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Amperometric flow-through biosensor for the determination of pesticides
ANALYTICA CHIMICA ACTA ELSEVIER
Analytica
Amperometric
Chimica Acta 308 (1995) 129-136
flow-through biosensor for the determination of pesticides
C. La Rosa, F. Pariente, L. Hernhdez, Dpto. de Quimica Analitica
y An&is
Instrumental,
E. Lorenzo
*
Facultad de Ciencius, Uniwrsidad Autcinoma de Madrid, Madrid 28049, Spain
Received 19 July 1994; revised manuscript
received 18 October 1994
Abstract An amperometric flow-through biosensor for the determination of pesticides is proposed. It is based on the inhibition of the acetyl cholinesterase-catalysed hydrolysis of 4-aminophenylacetate. The calibration graphs were linear from 5.0 X lo-’ to 1.0 x lop5 M and 5.0 X lo-’ to 5.0 X lo-’ M for paroxon and carbaryl, respectively. The detection limit (5% of inhibition) was 1.0 X lo-’ M pesticide. The relative standard deviations (R.S.D.) (n = 5) were 3.7% for 4.0 X lop5 M and 4.0% for 8.0 X 10ph M for either carbaryl or paroxon. Electroactive species such as uric and ascorbic acid and benzafdehyde that could be oxidized at the same potential as 4-aminophenol, do not interfere. However, compounds which strongly absorb onto the electrode surface, such as bovine serum albumin (BSA) and surfactants capable of denaturing the enzyme activity, cause an interference. The stability of the sensor was high and even after repetitive use for one month the electrode retained 90% of its original activity. The determination of carbaryl and paroxon was carried out in lagoon water and kiwi fruits. The lowest concentration of pesticide determined in lagoon water was 1.0 X lo-’ M for paroxon and 4.0 X lo-’ M for carbaryl. K~ywor~st Biosensors;
Flow system; Ampcrometry;
Pesticides
1. Introduction Organophosphorus compounds and carbamates represent a large number of pesticides widely employed in agriculture. In general, such substances are powerful inhibitors of a group of hydrolytic enzymes called esterases, but the inhibition of acetyl cholinesterase (AChE) is their principal action as pesticides. This enzyme hydrolyses the neurotransmitter acetylcholine in the synaptic membrane and
’ Corresponding 0003-2670/95/$09.50
author. 0 1995 Elscvier Science B.V. All rights reserved
SSDI 0003-2670(94)00529-X
plays a fundamental role in nerve function. A consequence of such hydrolysis reaction is the acetylation of the enzyme. However, this esteratic bond is weak and is rapidly hydrolysed during the recovery stage of the enzyme. Organophosphorus compounds and carbamates have some structural similarities with the acetylcholine but they prevent the enzyme function by basically different mechanisms. Carbamates block the active center of the enzyme by competition with the substrate. Organophosphorus compounds inactivate the enzyme by phosphorylation. These different inhibition patterns are very important in the design of experimental conditions for analysis of such pesticides using AChE.
130
C. La Rosa et ai. /Analytica
Chimica Acta 308 (19951 129-136
Enzymatic methods based on the inhibition of AChE activity have recently been the object of numerous investigations because their sensitivity, specificity and the possibility of employing alternative substrates to acetylcholine. Indeed, new substrates have been synthesized and applied with different analytical techniques [l-3]. In previous work [4,5] we synthesized and demonstrated the use of 4-aminophenyl acetate (PAPA) as an improved substrate for the determination of esterase activities via oxidation of 4-aminophenol (PAP), the product of the enzymatic reaction, and the possibility of using this system in inhibition studies of esterase activities by xenobiotic agents and the application of this inhibition for the determination of organophosphorus and carbamate pesticides. This method, contrary to most other ones previously described [6,7], requires short incubation periods between the enzyme and the pesticide before addition of the substrate and the amperometric measurement of the enzymatic reaction product. Taking advantage of the first, in this paper we describe the rapid flow-injection determination of organophosphorus and carbamate pesticides using an AChE electrode as an electrochemical detector which has a rapid response for PAPA.
Determinations on water from a lagoon of the Madrid county sampled by the authors and in kiwis were carried out. Water samples were stored at 4°C and analysed with prior filtering as the only treatment. Kiwis (mean weight 100 g) were treated by immersion in aqueous solution of carbaryl with a concentration of active compound of 200 mg/l for 2 h. Fruits were then placed in open plastic boxes and stored in 85-90% relative humidity at 4°C. Residues were determined 24 h after pesticide treatment. To determine the pesticide residue, the fruits were peeled. Samples of 8.20 g of peel were blended with 50 ml of methanol and sonicated for 2 min. The mixture was filtered, rinsed two times with 10 ml of ethanol and the filtrates were collected and evaporated until a volume of 20 ml in a rotary evaporator at 50°C bath temperature and brought to 25 ml in a volumetric flask with methanol.
2. Experimental
2.3. Preparation of the flow-type use based biosensor
2.1. Reagents Acetyl cholinesterase (EC 3.1.1.7; type III) from electric eel was purchased from Sigma as a solution containing 5 mg of ammonium sulfate per mg of protein and 0.8 IU per ~1 of solution. This enzyme 4preparation was stored frozen at -20°C. Nitrophenyl acetate (PNPA) was purchased from Aldrich and 4-aminophenyl acetate (PAPA) was synthesized by catalytic hydrogenation of the nitro group of PNPA as described previously [5]. 4-Aminophenol (PAP) was purchased from Merck. Glutaraldehyde (grade I 25% aqueous solution) was obtained from Sigma and stored below 0°C. Nylon filter meshes were obtained from Nytal. 2-Pyridinealdoxime methiodide was purchased from Aldrich. Deionized water from Milli-Q and Milli-RO systems (Millipore) was used to prepare all solutions. Pesticide preparation of carbaryl was obtained from Aragonesas
(Madrid) and paroxon (diethyl-4-nitrophenyl phosphate) from Aldrich. All other chemicals were of analytical reagent grade and were used as received. 2.2. Sampling and extraction procedure
acetylcholinester-
Acetylcholinesterase (AChE) was immobilized onto nylon grids (loo-150 pm thickness) as follows: 5-6 mm diameter disks were cut from a nylon net. The single disks were immersed in absolute methanol for 30 min, washed in buffer for 5 min and dried in air at room temperature. In sequential fashion 2.5 ~1 of 7% (v/v) glutaraldehyde, 2.0 ~1 of 10% (w/v) bovine serum albumin (BSA) and 0.75 /.~l of AChE (0.6 IU), were added and thoroughly mixed at room temperature. After 2 h at 4°C the disks were washed in phosphate buffer containing 1% (w/v) glycine and mounted in the flow system. After use the nylon disks were stored in 0.08 M phosphate buffer (pH 8.0) at 4°C. 2.4. Flow system and procedures The flow system used is shown schematically in Fig. 1. The flow type AChE biosensor was placed
C. La Rosa et al./Analytica
Chimica Acta 308 (1995) 129-136
131
where a, and a, are the enzyme activity before and after pesticide addition, respectively.
3. Results and discussion
Fig. 1. Arrangement for voltammetric FIA. C = Carrier stream; P = peristaltic pump; I = inhibitor injection valve; S = substrate injection valve; D = electrochemical detector; R = recorder; W = waste. Inset: Scheme of the amperometric flow-through biosensor.
(inset to Fig. 1) in the amperometric flow cell (Metrohm EA-1096). A constant potential ( + 0.25 V vs. Ag/AgCl) was applied with a Metrohm potentiostat (641 VA) and currents were measured with an Y-t recorder (Linseys L-6512). Carrier solution (0.08 M phosphate buffer at pH 8.0) from the reservoir was pumped with a peristaltic pump (Watson Marlow 202 U/l) at flow rates of 0.55 ml mini ’ or lower, to the injections valves I and S (Rheodyne Type 50) equipped with 500 and 150 ~1 sample loops, respectively. The initial enzyme activity was measured by starting the carrier stream of the flowinjection system and successive substrate injections via valve S. After injection of PAPA samples the enzymatic hydrolysis of the substrate proceeded in the immobilized acetylcholinesterase layer, and the PAP produced was monitored amperometrically. For the determination of pesticides the general procedure employed consisted of injection of either pesticide (using valve I) to give rise to enzyme inhibition. The electrode was rinsed with carrier solution for approximately 90 s. The remaining enzyme activity was subsequently determined by the injection of substrate (through valve S) and monitoring of the current due to PAP oxidation at 0.25 V (vs. Ag/AgCl). The percentage of inhibition, %I was expressed as: %I = ~ao - ar x 100 a0
The determination of organophosphorus compounds and carbamates is based on the inhibition of the immobilized acetylcholinesterase, so that the peaks resulting from the indicator reaction will decrease in relation to the inhibitor concentration. Therefore, the first experiments were conducted to establish the optimal conditions for the indicator reaction; the determination of PAPA with the flowtype AChE based biosensor. The purpose of transporting the sample solution through an automated analyzer is to treat the sample material in such a way that it will yield, when sensed by a detector, a response that is not only selective but also reproducible. If an amperometric detector is used to determine the original sample composition, the sample solution must be transported through the flow cell in an undiluted form in a highly reproducible manner. Thus, it is of primary interest to know how much the original sample solution is to be diluted on its way toward the detector, and how much time must elapse between sample injection and readout, to adjust the operational parameters to obtain optimal conditions for our procedure. The hydrodynamic direct current voltammograms were obtained for injection of 75 and 100 ~1 of 20 PM PAP. Under these conditions the voltammograms showed an increase in current until +0.20 V (vs. Ag/AgCl) and then the response leveled off. These results offer the possibility of holding the potential at relatively low values in the amperometric determination of PAP. This is most advantageous to avoid interferences from other compounds which could be oxidized at higher potentials (selectivity) and the low backgrounds obtained (detection limit). The dispersion (D) for flow-injection analysis (FIA) has been defined [8] as the ratio of concentration before and after the dispersion process has taken place in the element of fluid that yields the analytical readout. As the analytical readout is based on measurement of peak height, D = ho/h, where ho is the peak height recorded when the entire flow cell is filled with undiluted sample. We have obtained this
132
C. La Rosa et al./Analytica
parameter by two procedures; using PAP as carrier or injecting large volumes such as 1000 ~1. In our system, the calculated value for D for an injection volume of 75 ~1 of the product of enzymatic reaction (PAP) at a flow rate of 0.34 ml min-’ was 1.25 for the both methods. This, in accord with the general classification, is a limited dispersion (D = l-3). The peak height and peak width in flow systems depend on the dispersion of the sample plug during its passage from the point of injection to the detector; and in this case, where an enzymatic reaction is involved, on the response time of the kinetic reaction. Thus, variables such as injection volume, coil length and flow rate, which influence the dispersion of the sample and the possibility of reaction between enzyme and substrate, were adjusted according with the specific requirements of this system. Fig. 2, shows the effect of the injection volume of PAPA on the peak current. As the volume of PAPA injected was increased, the peak current increased, but the increase was small at higher concentrations. This could be due to a substrate saturation or to the fact that for large sample volumes, the peak height will become independent of sample volume in accord with theoretical aspects of FIA as established in the Sternberg equation 191.
3.0
2.5
2.0 3 4 1.5
I.0
,-
0.5
0.c b0
loo
200
300
400
500
Vi W1)
Fig. 2. Influence of injection volume on peak current for 1.2X lo-” M PAPA. The carrier stream was 0.08 M phosphate buffer (pH 8.0) at a flow rate of 0.80 ml min-‘.
Chimica Acta 308 (1995) 129-136
300
=f
6 L
-200
1.0-
g
-
150
-
loo
.\.,
‘----.
0.5 -
0.0
1 0.0
-50
I 0.5
1.0 Flux
1.5
2.0
2.5O
(ml/min.)
Fig. 3. Effect of flow rate on the peak current (0) and the peak width (m) (in seconds). Conditions as in Fig. 2 with a PAPA injection volume of 150 ~1.
The effect of flow-rate on the peak current and on the peak width were studied over the range of 0.3 to 2.0 ml mini’. The results, presented in Fig. 3, show that as the flow rate increased, the peak current decreased, but the decrease was smaller at higher flow rates. Thus, a faster flow results in smaller peak currents, as would be expected, since the PAPA is in contact with the enzyme for shorter time periods. Therefore, low flow rates would be desirable in order to maximize the response. On the other hand, a low flow rate results in broadened peaks so that flow rates below 0.50 ml min-’ required over 200 s for the current to reach its baseline value. Thus, 0.55 ml mini’ is recommended as the most suitable flow rate for achieving good sensitivity and reasonable sample throughput. When PAP (rather than PAPA) is injected and detected at a glassy carbon electrode that is not covered with a membrane, the peak current increased with flow rate and a plot of peak current vs. the square root of the flow rate was linear suggesting that mass transport is the limiting step in the reaction. However, when the electrode is covered with a membrane, the current was found to be independent
C. La Rosa et al. /Analytica
of flow rate, suggesting that diffusion through the membrane is likely rate controlling. In the enzymatic determination, the current due to PAP follows the enzymatic hydrolysis of PAPA. However, we saw above that under these conditions, the peak current actually decreased with increasing flow rate which indicates that in this case the limiting step is either the transport through the membrane or the enzymatic reaction itself.
Chimica Acta 308 (1995) 129-136
133
1%
60 -
40 -
3.1. Optimization
of inhibition reaction
The effect of different experimental conditions on the response of the biosensor in the presence of pesticides was studied in order to determine the optimal conditions. The inhibition of cholinesterase by organophosphorus and carbamate agents is well known [lo]. Several inhibition mechanisms have been suggested for acetylcholinesterase depending on the substrate [ll]. In a previous work [5], we demonstrated that the inhibition is a function of both inhibitor and substrate concentrations and that in the pesticide determination two approaches can be employed: inhibition in the presence of substrate or preincubation of the biosensor with the inhibitor. Because the preincubation method exhibited a lower detection limit and is less sensitive to possible interferences, it was selected for this work. The relationship between inhibition and preincubation time under quiescent conditions is described by the Aldridge equation [12]: log( lOO/%Z)
20
I
0'
00
0.3
0.6
0.9
Flow
1.2
1.5
1.9
Rate (ml/min.)
Fig. 4. Influence of injection volume of paroxon (10m4 M) on the percentage of inhibition, I%, at different flow rates. Injection volume of 1.2 x 1O-3 M PAPA: 150 ~1.
ing injection volumes of the pesticides, carbaryl and paroxon, was studied at a fixed concentration of substrate (1.2 mM of PAPA). The results, presented in Figs. 4 and 5, show that the inhibition, decreased linearly with increasing flow rate, for both carbaryl
= ZC,[r]t
where %Z is the percentage of inhibition, K, is the inhibition bimolecular rate constant [13], [I] the concentration of inhibitor and t the preincubation time. Thus, as the degree of enzyme inhibition is dependent on incubation time more sensitive determinations can be made by preincubations for large time periods. In the FIA approach proposed, the time that the inhibitor is in contact with the enzyme (incubation time) and therefore the extent of inhibition is determined by the flow rate and the sample injection volume. Thus, both parameters must be optimized. The inhibition study was made by observing the effect of different substrate and pesticide concentrations on the percentage of inhibition. The effect of flow-rate on the inhibition of the enzyme at increas-
01
0.0
0.3
0.6
0.9
1.2
1.5
1.8
21
Plow Rate (ml/min.) Fig. 5. Influence of injection volume of carbaryl in the percentage of inhibition, I%, at different flow rates. Conditions as Fig. 4.
134
C. La Rosa et al. /Analytica
and paroxon at all sample volumes employed. These results indicate that the percentage of inhibition, which is essentially the same as the binding between the enzyme and the inhibitor, is only dependent on the time that both are in contact. It should also be mentioned that at small sample volumes, the inhibition decreases linearly and sharply with increasing flow rate, for both carbaryl and paroxon but the decrease was small at large sample volumes. From these results it can be inferred that the influence of the flow rate will be small if large sample volumes are used. In this case, the time during which enzyme and inhibitor are in contact is compensated by the high concentration of inhibitor, [I], used. In plots of %I as a function of flow rate, the Y intercept represents the maximum inhibition obtained. It can be seen in Figs. 4 and 5 that the same Y intercept is obtained irrespective of the sample volume employed and in fact this value agrees well with the inhibition calculated for the same concentration of carbaryl or paroxon in a previous work under steady state conditions. Fig. 6 shows the effect of flow rate on the inhibition of the enzyme at the same concentration of
I 100
1% 80
60
L
y=48.7-31.9Y;r=0.9969
I
40
Chimica Acta 308 (1995) 129-136
1w
-
1%
60 -
40 -
I
0
0.0
0.2
04
06
I
0.6
I
1.0
I
1.2
I
1.4
I
1.6
I
1.6
Flow Rate (ml/&n.) Fig. 7. Influence of injection volume of substrate (1.2X 10-j M PAPA) on the percentage of inhibition at different flow rates. Injection volume of 1.0X lo-” M carbaryl: 500 /*l.
substrate employed in the experiments described above, and with increasing concentrations of carbaryl. It is clear that the flow rate is critical for the determination of small concentrations of inhibitor, and only with the lower flows could we detect carbaryl at concentrations below 10m6 M. However, as can be seen in Fig. 7, the injection volume of substrate has a minimal influence in the percentage of inhibition. The choice of optimum flow rate is a compromise between the time needed for the inhibition reaction to occur and the time needed for the current to return to baseline after injection, with the finality to obtain a reasonable sample throughput. Therefore, flow rates of 0.55 and 0.34 ml min. ’ for paroxon and carbaryl, respectively, were employed in all further studies. 3.2. Analytical characteristics
20
Flow Rate (mUmin.) Fig. 6. Influence of concentration of carbaryl on the percentage of inhibition, I%, at different flow rates. Injection volumes: 500 ~1 of carbaryl and 150 /Al of 1.2X lo-’ M PAPA.
As anticipated the signal peaks (peak current) decreased with increasing concentrations of pesticide. Linear calibration plots were obtained over the range of 5.0 X 10P7-1.0 X lop5 M and 5.0 X 10-7-5.0 x 1o-5 M for paroxon ( y = 433 + 70.00x; r = 0.9998) and carbaryl (y = 335 + 54.71 x; r = 0.99291, respectively. The detection lim-
C. La Rosa et al. /Analytica
its (defined as the concentration of inhibitor required to obtain 5% of inhibition) of the method is 1.0 X lo-’ M pesticide. Reproducibility was tested by repeated 500 ~1 injections of either pesticide at two different concentrations. The relative standard deviations were 3.7% for 4.0 X lop5 M and 4.0% for 8.0 X 1Omh M for either carbaryl or paroxon. Most of the electroactive species that could be oxidized at the same potential as PAP, do not interfere because with the preincubation procedure employed they are rinsed away with the carrier stream prior to determination of the remaining enzyme activity. In fact, in the determination of paroxon at 1.0 X 10m5 M we have observed no interference in the presence of uric and ascorbic acid nor benzaldehyde in the range of 1.2 X lop3 M to 1.5 X lo-* M. However, compounds which strongly absorb onto the electrode surface, such as 0.1% (w/v) of BSA, cause a decrease in the peak height (9% in case of BSA). The presence of surfactants capable of denature the enzyme activity, such as 0.1% of sodium dodecyl sulphate (SDS), causes a serious interference, given rise to a 50% decrement in the sensor response. The biosensor was used repeatedly to confirm its stability over a long time period. After use the inhibited enzyme membrane can be reactivated by immersion in a solution of 2-pyridinealdoxime methiodide (24.8 mg/lOO ml of phosphate buffer) for at least 4 h, rinsed and equilibrated for 2 h in phosphate buffer [5]. The stability of the sensor was high and even after repetitive use for one month the electrode retained 90% of its original activity when it was stored in the phosphate buffer while not in use. 3.3. Determination fruits
of pesticides
in lagoon water and
The determination of the pesticides carbaryl and paroxon was carried out in water. Linear calibration plots (1% vs. log(inhibitor)) in this matrix were obtained over the range of 5.0 X lo-‘-1.0 X lo-’ M and 5.0 X lo-‘-5.0 X lo-” M for paroxon (y = 377 + 59x; r = 0.9962) and carbaryl (y = 294 + 46x; r = 0.9932), respectively. Immobilized AChE was inhibited faster by the paroxon than by carbaryl. As a rapid and sensitive test was envisaged, a cycle time of 5 min for the slow one was needed. This
135
Chimica Acta 308 (1995) 129-136
included incubation with sample and determination of the remaining activity. Incubation with the lagoon water did not decrease AChE activity. A high concentration of paroxon (1.0 X 10d4 M) caused complete inhibition of the enzyme but at the same concentration carbaryl caused only an inhibition of 90%. The lowest concentration of pesticide determined was 1.0 X lo-’ M, for paroxon and 4.0 X lo-’ M for carbaryl. Longer incubation times, that means, lower flow rates resulted in greater inhibition, but low sample throughputs. The determination of carbaryl was also carried out in kiwi fruits. Carbamate insecticides such as carbaryl are often employed against Polychrosis botrana in some fruits including grapes, apples and kiwis. Kiwis were chosen for the present study in order to assess the validity of the proposed method to the detection of pesticides in real samples. For the residue determination of carbaryl in kiwi fruits, 2.5 ml of the ethanolic sample solution obtained as described in the Experimental section were brought to 25 ml with 0.08 M phosphate buffer (pH 8.0). 500 ~1 of this solution were subsequently injected in the FIA system and the determination carried out as described before. The validity of the method was assessed by comparing these results with those obtained by a UV method in which the absorbance of carbaryl was measured at 220 nm against an ethanolic extract of kiwi without pesticide, as blank. The calibration curve was constructed from absorbance measurements of carbaryl added to the kiwi blank solutions. An excellent correlation (r = 0.9972) was found over the range of 0.07 to 4.0 mg ll’. Similarly, a calibration curve for the determination of carbaryl in kiwi fruit extract with the amperometric biosensor described herein gave a correlation of (r = 0.9914). Upon comparison of the results obtained by both methods (Table l), an excellent agreement was found.
Table 1 Determination Pesticide
Carbaryl ’ Average
of carbaryl
in kiwi fruits
uv determination (mg 1-l)
Amperometric flow-through biosensor (mg I-
3.10 a
3.01 a
of three determinations.
‘)
C. La Rosa et al./Analytica
136
121K. Gibson and G.G. Guilbault, Anal. Chim. Acta, 76 (1975)
4. Conclusions The flow-injection device for the automated reaction of immobilized enzyme membranes permits rapid enzyme inhibition tests. The detection of pesticides based on the principle of acetylcholinesterase inhibition is achieved within a few minutes. The sensitivity of this assay is comparable to non-biological standard methods such as LC.
Acknowledgments This work was supported by the Comunidad Autonoma de Madrid, through grant No. 199/92. The authors acknowledge Dr. H.D. Abruiia for his contributions in critically reading this manuscript and the NATO Scientist Program.
References [I] H. Okabe, K. Sagehara,
N. Nakajima Chim. Acta, 80 (1977) 87.
Chimica Acta 308 (1995) 129-136
and A. Noma,
Clin.
245. [31 F. Mizutani and K. Tsuda, Anal. Chim. Acta, 139 (1982) 359. [41 F. Pariente, L. Hernlndez and E. Lorenzo, Anal. Chim. Acta, 273 (1993) 399. 151 C. La Rosa, F. Pariente, L. Hernlndez and E. Lorenzo, Anal. Chim. Acta, 295 (1995) 273. [61 C. Tran-Minh, P.C. Pandey and S. Kumaran, Biosensors Bioelectron., 5 (1990) 461. 171 M. Bernabei, C. Cremisini, M. Mascini and G. Palleschi, Anal. Lett., 24 (1991) 1317. [81 J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 99 (1978) 37. [91 J. Ruzicka and E.H. Hansen, Flow Injection Analysis, Chemical Analysis Vol. 62, Wiley, New York, 1981. Pesticides: Organic and Biologi1101 M. Eto, Organophosphorous cal Chemistry, CRC Press, Boca Raton, FL, 1974, pp. 131133. 1111 R.D. O’Brien, B.D. Hilton and L. Gilmour, Molec. Pharmacol., 2 (1966) 593. ml W.N. Aldridge, Biochem. J., 46 (19.50) 451. [131 P. Skladal and M. Mascini, Biosensors Bioelectron., 7 (1992) 335.
Analytica
Amperometric
Chimica Acta 308 (1995) 129-136
flow-through biosensor for the determination of pesticides
C. La Rosa, F. Pariente, L. Hernhdez, Dpto. de Quimica Analitica
y An&is
Instrumental,
E. Lorenzo
*
Facultad de Ciencius, Uniwrsidad Autcinoma de Madrid, Madrid 28049, Spain
Received 19 July 1994; revised manuscript
received 18 October 1994
Abstract An amperometric flow-through biosensor for the determination of pesticides is proposed. It is based on the inhibition of the acetyl cholinesterase-catalysed hydrolysis of 4-aminophenylacetate. The calibration graphs were linear from 5.0 X lo-’ to 1.0 x lop5 M and 5.0 X lo-’ to 5.0 X lo-’ M for paroxon and carbaryl, respectively. The detection limit (5% of inhibition) was 1.0 X lo-’ M pesticide. The relative standard deviations (R.S.D.) (n = 5) were 3.7% for 4.0 X lop5 M and 4.0% for 8.0 X 10ph M for either carbaryl or paroxon. Electroactive species such as uric and ascorbic acid and benzafdehyde that could be oxidized at the same potential as 4-aminophenol, do not interfere. However, compounds which strongly absorb onto the electrode surface, such as bovine serum albumin (BSA) and surfactants capable of denaturing the enzyme activity, cause an interference. The stability of the sensor was high and even after repetitive use for one month the electrode retained 90% of its original activity. The determination of carbaryl and paroxon was carried out in lagoon water and kiwi fruits. The lowest concentration of pesticide determined in lagoon water was 1.0 X lo-’ M for paroxon and 4.0 X lo-’ M for carbaryl. K~ywor~st Biosensors;
Flow system; Ampcrometry;
Pesticides
1. Introduction Organophosphorus compounds and carbamates represent a large number of pesticides widely employed in agriculture. In general, such substances are powerful inhibitors of a group of hydrolytic enzymes called esterases, but the inhibition of acetyl cholinesterase (AChE) is their principal action as pesticides. This enzyme hydrolyses the neurotransmitter acetylcholine in the synaptic membrane and
’ Corresponding 0003-2670/95/$09.50
author. 0 1995 Elscvier Science B.V. All rights reserved
SSDI 0003-2670(94)00529-X
plays a fundamental role in nerve function. A consequence of such hydrolysis reaction is the acetylation of the enzyme. However, this esteratic bond is weak and is rapidly hydrolysed during the recovery stage of the enzyme. Organophosphorus compounds and carbamates have some structural similarities with the acetylcholine but they prevent the enzyme function by basically different mechanisms. Carbamates block the active center of the enzyme by competition with the substrate. Organophosphorus compounds inactivate the enzyme by phosphorylation. These different inhibition patterns are very important in the design of experimental conditions for analysis of such pesticides using AChE.
130
C. La Rosa et ai. /Analytica
Chimica Acta 308 (19951 129-136
Enzymatic methods based on the inhibition of AChE activity have recently been the object of numerous investigations because their sensitivity, specificity and the possibility of employing alternative substrates to acetylcholine. Indeed, new substrates have been synthesized and applied with different analytical techniques [l-3]. In previous work [4,5] we synthesized and demonstrated the use of 4-aminophenyl acetate (PAPA) as an improved substrate for the determination of esterase activities via oxidation of 4-aminophenol (PAP), the product of the enzymatic reaction, and the possibility of using this system in inhibition studies of esterase activities by xenobiotic agents and the application of this inhibition for the determination of organophosphorus and carbamate pesticides. This method, contrary to most other ones previously described [6,7], requires short incubation periods between the enzyme and the pesticide before addition of the substrate and the amperometric measurement of the enzymatic reaction product. Taking advantage of the first, in this paper we describe the rapid flow-injection determination of organophosphorus and carbamate pesticides using an AChE electrode as an electrochemical detector which has a rapid response for PAPA.
Determinations on water from a lagoon of the Madrid county sampled by the authors and in kiwis were carried out. Water samples were stored at 4°C and analysed with prior filtering as the only treatment. Kiwis (mean weight 100 g) were treated by immersion in aqueous solution of carbaryl with a concentration of active compound of 200 mg/l for 2 h. Fruits were then placed in open plastic boxes and stored in 85-90% relative humidity at 4°C. Residues were determined 24 h after pesticide treatment. To determine the pesticide residue, the fruits were peeled. Samples of 8.20 g of peel were blended with 50 ml of methanol and sonicated for 2 min. The mixture was filtered, rinsed two times with 10 ml of ethanol and the filtrates were collected and evaporated until a volume of 20 ml in a rotary evaporator at 50°C bath temperature and brought to 25 ml in a volumetric flask with methanol.
2. Experimental
2.3. Preparation of the flow-type use based biosensor
2.1. Reagents Acetyl cholinesterase (EC 3.1.1.7; type III) from electric eel was purchased from Sigma as a solution containing 5 mg of ammonium sulfate per mg of protein and 0.8 IU per ~1 of solution. This enzyme 4preparation was stored frozen at -20°C. Nitrophenyl acetate (PNPA) was purchased from Aldrich and 4-aminophenyl acetate (PAPA) was synthesized by catalytic hydrogenation of the nitro group of PNPA as described previously [5]. 4-Aminophenol (PAP) was purchased from Merck. Glutaraldehyde (grade I 25% aqueous solution) was obtained from Sigma and stored below 0°C. Nylon filter meshes were obtained from Nytal. 2-Pyridinealdoxime methiodide was purchased from Aldrich. Deionized water from Milli-Q and Milli-RO systems (Millipore) was used to prepare all solutions. Pesticide preparation of carbaryl was obtained from Aragonesas
(Madrid) and paroxon (diethyl-4-nitrophenyl phosphate) from Aldrich. All other chemicals were of analytical reagent grade and were used as received. 2.2. Sampling and extraction procedure
acetylcholinester-
Acetylcholinesterase (AChE) was immobilized onto nylon grids (loo-150 pm thickness) as follows: 5-6 mm diameter disks were cut from a nylon net. The single disks were immersed in absolute methanol for 30 min, washed in buffer for 5 min and dried in air at room temperature. In sequential fashion 2.5 ~1 of 7% (v/v) glutaraldehyde, 2.0 ~1 of 10% (w/v) bovine serum albumin (BSA) and 0.75 /.~l of AChE (0.6 IU), were added and thoroughly mixed at room temperature. After 2 h at 4°C the disks were washed in phosphate buffer containing 1% (w/v) glycine and mounted in the flow system. After use the nylon disks were stored in 0.08 M phosphate buffer (pH 8.0) at 4°C. 2.4. Flow system and procedures The flow system used is shown schematically in Fig. 1. The flow type AChE biosensor was placed
C. La Rosa et al./Analytica
Chimica Acta 308 (1995) 129-136
131
where a, and a, are the enzyme activity before and after pesticide addition, respectively.
3. Results and discussion
Fig. 1. Arrangement for voltammetric FIA. C = Carrier stream; P = peristaltic pump; I = inhibitor injection valve; S = substrate injection valve; D = electrochemical detector; R = recorder; W = waste. Inset: Scheme of the amperometric flow-through biosensor.
(inset to Fig. 1) in the amperometric flow cell (Metrohm EA-1096). A constant potential ( + 0.25 V vs. Ag/AgCl) was applied with a Metrohm potentiostat (641 VA) and currents were measured with an Y-t recorder (Linseys L-6512). Carrier solution (0.08 M phosphate buffer at pH 8.0) from the reservoir was pumped with a peristaltic pump (Watson Marlow 202 U/l) at flow rates of 0.55 ml mini ’ or lower, to the injections valves I and S (Rheodyne Type 50) equipped with 500 and 150 ~1 sample loops, respectively. The initial enzyme activity was measured by starting the carrier stream of the flowinjection system and successive substrate injections via valve S. After injection of PAPA samples the enzymatic hydrolysis of the substrate proceeded in the immobilized acetylcholinesterase layer, and the PAP produced was monitored amperometrically. For the determination of pesticides the general procedure employed consisted of injection of either pesticide (using valve I) to give rise to enzyme inhibition. The electrode was rinsed with carrier solution for approximately 90 s. The remaining enzyme activity was subsequently determined by the injection of substrate (through valve S) and monitoring of the current due to PAP oxidation at 0.25 V (vs. Ag/AgCl). The percentage of inhibition, %I was expressed as: %I = ~ao - ar x 100 a0
The determination of organophosphorus compounds and carbamates is based on the inhibition of the immobilized acetylcholinesterase, so that the peaks resulting from the indicator reaction will decrease in relation to the inhibitor concentration. Therefore, the first experiments were conducted to establish the optimal conditions for the indicator reaction; the determination of PAPA with the flowtype AChE based biosensor. The purpose of transporting the sample solution through an automated analyzer is to treat the sample material in such a way that it will yield, when sensed by a detector, a response that is not only selective but also reproducible. If an amperometric detector is used to determine the original sample composition, the sample solution must be transported through the flow cell in an undiluted form in a highly reproducible manner. Thus, it is of primary interest to know how much the original sample solution is to be diluted on its way toward the detector, and how much time must elapse between sample injection and readout, to adjust the operational parameters to obtain optimal conditions for our procedure. The hydrodynamic direct current voltammograms were obtained for injection of 75 and 100 ~1 of 20 PM PAP. Under these conditions the voltammograms showed an increase in current until +0.20 V (vs. Ag/AgCl) and then the response leveled off. These results offer the possibility of holding the potential at relatively low values in the amperometric determination of PAP. This is most advantageous to avoid interferences from other compounds which could be oxidized at higher potentials (selectivity) and the low backgrounds obtained (detection limit). The dispersion (D) for flow-injection analysis (FIA) has been defined [8] as the ratio of concentration before and after the dispersion process has taken place in the element of fluid that yields the analytical readout. As the analytical readout is based on measurement of peak height, D = ho/h, where ho is the peak height recorded when the entire flow cell is filled with undiluted sample. We have obtained this
132
C. La Rosa et al./Analytica
parameter by two procedures; using PAP as carrier or injecting large volumes such as 1000 ~1. In our system, the calculated value for D for an injection volume of 75 ~1 of the product of enzymatic reaction (PAP) at a flow rate of 0.34 ml min-’ was 1.25 for the both methods. This, in accord with the general classification, is a limited dispersion (D = l-3). The peak height and peak width in flow systems depend on the dispersion of the sample plug during its passage from the point of injection to the detector; and in this case, where an enzymatic reaction is involved, on the response time of the kinetic reaction. Thus, variables such as injection volume, coil length and flow rate, which influence the dispersion of the sample and the possibility of reaction between enzyme and substrate, were adjusted according with the specific requirements of this system. Fig. 2, shows the effect of the injection volume of PAPA on the peak current. As the volume of PAPA injected was increased, the peak current increased, but the increase was small at higher concentrations. This could be due to a substrate saturation or to the fact that for large sample volumes, the peak height will become independent of sample volume in accord with theoretical aspects of FIA as established in the Sternberg equation 191.
3.0
2.5
2.0 3 4 1.5
I.0
,-
0.5
0.c b0
loo
200
300
400
500
Vi W1)
Fig. 2. Influence of injection volume on peak current for 1.2X lo-” M PAPA. The carrier stream was 0.08 M phosphate buffer (pH 8.0) at a flow rate of 0.80 ml min-‘.
Chimica Acta 308 (1995) 129-136
300
=f
6 L
-200
1.0-
g
-
150
-
loo
.\.,
‘----.
0.5 -
0.0
1 0.0
-50
I 0.5
1.0 Flux
1.5
2.0
2.5O
(ml/min.)
Fig. 3. Effect of flow rate on the peak current (0) and the peak width (m) (in seconds). Conditions as in Fig. 2 with a PAPA injection volume of 150 ~1.
The effect of flow-rate on the peak current and on the peak width were studied over the range of 0.3 to 2.0 ml mini’. The results, presented in Fig. 3, show that as the flow rate increased, the peak current decreased, but the decrease was smaller at higher flow rates. Thus, a faster flow results in smaller peak currents, as would be expected, since the PAPA is in contact with the enzyme for shorter time periods. Therefore, low flow rates would be desirable in order to maximize the response. On the other hand, a low flow rate results in broadened peaks so that flow rates below 0.50 ml min-’ required over 200 s for the current to reach its baseline value. Thus, 0.55 ml mini’ is recommended as the most suitable flow rate for achieving good sensitivity and reasonable sample throughput. When PAP (rather than PAPA) is injected and detected at a glassy carbon electrode that is not covered with a membrane, the peak current increased with flow rate and a plot of peak current vs. the square root of the flow rate was linear suggesting that mass transport is the limiting step in the reaction. However, when the electrode is covered with a membrane, the current was found to be independent
C. La Rosa et al. /Analytica
of flow rate, suggesting that diffusion through the membrane is likely rate controlling. In the enzymatic determination, the current due to PAP follows the enzymatic hydrolysis of PAPA. However, we saw above that under these conditions, the peak current actually decreased with increasing flow rate which indicates that in this case the limiting step is either the transport through the membrane or the enzymatic reaction itself.
Chimica Acta 308 (1995) 129-136
133
1%
60 -
40 -
3.1. Optimization
of inhibition reaction
The effect of different experimental conditions on the response of the biosensor in the presence of pesticides was studied in order to determine the optimal conditions. The inhibition of cholinesterase by organophosphorus and carbamate agents is well known [lo]. Several inhibition mechanisms have been suggested for acetylcholinesterase depending on the substrate [ll]. In a previous work [5], we demonstrated that the inhibition is a function of both inhibitor and substrate concentrations and that in the pesticide determination two approaches can be employed: inhibition in the presence of substrate or preincubation of the biosensor with the inhibitor. Because the preincubation method exhibited a lower detection limit and is less sensitive to possible interferences, it was selected for this work. The relationship between inhibition and preincubation time under quiescent conditions is described by the Aldridge equation [12]: log( lOO/%Z)
20
I
0'
00
0.3
0.6
0.9
Flow
1.2
1.5
1.9
Rate (ml/min.)
Fig. 4. Influence of injection volume of paroxon (10m4 M) on the percentage of inhibition, I%, at different flow rates. Injection volume of 1.2 x 1O-3 M PAPA: 150 ~1.
ing injection volumes of the pesticides, carbaryl and paroxon, was studied at a fixed concentration of substrate (1.2 mM of PAPA). The results, presented in Figs. 4 and 5, show that the inhibition, decreased linearly with increasing flow rate, for both carbaryl
= ZC,[r]t
where %Z is the percentage of inhibition, K, is the inhibition bimolecular rate constant [13], [I] the concentration of inhibitor and t the preincubation time. Thus, as the degree of enzyme inhibition is dependent on incubation time more sensitive determinations can be made by preincubations for large time periods. In the FIA approach proposed, the time that the inhibitor is in contact with the enzyme (incubation time) and therefore the extent of inhibition is determined by the flow rate and the sample injection volume. Thus, both parameters must be optimized. The inhibition study was made by observing the effect of different substrate and pesticide concentrations on the percentage of inhibition. The effect of flow-rate on the inhibition of the enzyme at increas-
01
0.0
0.3
0.6
0.9
1.2
1.5
1.8
21
Plow Rate (ml/min.) Fig. 5. Influence of injection volume of carbaryl in the percentage of inhibition, I%, at different flow rates. Conditions as Fig. 4.
134
C. La Rosa et al. /Analytica
and paroxon at all sample volumes employed. These results indicate that the percentage of inhibition, which is essentially the same as the binding between the enzyme and the inhibitor, is only dependent on the time that both are in contact. It should also be mentioned that at small sample volumes, the inhibition decreases linearly and sharply with increasing flow rate, for both carbaryl and paroxon but the decrease was small at large sample volumes. From these results it can be inferred that the influence of the flow rate will be small if large sample volumes are used. In this case, the time during which enzyme and inhibitor are in contact is compensated by the high concentration of inhibitor, [I], used. In plots of %I as a function of flow rate, the Y intercept represents the maximum inhibition obtained. It can be seen in Figs. 4 and 5 that the same Y intercept is obtained irrespective of the sample volume employed and in fact this value agrees well with the inhibition calculated for the same concentration of carbaryl or paroxon in a previous work under steady state conditions. Fig. 6 shows the effect of flow rate on the inhibition of the enzyme at the same concentration of
I 100
1% 80
60
L
y=48.7-31.9Y;r=0.9969
I
40
Chimica Acta 308 (1995) 129-136
1w
-
1%
60 -
40 -
I
0
0.0
0.2
04
06
I
0.6
I
1.0
I
1.2
I
1.4
I
1.6
I
1.6
Flow Rate (ml/&n.) Fig. 7. Influence of injection volume of substrate (1.2X 10-j M PAPA) on the percentage of inhibition at different flow rates. Injection volume of 1.0X lo-” M carbaryl: 500 /*l.
substrate employed in the experiments described above, and with increasing concentrations of carbaryl. It is clear that the flow rate is critical for the determination of small concentrations of inhibitor, and only with the lower flows could we detect carbaryl at concentrations below 10m6 M. However, as can be seen in Fig. 7, the injection volume of substrate has a minimal influence in the percentage of inhibition. The choice of optimum flow rate is a compromise between the time needed for the inhibition reaction to occur and the time needed for the current to return to baseline after injection, with the finality to obtain a reasonable sample throughput. Therefore, flow rates of 0.55 and 0.34 ml min. ’ for paroxon and carbaryl, respectively, were employed in all further studies. 3.2. Analytical characteristics
20
Flow Rate (mUmin.) Fig. 6. Influence of concentration of carbaryl on the percentage of inhibition, I%, at different flow rates. Injection volumes: 500 ~1 of carbaryl and 150 /Al of 1.2X lo-’ M PAPA.
As anticipated the signal peaks (peak current) decreased with increasing concentrations of pesticide. Linear calibration plots were obtained over the range of 5.0 X 10P7-1.0 X lop5 M and 5.0 X 10-7-5.0 x 1o-5 M for paroxon ( y = 433 + 70.00x; r = 0.9998) and carbaryl (y = 335 + 54.71 x; r = 0.99291, respectively. The detection lim-
C. La Rosa et al. /Analytica
its (defined as the concentration of inhibitor required to obtain 5% of inhibition) of the method is 1.0 X lo-’ M pesticide. Reproducibility was tested by repeated 500 ~1 injections of either pesticide at two different concentrations. The relative standard deviations were 3.7% for 4.0 X lop5 M and 4.0% for 8.0 X 1Omh M for either carbaryl or paroxon. Most of the electroactive species that could be oxidized at the same potential as PAP, do not interfere because with the preincubation procedure employed they are rinsed away with the carrier stream prior to determination of the remaining enzyme activity. In fact, in the determination of paroxon at 1.0 X 10m5 M we have observed no interference in the presence of uric and ascorbic acid nor benzaldehyde in the range of 1.2 X lop3 M to 1.5 X lo-* M. However, compounds which strongly absorb onto the electrode surface, such as 0.1% (w/v) of BSA, cause a decrease in the peak height (9% in case of BSA). The presence of surfactants capable of denature the enzyme activity, such as 0.1% of sodium dodecyl sulphate (SDS), causes a serious interference, given rise to a 50% decrement in the sensor response. The biosensor was used repeatedly to confirm its stability over a long time period. After use the inhibited enzyme membrane can be reactivated by immersion in a solution of 2-pyridinealdoxime methiodide (24.8 mg/lOO ml of phosphate buffer) for at least 4 h, rinsed and equilibrated for 2 h in phosphate buffer [5]. The stability of the sensor was high and even after repetitive use for one month the electrode retained 90% of its original activity when it was stored in the phosphate buffer while not in use. 3.3. Determination fruits
of pesticides
in lagoon water and
The determination of the pesticides carbaryl and paroxon was carried out in water. Linear calibration plots (1% vs. log(inhibitor)) in this matrix were obtained over the range of 5.0 X lo-‘-1.0 X lo-’ M and 5.0 X lo-‘-5.0 X lo-” M for paroxon (y = 377 + 59x; r = 0.9962) and carbaryl (y = 294 + 46x; r = 0.9932), respectively. Immobilized AChE was inhibited faster by the paroxon than by carbaryl. As a rapid and sensitive test was envisaged, a cycle time of 5 min for the slow one was needed. This
135
Chimica Acta 308 (1995) 129-136
included incubation with sample and determination of the remaining activity. Incubation with the lagoon water did not decrease AChE activity. A high concentration of paroxon (1.0 X 10d4 M) caused complete inhibition of the enzyme but at the same concentration carbaryl caused only an inhibition of 90%. The lowest concentration of pesticide determined was 1.0 X lo-’ M, for paroxon and 4.0 X lo-’ M for carbaryl. Longer incubation times, that means, lower flow rates resulted in greater inhibition, but low sample throughputs. The determination of carbaryl was also carried out in kiwi fruits. Carbamate insecticides such as carbaryl are often employed against Polychrosis botrana in some fruits including grapes, apples and kiwis. Kiwis were chosen for the present study in order to assess the validity of the proposed method to the detection of pesticides in real samples. For the residue determination of carbaryl in kiwi fruits, 2.5 ml of the ethanolic sample solution obtained as described in the Experimental section were brought to 25 ml with 0.08 M phosphate buffer (pH 8.0). 500 ~1 of this solution were subsequently injected in the FIA system and the determination carried out as described before. The validity of the method was assessed by comparing these results with those obtained by a UV method in which the absorbance of carbaryl was measured at 220 nm against an ethanolic extract of kiwi without pesticide, as blank. The calibration curve was constructed from absorbance measurements of carbaryl added to the kiwi blank solutions. An excellent correlation (r = 0.9972) was found over the range of 0.07 to 4.0 mg ll’. Similarly, a calibration curve for the determination of carbaryl in kiwi fruit extract with the amperometric biosensor described herein gave a correlation of (r = 0.9914). Upon comparison of the results obtained by both methods (Table l), an excellent agreement was found.
Table 1 Determination Pesticide
Carbaryl ’ Average
of carbaryl
in kiwi fruits
uv determination (mg 1-l)
Amperometric flow-through biosensor (mg I-
3.10 a
3.01 a
of three determinations.
‘)
C. La Rosa et al./Analytica
136
121K. Gibson and G.G. Guilbault, Anal. Chim. Acta, 76 (1975)
4. Conclusions The flow-injection device for the automated reaction of immobilized enzyme membranes permits rapid enzyme inhibition tests. The detection of pesticides based on the principle of acetylcholinesterase inhibition is achieved within a few minutes. The sensitivity of this assay is comparable to non-biological standard methods such as LC.
Acknowledgments This work was supported by the Comunidad Autonoma de Madrid, through grant No. 199/92. The authors acknowledge Dr. H.D. Abruiia for his contributions in critically reading this manuscript and the NATO Scientist Program.
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