Electrochemical Biosensors for Pollutants in the Environment

2015

Review

Electrochemical Biosensors for Pollutants in the Environment Michal Badihi-Mossberg, Virginia Buchner, Judith Rishpon* Faculty of Life Sciences, Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat-Aviv 69978, Israel *e-mail: [email protected] Received: May 14, 2007 Accepted: June 27, 2007 Abstract This article reviews recent advances in electrochemical biosensing and detection of environmental pollutants. Electrochemical biosensors offer precision, sensitivity, rapidity, and ease of operation for on-site environmental analysis. An electrochemical biosensor is an analytical device in which a specific biological recognition element (bioreceptor) is integrated within or intimately associated with an electrode (transducer) that converts the recognition event to a measurable electrical signal for the purpose of detecting a target compound (analyte) in solution. This approach not only provides the means for on-site analysis but also removes the time delay and sample alteration that can occur during transport to a centralized laboratory. We first address the basic principles of merging of electrochemistry and biology into a biosensing system, and then we discuss various environmental monitoring strategies involving this technology. Keywords: Environmental monitoring, Electrochemical biosensors, Contaminants, Pollutants DOI: 10.1002/elan.200703946

1. Introduction Environmental monitoring typically involves several steps such as sampling, sample handling, and sample transportation to specialized laboratories. The challenge of environmental monitoring in situ requires new and improved analytical devices featuring precision, sensitivity, specificity, rapidity, and ease of operation to detect decreasing concentrations of an ever growing array of pollutants. Such devices must be comparable to or better than traditional analytical systems, and must be simple to handle, small, cheap, able to provide reliable information in real-time, and must be sensitive and selective for the analyte of interest, and suitable for in situ monitoring. Biosensors not only fulfill all these requirements but also have a wide range of application in the areas of clinical diagnostics, forensic chemistry, pharmaceutical studies, food quality control, biological warfare detection, and environmental monitoring. Biosensors are small devices linking biological elements with signal providing mechanisms for fast and efficient warning of pollution incidents. The main advantage of biosensors is that they provide real-time, on-site detection and analysis in the field and often eliminate the need for sample collection, preparation, and laboratory analysis. The challenge of continuous in situ monitoring of environmental pollution in the field requires instruments that are robust and with sufficient sensitivity and long lifetime. Commonly used conventional methods are timeconsuming, expensive, require skilled operators, and lack the required selectivity. Biosensors have the advantage of being simple, uniform whole structures featuring direct transduction, high bioselectivity, high sensitivity, miniaturElectroanalysis 19, 2007, No. 19-20, 2015 – 2028

ization, electrical/ optoelectronic readout, continuous monitoring, ease of use, and cost effectiveness. User advantages include low price, reliability, no sample preparation, disposability, and clean technology. Hence, biosensors show the potential to complement both laboratory-based and field analytical methods for environmental monitoring [1 – 4]. The biocatalytic recognition element provides a high degree of selectivity for the analyte to be measured without complex sample processing. Hence, biosensors can detect biological or chemical species directly with an accuracy approaching that of traditional laboratory-based analyzers. This direct approach precludes not only the time delay but also the possibility of sample alteration that can occur during transport to a centralized analytical laboratory. Such devices are compact, portable, and cost-effective. Most biosensors are based on reactions catalyzed by macromolecules that are present in their original biological environment, previously isolated, or manufactured [5]. The “lock and key” conformation of enzyme-substrate, hormone-receptor, or antigen-antibody brings the ligands near to the working electrode. When such an agent is incorporated into the sensor, a successive consumption of substrate(s) can be accomplished. Miniaturized screenprinted electrodes in a multisensor array can be used for the parallel determination of several toxicants in real time. The high level of precision and reproducibility of screen-printing technology is ideal for measuring toxicants outdoors in a portable monitoring system. The biosensor can produce either individual or successive digital electronic signals that are equivalent to the concentration of a single analyte or a group of analytes to be monitored. Biosensors have been E 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2016 produced for many chemical compounds of environmental interest. Promising applications include groundwater monitoring, drinking-water analysis, and the rapid analysis of extracts of soils and sediments at hazardous waste sites [6]. Environmental monitoring requires rugged sensors for the detection of pollution and toxic chemicals, and automated and continuous, remote and in situ monitoring will be increasingly required. Today, electrochemical biosensors are at the forefront of a multidisciplinary science combining the fields of electrochemistry and biology. Devices combining the selectivity of biological molecules with the processing power of microelectronics offer a new approach for environmental monitoring that can be carried out in situ or online. An electrochemical biosensor comprises a biological recognition element immobilized on the surface of an electrode and a physicochemical detector component. The bioreceptor translates information from the analyte into a chemical or physical output signal with a defined sensitivity for quantifying the analyte to be monitored, and the transducer converts the recognition event to a measurable output signal. Concentration range, disposability, reusability, or renewability, accuracy and reproducibility, size of sensor, and size of analyte sample are important aspects for choosing electrochemical biosensors for environmental monitoring. The advantage of distinguishing oxidation states is highly important. The electrochemical approach can give a rapid answer, without digestion, as to the labile fraction of a given element in a particular oxidation state, and the experiment can be performed on-site in the field. The advantage of electrochemical biosensors is their high specificity, sensitivity, rapid response time, and ease of operation, thus fulfilling all the requisites for on-site environmental monitoring. Enzymes, antibodies, nucleic acids, hormone receptors, microorganisms, and tissue have been used widely in the construction of electrochemical biosensors, which have become indispensable for water and food quality control applications. Because they can be taken to the sampling area, electrochemical biosensing devices have a major impact upon the in situ monitoring of priority pollutants. The ability of such devices to provide rapid and reliable real-time information about the chemical composition of the surrounding environment is an important property for monitoring a variety of public health hazards. A group of chemicals known as persistent organic pollutants (POPs) can travel thousands of miles, accumulate in the food chain, and can resist degradation in the environment for centuries. Immobilization of the bioreceptor is crucial for electrochemical biosensing. The element can be immobilized in a thin layer at the transducer surface using such techniques as entrapment behind a membrane, within a polymeric matrix, or within self-assembled monolayers or bilayer lipid membranes; covalent bonding of receptors on membranes or surfaces activated by means of bifunctional groups or spacers; or the bulk modification of entire electrode material.

M. Badihi-Mossberg et al.

2. Enzyme Biosensors Enzyme biosensors are based on absorbing enzymes, whose products can be measured after degradation of the substrate, to the electrode surface. The electrode amperometrically or potentiometrically monitors changes by following the biocatalytic reaction. The current or potential measured is proportional to the rate-limiting step in the overall reaction. Enzyme biosensors are prepared by attaching to the electrode surface an enzyme whose products can be measured after the degradation of a substrate. Such systems usually involve the catalysis of redox reactions where either the substrate or the product is electrically charged. Many different types of enzyme biosensors have been developed for environmental monitoring. Environmental pollutants like parathion, nitrate, and formaldehyde can be detected by sulfite parathion hydrolase, nitrate reductase, and formaldehyde dehydrogenase [7 – 10]. Additionally, several biosensors for pesticides and toxic metals monitoring are based on the inhibition of enzymes [11 – 14].

2.1. Pesticides Pesticides account for the greatest number of reports for environmental biosensors. A pesticide is any substance or mixture of substances intended for preventing, destroying, repelling, or lessening the damage of any pest, as defined by the US Environmental Protection Agency (EPA). Of all the environmental pollutants, pesticides are the most abundant, present in water, the atmosphere, soil, plants, and food. 2.1.1. Organophosphorus (OP) Compounds Organophosphorus (OP) compounds are a group of chemicals that are widely used as insecticides in modern agriculture for controlling a wide variety of insect pests, weeds, and disease-transmitting vectors. The acute toxic properties of OPs are due to their ability to inhibit a group of hydrolytic enzymes called esterases [15]. Acetylcholine (ACh) is one of several chemicals (neurotransmitters) that are essential for the proper function of the nervous systems of both humans and insects. Acetylcholine causes muscles to contract. To prevent muscle paralysis and death, the enzyme acetylcholine esterase (AChE) immediately cleaves the neurotransmitter to enable muscle relaxation. Because OPs firmly bind to AChE to form a stable complex that disables its enzymatic activity, the ability to detect OP in the environment is vital. Direct, selective, rapid and simple determination of organophosphate pesticides has been achieved by integrating organophosphorus hydrolase with electrochemical and optical transducers. Organophosphorus hydrolase catalyzes the hydrolysis of a wide range of organophosphate compounds, releasing an acid and an alcohol that can be detected directly. Wang et al. [16] and Mulchandani et al. [17 – 21] developed applications of organophosphorus hydrolasebased potentiometric, amperometric and optical biosensors.

Electroanalysis 19, 2007, No. 19-20, 2015 – 2028 www.electroanalysis.wiley-vch.de E 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Electrochemical Biosensors for Pollutants

bilized on a carbon electrode. Enzymatic activity is detectable by its anodic oxidation. The current signal is linearly proportional to the parathion concentration, and the detection limit is lower than 1 ng mL1. A significant reduction in sample volume was achieved by using screenprinted electrodes and microflow-injection methods, which enhanced the sensitivity of the system; the use of pulsed techniques further increased the sensitivity.

2.2. Air Pollutants

Fig. 1. Enzyme activity in response to DDVP at different exposure times. (Adapted from [22] with permission.)

Several enzyme electrodes have been developed for the environmental monitoring of OPs based on pesticideinduced decreases in AChE activity. Neufeld et al. [22] developed a disposable enzymatic biosensor based on the AChE-acetylthiocholine-hexacyanoferrate(III) reaction. The sensor comprises an electrochemical cell consisting of screen-printed electrodes covered with an enzymatic membrane, which is placed in a home-made flow cell. The binding between AChE and an OP compound hinders the enzymatic degradation of acetylthiocholine chloride to thiocholine and acetic acid. The free thiocholine then reacts with hexacyanoferrate ion in the working solution. The ensuing reduction of ferricyanide to ferrocyanide and its subsequent reoxidation by the electrode generates very sharp, rapid, and reproducible electric signals that are proportional to the thiocholine concentration. The decrease in enzymatic activity in response to the OP compound dimethyl 2,2dichlorovinyl phosphate is presented in Figure 1. The system can trace small quantities of a desired analyte. The advantage this system offers lies in the ability to work with small amounts or volumes of samples, preventing risks that can threaten human health. 2.1.2. Parathion Parathion (O,O-diethyl-O-4-nitro-phenylthiophosphate), is a broad-spectrum OP pesticide having a wide range of applications against numerous insect species on several crops. Parathion is also used as a preharvest soil fumigant and foliage treatment for a wide variety of plants, both in the field and in the greenhouse [23]. Parathion is highly toxic by all routes of exposure – ingestion, skin adsorption, and inhalation, all of which have resulted in human fatalities. Like all OP pesticides, parathion irreversibly inhibits AChE. Sacks et al. [24] developed an amperometric organophosphorus hydrolase (OPH)-based biosensor for the direct measurement of parathion. The enzyme, which catalyzes the hydrolysis of parathion to form p-nitrophenol, was immo-

Air monitoring presents a special challenge because high selectivity, high sensitivity, real-time monitoring, and inexpensive analyses are required. Especially during emergencies, major incidents (fires and releases to the atmosphere) require real time data that can assist in making decisions about safety warnings and/or evacuations. For this purpose, emergency air monitoring mobile laboratories are designed to monitor a number of substances and to provide the public with an immediate snapshot of the air quality of the location monitored. Presently used chromatographic methods usually involve the use of a passive air pollutant sampling device and analysis in GC/MS systems that are expensive to purchase and maintain. Electrochemical biosensing systems would be ideal for use in such situations because they offer greater selectivity than many direct reading systems in use and are inexpensive to purchase and operate. 2.2.1. Formaldehyde The chemical compound formaldehyde (also known as methanal) is a gas with an acrid smell. Most formaldehyde is used in the production of toothpaste, polymers, chemicals, and permanent adhesives. The compound is used as the wet resin added to sanitary paper products, such as facial tissue, napkins, and roll towels. Formaldehyde is one of the hazardous air pollutants (HAP) that emerged from the industrial revolution. The compound, which is present in smoke from forest fires, in automobile exhaust, and in tobacco smoke, is toxic, allergenic, and accumulates in the air. In 2000, Herschkovitz et al. [25] presented an electrochemical biosensor for formaldehyde based on a flowinjection system using formaldehyde dehydrogenase and a Os(bpy)2-poly(vinylpyridine) (POs-EA) chemically modified, screen-printed electrode. The dehydrogenase enzymes oxidize a substrate by transferring one or more protons and a pair of electrons to an acceptor, usually NAD/NADP. The continuous flux of substrate to the system guaranteed by the flow cell prevents the accumulation or adsorption of product to the electrode. Another advantage of this system is the ability to work with low volumes of compounds and reagents, which is important when dealing with hazardous elements. The biosensor is stable over several days, disposable, and simple to operate. Flow-injection systems have proved to be practical applications in water and environmental control, agricultural, and pharmaceutical analysis.


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Fig. 2. Detection of formaldehyde. Amperometric response of the sensor to injections of formaldehyde. 0.1 M potassium phosphate buffer pH 8. Eapp ¼ 0.35 V. Numbers depict formaldehyde concentrations. (Adapted from [25] with permission.)

The system is a platform for using different dehydrogenase enzymes with their corresponding substrates. Approximately 250 NADH-dependent dehydrogenase enzymes have been identified [26]. The sensitivity of the formaldehyde dehydrogenase biosensor depends upon the efficiency of the electron transfer from the NADH via the POs-EA polymer mediator attached to the working electrode. The enzyme was immobilized onto a membrane on top of this mediator. This type of sensor is designed to measure the pollutant in aqueous solution. The detection limit of formaldehyde is 30 ng mL1 in the solution, which is equivalent to subparts per billion (ppb) concentrations of formaldehyde in the atmosphere. Figure 2 presents the rapid and sensitive response to successive additions of different concentrations of formaldehyde, ranging from 30 ng mL1 to 4.5 mg mL1. Excluding tails observed in low formaldehyde concentrations, the response to the pollutant is reproducible and linear. Formaldehyde is also an air pollutant; the gas must be transferred to an aqueous solution from an air sampling device. Formaldehyde can be removed from the atmosphere on site by pumping air through a glass coil together with an aqueous solution, which dissolves the formaldehyde and carries it to the electrode containing the enzyme, as described by Vianello et al. [27].


Fig. 3. Amperometric responses for different SO2 concentrations, and recovery after air exposure, at an s-type biosensor coated with 1.2 mg cytochrome c and 0.45 U SOD. (Adapted from [30] with permission.)

produces acidity in rain water and fogs, is a major source of corrosion for buildings and metal objects. Health concerns associated with exposure to high concentrations of SO2 include effects on breathing, respiratory sickness, and deterioration of cardiovascular disease [28]. During cold weather, SO2 in the air is associated with changes in both systolic and diastolic blood pressure [29]. An amperometric biosensor was developed by Hart et al. [30] for the measurement of SO2 in flowing gas streams. The biosensor is based on the enzyme sulfite oxidase with cytochrome c as the electron acceptor and a screen-printed transducer. Enzymatic reactions involve the sulfite ion (SO2 3 ), formed when SO2 gas is dissolved in the supporting electrolyte. Two types of the biosensor where established: an s-type biosensor, in which cytochrome c and sulfite oxidase were incorporated at the transducer surface; and a b-type biosensor, in which the components were mixed thoroughly with the same ink used to produce the screen-printed electrode. The modified ink was spread over the working electrode. The s-type biosensor was found to have the advantage of providing higher sensitivity and a faster response when compared with the b-type biosensor. Although both types showed linear responses in pollutant concentrations of 4 to 50 ppm, the sensitivity of the s-type was approximately twice that of the b-type biosensor. Figure 3 describes s-type biosensor response to different SO2 concentrations.

2.2.2. Sulfur Dioxide Sulfur dioxide (SO2) is a colorless gas that occurs as a contaminant in the atmosphere. Natural sources include releases from volcanoes, oceans, biological decay, and forest fires. Anthropogenic SO2 pollutants are products of fossil fuel combustion, smelting, manufacture of sulfuric acid, wood pulp industry oil and coal burning. Natural gas processing plants are responsible for close to half of the SO2 emissions in certain areas. Sulfur dioxide, which

3. Antibody Biosensors A biosensor having an antibody as its receptor is called an immunosensor. Antibody-based biosensors (immunosensor) are based on the principle that antigen – antibody interactions can be transduced directly into a measurable physical signal. Antibodies are immune system-related proteins (or immunoglobulins) that are secreted into the


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blood in response to stimuli by foreign substances (antigens). Specificity is the hallmark of the antibody-mediated immune response. Namely, the immune system responds to given antigenic substance by making a structurally unique antibody that specifically binds to and neutralizes only one site on the antigen. If an antigen has many sites, a different antibody is elicited for each site, collectively called polyclonal antibodies. An antibody produced against only one antigenic site is called a monoclonal antibody. Electrochemical immunosensors have been constructed using both types of antibodies. A hapten is a small molecule containing a single antigenic site that by itself cannot stimulate an antibody response but can do so when coupled to a large immunogenic molecule like a protein. The specific binding pairs employed in immunoassays are either an antigen or a hapten, and the antibody produced in an immune response to the antigen or hapten.

3.1. Coliforms Total coliform bacteria, represented by Klebsiella, and fecal coliforms, represented by Escherichia coli, are used as indicators of fecal pollution in the environment. As waterborne gastroenteric disease caused by high levels of these bacteria is a major public health problem, the quantitative determination of total and fecal coliforms is essential for water quality control. The presence of E. coli in an environmental sample implies the potential presence of a variety of pathogens originating from humans and warmblooded animals. Conventional microbiological methods for determining the number of coliforms in drinking water are time-consuming, with a long incubation period (from 24 to 48 hours) required to detect bacterial colonies. Mittelmann et al. [31] described a rapid and sensitive electroanalytical technique for the determination of total coliform bacteria and for the specific detection of E. coli. The sensor monitors the activity of b-d-galactosidase originating from the bacteria. The enzyme is often used as a general marker for total coliforms because its interference from nontarget positive bacteria is low and insignificant. The results are measured as colony forming units (cfu), with each unit representing one or more living bacterial cells. The electrodes were coated with polyclonal anti-E. coli antibodies prepared against the bacterial lysate. The detection of E. coli at the very low concentration of 1.2 cfu mL1 after 5 hours of incubation indicates that even very small concentrations of bacteria can be detected within a single working day using this sensitive technique. The results are presented in Figure 4.

Fig. 4. Detection of E. coli, at concentrations of 60 and 1.2 cfu/ mL, and K. pneumoniae, at concentrations of 60 and 1.3 cfu/mL, in 1 L of water after filtration, incubation in LB medium at 37 8C, and permeabilization. Each point represents the mean of three measurements þ standard deviation. (Adapted from [31] with permission.)

a mixture of individual compounds, primarily formed during the incomplete combustion of carbon-containing fuels. Their origin can be found in oil and coal industrial processes, fires, traffic, or heating. Some PAHs are known or suspected carcinogens and are linked to other health problems [32]. Polycyclic aromatic hydrocarbons that are present in nonprocessed foodstuffs are associated with environmental pollution from both human and industrial activities. [33] An amperometric biosensor for detecting PAHs was developed by Fahnrich [34]. The coating screen-printed carbon electrode antigen is phenanthrene-9-carboxaldehyde coupled to bovine serum albumin (BSA). The enzyme alkaline phosphatase (AP) was used, with the substrate p-aminophenyl phosphate (PAPP). The sensor was tested in tap and river water and showed only slightly decreased sensitivity compared to measurements carried out in buffer. Because many PAHs are very similar in electron density, molecular structure and weight, with a lack of side groups, producing antibodies specific for only one compound is impossible. Several antibodies have been raised against various PAH compounds, such as benzopyrene, pyrene, fluorene, phenanthrene (PHE), and anthracene, with benzopyrene certainly the most investigated. The biosensor is not specific for PHE, but shows cross-reactivity of varying degrees toward other PAH compounds.

3.3. Food Pathogens 3.2. Polycyclic Aromatic Hydrocarbons (PAHs) Polycyclic aromatic hydrocarbons (PAHs) are chemical compounds consisting of fused aromatic rings and do not contain heteroatoms or carry substituents. The PAHs, one of the most widespread organic pollutants, are always found as

Listeria monocytogenes, a bacterium motile by means of flagella, can be isolated from soil, silage, and other environmental sources. It has been associated with foods as raw milk, cheeses, raw vegetables, raw meats, and smoked fish. A relatively low percentage of the human population may be


2020 intestinal carriers of L. monocytogenes. Infection by the bacterium causes listeriosis, a general group of disorders that may include septicemia, meningitis encephalitis, pneumonia and cervical infections in pregnant women that could result in spontaneous abortion [35]. Once the bacterium enters the hostMs monocytes, macrophages, or polymorphonuclear leukocytes, it becomes blood-borne (septicemic) and can grow. Susmel et al. [36] demonstrated an immunosensor for the detection of the pathogenic bacteria; L. monocytogenes and Bacillus cereus, using screen printed gold electrodes (SPGEs). The gold electrode surface was modified with a thiol based self assembled monolayer (SAM) to expedite antibody immobilization. The SAMs are based on different chain lengths of thiols allowing optimum antibody immobilization, electrochemical response, orientation, and accessibility of the antigen binding site. The formation of the complex between the antigen and the antibody introduces a barrier for the electron transfer resulting in diffusion coefficient (D) changes of the redox probe. This change was measured chronocolometry. No change in the diffusion co-efficient was observed when a nonspecific antibody was immobilized and antigen added. A linear relationship between D and antigen concentration was observed.

3.4. Herbicides Enzyme immunoassays have been used for detecting herbicides contamination. Dzantiev et al. [37] combined the essential properties of antibody- and enzyme-based systems to construct an electrochemical immunoassay technique for chlorsulfuron determination. Chlorsulfuron is an herbicide used worldwide as an agrochemical for the selective control of weeds in wheat and barley [38]. The compound is part of a relatively new class of chemicals inhibiting the action of plant enzymes, stopping plant growth, and killing the plant [39]. The enzyme horseradish peroxidase was attached to a screen-printed electrode. On top of the enzyme, anti-chlorsulfuron antibodies were attached through a membrane. The assay is based on a competition for the available binding sites of the membraneimmobilized antibodies; between the monitored free pollutant chlorsulfuron and a chlorsulfuron-glucose oxidase conjugate. The addition of glucose to a solution containing both forms of the pollutant induced the generation of hydrogen peroxide by the glucose oxidase conjugate enzyme. The hydrogen peroxide reduced by the peroxidase on the electrode results in an electrical current change, due to the direct electron transfer of the enzyme, which reflects the chlorsulfuron content in the sample. The pollutant determination time was 15 minutes, and the detection range was 0.01 – 1 ng mL1. Yulaev et al. [40] presented a biosensor for simazine, Simazine, an herbicide of the triazine class, is used to control broad-leaved weeds and annual grasses. The biosensor is based on the potentiometric detection of the peroxidase activity after a competitive immune reaction on the


electrode surface. This biosensor detection limit was 3 ng mL1 of simazine. The pollutant was detected quantitatively without pre-treatment in meat extracts, milk and tomatoes, hence was found efficient for use in food quality control

4. Receptor Biosensors A receptor is a structure on the surface of a cell (or inside a cell) that selectively receives and binds a specific substance. Receptors can be adsorbed on the working electrode surface using several methods: capture behind a membrane, a polymeric matrix, or bilayer lipid membranes.

4.1. Endocrine-Disrupting Compounds The endocrine system consists of glands that secrete hormones for the control of growth, maturation, development, and regulation within the body, usually by binding to receptors. Endocrine disrupting chemicals are compounds that can mimic a hormone, entering the hormoneMs receptor in lieu of the hormone or blocking the normal passage of hormones into receptors. In the United States, the monitoring of water and food for the presence of endocrine disrupting chemicals is mandated by the Safe Drinking Water Act and the Food Quality Protection Act ((http:// www.epa.gov/safewater/sdwa/index.html; http://www.fda.gov/opacom/laws/foodqual/fqpatoc.htm). Estrogen is a fundamental hormone produced by the ovaries in the process of maturation of the female reproductive system. The hormone is also secreted by the adrenal glands and the male testes. Estrogen regulates cellular reactions through a specific intracellular receptor that functions as a ligand-inducible transcription factor. Xenoestrogens are endocrine disrupting chemicals that bind to the estrogen receptor and mimic estrogen activity. Xenoestrogens are commonly found as natural or synthetic in the environment. The lack of sufficient evidence for a clear-cut relation between xenoestrogen exposure and major human health concerns has created a need for highly sensitive screening systems to detect xenoestrogens in human and environmental samples for epidemiologic monitoring studies. Granek et al. [41] presented a novel impedance biosensor for monitoring such compounds based on a native estrogen receptor adsorbed to a synthetic lipid bilayer attached to gold electrodes. Estrogen or xenoestrogen binding to the receptor-modified electrode causes conformational alteration in the lipid layer, leading to electrical circuit changes detected by fast impedance measurements. Short galvanostatic pulses are applied and the changes in the potential are followed. The electrodes are left at open circuit potential for 2 min for equilibration, and then a constant current (1 mA) is applied. The values of the equivalent circuit components (Rs, Rp1, t1, C1, Rp2, and t2) for the monolayer, bilayer, bilayer-receptor, and bilayer receptor1 pg mL1 hormone are calculated. The capacitances, calculated by t1/Rp1, agree with the ac impedance data.


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Fig. 5. Nyquist plot for biolayer construction as drawn using the calculated capacitance and resistance values measured by galvanostatic pulses: (—) monolayer, (– –) bilayer, (- -) receptor, ( ····· ) 1 pg/mL estrogen. Inset: Equivalent circuit. (Adapted from [41] with permission.)

The obtained values of the capacitors and resistors were used to calculate Z’ and Z’’ in the complex plane plot, and the results are presented in Figure 5. Two types of xenoestrogens were monitored; bisphenol A, a synthetic xenoestrogen, and genistein, a phytoestrogen. The system was found to be highly sensitive, providing an efficient tool for monitoring small amounts of endocrine-disrupting chemicals. The effects of estrogen, bisphenol A, and genistein on the electrical properties of the bilayer-receptor modified electrode are summarized in Table 1. The bilayer – receptormodified electrode gave a similar response to either the natural hormone or the xenoestrogen. The Rs values increased, the Rp1 values decreased, the lifetime t1 values decreased, and accordingly the calculated C1 values decreased. These alterations can be attributed to the dimerization and conformational changes of the receptor, which increases its hydrophobicity and allows it to enter into the lipid layer. Schwartz-Mittelman et al. [42] tested the effects of various estrogens, xenoestrogens, phytoestrogens, and steroidal and nonsteroidal drugs on the estradiol-induced dimerization of the human estrogen receptor alpha (hERa). Most xenoestrogens have the ability to bind the ER and interfere with the natural function of the endocrine

system. The method used was a modified yeast two-hybrid (YTH) system with electrochemical detection, in which bgalactosidase activity is under estrogenic control through ER dimerization. [43] The YTH system is based on the observation that many eukaryotic transcription factors are divided into two separate functional domains that mediate DNA binding and transcriptional activation. Both domains contribute to dimerization. The YTH assay includes rebuilding b-galactosidase activity via protein – protein interactions. An ERa monomer is fused to the DNA-binding domain, and a second is fused to the activation domain of the same transcription factor. When both fused receptors are co expressed in yeast, ER dimerization leads to the reconstitution of a functional transcription factor, measured as bgalactosidase activity. The substrate used in this experiment was p-aminophenyl b-d-galactopyranoside. The product of this enzymatic reaction p-aminophenol (PAP) is oxidized at an electrode. The sensitive modified YTH electrochemical bioassay was used to characterize hERa dimerization induced by natural estrogens, phytoestrogens, xenoestrogens, EDCs, and the commonly used anticancer drug, tamoxifen, as well as for the antagonist activity of the analgesic drug, acetaminophen. This drug inhibited the 17b-estradiol-induced dimerization of human hERa at physiological concentrations of estradiol (1011 to 1012 M). The inhibition was determined by a reduction in the activity of the reporter enzyme, monitored by electrochemical measurements. Table 2 shows a summary of the results for yeast cells exposed overnight to various xenoestrogens and 17-bestradiol measured with the electrochemical two-hybrid system. For bisphenol-A, the lowest concentration measured was 106 M, whereas that of 17-b-estradiol was 1011 M. Diethylstilbestrol (DES), a synthetic estrogen, induced b-galactosidase activity expressed in yeast cell cultures at the same order of magnitude as that of 17-b-estradiol, implying a similar induction of receptor dimerization by both compounds. An influence of genistein and naringenin was also demonstrated.

5. Bacteriophage Biosensors A bacteriophage (phage) is an intracellular viral-like parasite that infects only one specific bacterial species. Hence, phages are useful for the identification of bacterial contaminants. Typically, bacteriophages consist of an outer protein shell enclosing genetic material and multiply by using the

Table 1. Summary of effects of exposure to different estrogen and xenoestrogen concentrations on electrical properties of bilayerreceptor-modified electrodes. ( Adapted from [41] with permission.)

0.015 pg/mL estrogen 44 pg/mL estrogen 30 ng/mL bisphenol A 3 mg/mL bisphenol A 1 ng/mL genistein

D% Rs ( MW)

D% t (s)

D% Rp ( MW)

D% C (mF )

no change 74.28 24.43 33.84 56.28

no change 19.96 56.63 63.26 21.71

no change 45.74 16.40 26.54 30.18

no change 28.98 47.34 48.69 9.78


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Table 2. Summary of results displayed as Dcurrent/Dtime (nA/s), for yeast cells exposed overnight to various xenoestrogens and 17-bestradiol measured with the electrochemical two-hybrid system. Each result represents the mean of three measurements. ( Adapted from [42] with permission.) Concentration ( M )

Naringenin

103 104 105 106 107 108 109 1010 1011

0.008 0.11 0.005 0.053 0.0014 0.03

BPA

Genistein

2,4-Dihydroxy BP

0.031 0.33 0.002 0.05

0.67 0.085 0.009 0.045 0.0035 0.035

0.0095 0.25 0.015 0.17 0.0099 0.051 0.0011 0.018

DES

0.034 0.58 0.078 0.54 0.025 0.42 0.015 0.17 0.0004 0.037

17-b-Estradiol

0.29 0.0027 0.11 0.027

hostMs biosynthetic machinery. This process begins when the phase attaches to specific receptors on the bacterial cell surface. The range of bacteria influenced is usually determined by the proteins on the bacterial cell surface. The phase can attach to proteins, lipopolysaccharides (LPS), pili, and lipoprotein presented on the outer membrane of the cell. Lytic or virulent phages can multiply in bacteria and kill the cell by lysis at the end of the life cycle, due to the accumulation of a phage lysis protein, and intracellular phage are released into the medium. The specific selectivity of the phage can be used for constructing a sensitive biosensor for bacteria, thereby precluding the need for timeconsuming conventional microbiological pretreatments. The linkage of phage-specific identification and the release of the inner enzymatic cell markers after the lysis of the cell provide a powerful tool as a highly specific detection method of a given bacterial strain.

5.1. E. coli A phage-based biosensor was developed by Neufeld et al. [44] , who combined specific phage detection and the release of intracellular enzymes to produce a highly specific marker for the bacterial strain E. coli K-12, MG1655. The virulent phage l vir serves not only as the specific recognition element for E. coli but also as the releasing agent of the enzyme b-d-galactosidase (which is widely used for identifying E. coli in water and food samples). The product of its enzymatic activity is measured amperometrically by monitoring its oxidation at the carbon anode. The amperometric detection enables the use of a wide range of bacteria concentrations, reaching as low as 1 cfu 100 mL1 within 6 – 8 hours, as shown in Figure 6. The electrochemical method can be applied to any type of bacterium – phage combination by measuring the enzymatic marker released by the lytic cycle of a specific phage. The detection of E. coli was also demonstrated using a phage based biosensor and the enzyme alkaline phosphatase. Plasmids are DNA molecules found in bacteria cells, separate from the bacterial chromosome, that are capable of autonomous replication. Neufeld et al. [45] constructed a

Fig. 6. Detection of low concentration of E. coli. A) 1 – 3, 10 cfu mL1; 4 – 6, 1 colony forming unit mL1; 7 – 8, without phage. B) 1 cfu 100 mL1. (Adapted from [44] with permission.)

bacteriophage containing a bacterial plasmid encoding for the enzyme alkaline phosphatase, known as a phagemid. In the bacteria, this enzyme reacts with the substrate, paminophenyl phosphate (PAPP), in the periplamic space separating the outer plasma membrane from the cell wall. Thus, the activity of the reporter enzyme can be measured directly without further treatment. The product of the enzymatic activity, p-aminophenol, diffuses out and is oxidized at the working electrode. Using a phagemid combines the advantages of the specific recognition contributed by bacteriophage and the effortless genetic manipulation of a plasmid, a concentration of 1 cfu mL1 E. coli from 50 mL of a contaminated water sample was detected rapidly within 2 – 3 hours. This method is specific and can be exercised for water or food borne bacterial contamination detection.


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6. Liposome Biosensors A liposome is an artificial spherical vesicle composed of a phospholipid bilayer surrounding an aqueous cavity, originally developed to study cell membranes. The ability to bear different molecules inside the cavity offered a great potential for using liposomes for diagnostics, drug delivery, and environmental monitoring. Different molecules can be associated with liposomes in several ways; encapsulation within the aqueous inner cavity, partitioning within the lipid tails of the bilayer, or covalent and electrostatic interactions with the polar head-groups of the lipids [46]. For surveying environmental contamination, liposomes enclosing a marker can be tagged on their surface with haptens, antibodies, or DNA [47]. The marker is released by lysing the liposomes.

6.1. Triazine Pesticides As a chemical family, the triazines are a group of pesticides with a wide range of uses. Their chemical structures are heterocyclic, composed of carbon and nitrogen in their rings. Herbicide members of this family include atrazine, hexazinone, metribuzin, prometon, prometryn, and simazine. Atrazine is one of the most used herbicides in Europe and the USA [48]. These compounds are known to have a moderate toxicity; yet can undergo transformation to more toxic, mutagenic, and carcinogenic forms. Such pollutants can be found in the human food chain or directly in drinking water, as a result of their presence in ground water [49]. Baumner et al. [50] developed a disposable amperometric sensor for the detection of triazine pesticides in water samples. The biosensor is based on the competition between the free pollutant and tagged liposomes. Thick film electrodes printed on PVC were used as strip-type transducers, and monoclonal antibodies against atrazine and terbutylazine attached on top served as the biorecognition element. Hapten-tagged liposomes entrapping ascorbic acid as a marker molecule were used to generate and amplify the signal. For signal detection on a graphite electrode, the liposomes were lysed by Triton X-100 and the released ascorbic acid was quantified at a potential of þ 300 mV vs. printed Ag/AgCl. The biosensor response time was 1 – 3 min, and the sensitivity of measurements in tap water was below 1 mg L1 of atrazine which correlates well with standard detection procedures.

6.2. Cholera Toxin The cholera toxin (CT) secreted by the bacterium Vibrio cholerae is a known causative agent of diarrhea, vomiting, and cramps, often leading to death in humans. When released from bacteria in the infected intestine, the cholera toxin binds to intestinal cells, triggering endocytosis of the toxin into the cell. Once inside, the toxin causing a dramatic efflux of ions and water from the infected cell, leading to watery diarrhoea. A sensitive biosensor for the detection of

Fig. 7. Calibration plot for cholera toxin. The inset shows a linear part of the main curve. (Adapted from [51] with permission.)

CT, described by Viswanathan et al., [51] is based on liposomes containing potassium ferrocyanide and labeled with highly specific recognition molecules for the analyte. The monitoring platform consists of a monoclonal antibody against the B subunit of the CT polymer coated on nafionsupported multi walled carbon nanotube on a glassy carbon electrode. The CT is first bound to the anti-CT antibody and then to the specific molecule attached to the liposome. The potassium ferrocyanide is released from the bounded liposomes by lysis with a methanolic solution of Triton X-100 and measured by adsorptive square-wave stripping voltammetry. The detection limit of this biosensor is 1016 g of cholera toxin. This device offers an effective tool for clinical diagnostics, food and water safety monitoring, and epidemic control. To determine the sensitivity of the immunosensor to the CT; analytical calibration at different concentrations of the target analyte was conducted. The calibration curve for the voltammetric detection of CT at optimum experimental conditions is presented in Figure 7.

7. Whole-Cell Biosensors In recent times, the use of whole cell biosensors in monitoring technologies has become more common. Such biosensors can monitor general toxicity or specific toxicity caused by one or more pollutants using whole cells as the biorecognition agent. A variety whole cell biosensors have been developed to enable the monitoring of pollutants by quantifying light, fluorescence, color, or electric current. Such biosensors can be used in a wide range of applications in the fields of pharmacology, medicine, cell biology, toxicology, and environmental monitoring. The biosensor is based on whole cell sensing systems carrying a genetically engineered reporter gene that is inserted into the cell under study and is expressed only upon exposure to a monitored toxicant, which can be quantitatively measured.


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Fig. 8. Monitoring genotoxin 4NQO. The assay is based on the response of E. coli to DNA-damaging agents using a strain which carries a lacZ gene fused to a gene promoter that responds to DNA damage (sfiA) [58] . b-Galactosidase activity was measured on-line with increasing concentrations of the genotoxin 4NQO. (Adapted from [57] with permission.)

Microorganisms, bacteria in particular, provide a good tool for monitoring because of their rapid growth, fast response, and ease of genetic adjustment. Two approaches to using whole bacterial cells as biosensors are used today; turn off or turn on mechanisms [52]. In the turn on mechanism, a signal appears following exposure to the monitored toxic compound, while in the turn off mechanism, a measurable signal decreases by toxicity, The second mechanism is more common, assembled by fusion of reporter genes to stress-response promoters, a regulatory region of DNA that provides a control point for gene transcription into RNA. [53, 54]. Such promoters, which can be activated by toxic or hazardous chemicals, can be fused to reporter genes to monitor the presence of those chemicals. The use of promoters sensitive to DNA damage, protein damage, and membrane-damage have been demonstrated in the past [55] . Promoters are sensitive elements located upstream of the translated gene; they control activation or repression and are sensitive to temperature, ionic strength, or compounds like metabolites or environmental stress agents. Hence, promoters can be useful for monitoring environmental pollution. Whole cell biosensors based on bacteria can be engineered by placing a reporter gene encoding reporter proteins like b-galactosidase (lacZ), green fluorescent (GFP), or alkaline phosphatase (AP, phoA), under a transcriptional control mediated by the monitored analyte. The thus engineered cell will then produce the reporter protein in the presence of the monitored analyte, which can be electrochemically detected and quantified [53, 54]. As a whole cell biosensor does not require pretreatment of the bacterial cells, the assay can be conducted using portable and simple equipment and disposable electrochemical electrodes. The detection can be carried out in situ

to monitor a wide range of toxicants and to determine promoter activities in the environment, as well as to understand complex microbial interactions [56].

7.1. Genotoxic Agents Genotoxic chemicals are capable of causing damage to DNA. General toxicity is often caused by genotoxic agents like mutagens and carcinogens that induce the cell regulatory systems, like stress response or the SOS DNA repair system, which allows bacteria to survive sudden increases in DNA damage. The drug 4-nitroquinoline 1-oxide (4NQO) is a genotoxic agent and model carcinogen that damages DNA, thereby inducing the SOS response in cells. Using E. coli as a model system, Paitan et al. [57] fused a lacZ gene encoding for the reporter enzyme b-galactosidase to the SOS promoter. The genetically engineered E. coli produced the protein b-galactosidase in response to the DNA damaged elicited by 4NQO. The amperometric monitoring of various concentration of the genotoxin 4NQO is shown in Figure 8.

7.2. Aromatic Hydrocarbons Pollution of water resources is an increasing problem worldwide. A biosensor based on genetically engineered cells can provide valuable information on the level of toxicity of wastewater and the quality of drinking water. Phenol is frequently used in oil refinery wastes and is also produced in the conversion of coal into gaseous or liquid fuels and in the production of metallurgical coke from coal. Phenol discharges can enter the environment from oil


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resulting in a signal proportional to the aromatic hydrocarbons concentration. This system was found to be sensitive enough to detect vapors of toluene and benzene in 35 and 25 min, respectively. Monitoring all three xylene stereoisomers – meta-, ortho-, and para-xylene in a short time (20 – 40 min) was achieved.

7.3. Heavy Metals

Fig. 9. E.coli sensor, pfabA pfabR response and induction by the phenol derivatives bisphenol A, DHBP, and nonylphenol at different toxicant concentrations. Reporter b-galactosidase activity is presented by DA/Dt. (Adapted from [59] with permission.)

refineries, coal conversion plants, municipal waste treatment plants, or spills. Recombinant bacteria are microorganisms whose genetic makeup has been altered by the deliberate introduction of new genetic elements. Neufeld et al. [59] used a recombinant E. coli containing two plasmids – pfabA encoding the protein b-galactosidase under the control of the fabA promoter; and pfabR, which encodes the repressor of this promoter. The recombinant microorganisms were exposed to the test chemicals in an electrochemical cell and the induced b-galactosidase activity was determined amperometrically. The product of the enzymatic reaction, PAP, is oxidized at the working electrode, resulting in signal that is proportional to the monitored analyte concentration. This biosensor is very sensitive to low concentrations of phenol (1.6 ppm), and results are obtained within a very short time 20 min. The sensor responds to phenol derivatives like nonylphenol, 4,4’biphenol (DHBP), toluene, hydrazine, and ethanol, while remaining insensitive to bisphenol A and the organophosphate DDVP. Characterization of the toxicity detection potential of the recombinant bacteria containing the two plasmids pfabA and pfabR, in the presence of phenol is presented in Figure 9. Other aromatic hydrocarbons of great concern to the environment are toluene and xylene. Paitan et al. [60] described a bacterial whole cell electrochemical biosensor that can be used for monitoring those aromatic hydrocarbons. The sensor is based on an aromatic compoundssensitive promoter that induces the production of analyte that can be monitored electrochemically at real-time and on-line. The promoter xylS was fused in E. coli, upstream to two DNA sequences containing two reporter genes, lacZ and phoA. The result is a whole cell electrochemical biosensor with b-galactosidase or AP as reporter genes, using their respective substrates p-aminophenyl-b-d-galactopyranoside (PAPG) and PAPP. The product of both enzymatic reactions, PAP, is oxidized at the electrode

Cadmium (Cd) is a chemical element ubiquitous in the environment. The concentrations of Cd in soils, plants, and other environmental media have escalated due to a growing use of the chemical in industrial processes. Cadmium can accumulate in specific organs of the human body; hence it is considered a cumulative poison. Cadmium is classified as a human carcinogen, and exposure to Cd has also been associated with renal dysfunction and bone diseases [61]. Biran et al. [62] constructed a biosensor for cadmium content in E. coli consisting of a lacZ gene that is expressed under the control of a cadmium- responsive promoter of zntA, which has been shown to be involved in the efflux of heavy metals [63]. A wide range of cadmium concentrations was monitored using an electrochemical assay of b-galactosidase activity, the reporter protein of the lacZ gene. The whole-cell biosensor could detect, within minutes, nanomolar concentrations of cadmium in water. Cadmium monitoring was also demonstrated using various types of growth media. Cadmium was detected at concentration as low as 25 nM in less than 1 hour. This biosensor was also used for the detection of Cd in soil samples. The signals obtained by the biosensor were proportional to the Cd concentration in the soil, as demonstrate in Figure 10 below. 600 ppb. (5.34 mM) of the metal could be detected without any pretreatment of the soil.

8. Biochip Based on Whole-Cell Biosensor Popovtzer et al. [64] presented a nano-biochip, which contains an array of nano volume electrochemical cells, based on silicon microsystem technology (MST). The whole cell bacteria used is a genetically engineered E. coli. The microorganisms integrated into the chip will express electrochemically detectable signals in the presence of toxicants, as described before by Biran et al. [65]. The device architecture includes an array of eight nano volume chambers functioning as electrochemical cells containing the bacteria. Using this method one can concurrently monitor eight different toxicants with the general stress responsive promoter. The E. coli strain used contains a deletion in the lacZ gene and carries recombinant plasmids that include fusions of the lacZ gene to promoters of heat shock genes coding for the GrpE and DnaK heat shock proteins. The promoter of those proteins responds to a variety of stresses, such as elevation in temperature and exposure to a variety of chemicals like ethanol or heavy metals [66]. In the presence of a toxin, the lacZ promoter is


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Fig. 10. Current signals obtained from the induction of the zntAlacZ fusion in cadmium-contaminated soil samples. The soil samples (30 mg) were added directly, without any pretreatment, to the bacterial culture in the electrochemical cells. The signals obtained 1 h after the additions of the soil samples are shown. Each point is the mean of four replicates from two separate experiments. The cadmium concentrations were also determined by ICP (Spectro), as shown by the numbers in the abscissa. 1 ppm 8.9 mM. (Adapted from [62] with permission.)

activated and induces the production of the reporter enzyme b-galactosidase. This enzyme reacts with the substrate PAPG to generate the product PAP; the oxidation of PAP creates a current which is monitored. To exemplify the nanobiochip ability to detect water toxicity, the authors tested various chemicals. The toxicants ethanol and phenol, inducers of the heat shock proteins, were introduced to genetically engineered E. coli in the presence of the PAPG substrate. The induced b-galactosidase activity was monitored electrochemically as presented in Figure 11. These results using this biosensor showed a direct correlation between the currents signals and the toxicant concentrations. Concentrations as low as 0.5% of ethanol and 1.6 ppm of phenol could be detected in less than 10 min. The novel technology described is a combination of biology and engineering, enabling multi analyte detection, high screening, and miniaturization in real time detection.

9. Conclusions The vast potential market for biosensors is only beginning to be exploited. A wide variety of laboratory-based biosensor techniques that could be applied to environmental measurement have been reported; and some have been commercialized. Electrochemical biosensors provide precise, rapid, sensitive, and easy to use tools for on-site environmental monitoring and analysis. The devices are ideal for environmental monitoring because only a small amount of the sample is needed for the analysis, and usually, pre


Fig. 11. Amperometric response curves for real-time monitoring of ethanol using the nano-biochip. The recombinant E. coli containing a promoterless lacZ gene fused to promoter grpE exposed to 0.5 – 2% concentration of ethanol. The bacteria cultures with the substrate PAPG and the ethanol were placed into the 100 nL volume electrochemical cells on the chip immediately after the ethanol addition (ca. 1 min) and were measured at 220 mV. (Adapted from [64] with permission.).

treatment of the sample is not required. Progress and breakthroughs in biotechnology, biochemistry, genetic engineering, and immunochemistry offer a wide range of platforms for recognition elements to be used in advanced biosensors. Using low cost materials as screen printed electrodes, enzymes and genetically engineered microorganisms provide an essential tool for monitoring pollutants in the environment. An electrochemical biosensor is a powerful tool for real time, on-site environmental analysis. One limitation of this approach is that often only a limited amount of information about the nature of pollutants is available for contaminated sites, thus monitoring methods must be capable of identifying expected as well as unexpected pollutants at low levels. At present, most biosensors are typically designed for specific applications that involve a narrow range of compounds. Nevertheless, as part of an integrated site study plan, the commensurate increase in the sampling frequency allowed by the lower cost of field screening analyses can reduce the overall uncertainty involved in characterizing the contaminated site. Once the key contaminating compounds have been identified, however, biosensor field screening methods could be used to map their spatial distribution. Analytical tasks associated with remediation and post-closure monitoring may require frequent and repetitive analysis at specific locations for particular compounds of interest. Biosensors are particularly well suited for this purpose because are optimized to rapidly measure a single compound or class of compounds.

10. Future Directions Biosensors and biosensor-related techniques that show potential for environmental applications must overcome a number of obstacles to become commercially viable in the



highly competitive area of field analytical methods. Some of the obstacles common to all field analytical methods include: the diversity of compounds and the complexity of matrices in environmental samples, the variability in data quality requirements among environmental programs, and the broad range of possible environmental monitoring applications. More specific to biosensor technology, these hurdles include: relatively high development costs for single analyte systems, limited shelf and operational lifetimes for premanufactured biorecognition components and relative assay format complexity for many potentially portable (but currently laboratory-based) biosensor systems. Nevertheless, there are a number of areas where the unique capabilities of electrochemical biosensors might be exploited to meet the requirements of environmental monitoring. Advances in areas such as toxicity, bioavailability, and multipollutant-screening, could widen the potential market and allow these techniques to be more competitive. Miniaturization, reversibility and continuous operation may allow biosensor techniques to be incorporated as detectors in chromatographic systems. Due to unique characteristics and flexibility in operational design, biosensors continue to show significant promise for use in environmental monitoring applications. Nevertheless, because of a variety of obstacles (many unique to the environmental monitoring area), the introduction (and early successes) of these devices into this commercial market will likely involve narrowly focused applications. Successful biosensors will likely incorporate some of the following features: sensor platforms that are versatile enough to support interchangeable recognition elements (to measure a number of analytes), miniaturization to allow automation and convenience at a competitive cost, and other capabilities not currently available such as automated, continuous and remote detection of multiple, complex organic analytes.

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