Dmitri Ivnitski

Biosensors for detection of pathogenic bacteria

Abstract This paper presents an overview of different physicochemical instrumental techniques for direct and indirect identification of bacteria such as: infrared and fluorescence spectroscopy, flow cytometry, chromatography and chemiluminescence techniques as a basis for biosensor construction. A discussion of publications dealing with emerging biosensors for bacterial detection is presented. The review presents recent advances in the development of alternative enzyme- and immunosensors for detection of pathogenic bacteria in a variety of fields (e.g. clinical diagnostics, food analysis and environmental monitoring). Depending on the biological element employed: enzyme; nucleic acid and antibody based biosensors are discussed. Depending on the basic transducer principles, recent advances in biosensing technologies that use electrochemical, piezoelectric, optical, acoustic and thermal biosensors for detection of pathogenic bacteria are overviewed. Special attention is paid to methods for improving the analytical parameters of biosensors including sensitivity and analysis time as well as automation of assay procedures. Recent developments in immunofiltration, flow-injection and flow-through biosensors for bacterial detection are overviewed from the system’s engineering point of view. Future directions for biosensor development and problems related to the commercialization of bacterial biosensors are discussed in the final part of this review.

Entrapment of Enzymes and Carbon Nanotubes in Biologically Synthesized Silica: Glucose Oxidase-Catalyzed Direct Electron Transfer

This work demonstrates a new approach for building bioinorganic interfaces by integrating biologically derived silica with single-walled carbon nanotubes to create a conductive matrix for immobilization of enzymes. Such a strategy not only allows simple integration into biodevices but presents an opportunity to intimately interface an enzyme and manifest direct electron transfer features. Biologically synthesized silica/carbon nanotube/enzyme composites are evaluated electrochemically and characterized by means of X-ray photoelectron spectroscopy. Voltammetry of the composites displayed stable oxidation and reduction peaks at an optimal potential close to that of the FAD/FADH(2) cofactor of immobilized glucose oxidase. The immobilized enzyme is stable for a period of one month and retains catalytic activity for the oxidation of glucose. It is demonstrated that the resulting composite can be successfully integrated into functional bioelectrodes for biosensor and biofuel cell applications.

Application of Electrochemical Biosensors for Detection of Food Pathogenic Bacteria

Current practices for preventing microbial diseases rely upon careful control of various kinds of pathogenic bacteria in food safety and environmental monitoring. The main disadvantages of conventional bacterial detection methods are the multistep procedure and long time requirements. This article gives an overview of alternative electrochemical biosensors for detection of pathogenic bacteria in the food industry. Focus has been on new microbial metabolism-based, antibody-based and DNA-based biosensors. The underlying principles and applications of these biosensors are discussed. Recent developments in flow-injection biosensor systems with an electrochemical detection are also presented.

Flow-through immunofiltration assay system for rapid detection of E. coli O157 :H7

A flow-through amperometric immunofiltration assay system based on disposable porous filter-membranes for rapid detection of Escherichia coli O157:H7 has been developed. The analytical system utilizes flow-through, immunofiltration and enzyme immunoassay techniques in conjunction with an amperometric sensor. The parameters affecting the immunoassay such as selection of appropriate filter membranes, membrane pore size, antibody binding capacity and the concentrations of immunoreagents were investigated and optimized. Non-specific adsorption of the enzyme conjugate was investigated and minimized. A sandwich scheme of immunoassay was employed and the immunofiltration system allows to specifically and directly detect E. coli cells with a lower detection limit of 100 cells/ml. The working range is from 100 to 600 cells/ml with an overall analysis time of 30 min. No pre-enrichment was needed. This immunosensor can be easily adapted for assay of other microorganisms and may be a basis for a new class of highly sensitive bioanalytical devices for rapid quantitative detection of bacteria.

High electrocatalytic activity of tethered multicopper oxidase–carbon nanotube conjugates

Multicopper oxidases linked to multiwall carbon nanotubes via the molecular tethering reagent, 1-pyrenebutanoic acid, succinimidyl ester, displayed high bioelectrocatalytic activity for oxygen reduction.

Highly sensitive flow-injection immunoassay system for rapid detection of bacteria☆

A flow-injection amperometric immunofiltration assay system for the rapid detection of total Escherichia coli and Salmonella was developed. The system is based on the use of disposable porous nylon membranes which act as a support for the immobilization of anti-E. coli or anti-Salmonella antibodies. The assay system consists of a flow-injection system, a disposable filter-membrane and an amperometric sensor. Parameters affecting the performance of the immunofiltration assay system such as membrane pore size, non-specific binding, conjugate concentration and sample volume were studied and optimized. A sandwich scheme of immunoassay was employed and the immunofiltration system was able to specifically and directly detect 50 cells/ml of total E. coli or 50 cells/ml of Salmonella with an overall analysis time of 35 min. This immunosensor can be easily adapted for the assay of other microorganisms and may be a basis for a new class of highly sensitive and automated bioanalytical devices for the rapid quantitative detection of bacteria.

Design Parameters for Tuning the Type 1 Cu Multicopper Oxidase Redox Potential: Insight from a Combination of First Principles and Empirical Molecular Dynamics Simulations

The redox potentials and reorganization energies of the type 1 (T1) Cu site in four multicopper oxidases were calculated by combining first principles density functional theory (QM) and QM/MM molecular dynamics (MD) simulations. The model enzymes selected included the laccase from Trametes versicolor, the laccase-like enzyme isolated from Bacillus subtilis, CueO required for copper homeostasis in Escherichia coli, and the small laccase (SLAC) from Streptomyces coelicolor. The results demonstrated good agreement with experimental data and provided insight into the parameters that influence the T1 redox potential. Effects of the immediate T1 Cu site environment, including the His(N(δ))-Cys(S)-His(N(δ)) and the axial coordinating amino acid, as well as the proximate H(N)(backbone)-S(Cys) hydrogen bond, were discerned. Furthermore, effects of the protein backbone and side-chains, as well as of the aqueous solvent, were studied by QM/MM molecular dynamics (MD) simulations, providing an understanding of influences beyond the T1 Cu coordination sphere. Suggestions were made regarding an increase of the T1 redox potential in SLAC, i.e., of Met198 and Thr232 in addition to the axial amino acid Met298. Finally, the results of this work presented a framework for understanding parameters that influence the Type 1 Cu MCO redox potential, useful for an ever-growing range of laccase-based applications.

Electrochemical biosensor based on supported planar lipid bilayers for fast detection of pathogenic bacteria

This paper presents a new ion-channel biosensor based on supported bilayer lipid membrane for direct and fast detection of Campylobacter species. The sensing element of a biosensor is composed of a stainless-steel working electrode, which is covered by artificial bilayer lipid membrane (BLM). Antibodies to bacteria embedded into the BLM are used as channel forming proteins. The biosensor has a strong signal amplification effect, which is defined as the total number of ions transported across the BLM. The total number of (univalent) ions flowing through the channels is 1010 ions s−1. The biosensor showed a very good sensitivity and selectivity to Campylobacter species.

A quantitative determination of organophosphate pesticides in organic solvents

An amperometric acetylcholinesterase AChE biosensor based on thiocholine-hexacyanoferrate reaction was developed for the . analysis of OPCs in pure organic solvents. The enzyme AChE was co-immobilized with an electron mediator, Prussian Blue, on the surface of a graphite electrode. The effect of organic solvents on acetylcholinesterase activity was estimated in the presence of polar . . hydrophilic and non-polar hydrophobic organic solvents in the range of 0.01-100%. The ability of the AChE biosensor to detect pesticides was demonstrated by quantitative determination of dichlorvos, fenthion and diazinon in ethanol solvent. The assay allows determination of OPCs in sub-micromolar concentration ranges with an overall assay time of 10 minutes. The sensing elements of the amperometric AChE biosensor can be stored in dry state for more than 2 months. The AChE biosensor possesses distinct advantages, including monitoring of hydrophobic substrates, elimination of microbial contamination, and relative ease of enzyme immobilization. Potential application areas include food analysis and environmental monitoring. q 2000 Elsevier Science S.A. All rights reserved.

Fast Amperometric Assay for E. coli O157:H7 Using Partially Immersed Immunoelectrodes

A novel amperometric immunoelectrode for fast and sensitive assay of E. coli O157:H7 is presented. Antibodies against E. coli O157:H7 were immobilized on the surface of carbon rods, which acted as both working electrode and sorbent surface. A sandwich scheme of immunoassay was used and 5-aminosalicylic acid was employed as a redox mediator for the amperometric detection of the enzyme-label (horseradish peroxidase). The immunoelectrodes were operated while being partly immersed in the detection cell which resulted in the acceleration of the diffusion-controlled rates of immunological, enzymatic and electrochemical reactions. The amperometric immunoelectrode allows the achievement of significantly lower detection limits (40 times lower) than that achievable with standard spectrophotometric detection ELISA method using the same immunochemicals. The immunoelectrode allows determination of E. coli cell concentrations in the range from 200 to 7000 cells/mL with an overall analysis time of 40 min. This immunoelectrode can be easily adapted for assay of other microorganisms and may be a basis for creating a new class of highly sensitive and rapid immunosensors.