Biosensors for determination of glucose with glucose oxidase immobilized on an eggshell membrane

A glucose biosensor using an enzyme-immobilized eggshell membrane and oxygen electrode for glucose determination has been fabricated. Glucose oxidase was covalently immobilized on an eggshell membrane with glutaraldehyde as a cross-linking agent. The glucose biosensor was fabricated by positioning the enzyme-immobilized eggshell membrane on the surface of a dissolved oxygen sensor. The detection scheme was based on the depletion of dissolved oxygen content upon exposure to glucose solution and the decrease in the oxygen level was monitored and related to the glucose concentration. The effect of glutaraldehyde concentration, pH, phosphate buffer concentration and temperature on the response of the glucose biosensor has been studied in detail. Common matrix interferents such as ethanol, d-fructose, citric acid, sodium benzoate, sucrose and l-ascorbic acid did not give significant interference. The resulting sensor exhibited a fast response (100 s), high sensitivity (8.3409 mg L⁻¹ oxygen depletion/mmol L⁻¹ glucose) and good storage stability (85.2% of its initial sensitivity after 4 months). The linear response is 1.0×10⁻⁵ to 1.3×10⁻³ mol L⁻¹ glucose. The glucose content in real samples such as commercial glucose injection preparations and wines was determined, and the results were comparable to the values obtained from a commercial glucose assay kit based on a spectrophotometric method.     

Biodegradation analyses of trichloroethylene (TCE) by bacteria and its use for biosensing of TCE

Trichloroethylene (TCE) is a toxic, recalcitrant groundwater pollutant. TCE-degrading microorganisms were isolated from various environments. The aerobic bacteria isolated from toluene- and tryptophan-containing media were Pseudomonas sp. strain ASA86 and Burkholderia sp. strain TAM17, respectively; these are necessary for inducing TCE biodegradation in a selective medium. The half-degradation time of TCE to a concentration of 1 mg/L was 18 h for strain ASA86 and 7 days for strain TAM17. While identifying toluene/TCE degradation genes, we found that in strain ASA86, the gene was the same as the todC1 gene product encoding toluene dioxygenase identified in Pseudomonas putida F1, and that in strain TAM17, the gene was similar to the tecA1 gene product encoding chlorobenzene dioxygenase identified in Burkholderia sp. PS12. A novel TCE biosensor was developed using strain ASA86 as the inducer of toluene under aerobic conditions. The TCE biosensor exhibited a linear relationship below 3 ppm TCE. Detection limit of the biosensor was 0.05 ppm TCE. The response time of the biosensor was less than 10 min. The biosensor response displayed a constant level during a 2 day period. The TCE biosensor displayed sufficient sensitivity for monitoring TCE in environmental systems.     

Speciation of chromium using chronoamperometric biosensors based on screen-printed electrodes

Chronoamperometric assays based on tyrosinase and glucose oxidase (GOx) inactivation have been developed for the monitoring of Cr(III) and Cr(VI). Tyrosinase was immobilized by crosslinking on screen-printed carbon electrodes (SPCEs) containing tetrathiafulvalene (TTF) as electron transfer mediator. The tyrosinase/SPC_TTFE response to pyrocatechol is inhibited by Cr(III). This process, that is not affected by Cr(VI), allows the determination of Cr(III) with a capability of detection of 2.0 ± 0.2 μM and a reproducibility of 5.5%. GOx modified screen-printed carbon platinised electrodes (SPC_PtEs) were developed for the selective determination of Cr(VI) using ferricyanide as redox mediator. The biosensor was able to discriminate two different oxidation states of chromium being able to reject Cr(III) and to detect the toxic species Cr(VI). Chronoamperometric response of the biosensor towards glucose decreases with the presence of Cr(VI), with a capability of detection of 90.5 ± 7.6 nM and a reproducibility of 6.2%. A bipotentiostatic chronoamperometric biosensor was finally developed using a tyrosinase/SPC_TTFE and a GOx/SPC_PtE connected in array mode for the simultaneous determination of Cr(III) and Cr(VI) in spiked tap water and in waste water from a tannery factory samples.     

Whole-cell Gluconobacter oxydans biosensor for 2-phenylethanol biooxidation monitoring

A microbial biosensor for 2-phenylethanol (2-PE) based on the bacteria Gluconobacter oxydans was developed and applied in monitoring of a biotechnological process. The cells of G. oxydans were immobilized within a disposable polyelectrolyte complex gel membrane consisting of sodium alginate, cellulose sulphate and poly(methylene-co-guanidine) attached onto a miniaturized Clark oxygen electrode, forming whole cell amperometric biosensor. Measured changes in oxygen concentration were proportional to changes in 2-PE concentration. The biosensor sensitivity was 864 nA mM⁻¹ (RSD = 6%), a detection limit of 1 μM, and the biosensor response towards 2-PE was linear in the range 0.02–0.70 mM. The biosensor preserved 93% of its initial sensitivity after 7 h of continuous operation and exhibited excellent storage stability with loss of only 6% of initial sensitivity within two months, when stored at 4 °C. The developed system was designed and successfully used for an off-line monitoring of whole course of 2-PE biooxidation process producing phenylacetic acid (PA) as industrially valuable aromatic compound. The biosensor measurement did not require the use of hazardous organic solvent. The biosensor response to 2-PE was not affected by interferences from PA and phenylacetaldehyde at concentrations present in real samples during the biotransformation and the results were in a very good agreement with those obtained via gas chromatography.     

Total internal reflectance fluorescence (TIRF) biosensor for environmental monitoring of testosterone with commercially available immunochemistry: antibody characterization, assay development and real sample measurements

Nowadays, little technology exists that can monitor various water sources quickly and at a reasonable cost. The ultra-sensitive, fully automated and robust biosensor River Analyser (RIANA) is capable of detecting multiple organic targets rapidly and simultaneously at a heterogeneous assay format (solid phase: bulk optical glass transducers). Commercialization of such a biosensor requires the availability of commercial high-affinity recognition elements (e.g. antibodies) and suitable commercial haptens (modified target molecules) for surface chemistry. Therfore, testosterone was chosen as model analyte, which is also a task of common analytical methods like gas chromatography-mass spectrometry (GC-MS), because they have to struggle with detecting sub-nanogram per liter levels in environmental samples. The reflectometric interference spectroscopy (RIfS) was used to characterize the commercially available immunochemistry resulting in a high-affinity constant of 2.6 ± 0.3 × 10⁹ mol⁻¹ for the unlabeled antibody. After the labeling procedure, necessary for the TIRF-based biosensor, a mean affinity constant of 1.2 × 10⁹ mol⁻¹ was calculated out of RIfS (1.4 ± 0.4 × 10⁹ mol⁻¹) and TIRF (1.0 ± 0.3 × 10⁹ mol⁻¹) measurements.Thereafter, the TIRF-based biosensor setup was used to determine the steroidal hormone testosterone at real world samples without sample pre-treatment or sample pre-concentration. Results are shown for rapid and ultra-sensitive analyses of testosterone in aqueous samples with at a remarkable limit of detection (LOD) of 0.2 ng L⁻¹. All real world samples, even those containing testosterone in the sub-nanogram per liter range (e.g. 0.9 ng L⁻¹), could be determined with recovery rates between 70 and 120%. Therefore, the sensor system is perfectly suited to serve as a low-cost system for surveillance and early warning in environmental analysis in addition to the common analytical methods. For the first time, commercially available immunochemistry was fully characterized using a label-free detection method (RIfS) and successfully incorporated into a TIRF-based biosensor setup (RIANA) for reliable sub-nanogram per liter detection of testosterone in aqueous environmental samples.     

An inhibition type amperometric biosensor based on tyrosinase enzyme for fluoride determination

In this study, a new biosensor based on the inhibition of tyrosinase for the determination of fluoride is described. To construct the biosensor tyrosinase was immobilized by using gelatine and cross-linking agent glutaraldehyde on a Clark type dissolved oxygen (DO) probe covered with a teflon membrane which is sensitive for oxygen. The phosphate buffer (50 mM, pH 7.0) at 30 °C were established as providing the optimum working conditions. The method is based on the measurement of the decreasing of dissolved oxygen level of the interval surface that related to fluoride concentration added into reaction medium in the presence of catechol. Inhibitor effect of fluoride results in decrease in dissolved oxygen concentration. The biosensor response depends linearly on fluoride concentration between 1.0 and 20 μM with a response time of 3 min.In the characterization studies of the biosensor some parameters such as reproducibility, substrate specificity and storage stability were carried out. From the experiments, the average value (x), Standard deviation (S.D) and coefficient of variation (C.V %) were found as 10.5 μM, ± 0.57 μM, 5.43%, respectively for 10 μM fluoride standard.     

Surface plasmon resonance immunosensor for bacteria detection

This work describes an approach for the development of two bacteria biosensors based on surface plasmon resonance (SPR) technique. The first biosensor was based on functionalized gold substrate and the second one on immobilized gold nanoparticles. For the first biosensor, the gold substrate was functionalized with acid-thiol using the self-assembled monolayer technique, while the second one was functionalized with gold nanoparticles immobilized on modified gold substrate. A polyclonal anti-Escherichia coli antibody was immobilized for specific (E. coli) and non-specific (Lactobacillus) bacteria detection. Detection limit with a good reproducibility of 10⁴ and 10³ cfu mL⁻¹ of E. coli bacteria has been obtained for the first biosensor and for the second one respectively. A refractive index variation below 5 × 10⁻³ due to bacteria adsorption is able to be detected. The refractive index of the multilayer structure and of the E. coli bacteria layer was estimated with a modeling software.     

Amperometric l-glutamate biosensor based on bacterial cell-surface displayed glutamate dehydrogenase

A novel l-glutamate biosensor was fabricated using bacteria surface-displayed glutamate dehydrogenase (Gldh-bacteria). Here the cofactor NADP⁺-specific dependent Gldh was expressed on the surface of Escherichia coli using N-terminal region of ice nucleation protein (INP) as the anchoring motif. The cell fractionation assay and SDS-PAGE analysis indicated that the majority of INP-Gldh fusion proteins were located on the surface of cells. The biosensor was fabricated by successively casting polyethyleneimine (PEI)-dispersed multi-walled carbon nanotubes (MWNTs), Gldh-bacteria and Nafion onto the glassy carbon electrode (Nafion/Gldh-bacteria/PEI-MWNTs/GCE). The MWNTs could not only significantly lower the oxidation overpotential towards NAPDH, which was the product of NADP⁺ involving in the oxidation of glutamate by Gldh, but also enhanced the current response. Under the optimized experimental conditions, the current–time curve of the Nafion/Gldh-bacteria/PEI-MWNTs/GCE was performed at +0.52 V (vs. SCE) by amperometry varying glutamate concentration. The current response was linear with glutamate concentration in two ranges (10 μM–1 mM and 2–10 mM). The low limit of detection was estimated to be 2 μM glutamate (S/N = 3). Moreover, the proposed biosensor is stable, specific, reproducible and simple, which can be applied to real samples detection.     

Fructose-selective calorimetric biosensor in flow injection analysis

A highly selective, interference free biosensor for the measurement of fructose in real syrup samples was developed. The assay is based on the phosphorylation of d(−)fructose to fructose-6-phosphate by hexokinase and subsequent conversion of fructose-6-phosphate to fructose-1,6-biphosphate by fructose-6-phosphate-kinase. The heat liberated in the second reaction is monitored using an enzyme thermistor. The major advantages of this biosensor are rapid and selective measurement of fructose without the need to eliminate glucose and inexpensive FIA-based, mediator-free calorimetric measurement suitable for regular fructose analysis. This method was optimised for parameters, such as pH, ionic strength, interference, operational stability and shelf life. Good and reproducible linearity (0.5-6.0 mM) with a detection limit of 0.12 mM was obtained. Fructose determination in commercial syrup samples and spiked samples confirmed the reliability of this set-up and technique. The biosensor gave reproducible results with good overall stability for continuous measurements over a period of three months besides a useful shelf life of six months. The method could be used for routine fructose monitoring in food samples.     

Biomimetic properties and surface studies of a piezoelectric caffeine sensor based on electrosynthesized polypyrrole

An approach for preparing a chemical sensor for caffeine through the combination of molecularly imprinted polypyrrole and a piezoelectric quartz transducer was proposed. The caffeine-imprinted polymer was synthesized using galvanostatic electropolymerization of pyrrole monomer directly onto one of the gold electrodes of a 9 MHz AT-cut quartz crystal in the presence of caffeine. The optimum conditions for the electrosynthesis of the reagent phase were established. Caffeine molecules were entrapped in the matrix of polymer film, and were removed by subsequent washing with water, leaving behind pores capable of recognizing the target analyte molecule.The caffeine sensor was fixed in a measuring cell and measurement of the resonant frequency of the quartz crystal as it comes in contact with the caffeine solution was carried out in a stopped flow mode. A steady-state response was achieved in about 10 min. The sensor exhibited a linear relationship between the frequency shift and the ln of caffeine concentration in the range of 0.1-10 mg/mL (correlation coefficient, r = 0.9882). The sensitivity of the sensor was about 255 Hz/ln concentration (mg/mL). A good repeatability, R.S.D. = 9 (n = 6) for 0.5 mg/mL caffeine solution was also observed. The use of the sensor can present a potential low-cost option for determining caffeine.Surface analytical techniques such as scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) were performed on the polymer coating in order to elucidate the imprinting process and rebinding of caffeine to the polymer matrix during the sensing process. The SEM micrographs and XPS spectra revealed features and structures that could support the imprinting and recognition of caffeine molecule by the imprinted polymer.     

Electrogenerated chemiluminescence biosensing for the detection of prostate PC-3 cancer cells incorporating antibody as capture probe and ruthenium complex-labelled wheat germ agglutinin as signal probe

A highly selective and sensitive electrogenerated chemiluminescence (ECL) biosensor for the detection of prostate PC-3 cancer cells was designed using a prostate specific antibody as a capture probe and ruthenium complex-labelled wheat germ agglutinin as a signal probe. The ECL biosensor was fabricated by covalently immobilising the capture probe on a graphene oxide-coated glassy carbon electrode. Target PC-3 cells were selectively captured on the surface of the biosensor, and then, the signal probe was bound with the captured PC-3 cells to form a sandwich. In the presence of tripropylamine, the ECL intensity of the sandwich biosensor was logarithmically directly proportion to the concentration of PC-3 cells over a range from 7.0 × 10² to 3.0 × 10⁴ cells mL⁻¹, with a detection limit of 2.6 × 10² cells mL⁻¹. The ECL biosensor was also applied to detect prostate specific antigen with a detection limit of 0.1 ng mL⁻¹. The high selectivity of the biosensor was demonstrated in comparison with that of a lectin-based biosensor. The strategy developed in this study may be a promising approach and could be extended to the design of ECL biosensors for highly sensitive and selective detection of other cancer-related cells or cancer biomarkers using different probes.     

Functional graphene-gold nano-composite fabricated electrochemical biosensor for direct and rapid detection of bisphenol A

In this research, the graphene with excellent dispersity is prepared successfully by introducing gold nanoparticle to separate the individual sheets. Various techniques are adopted to characterize the prepared graphene and graphene-gold nanoparticle composite materials. This fabricated new composite material is used as the support material to construct a novel tyrosinase based biosensor for detection of bisphenol A (BPA). The electrochemical performances of the proposed new enzyme biosensor were investigated by differential pulse voltammetry (DPV) method. The proposed biosensor exhibited excellent performance for BPA determination with a wide linear range (2.5 × 10⁻³–3.0 μM), a highly reproducible response (RSD of 2.7%), low interferences and long-term stability. And more importantly, the calculated detection limit of the proposed biosensor was as low as 1 nM. Compared with other detection methods, this graphene-gold nanoparticle composite based tyrosinase biosensor is proved to be a promising and reliable tool for rapid detection of BPA for on-site analysis of emergency BPA related pollution affairs.     

Amperometric choline biosensors prepared by layer-by-layer deposition of choline oxidase on the Prussian blue-modified platinum electrode

An amperometric choline biosensor was developed by immobilizing choline oxidase (ChOx) in a layer-by-layer (LBL) multilayer film on a platinum (Pt) electrode modified with Prussian blue (PB). 6-O-Ethoxytrimethylammoniochitosan chloride (EACC) was used to prepare the ChOx LBL films. The choline biosensor was used at 0.0 V versus Ag/AgCl to detect choline and exhibited good characteristics such as relative low detection limit (5 × 10⁻⁷ M), short response time (within 10 s), high sensitivity (88.6 μA mM⁻¹ cm⁻²) and a good selectivity. The results were explained based on the ultrathin nature of the LBL films and the low operating potential that could be due to the efficient catalytic reduction of H₂O₂ by PB. In addition, the effects of pH, temperature and applied potential on the amperometric response of choline biosensor were evaluated. The apparent Michaelis-Menten constant was found to be (0.083 ± 0.001) ×10⁻³ M. The biosensor showed excellent long-term storage stability, which originates from a strong adsorption of ChOx in the EACC multilayer film. When the present choline biosensor was applied to the analysis of phosphatidylcholine in serum samples, the measurement values agreed satisfactorily with those by a hospital method.     

Determination of catechins and caffeine in proposed green tea standard reference materials by liquid chromatography-particle beam/electron ionization mass spectrometry (LC-PB/EIMS)

Presented here is the quantitative analysis of green tea NIST standard reference materials (SRMs) via liquid chromatography-particle beam/electron ionization mass spectrometry (LC-PB/EIMS). Three different NIST green tea standard reference materials (SRM 3254 Camellia sinesis Leaves, SRM 3255 C. sinesis Extract and SRM 3256 Green Tea-containing Oral Dosage Form) are characterized for the content of caffeine and a series of catechin species (gallic acid, catechin, epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate (EGCG)). The absolute limits of detection for caffeine and the catechin species were determined to be on the nanogram level. A reversed-phase chromatographic separation of the green tea reference materials was carried out on a commercial C₁₈ column using a gradient of water (containing 0.1% TFA) and 2:1 methanol:acetonitrile (containing 0.1%TFA) at 0.9 mL min⁻¹ and an analysis time of 50 min. Quantification of caffeine and the catechin species was carried out using the standard addition and internal standard methods, with the latter providing appreciable improvements in precision and recovery.     

Preparation and characterization of Prussian blue nanowire array and bioapplication for glucose biosensing

Prussian blue nanowire array (PBNWA) was prepared via electrochemical deposition with polycarbonate membrane template for effective modification of glassy carbon electrode. The PBNWA electrode thus obtained was demonstrated to have high-catalytic activity for the electrochemical reduction of hydrogen peroxide in neutral media. This enabled the PBNWA electrode to show rapid response to H₂O₂ at a low potential of −0.1 V over a wide range of concentrations from 1 × 10⁻⁷ M to 5 × 10⁻² M with a high sensitivity of 183 μA mM⁻¹ cm⁻². Such a low-working potential also substantially improved the selectivity of the PBNWA electrode against most electroactive species such as ascorbic acid and uric acid in physiological media. A detection limit of 5 × 10⁻⁸ M was obtained using the PBNWA electrode for H₂O₂, which compared favorably with most electroanalysis procedures for H₂O₂. A biosensor toward glucose was then constructed with the PBNWA electrode as the basic electrode by crosslinking glucose oxidase (GOx). The glucose biosensor allowed rapid, selective and sensitive determination of glucose at −0.1 V. The amperometric response exhibited a linear correlation to glucose concentration through an expanded range from 2 × 10⁻⁶ M to 1 × 10⁻² M, and the response time and detection limit were determined to be 3 s and 1 μM, respectively.     

Sol-gel derived urease-based optical biosensor for the rapid determination of heavy metals

A urease optical biosensor for the determination of heavy metals based on sol-gel immobilization technique was developed. A fluorescent dye, FITC-dextran, was encapsulated and parameters including optical properties of the probe, relative enzyme activity, initial pH value and the buffer concentration for substrate preparation were investigated. In sol-gel immobilization, 1 mM Tris-HCl at pH 7.1 provided a sufficient buffer capacity for metal ion analysis as well as the enzyme activity maintenance. Also, two analytical procedures, incubated and un-incubated systems, were compared to understand the sensitivity and applicability to heavy metal analysis. The developed optical biosensor showed high reproducibility and the relative standard deviation (R.S.D.) of 5.1% (n=10) was obtained. Also, eight measurements can be completed automatically within 36 min. The biosensor has high sensitivity to Cu(II) and Cd(II) and an analytical range of 10-230 μM with a detection limit of 10 μM was achieved. Moreover, biological and environmental samples were examined to evaluate the applicability of the developed biosensor. A 19-82% of inhibition was observed when 20-45 μM metal ions were amended into tested samples, revealing that the developed system has the potential for the determination of heavy metals in real samples.     

Thermostable amperometric lactate biosensor with Clostridium thermocelluml-LDH for the measurement of blood lactate

The gene for Clostridium thermocelluml-lactate dehydrogenase enzyme was cloned into pGEX-4T-2 purification vector to supply a source for a thermostable enzyme in order to produce a stable lactate biosensor working at relatively high temperatures. The purified thermostable enzyme (t-LDH) was then immobilized on a gold electrode via polymerization of polygluteraldehyde and pyrrol resulting in a conductive co-polymer. t-LDH working electrode (t-LDHE) was used for determination of lactate in CHES buffer. Amperometric response of the produced electrodes was measured as a function of lactate concentration, at a fixed bias voltage of 200 mV in a three-electrode system. The linear range and sensitivity of the biosensor was investigated at various temperatures in the range of 25-60 °C. The sensitivity t-LDHE increased with increasing the temperature and reached its highest value at 60 °C. The calculated value was nearly 70 times higher as compared to the sensitivity value of the same electrode tested at 25 °C. The sensing parameters of t-LDHE were compared with the electrodes produced by commercially available rabbit muscle LDH (m-LDH). The sensitivity of t-LDHE was nearly 8 times higher than that of m-LDHE. t-LDHE was found to retain its activity for a week incubation at refrigerator (+5 °C), while m-LDHE lost its activity in this period. t-LDHE was also tested in the presence of human blood serum. The results showed that the current increased with increasing concentrations of lactate in the human blood serum and the biosensor is more sensitive to serum lactate as well as the commercial lactate dissolved in serum as compared to the commercial lactate dissolved in CHES buffer.     

Swellable molecularly imprinted polyN-(N-propyl)acrylamide particles for detection of emerging organic contaminants using surface plasmon resonance spectroscopy

Lightly crosslinked theophylline imprinted polyN-(N-propyl)acrylamide particles (ca. 300 nm in diameter) that are designed to swell and shrink as a function of analyte concentration in aqueous media were spin coated onto a gold surface. The nanospheres responded selectively to the targeted analyte due to molecular imprinting. Chemical sensing was based on changes in the refractive index of the imprinted particles that accompanied swelling due to binding of the targeted analyte, which was detected using surface plasmon resonance (SPR) spectroscopy. Because swelling leads to an increase in the percentage of water in the polymer, the refractive index of the polymer nanospheres decreased as the particles swelled. In the presence of aqueous theophylline at concentrations as low as 10⁻⁶ M, particle swelling is both pronounced and readily detectable. The full scale response of the imprinted particles to template occurs in less than 10 min. Swelling is also reversible and independent of the ionic strength of the solution in contact with the polymer. Replicate precision is less than 10⁻⁴ RI units. By comparison, there is no response to caffeine which is similar in structure to theophylline at concentrations as high as 1 × 10⁻² M. Changes in the refractive index of the imprinted polymer particles, as low as 10⁻⁴ RI units could be readily detected. A unique aspect of the prepared particles is the use of light crosslinking rather than heavy crosslinking. This is a significant development as it indicates that heavy crosslinking is not entirely necessary for selectivity in molecular imprinting with polyacrylamides.     

Modified epoxy coatings on mild steel: Tribology and surface energy

A commercial epoxy diglycidylether of bisphenol-A (DGEBA) was modified by adding fluorinated poly(aryl ether ketone) fluoropolymer and in turn metal micro powders (Ni, Al, Zn, and Ag) and coated on mild steel. Two curing agents were used; triethylenetetramine (a low temperature curing agent) and hexamethylenediamine (a high temperature curing agent) for understanding the curing temperature effect on the properties. Variations in tribological properties (dynamic friction and wear) and surface energies with varying amounts of metal powders and curing agents were evaluated. When cured at 30 °C, dynamic friction and wear decrease significantly due to phase separation reaction being favored between the fluoropolymer and the epoxy. However, when cured at 80 °C, friction and wear increase; this can be explained in terms of a crosslinking reaction favored at that temperature. There is a significant decrease in surface energies with the addition of modifiers.     

Use of bioluminescent bacterial sensors as an alternative method for measuring heavy metals in soil extracts

The luminescence based bacterial sensor strains Pseudomonas fluorescens OS8 (pTPT11) for mercury detection and Pseudomonas fluorescens OS8 (pTPT31) for arsenite detection were used in testing their application in detecting heavy metals in soil extracts. Three different soil types (humus, mineral and clay) were spiked with 1, 100 or 500 μg g⁻¹ Hg²⁺ or As³⁺. Samples were taken 1, 14 and 30 days and extracted with water, ammonium acetate, hydrogen peroxide and nitric acid to represent water soluble, bioavailable, organic matter bound and residual fractions, respectively. The lowest mercury-concentration measured using biosensor (0.003 μg kg⁻¹) was considerably lower than by chemical method (0.05 μg kg⁻¹). The sensor strain with pTPT31 appeared to have a useful detection range similar to that of chemical methods. Concentration results with chemical and biosensor analysis were very similar in the case of mercury-spiked samples. Although some of the arsenite samples showed higher variation between methods, it is concluded that the bacteria can be used as an alternative traditional methods for different types of samples.