Biomedical subjects

S R Mikkelsen

Publications and source records attributed to S R Mikkelsen.

7 recordsLinked to original sources

PubMed1996

Comparison of an intercalating dye and an intercalant-enzyme conjugate for DNA detection in a microtiter-based assay.

Two methods have been developed for the detection of DNA immobilized on the surface of microtiter wells. An intercalating dye, 3,6-diaminoacridine, is used in stain and rinse solutions, so that measured absorbance values (450 nm) reflect the sum of DNA-bound and free dye. With diaminoacridine, signal increases of 0.056 +/- 0.010 were achieved on immobilizing double-stranded calf thymus DNA. An intercalant-enzyme conjugate, consisting of an average of four daunomycin moieties covalently bound to each glucose oxidase, was shown to provide a 10-fold signal enhancement (optimum 0.25 microM, with rinsing and peroxidase-o-dianisidine detection) compared to diaminoacridine, due to catalytic amplification; signals of 0.50 +/- 0.05 were obtained. This conjugate possesses 56% of the activity of native glucose oxidase and was prepared using water-soluble carbodiimide and N-hydroxysuccinimide reagents. Single-stranded DNA was immobilized onto avidin-coated polystyrene plates and commercially available (Covalink) plates possessing secondary amine groups. Following hybridization with complementary DNA, detection was performed with the daunomycin-glucose oxidase conjugate. Both immobilization methods showed optimum DNA concentrations of 0.10 microgram/mL, and maximum signal intensities were obtained when > 0.5 microgram/mL complementary DNA was present in the hybridization solution. Some nonspecific binding of the intercalant-enzyme conjugate was suggested by results obtained with avidin-coated polystyrene plates, but not with Covalink plates.

Animals↗

PubMed1995

Enantioselective determination of oxprenolol and its metabolites in human urine by cyclodextrin-modified capillary zone electrophoresis.

A stereospecific capillary electrophoresis assay for oxprenolol enantiomers and their basic metabolites in human urine has been developed using hydroxypropyl-beta-CD as a chiral selector in the mobile phase. The bioassay method has been validated and the detection limit from spiked urine samples is 0.2 micrograms/ml. The calibration curves are linear from 0.4 to 16 micrograms/ml. Extraction recovery ranged from 84.7 to 96.4% for all the compounds studied. The influence of various parameters on the chiral separation of oxprenolol and its basic metabolites have been investigated. Urinary excretion profiles of oxprenolol enantiomers and those of two metabolites have also been studied, following a single oral dose of racemic oxprenolol.

Calibration↗

PubMed1994

Voltammetric DNA biosensor for cystic fibrosis based on a modified carbon paste electrode.

Carbon paste electrodes modified by the inclusion of either octadecylamine or stearic acid were used as solid phases to which DNA was covalently bound. Immobilized DNA was detected by voltammetry of solutions containing submillimolar quantities of Co(bpy)3(ClO4)3, Co(phen)3(ClO4)3, and Os(bpy)3-Cl2 (bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline), all of which associate reversibly with immobilized DNA and yield increased peak currents at DNA-modified electrodes. Immobilization onto octadecylamine-modified electrodes was performed using a water-soluble carbodiimide, and at high DNA concentrations in the reaction mixture, it resulted in visible polymerization of DNA on the surface. Optimization of the deoxyguanosine- (dG-) selective immobilization reaction for stearic acid-modified electrodes, using water-soluble carbodiimide and N-hydroxysulfosuccinimide reagents to activate carboxylate groups on the surface, yielded conditions of 4.5% (w/w) stearic acid and 10 micrograms/mL DNA. Polythymidylic acid of 4000-base average length (poly(dT)4000) was immobilized at stearic acid-modified electrodes following enzymatic elongation with dG residues at the 3'-end. These DNA-modified electrodes were used to study hybridization with analyte poly(dA)4000 by in situ voltammetry of 60 microM Co(bpy)3(ClO4)3 at low ionic strength (20 mM NaCl), and by voltammetry of the same complex, following exposure of the electrode to poly(dA)4000 in a separate hybridization step conducted at high ionic strength (0.5 M NaCl). Results indicate slow (> or = 1 h) hybridization at low ionic strength and fast (< or = 10 min) hybridization at high ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1993

Sequence-selective biosensor for DNA based on electroactive hybridization indicators.

Deoxyribonucleic acid was covalently immobilized onto oxidized glassy carbon electrode surfaces that had been activated using 1-[3-(dimethylamino)-propyl]-3-ethylcarbodimide hydrochloride and N-hydroxysulfosuccinimide. This reaction is selective for immobilization through deoxyguanosine (dG) residues. Immobilized DNA was detected voltammetrically, using tris (2,2'-bipyridyl)cobalt(III) perchlorate and tris (1,10-phenanthroline)cobalt(III) perchlorate (Co(bpy)3(3+) and Co(phen)3(3+). These complexes are reversibly electroactive (1e-) and preconcentrate at the electrode surface through association with double-stranded DNA. Voltammetric peak currents obtained with a poly(dG)poly(dC)-modified electrode depend on [Co(bpy)3(3+)] and [Co(phen)3(3+)] in a nonlinear fashion and indicate saturation binding with immobilized DNA. Voltammetric peak currents for Co(phen)3(3+) reduction were used to estimate the (constant) local DNA concentration at the modified electrode surface; a binding site size of 5 base pairs and an association constant of 1.74 x 10(3) M(-1) yield 8.6 +/- 0.2 mM base pairs. Cyclic voltammetric peak separations indicate that heterogeneous electron transfer is slower at DNA-modified electrodes than at unmodified glassy carbon electrodes. A prototype sequence-selective DNA sensor was constructed by immobilizing a 20-mer oligo (deoxythymidylic acid) (oligo(dT)20), following its enzymatic elongation with dG residues, which yielded the species oligo(dT)20(dG)98. Cyclic voltammograms of 0.12 mM Co(bpy)3(3+) obtained before and after hybridization with poly-(dA) and oligo(dA)20 show increased cathodic peaks after hybridization. The single-stranded form is regenerated on the electrode surface by rinsing with hot deionized water. These results demonstrate the use of electroactive hybridization indicators in a reusable sequence-selective biosensor for DNA.

Animals↗

PubMed1991

Rotating disc electrode characterization of immobilized glucose oxidase.

The kinetic properties of glucose oxidase (EC 1.1.3.4) which has been covalently immobilized to a rotating glassy carbon electrode surface have been investigated. Analysis of the rotation rate dependence of the hydrogen peroxide-derived current suggests that oxygen mass transport to the enzyme-electrode surface is rate controlling at low rotation rates. Only as the diffusion layer approaches zero thickness (i.e., infinitely fast rotation rate) does mass transport become unimportant. A diffusion-free glucose Km for air-saturated buffer is found to be 66 mM using this methodology. The importance of mass transport restrictions in two-substrate enzymes such as glucose oxidase is discussed in the context of biosensor design.

Electrochemistry↗

PubMed1989

Conductometric transducers for enzyme-based biosensors.

The use of alternating current conductometric transducers in biosensing devices has been investigated for urea and D-amino acid sensors using the enzyme systems urease and D-amino acid oxidase/catalase. Transducers with copper and platinum electrodes were constructed and characterized, and two enzyme immobilization methods were tested. Detection limits of 1 x 10(-6)M and linear ranges of 2 orders of magnitude were routinely achieved for these model sensors with enzymes covalently immobilized on collagen films.

Amino Acids↗

PubMedDate not supplied

Targeting glucose oxidase at aspartate and glutamate residues with organic two-electron redox mediators.

The bimolecular rate constants for the reactions of five organic two-electron redox mediators with reduced glucose oxidase (GOx) were determined by measuring voltammetric electrocatalytic currents at glassy carbon electrodes in the presence of excess glucose under anaerobic conditions. The mediators studied were thionine, brilliant cresyl blue, azure A, daunomycin, and dopamine, and the bimolecular rate constants for electron transfer between GOx and the oxidized mediator (M-1 s-1) are 1.6 x 10(4), 4.0 x 10(2), 9.8 x 10(2), 9.0 x 10(3), and 1.2 x 10(6), respectively. GOx was covalently derivatized using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and N-hydroxysulfosuccinimide to form amide bonds between the aliphatic primary amine groups on daunomycin and dopamine and carboxylate side chains of aspartate and glutamate residues. Derivatives with 2.5 +/- 0.1 daunomycin groups and 4 +/- 1 dopamine groups were obtained, with activities of 50% and 75%, respectively, relative to native GOx in a dye-peroxidase assay. Although the daunomycin derivative did not show measurable intramolecular electron-transfer rates, the dopamine derivative rapidly transfers electrons from active-site FADH2 groups to the oxidized (quinone) form of dopamine. Because the heterogeneous oxidation of dopamine is relatively slow, the currents measured at +0.75 V vs Ag/AgCl were not at their limiting (plateau) values, and only a minimum value of the intramolecular rate constant (4.5 s-1) could be determined. This value is > 20 times larger than values obtained for GOx-ferrocene derivatives in which surface lysine residues were covalently modified using identical coupling reagents and similar reaction conditions. This work shows that targeting GOx carboxylate groups with electron-transfer mediators may represent a promising approach to the design of reagentless glucose biosensors.

Anaerobiosis↗