial sensor response (compared to the ®rst measurement made witha sensor from a given batch) vs. the storage time at different tempera-tures.

Fig. 9. Calibration plots for a) trichlorfon, parathion, b) dichlorvos anddiazinon. Each sensor response is an average of four individual mea-surements obtained with different sensors from the same manufacturingbatch (SEM are shown).

Fig. 7. Effect of temperature during incubation (a) and inhibition (b)stages of the assay; overall effect (c).



plots are obtained under conditions (time of inhibition, pH andtemperature) optimized with the model analyte trichlorfon.

The lowest concentration of pesticide samples assayed with10 min of incubation of the electrode in inhibitor containingsolution was 50 ppb. This resulted in approximately 20 % of therelative inhibition signal. The detection limits for different OPCsare different because of the different ability of OPCs to inhibitcholinesterase. The detection limits for different OPCs can differin 3 orders of magnitude. In general, more toxic OPCs inhibitcholinesterase stronger than less toxic ones, and the detectionlimit for more toxic OPCs is lower than that for less toxic OPCs.Having in mind that the detection limit of our sensor for tri-chlorfon (less toxic OPC) is close to 1 ppb, one can expectsimilar or better performance with respect to dichlorvos to beachieved.

Figure 9 predicts much better performance of our systemcompared with the literature data. For example, trichlorfondetection by means of ISFET had a reported limit of detection ofca. 250 ppb [5], while conductometric sensor assay registeredtrichlorfon at ca. 25 ppb [6], still an order of magnitude higherthan the described sensor. An amperometric sensor was used todetect dichlorvos with a limit of detection of 350 ppb [21] and apotentiometric (pH-sensitive) sensor was shown to detect para-thion at 39 ppm and diazinon at 35 ppb [9].

The overall assay time includes the incubation of the electrodein the analyte solution and the duration of measurements ofelectrode activity before and after the incubation step. Theduration of the measurements of electrode activity does notexceed 5 min for each assay, therefore, the overall assay time isabout 20 min. Further development of electrode manufacturing inorder to improve the reproducibility of electrode performancewithin one manufacturing set of electrodes will avoid the need formeasurement of initial activity of each electrode. In this case, oneelectrode from the set can be used for determination of the initialactivity of the whole set. This will result in only one measure-ment of electrode activity for inhibitor assay and will lead to adecrease of the assay time.

4. Conclusions

This work demonstrates the potential for application ofpotentiometric enzyme electrodes based on mediatorless enzymeelectrocatalysis for fast and sensitive assay of organophosphoruspesticides. The sensing element based on screen-printed carbonmaterial permits mass fabrication of the electrodes at a low costwhich is essential for the disposable sensor concept. The bio-sensor does not require any low-molecular weight mediator andcan be arranged as an `all-solid-state' device. Such electrodes,being based on a potentiometric principle, are suitable for min-iaturization and arrangement in multi-sensor array for simulta-neous determination of several analytes in real samples. Thiswork presents the optimization of the assay parameters: itestablishes the optimal pH and temperature range for maximalelectrode response. The assay allows determination of OPCs insub-micromolar concentration ranges with an overall assay timeof 20 minutes. The enzyme sensor fabrication technologydemonstrates very long intrabatch reproducibility of the mainsensor analytical parameters. This is especially essential for atechnology aimed to disposable sensors allowing calibration to bedone by measurement of the performance of one sensor from thebatch. The shelf lifetime of the disposable sensing elements is

close to two months, while stored unpacked in refrigeratedconditions. Applicability of the assay is illustrated by detection ofseveral common organophosphorus pesticides. This sensortechnology provides a mean for estimation of the total OPCpesticide contamination of a given environmental sample, whichis a typical feature of all techniques based on cholinesteraseinhibition. It gives, however a promise for extremely low detec-tion limits which makes this technology advantageous especiallyin the context of increasing environmental regulations andrequirements for rapid detection of trace amounts of pesticides.Similarly to other disposable electrodes, reported elsewhere, thistechnology may ®nd application in rapid screening and ®eld siteenvironmental monitoring, thus reducing the volume and the costof the environmental analyses.

5. Acknowledgements

This research was supported in part by a grant from theDoE=Waste-Management Education and Research consortium ofNew Mexico. Authors express their gratitude to Prof. JosephWang (Department of Chemistry and Biochemistry, New MexicoState University, Las Cruces, NM) for providing the basic(untreated) screen-printed electrodes for the sensor construction.

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development of a disposable organophosphate biosensor

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