2000 Progress Report: Biosensors for Field Monitoring of Organophosphate Pesticides

EPA Grant Number: R828160
Title: Biosensors for Field Monitoring of Organophosphate Pesticides
Investigators: Mulchandani, Ashok , Chen, Wilfred , Wang, Joseph
Institution: University of California - Riverside , New Mexico State University - Main Campus
EPA Project Officer: Lasat, Mitch
Project Period: June 1, 2000 through May 31, 2002 (Extended to June 30, 2003)
Project Period Covered by this Report: June 1, 2000 through May 31, 2001
Project Amount: $227,169
RFA: Exploratory Research - Engineering, Chemistry, and Physics) (1999) RFA Text |  Recipients Lists
Research Category: Water , Land and Waste Management , Air , Engineering and Environmental Chemistry


The lack of sensors to perform discrete and real-time in situ measurement/detection of organophosphates (OPs) in the field has limited the ability to routinely monitor these highly neurotoxic, but widely used, pesticides/insecticides. The overall objective of this research is to develop, optimize, characterize, and validate biosensors for rapid, selective, sensitive, precise, accurate, simple, and low-cost discrete and real-time in situ monitoring of OPs in the field. The biosensors will be based on screen-printed electrodes (SPE), constructed using thick-film screen printing technology, modified with Escherichia coli cells displaying organophosphorus hydrolase (OPH) on the cell surface alone and together with p-nitrophenol (pNP)-monooxygenase. OPH catalyzes the hydrolysis of paraoxon, parathion, methyl parathion, fenitrothion, EPN, etc., at high rate and selectively to pNP, which will be detected directly at the SPE or converted to hydroquinone by pNP-monooxygenase and detected at SPE. Additionally, the biosensors will be coupled with micromachined electrophoresis chips for selective determination of different OPs in a mixture and real-time in situ measurement.

Progress Summary:

Research was performed on two fronts. The first involved the demonstration of the coupled micromachined capillary electrophoresis chip with thick-film amperometric detector to separate and detect individual organophosphate pesticides with nitrophenyl substituents in a mixture. The second was to identify and evaluate pNP metabolizing microorganisms as biological sensing elements for the detection of pNP formed as a result of organophosphorus hydrolase catalyzed hydrolysis of nitrophenyl substituent organophosphates. The accomplishments in these two areas are described below.

1. Detection of individual organophosphate pesticides in a mixture using capillary electrophoresis microchip.

Figure 1 shows the details of the integrated chip/detection microsystem. The glass microchip consisted of two crossed channels and three reservoirs, including a four-way injection cross and a 72 mm separation channel. The original waste reservoir was cut-off leaving the channel outlet at the end side of the chip, thus facilitating the end-column amperometric detection. A Plexiglas holder was fabricated for holding the separation chip and housing the detector and reservoirs. A short pipette tip was inserted into each of the three holes on the glass chip for solution contact between the channel on the chip and corresponding reservoir on the chip holder. The amperometric detector was placed in the waste reservoir (at the channel outlet side), and consisted of a Ag/AgCl wire reference, a platinum wire counter and a screen-printed carbon working electrode. The screen-printed working electrode (prepared from Acheson ink using a semi-automatic printer) was placed opposite to the channel outlet, at a 50 m distance (controlled by a plastic screw and a thin-layer spacer). Platinum wires, inserted into the individual reservoirs, served as contacts to the high-voltage power supply. The power supply had switchable voltage ports between running buffer and sample injections with a voltage range between 0 and +4000 V. Amperometric detection was performed with an Electrochemical Analyzer 621 (CH Instruments, Austin, TX) using the "Amperometric i-t Curve" Mode.

Figure 1. Figure 1. Capillary electrophoretic system with electrochemical detection. (A) Glass microchip, (B) separation channel, (C) injection channel, (D) pipette tip for buffer reservoir, (E) pipette tip for sample reservoir, (F) pipette tip for reservoir not used, (G) Plexiglass body, (H) buffer reservoir, (I) sample reservoir, (J) blocked (unused) reservoir, (K) detection reservoir, (L) screen-printed working-electrode strip, (M) screen-printed working electrode, (N) silver ink contact, (O) insulator, (P) tape (spacer), (Q) channel outlet, (R) counter electrode, (S) reference electrode, (T) high-voltage power electrodes, (U) plastic screw. For clarity, the chip, its holder, and the screen-printed electrode strip are separated, and dimensions are not in scale.
Figure 2. Figure 2. Separation and detection of organo-phosphorous nerve-agent compounds: (a) 1.0x10-5 M paraoxon, (b) 1.0x10-5 M methyl parathion, (c) 2.0x10-5 M fenitrothion, and (d) 4.0x10-5 M ethyl parathion. Separation buffer, 20 mM 2-(N-morpholino)-ethanesulfonic acid (MES) (pH 5.0) containing 7.5 mM dodecyl sodium sulfate (SDS); separation voltage, +2000 V; injection voltage, +1500 V; injection time, 3 sec; detection potential, -0.5 V (vs. Ag/AgCl wire) at bare carbon screen printed electrode.
Figure 3. Figure 3. Electrophorograms for river water sample before (A) and after (B) the addition of 1.4x10-5 M of paraoxon (a), 1.5x10-5 M methyl parathion (b), and 2.8x10-5 M fenitrothion (c). The river water was sampled from Rio Grand River at Las Cruces, NM. Other conditions, as in Figure 2. The untreated sample was filtered and spiked with the required amounts of MES hydrate, SDS, and sodium hydroxide (to yield 20mM and 7.5mM levels, and pH 5.0, respectively).


Variables, such as separation voltage, buffer type, pH, dodecyl sodium sulfate (SDS) concentration, and operating potential of the amperometric detector, affecting the separation and detection of paraoxon, parathion, methyl parathion, and fenitrothion in a mixture were optimized. A rapid (in 140 sec) and sensitive detection (lower detection limits of 0.21, 0.4, 4.48, and 1.06 ppm for paraoxon, methyl parathion, parathion, and fenitrothion, respectively) of the individual pesticides in the mixture was achieved operating under the following optimized conditions: 20 mM 2-(N-morpholino)-ethanesulfonic acid (MES) pH 5 running buffer containing 7.5 mM SDS at 2000 V separation voltage and ?0.5 V (vs. Ag/AgCl wire) (see Figure 2). For demonstration of application to real environmental samples, river water spiked with paraoxon, methyl parathion, and fenitrothion were evaluated. As shown in Figure 3, the sensor detected the pesticides rapidly and sensitively. Lower detection limits for the three pesticides in river water were slightly higher than those observed for spiked buffers (0.31, 0.8, and 1.34 ppm for paraoxon, methyl parathion, and fenitrothion, respectively).

2. Evaluation of pNP metabolizing microorganism as biological detection element for pNP detection.

pNP-metabolizing microorganism, Moraxella sp. (obtained from Dr. Jim Spain, Armstrong Laboratory, Tyndall Air Force Base, FL) was grown overnight in tryptic soy broth at 30oC and 300 rpm. The cells then were transferred (final O.D.600 = 0.1) into minimal salt medium supplemented with 0.1 percent yeast extract and 0.4 mM pNP and incubated at 30oC and 300 rpm. Following the disappearance of the yellow color, attributed to pNP, indicating its complete metabolism (usually in approximately 5 hours), the culture broth was supplemented with additional 0.4 mM pNP; this was repeated three times. Moraxella cells adapted to pNP were harvested, washed, and then adsorbed on a polycarbonate membrane (performed by dropping the cell suspension on a small spot on the membrane under low suction). The cell retaining membrane then was placed (cells on the inside) on the top of the Teflon membrane of a dissolved oxygen electrode and held in place by an o-ring. The cell modified oxygen electrode, biosensor, was introduced through the hole in the rubber cork enclosing a temperature controlled cell (Fig. 4). Variables, amount of cells immobilized, operating

Figure 4. Figure 5.
Figure 4. Schematic of whole cell biosensor (1) oxygen electrode, (2) digital multimeter,
(3) recorder, (4) beaker, (5) thermostat, (6) magnetic stirrer
Figure 5. Accuracy of microbial biosensor 0.2 mg cell dry weight, pH 7.5, 25oC

buffer pH and temperature, affecting the biosensor response were optimized. Operating under optimized conditions, 0.29 mg of cell dry weight, 7.5 pH and 25oC, the biosensor detected pNP selectively (no interference from other nitrophenols, phenol, sugars, acetate, and succinate), sensitively (lower detection limit 14 ppb) and rapidly (in < 3 minutes). The biosensor had excellent accuracy (Figure 5), reproducibility between multiple samples (RSD = 4.8 percent, n = 8) and between multiple electrodes (RSD = 4.6 percent, n = 5) and was stable for 2 weeks.

Future Activities:

In the second year we plan to: (1) incorporate OPH enzyme and pNP oxygenase in the detection scheme of the microchip separation and detection to improve the selectivity and sensitivity of the analytical device, (2) incorporate OPH enzyme or cells expressing OPH with the pNP metabolizing microorganism to extend the pNP sensor for detection of organophosphate pesticides, and (3) develop and evaluate amperometric biosensor incorporating OPH and purified pNP oxygenase or whole cells based on the amperometric detection of metabolic product(s) produced during pNP oxidation.

Journal Articles on this Report : 4 Displayed | Download in RIS Format

Other project views: All 5 publications 4 publications in selected types All 4 journal articles
Type Citation Project Document Sources
Journal Article Mulchandani A, Chen W, Mulchandani P, Wang J, and Rogers KR. Biosensors for direct determination of organophosphate pesticides. Biosensors and Bioelectronics 2001;16(4-5):225-230. R828160 (2000)
not available
Journal Article Mulchandani P, Chen W, Mulchandani A, Wang J, and Chen L. Amperometric microbial biosensor for direct determination of organophosphate pesticides using recombinant microorganisms with surface expressed organophosphorus hydrolase. Biosensors & Bioelectronics 2001;16(7-8):433-437. R828160 (2000)
not available
Journal Article Wang J, Chatrathi MP, Mulchandani A, Chen W. Capillary electrophoresis microchips for separation and detection of organophosphate nerve agents. Analytical Chemistry 2001;73(8):1804-1808. R828160 (2000)
not available
Journal Article Wang J, Krause R, Block K, Musameh M, Mulchandani A, Schoning MJ. Flow injection amperometric detection of OP nerve agents based on an organophosphorus-hydrolase biosensor detector. Biosensors & Biolectronics 2003;18(2-3):255-260. R828160 (2000)
not available

Supplemental Keywords:

neurotoxic, nerve agents, capillary electrophoresis, environment, agriculture., RFA, Scientific Discipline, Air, Toxics, Ecosystem Protection/Environmental Exposure & Risk, Environmental Chemistry, Chemistry, pesticides, Monitoring/Modeling, Engineering, Engineering, Chemistry, & Physics, environmental monitoring, Escherichia coli cells, neurotoxic, screen-printed electrodes, amperometric, field monitoring, biosensing system, environmental engineering, insecticides, agriculture, organophosphorus hydrolase, biosensor, biosensors, herbicides, biocatalytic actions, capillary zone

Progress and Final Reports:

Original Abstract
  • 2001
  • 2002
  • Final