All experiments were performed using a laboratory-built unit. The unit consists of a ± 30 kV high voltage power supply (EH 30P3, Glassman, USA) and a UV-Vis detector (870-CE, Jasco, Japan). Electropherograms were recorded and processed with a CT-21 data processor (Peak-ABC, Singapore). The fused silica capillaries (Polymicro Technologies, Phoenix, AZ, USA) had a 75-μm i.d. and a 375-μm o.d. with a total length of 75 cm and a distance of 55 cm between the injection point and the detection window.

BSA, disodium 2,2’-biquinoline-4,4’-dicarboxylate (Na₂BCA) and tetraethyl orthosilicate (TEOS) were obtained from Sigma (St. Louis, MO, USA). Cupric sulfate, sodium carbonate and sodium tartrate were obtained from Acros (Geel, Belgium). Purified water (18 MΩ-cm) from a Milli-Q water purification system (Millipore, Bedford, MA, USA) was used to prepare all solutions.

TEOS sols were prepared by sonicating a mixture of TEOS (4.5 mL), pure H₂O (1.4 mL) and 0.1 M HCl (100 μL) for 1 h at ambient temperature until it was visibly homogeneous. The sols were stable at −20˚C for as long as two weeks. Xerogels were prepared by mixing TEOS sol (2.4 mL) with 3.9 mL phosphate buffer (50 mM, pH 7) containing 30 μg/mL BSA for protein doped preparations. The mixtures were stored at 4˚C until gelation occurred . The gels were then ground into fine powders, at which point they were ready for use.

Reagent A was an aqueous solution (100 mL, pH 11.25) of sodium carbonate monohydrate (0.8 g) and sodium tartrate (1.6 g). Reagent B was prepared by dissolving BCA (4 g) in pure water and diluting to the mark (100 mL). Reagent C was cupric sulfate pentahydrate (0.4 g) dissolved in water (10 mL). The assay reagent was prepared to have a volume ratio of reagent A:B:C equal to 26:25:1. Equivalent volume ratios of assay reagent relative to protein standard were prepared and incubated at 60˚C for 1 h. After cooling to the room temperature, the absorbance was measured at 562 nm to construct the calibration curve.

Protein release studies were conducted with various sol-gel particles (30 mg). They were immersed in phosphate buffer (50 mM, pH 7.0). At predetermined time points, the sample was filtered and the supernatant was injected hydrostatically into the CE system. In each experiment, the sample was analyzed in triplicate.

A fused-silica capillary was flushed prior to use with 0.1 M NaOH, followed by pure water, for at least 30 min each. The capillary was then flushed with 0.1 M HCl for 30 min followed by pure water for an additional 30 min.

Before analysis, the coated columns were preconditioned with the running buffer. Between sample runs, the columns were rinsed with pure water and running buffer for 1 or 2 min intervals. The samples were injected by siphoning at a height difference of 20 cm for 10 s. The samples were monitored at 278 nm with the UV absorbance detector.

We have adopted the method for protein assays because BCA better tolerates the presence of compounds that interfere with the Lowry assay. It is not affected by various detergents and denaturing agents, such as urea and guanidinium chloride, although it is more sensitive to the presence of reducing sugars. The calibration curve was prepared over a sample concentration range from 0.4 μg/ mL to 20 μg/mL. The formula of the calibration curve as derived from the least square method was y = 0.0436x + 0.1613.

The sol-gel reaction involves the following steps [25]: 1) hydrolysis of alkoxysilane; 2) condensation of hydrated silica to form siloxane bonds; and 3) polycondensation linkage of additional silanol groups to form cyclic oligomers. Protein molecules are trapped within the silicate network as it forms.

The release buffer was similar to the BGE of CE in each case. The rate of protein release from the sol-gel was determined in buffer solutions at different pH values (pH 5, 7, 8 and 9). Increasing the buffer pH enhances the electrophoretic mobility of the BSA macromolecule. Greater EOF would also occur at higher pH. The net result revealed a faster migration of the BSA macromolecule (Figure 1). Interestingly, there are two sample peaks which were assigned to be the isoforms of BSA at phosphate buffer concentration of 20 mM and pH above 5. At buffer pH 9, much greater amount of BSA was released by comparison with those at lower pH (Figures 1(c) vs. (a) & (b)). Meanwhile, a slightly different electropherogram from that shown in Figure 1(b) was demonstrated. There are two possible explanations for the phenomena. Either the migration order of the isoforms reversed or the amount of the isoforms altered as the condition changed. Conformational transitions of BSA dependent on pH and ionic strength might be the reason [11,26-28].

Figure 2 shows the release behavior of BSA encapsulated in the sol-gel. Most of the BSA was released in the first 5 h. A slower release rate was then observed, and some BSA remained encapsulated after 25 h. Gao et al. [29] have investigated the mechanism by which encapsulated BSA is released from polylactic acid-hyperbranched polyglycerol conjugate nanoparticles. The overall mechanism of BSA release from the nanoparticles is heavily dependent on copolymer relaxation, protein diffusion, protein dissolution or combinations thereof. The cumulative BSA release curves exhibited a burst effect followed by a slowly continuous release phase. Similar release behavior (Figure 2) was observed in our own work.

The effect of buffer concentration on the protein release behavior was also investigated. In each case, the buffer concentration was varied over 20, 40, 50, 60, 80 and 100 mM at definite pH value. The sample peak in-

tensity increased significantly as the releasing buffer concentration increased. This relationship was not maintained, however, when the buffer concentration was greater than 50 mM. A summary (3D plot) of the release profile of the encapsulated protein across various buffer compositions is shown in Figure 3. The peak corresponding to maximum protein release clearly occurs at

pH 7 with buffer concentrations of approximately 50 mM.

The protein loading capacities of the sol-gel (1.3 mg) were also evaluated. For BSA (1 mL) loadings of 20, 25, 30 and 40 μM, the amount of protein released after 5 h immersed in 1 mL phosphate buffer (50 mM, pH 7) was 54.01%, 45.52%, 39.92% and 34.81%, respectively.