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Whole-cell biosensors can form the basis of affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing, but to date the detection of analytes such as proteins that cannot easily diffuse across the cell membrane has been challenging. Here we developed a novel biosensing platform based on cell agglutination using an E. coli whole-cell biosensor surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design to detect a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations using straightforward design rules and a mathematical model. Finally, we re-engineer our whole-cell biosensor for the detection of a medically relevant biomarker by the display of two different nanobodies against human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for field-testing in the developing world, emergency or disaster relief sites, as well as routine medical testing and personalized medicine.

The catalytic properties and the inter-domain electron transfer of cellobiose dehydrogenase (CDH) from the ascomycete fungus Myriococcum thermophilum adsorbed on graphite and thiol (SAM) modified gold electrodes were investigated using cyclic voltammetry, flow injection amperometry and UV-Vis spectroelectrochemistry. The fabrication and characterisation of a noncompartmentalised, mediator and cofactor free glucose–oxygen biofuel cell well-operating in glucose-containing buffers and human blood serum was performed. The biofuel cell was based on adsorbed enzymes exhibiting direct bioelectrocatalysis, viz. CDH from Dichomera saubinetii and Corynascus thermophilus used as anodic bioelements and laccase (Lc) from Trametes hirsuta and bilirubin oxidase (BOD) from Myrothecium verrucaria used as cathodic bioelements, respectively. Different bacterial strains of E. coli and B. subtilis were immobilised on the electrode surface and tested for electrical communication using soluble and polymeric mediators. In the case of E. coli, the introduction of cytochromes in the inner membrane facilitated the electrochemical communication when using artificial mediators, while in the case of B. subtilis, the results demonstrated that mediators did not have to pass the cytosolic membrane to bring about an efficient electronic communication between bacterial cells with a thick cell wall and electrodes.

A rapid biological method for the determination of the bioavailability of naphthalene was developed and its value as an alternative to extraction-based chemical approaches demonstrated. Genetically engineered whole-cell biosensors are used to determine bioavailable naphthalene and their responses compared with results from Tenax extraction and chemical analysis. Results show a 1:1 correlation between biosensor results and chemical analyses for naphthalene-contaminated model materials and sediments, but the biosensor assay is much faster. This work demonstrates that biosensor technology can perform as well as standard chemical methods, though with some advantages including the inherent biological relevance of the response, rapid response time, and potential for field deployment. A survey of results from this work and the literature shows that bioavailability under non-equilibrium conditions nonetheless correlates well with Koc or K. A rationale is provided wherein chemical resistance is speculated to be operative.Whole-cell biosensing and mild extraction followed by chemical analysis quantify bioavailable naphthalene in sediment materials equally well.

The promoter probe mini-Tn5-luxAB-tet was used to create a luxAB transcriptional fusion responding to fluorene in the fluorene utilising bacterium Sphingomonas sp. LB126. The mutant strain, named L-132, was impaired in fluorene utilisation and strongly emitted light upon addition of fluorene to the growth medium. L-132 was initially characterised and examined for its potential use as a whole-cell biosensor in the perspective of quantifying fluorene in environmental samples. Activity of the reporter gene as a response to fluorene was detectable after 30 rain and was optimal after 4 h. A linear response to fluorene concentrations within the water solubility range was achieved, with a detection limit of 200 mug per litre. Besides fluorene, L-132 weakly responded to the polycyclic aromatic hydrocarbons phenanthrene and dibenzothiophene, whereas strong responses were obtained with 9-fluorenone, 9-hydroxyfluorene, phthalic acid and protocatechuic acid. The latter four compounds are metabolites formed in course of fluorene degradation, which suggested that a fluorene metabolite rather than fluorene itself was the true inducer of the luxAB fusion in L-132. (C) 2001 Editions scientifiques et medicales Elsevier SAS.