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.
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•Cell entrapment by phase separation.•PES-coated electrodes containing immobilized bacteria.•Polyethersulfone bacteria biosensor for amperometric toxicity testing.
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.
Aquatic microorganisms have the ability to adhere onto any solid surface. They are able to re-organise as biofilms when environmental conditions change and put their life at risk. Biofilms allow ...bacteria to remain inside water pipes without being eliminated by biocides. Among other properties, biofilms are electrically insulating. Because of this, as they grow on a metal transducer surface, biofilms produce changes in the electrode–solution interface properties. These changes have been monitored using impedance measurements and microchips as electrical transducers. Biofilm formation has been characterised using on-chip gold working electrodes and the various growth phases have been related to specific impedance changes.
Measurements employing new and reused chips of bacterial and non-bacterial solutions have been performed. Although differences between new and reused chips have been found, both kinds of electrodes can be used to evaluate biofilm formation.
The effect of several biocides on biofilms has also been studied. The disinfecting properties of peroxides, strong acid/bases and alcohols have been compared as well as their ability to remove adhered substances from chip surfaces.
There is an increasing demand on alternatives methods to animal testing. Numerous health parameters have been already studied using in vitro devices able to mimic the essential functions of the ...organs, being the real-time monitoring and response to stimuli their main limitations. Regarding the health of the gut, the short chain fatty acids, and particularly acetate, have emerged as key biomarkers to evaluate gut healthiness and disease development, although the number of acetate biosensors is still very low. This article presents a microbial biosensor based on fully biocompatible materials which is able to detect acetate in aerobic conditions in the range between 11 and 50 mM, and without compromising the viability and function of either bacteria (>90% viability) or mammalian cells (>80% viability). The detection mechanism is based on the metabolism of acetate by Escherichia coli bacteria immobilized on the transducer surface. Ferricyanide is used as a redox mediator to transfer electrons from the acetate metabolism in the bacterial cells to the transducer. High bacterial concentrations are immobilized in the transducer surface (109 cfu mL−1) by electrodeposition of conductive alginate hydrogels doped with reduced graphene oxide. The results show successful outcomes to exploit bacteria as a biosensing tool, based on the use of inkjet printed transducers, biocompatible materials and cell entrapment technologies.
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•Escherichia coli uses acetate as carbon source in aerobic conditions.•Stable, biocompatible, conductive alginate hydrogel is electrodeposited on inkjet printed sensor's working electrode.•Immobilization of high bacterial concentrations of E. coli within alginate hydrogel.•Bacterial metabolism oxidizes acetate, enabling amperometric measurement of electron flow via ferricyanide reduction.•Successful proof-of-concept demonstration of bioelectrochemical acetate detection is performed.
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.
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•Electro-addressable conductive alginate hydrogels.•Live bacteria electrotrapping for biosensing.•Enhanced general toxicity assessment through ferricyanide respirometry.
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.
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•Paper-based microbial toxicity bioassay.•Chromatic analysis of bacterial ferricyanide reduction.•Low cost and simple analysis with minimum instrumentation.
This work describes the resolution of binary mixtures of microorganisms using electrochemical impedance spectroscopy (EIS) and artificial neural networks (ANNs) for the processing of data.
...Pseudomonas aeruginosa,
Staphylococcus aureus and
Saccharomyces cerevisiae were chosen as models for Gram-negative bacteria, Gram-positive bacteria and yeasts, respectively. In this study, best results were obtained when entering the imaginary component of the impedance at each frequency (strongly related to the capacitive elements of the electrical equivalent circuit) into backpropagation neural networks made up by two hidden layers. The optimal configuration of these layers respectively used the
radbas and the
logsig transfer functions with 4 or 6 neurons in the first hidden layer and 10 neurons in the second one. In all cases, good prediction ability was obtained with correlation coefficients better than 0.989 when comparing the predicted and the expected values for a set of six external test samples not used in the training process.
A miniaturized calorimeter, based on silicon integrated thermopile chips, has been developed for the determination of growth-related heat production in microbial cultures. The calorimetric vessels ...consists of two independent sensors located within a thermostated aluminum frame, the heat sink, and covered by a 0.6
ml reaction chamber made of PTFE for improved thermal insulation. The second sensor was used as a reference to minimize temperature perturbations on the output signal. Baseline stability was better than 0.08
μW
h
−1. The Si thin-film membrane which supports the Al–Si thermopiles enabled an excellent dynamic response and a temperature resolution of 50
μK. The sensitivity for the heat power measurement was 0.39
V
W
−1. Batch measurements of
Escherichia coli activity under different conditions have been performed. The thermal profiles matched the exponential growth kinetics usually found in batch cultures of bacteria. A simplified model based in the Monod equation is used to analyze the influence of oxygen depletion on cell growth.
This paper describes an approach for quantifying low concentrations of bacteria, particularly Escherichia coli, based on the measurement of the initial attachment of bacteria to platinum surfaces, ...using impedance spectroscopy. The value of the interface capacitance in the pre-attachment stage (before 1min of attachment) showed correlation with suspended concentration of bacteria from 101 to 107CFUmL−1 (colony forming units per mL). This method was found to be sensitive to the attachment time, to the applied potential and to the size of the counter electrode. The sensor lifetime was also evaluated.
The detection of living organisms at very low concentrations is necessary for the early diagnosis of bacterial infections, but it is still challenging as there is a need for signal amplification. ...Cell culture, nucleic acid amplification, or nanostructure-based signal enhancement are the most common amplification methods, relying on long, tedious, complex, or expensive procedures. Here, we present a cyanotype-based photochemical amplification reaction enabling the detection of low bacterial concentrations up to a single-cell level. Photocatalysis is induced with visible light and requires bacterial metabolism of iron-based compounds to produce Prussian Blue. Bacterial activity is thus detected through the formation of an observable blue precipitate within 3 h of the reaction, which corresponds to the concentration of living organisms. The short time-to-result and simplicity of the reaction are expected to strongly impact the clinical diagnosis of infectious diseases.
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