Biomedical subjects

C Zaborosch

Publications and source records attributed to C Zaborosch.

7 recordsLinked to original sources

PubMed2000

Analysis of ternary mixtures with a single dynamic microbial sensor and chemometrics using a nonlinear multivariate calibration.

An amperometric biosensor based on immobilized bacterial cells of Alcaligenes eutrophus KT02 and an oxygen electrode was integrated in a flow-through system. Because microorganisms metabolize various organic analytes in a specific manner, the sensor shows for different pure analytes distinct time-dependent oxygen consumption rates that can be treated as characteristic patterns. This behavior is conserved also when the biosensor is exposed to a mixture of these organic analytes; the sensor with a particular type of microorganisms responds with a total signal. The respiration curves as time-dependent amplitudes were subdivided into several time channels. This procedure creates an additional data dimension and makes the single sensor "dynamic". Using multivariate calibration models with only one single biosensor, simultaneous quantitative analysis of ternary mixtures of acetate, L-lactate, and succinate was realized. A nonlinear algorithm that compensated for conceivable interactions of the analytes was superior to a partial least-squares algorithm. Each analyte was predicted more precisely by the nonlinear approach resulting in root-mean-square errors of prediction of 0.20 mg/L for acetate, 0.43 mg/L for L-lactate, and 0.73 mg/L for succinate.

Alcaligenes↗

PubMed2000

Multimicrobial sensor using microstructured three-dimensional electrodes based on silicon technology.

Two microbial strains with different substrate spectra were immobilized separately within a single biosensor chip featuring four individually addressable platinum electrodes. These were sputtered onto the inner surface of four isolated pyramidal cavities ("containments") micromachined on a silicon wafer. The biosensor chip was integrated into a flow-through system to measure the oxygen consumption of the immobilized microorganisms in the presence of assimilable analytes. As a model system, a yeast for the determination of biochemical oxygen demand (BOD) and a strain capable of degrading polycyclic aromatic hydrocarbons (PAH) were chosen. It was shown that the simple and mass-producible containment sensor exhibits good performance data: lower detection limit 0.1 mg/L naphthalene and 1 mg/L sensor-BOD; calibration range up to 30 mg/L; precision 3-6%; response time 2-3 min; service life up to 40 days; shelf life at 4 degrees C 6 months. The versatility of the multimicrobial sensor was demonstrated by measuring ordinary municipal wastewater samples as well as various aqueous samples contaminated with PAH. The concept of a multimicrobial sensor not only enlarges the substrate spectrum for sum parameters such as BOD but leads to additional information which allows for a more differentiated and immediate knowledge of sample composition. Using chemometrical data analysis, the multimicrobial sensor lays a foundation for developing an "electronic tongue".

Biodegradation, Environmental↗

PubMed2000

Screen-printed enzyme sensors for l-lysine determination.

Sensors for the determination of L-lysine in samples of fermentation broth have been developed. Low-cost screen-printed sensors comprising a platinum working electrode, an Ag/AgCl pseudo reference and a carbon counter electrode were used as transducers for the enzyme sensors. L-lysine-(alpha)-oxidase from Trichoderma viride has been immobilized by entrapment into a polyurethane hydrogel. Sensors were characterized for L-lysine with respect to pH value, linear range, reproducibility, repeatability, storage and working stability. The sensitivities to other amino acids were also determined. A batch system with two working electrodes, one with immobilized enzyme and one without was adapted for the determination of L-lysine by differential measurements. Good agreement was found between L-lysine concentrations measured by the enzyme sensors and by a conventional amino acid analyzer.

Journal Article↗

PubMed2000

Microbial and cytoplasmic membrane-based potentiometric biosensors for direct determination of organophosphorus insecticides.

Potentiometric biosensors for the determination of organophosphorus (OP) insecticides were developed by applying either immobilized whole cells or cytoplasmic membrane fractions of wild-type Flavobacterium sp. on the surface of a glass pH electrode. The ability of Flavobacterium sp. to degrade OP compounds as sole carbon source was demonstrated for parathion with a degradation rate of almost 100% after 30 min and for chlorpyrifos of 33% after 48 h incubation. The products of hydrolysis of these compounds, p-nitrophenol and 3,5,6-trichloro-2-pyridinol, were accumulated in the medium and not used as substrates for growth by Flavobacterium sp. In the course of hydrolysis, which is catalyzed by organophosphorus hydrolase, two protons are released for each substrate molecule hydrolyzed. This stoichiometry forms the electrochemical basis of the potentiometric biosensors. Direct determination without previous extraction of OP was carried out in a stirred measuring cell with a pH electrode as transducer. Poly(carbamoyl sulfonate) (PCS) prepolymer, a hydrogel with good adhesive properties, was used for immobilization of whole cells and membrane-associated organophosphorus hydrolase. The sensor with cytoplasmic membrane fractions was superior to the one with whole cells and showed a linear range for paraoxon from 0.01 to 0.47 mM and 3 weeks' working stability.

Aryldialkylphosphatase↗

PubMed2000

Disposable potentiometric enzyme sensor for direct determination of organophosphorus insecticides.

A potentiometric disposable enzyme sensor for the direct and fast determination of organophosphorus (OP) insecticides was developed by using an organophosphorus hydrolase (OPH) immobilized on an ion-selective electrode. The disposable screen-printed transducer was based on double matrix membrane technology which allows easy mass production. The potentiometric device consisted of a H(+)-sensitive electrode with integrated Ag/AgCl reference electrode. The electrodes were prepared with N,N-dioctadecylmethylamine as H(+)-sensitive ionophore and pH calibration resulted in slopes of 55 mV decade-1 over a pH range from 11 to 6. OPH was isolated from recombinant Escherichia coli DH5 alpha and immobilized within poly(carbamoyl sulfonate) prepolymer on the surface of the H(+)-sensitive electrode without any further fixation membrane. OPH catalyzes the hydrolytic cleavage of OP compounds which releases protons in a concentration proportional to hydrolyzed substrate. Sensor performance was investigated with regard to enzyme load, concentration, pH and temperature of the measuring buffer using paraoxon as analyte. Best sensitivity and response time were obtained with sensors prepared with 250 U of OPH and measuring at 37 degrees C in 1.0 mM HEPES buffer, pH 9.3, containing 100 mM NaCl. The enzyme sensor exhibited a linear calibration range of 0.01-0.15 mM chlorpyrifos, 0.05-0.35 mM diazinon, 0.05-0.4 mM paraoxon and 0.007-0.05 mM parathion, respectively. For all these analytes response times to reach 95% of maximum change in potential did not exceed 5 min. Sensors stored under dry conditions at 4 degrees C still showed 60% of initial hydrolytic rate after 70 d. The sensors even when stored dry were ready for measurements after 5 min incubation in measuring buffer. A range of putative interfering substances did not influence sensor response, and suitability of measuring OPs in soil extracts was ascertained.

Biosensing Techniques↗

PubMed1990

Cloning and nucleotide sequences of the genes for the subunits of NAD-reducing hydrogenase of Alcaligenes eutrophus H16.

The genes hoxF, -U, -Y, and -H which encode the four subunit polypeptides alpha, gamma, delta, and beta of the NAD-reducing hydrogenase (HoxS) of Alcaligenes eutrophus H16, were cloned, expressed in Pseudomonas facilis, and sequenced. On the basis of the nucleotide sequence, the predicted amino acid sequences, and the N-terminal amino acid sequences, it was concluded that the structural genes are tightly linked and presumably organized as an operon, denoted hoxS. Two pairs of -24 and -12 consensus sequences resembling RpoN-activatable promoters lie upstream of hoxF, the first of the four genes. Primer extension experiments indicate that the second promoter is responsible for hoxS transcription. hoxF and hoxU code for the flavin-containing dimer (alpha and gamma subunits) of HoxS which exhibits NADH:oxidoreductase activity. A putative flavin-binding region is discussed. The 26.0-kilodalton (kDa) gamma subunit contains two cysteine clusters which may participate in the coordination of two [4F3-4S]centers. The genes hoxY and hoxH code for the small 22.9-kDa delta subunit and the nickel-containing 54.8-kDa beta subunit, respectively, of the hydrogenase dimer of HoxS. The latter dimer exhibits several conserved regions found in all nickel-containing hydrogenases. The roles of these regions in coordinating iron and nickel are discussed. Although the deduced amino acid sequences of the delta and beta subunits share some conserved regions with the corresponding polypeptides of other [NiFe] hydrogenases, the overall amino acid homology is marginal. Nevertheless, significant sequence homology (35%) to the corresponding polypeptides of the soluble methylviologen-reducing hydrogenase of Methanobacterium thermoautotrophicum was found. Unlike the small subunits of the membrane-bound and soluble periplasmic hydrogenases, the HoxS protein does not appear to be synthesized with an N-terminal leader peptide.

Alcaligenes↗

PubMed1989

Comparison of the NH2-terminal amino acid sequences of the four non-identical subunits of the NAD-linked hydrogenases from Nocardia opaca 1b and Alcaligenes eutrophus H16.

The cytoplasmic, NAD-linked hydrogenase of the Gram-positive hydrogen-oxidizing bacterium Nocardia opaca 1b was compared with the analogous enzyme isolated from the Gram-negative bacterium Alcaligenes eutrophus H16. The hydrogenase of N. opaca 1b was purified by a new procedure applying chromatography on phenyl-Sepharose and DEAE-Sephacel with two columns in series. A homogeneous enzyme preparation with a specific activity of 74 mumol H2 oxidized.min-1.mg protein-1 and a yield of 32% was isolated. The A. eutrophus enzyme was purified as previously published. Both enzymes are tetrameric proteins composed of four non-identical subunits (alpha, beta, gamma, delta). The four subunits of both of these enzymes were separated and isolated as single polypeptides by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Immunological comparison of the four subunits of the Nocardia hydrogenase with those of the Alcaligenes enzyme showed that the alpha, beta, gamma, and delta subunits of one organism were serologically related to the analogous subunits of the other organism. Among themselves, the four subunits do not have any serological relationship. The eight individual polypeptides were also compared with respect to the NH2-terminal amino acid sequences determined by automated Edman degradation and to the amino acid compositions. Strong sequence similarities exist between the analogous subunits isolated from the two bacteria. Within the established N-terminal sequences the similarities between both alpha, beta, gamma and delta subunits amount to 63%, 79%, 80% and 65%, respectively. No similarities exist between the different, non-analogous subunits alpha, beta, gamma and delta.

Alcaligenes↗