A dual enzyme electrochemical assay for the detection of organophosphorus compounds using organophosphorus hydrolase and horseradish peroxidase

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Abstract

Neurotoxic organophosphorus (OP) compounds are commonly used as chemical warfare agents and pesticides. Due to their high toxicity, rapid and sensitive field detection of these compounds has been an ongoing topic of interest. Biosensors made with organophosphate hydrolase enzyme (OPH) are generally designed to either amperometrically detect an electroactive leaving group produced following enzymatic cleavage, or to potenitometrically detect the pH change that occurs during cleavage. Since OPs are more likely to have phenolic leaving groups as compared to electroactive leaving groups, we have developed a new amperometric dual enzyme electrochemical assay that enables the detection of a broad class of OP compounds using the OPH enzyme combined with horseradish peroxidase (HRP). The assay has been applied to the detection of dichlofenthion, which does not have an electroactive leaving group and is not a commonly investigated OPH substrate. Using reverse phase HPLC, we have determined the Michaelis–Menten kinetic parameters of an engineered OPH enzyme to be KM = 0.11 ± 0.02 mM and kcat = 0.046 ± 0.003 s−1 with dichlofenthion as the substrate. Detection of the phenolic leaving group from the OPH enzyme reaction using the HRP electrode is carried out at −50 mV vs. Ag/AgCl where the noise and background are low and interferences are negligible. After optimization of the solution pH, the dual enzyme biosensor was found to have a limit of detection (LOD) of 24 μM (7.6 ppm), and a sensitivity of 0.095 ± 0.024 nA/μM for dichlorofenthion. By detecting the phenolic leaving groups from the OP targets using the HRP electrode, biosensors made using this new platform have the potential to detect a broad range of important OP compounds.

Introduction

Organophosphorus compounds (OPs) constitute a large fraction of the pesticides that are commercially available. They work by inhibiting acetylcholinesterase (AChE), resulting in an acute neurotoxic response in targeted pests [1]; however, these pesticides can be highly toxic to non-targeted species as well. Nerve agents such as sarin, soman and VX also belong to this class of compounds. Sarin was used in terrorist attacks to the subway system in Tokyo in 1999 and resulted in hospitalization of 5500 victims, with twelve fatalities [2]. Tabun and sarin were also used by Iraq in the first Persian Gulf War and against Kurdish rebels [3]. In 1997, a chemical weapons convention asked all its members to destroy all chemical weapons within a 10 year period. This resolution was signed by 170 countries and ratified by 139 [3]. However, the production of all OPs could not be banned due to their common use as pesticide. Due to their adverse environmental effects and high toxicity, sensitive, selective, portable and fast detection of OPs has been a topic of interest.

Gas and liquid chromatography, enzyme-linked immunoabsorbant assays (ELISAs) and various types of spectroscopy are some of the analytical techniques that have been utilized for detection of OPs [4], [5], [6], [7], [8]. These methods can have very low limits of detection (LOD) and they are highly selective and sensitive, however, they require trained personnel, expensive instruments, and elaborate sample preparation; thus they are not readily employable for field detection.

Numerous biosensors have been developed in an attempt to satisfy these needs; and electrochemical biosensors are especially desirable for field applications since they are easy to manufacture and deploy, and potentially can be engineered to be highly selective and sensitive. Reported electrochemical OP sensors are based on either indirect detection by cholinesterase enzyme inhibition or by direct detection by the organophosphorus hydrolase enzyme (OPH) [9].

Potentiometric and amperometric cholinesterase-based sensors have been reported. Cholinesterase sensors based on potentiometry rely on the pH change caused by the production of acetic acid during hydrolysis [10], [11]. Amperometric AChE sensors are based on the change in concentration of the electroactive product thiocholine, produced as a result of hydrolysis of acetylcholine [12], [13], [14]. When OPs are present, AChE is inhibited and therefore less thiocholine is produced. AChE sensors coupled with choline oxidase (ChO) are also commonly employed. The product of this coupled enzyme reaction is hydrogen peroxide which is a very common analyte and can be detected using peroxidases such as horseradish peroxidase (HRP) [15]. Cholinesterase-based biosensors have LODs comparable to the laboratory techniques listed previously, but they can suffer from time consuming sample preparation, slow response, and poor selectivity [5], [16].

Direct detection using OPH can be utilized for a broad class of OPs since OPH has been reported to hydrolyze a wide range of pesticides such as paraoxon, parathion and nerve agents such as sarin, soman, tabun and VX [17], [18]. Amperometric and potentiometric electrochemical OPH biosensors have been reported in the literature. Amperometric OPH biosensors rely on the detection of the electroactive product of the hydrolysis reaction [19], [20], [21], [22], [23], [24]. These sensors can achieve very low LODs but their use is limited to the select few OPs with electroactive leaving groups. Most of the amperometric OPH biosensors reported in literature are based on anodic detection of the hydrolysis product p-nitrophenol at high overpotentials. Due to high operating potentials, interferences constitute a problem as well as surface fouling caused by the phenolic groups [25]. Potentiometric OPH biosensors rely on detecting the change in local pH as result of the hydrolysis reaction [19], [26], [27], [28]. These sensors can be used for all OPs that can be hydrolyzed by OPH, but they do not have low LODs. Therefore, an electrochemical biosensor for field detection of OPs with low LOD and broad substrate selectivity to various OPs is needed.

The dual enzyme electrochemical assay presented here is a new approach to the electrochemical detection of OPs. The main goal of this paper is to introduce this new method and demonstrate its application to an under investigated pesticide that does not have an electroactive leaving group. Since many OPs, such as dichlofenthion, prothiofos and EPBP (s-seven) have aromatic hydrolysis products, a dual enzyme electrode consisting of OPH and HRP can be used to detect these OPs [29]. In this concept, the OPH hydrolyzes the OP, and the phenolic hydrolysis products serve as mediators for electron transfer (see below), and are thus detected at the HRP electrode as shown in Fig. 1. This concept can also be applied to many other enzyme electrodes that have mediated electron transfer such as laccase, tyrosinase and other peroxidase-modified electrode structures [30], [31], [32].

HRP-based biosensors have been widely studied for detection of hydrogen peroxide since it is an important analyte produced in many coupled enzyme reactions. The peroxidase cycle for HRP is shown below:HRP+H2O2k1Compound I+H2OCompound I+AH2k2Compound II+AH*Compound II+AH2k3HRP+AH*+H2O

In reaction (1a), HRP is oxidized to Compound I in the presence of hydrogen peroxide. In the following reactions, Compound I is first reduced to Compound II and then back to native HRP. The electrons necessary for the reduction are either provided directly by the electrode surface or indirectly by means of electron mediators, shown as AH2 in reactions (1b), (1c). These mediators shuttle electrons between the electrode surface and HRP. Phenols [33], [34], [35], [36], aromatic amines [33], [36], [37], ferrocyanide [38], ferrocenes [39] and iodine can act as mediators for the HRP electrode. The change in the concentration of the mediators can be detected in the presence of a constant amount of hydrogen peroxide. This principle is widely used for detection of phenolic compounds and it can be carried out at operating potentials as low as −50 mV vs. Ag/AgCl. The large number of phenols listed in the literature as mediators for HRP [29] suggest that this approach should work for a large class of OPs. Table 1 shows a sample list of organophosphates that have electroactive leaving groups and phenolic leaving groups. There are only three OPs with electroactive leaving groups as opposed to 11 with phenolic leaving groups of which 6 have been reported to be detected by HRP biosensors. Therefore, biosensors made using the dual enzyme have the potential to detect a large class of OPs. The dual enzyme method also has low operating potentials where the background and interferences can be rendered negligible.

A significant challenge in the development of new OPH-based sensors is the OPH enzyme itself. This enzyme is generally not obtainable commercially, and it is a difficult protein to express in Escherichia coli. This has resulted in a wide range of reported kinetic parameters for the enzyme as it can be difficult to obtain protein in large amounts with high activity. We have recently created OPH fusion proteins where we have added helical appendages to the enzyme to enable it to self-assemble into enzymatically active biomaterials [40]. A benefit of this effort is that the mutant OPH enzymes express at significantly higher levels under standard culturing conditions, with high activity. Therefore, this new OPH fusion protein was used to create the dual enzyme biosensors.

To demonstrate the possible broad detection capabilities of the dual enzyme electrochemical assay we chose a rarely studied model OP compound, dichlofenthion, a pesticide and nematicide, which can be hydrolyzed by OPH to produce the mediator, 2,4-dichlorophenol, as shown in reaction (2). The mediator can then be detected using HRP electrodes [29], [41].

Dichlofenthion hydrolysis by OPH has not been reported in the literature to the best of our knowledge. Thus, to confirm enzymatic activity of OPH with dichlofenthion we have also conducted kinetic studies using reverse phase high performance liquid chromatography (HPLC). The results of the kinetics studies and optimization of the dual enzyme electrochemical assay for detection of dichlofenthion are presented in this paper.

Section snippets

Chemicals and reagents

Dichlofenthion, 2,4-dichlorophenol (Fluka, 99% purity), horseradish peroxidase (HRP) (Type VI-A), sodium phosphate monobasic and sodium phosphate dibasic were purchased from Sigma–Aldrich (99% purity). Hydrogen peroxide (30%, w/v solution) was purchased from Fisher Scientific. The OPH fusion protein was expressed and purified in-house as described in Lu et al. [40]. Lyophilized OPH was hydrated to an enzyme concentration of 75 mg/mL before use in electrochemical experiments and the activity was

Kinetics

The hydrolysis of dichlofenthion to 2,4-dichlorophenol by OPH follows simple Michaelis–Menten kinetics. The kinetic data for various concentrations of dichlofenthion shown in Fig. 2 were fit to the Michaelis–Menten equation using non-linear regression software (Sigma Plot, San Jose, CA). Since the pH optima for OPH and the dual enzyme sensor are 10.5 and 8.0, respectively [40], all kinetic data were collected at an intermediate pH of 9.0. Similar experiments were conducted using parathion

Conclusions

A new electrochemical assay based on the detection of phenolic products of hydrolysis reaction of OPs with OPH is demonstrated. Specifically, we exploit the fact that multiple phenolic compounds are electron mediators for the HRP electrode. This dual-enzyme method can be extended to other compounds when the OPH hydrolysis product is an electron mediator. Hydrolysis kinetics of a model OP, dichlofenthion, with OPH was studied using reverse phase HPLC. The Michaelis–Menten parameters KM and kcat

Acknowledgments

This research was supported by U.S. Army Corps of Engineers Applied Research program with funding from the U.S. Army Engineering Research and Development Center, Construction Engineering Research Laboratory (ERDC-CERL). The authors would also like to thank Mr. Hoang D. Lu and Dr Zengmin Li for their valuable contributions.

Asli Sahin is a 3rd year PhD candidate co-advised by Professor Alan C West and Professor Scott Banta at Columbia University. She received her MS in chemical engineering from Columbia University in 2010 and BS in Chemical and Biomolecular Engineering and Material Science Engineering from University of Pennsylvania in 2008. Her research is focused on electrochemical biosensors and biofuel cells.

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      Reports [64,65] show that the phenolic leaving groups from the OPs can be detected by HRP. According this theory, researchers [63] proposed dual enzyme biosensors based on OPH and HRP. With the aid of OPH, dichlofenthion (one kind of OPs) can be hydrolyzed into 2,4-dichlorophenol, which is an electrode media for the HRP.

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    Asli Sahin is a 3rd year PhD candidate co-advised by Professor Alan C West and Professor Scott Banta at Columbia University. She received her MS in chemical engineering from Columbia University in 2010 and BS in Chemical and Biomolecular Engineering and Material Science Engineering from University of Pennsylvania in 2008. Her research is focused on electrochemical biosensors and biofuel cells.

    Kevin Dooley is a PhD candidate at Columbia University in the department of chemical engineering. He received his BS and MS in chemical engineering from Manhattan College in 2008 and 2009. His research is focused in protein engineering with applications in biosensors and biomaterials.

    Donald M. Cropek is a research chemist for the U.S. Army Corps of Engineers. He received his PhD in analytical chemistry at the University of Illinois Urbana-Champaign in 1991. His research focuses on biosensors and microfluidics primarily for environmental analysis and toxicity monitoring

    Alan C. West is a professor of chemical engineering at Columbia University. He received his PhD in chemical engineering at the University of California, Berkeley in 1989. His research is focused on engineering applications of electrochemistry, including microfabrication processes, sensors, fuel cells, and batteries.

    Scott Banta is an associate professor of chemical engineering at Columbia University. He received his PhD in chemical and biochemical engineering from Rutgers University in 2002 and he was a postdoctoral fellow at Harvard Medical School until 2004. His research is focused in the areas of protein and metabolic engineering with applications in targeted delivery, biomaterials, bioenergy, biosensors, and bioelectrocatalysis.

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