Tiemin Huang

Microfabrication of a tapered channel for isoelectric focusing with thermally generated pH gradient.

A simple microfabrication technique for the preparation of a tapered microchannel for thermally generated pH gradient isoelectric focusing (IEF) has been demonstrated. The tapered channel was cut into a plastic sheet (thickness was 120 νm), and the channel was closed by sandwiching the plastic sheet between two glass microscope slides. The length of the microchannel was 5 cm. The width of the separation channel was 0.4 mm at the narrow end and 4 mm at the wide end. The channel was coated with polyacrylamide to prevent electroosmotic flow (EOF) during focusing. Two electrolyte vials were mounted on top of each end of the channel with the wide end of the channel connected to the cathodic vial and the narrow to the anodic vial. The feasibility of the thermally generated pH gradient in a tapered channel was demonstrated. Important parameters that determined the feasibility of using a thermally generated pH gradient in a tapered channel were analyzed. Parameters to be optimized were control of EOF and hydrodynamic flow, selection of power supply mode and prevention of local overheating and air bubble formation. Tris‐HCl buffer, which has a high pKa dependence with temperature, was used both to dissolve proteins and as the electrolyte. The thermally generated pH gradient separation of proteins was tested by focusing dog, cat and human hemoglobins with a whole column detection capillary IEF (CIEF) system.

Axially illuminated fluorescence imaging detection for capillary isoelectric focusing on Teflon capillary

An axially illuminated laser induced fluorescence (LIF) whole column imaging detection (WCID) method was developed for capillary isoelectric focusing (CIEF). In this method, an argon ion laser was used as the excitation source and the isoelectric focusing process was monitored dynamically by a thermoelectrically (TE) cooled (charge coupled device) CCD camera. Low refractive index PTFE capillary was used in the experiment, and electroosmotic flow (EOF) and protein adsorption were minimized by dynamic conditioning of the capillary with methyl cellulose (MC). With the addition of a small amount of high refractive index organic solvent such as glycerol in the sample mixture, the laser beam was axially propagated with total internal reflection. Stray light and scattering light originating from the wall of the capillary were minimized. The instrumentation is simple and the method is sensitive. Two naturally fluorescent proteins (R-phycoerythrin and green fluorescent protein) were separated, and a concentration detection limit (LOD) of 10−13 M or mass LOD of 10−19 mol for R-phycoerythrin was obtained.

Side-by-side comparison of disposable microchips with commercial capillary cartridges for application in capillary isoelectric focusing with whole column imaging detection

Simple-structured, well-functioned disposable poly(dimethylsiloxane) (PDMS) microchips were developed for capillary isoelectric focusing with whole column imaging detection (CIEF-WCID). Side-by-side comparison of the developed microchips with well-established commercial capillary cartridges demonstrated that the disposable microchips have comparable performance as well as advantages such as absence of lens effect and possibility of high-aspect-ratio accompanied with a dramatic reduction in cost.

Laser‐induced fluorescence detection of non‐covalently labeled protein in capillary isoelectric focusing

Non-Covalent labeling for fluorescence detection of proteins has been investigated to increase the sensitivity of capillary isoelectric focusing using laser-induced fluorescence (LIF) detection. Non-covalently labeling fluorescent dyes, NanoOrange, Sypro red, Sypro orange, and Sypro tangerine were explored for the coupling of bovine serum albumin (BSA) and hemoglobin. Labeled proteins were studied by two complementary detection methods, viz. whole column UV and LIF detection instruments. The studies using a commercial capillary isoelectric focusing (CIEF) instrument with UV detection gave accurate pI point determination of the labeled protein, and it was confirmed that non-covalently labeled BSA focused to well characterized peaks and the related calculated pI values did not change significantly. The axial LIF detection system confirmed the formation of fluorescent labeled BSA, and an improvement of detection sensitivity of at least 10 times was achieved using LIF as compared to the UV absorption instrument.

Analysis of proteins by CE, CIEF, and microfluidic devices with whole-column-imaging detection.

The recently developed whole-column-imaging detection technique for capillary electrophoresis (CE) and capillary isoelectric focusing (CIEF), a commercial whole-column-imaged CIEF instrument and its standard operation protocol are introduced. Furthermore, new developments and applications of whole-column-imaging detection in protein-protein interaction study, in protein separation using microfluidic devices and CIEF methods without carrier ampholytes, as well as in 2D separation techniques are reviewed. Miniaturization of whole-column-imaging CIEF and axially illuminated fluorescence whole-column-imaging CIEF are also discussed.