Fast fabrication of reusable polyethersulfone microbial biosensors through biocompatible phase separation.

In biosensors fabrication, entrapment in polymeric matrices allows efficient immobilization of the biorecognition elements without compromising their structure and activity. When considering living cells, the biocompatibility of both the matrix and the polymerization procedure are additional critical factors. Bio-polymeric gels (e.g. alginate) are biocompatible and polymerize under mild conditions, but they have poor stability. Most synthetic polymers (e.g. PVA), on the other hand, present improved stability at the expense of complex protocols involving chemical/physical treatments that decrease their biological compatibility. In an attempt to explore new solutions to this problem we have developed a procedure for the immobilization of bacterial cells in polyethersulfone (PES) using phase separation. The technology has been tested successfully in the construction of a bacterial biosensor for toxicity assessment. Biosensors were coated with a 300  µm bacteria-containing PES membrane, using non-solvent induced phase separation (membrane thickness ≈ 300 µm). With this method, up to 2.3 × 106 cells were immobilized in the electrode surface with an entrapment efficiency of 8.2%, without compromising cell integrity or viability. Biosensing was performed electrochemically through ferricyanide respirometry, with metabolically-active entrapped bacteria reducing ferricyanide in the presence of glucose. PES biosensors showed good stability and reusability during dry frozen storage for up to 1 month. The analytical performance of the sensors was assessed carrying out a toxicity assay in which 3,5-dichlorophenol (DCP) was used as a model toxic compound. The biosensor provided a concentration-dependent response to DCP with half-maximal effective concentration (EC50) of 9.2 ppm, well in agreement with reported values. This entrapment methodology is susceptible of mass production and allows easy and repetitive production of robust and sensitive bacterial biosensors.

Electro-addressable conductive alginate hydrogel for bacterial trapping and general toxicity determination.

In biosensors development, alginate hydrogels are a first choice for enabling stable biomolecules entrapment in biocompatible membranes obtained under soft physiological conditions. Although widely exploited, most alginate membranes are isolating and poorly repetitive, which limit their application in biosensing. Significant steps forward on improving repeatability and conductivity have been performed, but to date there is no single protocol for controlled deposition of live cells in replicable conductive alginate layers. Here, cell electrotrapping in conductive alginate hydrogels is examined in order to overcome these limitations. Conductive alginate-coated electrodes are obtained after potentiostatic electrodeposition of graphite-doped alginate samples (up to 4% graphite). The presence of graphite reduces electrode passivation and improves the electrochemical response of the sensor, although still significantly lower than that recorded with the naked electrode. Bacterial electrotrapping in the conductive matrix is highly efficient (4.4 × 107 cells per gel) and repetitive (CV < 0.5%), and does not compromise bacterial integrity or activity (cell viability = 56%). Biosensing based on ferricyanide respirometry yielded a four times increase in biosensor response with respect to non-conductive alginate membrane, providing toxicity values completely comparable to those reported. Cell electrotrapping in conductive hydrogels represents a step forward towards in high-sensitive cell-based biosensors development with important influence in environmental analysis, food and beverage industry as well as clinical diagnosis.

Paper-based chromatic toxicity bioassay by analysis of bacterial ferricyanide reduction.

Water quality assessment requires a continuous and strict analysis of samples to guarantee compliance with established standards. Nowadays, the increasing number of pollutants and their synergistic effects lead to the development general toxicity bioassays capable to analyse water pollution as a whole. Current general toxicity methods, e.g. Microtox(®), rely on long operation protocols, the use of complex and expensive instrumentation and sample pre-treatment, which should be transported to the laboratory for analysis. These requirements delay sample analysis and hence, the response to avoid an environmental catastrophe. In an attempt to solve it, a fast (15 min) and low-cost toxicity bioassay based on the chromatic changes associated to bacterial ferricyanide reduction is here presented. E. coli cells (used as model bacteria) were stably trapped on low-cost paper matrices (cellulose-based paper discs, PDs) and remained viable for long times (1 month at -20 °C). Apart from bacterial carrier, paper matrices also acted as a fluidic element, allowing fluid management without the need of external pumps. Bioassay evaluation was performed using copper as model toxic agent. Chromatic changes associated to bacterial ferricyanide reduction were determined by three different transduction methods, i.e. (i) optical reflectometry (as reference method), (ii) image analysis and (iii) visual inspection. In all cases, bioassay results (in terms of half maximal effective concentrations, EC50) were in agreement with already reported data, confirming the good performance of the bioassay. The validation of the bioassay was performed by analysis of real samples from natural sources, which were analysed and compared with a reference method (i.e. Microtox). Obtained results showed agreement for about 70% of toxic samples and 80% of non-toxic samples, which may validate the use of this simple and quick protocol in the determination of general toxicity. The minimum instrumentation requirements and the simplicity of the bioassay open the possibility of in-situ water toxicity assessment with a fast and low-cost protocol.

Fast and sensitive optical toxicity bioassay based on dual wavelength analysis of bacterial ferricyanide reduction kinetics.

Global urban and industrial growth, with the associated environmental contamination, is promoting the development of rapid and inexpensive general toxicity methods. Current microbial methodologies for general toxicity determination rely on either bioluminescent bacteria and specific medium solution (i.e. Microtox(®)) or low sensitivity and diffusion limited protocols (i.e. amperometric microbial respirometry). In this work, fast and sensitive optical toxicity bioassay based on dual wavelength analysis of bacterial ferricyanide reduction kinetics is presented, using Escherichia coli as a bacterial model. Ferricyanide reduction kinetic analysis (variation of ferricyanide absorption with time), much more sensitive than single absorbance measurements, allowed for direct and fast toxicity determination without pre-incubation steps (assay time=10 min) and minimizing biomass interference. Dual wavelength analysis at 405 (ferricyanide and biomass) and 550 nm (biomass), allowed for ferricyanide monitoring without interference of biomass scattering. On the other hand, refractive index (RI) matching with saccharose reduced bacterial light scattering around 50%, expanding the analytical linear range in the determination of absorbent molecules. With this method, different toxicants such as metals and organic compounds were analyzed with good sensitivities. Half maximal effective concentrations (EC50) obtained after 10 min bioassay, 2.9, 1.0, 0.7 and 18.3 mg L(-1) for copper, zinc, acetic acid and 2-phenylethanol respectively, were in agreement with previously reported values for longer bioassays (around 60 min). This method represents a promising alternative for fast and sensitive water toxicity monitoring, opening the possibility of quick in situ analysis.