Biomedical subjects

Nathan A Lacher

Publications and source records attributed to Nathan A Lacher.

5 recordsLinked to original sources

PubMed2006

Microchip capillary electrophoresis: application to peptide analysis.

The development of analytical methodologies to elucidate mechanisms of peptide transport and metabolism is important for the understanding of disease states and the design of effective drug therapies. Interest in the use of microchip capillary electrophoresis (CE) devices for peptide analysis stems from the ability to perform fast, highly efficient separations combined with small sample volume requirements. Many of the separation modes developed on conventional systems, including electrochromatography, isoelectric focusing, and electrophoretic bioaffinity assays, have been demonstrated on microchip devices. Steps that include sample preparation and labeling can also be integrated onto the microchip platform. This chapter will discuss considerations for peptide analysis using microchip CE and will focus on different approaches to sample preparation, separation, and detection.

Affinity Labels↗

PubMed2004

Development of a microfabricated palladium decoupler/electrochemical detector for microchip capillary electrophoresis using a hybrid glass/poly(dimethylsiloxane) device.

The fabrication and evaluation of a palladium decoupler and working electrode for microchip capillary electrophoresis (CE) with electrochemical detection is described. The use of the Pd decoupler allows the working electrode to be placed directly in the separation channel and eliminates the band-broadening characteristic of the end-channel configuration. The method used for fabrication of the decoupler and working electrode was based on thin-layer deposition of titanium followed by palladium onto a glass substrate. When employed as the cathode in CE, palladium absorbs the hydrogen gas that is generated by the hydrolysis of water. The effect of the decoupler size on the ability to remove hydrogen was evaluated with regard to reproducibility and longevity. Using boric acid and TES buffer systems, 500 microm was determined to be the optimum decoupler size, with effective voltage isolation lasting for approximately 6 h at a constant field strength of 600 V/cm. The effect of distance between the decoupler and working electrode on noise and resolution for the separation of dopamine and epinephrine was also investigated. It was found that 250 microm was the optimum spacing between the decoupler and working electrode. At this spacing, laser-induced fluorescence detection at various points around the decoupler established that the band broadening due to pressure-induced flow that occurs after the decoupler did not significantly affect the separation efficiency of fluorescein. Limits of detection, sensitivity, and linearity for dopamine (500 nM, 3.5 pA/microM, r(2) = 0.9996) and epinephrine (2.1 microM, 2.6 pA/microM, r(2) = 0.9996) were obtained using the palladium decoupler in combination with a Pd working electrode.

Dimethylpolysiloxanes↗

PubMed2003

Comparison of the performance characteristics of poly(dimethylsiloxane) and Pyrex microchip electrophoresis devices for peptide separations.

A comparative study of electrophoretic separations of fluorescently labeled peptides and amino acids on poly(dimethylsiloxane) (PDMS) and Pyrex microchips is presented. The separation parameters for each microchip substrate were compared, including electroosmotic flow, plate numbers, resolution, and limits of detection. The effect of buffer composition on the separation was also investigated. Acceptable separations were obtained for most peptides with both substrates; however, PDMS chips exhibited much lower separation efficiencies and longer analysis times.

Amino Acids↗

PubMed2002

Detection of homocysteine by conventional and microchip capillary electrophoresis/electrochemistry.

A method based on capillary electrophoresis (CE) with electrochemical (EC) detection for the determination of both total homocysteine (tHcy) and protein-bound homocysteine (pbHcy) in plasma is described. Both end-column and off-column amperometric detection were investigated. Off-column detection resulted in a more sensitive assay for the determination of homocysteine (Hcy). The detection limit for homocysteine was 500 nM using off-column EC detection and the response was linear over the range 1-100 microM. Therefore, this assay is appropriate for the quantification of Hcy over the physiological concentration ranges found in all disease states. Methodologies for the determination of tHcy and pbHcy in human plasma were investigated and optimized and the concentrations of both pbHcy and tHcy in plasma obtained from a healthy individual were determined to be 2.79+/-0.31 nuM (n = 4) and 3.37+/-0.15 microM (n = 3), respectively. The methodology was then transferred to a microchip CE-EC format and Hcy and reduced glutathione (GSH) were detected. Future work will focus on the development of ancillary methodologies to identify the other forms of Hcy in vivo.

Electrochemistry↗

PubMed2002

Separation and detection of angiotensin peptides by Cu(II) complexation and capillary electrophoresis with UV and electrochemical detection.

The use of capillary electrophoresis (CE) with on-capillary Cu(II) complexation for the determination of angiotensin and its metabolites is described. The resulting copper-peptide complexes can be detected using either UV or electrochemical (EC) detection. Optimal reaction and separation conditions for the angiotensin peptides were first determined using CE with UV detection. With UV detection, the limit of detection (signal-to noise ratio S/N = 3) for native angiotensin II was 18 microM, while the limit of detection (LOD) obtained for the copper-angiotensin II complex is 2 microM. CE with EC detection was then evaluated, yielding significantly lower LODs--2 microM for native angiotensin II and 200 nM for the copper-angiotensin II complex. The addition of copper to the run buffer improved the separation and sensitivity for both CE-UV and CE-EC detection. The method was demonstrated by monitoring the conversion of angiotensin I to angiotensin II in plasma via angiotensin-converting enzyme (ACE) and subsequent inhibition of ACE by captopril.

Angiotensin I↗