Fiber-Optic Immunosensors for Detection of Pesticides - ACS

Chapter 14

Fiber-Optic Immunosensors for Detection of Pesticides 1

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Mohyee E. Eldefrawi , Amira T. Eldefrawi , Nabil A. Anis , Kim R. Rogers , Rosie B. Wong , and James J. Valdes Immunoanalysis of Agrochemicals Downloaded from pubs.acs.org by NATL TAIWAN UNIV on 07/01/15. For personal use only.

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Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201 Biotechnology Division, U.S. Army Research Development and Engineering Center, Edgewood, MD 21010 Exposure Assessment Research Division, Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency, P.O. Box 94378, Las Vegas, NV 89193 American Cyanamid Agricultural Research Division, P.O. Box 400, Princeton, NJ 08543 2

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A reusable fiber optic enzyme biosensor provided rapid detection of acetylcholinesterase (AChE) inhibitors and fast regeneration of the sensor for reuse. However, while highly sensitive in detection of oxyphosphate AChE inhibitors, it was insensitive in detection of the less active thiophosphates. It was generic in its identification and did not identify the chemical structure of the analyte. A fiber optic immunosensor, using polyclonal antiparathion antibodies (Abs) was very selective (could differentiate between parathion and paraoxon) and more sensitive, but too slow and nonreusable. A new strategy was developed, using fluorescent pesticide derivatives and polyclonal or monoclonal Abs to construct reusable biosensors with faster turn around time. An immunosensor was developed to assay for imazethapyr herbicide, that was highly sensitive and selective for imidazolinone compounds, unaffected by soil extract matrix and capable of repeated usage. Advantages of biosensors over ELISA are simplicity, speed and reduced need for sample pretreatment. Immunoassays have been used for years to detect pesticide residues in soil, water and plants. However, the ELISA type assays used are usually time consuming and many of the antibody (Ab) types utilized are either polyclonal or are not highly selective. There is a growing need for assays that are rapid, cost effective and highly sensitive, without giving false negative or false positive results. Two major technological developments are making these objectives within reach. One is in the biosensor field, with advances in a variety of transducers and biological sensing elements, which makes it technically feasible to detect almost any analyte. The other is in the field of molecular immunology, which allows the engineering of an Ab with the exact affinity needed for each pesticide or metabolite of interest, that would provide sensitivity, selectivity and reversibility, and guarantees a stable continuous supply by transfecting the whole or fragments of the Ab gene(s) in E. coli (Ward, 1992). 0097-6156/95/0586-0197$12.00/0 © 1995 American Chemical Society

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IMMUNOANALYSIS OF AGROCHEMICALS

Immunoanalysis of Agrochemicals Downloaded from pubs.acs.org by NATL TAIWAN UNIV on 07/01/15. For personal use only.

Acetylcholinesterase-Based Biosensors We have utilized a fiber optic evanescent fluorosensor (Rogers et al., 1991) and a potentiometric sensor (Fernando et al., 1993) to detect acetylcholinesterase (AChE) inhibitor insecticides. The fiber optic biosensor has the advantage of no direct electrical connections, no drift problem, suitability for continuous monitoring and easier applicability for field use. 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 Block and Hirschfeld (1986) and 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, onto which are immobilized the sensing elements, are 1 mm in diameter and 6 cm long with polished ends. The fiber optic evanescent fluorosensor makes use of the evansecent wave effect by exciting a fluorophore just 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 through the fiber. This is detected after transmission through 510 nm LP and 530/30 nm filters. The quartz fiber is inserted in a flow cell which allows its center 47 mm to be immersed in 46 ml, which is exchanged every 14 sec. Initially, a fluorosensor was developed, with immobilized nicotinic acetylcholine receptor on quartz fibers. It was found to be effective in detecting fluorescein isothiocyanate (FITC)-labeled receptor-specific α-neurotoxins (e.g. oc-bungarotoxin, oc-cobratoxin), as well as receptor agonists (e.g. nicotine) and antagonists (e.g. dtubocurarine) (Rogers et al., 1991). The pH-dependence of the quantum yield of fluorescence, suggested that fluorescein may also be used as a proton (H+) sensor. Because AChE hydrolysis of acetylcholine (ACh) produces acetate and protons, we developed a potentiometric biosensor to detect AChE inhibitors in Krebs physiological solution buffered with fairly low molarity (0.1 mM) Na phosphate (Dawson and Elliot, 1959). This increases sensitivity since it is the generated protons that quench the fluorescence. The biosensor was constructed by immobilizing FITCtagged AChE on the quartz fiber (Fig. 1) and monitoring its activity. The pHdependent fluorescent signal, in the evanescent zone on the fiber surface, was quenched by the protons produced during ACh hydrolysis (Fig. 2). The addition of the substrate ACh to the buffer perfusion medium resulted in quenching of the steadystate fluorescence. The AChE activity was assayed by interrupting the flow of the perfusate around the quartz fibers by turning the pump off, which allowed the local pH in the vicinity of the FITC-labeled enzyme to drop (Fig. 2B), and measuring the percent decrease in the baseline fluorescence during a 2 min period. The reduction in fluorescence was dependent upon the presence of the substrate ACh (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 same fiber without loss in AChE activity (Fig. 2C). The specific activity of the soluble FITC-AChE was 680 μιτιοί min-1 mg-1 for the hydrolysis of achetylthiocholine. Assuming that the specific activity of FITCAChE did not change upon immobilization, the assay of the immobilized enzyme activity by the method of Ellman et al. (1961) yielded a value of 0.48 pmol catalytic sites immobilized per fiber. There was excellent substrate specificity, using various choline esters, compared to manometric data. Whereas the reversible AChE inhibitor edrophonium (0.1 mM) reducedfluorescencequenching, that recovered immediately


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Fiber-Optic Immunosensors for Pesticide Detection 199

Figure 1. Schematic presentation of AChE biosensor with the optical system to measure fluorescence, showing the lenses, detector and the flow cell with the optic fiber. Inset shows the fiber with immobilized FITC-AChE and the chemical reaction that occurs. f=focal length (Reproduced with permission from Reference 9. Copyright 1991 Society of Toxicology.)

Β Krebs

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OFF ON

Figure 2. The change in fluorescence as a result of AChE activity. (A) Steadystate fluorescence in the absence of ACh was unaffected by interruption in the beffer flow. (B) In the presence of 1 mM ACh, fluorescence was quenched when the pump was turned off and protons accumulated. The baselinefluorescencewas quickly reestablished when the pump was turned on and the excess protons were removed by the perfusing substrate solution. Enzyme activity was measured by the amplitude of signal quenching after 2 min. (C) The response was reproducible after 2 h. (Reproduced with permission from Reference 9. Copyright 1991 Society of Toxicology.)


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upon its removal (Fig. 3), the carbamate insecticides bendiocarb and methomyl inhibited the biosensor response, but recovery was much slower. Pre-exposure of the fiber to the organophosphate (OP) antiChEs echothiophate and paraoxon irreversibly inhibited AChE and accordingly the quenching (Fig. 4). However, the OP-inhibited AChE biosensor, could be reactivated by the nucleophilic 2-pralidoxime (2-PAM), which reactivates the phosphorylated AChE by dephosphorylating it. This makes the biosensor reusable for detecting AChE inhibitors and distinguishing inhibition by OPs from that caused by unrelated dénaturants, such as heavy metals, whose inhibition is not reversed by 2-PAM. These effects reflected the mechanisms of action of the inhibitors with AChE. The inhibition (Fig. 5) constant values, obtained by the fiber optic enzyme biosensor, were comparable to those obtained by the colorimetric method (Table I). Antibody-Based Biosensors Using the FITC-AChE biosensor, neither malathion, parathion nor dicrotophos could be detected even at 1 mM concentration, since they require bioactivation to malaoxon, paraoxon and dicrotophos oxon, respectively. This failure led to the development of a fiber optic immunosensor using, as the biological sensing element, rabbit Abs raised against bovine serum albumin-parathion conjugate (Anis et al., 1992). [In the immunosensor assay for parathion, a sandwich strategy was used, similar to that of the ELISA assay. The casein-parathion conjugate was attached to the glass fiber and exposed to the sample containing rabbit anti-parathion sera, in absence or presence of parathion. A second step was then needed to develop fluorescence by the addition offluorescein-taggedgoat anti-rabbit Ab]. FITC goat antirabbit IgG was used to generate the optical signal which was reduced, in a dose dependent manner, by the presence of parathion in the sample (Fig. 6). Parathion inhibited binding of the Ab to the fiber, thereby reducing subsequent fluorescence. This biosensor could detect 0.3 ppb parathion despite its poor potency in inhibiting AChE and was a 100 fold more selective for parathion than paraoxon. This is unlike the AChE optic fiber biosensor, which was highly selective for paraoxon. The AChEbased biosensor was generic in its detection capabilities and could not identify the chemical structure of the AChE inhibitor. In order to simplify and speed up the detection process, a one step competitive Ab binding assay was developed. This strategy is based on the competition between the analyte (e.g. parathion) and afluoresceinconjugate of the analyte for binding to the immobilized anti-analyte Abs (Fig. 7). This speeds up detection significantly. We used it to detect the herbicide imazethapyr in soil extracts. The Ab against imazethapyr conjugate was immobilized directly onto the quartz fiber, and its binding offluorescein-taggedimazethapyr (FHMI) in buffer solution resulted in increased total internalfluorescence(Fig. 8). The presence of imazethapyr in the sample competed with FHMI for the bound Ab, thereby reducing the rate of fluorescence in a time- and concentration-dependent manner from 0.1 to 100 μΜ imazethapyr (Fig. 9A). The rate of association was calculated from the slope of the fluorescence signal plot during the initial 20 second segment of the response. The IC50 of the dose-response curve (Fig. 9B) was calculated to be 2 μΜ. An alternate displacement mode was used which improved sensitivity. Rather than determining the concentration of the analyte by the degree of reduction in the rate offluorescenceincrease, it was determined by the reduction influorescenceafter it reached a steady state. Thus, thefluorescenceresulting from binding of FHMI in buffer to the Ab coated fiber, that reached a steady state in about 2 minutes, was reduced by the addition of imazethapyr almost immediately. It was concentration -dependent and more sensitive for imazethapyr, being effective at 0.001 to 100 μΜ, giving an IC50 of 0.3 μΜ (Anis et al, 1993).

14. ELDEFRAWI ET AL.

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Figure 3. Reversible inhibition of the fluorescent signal generated by the biosensor in presence of 1 mM ACh (A), and after 2 min perfusion with 0.1 mM edrophonium + 1 mM ACh (B). After removal of edrophonium and reperfusion with 1 mM ACh (C) the signal was restored. Arrows indicate times when the pump was turned off. The pump was turned on again after 2 min in each case. Three measurements, 2 min apart, were recorded for each condition. (Reproduced with permission from Reference 9. Copyright 1991 Society of Toxicology.)

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Figure 4. Inhibition of the AChE biosensor by echothiophate. (A) Control responses of the biosensor to ACh (lmM). Echothiophate (0.1 mM) was then added to the ACh-Krebs solution and after a 10-min perfusion the biosensor signal was recorded (B). Echothiophate was replaced with 1 mM 2-PAM in the AChKrebs solution and after a 10 min perfusion 2-PAM was removed and the biosensor response was recorded (C). (Reproduced with permission from Reference 9. Copyright 1991 Society of Toxicology.)

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-LOG INHIBITOR CONC (M) Figure 5. Concentration-dependent inhibition of the AChE biosensor by echothiophate ( · ) ; paraoxon (O); bendiocarb (•); methomyl (•); dicrotophos (A); parathion (X); and malathion ( Δ ). The AChE biosensor was exposed to the indicated compound for 10 min prior to the introduction of ACh and subsequent assay of activity. The symbols are means of at least three measurements. (Reproduced with permission from Reference 9. Copyright 1991 Society of Toxicology.) Table I. Comparative Inhibition of Immobilized and Soluble AChE by Organophosphates and Carbamates, Assayed by the Fiber-Optic Biosensor and Colorimetric Assays. (Reproduced with permission from Rogers et al., 1991). Fiber-optic Colorimetric Compound biosensor assay assay ^ IC50(M) IC50(M; a

Echothiophate Paraoxon Bendiocarb Methomyl Dicrotophos a

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AChE biosensor was incubated in the presence of each compound for 10 min prior to introduction of ACh (1 mM) and subsequent assay of activity in the presence of inhibitior. b Soluble AChE was incubated in the presence of each compound for 10 min and then assayed using the method of Ellman et al. (1961).


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Figure 6. Use of optic fiber immunosensor for detection of parathion. (A) Inhibition of the optical signal generated by binding of FITC goat anti-rabbit IgG to fibers precoated with casein-parathion then incubated in the rabbit antiparathion IgG (1/500 diluted serum), by different concentraions of parathion. Control fiber was coated with casein-parathion but not incubated with the immune serum. The amount of parathion in the sample is reflected in reduction of fluorescence. (B) The dose effect of the presence of parathion ( · ) or paraoxon (O) in the medium, which competes for the fluorescent-labeled complex and prevents its binding, on the signal generated by binding of FITC-goat antirabbit IgG to the antigen and Ab coated fiber. The 100% control level is the rate recorded in absence of parathion or paraoxon. Symbols are means of triplicate measurements, made on three separate fibers, with standard errors of

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Figure 11. Concentration-dependent FHMI displacement by imidazolinone and non-imidazolinone compounds, whose chemical structures are shown. Three concentrations (1, 10, and 100 μΜ) of each compound were used for displacement of signal from steady-state bouund levels of fluorescence (Top). The imidazolinones displaced the signal in a concentration-dependent manner, while the non-imidazolinone, even at 100 μΜ concentration, did not. Each symbol represents a mean of three measurements (SD +

Fiber-Optic Immunosensors for Detection of Pesticides - ACS

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