Biomedical subjects

Werner G Kuhr

Publications and source records attributed to Werner G Kuhr.

7 recordsLinked to original sources

PubMed2003

Performance of pyrolyzed photoresist carbon films in a microchip capillary electrophoresis device with sinusoidal voltammetric detection.

Pyrolyzed photoresist films (PPF) are introduced as planar carbon electrodes in a PDMS-quartz hybrid microchip device. The utility of PPF in electroanalytical applications is demonstrated by the separation and detection of various neurotransmitters. PPF is found to form a stable, low-capacitance, durable layer on quartz, which can then be used in conjunction with a microchip capillary electrophoretic device. Sinusoidal voltammetric detection at PPF electrodes is shown to be very sensitive, with a detection limit (S/N = 3) of 100 nM for dopamine, corresponding to a mass detection limit (S/N = 3) of 2 amol. The selectivity of analysis in the frequency domain is demonstrated by isolating each individual signal in a pair of analytes that are chromatographically unresolved. Effectively decoupling the electrophoresis and electrochemical systems allows the electrodes to be placed just inside the separation channel, which results in efficient separations (80 000-100 000 plates/m).

Journal Article↗

PubMed2003

A microchip electrophoresis device with integrated electrochemical detection: a direct comparison of constant potential amperometry and sinusoidal voltammetry.

A microchip electrophoresis system with integrated electrochemical detection is described in this work. The hybrid device utilizes poly(dimethylsiloxane) as the electrophoresis channel substrate and a planar gold electrode lithographically fabricated onto a glass slide for electrochemical detection. The system is characterized by the separation and detection of various neurotransmitters. The gold working electrode is placed just inside the separation channel without adverse effects on the detection sensitivity, due to the electrical decoupling of the detection and electrophoresis systems. The close proximity of the working electrode to the exit of the separation channel results in symmetric peak shapes and efficient separations (50,000-100,000 plates/m). A direct comparison between the frequency-based electrochemical technique, sinusoidal voltammetry, and the more commonly used constant potential (DC) amperometry is made. Sinusoidal voltammetry is found to be roughly an order of magnitude more sensitive than DC amperometry, with calculated mass detection limits (S/N = 3) of 12 amol and 15 amol for dopamine and isoproterenol, respectively.

Electrochemistry↗

PubMed2003

Measurements of electron-transfer rates of charge-storage molecular monolayers on Si(100). Toward hybrid molecular/semiconductor information storage devices.

Redox kinetics were measured for two electroactive molecules attached to Si(100) surfaces, a ferrocene (Fc-BzOH) and a Zn(II) trimesitylporphyrin (Por-BzOH). Each molecule was derivatized with a benzyl alcohol linker for attachment to the Si surface via the formation of a Si-O bond. A complete protocol was developed for the preparation of stable Si(100) surfaces derivatized with the electroactive molecules. The redox-kinetic measurements were performed on the resulting Fc-BzOH and Por-BzOH monolayers to probe (1) the rate of electron transfer (k0) for oxidation in the presence of applied potentials and (2) the rate of charge dissipation after the applied potential is disconnected (in the form of a charge-retention half-life t1/2). The k0 values for the two types of monolayers were found to be similar to one another as were the t1/2 values. Perhaps more importantly, the electron-transfer rates for both the Fc-BzOH and the Por-BzOH monolayers differ from the charge-dissipation rates by approximately 6 orders of magnitude and are strongly dependent on the surface concentration of the electroactive species. For the Por-BzOH monolayers on Si(100), the k0 and t1/2 values and their trends as a function of surface coverage were determined to be similar to those previously measured for the analogous thiol-derivatized molecule assembled on Au(111). In contrast, the Fc-BzOH monolayers on Si(100) were found to exhibit much slower electron-transfer and charge-dissipation rates than those in the corresponding thiol-Au(111) case. Two alternative hypotheses are advanced to explain both the diminution in rates with increased surface coverage and the contrasting behavior with the analogous thiols on Au, one based on space-charge effects at the monolayer-solution interface, and a second relying on changes in distance of the redox centers from the surface as modulated by the orientation of the linking chains. Collectively, the ability to prepare and study stable, electroactive molecular media on Si(100) is likely to be key in the development of hybrid molecular/semiconductor devices.

Journal Article↗

PubMed2003

Use of an electrochemically labeled nucleotide terminator for known point mutation analysis.

A novel single nucleotide polymorphism (SNP) assay utilizing an electrochemically tagged chain terminator is described. The system employs the single-base extension (SBE) technique coupled to capillary gel electrophoresis with end-column electrochemical detection. A redox-labeled chain terminator, ferrocene-acycloATP, is used in the SBE reaction. When the mutation site corresponds to the labeled chain terminator, the extension product is rendered electroactive. The reaction mixture is subsequently separated by capillary gel electrophoresis and the extension product detected at the separation anode with sinusoidal voltammetry. This work demonstrates the first known SNP assay utilizing redox-active chain terminators coupled to electrochemical detection. The methodology presented could lead to a fast, simple, and cost-effective SNP scoring system.

Adenosine Triphosphate↗

PubMed2003

Independently-addressable micron-sized biosensor elements.

With the continuing development of micro-total analysis systems and sensitive biosensing technologies, it is often desirable to immobilize biomolecules onto a surface in a small well-defined area. A novel method was developed to electrochemically attach DNA probes to micron-sized regions of a gold surface using biotin-LC-hydrazide (BH). Previously, we have found that the radical produced during the oxidation of BH will attach to a wide variety of electroactive surfaces. An array of micron-sized gold band electrodes (75 microm wide) was fabricated onto glass microscope slides and BH was deposited onto each electrode through the application of an oxidizing potential. Subsequent attachment of avidin to the biotinylated surface created the 'molecular sandwich' architecture necessary for further immobilization of biotinylated biomolecules to the surface. In this work, we utilized biotinylated DNA probes of varying sequence to illustrate the specificity of the attachment scheme. The immobilization of avidin, DNA probe, and hybridization of DNA target is visualized with fluorescence tags and the spatially selective attachment and hybridization of unique DNA sequences is demonstrated.

Avidin↗

PubMed2002

A single base extension technique for the analysis of known mutations utilizing capillary gel electrophoreisis with electrochemical detection.

A novel single nucleotide polymorphism (SNP) detection system is described in which the accuracy of DNA polymerase and advantages of electrochemical detection are demonstrated. A model SNP system is presented to illustrate the potential advantages in coupling the single base extension (SBE) technique to capillary gel electrophoresis (CGE) with electrochemical detection. An electrochemically labeled primer, with a ferrocene acetate covalently attached to its 5' end, is used in the extension reaction. When the Watson-Crick complementary ddNTP is added to the SBE reaction, the primer is extended by a single nucleotide. The reaction mixture is subsequently separated by CGE, and the ferrocene-tagged fragments are detected at the separation anode with sinusoidal voltammetry. This work demonstrates the first single base resolution separation of DNA coupled with electrochemical detection. The unextended primer (20-mer) and the 21-mer extension product are separated with a resolution of 0.8.

Base Pairing↗

PubMed2002

Microchip capillary electrophoresis coupled to sinusoidal voltammetry for the detection of native carbohydrates.

The development of a microchip electrophoresis system involving integrated frequency based electrochemical detection is described. The use of poly(dimethylsiloxane) (PDMS) as the channel substrate greatly simplifies the fabrication process while decreasing cost and time consumption. Characterization of this system is accomplished through the detection of native carbohydrates at planar copper electrodes. Various photolithographic techniques are explored in the optimization of electrode area. Separation efficiency of 1 x 10(5) theoretical plates per meter is demonstrated. Sinusoidal voltammetry utilizes information in the frequency domain to achieve sensitive detection through either of two approaches, maximization of signal or minimization of noise. Mass detection limits (S/N = 3) of less than 200 amol have been accomplished for glucose and sucrose. Sinusoidal voltammetry also facilitated the selective isolation of an analyte signal from a pair of chromatographically unresolved species through the use of phase discrimination.

Carbohydrates↗