Use of Charge Coupled Devices for the Simultaneous Detection of

Chapter 16

Use of Charge Coupled Devices for the Simultaneous Detection of Multiple Pesticides by Imaging ELISA Techniques Ioana Surugiu, Anatoli Dzgoev, Kumaran Ramanathan, and Bengt Danielsson Downloaded by UNIV OF MICHIGAN ANN ARBOR on October 12, 2015 | http://pubs.acs.org Publication Date: August 15, 2000 | doi: 10.1021/bk-2000-0762.ch016

1

Department of Pure and Applied Biochemistry, Box 124 Center for Chemistry and Chemical Engineering, Lund University, S-221 00 Lund, Sweden

The chemiluminescent reaction between Horse Radish Peroxidase (HRP)/ Alkaline Phosphatase (AP) and the luminol/CSPD/hydrogen peroxide substrate is used i n a multianalytical E L I S A approach to simultaneous analysis of different pesticides. The pesticides included i n the present study were 2,4-D, Atrazine and Simazine. A novel variant of peroxidase (from transgenic tobacco, TOP) has also been investigated. The microformat E L I S A previously described was employed using thick film hydrophobic pattern on glass plates with flat wells of 2 µL capacity. In addition, sol-gel modified glass capillaries were also employed. As detection system for the chemiluminescent reaction we used a PhotoMultiplier Tube (PMT) or a Charge Coupled Device (CCD) camera. For the P M T / C C D camera based assay the monoclonal antibodies (mAbs) were diluted 1:1000 and bound to the surface during an over night incubation at 4 °C. Non bound antibodies were removed by washing with PBST buffer and the free space was blocked with 2.7 mg mL of the gelatin-based blocking reagent. For 2,4-D a detection range of 0.1-100 ng mL was obtained. Work with real samples and with mixtures of pesticides is under way. -1

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Pesticides is a term used i n a broad sense for chemicals, synthetic or natural, which are used for the control of insects, fungi, bacteria, weeds, nematodes, rodents, and other pests (1,2). The use of pesticides must be regulated i n such a manner that Corresponding author. © 2000 American Chemical Society

223

In Chemical and Biological Sensors for Environmental Monitoring; Mulchandani, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

224 the intake of a pesticide residue does not exceed the acceptable daily intake. Thereby, the monitoring of pesticides in food, soil, water etc is one of the most important aspects of minimizing potential hazards to human health (3). Several different forms of pesticides have been described i n the literature. See Table 1.

Downloaded by UNIV OF MICHIGAN ANN ARBOR on October 12, 2015 | http://pubs.acs.org Publication Date: August 15, 2000 | doi: 10.1021/bk-2000-0762.ch016

Table 1: Examples for varions categories of Pesticides Examples Pesticides Organochlorine Insecticides Organo phosphorus Insecticides and Paraoxon,Aldicarb, carbofuran, dimethoate, bromophos,ethylparathion,chlorofenvinphs, carbamate pyrimicarb,memylparatlno,diethoxy phosphoryl cyanide, pyrethroid insecticides, methyl phosphonic acid, p-aminophenyl l,2,2,trimethyl-propyl diester (MATP). Ammonium ion based fertilizers

Propazine, Atrazine, Simazine.

Herbicides

Glyphosate,sulfonylurea,2,4-dichloro phenoxy acetic acid (2,4-D), chlortoluron.

Thiocarbamate insecticides

Dimemoate,triallate,femtrotrdon,methiocar, fenthion,methomyl, thiocarbamate, aldicarb sulfone,aldicarbflfoxe, oxamyl, mathrocarb, eptam, triasulfuron.

Organophosphorus compounds are powerful inhibitors of the enzymes involved in the nerve function. Such compounds can form stable complexes with acetylcholinesterase thus preventing, by phosphorylation its function. One of the most used herbicide is 2,4-dichlorophenoxyacetic acid (2,4-D), a pesticide with a wide range of biological effects and very toxic to mammals. The human oral lethal dose is 0.1 ppm (4). This is also the basis for the most common approach to detect cholinesterase inhibitors (5). Numerous electrochemical assays have also been proposed. The acetylcholine-esterase activity can also be assayed by measuring the choline with choline oxidase resulting in sensitivities i n the 10 ppm range (6). Similar experiments can be carried out with thermometric sensing. The most sensitive assay realised with immobilized choline oxidase/catalase column was used in the enzyme thermistor (ET) and the acetylcholine esterase (AchE) was recently immobilized in a precolumn with ConA-Sepharose (7). The advantages of immunoassays as a selective, sensitive, and cost-effective method for assaying/screening clinical and environmental samples are now widely acknowledged (8). Despite the success of the microtitre plate based enzyme-linked immunosorbent assay (ELISA) (9), its extensive use is limited due to the requirement of trained personnel and long assay time. These drawbacks have initiated the



225 development of a variety of other formats such as; surface plasmon resonance (10% optical fibres (11), planar (12)1 surface acoustic waveguides (13) and piezoelectric devices (14). Another class of immunosensors utilizes enzyme conjugates with antigens at the surface of antibody coated capillaries or thick film patterned glass plates (15). This report describes a P M T and C C D camera based immunosensor for the herbicide (2,4D) and fertilizers (Atrazine and Simazine). The detection principle improves the sensitivity of the sensor by atleast three orders of magnitude compared to a microtitre plate assay. Capillary based chemiluminescent assays offer the advantage of high surface to volume interaction and efficient signal to noise ratio, compared to microtitre plate assays. Although capillary based chemiluminescent assays have proven to be very successful with clinical analytes (16) there are no reports of them being used i n immunosensors for pesticide detection. Several recent reports, however, do describe other types of chemiluminescence based sensors for pesticides. Ayyagari et al (17) reported on the development of chemiluminescence assays for organophosphorus compounds based on the inhibition of alkaline phosphatase. Gao et al (18) investigated the effect of different tapered fibre tip construction on the chemiluminescent intensity. Heineman et al (19) reported on the use of capillary enzyme immunoassay for electrochemical detection for the determination of atrazine in water. ci 2,4-D

CI

Atrazine

CI

Siniazine H

H

Figure 1: Structures of the pesticides: 2,4-D, Atrazine and Simazine.



226 We have chosen chemiluminescence based detection of 2,4-D, Atrazine and Simazine (See Figure 1) using the capillary based immunosensor since a) mere is a need for a fast, sensitive, reliable, and cost-effective means to detect pesticides for environmental monitoring and the food industry, and b) we have previously investigated the C C D camera based immunosensor schemes using microformat and "patterned thick film" based immunoassay formats. The analytical figures of merit for various chemiluminescence based sensors previously reported in the literature for pesticides are summarized in Table II. It is clear from Table II that there is a trade off between detection limit and kind of assay format chosen. In contrast, the P M T and C C D based immunosensors offer high sensitivity as well as faster analysis time. Limits of detection (S/N>3) using the C C D and P M T based immunosensors for 2,4-D are about 6 pg. The total assay time (measuring time excluding the preparation) is less than 120 sec using both the approaches.

Experimental Materials Chemicals, Immunoreagents and Standards: The 2,4-dichlorophenoxyacetic acid (2,4-D), N-hydroxysuccinimide (NHS), N,N'-dicyclohexylcarbodiimide (DCC), Tween 20 and bovine serum albumin (BSA) were purchased from Sigma chemical Co. (St. Louis, MO). The ðanolamine was obtained from Merck-Schuchardt (Hannover, Germany). The 1,4-dioxan was bought from Riedel-De Haen A G (Hannover, Germany). The disodium 3-(4-methoxy spiro[l,2- dioxetane- 3,2'- (5'-chloro) tricyclo[3.3.1]decan]-4-yl) phenyl phosphate (CSPD), 11.6 mg mL" stock solution and Emerald II enhancer, 10 mg mL" stock solution were purchased from Tropix Inc. (Bedford, M A ) . Calf intestinal alkaline phosphatase (EIA grade) and 4-nitrophenyl phosphate (4-NPP) were obtained from Boehringer Mannheim (Mannheim, Germany). The anti-2,4-D/Atrazme/Smiazine mAbs (clone 1/F6/C10) were raised at the Veterinary Research Institute (Brno, Czech Republic) and kindly provided by Dr. M i a n Franek and Dr. Sergei Eremin, Moscow State University, Russia. Rabbit anti-mouse IgG-AP was purchased from D A K O (Copenhagen, Denmark). The HRP, A P and T O P were obtained from Boehringer Mannheim (Germany). Buffers and standards were prepared using distilled and deionized water. Phosphate-buffered saline (PBS), p H 7.4, contained 0.13 M NaCl, 2.6 m M KC1, 4.0 m M N a H P 0 . 7 H 0 , and 1.0 m M K H P 0 . Washing buffer solution (PBST) contained PBS with 0.1% Tween-20. Blocking solution contained PBST with 27 mg/ml blocking reagent for E L I S A (Boehringer Manheim). Sodium carbonate buffer was used for coating and contained 13 m M N a C 0 and 85 m M N a H C 0 , p H 9.6. A stock solution of 2,4-D (10 mg m L ' ) was prepared in methanol. For calibration, a serial dilution of the stock solution with PBS was prepared from 0.001 to 1000 ng mL" . The C C D camera employed i n this study was a Photometrix 200 (Photometrix, Tucson, A Z ) . The camera was thermoelectrically cooled to -45 °C, equipped with a Thompson T H 7895 chip which was 512 x 512 pixels, dark current 0.3 electrons s" 1

2

4

1

2

2

4

2

3

3

1

1

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227 Table EkApplication of various chemiluminescent assays for pesticide analysis Pesticide Exposure Reference Method of Detection time detection limit #4 Paraoxon AchE Roda et al (20) 0.75 \ig L inhibition #4 Aldicarb Roda et al (20) 4 ngL" Pyricarb 5 min Moris et al (21) ngL' (carbamate) Paraoxon Ayyagari et al A P inhibition 30 sec 50ppb (22) u Methyl parathion ?mdeetal(23) 500-700 ppb 30 sec #4 N H 4 ions in fert N-compounds Halvatzis et al 0.03 ng m L ' ilizers (glyphosate) using C L * . (24) #4 Karen Chang et Thiocarbamate S-compounds 4pg al(25) using C L * . Navaz Diaz et al Dichlorprop methyl Agrochemical 600 sec O.llngmL ester (DME) products by (26) immunoassay. 2,4Dzgoev et al (27) 90 sec 6pg dichlorophenoxy acetic acid (2,4-D) a Erhd>r&etal(28) Methyl phosphonic 5 min 10-6 fjmol acid (MATP) a Chlortoluron Fawaz et al (29) 40 sec 0.1 ^igL u Triasulfuron Schlaeppi et al 7.5 min 0.02 ng L" (30) AchE CO Acetylcholine acetate + choline betaine + H 0 HRP Luminol + 2 H 0 + OH" aminophthaleine anion + N + 3 H 0 + light. AchE=Acetylcholinesterase; CO=Choline oxidase. N-compound + 0 — - N O * + other products NO* + 0 N0 + 0 N 0 + 0 + light. *excited state 1

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1


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2

3

1

1

1

2

2

2

2

2

2

2

3

2

S O ^ + Oacg)

2

2

2

SQ2*(g)+Q2

Use of Charge Coupled Devices for the Simultaneous Detection of

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