Detection of organophosphate and carbamate pesticides using disposable biosensors based on chemically modified electrodes and immobilized cholinesterase

281

Anafytzca Chca Acta, 269 (1992) 281437 Elsevler Science Pubhshers B V , Amsterdam

Detection of organophosphate and carbamate pesticides using disposable biosensors based on chemically modified electrodes and immobilized cholinesterase Petr Sklldal Departmentof Bwchemrsiry, Masaryk Unwersrty, KotI&skL 2, 61137 Bmo (Czechoslovakd (Recerved 7th Apnl1992, revised manuscrrpt received 23rd June 1992)

AhStIBCt

An ampezometnc blosensor for the detection of organophosphate and carbamate pesticides was constructed as a disposable stnp contaming a cobalt phthalocyanme-modlfed carbon composite electrode and a cross-lmked chohnesterase layer With butyrylthmchohne as substrate, enzymatlcally produced thmchohne was oxldned at + 250 mV The steady-state current, I,, was used as a measure of the enzyme actlvlty In the presence. of pestlades, an u-reversible mhlbltlon of chohnesterase occurred, resulting m a decrease m the rate of current change, dI/dr The ratio (dl/dr)/Z,, was used for evaluations The Influence of the chohnesterase loading and the cholmesterase to glutaraldehyde ratio on the blosensor response was studled and the measuring condltlons (pH, temperature, substrate concentration) were optmnzed Detection hmlts of 0 30, 12 and 11 nmol 1-l for paraoxon, dlchlorvos and carbaryl, respectively, were achieved The time of mhlbltlon varied from a few seconds (high pestlclde concentrations) to 6 mm reqmred for rehable measurements at levels close to the detection hmlt When the analysis was performed by 10 mm premcubatlon of the bmsensor or free chohnesterase mth sample, paraoxon concentrations above 3 0 nmol I-’ could be detected Keywords Amperometry,

Blosensors,

Carbamate

pestlades,

Momtormg of organophosphate and carbamate pestlades IS of considerable importance These pestlcldes are preferred m agnculture for their relatively low persistence m the envlronment, but some of them exhlblt fairly high acute tomcIty [l] Consequently, detectlon systems are required for the protection of hvmg orgamsms and for the ldentlficatlon of pesticide resrdues m food products Blosensors for the detection of these substances are mostly based on acetylchohnesterase (AChE) (E C 3 1 17) or cholmesterase (ChE) Correspondence to P Sklidal, Department of Bmchemstry, Masaryk Umversity, KotlGski 2, 611 37 Bmo (Czechoslovalua) 0003-2670/92/$05

Organophosphorus

pestlcldes,

Pestlcldes

(E C 3 1 1 8) In the reaction of chohnesterases with mhlbltors, first an enzyme-mhlbltor complex IS formed, which IS subsequently converted mto an inactive phosphorylated (or carbamylated) form of enzyme [2] This mechanism explams the mhlbltory action of pesticides and it can also be used as a basis for blochemlcal analysis Chohnesterases as molecular recogmtlon elements can be combined with a variety of transducers Thus, pH electrodes [3-71 and pH-sensltlve ISFETs [8,9] are very popular, various optical spectrophotometrlc [lo-121 and fluorlmetrlc [ 131 flow-through systems have been utilized and conductlmetrlc [ 141, voltammetrlc [ 151 and plezoelectrlc [16] systems have been Investigated When ChE or AChE 1s used together with choline 0x1-

00 0 1992 - Elsevler Science Publishers B V All nghts reserved

282

dase, the resulting enzyme system can be linked with 0, or H,O, probes, thus provldmg amperometrrc sensors suitable for analyses for pesticides [ 17- 191 When acetyl- or butyrylthlocholme (BTCh) 1s chosen as a substrate, thlochohne produced m the enzymatic reaction can be anodlcally oxldlzed on platinum [20,21] or mercury [15] electrodes, but a chemically modified carbon electrode (CME) 1s better suited for this purpose [22-241 So far, tetracyanoqumodlmethane [22] and cobalt phthalocyanme (CoPC) [23,241 have been tested as possible modifiers, both of these mediators permitted the oxldatlon of thlochohne at potentrals substantially lower m comparison with metallic electrodes The previous versions of CoPC composite electrodes were linked with a separate enzyme membrane In this work, a compact disposable enzyme electrode based on CoPC-CME with increased sensltlvlty was developed and various approaches to the determination of pesticides were mvestlgated

EXPERIMENTAL Chemicals

Chohnesterase from horse serum (specific actrvlty 147 nkat mg-’ protem) was obtained from USOL (Prague), paraoxon from Sigma (St LouIs, MO) and dlchlorvos and carbaryl were kindly provided by Dr B Safiii (Institute of Analytical Chemistry, Brno) Cobalt phthalocyamne was supplied by Aldrich (Milwaukee, WI), glutaraldehyde by Reanal (Budapest) and graphite powder (particle size < 50 pm> by Merck (Darmstadt, Germany) All other reagents were supplied by Lachema (Bmo) Construction of bzosensors Plastic sheets (10 x 35 x 0 8 mm) covered with

copper foil (commonly used for electronic hnks) were used as a support for the blosensors The copper foil was partially etched away m order to obtain the desired conductwe pattern, 1 e , a 5-mm diameter disc at one end Joined with a l-mm wide conducting line with the opposite end of the strip The line was isolated with a layer of epoxy glue,

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Acta 269 (1992) 281-287

leaving free only a small end part for contact The remammg free surface of disc was then completely covered with an electrode layer, which was prepared by deposltmg 10 ~1 of a suspension obtained by mung 5 mg of CoPC, 100 mg of graphite powder and 300 ~1 of 15% acetylcellulose solution m acetone-cyclohexanone (1 + 0, slmllarly to [25] The resulting electrode had a diameter of 8 mm The enzyme layer was made by spreading on the dried electrode surface 20 ~1 of 50 mmol 1- ’ phosphate buffer solution (pH 7 3) containing 125 pg of ChE and 25 pg of glutaraldehyde (the optumzed composltlon, see below) Complete blosensors were ready for use the next day, and were stored dry at 4°C Instrumentation and procedures

A three-electrode system, conslstmg of an Ag/AgCl/saturated KC1 reference electrode, platinum counter electrode and the blosensor as workmg electrode, was used for amperometrlc measurements The potential of the workmg electrode was set at 250 mV using an ADLC 2 detector (Laboratory Instruments, Prague) as a potentlostat The detector was connected through a 12-bit A/D converter to a PC/AT compatible computer, which was equipped with its own software for data acqulsltlon and evaluation of results The experiments were done m a thermostated vessel containing 3 5 ml of 50 mmol 1-l phosphate (pH 7 3), stirred at 300 rpm The activity of free chohnesterase was determined amperometrlcally with 2 mm01 1-l butyrylthlochohne, the bare CoPC modified electrode served for the detection of thlochohne Two methods were utilized for the determmatlon of pesticides using blosensors The previously described [23,24] mhlbltlon m the presence of substrate (0 50 mmol 1-l butyrylthlocholme) provided the relative mhlbltlon parameter RI, which was calculated as RI= (dI/dt)/I,,

(1)

where I,, 1s the blosensor steady-state current with substrate and d I/dt 1s the rate of decrease of the current observed after the addition of a sample contammg pesticide (an mhlbltor) The other method was based on a lo-mm premcuba-

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Chm Acta 269 (1992) 281-287

283

tlon of the blosensor or free cholmesterase m the presence of mhlbltor The percentage mhlbltlon was calculated according to h

I(%) = lOO(L

-L2)/L

= lOO(a, -ad/a,

L

(2)

B0

c

Steady-state currents or enzyme actlvltles (a) were determined with 2 mm01 I-’ butyrylthlochohne, the subscripts 1 and 2 correspond to rneasurements performed before and after mcubatlon, respectively

-2

u

,

0

0

0

20

20 ChE

RESULTS

AND DISCUSSION

Development of the enzyme layer

For substrate blosensors, diffusion control of the response 1s preferred, because it provides a wider linear workmg range Dlffuslonal hmltatlon 1s usually achieved for enzyme layers contammg high loadings of activity, it can also be realized with the help of an additional control membrane 1261 With blosensors for mhlbltors, kmetlc control of the response 1s desired and the signal of the blosensor should be proportional to the content of activity m the enzyme layer Generally, the lower the enzyme loading, the greater 1s the sensltlvlty to mhlbltors observed [6,17-19,241 Here, enzyme layers were prepared by crosslmkmg of ChE by glutaraldehyde This method 1s simple and 1s mdely used m various modlficatlons [3,6,22,231 However, it has to be used carefully, otherwise layers contammg high portions of latent activity could be obtained During the nnmoblhzatlon, the absolute ChE loading on the sensor (Fig 1) and the ChE (protem) to glutaraldehyde ratio (Fig 2) were optlmlzed The influence on substrate reaction (Fig 1, inset) was evaluated as the slope of the cahbratlon graph, dl/dc, for BTCh It increased up to 40 nkat cm-*, then a sharp decrease occurred, which was probably due to the unfavourable thickness of the layer For low concentrations of mhlbltor (paraoxon, 9 nmol l-‘, 2 5 pg l-l), maximum mhlbltlon was achieved for sensors containing 37 nkat cm-* ChE (Fig 1) The fact that maximum sensltlvlty to rnhlbltlon was not achieved for the lowest loading tested

,40

00

00,

40

60

0

60_

(nkat cm-*)

Ftg 1 Effect of ChE content m the enzyme layer on sensor responses to substrate (mset, slope of cahbratron graph d I/de, + 1 and mhtbttor [mhtbtttons obtamed for 9 0 nmol 1-l (01, RI;, or 3 6 pmol 1-l (A), RI,, paraoxon] The ChE to glutaraldehyde wetght ratto of 1 1 was constant for all enzyme layers being prepared

corresponds with the previous results with ChE cross-linked on nylon nets [23] For 3 6 pmol 1-l (1 mg 1-l) paraoxon, RI increased throughout the whole investigated interval of ChE loadings The influence of the ChE to glutaraldehyde ratio on the sensltlvlty for the substrate 1s shown m Fig 2 A maximum 1s found for the ratio 3 2

A

10

A-,

A

4

i

-

‘in

m

$

7

05 c

28

-J 0:

-= Dc

0

0 0

4

2 mChE

mGA

Ftg 2 Effect of ChE to glutaraldehyde ratro on responses to substrate (inset graph) and mhtbttor Symbols as u-rFtg 1 18 nkat of ChE were used for all btosensors

284

P Sklridal/Anal

However, for low pestlcrde concentrations, the highest RZ was determined for layers with the lowest glutaraldehyde content (5 1 ratio) Inhlbltlons by higher paraoxon concentrations were not affected For further measurements, the enzyme layers were prepared from 125 pg of ChE (maximum of RI,, Fig 1) and 25 pg of glutaraldehyde A significant saving of chohnesterase was achieved m comparison with the previously used cross-lmkmg on nylon nets [24] For the resulting blosensor, the apparent K, was 4 08 f 0 63 mmol I- ’ and Zmax51 1 f 3 2 MA (calculated using non-hnear regression, n = 15) For soluble ChE, the K, value was 0 199 * 0 024 mm01 1-l (n = 7), which Indicates the existence of a dlffuslonal barrier of the sensor even after the optnmzatlon with respect to mhlbltlon Optlmtzatron

Chm

Acta 269 (1992) 281-287

15

10

3 -8

5

A / A/ /

A-

-A

Y_

0

10

20

50

40

50

Fig 3 Temperature dependences of substrate (0) (0 5 mmol 1-l butyrylthmchohne) and mhlbltor (A) (3 6 pmol I-’ paraoxon) reactlons of blosensor

of the operatmg condrtlons

Workmg potentials from 0 to 500 mV (vs Ag/AgCl) were tested The useful signal resultmg from thiochohne oxldatlon increased up to 300 mV, for further measurements, a smaller value of 250 mV was selected because it ensured a lower background current (0 63 PA) Iodide Ions did not interfere at this low potential The optunum pH region from 7 2 to 8 0 was slmdar to that with previous versions of this sensor [23,24] and with the soluble enzyme [2] pH 7 3 was selected, which mmmnzes spontaneous hydrolysis of butyrylthlochohne Phosphate and borate buffers were studied as workmg solutions The sensor current decreased with mcreasmg buffer concentration, a 15 times higher response to substrate could be obtained m 5 mmol 1-l than 50 mmol I- ’ phosphate However, when using the lower concentration, the steady-state current was unstable, slmllar behavlour was also observed when working m borate buffers Consequently, 50 mmol l- ’ phosphate (pH 7 3) was selected as a suitable compromise An mterestmg temperature dependence of blosensor responses to substrate and mhlbltor was observed (Fig 3) Different temperature optima were found for the reaction with substrate and for mhlbltlon, the former being about 3040°C and the latter reachmg its maxnnum at 50°C At this temperature, ChE became ther-

mally denatured Subsequent analyses were performed at 30°C From Fig 3 it follows that a constant temperature is very important for precise measurements, because the possible temperature fluctuations could be misinterpreted as false mhlbltlons For the determination of mhlbltors, the substrate concentration 1s an important factor Substrates and mhlbltors (organophosphates and carbamates) bmd to the same actlve site of chohnesterase, so that competltlon exists between them [2] In the case of soluble enzyme, the rate of ChE mhlbltlon m the presence of substrate could be expressed as [27] d[EPl/dt=k,K,[Il([El,

- N’l)

/Kn[Il+ Kn& +&PI)

(3)

where K, 1s the dlssoclatlon constant of the enzyme-mhlbltor complex EI, k, IS the rate constant of transformation of EI to the phosphorylated enzyme EP, K, IS the Mlchaehs constant for ChE, [II and [S] are concentrations of mhlbltor and substrate, respectively, and [El, 1s the mltlal ChE concentration For kmetlcally controlled operation of blosensors, dZ/dt is proportlonal to d[EPl/dt and Z,, to [El, - [EPI, even if the constants of proportlonahty are unknown, they could be assumed to

P Skkidal /Anal

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Acta 269 (1992) 281-287

be equal Then, for the proposed measurement pestlcldes, the Eqn 3 may be rearranged to

wm[w(hI[Il+ G& + &PI) Thiscould be used for the interpretation

of

(4)

Rz =

of the results m Fig 4, where dependences of RI and the absolute rate of current decrease, d I/dt on the substrate concentration are presented The observed hyperbolic dependence of RZ on [S] 1s m good agreement with Eqn 4, a more detailed study of this problem ~11 be published elsewhere From the practical pomt of view, the lowest substrate concentrations are not useful because the signal 1s small m comparison with the background current For analysis, 0 5 mm01 1-l butyrylthlocholine was chosen as a reasonable compromise Varzous methods for pestzczde determznatzon Three approaches to the analysis were compared, premcubatlon of the blosensor or free ChE with mhlbltor and the proposed mhlbltlon m the presence of substrate The mcubatlon approach 1s based on the widely used equation of Aldrldge [28] A In a = k,[I]t

(5) where k, 1s the mhlbltlon bimolecular rate constant (k, = kZ/Kd) and A In a 1s the difference m the logarithms of ChE enzyme actlvltles (m chosen units) measured before and after the mcuba-

3

2 CI

-2 L t

i

0

3

c B

1 i; \ -6

1

0

0

00

05

10

15

[S] (mm01 I-‘) Fig 4 Influence of substrate concentratton on the mhtbttton by paraoxon (9 0 nmol 1-l) The effects on the absolute rate of current decrease, d I/dt (o), or RI (01 are demonstrated

100 60

20

-6

-7 log [I]

-6

-5

-4

(mol I-‘)

FIN 5 Cahbratton graphs for determmattons of paraoxon using lo-mm premcubatton with sample The measurements wtth the btosensor (0) or wtth an equal amount (18 nkat) of free ChE (0) are compared Inhtbtttons were calculated according to Eqn 2 Expenmental points represent averages of three measurements, standard devtatlons are shown as error bars

tlon with mhlbltor for time t It 1s evident that the extent of mhlbltlon 1s directly proportional to the time of mcubatlon and this fact 1s often used to make detection more sensltlve Thus, analyses are performed for 30-60-mm mcubatlon [5,17] The mcubatlon methods have recently been automated by usmg stopped flow techniques [7,11,12] Here, lo-mm mcubatrons with samples were performed Cahbratlon graphs for the blosensor and for an equal amount of free ChE are shown m Fig 5 Both methods exhibit comparable detection limits (defined as the concentration required for 5% mhlbltlon) equal to 3 0 nmol l- ’ paraoxon The advantage of lmmoblhzed ChE 1s the much wider workmg range For free ChE, 0 6 pmol 1-l paraoxon represents the upper limit for measurements, whereas up to 0 12 mmol l- ’ could be determined with the blosensor, which could even be used for several analyses provided that zero or very low concentrations of mhlbltors had been detected durmg previous measurements The experimental traces obtained with the other method are shown in Fig 6 As can be seen, the steady state after the addition of substrate 1s established quickly (95% of steady state

P Skl&l/Anal

286

after 45 s) The total time required for a complete analysis depends also on the washing time (less than 3 mm) and especially on the period required for reliable determmatlon of dZ/dt This can range from several seconds for high pestlclde concentrations up to ca 6 mm necessary for the lowest nanomolar levels, when the influence of noise has to be considered A hmltmg value of RZ 1s ca 5 x 10m5 s, it IS also used for the defmltlon of the hmlt of detection For measurements of low concentrations, when the current decrease IS very small compared with the mltlal Z,,, the approach has to be slightly dtierent than m Fig 6, the relatively high Z, IS almost completely compensated for and a greater sensltlvlty of the detector IS utlhzed Cahbratlon graphs for selected pesticides are shown m Fig 7 The shape of these graphs corresponds with Eqn 4 only for higher mhlbltor concentrations, below mlcromolar levels, higher RZs were obtained than expected, probably owing to the adsorption of pestlcldes m the more hydrophobic environment of the enzyme layer Based on the above-stated RZ limit, 0 30, 12 and 11 nmol I- ’ concentrations of paraoxon, dlchlorvos and carbaryl could be detected This method therefore exhibits better sensltlvlty than the mcu-

I

Chm Acta 269 (1992) 281-287

I

I

-9

-6

-7 log [I]

-6

-5

-4

(mol I-‘)

Fig 7 Cahbratlon graphs for (0) paraoxon, (0) dlchlorvos and (A) carbaryl determmed by measurmg the mhlblhon m the presence of substrate Experimental pomts represent averages of three measurements, standard devlatlons are shown as error bars

batlon approach The detection hmlt 1s agmflcantly improved by 1000 and 18 times m companson with the carbon paste and cross-linked enzyme membrane [23] and with the sensor based on ChE covalently bound to nylon net [241, respectively The practical use of a strip sensor IS also easier than working with separate electrodes and enzyme membranes The proposed method achieves the published detection limits for paraoxon obtained by mcubatlon approaches, for which concentrations from 0 1 nmol l- ’ to mlcromolar levels have been reported [5-7,1012,17,18] This blosensor can be used as a basis for the construction of continuously workmg monitoring systems for pesticides and for measurements of separate samples The most important advantages are the short time of analysis and possible apphcation in various analytlcal arrangements

REFERENCES

Rg 6 Examples of experlmental traces obtained durmg determmatlons of pestlcldes S mdlcates addltlon of substrate and A-F show addltlons of paraoxon to final concentrations of 32, 160 and 800 nmol I-’ and 4, 20 and 100 pmol I-‘, respectively

R Cremlyn, Pestlcldes - Preparation and Mode of Action, Wiley, ChIchester, 1978, p 213 M Eto, Organophosphorus Pestlcldes Orgamc and BIOloglcal Chemistry, CRC, Boca Raton, FL, 1974, p 123 P Durand, J Mallevlalle and J M Nlcaud, J Anal Toxlcol, 8 (1984) 112

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4 C Tran-Mmh, Ion-Se1 Electrodes Rev, 7 (1985) 41 5 C Tran-Mmh, PC Pandey and S Kumaran, Blosensors Bloelectron , 5 (1990) 461 6 S Kumaran, H Meler, AM Danna and C Tran-Mmh, Anal Chem, 63 (1991) 1914 7 S Kumaran S and C Tran-Mmh, Anal Bmchem, 200 (1992) 187 8 J Janata, R J Huber, R Cohen and E S Kolesar, Avlat Space Environ Med , 52 (1981) 666 9 KR Rogers, M Foley, S Alter, P Koga and M Eldefrawl, Anal Lett , 24 (1991) 191 10 0 Wolfbels and E Koller, m R D Schmld and F Scheller (Eds 1, GBF Blosensor Workshop, GBF Monographs, Vol 13, VCH, New York, 1989, p 221 11 R Kmdervater, W Kunnecke and R D Schmld, Anal Chum Acta, 234 (1990) 113 12 M F Leon-Gonzales and A Townshend, Anal Chum Acta, 236 (1991) 267 13 K R Rogers, C J Cao, J J Valdes, AT Eldefrawl and M Eldefravq Fundam Appl Toxlcol , 16 (1991) 810 14 J M Wallach, Gov Rep Announce Index (U S >, 90 (1990) Abstr 039 166 15 E P Medyantseva, G K Budmkov and S S Babkma, Zh Anal Khnn , 45 (1990) 1386 16 G G Gudbault and J Ngeh-Ngwambl, m G G Gudbault

17 18 19 20 21 22 23 24 25 26

27 28

and M Mascuu (Eds ), NATO ASI Ser C Mathematical and Physical Sciences, Vol 226, Reldel, Dordrecht, 1989, p 187 M Bemabel, C Cremisuu, M Mascuu and G Palleschl, Anal Lett ,24 (1991) 1317 L Campanella, M Achdh, M P Sammartmo and M Tomassettl, Bmelectrochem Bloenerg ,26 (1991) 237 U Wollenberger, K. Setz, F Scheller, U Loffler and W Gopel, Sensors Actuators B, 4 (1991) 257 L H Goodson and W B Jacobs, Methods Enzymol , 44 (1976) 647 R Gruss, F Scheller, M J Shao and CC Lm, Anal Lett , 22 (1989) 1159 J Kulys and E J D’Costa, Blosensors Bloelectron ,6 (1991) 109 P Sklidal, Anal Chum Acta, 252 (1991) 11 P Sklhdal and M Mascuu, Blosensors Bloelectron , 7 (1992) 335 S A Wring, J P Hart and J B Birch, Analyst, 116 (1991) 123 LX Tang and P M Vadgama, m D L Wise (Ed 1, Blomstrumentatlon, Research, Development and Apphcatlons, Dekker, New York, 1991, p 211 I M Kovach, J Enzyme Inhlb , 4 (1991) 201 W N AIdrIdge, Blochem J ,46 (1950) 451