On-line and in situ biosensors for monitoring environmental pollution

Efficient tools for on-line and in situ monitoring of environmental pollutants are required to provide early warning systems. In addition, such tools can contribute important information on the progress of various remediation treatments. One of the recently developed monitoring technologies involves the use of whole-cell biosensors. Such biosensors could be constructed to detect general toxicity or specific toxicity caused by one or more pollutants. Currently, a large spectrum of microbial biosensors have been developed that enable the monitoring of pollutants by measuring light, fluorescence, color or electric current. Electrochemical monitoring is of special interest for in situ measurements as it can be performed using simple, compact and mobile equipment and is easily adaptable for on-line measurements. Here we survey the potential application of electrochemical biosensors in monitoring of general toxicity as well as hydrocarbons and heavy metals.     

Development of a novel,bioluminescence-based,fungal bioassay for toxicity testing

Naturally bioluminescent fungi, Armillaria mellea and Mycena citricolor, were used to develop a novel, bioluminescence-based bioassay for toxicity testing. Bioassays were carried out to assess the toxicity of 3,5-dichlorophenol (3,5-DCP), pentachlorophenol (PCP), copper and zinc. The results suggested that 60 min was a suitable exposure time for the bioassay. Light reduction was observed in response to 3,5-DCP, PCP and Cu for both A. mellea and M. citricolor, but to Zn only for A. mellea. Armillaria mellea was significantly less sensitive to 3,5-DCP and PCP than M. citricolor. The EC50 values for A. mellea and M. citricolor were similar to EC50 values for 3,5-DCP, PCP and Cu (but not Zn) of bioluminescence-based bacterial biosensors. They were also similar to EC50 values for Cu and Zn of a bioluminescence-based yeast biosensor. The results highlighted the importance of using both prokaryotic and eukaryotic biosensors. The novel bioassay provides a rapid and sensitive method to assess bioavailability of pollutants as well as a method to determine their toxicity to filamentous fungi. It also expands the range of organisms that can be used for bioluminescence-based toxicity testing by complementing existing biosensors.     

Genetically engineered microbial biosensors for in situ monitoring of environmental pollution

Microbial biosensors are compact, portable, cost effective, and simple to use, making them seem eminently suitable for the in situ monitoring of environmental pollution. One promising approach for such applications is the fusion of reporter genes with regulatory genes that are dose-dependently responsive to the target chemicals or physiological signals. Their biosensor capabilities, such as target range and sensitivity, could be improved by modification of regulatory genes. Recent uses of such genetically engineered microbial biosensors include the development of portable biosensor kits and high-throughput cell arrays on chips, optic fibers, or other platforms for on-site and on-line monitoring of environmental pollution. This mini-review discusses recent advances in microbial biosensors and their future prospects, with a focus on the development and application of genetically modified microbial biosensors for in situ environmental monitoring.     

Modeling and measurement of a whole-cell bioluminescent biosensor based on a single photon avalanche diode

Whole-cell biosensors are potential candidates for on-line and in situ environmental monitoring. In this work we present a new design of a whole-cell bioluminescence biosensor for water toxicity detection, based on genetically engineered Escherichia coli bacteria, carrying a recA::luxCDABE promoter-reporter fusion. Sensitive optical detection is achieved using a single photon avalanche photodiode (SPAD) working in the Geiger mode. The present work describes a simple mathematical model for the kinetic process of the bioluminescence based SOS toxin response of E. coli bacteria. We find that initially the bioluminescence signal depends on the time square and we show that the spectral intensity of the bioluminescence signal is inverse proportional to the frequency. We get excellent agreement between the theoretical model and the measured light signal. Furthermore, we present experimental results of the bioluminescent signal measurement using a SPAD and a photomultiplier, and demonstrate improvement of the measurement by applying a matched digital filter. Low intensity bioluminescence signals were measured after the whole-cell sensors were exposed to various toxicant concentrations (5, 15 and 20ppm).     

Electrochemical DNA biosensor for screening of chlorinated benzene pollutants

In this work, an electrochemical DNA biosensor based on double-stranded DNA modified Au electrode (dsDNA/Au) was proposed for the rapid screening and detection of chlorinated benzenes pollutants, in which redox-active methylene blue (MB) was used to amplify the interaction between dsDNA and the target analyte. Using hexachlorobenzene (HCB) as a model analyte of chlorinated benzenes, the biosensor demonstrated a linear response with the logarithm of HCB concentrations from 100 pmol L(-1) to 100 nmol L(-1). The obtained detection limit was 30 pmol L(-1), which was remarkably superior to other biosensors. The interaction mechanism of the biosensor with HCB was proposed based on systematical characterization by cyclic voltammetry (CV), differential pulse voltammetry (DPV), UV-vis spectrometry and electrochemical quartz crystal microbalance (EQCM). Further studies revealed that the biosensor could screen chlorinated benzenes in the presence of 100 fold amount of other co-existing chemicals (ethyl acetate and sodium oxalate, etc.), and the response signal of the biosensors for different chlorinated benzenes was correlative to their respective toxicity. The proposed biosensor proved to be a promising "alarm" tool for rapid screening of chlorinated benzenes in real water samples.     

Microbial biosensors.

A microbial biosensor consists of a transducer in conjunction with immobilised viable or non-viable microbial cells. Non-viable cells obtained after permeabilisation or whole cells containing periplasmic enzymes have mostly been used as an economical substitute for enzymes. Viable cells make use of the respiratory and metabolic functions of the cell, the analyte to be monitored being either a substrate or an inhibitor of these processes. Bioluminescence-based microbial biosensors have also been developed using genetically engineered microorganisms constructed by fusing the lux gene with an inducible gene promoter for toxicity and bioavailability testing. In this review, some of the recent trends in microbial biosensors with reference to the advantages and limitations are been discussed. Some of the recent applications of microbial biosensors in environmental monitoring and for use in food, fermentation and allied fields have been reviewed. Prospective future microbial biosensor designs have also been identified.     

A novel design of multifunctional integrated cell-based biosensors for simultaneously detecting cell acidification and extracellular potential

The paper discussed a novel design of multifunctional cell-based biosensors for simultaneously detecting cell acidification and extracellular potential. Employing living cells such as cardiac myocytes as a source for the light addressable potentiometric sensor (LAPS) array, this cell-based biosensor was able to monitor both the acidification and extracellular potential in parallel. For LAPS array fabrication, part of the silicon base was heavily doped with boron to form separate testing areas. Detecting system was built involving lock-in amplifier and digital demodulation with FFT methods. This LAPS array showed a good sensitivity of 53.9 mV/pH to H(+) with good linearity. Each testing area for extracellular potential detection was decreased to 200 microm x 200 microm in size to obtain a better sensitivity. Experiment results showed that this LAPS array could monitor the acidification of cells as well as the extracellular potential with good sensitivity. This novel integrated biosensor will be useful for multi-parameter extracellular monitoring and can possibly be a platform for drug screening.     

Alginate beads as a storage, delivery and containment system for genetically modified PCB degrader and PCB biosensor derivatives of Pseudomonas fluorescens F113

Aims: Pseudomonas fluorescens F113Rifpcb is a genetically engineered rhizosphere bacterium with the potential to degrade polychlorinated biphenyls (PCBs). F113Rifpcbgfp and F113L::1180gfp are biosensor strains capable of detecting PCB bioavailability and biodegradation. The aim of this paper is to evaluate the use of alginate beads as a storage, delivery and containment system for use of these strains in PCB contaminated soils. Methods and Results: The survival and release of Ps. fluorescens F113Rifpcb from alginate beads were evaluated. Two Ps. fluorescens F113‐based biosensor strains were encapsulated, and their ability to detect 3‐chlorobenzoate (3‐CBA) and 3‐chlorobiphenyl (3‐CBP) degradation in soil was assessed. After 250 days of storage, 100% recovery of viable F113Rifpcb cells was possible. Amendments to the alginate formulation allowed for the timed release of the inoculant. Encapsulation of the F113Rifpcb cells provided a more targeted approach for the inoculation of plants and resulted in lower inoculum populations in the bulk soil, which may reduce the risk of unintentional spread of these genetically modified micro‐organisms in the environment. Encapsulation of the biosensor strains in alginate beads did not interfere with their ability to detect either 3‐CBA or 3‐CBP degradation. In fact, detection of 3‐CBP degradation was enhanced in encapsulated biosensors. Conclusions: Alginate beads are an effective storage and delivery system for PCB degrading inocula and biosensors. Significance and Impact of the Study: Pseudomonas fluorescens F113Rifpcb and the F113 derivative PCB biosensor strains have excellent potential for detecting and bioremediation of PCB contaminated soils. The alginate bead delivery system could facilitate the application of these strains as biosensors.     

A New,Sensitive Marine Microalgal Recombinant Biosensor Using Luminescence Monitoring for Toxicity Testing of Antifouling Biocides

In this study, we propose the use of the marine green alga Ostreococcus tauri, the smallest free-living eukaryotic cell known to date, as a new luminescent biosensor for toxicity testing in the environment. Diuron and Irgarol 1051, two antifouling biocides commonly encountered in coastal waters, were chosen to test this new biosensor along with two degradation products of diuron. The effects of various concentrations of the antifoulants on four genetic constructs of O. tauri (based on genes involved in photosynthesis, cell cycle, and circadian clock) were compared using 96-well culture microplates and a luminometer to automatically measure luminescence over 3 days. This was compared to growth inhibition of O. tauri wild type under the same conditions. Luminescence appeared to be more sensitive than growth inhibition as an indicator of toxicity. Cyclin-dependent kinase (CDKA), a protein involved in the cell cycle, fused to luciferase (CDKA-Luc) was found to be the most sensitive of the biosensors, allowing an accurate determination of the 50% effective concentration (EC₅₀) after only 2 days (diuron, 5.65 ± 0.44 μg/liter; Irgarol 1015, 0.76 ± 0.10 μg/liter). The effects of the antifoulants on the CDKA-Luc biosensor were then compared to growth inhibition in natural marine phytoplankton. The effective concentrations of diuron and Irgarol 1051 were found to be similar, indicating that this biosensor would be suitable as a reliable ecotoxicological test. The advantage of this biosensor over cell growth inhibition testing is that the process can be easily automated and could provide a high-throughput laboratory approach to perform short-term toxicity tests. The ability to genetically transform and culture recombinant O. tauri gives it huge potential for screening many other toxic compounds.     

A new helicase assay based on graphene oxide for anti-viral drug development

Recently, graphene oxide (GO), one of the carbon nanomaterials, has received much attention due to its unique physical and chemical properties and high potential in many research areas, including applications as a biosensor and drug delivery vehicle. Various GO-based biosensors have been developed, largely based on its surface adsorption properties for detecting biomolecules, such as nucleotides and peptides, and real-time monitoring of enzymatic reactions. In this review, we discuss recent advances in GO-based biosensors focusing on a novel assay platform for helicase activity, which was also employed in high-throughput screening to discover selective helicase inhibitors.     

3D-biohybrid systems: applications in drug screening

Biotechnology demands powerful methods for the functional characterisation and monitoring of molecular alterations in tissues in response to various stimuli. Currently, cellular biosensors provide information about cell and tissue internal transduction pathways. In this article, recent biosensor systems are briefly described and the use of 3D tissue aggregates as recognition elements is discussed. An example of an innovative approach for drug testing using 3D heart muscle aggregates, as well as tumor models, positioned in capillary systems for electrical potential recording and impedance measurement is described. The effectiveness of drugs and therapies can be tested and monitored in a short time using such biohybrid sensors.     

Bio-smart hydrogels: co-joined molecular recognition and signal transduction in biosensor fabrication and drug delivery

Two classes of polymers that are currently receiving widespread attention in biosensor development are hydrogels and conducting electroactive polymers. The present study reports on the integration of these two materials to produce electroactive hydrogel composites that physically entrap enzymes within their matrices for biosensor construction and chemically stimulated controlled release. Enhanced biosensing capabilities of these membranes have been demonstrated in the fabrication of glucose, cholesterol and galactose amperometric biosensors. All biosensors displayed extended linear response ranges (10(-5)-10(-2) M), rapid response times (<60 s), retained storage stabilities of up to 1 year, and excellent screening of the physiological interferents ascorbic acid, uric acid, and acetaminophen. When the cross-linked hydrogel components of these composite membranes were prepared with the amine containing dimethylaminoethyl methacrylate monomer the result was polymeric devices that swelled in response to pH changes (neutral to acidic). Entrapment of glucose oxidase within these materials made them glucose-responsive through the formation of gluconic acid. When insulin was co-loaded with glucose oxidase into these "bio-smart" devices, there was a twofold increase in insulin release rate when the devices were immersed in glucose solutions. This demonstrates the potential of such systems to function as a chemically-synthesized artificial pancreas.     

Strategies to develop malic acid biosensors based on malate quinone oxidoreductase (MQO)

An amperometric biosensor based on malate quinone oxidoreductase (MQO) was developed for monitoring of the malolactic fermentation of wines. Screen-printed electrodes coupled with appropriate mediators were used as transducers for this novel biosensor. MQO was immobilized by physical entrapment in a photo-cross-linkable poly(vinyl alcohol) polymer (PVA-SbQ) on the surface of the working electrode. Several electrochemical mediators were studied in order to lower the applied potential and minimise the matrix effects. Among them, 2,6-dichlorophenol indophenol (DPIP) and phenazine methosulfate (PMS) were chosen for further development. The working conditions (mediator concentration, applied potential and pH) were optimised for both DPIP and PMS. Detection limits for both types of biosensors were of 5 μM malic acid. Sensitivities obtained for the linear part of the calibration curve were 0.85 and 1.7 mA/M for the biosensors based on DPIP and PMS, respectively. Interferences due to non-specific oxidations were shown to be negligible when using PMS as mediator.     

Principles of affinity-based biosensors

Despite the amount of resources that have been invested by national and international academic, government, and commercial sectors to develop affinity-based biosensor products, little obvious success has been realized through commercialization of these devices for specific applications (such as the enzyme biosensors for blood glucose analysis). Nevertheless, the fastest growing area in the biosensors research literature continues to involve advances in affinity-based biosensors and biosensor-related methods. Numerous biosensor techniques have been reported that allow researchers to better study the kinetics, structure, and (solid/liquid) interface phenomena associated with protein-ligand binding interactions. In addition, potential application areas for which affinity-based biosensor techniques show promise include clinical/diagnostics, food processing, military/antiterrorism, and environmental monitoring. The design and structural features of these devices—composed of a biological affinity element interfaced to a signal transducer—primarily determine their operational characteristics. This paper although not intended as a comprehensive review, will outline the principles of affinity biosensors with respect to potential application areas.     

Fiber optic monooxygenase biosensor for toluene concentration measurement in aqueous samples

Measurements of pollutants such as toluene are critical for the characterization of contaminated sites and for the monitoring of remediation processes and wastewater treatment effluents. Fiber optic enzymatic biosensors have the potential to provide cost-effective, real time, continuous, in situ measurements. In this study, a fiber optic enzymatic biosensor was constructed and characterized for the measurement of toluene concentrations in aqueous solutions. The biological recognition element was toluene ortho-monooxygenase (TOM), expressed by Escherichia coli TG1 carrying pBS(Kan)TOM, while an optical fiber coated with an oxygen-sensitive ruthenium-based phosphorescent dye served as the transducer. Toluene was detected based on the enzymatic reaction catalyzed by TOM, which resulted in the consumption of oxygen and changes in the phosphorescence intensity. The biosensor was found to have a limit of detection of 3 μM, a linear signal range up to 100 μM, and a response time of 1 h. The performance was reproducible with different biosensors (RSD=7.4%, n=8). The biosensor activity declined with each measurement and with storage time, particularly at elevated temperatures. This activity loss could be partially reversed by exposure to formate, suggesting that NADH consumption was the primary factor limiting lifetime. This is the first report of an enzymatic toluene sensor and of an oxygenase-based biosensor. Since many oxygenases have been reported, the design concept of this oxygenase-based biosensor has the potential to broaden biosensor applications in environmental monitoring.     

Monitoring the dynamics of Src activity in response to anti-invasive dasatinib treatment at a subcellular level using dual intravital imaging

Optimising response to tyrosine kinase inhibitors in cancer remains an extensive field of research. Intravital imaging is an emerging tool, which can be used in drug discovery to facilitate and fine-tune maximum drug response in live tumors. A greater understanding of intratumoural delivery and pharmacodynamics of a drug can be obtained by imaging drug target-specific fluorescence resonance energy transfer (FRET) biosensors in real time. Here, we outline our recent work using a Src-FRET biosensor as a readout of Src activity to gauge optimal tyrosine kinase inhibition in response to dasatinib treatment regimens in vivo. By simultaneously monitoring both the inhibition of Src using FRET imaging, and the modulation of the surrounding extracellular matrix using second harmonic generation (SHG) imaging, we were able to show enhanced drug penetrance and delivery to live pancreatic tumors. We discuss the implications of this dual intravital imaging approach in the context of altered tumor-stromal interactions, while summarising how this approach could be applied to assess other combination strategies or tyrosine kinase inhibitors in a preclinical setting.     

Fabrication of a carbon fiber paper as the electrode and its application toward developing a sensitive unmediated amperometric biosensor

Carbon fiber paper (CFP), a material frequently used as the diffusion layer in fuel cells, was found recently to exhibit a potential as an electrode for the development of sensitive, unmediated biosensors. After nitrogen plasma treatment, the CFP exhibited a quasi-reversible behavior to the redox couple (e.g., ferricyanide) with an electron transfer rate constant of 7.2 × 10(-3)cms(-1). This rate constant is approximately double that of a Pt-electrode and is much higher than that of many carbon-based electrodes. The unmediated CFP-based tyrosinase biosensor fabricated for this study exhibited an optimal working potential and operating pH value of -0.2V and 6.5, respectively. Compared to other unmediated tyrosinase biosensors, the CFP-based tyrosinase biosensor offers a high sensitivity for the monitoring of phenolic compounds (17.8, 7.1, 5.2 and 3.7 μA μM(-1)cm(-2) for catechol, phenol, bisphenol and 3-aminophenol, respectively). The lowest detection limit for catechol, phenol, bisphenol and 3-aminophenol was 2, 5, 5 and 12 nM, respectively. Furthermore, this biosensor exhibited a good repeatability, a fast response time (around 10s), and a wide linear dynamic range of detection for phenolic compounds.     

Twenty years research in cholinesterase biosensors: from basic research to practical applications

Over the last decades, cholinesterase (ChE) biosensors have emerged as an ultra sensitive and rapid technique for toxicity analysis in environmental monitoring, food and quality control. These systems have the potential to complement or replace the classical analytical methods by simplifying or eliminating sample preparation protocols and making field testing easier and faster with significant decrease in costs per analysis. Over the years, engineering of more sensitive ChE enzymes, development of more reliable immobilization protocols and progress in the area of microelectronics could allow ChE biosensors to be competitive for field analysis and extend their applications to multianalyte screening, development of small, portable instrumentations for rapid toxicity testing, and detectors in chromatographic systems. In this paper, we will review the research efforts over the last 20 years in fabricating AChE biosensors and the recent trends and challenges encounter once the sensor is used outside research laboratory for in situ real sample applications. The review will discuss the generations of cholinesterase sensors with their advantages and limitations, the existing electrode configurations and fabrication techniques and their applications for toxicity monitoring. We will focus on low-cost electrochemical sensors and the approaches used for enzyme immobilization. Recent works for achieving high sensitivity and selectivity are also discussed.     

Development of an automated water toxicity biosensor using Thiobacillus ferrooxidans for monitoring cyanides in natural water for a water filtering plant

An on-line biosensor consisting of immobilized Thiobacillus ferrooxidans and an oxygen electrode was developed for automated monitoring of acute toxicity in water samples. T. ferrooxidans is an obligatory acidophilic, autotrophic bacterium and derives its energy by the oxidation of ferrous ion, elemental sulfur, and reduced sulfur compounds including metal sulfides. The assay is based on the monitoring of a current increase by addition of toxicoids, which is caused by the inhibition of bacterial respiration and decrease in oxygen consumption. Optimum cell number on the membrane was 5.0 x 10(8) cells. The steady-state current was obtained when concentration of FeSO4 was above 3.6 mM at pH 3. The sensor response of T. ferrooxidans immobilized membrane for 5.0 microM KCN was within an error of 10% for 30 membranes. A linear relationship was obtained at KCN concentration in the range of 0.5-3.0 microM in a flow-type monitoring system. Minimum detectable concentrations of KCN, Na2S, and NaN3 were 0.5, 1.2, and 0.07 microM, respectively. The monitoring system contained two biosensors and these sensors were cleaned with sulfuric acid (pH 1.5) twice a day. This treatment could remove fouling on microbial immobilized membrane by natural water and ferrous precipitation in the flow cell. This flow-type monitoring sensor was operated continuously for 5 months. Also, T. ferrooxidans immobilized membrane can be stored for one month at 4 degrees C when preserved with wet absorbent cotton under argon gas.     

Biosensors for phenolic compounds: The catechol as a substrate model

The behaviour of three different laccase-based graphite biosensors was studied in view of their use in agricultural or industrial waters polluted by phenolic compounds. Catechol was used as a substrate model. Laccase from Trametes versicolor was immobilized on one biosensor (type A electrode) by adsorption while, on the other two biosensor types, laccase was covalently bound through the carboxylic groups created on the graphite by means of treatment with an electric potential difference (type B electrode) or with nitric acid (type C electrode). In the latter two cases, hexamethylenediamine and glutaraldehyde were used as the spacer and the coupling agent, respectively. The extension of linear response range and the sensitivity and time stability of each biosensor type were investigated. The type C biosensor gave the best results and its electrochemical properties proved comparable to those reported by other authors.