Flow injection analysis of paraoxon with the use of an immobilized acetylcholinesterase reactor

ANALYTIcA CHIMtcA ACTA Analytica Chimica Acta 324 (1996) 21-27

Flow injection analysis of paraoxon with the use of an immobilized acetylcholinesterase reactor Renbing Shi ‘, Kathrin Stein

*

Institutfir Anorganische und Anulytische Chemie, TU Clausthal, Paul-Ernst Str. 4.38678 Clausthal-Zellerfeld, Germany Received 15 December 1994; revised 20 November 1995; accepted 10 December 1995

Abstract A flow-injection procedure for the determination of paraoxon as an example of organophosphorus pesticides based on inhibition of acetylcholinesterase immobilized on the polymer carrier VA Epoxy Biosynth was studied. The detection was

carried out spectrophotometrically by means of enzymatic hydrolysis of acetylthiocholine iodide and reaction of the thiocholine from the enzymatic reaction with 5,5’-dithiobis(2-nitnzoic acid). Under optimal conditions for an inhibition time of 30 min the calibration graph was linear from 0.05 to 0.5 pg 1-l (r = 0.998, n - 5) with a relative standard deviation of 4.1% at 0.1 pg l- ‘. For an inhibition time of 3 min the calibration graph was linear from 2 to 20 pg l- ’ with relative standard deviation (n - 3) of 1.2% at 5 pg l- ‘. The inhibited enzyme was reactivated by 0.01 mol l- ’ 2-pyridinealdoxime methiodide. The recoveries from the samples to which paraoxon had been added (water, soil) were 104% and 94%, respectively. Keywords: Flow injection; Enzymatic methods; Acetylcholmesterase; Paraoxon; Organophosphorus pesticides

1. Introduction The use of pesticides to protect crops and plants from harm caused by insects and other organisms is unavoidable. Unfortunately, these compounds, such as organophosphates and N-methylcarbamates, which possess high biological toxicity by inhibition of cholinesterase, have been found to contaminate the environment, especially water supplies. Therefore, rapid and sensitive screening methods are required to ascertain the presence of these compounds. Indeed, gas chromatography (CC), high-performance liquid

* Corresponding author. ’ Permanent address: Department of Economic Forest, CentralSouth Forestry University, Zhuzhou, VR China. COO3-2670/%/$15.00 0 1996 Elsevier Science B.V. PII SOOO3-2670(96)00018-9

chromatography H-IPLC) and GC coupled with mass spectrometry (CC-MS) are the best available techniques. However, they are expensive and need a number of sample-handling steps. Accordingly, a lot of effort has been devoted to developments of relatively inexpensive enzymatic methods. These developments fall into two branches, namely, enzyme-biosensors and bioreactors. In the first branch, a great deal of work has been done [l-l 11. According to Schwedt and Hauck, the carbamate- and phosphor acid ester insecticide can be detected by a pH electrode modified with an acetylcholinesterase (AChE) layer [ 121. Afterwards, Stein and Schwedt developed the technique of the acetylcholinesterase-biosensor based on the combination of a pH electrode with an enzyme membrane to detect pesticides in drinking water [ 13,141. In particular, an application of the

22

R. Shi, K. Stein/Andytica

AChE biosensor was put forward in a flow-through system [ 151. By employing an enzyme reactor to determine pesticides two flow-injection methods (continuous-flow and stopped-flow) were proposed by Leon-Gonzalez and Townshend [16], which are based on the inhibition of acetylcholinesterase. The detection was carried out spectraphotometrically after conversion of cr-naphthyl acetate by the enzyme. The irreversibly inhibited enzyme was reactivated by either pyridine-2-aldoxime methiochloride (2-PAM) or l ,l’-trimethylene-bis(4-formylpyridinium bromide) dioxime [ 17,181. In order to employ this technique for the determination of pesticides in water and soil, we immobilized AChE on a polymer carrier VA Epoxy Biosynth. The use of this immobilized AChE reactor in the FIA system to determine paraoxon, as a representative of organophosphorus pesticides, has been systematically studied [ 171..The sensitivity was here enhanced so as to monitor trace residue of pesticides in soil and water and meet the demand of the ‘Prescription of drinking water’ in Germany [19]. The procedure is described below.

Chimica Acta 324 (1996) 21-27

tilled water, whose pH was adjusted to 8.3 with HCl (0.01 mol l- *> at room temperature. The reactivating solution used was 0.01 mol l- ’ 2-PAM in the carrier solution. All solutions were prepared daily. 2.2. Immobilization of acetylcholinesterase

2. Experimental

The acetylcholinesterase was immobilized on polymer carrier VA Epoxy Biosynth, by a modification described earlier [20]. In a flask 288 mg (100 U) of AChE were first suspended in 6 ml 1 mol l- ’ phosphate buffer (pH 8.0) and then 120 mg VA Epoxy Biosynth was added. After shaking the mixture at room temperature for 50 h, 10 ml 1 mol 1-l phosphate buffer (pH 8.0) were added to wash the immobilized preparation. Afterwards, the solution was carefully poured out and the immobilized AChE was finally stored in 10 ml 1 mol l- ’ phosphate buffer (pH 8.0) at 4°C. The enzyme reactor was obtained by packing a proper amount of the immobilized product into a glass column, whose size is 2 mm id, 45 mm length. When not in use, the reactor was kept in 0.05 mol 1-l Tris buffer (pH 7.4) containing 6.64 g l- ’ NaCl and stored refrigerated.

2.1. Reagents

2.3. Flow system and procedures

All reagents were commercially obtained. Acetylcholinesterase (E.C 3.1.1.7) Type XII-S from bovine erythrocytes, 0.35 units mg-’ solid supplied by Sigma, tris(hydroxymethyl)-aminomethane (Tris) from Fluka, acetylthiocholine iodide and 5,5’-dithioacid) from Serva, 2bis-(2-nitrobenzoic pyridinealdoxime methiodide (2-PAM), 99%, from Aldrich, paraoxon-ethyl, certified 0.1 g 97.5% from Labor Dr. Ehrenstorfer, VA Epoxy Biosynthe from Riedel-de Ha&. All other chemicals used were of analytical grade. A stock solution of paraoxon (1 g 1-l ) was prepared in acetone. The working solutions were obtained by proper dilution with bidestilled water and adjusted to pH 6.0 with 0.01 mol 1-l NaOH. A solution of 5,5’-dithiobis(2-nitobenzoic acid) (DTNB) was made by dissolving 0.019 g of DTNB in 100 ml 0.1 mol l- ’ phosphate buffer (Na, IWO,, pH 7.0). Carrier solution contained 0.001 mol Tris; 6.64 g NaCl; and 1 g MgCl, .6H,O per 1 1 bides-

2.3.1. Apparatus

Lambda FIA-System (Perkin Elmer) equipped with an autosampler AS 90, PC with PEFIAS-control software and a flow cell with a volume of 18 ~1 and light path of 10 mm was used for the measurements. A 100 ~1 sample loop (Teflon tube id 0.5 mm) and Tygon-pump tubes with colour codings white/white (1.016 mm id for carrier, inhibitor and reactivator), white/orange (0.635 id for DTNB) and violet/violet (2.06 id for substrate) was used. The flow system is illustrated in Fig. 1. ‘Acetylthiocholine used as substrate was injected into the carrier stream (Tris buffer) and passed through the AChE reactor, where the enzymatic hydrolysis took place. The flow leaving the reactor was mixed with DTNB-solution (Ellman’s reagent). The yellow colour developed by the reaction of thiocholine released in the hydrolysis with DTNB was measured at 412 nm. The programming of the pumps and valve is given in Table 1. Inhibition was carried

R. Shi, K. Stein/Analytics

23

Chimica Acta 324 (1996) 21-27

substrate (P2)

y;,yP

I

reaction coil b

waste

AChE-reactor photometric measurement l

flow-direction Fig. 1. Schematic diagram of the flow injection system. C: Carrier solution, S: Sample, Rl: reactivation reagent, R2: DTNB solution.

Reactivation of the inhibited AChE reactor was carried out by passing 0.01 mol 1-l 2-PAM solution through it at 1.32 ml min- ’ for 1.5 min. The procedure for the determination is given in Table 2. The enzyme reactor was thermostated at 30°C in a water-bath.

by passing the sample through the reactor instead of the carrier for 3 or 30 min. According to the difference of the absorbances before and after the solution of paraoxon passed the reactor, the percentage inhibition (1%) can be calculated using the following equation:

out

I%=

(AO-Ai)

x loo

2.4. Sample handling

where A, is the peak height absorbance of the uninhibited reactor and Ai is the peak height absorbance after inhibition.

Three samples were measured with the system developed; one was water from a pond near Clausthal, the second was drinking water of Clausthal-Zeller-

Table 1 Programming of the pumps and valve Step

Time (s)

Pump1 (PI) (rprn)

Pump 2 (P2) (rpm)

Valve

Reading Time k.)

P&-ill 1 2 3

10 8 72 0

100 30 (optimum) 30 (optimum)

100 100 0

Fill Fill Inject

0 72

Table 2 Procedure for the determination of paraoxon

Time step (s)

Measurement

Inhibition

Measurement

Reactivation

Rinsing

90

180 (1800)

90

90

90

R. Shi, K. Stein /Analytica

24

Chimica Acta 324 (19%) 21-27

(Fig. 2b). A high pH is not favourable for the enzyme, so pH 8.3 was chosen.

“*:g, +b

0,02-

ONJ 6,O

*)a

,,,,,,,(,,,,,,,,

8.5

7,o

,,,,

7.5

9,o

8.5

,,,,,_ 9.0

9,5

PH

Fig. 2. Effect of pH at 20°C. Carrier: 1 mmol 1-l Tris buffer containing 6.64 g I-’ NaCl, 1.32 ml min- ‘, c(DTNB) = 0.099 g I- ‘, in 100 mmol I-’ Na,HPO,, 0.56 ml min- ‘, c(Acetylthiocholine iodide) = 0.1 g 1-l. a: Varying the pH of the DTNB solution by fixed pH of the carrier (pH 7.4). b: Varying the pH of the carrier solution by a DTNB solution, pH 7.0.

feld and the third was soil from the woodland of Clausthal. The water sample was measured directly. The soil was handled as follows: 20 g soil was suspended in 200 ml bidestilled water and shaken for 24 h. The extract was finally filtrated by blue band filter paper.

3. Results and discussion 3.1. Optimisation of experimental conditions for activity determination 3.1.1. EfSect of pH The effect of pH of the phosphate buffer on the reaction between DTNl3 and thiocholine was first studied in the range pH 6.0 to 9.4. The absorbance remained almost constant from pH 6.5 to 7.9 (Fig. 2a), a small decrease was observed at pH > 7.9. Therefore, pH 7.0 was chosen for the following optimization procedure. The dependence of the enzyme activity on the pH was studied by varying the pH of the carrier solution from 6.5 to 9.5, because the pH-optimum for immobilized enzyme changed with buffer type and immobilizing technique applied. In this case, the absorbances increased with increasing pH from 6.5 to 8.0 and remained almost constant from 8.0 to 9.5

3.1.2. EfSect of Tris buffer concentrations Tris buffer concentration shows a relationship with buffer concentration. When used as carrier solution, it affects not only the enzymatic hydrolysis in the reactor, but also the subsequent reaction of DTNEl with thiocholine. If the buffer concentration is too high, the pH of the following phosphate buffer will be influenced. The results showed that the absorbance increased slowly with decreasing Tris buffer concentration, from 50 to 1 mol l- ‘. Therefore, 1 mm01 l- ’ Tris buffer was chosen for the following studies. 3.1.3. ESfect of flow-rates The absorbances decreased linearly with increasing flow rate over the range of pump speed, 30-50 rpm. A pump-rate of 30 rpm (1.32 ml min- ’ for carrier and 0.56 ml min- ’ for DTNB solution) resulted in the largest absorbance measured and so it was used for further studies in order to achieve sufficient contact time between the substrate and immobilized enzyme. 3.1.4. Effect of DTNB concentrations The effect of DTNB concentration on the reaction of DTNB with thiocholine formed was investigated over the range 0.02 to 1 g 1-l. The absorbance increased slightly as the DTNB concentrations decreased. If DTNl3 concentration was too low ( < 0.05 g l-‘) to react sufficiently with thiocholine, the absorbance decreased. Therefore a concentration of 0.1 g 1-l DTNB was selected for this work. Using these optimised conditions the calibration for acetylthiocholine was carried out. The calibration graph shows linear response up to a acetylthiocholine iodide concentration of 100 mg 1-l (Fig. 3). It has been reported that the inhibition is a function of both paraoxon and substrate concentrations [2]. Thus, for inhibition measurements, the acetylthiocholine concentration must be fixed. According to Stein, the optimal substrate concentration for inhibition is that concentration, where the reactor first showed saturation [ 141. In this way, an acetylthiocholine iodide concentration of 100 mg 1-l was chosen for the inhibition studies.

25

R. Shi. K. Stein / Analytica Chimica Acta 324 (19%) 21-27

0.20,

1

0.15

i

/,,,//f

0.10

an inhibition time of 30 min the calibration plot was linear from 0.05 to 0.5 pg 1-l paraoxon with a correlation coefficient of 0.998. For the shorter inhibition time (3 min) the calibration graph was linear from 2 to 20 pg l- ’ paraoxon with a correlation coefficient of 0.999.

0,05 t

owC ’

0

10

20

30

40

conc.wWallon

Inhibition [%] 50

90

70

30

of acetybthiocholine

90

loo

110 120 130 140

25ti

iodide [mgl”]

Fig. 3. Calibration for aceIylthiccholine iodide at 30°C; Carrier: 1 mmol I-’ Tris buffer pH 8.3 containing 6.64 g l- * NaCl and 1 g I-’ MgCl,.6H,O, 1.32 ml mit-‘, c(DTNB)= 0.1 g 1-l in 100 mm01 I-’ Na,HPO,, pH 7.0, 0.56 ml min-‘.

3.2. Optimisation of experimental conditions for inhibition

Inhibition [%]

3.2.1. E#ect of temperature Temperature has a large influence on enzymatic catalysis, so its effect on both hydrolysis and inhibition was examined. At higher temperatures the absorbances increased, at 37°C the absorbance reached a maximum in the temperature range investigated. However, the optimum temperature for inhibition by paraoxon was at 3O”C, which was selected (Fig. 4a). 3.2.2. Effect of pH on the inhibition The pH influences not only the determination of the enzymatic activity but also the inhibition. As shown in Fig. 4b, the pH range from 5.8 to 6.5 is most favourable for the inhibition, so pH 6.0 was used.

’ 5,0

6,0

5,5

6.5

7.0

7,5

6,0

PH Inhibition [%] 20t

::‘1” 7 5;

3.2.3. Effect of jlowthrough time Solutions with different concentration of paraoxon (0.1 and 5 pg 1-l ) were tested. Both measurements show, the longer the flow through time, the higher the achieved inhibition. For the higher concentration, after an inhibition time of 3 min over 15% inhibition was observed, but for the lower concentration (0.1 pg 1-l) an inhibition time of 30 min was necessary to achieve 15% inhibition (Fig. 4c). The calibration graph for paraoxon obtained under the optimised conditions is presented in Fig. 5. For

! (c) 0

~0

.,.,‘..,

”

5

‘.’

10

3 “”

15

20

“’

25

J

“‘I

30

35

40

flow through time [min]

Fig. 4. Effect of (a) temperature. (b) pH and (c) flow through time on inhibition, experimental condition see Fig. 4, c(acetylthiocholine iodide)= 100 mg I-‘. (1) paraoxon 0.1 pg I-’ in bidestilled water pH 5.8, 1.32 ml min- ‘, (2) paraoxon 5 Fg 1-l in bidestilled water, pH 5.8, 1.32 ml min- ‘, (a) flow through time for 0.1 Pg 1-l paraoxon 30 min, for 5 pg I-’ paraoxon 3 min, (b) paraoxon 5 pg 1-l in bidestilled water, 1.32 ml min- ‘, flow through time 3 min at 30°C and (c) the measurements were carried out at 30°C.

26

R. Shi, K. Stein/Analytics

Chimica Acta 324 (1996) 21-27

3.3. Reactivation and stability The enzyme reactor is strongly irreversibly inhib-

ited, by paraoxon. Therefore, enzyme reactivation is a key step for practicable application. In this experiment, pyridine-2-aldoxime- 1-methiodide (2-PAM) was used for this purpose. Different 2-PAM concentrations, viz. 2.5 X IO-’ 1.0 X 10m3, 5.0 X 10m3 and 10 X 10m3 mol l-‘, were examined. A 2-PAM 10 X 10m3 mol 1-l solution passing through an inhibiting reactor for a time of 1.5 min was sufficient to achieve over 90% of the starting activity, solutions with lower concentrations needed longer reactivation time. In addition, appropriate ionic strength is favourable for enzyme catalysis. It was found that by addition of NaCl and MgCl, the AChE reactor was activated, thus shorting the reaction time and raising the stability. In the absence of paraoxon over 700 measurements of substrate conversion did not give rise to decreasing activity, when the Tris buffer containing 6.64 g I- ’ NaCl was used as carrier solution. The reactor could be used more than 30 times for inhibition, when 1 g MgCl, - 6H,O was added to 1 1 of the carrier solution. However, if the amount of MgCl,. 6H,O is over 3 g l-‘, a decreased activity of the reactor was observed. When not in use, storing the reactor in Tris buffer (pH 7.4)

lnhibiiion [%] 5oc

70 50 50

L

;I /.A ,o

,r( 10,o

5,o

15,o

20,o

!25,0

30,o

c(para0xon) CgLq

Fig. 5. Calibration for paraoxon, conditions as in Fig. 4, pH of the samples = 6.0. (a) %Inhibition = 122.69 X c(paraoxon) + 2.38, linear range: 0.005 to 0.5 lg l- ‘, r = 0.998. (b) %Inhibition = 2.97 X c(paraoxon)+ 1.78, linear range: 2 to 20 pg I-‘, r = 0.999.

containing 6.64 g 1-l NaCl did not result in loss of activity. After 8 months storage a reactor showed an activity of 85% of the original. 3.4. Reproducibility Comparing to enzyme biosensors, the enzyme reactor has a great advantage in the reproducibility

Table 3 Reproducibility of the inhibition at pH 6 Reactor (3 mm) No. 1 1.2.3 1

c(Paraoxon) ( cLg/l)

Inhibition time (min)

Inhibition (%I

Standard deviation (%)

Highest/lowest inhibition (%I

0.1 0.1 5

30 30 3

14.6(n=5) 14.5 (n = 3) 16.2(n=3)

0.6 0.3 0.2

14.9/14.2 14.8/14.2 16.4/16.0

Table 4 Results of the samples (PH 6.0. n = 3) Sample

Inhibition time (min)

Inhibition (a/o)

Paraoxon added ( pg/L)

Paraoxon found ( pg/L)

Recovery (a)

Bidestilled water Water from pond Soil from woodland Drinking water

30 30 30 3

0

0.1 0.1 0.1 5.0

0.100 0.104 0.094 5.2

100 104 94 104

Experimental conditions used in Fig. 5. DL: detection limit.

R. Shi, K. Stein / Analytica Chimica Acta 324 (1996) 21-27

of its responses. Inhomogeneous enzyme membranes lead to incomparable results for different enzyme biosensors. For enzyme reactors, this drawback can be overcome by packing the immobilized enzyme reproducibly. Great reproducibility was realized in these experiments (Table 3).

27

For an inhibition time of 30 min the linear calibration range is 0.05 to 0.5 pg 1-l paraoxon, but for a shorter inhibition time of 3 min it is 2 to 20 pg 1-l paraoxon with a sample throughput rate of 6 h- ’ .

References 3.5. Samples assay Ill P. Durand, J.M. Nicaud and J. Mallevialle, J. Anal. Toxicol., After any necessary pretreatment of the samples the assay was carried out under the calibration conditions. The pH of all samples was adjusted to pH 6.0 and the samples were measured before and after spiking with paraoxon. The results (Table 4) show that paraoxon added to the samples also led to a similar inhibition compared with inhibition by paraoxon in bidestilled water. The recoveries of paraoxon added to sample types (water/soil) were 104% and 94%, respectively. This means that the matrix of water and soil did not interfere in the determinations.

4. Conclusions In this work acetylcholinesterase was immobilized in a simple way on polymer carrier VA Epoxy Biosynth and packed in a column. The immobilized AChE was very stable, after 8 months storage a reactor shows residual activity of 85% of the original. For the use of this reactor in a flow injection system to determine paraoxon the working conditions for the enzyme reactions (substrate and inhibition) and the photometric detection with Ellmans reagent were optimized so that paraoxon can successfully be detected in the trace range in soil and water sample matrices. After inhibition the enzyme can be reactivated completely with a solution containing 2-PAM and Mg2+. This ensures that the enzyme reactor can be used more than 30 times.

8 (1984) 112.

Dl P. Durand and D. Thomas, J. Envrion. Pathol. Toxicol., 5 (1984) 51. [31 R. Grass, F. Scheller, M.J. Sao and C.C. Liu, Anal. Lett., 22 (1989) 1159. [41 L.P. Kuznetsova, L.I. Kugusheva, E.B. Nikol’skaya and O.V. Yagodina, J. Anal. Chem. USSR, 45 (1991) 1033. [51 H. Lay and U. Draeger, Deutsche Lebensmittel-Rundschau, 88 ( 1992) 349. [61 J. Manem, J. Mallevialle, P. Durant and E. Chabert, L’Eau, l’industrie, les nuisances, 74 (1983) 31. [71 P. Skladal, Anal. Chim. Acta, 252 (1991) 11. 181P. Skladal, Anal. Chim. Acta, 269 (1992) 28 1. [91 C. Tran-Minh, P.C. Pandey and S. Kumuran, Biosens. Bioelectron., 5 (1990) 461. [lOI D. Martorell, F. Ctspedes, E. Martinez-F&bre.gas and S. Alegret, Anal. Chim. Acta, 290 (1994) 343. 1111C. Larosa, F. Pariente, L. Hemandez and E. Lorenzo, Anal. Chim. Acta, 295 (1994) 273. [121 G. Schwedt and M. Hauck, Fresenius’ Z. Anal. Chem., 331 (1988) 316. 1131 K. Stein and G. Schwedt, Anal. Chim. Acta, 272 (1993) 73. 1141 K. Stein and G. Schwedt, Vom Wasser, 79 (1992) 211. [151 K. Stein, CLB, 44 (1993) 453. [161 M.E. Leon-Gonzales and A. Townshend, Anal. Chim. Acta, 236 (1990) 267. I171 I.A. Takrmri, A.M. Almuaibed and A. Townshend, Anal. Chim. Acta, 282 (1994) 307. [181 C.G. De Maria, T.M. Muiioz and A. Townshend, Anal. Chim. Acta, 295 (1994) 287. 1191Verordnung ilber Trinkwasser und ilber Wasser Wr Lebensmittelbetriebe (Trinkwasserverordnung-TrinkwV), vom 22. Mai 1986, BGBl, Bd. I, 760. 1201G. Schwedt, R. Shi and K. Stein, Deutsche LebensmittelRundschau, 90 (1994) 178.