B IOSENSORS M ETHODS FOR P ESTICIDES Several types of biosensors have been developed for measuring pesticides in various

4.3.3 B IOSENSORS M ETHODS FOR P ESTICIDES Several types of biosensors have been developed for measuring pesticides in various

sample media. However, the use of biosensors for obtaining environmental meas- urements is not as common as for immunoassay. This section presents the applica- tion of biosensor techniques for detecting pesticides and illustrates the potential of various sensor designs for environmental monitoring.

4.3.3.1 Potentiometric, Light Addressable Potentiometric Sensor, and Amperometric Detection

Molecular devices employ the use of a ‘‘Light Addressable Potentiometric Sensor’’ (LAPS) for detection on large arrays. The samples are captured on membranes via vacuum filtration into discreet spots on a membrane [81]. The detection is pH-based using a sensitive LAPS method that can detect the urease enzyme conversion of urea in a pH-sensitive manner (potentiometric readings). This technique has been applied to the herbicide atrazine. As atrazine is a small molecule, a competitive assay format was developed. Fluorescein-labeled anti-atrazine antibodies and atrazine covalently linked to biotin-DNP were used as reagents. When the fluorescein-labeled antibody is bound to the biotinylated atrazine, the complex will bind to the streptavidin-coated membrane. If nonbiotinylated atrazine (from the sample) is added to the mix, any antibody bound to this species will be washed away. Thus, in this competitive assay format, the fluorescein-labeled anti-atrazine antibody can either bind to the nonla- beled or biotin-labeled atrazine. A species-specific secondary antibody labeled with urease reacts with the bound anti-atrazine antibody to generate a pH flux, providing the signal for the LAPS sensor. In this mode, there is an inverse relationship between signal and amount of nonlabeled analyte found in solution. The largest signal output is seen when there is no atrazine present and the lowest signal is observed when a large quantity of nonlabeled atrazine is present. Thus, if there is a large amount of environmental atrazine measured, the signal will be low. The result is a sigmoidal curve similar to the one shown in Figure 4.3 for the ELISA to detect 3-PBA. Note that the detection range tends to be narrow using this format (due to the sigmoidal curve) and the sensitivity can be limited. This assay would be classified as a biosensor as eight simultaneous assays can be performed using this system.

In addition to using a fluorogenic substrate for detection, other means may be used to detect the presence of pesticide analytes in environmental samples. One of the simplest techniques is a potentiometric sensor based on pH changes. In this case,

a simple biosensor that is sensitive to changes in pH would be adequate. The enzyme

Immunoassays and Biosensors 113 organophosphorus hydrolase needs only to be attached to the electrode, encom-

passed in a polymer and attached to a bioresin over the electrode for OP detection. Organophosphorus hydrolase catalyzes the hydrolysis of a wide range of OP pesti- cides (e.g., coumaphos, diazinon, dursban, ethyl parathion, methyl parathion, and paraoxon). The attached or trapped hydrolase then acts on the OP compound to produce an alcohol and an acid. The resulting acid compound is monitored as a pH change at the electrode. This is a very simple system to use and is similar to LAPS detection.

Mulchandani et al. [82] developed an assay where organophosphorus hydrolase was placed onto an electrode. The phosphate hydrolysis product was monitored by measuring the current produced at the electrode. The output of the amperometric sensor could be correlated to the concentration of pesticide in sample solutions of soil and vegetation. This detection method can be incorporated into large arrays, such as the one used by CombiMatrix on electroactive electrode arrays.

Another biosensor method is applicable to other OP compounds that produce PNP as a releasing compound. These compounds include ethyl parathion, methyl parathion, paraoxon, fenithrothion, and O-ethyl O-(4-nitrophenyl) phenylphospho- nothioate (EPN). The released PNP is oxidized at the anode to insert a hydroxyl group that is ortho to the nitro group. In this case, the oxidation current is measured amperometrically at a fixed potential. The signal is linear to the concentration of PNP present. The analysis relies on the OP compound to be trapped or conjugated to material over the electrode.

4.3.3.2 Piezoelectric Measurements Many pesticides (e.g., organophosphates and carbamates) or their metabolites are

cholinesterase inhibitors. This phenomenon can be used to develop sensors for the detection of these types of compounds. Using a piezoelectric sensor format, para- oxon was bound to an electrode (gold on a piezo=quartz surface) as the recognition element [83]. The analysis was performed by allowing a cholinesterase to interact with the modified electrode surface and with free paraoxon in a standard or sample. An oscillation change can be observed in terms of hertz or an electronic occurrence.

A competitive assay was developed that allowed competition for cholinesterase between a cholinesterase inhibiting pesticide in solution and the inhibitor bound to the electrode surface. The ability of cholinesterase to bind to the paraoxon immo- bilized on the electrode is minimized or prevented in the presence of free inhibitor (analyte) in solution. In this case, the cholinesterase remains in solution bound to the pesticide in the sample. The sensing surface can be regenerated for reuse. The format can be used to develop better inhibitors and to quantitate OP compounds in solutions of environmental samples.

4.3.3.3 Surface Plasmon Resonance SPR technology has been used in the biosensor field for some time and many sensors

of this type are commercially available. The technique depends on the change in the reflectance angle (Plasmon) due to mass changes at the surface. Binding of proteins

and small materials change the mass number at the surface and the reflectance angle

114 Analysis of Pesticides in Food and Environmental Samples is altered [84,85]. SPR detection has demonstrated the usage of many types of

compounds. Initially, the technique was applied only to large molecules but as the technology has matured so has its potential for monitoring various pesticides, including photosynthetic inhibitors.

The crux of the system is a gold film on a glass surface. Attached to the gold film are self-assembled monolayers (SAMs) and capture reagents. These capture reagents may be antibodies, receptors, enzymes, ssDNA, streptavidin, and protein

A or G (dependent on the type of antibody used) as well as other reagents. As the specific species is captured, the mass on the chip surface increases and changes the specific reflection angle. In this technique, a herbicide such as atrazine may be detected in several modes. The simplest mode would be to attach an anti-atrazine antibody (as a whole or in parts) to the chip surface. If the solution under test shows the presence of atrazine, a signal response on the chip would be detected.

Another option would be to attach the photosynthetic reaction center (RC) from

a purple bacterium to the sensing chip. This can be accomplished in a number of ways, but literature evidence suggests that histidine (His) tags can be conveniently used. The system can easily be reused as the RC can be removed and the chip regenerated once the assay is completed. Samples of atrazine are introduced and the signal is monitored. A positive response can be quantitated and the chip can be reactivated for the next sample.

4.3.3.4 Conductive Polymers One way to increase the use of electrochemical detection methods is to use conduct-

ive polymers [86]. The concept is that the interference from sample components is limited and many conductive polymers can be formed in situ directly over the electrode. Most of the polymers that have been used are electrochemically derived (synthesized in situ), formed by a host of starting materials. Additionally, many can

be tethered to electrochemical conducting wires or even be encapsulated in a biopolymer matrix such as microgels [86 –91]. A sensor using an electrodeposited conductive layer was able to detect the herbicide diruon [92] and could be applied to other substituted urea compounds.

For this technique to function, an enzymatic system is often used, such as glucose oxidase. Other enzymes may be employed, dependent on the nature of the biosensor developed and the anticipated monitoring applications. One application that appears to dominate for commercial development is that of a glucose sensor. Glucose is converted to gluconic acid and amperometric signals are observed based on the production of hydrogen peroxide. The polymer may encapsulate the electrode or be placed on the electrode using microparticle slurries.

Another polymer that can be used is a water-soluble Os-poly(vinyl imidazole) redox hydrogel. Again, the electron transfer is very efficient and necessitates a redox enzyme placed in the gel. A polypyrrole film has also been used in conjunction with

NADH þ ferro-=ferricyanide redox chemistries. An enzyme is required whose func- tion is to use NADP þ in conjunction with an enzymatic substrate to release a product and the cofactor, NADPH. The ferricyanide is present to efficiently shuttle the electrons.

Immunoassays and Biosensors 115 There are also reports on the use of PVPOs(bpy) polymer and poly(mercapto-

p -benzoquinone) on gold electrodes or within conducting hydrogels. For these systems, the redox enzyme horseradish peroxidase is used or the CV of the substrate, sulfo-p-benzoquinone (SBQ) is monitored. The types of solid supports and electro- chemical methods are almost limitless.