Electrochemical As(III) whole-cell based biochip sensor.

The development of a whole-cell based sensor for arsenite detection coupling biological engineering and electrochemical techniques is presented. This strategy takes advantage of the natural Escherichia coli resistance mechanism against toxic arsenic species, such as arsenite, which consists of the selective intracellular recognition of arsenite and its pumping out from the cell. A whole-cell based biosensor can be produced by coupling the intracellular recognition of arsenite to the generation of an electrochemical signal. Hereto, E. coli was equipped with a genetic circuit in which synthesis of beta-galactosidase is under control of the arsenite-derepressable arsR-promoter. The E. coli reporter strain was filled in a microchip containing 16 independent electrochemical cells (i.e. two-electrode cell), which was then employed for analysis of tap and groundwater samples. The developed arsenic-sensitive electrochemical biochip is easy to use and outperforms state-of-the-art bacterial bioreporters assays specifically in its simplicity and response time, while keeping a very good limit of detection in tap water, i.e. 0.8ppb. Additionally, a very good linear response in the ranges of concentration tested (0.94ppb to 3.75ppb, R(2)=0.9975 and 3.75 ppb to 30ppb, R(2)=0.9991) was obtained, complying perfectly with the acceptable arsenic concentration limits defined by the World Health Organization for drinking water samples (i.e. 10ppb). Therefore, the proposed assay provides a very good alternative for the portable quantification of As (III) in water as corroborated by the analysis of natural groundwater samples from Swiss mountains, which showed a very good agreement with the results obtained by atomic absorption spectroscopy.

Activation of the heat shock response in plants by chlorophenols: transgenic Physcomitrella patens as a sensitive biosensor for organic pollutants.

The ability to detect early molecular responses to various chemicals is central to the understanding of biological impact of pollutants in a context of varying environmental cues. To monitor stress responses in a model plant, we used transgenic moss Physcomitrella patens expressing the beta-glucuronidase reporter (GUS) under the control of the stress-inducible promoter hsp17.3B. Following exposure to pollutants from the dye and paper industry, GUS activity was measured by monitoring a fluorescent product. Chlorophenols, heavy metals and sulphonated anthraquinones were found to specifically activate the hsp17.3B promoter (within hours) in correlation with long-term toxicity effects (within days). At mildly elevated physiological temperatures, the chemical activation of this promoter was strongly amplified, which considerably increased the sensitivity of the bioassay. Together with the activation of hsp17.3B promoter, chlorophenols induced endogenous chaperones that transiently protected a recombinant thermolabile luciferase (LUC) from severe heat denaturation. This sensitive bioassay provides an early warning molecular sensor to industrial pollutants under varying environments, in anticipation to long-term toxic effects in plants. Because of the strong cross-talk between abiotic and chemical stresses that we find, this P. patens line is more likely to serve as a direct toxicity bioassay for pollutants combined with environmental cues, than as an indicator of absolute toxicity thresholds for various pollutants. It is also a powerful tool to study the role of heat shock proteins (HSPs) in plants exposed to combined chemical and environmental stresses.

Compact portable biosensor for arsenic detection in aqueous samples with Escherichia coli bioreporter cells.

We present a compact portable biosensor to measure arsenic As(III) concentrations in water using Escherichia coli bioreporter cells. Escherichia coli expresses green fluorescent protein in a linearly dependent manner as a function of the arsenic concentration (between 0 and 100 μg/L). The device accommodates a small polydimethylsiloxane microfluidic chip that holds the agarose-encapsulated bacteria, and a complete optical illumination/collection/detection system for automated quantitative fluorescence measurements. The device is capable of sampling water autonomously, controlling the whole measurement, storing and transmitting data over GSM networks. We demonstrate highly reproducible measurements of arsenic in drinking water at 10 and 50 μg/L within 100 and 80 min, respectively.

Development of a microfluidics biosensor for agarose-bead immobilized Escherichia coli bioreporter cells for arsenite detection in aqueous samples.

Contamination with arsenic is a recurring problem in both industrialized and developing countries. Drinking water supplies for large populations can have concentrations much higher than the permissible levels (for most European countries and the United States, 10 μg As per L; elsewhere, 50 μg As per L). Arsenic analysis requires high-end instruments, which are largely unavailable in developing countries. Bioassays based on genetically engineered bacteria have been proposed as suitable alternatives but such tests would profit from better standardization and direct incorporation into sensing devices. The goal of this work was to develop and test microfluidic devices in which bacterial bioreporters could be embedded, exposed and reporter signals detected, as a further step towards a complete miniaturized bacterial biosensor. The signal element in the biosensor is a nonpathogenic laboratory strain of Escherichia coli, which produces a variant of the green fluorescent protein after contact to arsenite and arsenate. E. coli bioreporter cells were encapsulated in agarose beads and incorporated into a microfluidic device where they were captured in 500 × 500 μm(2) cages and exposed to aqueous samples containing arsenic. Cell-beads frozen at -20 °C in the microfluidic chip retained inducibility for up to a month and arsenic samples with 10 or 50 μg L(-1) could be reproducibly discriminated from the blank. In the 0-50 μg L(-1) range and with an exposure time of 200 minutes, the rate of signal increase was linearly proportional to the arsenic concentration. The time needed to reliably and reproducibly detect a concentration of 50 μg L(-1) was 75-120 minutes, and 120-180 minutes for a concentration of 10 μg L(-1).

An ultra-sensitive impedimetric immunosensor for detection of the serum oncomarker CA-125 in ovarian cancer patients.

Effective treatment of ovarian cancer depends upon the early detection of the malignancy. Here, we report on the development of a new nanostructured immunosensor for early detection of cancer antigen 125 (CA-125). A gold electrode was modified with mercaptopropionic acid (MPA), and then consecutively conjugated with silica coated gold nanoparticles (AuNP@SiO2), CdSe quantum dots (QDs) and anti-CA-125 monoclonal antibody (mAb). The engineered MPA|AuNP@SiO2|QD|mAb immunosensor was characterised using transmission electron microscopy (TEM), atomic force microscopy (AFM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Successive conjugation of AuNP@SiO2, CdSe QD and anti-CA-125 mAb onto the gold electrode resulted in sensitive detection of CA-125 with a limit of detection (LOD) of 0.0016 U mL(-1) and a linear detection range (LDR) of 0-0.1 U mL(-1). Based on the high sensitivity and specificity of the immunosensor, we propose this highly stable and reproducible biosensor for the early detection of CA-125.

Measurement of biologically available naphthalene in gas and aqueous phases by use of a Pseudomonas putida biosensor.

Genetically constructed microbial biosensors for measuring organic pollutants are mostly applied in aqueous samples. Unfortunately, the detection limit of most biosensors is insufficient to detect pollutants at low but environmentally relevant concentrations. However, organic pollutants with low levels of water solubility often have significant gas-water partitioning coefficients, which in principle makes it possible to measure such compounds in the gas rather than the aqueous phase. Here we describe the first use of a microbial biosensor for measuring organic pollutants directly in the gas phase. For this purpose, we reconstructed a bioluminescent Pseudomonas putida naphthalene biosensor strain to carry the NAH7 plasmid and a chromosomally inserted gene fusion between the sal promoter and the luxAB genes. Specific calibration studies were performed with suspended and filter-immobilized biosensor cells, in aqueous solution and in the gas phase. Gas phase measurements with filter-immobilized biosensor cells in closed flasks, with a naphthalene-contaminated aqueous phase, showed that the biosensor cells can measure naphthalene effectively. The biosensor cells on the filter responded with increasing light output proportional to the naphthalene concentration added to the water phase, even though only a small proportion of the naphthalene was present in the gas phase. In fact, the biosensor cells could concentrate a larger proportion of naphthalene through the gas phase than in the aqueous suspension, probably due to faster transport of naphthalene to the cells in the gas phase. This led to a 10-fold lower detectable aqueous naphthalene concentration (50 nM instead of 0.5 micro M). Thus, the use of bacterial biosensors for measuring organic pollutants in the gas phase is a valid method for increasing the sensitivity of these valuable biological devices.

A new green fluorescent protein-based bacterial biosensor for analysing phenanthrene fluxes.

The polycyclic aromatic hydrocarbon (PAH)-degrading strain Burkholderia sp. RP007 served as host strain for the design of a bacterial biosensor for the detection of phenanthrene. RP007 was transformed with a reporter plasmid containing a transcriptional fusion between the phnS putative promoter/operator region and the gene encoding the enhanced green fluorescent protein (GFP). The resulting bacterial biosensor--Burkholderia sp. strain RP037--produced significant amounts of GFP after batch incubation in the presence of phenanthrene crystals. Co-incubation with acetate did not disturb the phenanthrene-specific response but resulted in a homogenously responding population of cells. Active metabolism was required for induction with phenanthrene. The magnitude of GFP induction was influenced by physical parameters affecting the phenanthrene flux to the cells, such as the contact surface area between solid phenanthrene and the aqueous phase, addition of surfactant, and slow phenanthrene release from Model Polymer Release System beads or from a water-immiscible oil. These results strongly suggest that the bacterial biosensor can sense different phenanthrene fluxes while maintaining phenanthrene metabolism, thus acting as a genuine sensor for phenanthrene bioavailability. A relationship between GFP production and phenanthrene mass transfer is proposed.

A fluorescent hormone biosensor reveals the dynamics of jasmonate signalling in plants.

Activated forms of jasmonic acid (JA) are central signals coordinating plant responses to stresses, yet tools to analyse their spatial and temporal distribution are lacking. Here we describe a JA perception biosensor termed Jas9-VENUS that allows the quantification of dynamic changes in JA distribution in response to stress with high spatiotemporal sensitivity. We show that Jas9-VENUS abundance is dependent on bioactive JA isoforms, the COI1 co-receptor, a functional Jas motif and proteasome activity. We demonstrate the utility of Jas9-VENUS to analyse responses to JA in planta at a cellular scale, both quantitatively and dynamically. This included using Jas9-VENUS to determine the cotyledon-to-root JA signal velocities on wounding, revealing two distinct phases of JA activity in the root. Our results demonstrate the value of developing quantitative sensors such as Jas9-VENUS to provide high-resolution spatiotemporal data about hormone distribution in response to plant abiotic and biotic stresses.

Multi-analyte detection for biological fluids. Towards continous monitoring of glucose, ionized calcium and pH using a viscometric affinity biosensor.

We present a viscometric affinity biosensor that can potentially allow continuous multi-analyte monitoring in biological fluids like blood or plasma. The sensing principle is based on the detection of viscosity changes of a polymeric solution which has a selective affinity for the analyte of interest. The chemico-mechanical sensor incorporates an actuating piezoelectric diaphragm, a sensing piezoelectric diaphragm and a flow-resisting microchannel for viscosity detection. A free-standing Anodic Alumina Oxide (AAO) porous nano-membrane is used as selective interface. A glucose-sensitive sensor was fabricated and extensively assessed in buffer solution. The sensor reversibility, stability and sensitivity were excellent during at least 65 hours. Results showed also a good degree of stability for a long term measurement (25 days). The sensor behaviour was furthermore tested in fetal bovine serum (FBS). The obtained results for glucose sensing are very promising, indicating that the developed sensor is a candidate for continuous monitoring in biological fluids. Sensitive solutions for ionized calcium and pH are currently under development and should allow multi-analyte sensing in the near future.

Miniaturized bacterial biosensor system for arsenic detection holds great promise for making integrated measurement device.

Combining bacterial bioreporters with microfluidics systems holds great promise for in-field detection of chemical or toxicity targets. Recently we showed how Escherichia coli cells engineered to produce a variant of green fluorescent protein after contact to arsenite and arsenate can be encapsulated in agarose beads and incorporated into a microfluidic chip to create a device for in-field detection of arsenic, a contaminant of well known toxicity and carcinogenicity in potable water both in industrialized and developing countries. Cell-beads stored in the microfluidics chip at -20°C retained inducibility up to one month and we were able to reproducibly discriminate concentrations of 10 and 50 μg arsenite per L (the drinking water standards for European countries and the United States, and for the developing countries, respectively) from the blank in less than 200 minutes. We discuss here the reasons for decreasing bioreporter signal development upon increased storage of cell beads but also show how this decrease can be reduced, leading to a faster detection and a longer lifetime of the device.

In vivo electrochemical corrosion study of a CoCrMo biomedical alloy in human synovial fluids.

The present study was initiated with the aim to assess the in vivo electrochemical corrosion behaviour of CoCrMo biomedical alloys in human synovial fluids in an attempt to identify possible patient or pathology specific effects. For this, electrochemical measurements (open circuit potential OCP, polarization resistance Rp, potentiodynamic polarization curves, electrochemical impedance spectroscopy EIS) were carried out on fluids extracted from patients with different articular pathologies and prosthesis revisions. Those electrochemical measurements could be carried out with outstanding precision and signal stability. The results show that the corrosion behaviour of CoCrMo alloy in synovial fluids not only depends on material reactivity but also on the specific reactions of synovial fluid components, most likely involving reactive oxygen species. In some patients the latter were found to determine the whole cathodic and anodic electrochemical response. Depending on patients, corrosion rates varied significantly between 50 and 750mgdm(-2)year(-1).

An automated microreactor for semi-continuous biosensor measurements.

Living bacteria or yeast cells are frequently used as bioreporters for the detection of specific chemical analytes or conditions of sample toxicity. In particular, bacteria or yeast equipped with synthetic gene circuitry that allows the production of a reliable non-cognate signal (e.g., fluorescent protein or bioluminescence) in response to a defined target make robust and flexible analytical platforms. We report here how bacterial cells expressing a fluorescence reporter ("bactosensors"), which are mostly used for batch sample analysis, can be deployed for automated semi-continuous target analysis in a single concise biochip. Escherichia coli-based bactosensor cells were continuously grown in a 13 or 50 nanoliter-volume reactor on a two-layered polydimethylsiloxane-on-glass microfluidic chip. Physiologically active cells were directed from the nl-reactor to a dedicated sample exposure area, where they were concentrated and reacted in 40 minutes with the target chemical by localized emission of the fluorescent reporter signal. We demonstrate the functioning of the bactosensor-chip by the automated detection of 50 μgarsenite-As l(-1) in water on consecutive days and after a one-week constant operation. Best induction of the bactosensors of 6-9-fold to 50 μg l(-1) was found at an apparent dilution rate of 0.12 h(-1) in the 50 nl microreactor. The bactosensor chip principle could be widely applicable to construct automated monitoring devices for a variety of targets in different environments.