Analysis of Lipoproteins by Capillary Zone Electrophoresis in Microfluidic Devices:  Assay Development and Surface Roughness Measurements

Abstract

The development of a new assay for lipoproteins by capillary electrophoresis in fused-silica capillaries and in glass microdevices is described in this paper. The separation of low-density (LDL) and high-density (HDL) lipoproteins by capillary zone electrophoresis is demonstrated in fused-silica capillaries with both UV absorption and laser-induced fluorescence detection. This separation was accomplished using Tricine buffer (pH 9.0) with methylglucamine added as a dynamic coating. With UV detection, LDL eluted as a relatively sharp peak with a migration time of approximately 11 min and HDL eluted as a broad peak with a migration time of 12.5 min. Fluorescence detection of lipoproteins stained with NBD-ceramide was used with the same buffer system to give comparable results. Furthermore, fluorescence staining of human serum samples yielded results similar to the fluorescently stained LDL and HDL fractions, showing that this method can be used to quantify lipoproteins in serum samples. The method was also used to detect lipoproteins in glass micro-CE devices. Very similar results were obtained in microdevices although with much faster analysis times, LDL eluted as a sharp peak at approximately 25 s and HDL as a broad peak at slightly longer time. In addition, higher resolution was obtained on chips. To our knowledge, these results show the first separation and detection of lipoproteins in a microfluidic device using native serum samples. Atomic force microscopy was used to characterize the rms surface roughness (Rq) of microfluidic channels directly. Devices with different surface roughness values were fabricated using two different etchants for Pyrex wafers with a polysilicon masking layer. Using 49% HF, the measured roughness is Rq = 10.9 +/- 1.6 nm and with buffered HF (NH4F + HF) the roughness is Rq = 2.4 +/- 0.7 nm. At this level of surface roughness, there is no observable effect on the performance of the devices for this lipoprotein separation.

Characterization and performance of injection molded poly(methylmethacrylate) microchips for capillary electrophoresis

“…The roughness of the Ni master was found to have a maximum height of ~80 μm and an average roughness (R) of 170 nm for the Ni disk itself, 210 nm for the electroplated pattern, and 52 nm for the polished electroplated pattern. The resulting surface roughness of 52 nm is higher than the cases for wet-etched fused-silica capillary or glass CE chip shown in [46]. However, the surface roughness that we have found seems in an acceptable range since we did not experience any experimental difficulties.…”

“…The surfaces of the microchannels typically have surface roughness values of a few nanometers to a few micrometers [45]. For example, it was found that surface roughness was from 1-11 nm for a fused-silica capillary [46]. Surface roughness does not affect the separation efficiency, however, it increases the relative standard deviation (RSD) of the migration times and peak areas.…”

Microchip‐based small, dense low‐density lipoproteins assay for coronary heart disease risk assessment

“…Potential advantages of microchip CE include miniaturization, integration, high-throughput analysis, low reagent consumption, and low cost [12]. Weiller et al [13,14] first utilized microchip CE to separate high-density lipoproteins (HDLs) and LDLs. However, HDL and LDL were not baselineresolved and obtained poor reproducibility.…”

Coating of open tubular capillaries with discoidal and spherical high‐density lipoprotein particles in electrochromatography

“…In most of the studies reported in the literature, successful analysis of lipoproteins has been achieved only after permanent [12] or dynamic [13] coating of the capillary wall has been applied to suppress adsorption of the particles. Adopting a quite different approach, we have recently demonstrated the usefulness of low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) particle coatings in lipoprotein oxidation studies by CEC [14,15].…”