Biosensom & Bioelectmnicr 6 (1991) 239-243

Flow injection immunoanalysis (FIIA) - a new immunoassay format for the determination of pesticides in water Petra Kr4mer & Rolf Schmid GBF - Gesellschaft fur Biotechnologische Forschung mbH, Abtl. Enzymtechnologie, 3300 Braunschweig, FRG

Mascheroder Weg 1,

(Received 23 July 1990; revised version received 20 September 1990, accepted 24 September 1990)

Abstract: This paper presents the development of a new heterogeneous enzyme immunoassay format for the detection of pesticides. It uses the technique of a flow injection system and is named flow injection immunoanalysis (FIIA). Results are demonstrated for the measurements of the herbicide atrazine, which belongs to the triazines, and the potential of this method compared with another immunoassay format (ELISA) is discussed. Keywords: flow injection immunoanalysis, immunoassay, pesticides, triazines, herbicides, simazine, atrazine, propazine, water.

water, surface water and drinking water are HPLC (high performance liquid chromatography) and GC/MS (gas chromatography with mass spectrometry). The detection of polar compounds at this critical concentration is extremely difftcult (Schlett, 1990).Normally pretreatment (derivatization) and preconcentration are necessary before measurement (Lee & Chau, 1983; Oehmichen et al., 1987).Although it is possible to determine several pesticides within one run, all these methods are time consuming and expensive. Because large numbers of samples have to be measured, the development of fast, automated and inexpensive tests is of great interest to water supply stations. As a result, immunoassays for pesticide detection are of increasing interest, because they are sensitive, specific, fast and inexpensive (Hammock et al., 1987; Mumma 8z Brady, 1987; Haberer &

INTRODUCTION The appearance of pesticides in groundwater has posed considerable problems in the control of drinking water quality (Haberer et al., 1988u,b; Klein & Klein, 1990). Regulations governing pesticide concentration in drinking water are very strictly enforced with a maximum allowable pesticide concentration of O-5&itre, including metabolites (EEC Drinking Water Directive, 1980; TrinkwV, 1986). 232 pesticides are permitted in the FRG (BBA, 1990), and these pesticides are used in approximately 1800 different formulations. Beside the pesticide residues there are a large number of metabolites that also have to be analysed (Haberer, 1989). The methods used in water supply stations to monitor the pesticide concentration in ground239 Bkwensors & Bioelectrvnics 095~5663191/$03.50Q

1991 Elsevier Science Publishers Ltd. England. Printed in Great Britain

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Petra Ktimer, Roif Schmid

K&imer, 1988;Hock, 1989;Jung et al., 1989; Van Emon et al., 1989). Normally pretreatment of samples is not necessary. Nowadays immunoassays are carried out on microtiter plates or in tubes (W&t et al., 1990).In both cases a lot of manual work is involved, To improve automation (Ruzicka, 1988)we developed a new form of heterogeneous enzyme immunoassay for the detection of pesticides. Similar techniques also developed with a flow injection system have already been presented by Heineman and Halsall(1985), by Wehmeyer epal. (1986)with electrochemical detection, by Lee and Meyerhoff (1988) with an ion-selective electrode as detector and by Plant er al. (1988), who used fluorescence detection and liposomes for signal enhancement. No other system of flow injection immunoanalysis (FIIA) that is used for the detection of pesticides is known to the authors. MATERIALS AND METHODS Immobilization of antibudies to the membrane The general procedure for binding proteins on

PALL Immunodyne”” Immunoaflinity membranes

(PALL Corp., Glen Cove, NY) was slightly modified for the immobilization of antise~m. Sections of dry membrane (31.lm pore size, 50 mm X 60 mm) were immersed in antiserum dilution (protein concentration 60 &ml, 10,!& cm* membrane, 80 mmol/litre phosphate-buffered saline (PBS), pH 7.3) and slightly agitated for 2 h or overnight at room temperature. In the next step the membrane was placed for 1 h at 65°C into 1% bovine serum albumin solution to block nonspecific protein binding. Then the membrane was washed in buffer (PBS; 3 X 5 min). Before use the membrane had to be completely dry. The membrane with immobilized antibodies was stored at 4°C. Principle of flow injection immunoanalysis The FIIA set-up is shown in Fig. 1. The principle of this format is based on a sequential competitive enzyme immunoassay (Tijssen, 1985),where the hapten (herbicides of the triazine group) competes with the corresponding enzyme-labelled hapten (modified atrazine conjugated to peroxidase) for the limited binding sites of the anti-atrazine antibodies. These antibodies are bound to the

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Fig. 1. Set-up of FEY flow injection immunoanalys&)for the monitoring ofpesricider. Four pumps with d@rent magents are working in a time-cont<dsequmce. All reagents have topas the antibody reactor where the specific antibodim am locattxi. These antibodies are immobilized on a membrane which is changed apet each tart. Thejluonsence of the product of the enzyme reaction is measured with a jluorimeter and the peak height and anxt aIy? @SW& with an integmtor. HPPA, H@nqyphenyl propionic acid; KID, homeradish penxidase: w, waste.

241

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membrane, which is automatically changed after each assay. The reagents are pumped one after the other in a ‘stop and go’ sequence in a cross flow over the membrane with antibodies, located in the antibody reactor (Fig. 1).The standards of different concentrations of a tiazine herbicide (atrazine, simazine, propazine) are pumped for 3 min over the membrane (flow rate, @78 mUmin). After a short rinsing step with buffer (flow rate 0.65 ml/min, 80 mmol/litre PBS, pH 7.3) a ‘stop and go’ cycle follows with the peroxidase-labelled hapten (40~1): 5 X 20 s. &r another short rinsing step with buffer the substrates are injected, mixed and the flow stops for an incubation time of 2 min above the membrane. For this detection hydroxyphenyl propionic acid (HPPA, 5 mM, 40~1) was used; a fluorogenic substrate of peroxidase (Zaitsu & Ohkura, 1980) and Hz02 (2 mM, 40 ~1). The enzyme-generated product is measured downstream in a flow-through cuvette of a fluorescence detector (&., 320 nm, A,,,, 404 nm, Hitachi, F 1000)and the peak height and area are registered with an integrator (Shimadzu, C-R6A Chromatopac). The result is inversely proportional to the pesticide concentration; which means that the higher the peak, the lower the pesticide concentration. The system is controlled by a time processor (Alphotronic, FRG) and the determination of one concentration (standard/sample) needs 15 min. After each concentration, the tube with the pesticide has to be rinsed with ethanol (absolute, flow rate 078 ml/min) for 1 min, followed by rinsing with the next standard/sample for 1 min before starting the next test. Enzyme-linked immunosorbent assay (ELBA)

ELISA was carried out according to the procedure of Wittmann and Hock (1989).

RESULTS AND DISCUSSION As an example Fig. 2 shows a comparison of standard curyes for the herbicide atrazine gained with ELISA (top) and FIIA (bottom). The limit of detectability was with ELISA @Ol&litre and with FIIA 002 &litre. In this case the detection limit is defined as a practical limit of quantitation;

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Fig. 2. Comparison of stanaki curvesfor atmzine gained with (a) ELBA and (b) FIU. With both metho& it is posible to meawre in the linear mnge of the cuwe (EL&I: 001-I @litnz FI..: OO243pgAitw). (a) Data points are means of three replcates on the same plate. (b) Data points are means offour wplicates (with a mixtureof th~di&vnt sections of membmne). The dilutions of an&rum and hapten-enzyme conjugate are di&vnt in both methoak

that is, the lowest concentration observed on the linear portion of the standard curve which significantly differs from the zero sample. The measurements should be carried out within the linear range of the curve, which is slightly shorter in FM (ELISA 001-l &itre; FIIA 00243 &litre). With FIIA the slope of the standard curye is steeper. In general, FIIA has a higher coefficient of variation (CV%) than ELISA (low concentration: FIIA lO-20%, ELISA l-2%; mid-concentration: FIIA 2040%, ELISA 3-4%; high concentration: FIIA lo-20%, ELISA 0*5-l%). This is due to several facts. First, FIIA is a sequential assay, that means that analyte and enzyme-labelled hapten are not competing for the binding sites of the antibodies at the same time. Second, the immunochemical reaction and the enzyme reaction are measured under non-equilibrium conditions. Third, the immobilization of antibodies to the membrane and the saturation of non-specific

242

binding is probably not as even as on microtiterplates. Studies are in progress to improve the immobilization technique. In FIIA all reagents are pumped one after the other. Compared with the ELISA procedure, FIIA uses very short incubation times (ELISA: 1.5 h; FIti 6.5 min). In comparison with ELISA, FIIA needs about 100 times more antiserum and about 10 times more hapten-enzyme conjugate (Fig. 2). On the other hand, FIIA has the potential to be used as an automated unit for pesticide control. This is a great advantage, especially if this system were used as a unit for environmental control. The polyclonal antiserum used in this test has significant cross reactivities with three herbicides of the triazine group; namely, atrazine (lOO%), propazine (195%)and simazine (20%)(Wittmann & Hock, 1990). The sensitivity is highest for propazine (data not shown) but propazine is not in use in the FRG. Therefore it is better to calibrate the system with atrazine and then express the results as ‘equivalents of atrazine’. One also has to take into account that in environmental samples there is often a mixture of different pesticides. If those pesticides belong to the same group, for example to the triazines, it is assumed that the test will be affected. In this case the competition will be between all similar chemical structures in the water sample and the enzyme-labelled hapten for the limited binding sites of the antibodies. Therefore immunochemical tests are especially useful if one uses monospecific antibodies for one special pesticide and screens for this chemical in a high number of samples (Hammock et al., 1987). Further on one has to take into account the fact that the matrix of environmental samples is not uniform. The immunochemical reaction will be affected by different matrices in a certain way. This means that the system should be calibrated with the special sample matrix. Besides water supply stations, another eflicient operation of FIIA would therefore be in the control of eflluent in pesticide production plants, where it could be used as an alarm system. Immunoassays for different pesticides have been under development for about 10 years now. However, until now it has neither been possible to buy the pesticide-specific antibodies, nor the haptens that are needed to conjugate to the enzyme to be used in this competitive test. This means, for the development of FIIA, that the

??etm Ktimer, Roif Schmid

reagents needed are not available in ‘unlimited amounts. On this side of the development, it would be important to get the chemical industry involved. ACKNOWLEDGEMENTS The authors thank Professor B. Hock and his coworkers (TU Mdnchen, FRG) for the supply with specific polyclonal antiserum and the haptenenzyme conjugate. REFERENCES BBA (1990). Liste aller in zugelassenen Pmparaten vorkommenden Wirkstoffe. Biologische Bundesanstalt fzlr Land- und Forstwimchafr Braunschweig 11 June, 1990. EEC Drinking Water Directive (1980). Europlische Gemeinschaften: Richtlinie des Rates vom 15. Juli 1980 ttber die Qualit& von Wasser fur den menschlichen Gebrauch (80/778/EWG). Amtsblatt der EG Nr. L 229 (30 August 1980), pp. 1l-29. Haberer, K (1989). Pflanzenschutzmittel und Rheinwasserwerke. GWF Wasser Abwasq 130 (lo), 510-16. Haberer, K. & Kramer, P. (1988). Verfngbarkeit immunochemischer Nachweisverfahren filr Pflanzenschubmittel im Wasser. Vom Waster, 71, 231-44. Haberer, K., Normann, S. & Schmitz, M. (19880). Pesticides in the view of the drinking water supply - Part 1: Variety of properties, applications and behaviour of pesticides in soil and water. Wasser + B&en, 4, 177-83. Haberer, K., Normann, S. & Schmitz, M. (19886). Pesticides in the view of the drinking water supply - Part 2: Pesticides are menacing water resources and drinking water. Warer + Boden, 5,258~64. Hammock, B. D., Gee, S. J., Cheung, P. Y. K, Miyamoto, T., Goodrow, M. H., Van Emon, J. & Seiber, J. N. (1987). Utility of immunoassays in pesticide trace analysis. In Pesticide Science and Biotechnology. 6th Int. IUPAC Congress of Pesticide Chemistq ed. R Greenhalgh & R Roberts, Blackwell Scientific, Oxford, 309-16. Heineman, W. R & Halsall, H. B. (1985). Strategies for electrochemical immunoassay. Anal Chem., 57 (12) 1321A-31k Hock, B. (1989). Enzymimmunoassays zur Bestimmung von Pflanzenschutzmitteln im Wasser. Z. WarserAbwasser-Forsch., 22 (2), 78-84. Jung, F., Gee, S. J., Harrison, R O., Goodrow, M. H., Karu, A. E., Braun, A L., Li, Q. X & Hammock B. D. (1989). Use of immunochemical techniques for the analysis ofpesticides. Pestik SC& 26,303-17.

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243 Burdon & P. H. Knippenbetg. Elsevier, Amsterdam, pp. 142-5. TrinkwV (Trinkwasserverordnung) (1986). Verordnung tiber Trinkwasser und Uber Wasser ftir Lebensmittelbetriebe vom 22. Mai 1986, Bundesgesetzblatt 1 I, 760-73. Van Emon, J. M., Seiber, J. N. & Hammock B. D. (1989). Immunoassay Techniques for Pesticide Analysis ed. J. Sherma, Analytical Methods for Pesticides and Plant Growth Regulators, Vol. XVII, Academic Press, New York pp. 217-63. Wehmeyer, K. R, Halsall, H. B., Heineman, W. R, Volle, C. P. & Chen, I.-W. (1986). Competitive heterogeneous enzyme immunoassay for digoxin with electrochemical detection. Anal. Chem., 58, 135-9. Wittmann, C. & Hock, B. (1989). Improved enzyme immunoassay for the detection of s-triazines in water samples. Food h Agricultural Immunology, 1, 211-24. Wittmann, C. & Hock, B. (1990). Ein ELISA zur Bestimmung von Atmzin und Atrazin-Metaboliten in Wasser. Paper presented at the Jahrestagung der GDCh Fachgruppe Wasserchemie, Timmendorfer Strand, FRG, May 1990. Whist, S., Dohf U., Giersch, Th., Wittmann, Ch. & Hock B. (1990). Sensitiver s-Triazin-Enzymimmuno assay fur Wasserproben in PolystyrolRohrchen. Sensitive s-triazine enzyme immunoassay for water samples in polysterene tubes. GIT Fachz. Lab., 2,99-106. Zaitsu, K. & Ohkura, Y. (1980). New fluorogenic substrates for horseradish peroxidase: rapid and sensitive assays for hydrogen peroxide and the peroxidase. Analytical Biochemistry, 109, 10913.