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Mediated bioelectrochemical system for biosensing the cell viability of Staphylococcus aureus
(2016)
Staphylococcus aureus is one of the most dangerous human pathogens and is the cause of numerous illnesses ranging from moderate skin infections to life-threatening diseases. Despite advances made in identifying microorganisms, rapid detection methods for the viability of bacteria are still missing. Here, we report a rapid electrochemical assay for cell viability combining the use of double redox mediators and multiwall carbon nanotubes-screen printed electrodes (MWCNTs-SPE), ferricyanide (FCN) and 2,6-dichlorophenolindophenol (DCIP), which served as electron shuttle to enable the bacterial-electrode communications. The current originating from the metabolically active cells was recorded for probing the activity of the intracellular redox centers. Blocking of the respiratory chain pathways with electron transfer inhibitors demonstrated the involvement of the electron transport chain in the reaction. A good correlation between the number of the metabolically active cells and the current was obtained. The proposed assay has been exploited for monitoring cell proliferation of S. aureus during the growth. The sensitivity of the detection method reached 0.1 OD600. Therefore, the technique described is promising for estimating the cell number, measuring the cell viability, and probing intracellular redox center(s).
Wastewater samples from a Swedish chemi-thermo-mechanical pulp (CTMP) mill collected at different purification stages in a wastewater treatment plant (WWTP) were analyzed with an amperometric enzyme-based biosensor array in a flow-injection system. In order to resolve the complex composition of the wastewater, the array consists of several sensing elements which yield a multidimensional response. We used principal component analysis (PCA) to decompose the array's responses, and found that wastewater with different degrees of pollution can be differentiated. With the help of partial least squares regression (PLS-R), we could link the sensor responses to the toxicity parameter, as well as to global organic pollution parameters (COD, BOD, and TOC). From investigating the influences of individual sensors in the array, it was found that the best models were in most cases obtained when all sensors in the array were included in the PLS-R model. We find that fast simultaneous determination of several global environmental parameters characterizing wastewaters is possible with this kind of biosensor array, in particular because of the link between the sensor responses and the biological effect onto the ecosystem into which the wastewater would be released. In conjunction with multivariate data analysis tools, there is strong potential to reduce the total time until a result is yielded from days to a few minutes.
For the first time a molecularly imprinted polymer (MIP) with direct electron transfer (DET) and bioelectrocatalytic activity of the target protein is presented. Thin films of MIPs for the recognition of a hexameric tyrosine-coordinated heme protein (HTHP) have been prepared by electropolymerization of scopoletin after oriented assembly of HTHP on a self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) on gold electrodes. Cavities which should resemble the shape and size of HTHP were formed by template removal. Rebinding of the target protein sums up the recognition by non-covalent interactions between the protein and the MIP with the electrostatic attraction of the protein by the SAM. HTHP bound to the MIP exhibits quasi-reversible DET which is reflected by a pair of well pronounced redox peaks in the cyclic voltammograms (CVs) with a formal potential of -184.4 +/- 13.7 mV vs. Ag/AgCl (1 M KCl) at pH 8.0 and it was able to catalyze the cathodic reduction of peroxide. At saturation the MIP films show a 12-fold higher electroactive surface concentration of HTHP than the non-imprinted polymer (NIP).
Phenothiazine-modified redox hydrogels were synthesized and used for the wiring of the aldehyde oxidoreductase PaoABC to electrode surfaces. The effects of the pH value and electrode surface modification on the biocatalytic activity of the layers were studied in the presence of vanillin as the substrate. The enzyme electrodes were successfully employed as bioanodes in vanillin/O-2 biofuel cells in combination with a high potential bilirubin oxidase biocathode. Open circuit voltages of around 700 mV could be obtained in a two compartment biofuel cell setup. Moreover, the use of a rather hydrophobic polymer with a high degree of crosslinking sites ensures the formation of stable polymer/enzyme films which were successfully used as bioanode in membrane-less biofuel cells. (C) 2015 Elsevier B.V. All rights reserved.
The creation of electron transfer (ET) chains based on the defined arrangement of enzymes and redox proteins on electrode surfaces represents an interesting approach within the field of bioelectrocatalysis. In this study, we investigated the ET reaction of the flavin-dependent enzyme fructose dehydrogenase (FDH) with the redox protein cytochrome c (cyt c). Two different pH optima were found for the reaction in acidic and neutral solutions. When cyt c was adsorbed on an electrode surface while the enzyme remained in solution, ET proceeded efficiently in media of neutral pH. Interprotein ET was also observed in acidic media; however, it appeared to be less efficient. These findings suggest that two different ET pathways between the enzyme and cyt c may occur. Moreover, cyt c and FDH were immobilized in multiple layers on an electrode surface by means of another biomacromolecule: DNA (double stranded) using the layer -by -layer technique. The biprotein multilayer architecture showed a catalytic response in dependence on the fructose concentration, indicating that the ET reaction between both proteins is feasible even in the immobilized state. The electrode showed a defined response to fructose and a good storage stability. Our results contribute to the better understanding of the ET reaction between FDH and cyt c and provide the basis for the creation of all-biomolecule based fructose sensors the sensitivity of which can be controlled by the layer preparation.
Microelectrodes modified with electropolymerized plumbagin (PLG) were used for the generation of superoxide radical (O-2(center dot-)) and hydrogen peroxide (H2O2) during oxygen reduction reaction (ORR) in an aqueous medium, specifically in serum-free cell culture media. This is enabled by the specific design of a polymer film on the microelectrode. The generation and diffusion of O-2(center dot-) during electrocatalytic ORR at a positionable PLG polymer-modified microelectrode was followed by fluorescence microscopy with the selective dye 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and by amperometric detection using a cytochrome c-modified electrode at + 0.13 V. H2O2 production, either by direct oxygen reduction or as product of O-2(center dot-) disproportionation, was monitored by the reaction with Amplex UltraRed. The PLG polymer-modified microelectrodes were used to expose mammalian B6-RPE07 retinal cells to defined local fluxes of reactive oxygen species (ROS), and cellular responses and morphological alterations were observed. The use of a controllable source of ROS opens many possibilities to study how living cells respond to the presence of a certain flux of specific ROS.