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Here we present a unique fabrication of capillary coatings by using a native protein-phospholipid assembly, human low-density lipoprotein (LDL). By taking advantage of its constituents of apoproteins and phospholipids, two types of LDL coatings (non-covalent and covalent) were constructed. The non-covalent LDL coatings were prepared through physical adsorption of its phospholipids on fused silica, and the covalent coatings were prepared through covalent bonding of its apoproteins with the aminopropyl-modified silica. These coatings were subjected to CE separation of basic proteins (lysozyme, cytochrome c, ribonuclease A, and alpha-chymotrypsinogen A). Although both types of the coatings display efficient separation of the four proteins while effectively inhibiting the undesirable adsorption, the covalent LDL coatings provide higher separation efficiency (0.67-2.01 x 10(5) theoretical plates/m) and longer coating-life. Results also reveal that the LDL coatings can hydrophobically interact with proteins and provide options for tuning the separation selectivity of proteins besides their inherent electrophoretic mobility. By using SYPRO Orange staining and fluorescence imaging, the coverage and homogeneity of the LDL coatings along the capillary were further visually characterized. It was found that the coverage of LDL particles in the covalent coatings was much higher than that in the non-covalent coatings. To the best of our knowledge, this is the first report describing the enhanced CE protein separation by lipoprotein coating. In virtue of the apoproteins-phospholipids structure, the LDL coatings are potentially useful in various LDL-related binding studies and complex bio-analysis.

Multiple labeling of nucleic acids by intercalative dyes is a promising method for ultrasensitive nucleic acid assays. The properties of the fast dissociation and instability of dye-DNA complexes may prevent from their wide applications in CE-LIF nucleic acid analysis. Here, we describe an optimum CE focusing method by using appropriately paired sample and separation buffers, Tris-glycine buffer and Tris-glycine-acetic acid buffer. The developed method was applied in both uncoated and polyacrylamide coated fused-silica capillary-based CE-LIF analysis while the sample and separation buffers were conversely used. The complexes of intercalative dye benzoxazolium-4-pyridinium dimer and dsDNA were greatly focused (separation efficiency: 1.8 million theoretical plates per meter) by transient isotachophoresis mechanism in uncoated capillary, and moderately focused by transient isotachophoresis in combination of field amplified sample stacking and further stabilized by the paired buffer in polyacrylamide coated capillary. Based on the developed focusing strategy, an ultrasensitive DNA assay was developed for quantitation of calf thymus dsDNA (from 0.02 to 2.14 pM). By the use of an excitation laser power as low as 1 mW, the detection limits of calf thymus dsDNA (3.5 kb) are 7.9 fM in concentration and 2.4x10(-22) mol (150 molecules) in mass. We further demonstrate that the non-gel sieving CE-LIF analysis of DNA fragments can be enhanced by the same strategy. Since the presented strategy can be applied to uncoated and coated capillaries and does not require special device, it is also reasonable to extend to the applications in chip-based CE DNA analysis.

The measurement of protein binding of highly hydrophobic molecules is challenging due to poor solubility and strong adsorption, and further complicated by the competition of lipophilic partition in biological systems. Here, an attempt is presented to simultaneously simulate protein binding and lipophilic partition of hydrophobic molecules in a single system using CE. In this system, the incorporated biocompatible micelles also facilitate the protein binding study of hydrophobic molecules by enhancing their solubility (27 to 10(4) times) and eliminating the problematic adsorption. An equation is derived to describe the competition of protein binding and lipophilic partition and to estimate the protein binding constants in nonmicellar aqueous solution. Five polycyclic aromatic hydrocarbons (PAHs) and HSA were used as model hydrophobic compounds and protein, respectively. The study of the competition between lipophilic partition and protein binding reveals that the binding of the PAHs to HSA is governed by hydrophobic interactions and such binding (except naphthalene) can be eliminated by the lipophilic partition in the nonionic surfactant Tween-20. The developed method may be extended to evaluate the interactions of various macromolecules (receptors, enzymes, proteins, and DNA/RNA) and hydrophobic molecules.

An on-line SPE-HPLC method, using a monolithic poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (poly(GMA-EDMA)) based weak cation-exchange (WCX) column, was developed for simultaneous determination of alpha1-adrenergic receptor antagonists in human plasma. The monolithic WCX column was prepared by an in-situ polymerization protocol and modified stepwise with ethylenediamine and chloroacetic acid. On connecting this column to an injection valve, an on-line SPE protocol could be established for removal of matrices (mainly proteins and lipids) and preconcentration of four alpha1-adrenergic receptor antagonists in human plasma. This method was validated and then used for determination of terazosin, alfuzosin, prazosin, and doxazosin in clinical plasma samples. For all analytes, each calibration curve was found to be linear over a range of 0.005-5 microg/mL (R>0.997), and the limit of detection for each analyte was 0.5 ng/mL. Recovery (>80%) and precision (RSD<15%) for inter- and intra-day assay were tested at three concentration levels of each analyte and showed acceptable results for quantitative assay. Real samples from hypertensive patients were monitored and results were in agreement with those of the conventional liquid-liquid extraction-HPLC method. Furthermore, due to its good permeability and biocompatibility, the monolithic WCX sorbent could be reused more than 300 times. The proposed method was especially appropriate for multi-analyte monitoring in plasma samples.