Biosensors for Direct Monitoring of Environmental Pollutants in Field

Research on indoor air pollution is motivated by the following three maxims (1) individuals spend up to 95 percent of their time indoors in residences, work place, intransit and in entertainment environments; (2) indoor air pollutant concentrations are frequently higher than corresponding outdoor pollutant concentrations; and (3) if all ambient air pollution standards were attained, health effects associated with air pollution would not be eliminated because exposure to pollutants generated indoors would remain at the same levels. Biosensor monitoring for indoor air pollution research and exposure assessment is inspired by a strong desire for direct exposure monitoring because, presently, the majority of exposure sampling studies combine area monitoring with time budget information to estimate exposure to air pollutants as the initial step to exposure assessment.The objective of this paper is to identify the potential for direct monitoring biosensors for one of the newest disciplines of environmental research: Indoor air quality and exposure assessment. An historical perspective is used to elaborate on the three maxims of indoor air and exposure research, to identify priorities of research in the discipline, to set quality control requirements for biosensor samplers, and to list unique attributes of pollutant sampling devices applicable only to indoor environments. Biosensors for direct air sampling and exposure assessment must have the following attributes: (1) uniquely, they must be personal samplers; (2) ideally, they must measure both pollutant concentrations and time spent at each of several microenvironments; and (3) hopefully, they must measure several variables simultaneously including multiple pollutants and physical parameters. This paper introduces the present state of the art of indoor pollutant and exposure sampling, and challenges the biosensor scientific community to improve on current capabilities by using biosensor technology.

The determination of VOCs in the different air environments has attracted attention from the scientific community the last ten years because VOCs create photochemical pollution and cause many health problems. The on-line determination of VOCs in indoor/outdoor field measurements can be carried out using different analytical methods and instrumentation. The main category of methods concerns automated gas chromatographs GCs equipped either with flame ionization detector (FID) or photoionization detector (PID). For outdoor measurements spectrometric methods can often be used such as Differential Optical Absorption Spectrometry (DOAS) and Fourier Transform IR (FT-IR). Recent advances have made available methods such as Direct MS analysis (D-MS) and Membrane Introduction MS analysis (MI-MS) for the direct determination of VOCs. Recently, the use of biosensors for the in-situ determination of VOCs has been grown up.

This paper concerns the application to the sensor field of the various modes of computational exploration, representation and prediction of the nature of intermolecular interactions that lie at the heart of the response selectivity of chemical and biosensors to analyte species. Such a strategy is expected to be helpful in the understanding of response associated with existing sensor-receptor combinations, and in the possible design of new surfaces for the detection of environmental molecules. A key issue in this area is the role of shape complementarity between the receptor site and the analyte molecule and its connection to the interaction energies of instigated hydrogen bonds. We have used three distinct methods for the study of such molecular interactive systems; these are molecular mechanics and semi-empirical and ab initio quantum mechanical models. These methods applied to the problem of intermolecular interactions must accurately reproduce the molecular geometry of the separate receptor and analyte entities, and the hydrogen-bonded complex. In addition, reasonable values for the relative energies of these moieties must be available.In present paper we will describe an explicit comparison of the results of computational methods applied to a receptor-analyte combination with experimental measurements of responses of SAW and TSM acoustic wave sensors to organonitrocontaining compounds. The system involved concerns the hydrogen-bonding of nitro functional group species to amino-silanes bound on the sensor surface. The correlation between theory and experiment clearly demonstrates the difficulties in facile interpretation of the results of calculation on the one hand, but great utility for the redesign of new surface receptors on the other.

Chemical sensors are ideally suited to miniaturisation and reformatting in order to conform to particular types of flowing, static and low volume samples. An important additional need is the complementary tailoring of a measurement cell in order to create a functional, integrated sensor-based measuring system. In this context the importance of sampling, membrane barriers, electrode modification and transduction strategy have been outlined and specific examples provided. A description of the sampling technique, open micoflow, is given which demonstrates how measurements can be performed in colloid containing samples without associated sensor fouling. The permselectivity and porosity of microporous and homogeneous barrier membranes can be tailored by addition of suitable modifiers (eg. surfactant) to alter hydrophilic/hydrophobic properties. Specifically, this has facilitated the control of solute flux and permselectivity towards neutral, charged, polar or non-polar species. Ultrathin non-conducting electropolymerised films are an alternative route in creating permselectivity barriers. It is shown that by judicious choice of monomer derivative both permselectivity and functionality can be achieved. Impedance spectroscopy and spectral reflectance as sensor transduction strategies have been explored in specific relation to ligand containing conducting polymer films. Such techniques have enabled enhanced sensitivity and selectivity to be achieved which can be extended to a wide range of chemical sensor applications.

A microformat ELISA has been developed for the simultaneous determination of multiple 2,4-dichlorophenoxyacetic acid (2,4-D) samples using a CCD-camera and highly effficient chemiluminescent enzyme substrates. The linear range was 4.5×10−10 – 4.5×10−7 M with a lower detection limit of 4.3×10−10 M, corresponding to 96 pg/ml or 192 pg/well with a CV of 12.5%. The use of gold coated silicon wafers and glass capillaries as solid phase supports in the imaging ELISA is also described. The highly reflective gold surfaces improved the linear range and the sensitivity as compared to thick-film patterned surfaces. Sampling in capillary tubes reduces the sensitivity and the linear range, but there are indications that the capillaries can act as light guides and thereby improve the light collection efficiency.

A biosensing system based on total internal reflection fluorescence (TIRF) experiments on the surface of a fused silica optical fiber can be used for detection of hybridization of DNA and RNA. Preliminary work has shown that the approach can provide quantitative results in minutes at low concentrations of target nucleic acid strands.

An electrochemical DNA-biosensor consisting of a glassy carbon electrode modified with DNA was developed to evaluate and to predict DNA interactions with and damage by health hazardous compounds, for example carboplatin and nitroimidazoles. This electrode was successfully used for the electrochemical determination of carboplatin and to study the electrochemical reduction of nitroimida7ole compounds. The DNA-biosensor enables preconcentration of the sample onto the electrode surface, and in situ electrochemical detection of the damage they cause to DNA on the electrode surface.

A nucleic acid based optical bioprobe for environmental monitoring is described. The sensor employs the long wavelength intercalating fluorophore ToPro-3. Compounds which interact with the ToPro-3:nucleic acid complex are detected indirectly by measuring changes in the fluorescent signal intensity. The scheme attempts to combine the broad range detection capability of whole cell assays with the speed and simplicity of specific immunoassays and compliments both strategies. Previously, we have shown that the assay scheme is capable of detecting compounds with affinity for nucleic acids ranging from carcinogens to natural products. Here we report application of nucleic acid based sensing for detecting organic solvents and heavy metal ions. In general, the solvent sensitivity of the assay follows the polarity of the solvent, i.e. toluene> DMF> DMSO> methanol. The detection limit for toluene was 0.025% or 25 ppm. A variety of metal ions have been tested including: cadmium, cerium, chromium, iron, lead, magnesium, manganese, nickel and zinc. Of these only cerium and chromium were detected. In addition, the ability of the assay to discriminate between structurally related acridine compounds was investigated. The interaction profiles follow closely what would be expect from the chemical structure of the various dyes. The assay is also capable of detecting structural differences in a series of related compounds which indicates that the assay could be useful in for determining structure activity relationships (SAR).

The nature of the interactions between biomolecules, like proteins and enzymes, and smaller molecules or ligands has prompted the development of novel recognition elements for ion-selective electrodes. This article will focus on biomimetic ionophore design and polymer imprinting as approaches to incorporate biorecognition elements into ISEs. From the interaction of arginine residues in proteins with oxoanions, guanidinium functionalities were incorporated in simpler and sturdier organic compounds to result in ionophores selective to hydrogen sulfite and to salicylate. The preparation of an imprinted polymer complimentary in size, shape and charge to the analyte resulted in the development of polypyrrole-based nitrate-selective electrodes that were later incorporated in a gas sensor for NO_x.

A general survey of the modification of electrodes by lipids and proteins (enzymes) for potential application in environmental analysis is considered. Advantages of mixing both lipids and enzymes in the matrix of the carbon paste electrode are highlighted. Mercury, and noble metal electrodes may spontaneously adsorb lipids giving rise to organized membrane like structures. The latter are obtained by lipid vesicle fusion, by pre-formed films or by a “painting” technique. The resulting supported lipid bilayer membranes (sBLM) may insert proteins and enzymes either spontaneously, by chemical reactions, or by formation of a complex with polyelectrolytes. Examples from the literature are reviewed.

Analytical parameters of any immunochemical assay are based primarily on binding characteristics of the antibody employed. In this laboratory, a wide variety of immunogens, antibodies and tracer conjugates have been developed to incorporate them into competitive enzyme linked immunoadsorbent assays (ELISAs) and other immunochemical formats including biosensors. The antibody based assays, working on competitive principle, provided sensitive responses to the presence of ppt-ppb amounts of toxic substances measured in terms of changes of radioactive, color, fluorescent, or electric signals. Thus, a sensitive and highly selective immunochemical analysis of 2,4-dichlorophenoxyacetic acid (2,4-D), s-triazines and polychlorinated biphenyls (PCBs) can be performed under both laboratory and field conditions. Favorable correlation between immunochemical and instrumental techniques has been found. Several interrelated issues concerning the conventional immunoassays and immunosensors are compared and discussed in terms of needs and methodological maturation.

Three different electrochemical approaches for the detection of naphthol (amperometry, chronoamperometry and differential pulse voltammetry), using screen-printed carbon electrodes, were compared with respect to measurable range, detection limit, reproducibility, sample throughput and suitability for in-field use. The highest sensitivity (252 nA/μM) with a calculated detection limit of 0.1 µM, combined with a remarkable ‘time for sample measurement’ (5.5 seconds) was achieved with differential pulse voltammetry. The standard error in the all measured range (0.5–100 μM) was below 5%. The scan speed and the pulse amplitude of the differential pulse voltammetry were optimised using the simplex method. The optimised electrochemical procedure was then used to evaluate the detection of low levels of alkaline phosphatase: this enzyme catalyses the hydrolysis of naphthyl phosphate to naphthol. Using 2 minutes as incubation time for the enzyme reaction, a calibration curve in the range 1–10−5 U/ml was obtained with a calculated detection limit of 2.1×10−6 U/ml. The standard error for the 10−2 U/ml enzyme standard was 6.8% Finally a competitive enzyme immunoassay for polychlorinated biphenyls based on alkaline phosphatase was performed using the optimised electrochemical detection of naphthol. The assay measurable range was 0.01–10 gg/ml, and the detection limit was 0.01 µg/ml.

Plastic, fully screen-printed, disposable pH sensors based on ruthenium dioxide are described. Application of thick-film technology resulted in cheap and highly reproducible fabrication of the sensors. The electrodes enable fast and selective measurements, with good sensitivity, in acidic and neutral range of pH. The possibility of monitoring pH changes caused by enzymatic processes make them useful as transducers in pH-based biosensors. Moreover, the surface of carbon electrodes doped with ruthenium dioxide offer a good support for enzyme adsorption. These electrodes with immobilised acetylcholinesterase can be used for determination of some enzymatic inhibitors. The biosensors enable easy and fast detection of carbofuran at the sub-ppb level. Only a few minutes are required for pesticide determination, including the biosensor incubation in a sample. Other anticholinesterase active substances, which belong to the group of carbamates and phosphoorganic compounds might be determined in similar way

Immunochemical biosensors for pesticides based on piezoelectric quartz crystals as transducers are described. The changes of resonant frequency of the crystal placed in a flow cell allow to measure mass changes on its surface resulting from binding of biomolecules. In this way, no labeling procedures are required. The biospecific interactions can be followed directly in real time using an inexpensive measuring system — frequency counter, and valuable kinetic characteristics can be obtained. The construction of immunosensors for 2,4-dichlorophenoxyacetic acid (2,4-D), atrazine and parathion uses competitive measuring procedures. The modified analyte is immobilized as a ligand on the transducer. The mixture of antibody and sample containing free analyte is injected and mass changes are recorded. The limits of detection of piezoelectric immunosensors for pesticides are close to 0.1 μg/l. A typical measurement lasts 15 min, one sensor can be used 20 to 50 times. The initial experience regarding the use of immunosensors in the presence of organic solvents is discussed.

Monoclonal antibodies against a derivative of diphenylurea and the derived Fab fragments were investigated using a BIACORE® system with respect to binding kinetics, thermodynamics and assay characteristics. The chloroacetyl group of the hapten II used for immunization and immobilization was involved in the antibody binding. The binding of Fab to immobilized hapten was enthalpy driven below 37°C. Ethanol led to decreased affinities, but shifted the crossreactivity in favour of hapten II.

Biosensors for the detection of low levels of pesticides were prepared by entrapment of different enzymes in polyvinyl-alcohol bearing styryl pyridinium groups (PVA-SbQ). The inhibition of aldehyde dehydrogenase (EC 1.2.1.5), acetolactate synthase (EC 4.1.3.18) and acetyl cholinesterase (EC 3.1.1.7) allows respectively the detection of dithiocarbamate fungicides (10−8 M), sulfonylurea herbicides (10−6 M) and organophosphorus insecticides (10−7 M). The detection is amperometrically carried out.

One of the pronounced trends in the development of instrumentation for chemical analysis in recent years is the design of analytical instruments that makes possible their operation in situ in the direct vicinity of the material to be analysed. The objectives are to allow operation in rugged areas without electricity or consumables, and to limit or even eliminate difficult problems associated with sampling, transport of the samples, and sample pretreatment. This approach is the basic for the intensive developments in areas of chemistry such as in process analysis, in vivo analysis, and field analysis.Field analysis is being developed particularly for environmental, military, industrial and agriculture aims. For these purposes, various types of instrumentation designs are utilized in order to miniaturize the analytical measuring device. The rapid technological progress in material science, electronics and optoelectronics allows the constrrction of portable chromatographs, mass spectrometers, FT-IR, and X-ray spectrometers. The classical methods of wet analysis have also been used in procedures based on various systems with layers of solid reagents. Specific field tests dependent on immunochemical reactions have replaced with great success the more complex chromatographic method.Is there in this race a place for field devices based on flow-injection analysis (FIA) methodology? FIA has been widely accepted in analytical laboratories using wet procedures, and there has been an increasing number of applications reported over recent years in process analysis. The miniaturization of fluid transport systems for liquids and gases, and also detectors, is now in advanced stage of development for use in field portable instrumentation. The use of appropriate biocatalytic and immunochemical steps provides the means for obtaining sufficient selectivity, and thus eliminates sample pretreatment steps. Appropriately designed FIA systems can be operated with very small sample volumes in closed-loop systems allowing re-circulation of reagents.FIA systems also allow the simple dosing of small volumes of sample and reagent solutions without additional accessories, which are troublesome in field usage. Thus, we can conclude that field FIA systems in comparison with solid reagent systems are especially useful for environmental analytes, both gaseous and liquid, which normally would require pre-concentration of trace quantities from large volumes of the original material or separation from complex matrices.

The pollution of the environment is a problem of increasing importance. Monitoring techniques may help to continuously detect pollutants and thus to lower their emision into the environment. Nitrate, phenol, ortho phosphate and 2,4-D were chosen as model substances. They were determined by chemical, enzymatical and immunological sensor systems. Sensors as well as part of the flow systems were realzied in microsystem technology showing the high potential of this technology. Due to miniaturization these systems require only small amounts of chemicals, they offer the chance of mass production, and, in the future, will provide the basis for multianalyte detection.

Electrochemical sensors based on bilayer lipid membranes (BLMs) and related thin film technology offer an attractive route for the construction of devices for the detection of a large number of environmental pollutants in field. Mechanically stabilized bilayer lipid membrane assemblies (stabilized on ultrafiltration membranes or supported on metal surfaces) have been used for the rapid sensitive and selective detection of toxins, triazine herbicides and gases.Filter-supported BLMs were used for the electrochemical flow-injection monitoring of triazine herbicides (simazine, atrazine and propazine) in protein-free water samples.These filters were found to enhance the mechanical stability of BLMs for flow injection experiments. Injections of the analytes were made into a flowing stream of a carrier electrolyte solution. The mechanism of signal generation is related to a rapid adsorption of the analyte followed by a slow aggregative process of the analyte at the surface of BLMs with a consequent rapid reorganization of the membanne electrostatics. The transient response for triazine herbicides increase to the order of simazine, atrazine and propazine which allows selective detection and analysis of these triazines in mixtures. The system is regenerable and can be used for repetitive cycles of injections.Self-assembled metal supported BLMs (s-BLMs) were used for the rapid and sensitive electrochemical detection of gases such as NH₃ and CO₂ using gramicidin D and hemoglobin, respectively, as ionophores. The use of s-BLMs has allowed the electrochemical investigation of the reversibility of response to analytes and of ionophore binding to lipid membranes. Semisynthetic platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, AGEPC) was used to improve the response characteristics of the sensors (i.e., of sensitivity and selectivity). The sensors exhibit good mechanical stability and longevity (over 48 hours), and constant sensitivity and response to a given concentration of analyte in solution. The ammonium sensor also exhibits good selectivity towards volatile amines. The sensors can be fabricated at low cost and provide the advantages of fast response times (in the order of seconds) to alterations of analyte concentration, low detection limits (ca. 10-6 M for ammonium ions and ca. 10-9 M for carbon dioxide), capability of format) applications.Self-assembled metal supported BLMs were also used for rapid and sensitive screening of the triazine herbicides simazine, atrazine and propazine. The interactions of triazines with s-BLMs produced electrochemical ion current increases which reproducibly appeared within ca. 10 s after exposure of the lipid membranes to the herbicides. The sensitivity of the response was maximized by use of BLMs composed of 35% (w/w) DPPA, and by alteration of the phase distribution within membranes by the introduction of 1.0 mM calcium ions in bulk solution. The mechanism of signal generation could be a result of rapid adsorption of the triazine on the surface of s-BLMs with a consequent rapid reorganization of the electrostatics of the membrane. The magnitude of the current signal was linearly related to the herbicide concentration, which could be determined at nanomolar level. The present one-shot sensor has exhibited constant response characteristics (i.e., sensitivity and response to a given concentration of triazine in solution), fast response times, and low detection limits. The sensor can be simply and reliably fabricated at low cost. Studies have shown high selectivity for triazines in the presence of insecticides and pesticides.

Enzymatic sensors based on the ion sensitive field effect transistor (ISFET) and semiconductor structures for detection of phosphororganic pesticides were developed. Their characteristics were optimised to control these pollutants in field. Acetyl-(AChE), butyril- (BChE) cholinesterase’s (ChE) and crude substances contained both forms of ChE were used as chemically sensitive substances to the phosphororganic pesticides. Detectable phosphororganic components were o,o-diethyl o-3,5,6-trichloro-2 pyridil phosphortionate, 2,2-dichlorovinyl dimethyl phosphate and phasolone. They are irreversible inhibitors for above mentioned ChE’s. It is shown that the usage of replaceable enzymatic membrane is more preferable for repeated analysis than its reactivating. Alginate gel and nitrocellulose (NC) strips are very suitable for creation of replaceable enzymatic membrane. The standard deviation of sensor responses for series measurements and for different membrane castings did not exceed 10%. The working characteristics of enzymatic sensors based on the ISFETs depend on medium condition of samples to be analysed. Vegetable sap influences on the value of sensor response. To prevent dependence of sensor signal on medium of analysed samples, the measurements should be performed in standard solution (3–5 mmo1/1, pH 7.3 tris-HCL buffer, contained 140 mmo1/1 sodium chloride). The sensitivities of enzymatic sensors based on the ISFETs to above mentioned pesticides were within the range 10−5 – 10−7 mol/l. ChE’s are reversible inhibited by heavy metal ions. The activity of ChE’s is significantly reduced at their concentration 10−3mo1/l and higher. To diminish nonspecific signal which is generated by ChE’s in the presence of heavy metal ions, it is necessary to have information from urease sensor. The usage of urease and any ChE as well as transducer in form of Si-SiO₂-Si₃N₄-Ta₂O₅-electrolyte metal allows to get multichannel sensor for pesticide analysis. The detectable concentration of phasolone by multi-enzymatic sensor was about 10−7 mol/1. The sensitivity of analysis by both developed sensors is significantly higher than permissible limit of pesticide concentration in water and in vegetable food.

A bioelectrochemical method for the determination of heavy metals has been developed using oxidase enzymes and screen-printed electrodes. The activity of the oxidase enzymes has been measured following H₂O₂ production in the presence of the substrate with a Ru/graphite working electrode polarized at +700 mV vs. Ag/AgCI.Interfence signals due to the oxidation of the enzyme substrates have been minimized by the use of a cellulose acetate membrane placed onto the electrode surface. Inhibition of the activity of enzymes such as alcohol oxidase, sarcosine oxidase and glycerol-3-P oxidase by the metal ions Hg(II), V(V), Cu(II), Ni(II) resulted in the costruction of calibration curves for the metals in the low ppm range. Total analysis time was 10 min (5 min incubation + 5 min measuring) with CVs of 10–17%.

Environmental discharge is a global problem. Among many others, the Water Industries seek new broad sreening devices able to provide early warning of toxic discharge at presently “unprotected” sites. The search for broad screening presents different problems to the detection of a specific known analyte and screening procedures with a broad specificity must be chosen. This paper describes a broad band screen for herbicides active on the photosynthetic system and explores whether analysis of the signal detail can give more information about the analyte. It also looks critically at the construction of a biosensor which would use this photoelectrochemical mechanism, employing a reagent phase immobilised next to an electrode and debates the possibility of other formats. An inverted format sensor is then exploited to devise a single analyte sensor specific for formaldehyde monitoring in the gas phase and operational over an eight hour work shift period.

Various potentiometric and amperometric biosensors based on immobilized cholinesterases have been investigated for the determination of inhibitors in laboratory and field conditions. The role of factors affecting the analytical characteristics of inhibitor biosensors and their selectivity, i.e. the influence of membrane material, the optimization of working conditions, pretreatment of the sample tested has been reviewed and discussed.

Various types of carbon-containing sensors (electrodes) — glassy carbon, impregnated graphite, thick-film graphite and modified sensors — and two versions of stripping voltammetry — differential pulse voltammetry and fast linear scan response differentiation voltammetry — are described. By way of example, concentrations of Cu, Pb and Cd were determined with in situ mercury plated electrode, those of Pb and Cd on modified electrodes in the absence of metallic mercury or soluble mercury salts, and those of Cr, W and Ni using adsorptive stripping voltammetry. The fast linear scan response differentiation voltammetry is shown to have certain advantages, such as elimination of the need for removal of oxygen from the solution and well defined responses of reversible and quasireversible processes, specifically, from copper.

A new method for the chemiluminescent detection of halophenols is presented. It is based on the ability of certain substituted phenols to enhance the chemiluminescence reaction of luminol catalyzed by horseradish peroxidase. The magnitude of enhancement depends on the particular enhancer employed and also on the enhancer concentration. Taking advantage of the properties of these enhancers, a flow injection analysis (FIA) method for halophenols involving a fiber optic sensor and immobilized peroxidase has been developed. We mainly focussed on the detection of chlorophenols due to their toxicity and persistence in the environment. The composition of the reaction medium have been optimized. Among the ten chlorophenols tested, six of them could be determined with the present method with a detection limit varying from 0.5 μM to 100 μM. The greatest sensitivity was obtained for 4-chloro-3-methylphenol whereas for the others the sensitivity decreased according to the following order: 4chlorophenol > 2,4-dichlorophenol > 2-chlorophenol = 3-chlorophenol > 2.4,5trichlorophenol. Pentachlorophenol, 2,4,6-trichlorophenol, 2,6-dichlorophenol and 2amino-4-chlorophenol could not be detected by this chemiluminescent method.

Selective biosensor systems for determination of heavy metal ions based on ion sensitive field effect transistors and thin-film interdigitated planar (conductometric) electrodes have been developed. Glucose oxidase, alcohol oxidase and butyril cholinesterase immobilised on the transducer surfaces have been used as bioactive elements for preparation of Ag+, Hg2+ and Pb2+ sensitive biosensors. The biosensors developed exhibit a dynamic range of 1 – 100 μM for these heavy metal ions. The performance characteristics of the biosensors are discussed.

Organic-phase biosensors have been developed for the detection of environmental pollutants which are either enzyme substrates or enzyme inhibitors. The enzyme substrates are phenols for tyrosinase-based biosensors and peroxides for horseradish peroxidase (HRP)-based sensors. Organic-phase inhibition biosensors for thiourea (THU), ethylenethiourea (ETU), mercaptoethanol (MCE), hydroxylamine (HLA), methylisothiocyanate (MeSNC) and cyanide were based on a peroxidase biosensor, while diethyldithiocarbamate (DEDTC) was detected with a tyrosinase biosensors. The organic-phase HRP and tyrosinase biosensors were prepared by simple immobilisation techniques. The enzymes were entrapped in a cation exchange polymer (Eastman AQ polymer), or in an Os-polymer-enzyme electrostatic complex in the presence of bifunctional glutaraldehyde or poly(ethylene glycol) as a cross-linking agent. A cyanide biosensor was based on the entrapment of HRP in methyltriethoxysilane (MTEOS) sol-gel. The sensors were applied in organic media (such as acetonitrile, methanol, acetone, THF, propanol and 2-butanol) for the detection of peroxides, phenols and the other pollutants (inhibitors). The kinetic parameters for the peroxide and phenol biosensors were determined in the presence and absence of the inhibitors. More efficient organic-phase enzyme electrodes (OPEEs) for the detection of the pollutants, which might be in very small quantities, were developed by employing chemically modified enzymes in the biosensor fabrication. For example, HRP electrodes were prepared with enzymes in which the ε-lysine residues were derivatised with bifunctional N-hydroxysuccinimide esters (NHS). The bis-sucinimide-derivatised HRP biosensor exhibited greater organo-tolerance, longer storage/thermal stability and enhanced sensitivity for both peroxides and inhibitors than sensors containing the native enzyme.

The possibilities and advantages of using reversed micelles as appropriate working media for the development of amperometric enzyme biosensors with analytical purposes is discussed. The influence of the composition of reversed micelles on the amperometric responses obtained at the enzyme electrodes is illustrated. Some analytical applications such as the determination of phenolic pollutants, of the antioxidant BHA and of dimethyl- and diethyldithiocarbamates are shown.

The paper reports the significance of bilayer lipid membranes on a solid support (sBLM) for the construction of biosensors. The methods of formation of lipid membranes on different solid supports including different metals (silver, gold, stainless steel), agar and conducting polymers are presented. Several examples of the application of electrostriction and dielectric relaxation methods for the study of mechanical properties and dynamics of solid supported bilayers have been shown. We demonstrated that these methods are usefull for determination of the binding of enzymes and antibodies to sBLM and for study physical properties of modified supported membranes.

Due to the expanding scope and budget for environmental monitoring in the U.S., as well as the high cost and slow turnaround times typically associated with the measurement of regulated pollutants, there exists a clear need for novel environmental diagnostics which are fast, portable, and cost-effective. Although there is a clear opportunity for innovative technologies, methods developed to fill this need must overcome a number of challenges before they find widespread acceptance and use for environmental applications. Some of these challenges include: the number of potential pollutants and range of their chemical classes, the complexity of environmental matrices, and the variety of possible co-contaminants. Due primarily to their versatile operating formats, biosensors show the potential to provide solutions for certain environmental screening and monitoring applications. These analytical devices have been used to measure a wide variety of environmentally related chemical and biological parameters including: organics, heavy metals, bacteria, biological oxygen demand. toxicity, and DNA hybridization. Prototype biosensors have been demonstrated using laboratory standards in buffer solutions as well as matrices such as waste water. surface water, and mixed organic solvents. In addition, several of these devices are undergoing field trials in environmental settings.

Miniaturization of biosensors has increased the importance of microsystem technology particulary of microelectronics, micromachining and thin film technology. Thin-film technology microelectrodes and microsystems serve as well-defined and reproducible interfaces between sensing, recognition and transduction sites. Solving the compatibility of bilayer lipid membranes with planar thin-film microsystems is esential for future applications of that very “adaptive” material in the field of microbiosensors. Microsystem technology gives comprehensive and synergetic effect on the innovation and mass-production of microinstruments for biomedical analysis and enviromental monitoring.