Microbial biosensors: a review

A microbial biosensor is an analytical device which integrates microorganism(s) with a physical transducer to generate a measurable signal proportional to the concentration of analytes. In recent years, a large number of microbial biosensors have been developed for environmental, food, and biomedical applications. Starting with the discussion of various sensing techniques commonly used in microbial biosensing, this review article concentrates on the summarization of the recent progress in the fabrication and application of microbial biosensors based on amperometry, potentiometry, conductometry, voltammetry, microbial fuel cell, fluorescence, bioluminescence, and colorimetry, respectively. Prospective strategies for the design of future microbial biosensors will also be discussed.     

Whole-cell bacterial biosensors for the detection of aromatic hydrocarbons and their chlorinated derivatives (Review)

The review summarizes the data on new directions in biosensor technologies based on whole bacterial cells. Biosensors for the monitoring of mono(poly)aromatic hydrocarbons and their chlorinated derivatives, which are constructed with genetically modified bacterial cells bearing a reporter gene fusion, are considered. The operating principle of these biosensors is based on the expression of reporter genes (luc, lux, gfp, rfp) under the control of a promoter and a regulator that specifically respond to a detected compound.     

Novel BOD (biological oxygen demand) sensor using mediator-less microbial fuel cell

A microbial fuel cell type of biosensor was used to determine the biochemical oxygen demand (BOD) of wastewater. The biosensor gave a good correlation between the BOD value and the coulomb produced. The BOD sensor has been operated for over 5 years in a stable manner without any servicing. This is much longer that that of previously reported BOD biosensors.     

The response of Moina brachiata (Jurine) 1820 to biological oxygen demand,oxygen, and light

Summary A steady-state gradient apparatus was used to study population dynamics and to investigate chemical, physical, and behavioral characteristics influencing populations. Preferential localization of the cladoceranMoina brachiata (Jurine) was observed within the experimental gradient. Laboratory and field studies suggest thatM. brachiata responds to light as a discriminated stimulus when light is paired with a reinforcer, food. The cladocerans preferred as a medium water having a five-day biological oxygen demand (BOD) of approximately 14–16 ppm. Preference for BOD levels appears to be more restrictive than preference for levels of dissolved oxygen. The relationship of space and time with respect to ponds and gradient is discussed.Present address: University of Negev, P.O.B. 2053, Beer-sheva, Israel.     

PNA biosensors for nucleic acid detection

Biosensor devices, based on the conversion of nucleic acid recognition reactions into useful electrical signals, offer considerable promise for DNA diagnostics. The unique hybridization properties of solution-phase PNA can be extrapolated onto transducer surfaces in connection with the design of remarkably specific DNA biosensors. This article reviews the development of PNA biosensors, and discusses common PNA-biosensing protocols along with their prospects in DNA biosensor technology.     

Sediment oxygen demand in man-made Lake Ton-Ton (Uruguay)

A study on sediment metabolism was carried out during 1986 in Lake Ton-Ton, Uruguay. Sediment oxygen demand (SOD) from chemical and biological origin was measured in undisturbed sediment cores taken from the deepest part of the lake. Mean SOD rate for the study period (51.56 mgO₂ m^–2 h^–1) corresponded well with the eutrophic state of the lake. During stratification, SOD from chemical origin accounted for 69–87% of total SOD, while SOD from biological origin was dominating for the rest of the year, except in July. Biological respiration was principally of microbial origin. Hypolimnetic temperature was the main factor controlling SOD rates (r = 0.771,p < 0.001). Nevertheless, freshly sedimented phytodetritus from anAnabaena bloom, together with a renewed input of oxygen to bottom water were responsible for the maximum SOD values, recorded at the beginning of a mixing period in April (72.51 mgO₂ m^–2 H^–1).     

Application of biochemical oxygen demand (BOD) biosensor for optimization of biological carbon and nitrogen removal from synthetic wastewater in a sequencing batch reactor system

A bench scale reactor using a sequencing batch reactor process was used to evaluate the applicability of biosensors for the process optimization of biological carbon and nitrogen removal. A commercial biochemical oxygen demand (BOD) biosensor with a novel microbial membrane was used to determine the duration of each phase by measuring samples in real time in an SBR cycle with filling/anoxic-anaerobic/aerobic/sludge wasting/settling/withdrawal periods. Possible strategies to increase the efficiency for the biological removal of carbon and nitrogen from synthetic wastewater have been developed. The results show that application of a BOD biosensor enables estimation of organic carbon, in real time, allowing the optimization or reduction the SBR cycle time. Some typical consumption patterns for organic carbon in the non-aeration phase of a typical SBR operation were identified. The rate of decrease of BOD measured using a sensor BOD, was the highest in the initial glucose breakdown period and during denitrification. It then slowed down until a 'quiescent period' was observed, which may be considered as the commencement of the aeration period. Monitoring the BOD curve with a BOD biosensor allowed the reduction of the SBR cycle time, which leads to an increase in the removal efficiency. By reducing the cycle time from 8 to 4 h cycle, the removal efficiencies of nitrate, glucose, and phosphorus in a given time interval, were increased to nearly double, while the removal of nitrogen ammonium was increased by one-third.     

Ceriometry,a combined method for chemical oxygen demand and dissolved oxygen (with a discussion on the precision of the winkler technique)

A ceriometric method is described in which Ce³⁺ salts are used for the determination of dissolved oxygen and Ce⁴⁺ salts for the determination of the chemical oxygen demand. The interference of COD in the O₂ determination, a common feature of most Winkler determinations, is corrected. The standard deviation is typically about 1% of the mean, and bias (inaccuracy) is very small.The method is simple, quick and reliable.The precision and accuracy of the Winkler method is discussed and compared with that of the Cerium method.     

Electrical impedimetric biosensors for liver function detection

In this study, electrical impedimetric biosensors composed of Au-electrodes were fabricated for the quantitative detection of human serum albumin (HSA), an essential biomarker of liver function. The Au-electrodes were fabricated via a single-step photolithography process, and can be easily integrated in biochips for assessing liver function in the future. The glass sensing surface between two adjacent Au-electrodes was modified with 3-aminopropyltriethoxysilane (APTES) to improve the biocompatibility for its subsequent binding to anti-human serum albumin (AHSA). The sensing surface without AHSA binding was blocked using skim milk powders, preventing possible non-specific bonding HSA conjugation. Biosensors were used to measure HSA concentration for liver function detection. The impedance between two adjacent Au-electrodes of the biosensors applied with various HSA concentrations was directly measured, and quantified using an electrochemical impedance spectroscopy system under AC conditions. The results of plotting both values in log scales indicated the impedance increased linearly with HSA conjugation increase. The limit of HSA detection was about 2'10(-4)mg/ml using the electrochemical impedimetric biosensor proposed in this work. This study demonstrates the feasibility of using electrochemical impedimetry as a bio-sensing mechanism to quantify human serum albumin concentration. The sensor proposed in this work also displays great potential for assessing liver function because of its simple detection mechanism, ease of biochip integration, and low cost.     

Fluorescent labels in biosensors for pathogen detection

Infectious diseases caused by pathogens have become a life-threatening problem for millions of people around the world in recent years. Therefore, the need of efficient, fast, low-cost and user-friendly biosensing systems to monitor pathogen has increased enormously in the last few years. This paper presents an overview of different fluorescent labels and the utilization of fluorescence-based biosensor techniques for rapid, direct, sensitive and real-time identification of bacteria. In these biosensors, organic dyes, nanomaterials and rare-earth elements are playing an increasing role in the design of biosensing systems with an interest for applications in bacterial analysis.     

Disposable sensor for biochemical oxygen demand

 Disposable-type microbial sensors were prepared for the determination of biochemical oxygen demand (BOD). The yeast, Trichosporon cutaneum, was directly immobilized on the surface of miniature oxygen electrodes using an ultraviolet crosslinking resin (ENT-3400). The oxygen electrodes (15 mm× 2 mm×0.4 mm) were made on silicon substrates using micromachining techniques. They were Clark-type two-electrode systems with−1021 mV applied to the working electrode. Typical response times of the BOD sensors were in the range of 7–20 min. At 20°C, the sensors’ dynamic range was from 0 to 18 mg/l BOD when a glucose/glutamate BOD standard solution was used. The lower limit of detection was 0.2 mg/l BOD. This value was one order of magnitude lower than that of sensors previously reported. The sensors’ operational lifetime of 3 days was satisfactory for a disposable type. The sensors’ responses were reproducible to within 8% relative standard deviation. The BOD sensors’ were applied to untreated and treated waste waters from industrial effluents and municipal sewage. BOD values determined using these sensors correlated well with those determined by the conventional 5-day BOD determination method. Received: 22 December 1995/Received revision: 19 February 1996/Accepted: 17 March 1996     

Sol-gel based glucose biosensors employing optical oxygen transducers, and a method for compensating for variable oxygen background

Various types of thin-film glucose biosensors based on the use of the enzyme glucose oxidase (GOx) have been developed. The luminescent oxygen probe Ru(dpp)--whose emission is quenched by oxygen--is used to measure the consumption of oxygen. Three different combinations of oxygen transducer and sol-gel immobilized GOx were tested. In the first, GOx was sandwiched between a sol-gel layer doped with Ru(dpp) and a second sol-gel layer composed of pure sol-gel (the 'sandwich' configuration). In the second, a sol-gel layer doped with Ru(dpp) was covered with sol-gel entrapped GOx (the 'two-layer configuration'). In the third, both GOx and a sol-gel powder containing GOx were incorporated into a single sol-gel phase (the 'powder configuration'). In all cases, it was found to be essential to add sorbitol which results in a more porous sol-gel in which diffusion is not impaired. The sandwich configuration provides the highest enzyme activity and the largest dynamic range (0.1-15 mM), but suffers from a distinct decrease in sensitivity upon prolonged use. The two-layer configuration has the fastest response time (t90 = 50 s), while the 'powder configuration' provides the best operational lifetime. The storage stability of all configurations exceeds 4 months if stored at 4 degrees C. In an Appendix, equations are derived which describe the response of such sensors, how the effect of varying oxygen supply can be compensated for by making use of two sensors, one sensitive to oxygen only, the other to both oxygen and glucose, and how such sensors can be calibrated using two calibrators only.     

A novel electrochemical detection method for aptamer biosensors

A beacon aptamer-based biosensor for the detection of thrombin was developed using electrochemical transduction method. Gold surface was modified with a beacon aptamer covalently linked at 5'-terminus with a linker containing a primary aliphatic amine. Methylene blue (MB) was intercalated into the beacon sequence, and used as an electrochemical marker. When the beacon aptamer immobilized on gold surface encounters thrombin, the hairpin forming beacon aptamer is conformationally changed to release the intercalated MB, resulting a decrease in electrical current intensity in voltamogram. The peak signal of the MB is clearly decreased by the binding of thrombin onto the beacon aptamer. The linear range of the signal was observed between 0 and 50.8 nM of thrombin with 0.999 correlation factor. This method was able to linearly and selectively detect thrombin with a detection limit of 11 nM.     

Microbial alpha-galactosidase (a review)

The review discusses properties, distribution and potential use of microbial alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22), the enzyme catalyzing degradation of alpha-D-galactoside bonds. Recent years have witnessed many publications describing microbial alpha-galactosidase which, in contrast to the similar enzyme from higher plants, has been poorly studied. The microbial enzyme has certain specificities: a smaller substrate specificity, existence in one molecular form, etc. The present communication is an attempt to systematize the data about microbial alpha-galactosidase and to outline the most important investigations for the future.     

Microbial and cytoplasmic membrane-based potentiometric biosensors for direct determination of organophosphorus insecticides

Potentiometric biosensors for the determination of organophosphorus (OP) insecticides were developed by applying either immobilized whole cells or cytoplasmic membrane fractions of wild-type Flavobacterium sp. on the surface of a glass pH electrode. The ability of Flavobacterium sp. to degrade OP compounds as sole carbon source was demonstrated for parathion with a degradation rate of almost 100% after 30 min and for chlorpyrifos of 33% after 48 h incubation. The products of hydrolysis of these compounds, p-nitrophenol and 3,5,6-trichloro-2-pyridinol, were accumulated in the medium and not used as substrates for growth by Flavobacterium sp. In the course of hydrolysis, which is catalyzed by organophosphorus hydrolase, two protons are released for each substrate molecule hydrolyzed. This stoichiometry forms the electrochemical basis of the potentiometric biosensors. Direct determination without previous extraction of OP was carried out in a stirred measuring cell with a pH electrode as transducer. Poly(carbamoyl sulfonate) (PCS) prepolymer, a hydrogel with good adhesive properties, was used for immobilization of whole cells and membrane-associated organophosphorus hydrolase. The sensor with cytoplasmic membrane fractions was superior to the one with whole cells and showed a linear range for paraoxon from 0.01 to 0.47 mM and 3 weeks' working stability. Received: 11 February 2000 / Received revision: 25 May 2000 / Accepted: 26 May 2000     

Novel biosensors for the detection of estrogen receptor ligands

There exists a significant need for the detection of novel estrogen receptor (ER) ligands for pharmaceutical uses, especially for treating complications associated with menopause. We have developed fluorescence resonance energy transfer (FRET)-based biosensors that permit the direct in vitro detection of ER ligands. These biosensors contain an ER ligand-binding domain (LBD) flanked by the FRET donor fluorophore, cyan fluorescent protein (CFP), and the acceptor fluorophore, yellow fluorescent protein (YFP). The ER-LBD has been modified so that Ala 430 has been changed to Asp, which increases the magnitude of the FRET signal in response to ligand-binding by more than four-fold compared to the wild-type LBD. The binding of agonists can be distinguished from that of antagonists on the basis of the distinct ligand-induced conformations in the ER-LBD. The approach to binding equilibrium occurs within 30min, and the FRET signal is stable over 24h. The biosensor demonstrates a high signal-to-noise, with a Z' value (a statistical determinant of assay quality) of 0.72. The affinity of the ER for different ligands can be determined using a modified version of the biosensor in which a truncated YFP and an enhanced CFP are used. Thus, we have developed platforms for high-throughput screens for the identification of novel estrogen receptor ligands. Moreover, we have demonstrated that this FRET technology can be applied to other nuclear receptors, such as the androgen receptor.     

Fully reversible optical biosensors for uric acid using oxygen transduction

An optical biosensor is presented for continuous determination of uric acid. The scheme is based on the measurement of the consumption of oxygen during the oxidation of uric acid that is catalyzed by the enzyme uricase. The enzyme is immobilized in a polyurethane hydrogel next to a metal-organic probe whose fluorescence is quenched by oxygen. The consumption of oxygen was followed by measurement of changes of luminescence intensity of two kind of probes and can be related to the concentration of uric acid. Analytical ranges (0-2mM), the response times (80-100s), reproducibility, and long-term stability were investigated. The biosensors are stable for at least 1 month and are not interfered by common interferents. One kind of biosensor was applied to the determination of uric acid in human blood serum. The results agree with those of a commercial colorimetric detection kit.