Biomedical subjects

Jan Tkac

Publications and source records attributed to Jan Tkac.

11 recordsLinked to original sources

PubMed2006

The use of single walled carbon nanotubes dispersed in a chitosan matrix for preparation of a galactose biosensor.

Chitosan was chosen as a natural polymer for dispersion of single walled carbon nanotubes (SWNT) based on its ability to efficiently solubilize SWNTs to form a stable dispersion. Moreover, chitosan films deposited on a surface of a glassy carbon (GC) electrode are mechanically stable. Further stabilisation of the chitosan film containing SWNT (CHIT-SWNT) was done by chemical crosslinking with glutaraldehyde and free aldehyde groups produced a substrate used for covalent immobilisation of galactose oxidase (GalOD). Different galactose biosensor configurations were tested with optimisation of composition of inner and outer membrane; and enzyme immobilisation procedure, as well. Detection of oxygen uptake by GalOD on CHIT-SWNT layer at -400 mV is robust and, when flow injection analysis (FIA) was applied for assays, a low detection limit (25 microM) and very high assay throughput rate (150 h-1) was achieved. This new galactose biosensor offers highly reliable detection of galactose with R.S.D. well below 2% and it has been successfully applied to assaying galactose in a blood sample with recovery index between 101.2 and 102.7%.

Biosensing Techniques↗

PubMed2006

Electrical wiring of Pseudomonas putida and Pseudomonas fluorescens with osmium redox polymers.

Two different flexible osmium redox polymers; poly(1-vinylimidazole)12-[Os-(4,4'-dimethyl-2,2'-di'pyridyl)2Cl2](2+/+) (osmium redox polymer I) and poly(vinylpyridine)-[Os-(N,N'-methylated-2,2'-biimidazole)3](2+/3+) (osmium redox polymer II) were investigated for their ability to efficiently "wire" Pseudomonas putida ATCC 126633 and Pseudomonas fluorescens (P. putida DSM 6521), which are well-known phenol degrading organisms, when entrapped onto cysteamine modified gold electrodes. The two Os-polymers differ in redox potential and the length of the side chains, where the Os(2+/3+)-functionalities are located. The bacterial cells were adapted to grow in the presence of phenol as the sole source of organic carbon. The performance of the redox polymers as mediators was investigated for making microbial sensors. The analytical characteristics of the microbial sensors were evaluated for determination of catechol, phenol and glucose as substrates in both batch analysis and flow analysis mode.

2,2'-Dipyridyl↗

PubMed2005

Intracellular monitoring of superoxide dismutase expression in an Escherichia coli fed-batch cultivation using on-line disruption with at-line surface plasmon resonance detection.

An on-line cell disruption system for at-line monitoring of the intracellular concentration of recombinant human superoxide dismutase (rhSOD) in a genetically modified Escherichia coli strain, HMS174(DE3) (pET11a/rhSOD), in bioreactor cultivations is described. The sampled bacteria were disrupted on-line by rapid mixing with a nonionic detergent. The recombinant protein content of the lysed bacterial sample was quantitated by a subsequent surface plasmon resonance biosensor with a specific monoclonal antibody. Extraction efficiency of the monitoring system was optimized with respect to the flow rate ratio of the cell suspension and the detergent at relevant cell densities with the aim to attain rapid monitoring. Monitoring was demonstrated for a shake flask culture and a glucose-limited fed-batch cultivation. The results are compared with a traditional enzyme-linked immunosorbent assay method showing a correlation coefficient of R2 = 0.97. Extraction efficiency of rhSOD reached 95-99% at a total processing time of 1.8-2.6 min and a contact time of 0.8-1.4 min. The possibility of extending the monitoring system to other intracellular proteins is discussed.

Bacteriological Techniques↗

PubMed2004

Direct electron transfer between copper-containing proteins and electrodes.

The electrochemistry of some copper-containing proteins and enzymes, viz. azurin, galactose oxidase, tyrosinase (catechol oxidase), and the "blue" multicopper oxidases (ascorbate oxidase, bilirubin oxidase, ceruloplasmin, laccase) is reviewed and discussed in conjunction with their basic biochemical and structural characteristics. It is shown that long-range electron transfer between these enzymes and electrodes can be established, and the mechanistic schemes of the DET processes are proposed.

Adsorption↗

PubMed2004

Off-line monitoring of bacterial stress response during recombinant protein production using an optical biosensor.

A surface plasmon resonance (SPR) biosensor was used to monitor the profiles of the heat-shock protein (DnaK) and the expression of a heterologous protein to map the dynamics of the cellular stress response in Escherichia coli. As expression system was used an E. coli strain overproducing human recombinant superoxide dismutase (rhSOD). Expression of DnaK showed complex patterns differing with strength of induction. The strong up-regulation of DnaK expression was observed in all cultivations which over-produced of rhSOD. Similar patterns were not observed in non-induced reference cultures. Differences in DnaK concentration profiles were correlated with induction strength. Presented data, carried out in shake flask and glucose limited fed-batch cultivation, show a good consistency with previously published transcriptional profiling results and provide complementary information to understand stress response related to overproduction of recombinant protein. The study also demonstrates the feasibility of using the SPR as a two channel protein array for monitoring of intracellular components.

Bioreactors↗

PubMed2004

Evaluation of disruption methods for the release of intracellular recombinant protein from Escherichia coli for analytical purposes.

The aim of the present study was to find disruption methods that allow fast and reproducible measurement of intracellular recombinant proteins with potential for on-line application. Production of rhSOD (recombinant human superoxide dismutase) by Escherichia coli was used as a model. Three methods of cell disruption, sonication, osmotic shock and chemical treatment using a non-ionic surfactant, were critically compared with respect to efficiency and reproducibility of the release of rhSOD. The release of the recombinant protein was monitored by (i) measurement of the protein content in cell-culture extracts using an SPR (surface plasmon resonance) biosensor, and (ii) assaying the enzyme activity with a colorimetric reagent using a spectrophotometer. Disruption by the non-ionic surfactant showed the best performance in terms of simplicity, reproducibility and efficiency of sample treatment. The surfactant did not interfere with the rhSOD binding to the antibody immobilized on the SPR chip or with the rhSOD activity assay. When comparing the two detection methods during monitoring of an E. coli cultivation, comparable results were obtained.

Bacteriolysis↗

PubMed2003

Monitoring of the heat-shock response in Escherichia coli using an optical biosensor.

A surface plasmon resonance (SPR) method for monitoring the concentration of the chaperone DnaK and its relation to physiological stress response in a recombinant Escherichia coli strain subjected to heat shock is described. The DnaK protein, an abundantly occurring representative of the heat-shock proteins, was used as a marker of physiological stress. The SPR biosensor instrument was used for label-free immunoaffinity detection directly in cell culture lysates using an anti-DnaK monoclonal IgG antibody immobilized on the sensor surface. The SPR method provides a fast response (<8 min) and a reproducible (RSD<2%), accurate (comparison to the direct enzyme-linked immunosorbent assay), and sensitive (LOD<1 nM) assay for determination of the DnaK level in cell culture lysates. The operational stability of the method was high compared to that of other SPR assays; the sensitivity decreased at only 2.7%/h. This allowed measurement of more than 220 samples per sensor surface. Storage stability was determined at 25 degrees C (100% after 17 h) and 10 degrees C (101% after 1 month). The method was validated by standard additions of DnaK (30, 60, and 120 nM) with recovery indices in the range 95.7-103.7%.

Biochemistry↗

PubMed2003

Improved selectivity of microbial biosensor using membrane coating. Application to the analysis of ethanol during fermentation.

A ferricyanide mediated microbial biosensor for ethanol detection was prepared by surface modification of a glassy carbon electrode. The selectivity of the whole Gluconobacter oxydans cell biosensor for ethanol determination was greatly enhanced by the size exclusion effect of a cellulose acetate (CA) membrane. The use of a CA membrane increased the ethanol to glucose sensitivity ratio by a factor of 58.2 and even the ethanol to glycerol sensitivity ratio by a factor of 7.5 compared with the use of a dialysis membrane. The biosensor provides rapid and sensitive detection of ethanol with a limit of detection of 0.85 microM (S/N=3). The selectivity of the biosensor toward alcohols was better compared to previously published enzyme biosensors based on alcohol oxidase or alcohol dehydrogenases. The biosensor was successfully used in an off-line monitoring of ethanol during batch fermentation by immobilized Saccharomyces cerevisiae cells with an initial glucose concentration of 200 g l(-1).

Biofilms↗

PubMed2002

Amperometric urea biosensor based on urease and electropolymerized toluidine blue dye as a pH-sensitive redox probe.

The electropolymerized toluidine blue film deposited on the glassy carbon electrode show amperometrically detectable pH sensitivity. This feature of polytoluidine blue (PTOB) film was used for a construction of an amperometric urea biosensor. We have observed a linear shift of the formal redox potential with increasing pH value between 4 and 8 giving the slope of 81 mV(Delta) pH(-1). Polytoluidine blue film has had a significantly increased stability and higher electrochemical activity compared to the adsorbed monomeric dye. The polytoluidine blue urea biosensor has been operating at a working potential of -200 mV vs. SCE. The sensitivity of the biosensor was 980 nA mM(-1) cm(-2). The biosensor showed linearity in concentration range up to 0.8 mM with the detection limit of 0.02 mM (S/N=3).

Biosensing Techniques↗

PubMed2002

Indirect evidence of direct electron communication between the active site of galactose oxidase and a graphite electrode.

Bi-enzymatic biosensor based on galactose oxidase (GalOD) and horseradish peroxidase (HRP) using ferrocene as an efficient mediator was constructed. When a dependence of a working potential on the sensor performance was examined, an unusual behaviour was observed. With increasing of an applied working potential a lower concentration of substrate to attain full linear range was needed. A fully linear dependence from the first substrate addition was observed at and above the working potential of 150 mV. This activation of the biosensor response by an applied working potential very well corresponds with a formal potential of GalOD (156 mV). When a membrane prevented GalOD access to the electrode surface was applied, no activation effect of a working potential on the sensor performance was observed. Thus, it can be assumed that direct electron communication between GalOD and the electrode occurred.

Binding Sites↗

PubMed2002

Monitoring of ethanol during fermentation using a microbial biosensor with enhanced selectivity.

The present study is concerning the construction of ferricyanide-mediated Gluconobacter oxydans cell ethanol biosensor. The size exclusion effect of a cellulose acetate membrane was used for elimination of glucose interferences during ethanol assays in real samples. A typical response time of the biosensor was 13 s with a high sensitivity of 3.5 microA mM(-1). The microbial biosensor exhibits a very low detection limit of 0.85 microM and a wide linear range from 2 to 270 microM. The operational stability was excellent. During 8.5 h of repetitive ethanol assays, no decrease in the sensor sensitivity was observed. The biosensor was successfully used in the off-line monitoring of ethanol fermentation with a good agreement with HPLC measurements (R(2)=0.998).

Biosensing Techniques↗