Biomarkers of Human Exposure to Pesticides - American Chemical

Chapter 7

Downloaded by UNIV OF GUELPH LIBRARY on September 9, 2012 | http://pubs.acs.org Publication Date: November 23, 1993 | doi: 10.1021/bk-1992-0542.ch007

A Biosensor for Monitoring Blood Cholinesterases as a Biomarker of Exposure to Organophosphorus Anticholinesterase Pesticides 1

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Mohyee E. Eldefrawi , Kim R. Rogers , Nabil A. Anis , Roy Thompson , and J. J. Valdes 3

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Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201 Exposure Assessment Research Division, Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency, P.O. Box 93478, Las Vegas, NV 89193-3478 Biotechnology Division, U.S. Army Research Development and Engineering Center, Edgewood, MD 21010 2

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A fiber optic evanescent fluorosensor was developed for the rapid detection of anticholinesterases (AntiChEs) and was modified to measurecholinesterase(ChE) activities in whole blood. Quartz fibers coated with fluorescein isothiocyanate (FITC)-tagged acetylcholinesterase (AChE), detected AntiChEs by their reduction of quenching of fluorescence that was produced by protons generated during acetylcholine (ACh) hydrolysis. Blood ChE activity was detected by quenching the fluorescence of FITC bovine serum albumin immobilized on the quartz fiber. High ChE activity in blood samples produced strong fluorescence quenching and exposure to antiChEs reduced quenching. Fluorometric measurements were made in seconds to minutes by evanescent waveguide fluorometer on 200 μl blood samples. A 2-minute rinse in Krebs buffer was sufficient to prepare thefiberfor another measurement. Biosensors have captured the imagination of the world's scientific and commercial communities by combining interdisciplinary skills of biologists, physicists, chemists and engineers to provide innovative solutions to analytical problems. Biosensors are applicable to clinical diagnostics, food analysis, cell culture monitoring, environmental pollutants and should also be applicable to detection of drug residues in milk and animaltissuesand drugs of abuse and metabolites in urine, blood and sweat. Biosensors are analytical devices made of biological recognition materials (e.g. enzymes, receptors or antibodies) coupled to transduction elements that 0097-6156/94/0542-0114S06.00/0 © 1994 American Chemical Society

In Biomarkers of Human Exposure to Pesticides; Saleh, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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A Biosensor for Monitoring Blood Cholinesterases

range from glass electrodes to silicon chips and opticfibers(Wise, 1989). The high specificities of the binding sites of these proteins for certain drugs, toxicants and toxins is the basis for their use in detection. Biosensors are possible successors to a wide range of analytical techniques in process control, clinical laboratories, veterinary health care and in food industry (McCann, 1987; Robinson, 1991). Although the functions of complex proteins are typically affected by pH, ionic strength, detergents, and dénaturants, biosensors can function within a wide range of conditions. Receptors, enzymes and antibodies bind their specific ligands, substrates and antigens, respectively with high affinities in cellular and extracellular media in presence of ions, lipids and other proteins. Enzyme-, Receptor- and Immuno-Sensors Enzyme (by far the most widely used biological recognition material tested) biosensors have led the developments in thefield(Guilbault, 1981; Clark, 1987). A good example is glucose oxidase, which is used to construct glucose sensitive electrodes (Fortier et al., 1990), fiber optic biosensors (Mansouri and Schultz, 1984) and chemical field effect transistor sensors (Murakami et al., 1986). Another example is acetylcholinesterase (AChE), which has been used to construct enzyme electrodes (Baum and Ward, 1971; Durand et al., 1984), fiber optic biosensors (Rogers et al., 1991a; Hobel and Polster, 1992) and potentiometric biosensors (Rogers et al., 1991b) for detecting anticholinesterases (antiChEs). The approach used in thefiberoptic biosensor is to label AChE with the pH-sensitive fluorescein-labeled-isothiocyanate and immobilize them on optic fibers. Hydrolysis of ACh by AChE releases acetate, which changes the pH thereby quenching the fluorescence. AntiChE values obtained with this biosensor were in good agreement with those obtained by the Ellman et al. (1961) spectrophotometric method using soluble AChE. Even though the biosensor is fast with results obtainable in seconds to minutes, a disadvantage is that many antiChEs require bioactivation and thus the less active parent compounds are detectable only at high concentrations. Since the antiChE-inhibited biosensor can be reactivated by oximes the biosensor can be used repeatedly. Also, inactivation of the biosensor by unrelated dénaturants such as Hg can be determined. The use of neurotransmitter or hormone receptors to construct biosensors has been utilized mostly with the nicotinic ACh receptor (Gotoh et al., 1987; Eldefrawi et al., 1988; Taylor et al., 1988; Rogers et al., 1989). The ACh receptor fiber optic evanescent fluorosensor has a wide detection range, and shows excellent sensitivity and pharmacological specificity (Rogers et al., 1991c). Immunosensors utilize antibodies for the detection of specific antigens. In contrast to a receptor or enzyme, an antibody is specific for a single epitope or antigen. As a consequence, immunosensors are well suited for detecting a specific compound in relatively complex media, which may contain structurally related chemicals. Antibodies have been used as sensing elements in surface plasmon resonance (Morgan and Taylor, 1992), piezoelectric (Guilbault et al., 1992), capacitive and ISFET biosensors (Gotoh et al, 1987; Eldefrawi et al., 1988). Optical immunosensors, which utilize antibodies (Ab) for the detection of specific antigens, are under intensive investigation, in a number of laboratories (Robinson, 1991). Evanescent excitation with collection of the fluorescence that In Biomarkers of Human Exposure to Pesticides; Saleh, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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tunnels back into trapped modes of the waveguide was described by Hirschfeld and Block (1984) and Andrade et al. (1985). Fiber optic evanescent waveguide fluorosensors are superior in sensitivity to distal end sensors and have been used successfully (Rogers et al., 1989, 1991a, b; Anis et al., 1992).


AChE-Biosensor for Detection of Anticholinesterases The fiber-optic evanescent fluorosensor instrument, designed and built by ORD, Inc. (North Salem, NH), is a portable fluorometer that is adaptable to field work. Components of this instrument which were described in detail by Glass et al. (1987) include a 10-W Welch Allyn quartz halogen lamp, a Hamamatsu S-1087 silicon detector, an Ismatec fixed speed peristaltic pump, a Pharmacia strip chart recorder, and bandpass filters and lenses as indicated in Fig. 1. The quartz fibers are 1 mm in diameter and 6 cm long with polished ends. The fiber-optic evanescent fluorosensor makes use of the evanescent wave effect by exciting a fluorophorejust outside the waveguide boundary (excitation wavelength = 485/20 nm; the latter number representing full width at half maximum). A portion of the resultant fluorophore emission then becomes trapped in the waveguide and is transmitted back up the fiber. This is detected after transmission through 510 nm LP and 530/30 nm filters. The flow cell allows the center 47 mm of a 60-mmlong fiber to be immersed in 46 μΐ, which was exchanged every 14 sec. An optical biosensor for antiChEs was constructed by immobilizing fluorescein isothiocyanate (FITC)-tagged AChE from electric eel on the quartz fiber and monitoring its activity in presence of the substrate ACh and in presence or absence of antiChEs. Fluorescence emission by the pH-sensitive dye, fluorescein, was reduced in presence of ACh that was hydrolyzed to choline and acetate and the latter reduced the pH of the medium significantly. A fairly low molarity (0.1 mM) phosphate-buffered Krebs was used to increase sensitivity. The fluorescent signal generated by the FITC-AChE immobilized on the fiber in the evanescent zone was extremely stable and showed little drift for several hours. Addition of the substrate ACh to buffer perfusion medium resulted in a readjustment of the steady-state fluorescence. The enzyme activity was assayed by interrupting the flow of the perfusate and measuring the percent decrease in the baseline fluorescence during a 2 min period. The magnitude of signal reduction was dependent on buffer capacity, and the biosensor function was dependent on the presence of the substrate ACh. In its presence (Fig. 2A), interruption of the buffer flow by turning the pump off, allowed the local pH in the vicinity of the FITC-labeled enzyme to drop resulting in fluorescence quenching (Fig. 2B). Resuming the buffer flow allowed the equilibrium to be reestablished. The assay was very stable and could be repeated numerous times on the samefiberwithout loss in enzyme activity (Fig. 2C). Several factors most likely influenced the rate of fluorescence decrease upon interruption of the buffer flow. These include the decrease in substrate and increase in product in the local environment of AChE, the effect of local pH changes on the turnover number, and the nonlinear relationship between proton concentration and quantum yield of FITC. Kinetic analysis of ACh hydrolysis by the immobilized FITC-AChE, yielded an apparent ^ (i.e., K ) value of 0.42 mM and a of 400 mV/2 min assay. By comparison, the immobilized and w


7. ELDEFRAWI ET A L

A Biosensor for Monitoring Blood Cholinesterases


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DETECTOR

Figure 1. Schematic presentation of the optical system used to measure fluorescein. Fiber inset illustrates biochemical model for A C h E biosensor usai for detection of antiChEs. f» focal length. (Reproduced with permission from ref. 22).

A

Β

Krebs

Krebs.ACh

A f t e r

6 0

^

m i n

C

Krebs.ACh

PUMP

OFF ON

OFF

ON

Figure 2. The change in total internal reflection fluorescence as a result of A C h E activity. (A) Steady-state fluorescence in the absence of A C h was unaffected by interruption in the buffer flow. (B) In the presence of 1 m M A C h , fluorescence was quenched when the pump was turned off and protons accumulated. The baseline fluorescence was quickly reestablished when the pump was turned on and the excess protons were removed by the perfusing substrate solution. (C) Enzyme activity was measured by the amplitude of signal quenching after 2 min. This response was stable after 2 h. (Reproduced with permission from ref. 22).


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soluble FITC-AChE yielded respective values of 80 and 50 μΜ for hydrolysis of acetylthiocholine measured by the method of Ellman et al. (1961). The specific activity of the soluble FITC-AChE was 680 μπιοί min" mg" for hydrolysis of ACh. Assuming that the specific activity of FITC-AChE did not change upon immobilization, assay of the immobilized enzyme by the method of Ellman et al. (1961) yielded a value of 0.48 pmol catalytic sites immobilized per fiber. The biosensor showed excellent substrate specificity (Table I) for several of the substrates used by Adams (1949) for characterization of human erythrocyte AChE. The differences in hydrolysis rate of butryrylcholine and n-butylacetate may be due to differences between erythrocyte AChE used by Adams and eel AChE, which was used in this study. The addition of 0.1 mM of the reversible AntiChE, edrophonium, to the substrate-Krebs solution resulted in a 79% reduction in enzyme activity (Fig. 3B). Quenching of the fluorescent signal was stable for as long as edrophonium remained in the perfusion solution. However, upon removal of edrophonium by switching to edrophonium-free Krebs solution, the signal recovered totally within 2 min of wash.


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Table I. Effect of AChE Substrates on the Response of the AChE Fiber-Optic Biosensor (Reproduced with permission from ref. 22)

Substrate

Relative rate of hydrolysis Fiber-optic Manometric sensor (Warburg)

Acetylcholine Acetyl-0-methylcholine Benzyl acetate Butyrylcholine Benzoylcholine /i-Butyl acetate Carbamylcholine

100 ± 0 63 ± 11 22 ± 7 21 ± 6

Biomarkers of Human Exposure to Pesticides - American Chemical

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