Single-step approach for fabrication of vancomycin-bonded silica monolith as chiral stationary phase.

A vancomycin-bonded silica monolithic column for capillary electrochromatography (CEC) was prepared by a single-step in situ sol-gel approach. This sol-gel process incorporates a synthetic sol-gel precursor which contains a macrocyclic antibiotic, vancomycin, to form a porous silica network inside a fused-silica capillary. To avoid degradation of vancomycin during the column fabrication, a mild step was adopted into the sol-gel process. The performance of the vancomycin chiral stationary phase was investigated by CEC in both the reversed-phase mode and the normal-phase mode. The vancomycin chiral stationary phase was optimized with respect to vancomycin loading in the reversed-phase mode for chiral separation of thalidomide enantiomers. The best efficiency and resolution values of 94600plates/m and 5.79, respectively, were achieved. The optimized column was further applied to chiral separation of alprenolol enantiomers. A plate height of less than 7µm for the first eluted enantiomer of alprenolol was obtained in an aqueous mobile phase at a flow rate of 0.74mm/s. Using enantiomers of seven ß-blockers and some other basic enantiomers as test analytes, separation efficiencies of up to 148100plates/m in the reversed-phase mode and up to 138100plates/m in the normal-phase mode were achieved.

Fiber-optic particle plasmon resonance sensor for detection of interleukin-1ß in synovial fluids.

A facile and label-free biosensing method has been developed for determining an osteoarthritis concerned cytokine, interleukin-1ß (IL-1ß), in synovial fluids. The biosensing technique, fiber-optic particle plasmon resonance (FOPPR), is based on gold nanoparticles-modified optical fiber where the gold nanoparticle surface has been modified by a mixed self-assembled monolayer for further conjugation of anti-IL-1ß antibody and minimization of nonspecific adsorption. Upon binding of IL-1ß to anti-IL-1ß on the gold nanoparticle surface, the absorbance of the gold nanoparticle layer on the optical fiber changes and the signal change is enhanced through multiple total internal reflections along the optical fiber. Results show that the detection of IL-1ß in synovial fluid by this sensor agrees quantitatively with the clinically accepted enzyme-linked immunosorbent assay (ELISA) method but a much shorter analysis time is required (<10 min). The sensor response versus log concentration of IL-1ß was linear (r=0.9947) over the concentration range of 0.050-10 ng/mL and a limit of detection (LOD) of 21 pg/mL (1.2 pM) was achieved. Such a LOD for IL-1ß (17 kDa) represents a major advancement in the field of real-time monitoring of low molecular weight proteins in complex biological fluids.

Hybrid surface-enhanced Raman scattering substrate from gold nanoparticle and photonic crystal: maneuverability and uniformity of Raman spectra.

A novel hybrid surface-enhanced Raman scattering (SERS) substrate based on Au nanoparticles decorated inverse opal (IO) photonic crystal (PhC) is presented. In addition to the enhancement contributed from Au nanoparticles, a desired Raman signal can be selectively further enhanced by appropriately overlapping the center of photonic bandgap of the IO PhC with the wavelength of the Raman signal. Furthermore, the lattice structure of the IO PhC provides excellent control of the distribution of Au nanoparticles to produce SERS spectra with high uniformity. The new design of SERS substrate provides extra maneuverability for ultra-high sensitivity sensor applications.

Single-step approach to beta-cyclodextrin-bonded silica as monolithic stationary phases for CEC.

A novel single-step sol-gel approach for the preparation of beta-CD-bonded silica monolithic electrochromatographic columns is established. The porous silica networks were fabricated inside fused-silica capillaries using sol-gel processing of tetramethoxysilane and an organfunctional silicon alkoxide that contains beta-CD. Scanning electron micrographs and nitrogen adsorption-desorption data showed that these functional monolithic columns have double pores structures with micrometer-size co-continuous through-pores and silica skeletons with open mesopores. The beta-CD monolithic columns have successfully been applied to the separation of several neutral and negatively charged isomers by CEC. The column performance was evaluated by using positional isomers of naphthalenedisulfonic acid as model compounds. A plate height of less than 10 mum for the first eluted isomer of naphthalenedisulfonic acid was obtained at an optimal flow rate (0.47 mm/s) of the mobile phase. Moreover, the columns have been proved to be stable for more than 100 runs during 3 months period and show reasonable column reproducibility.

Identification of a new chromophoric substrate in the library of amino acid p-nitroanilides for continuous assay of VanX, a D,D-dipeptidase essential for vancomycin resistance.

As one of key bacterial proteins involved in vancomycin resistance, VanX is a D,D-dipeptidase that impedes bacterial cell wall biosynthesis by hydrolyzing the essential D-Ala-D-Ala dipeptide. Based on a report by Crowder and co-workers that L-alanine-p-nitroanilide (L-Ala-pNA) was a useful substrate for continuous assay of VanX, we constructed a library of 35 L- and D-amino acid p-nitroanilides to provide the needed diversity to discover new substrates that are more specific than L-Ala-pNA. We report here that, among all compounds tested, D-leucine-p-nitroanilide (D-Leu-pNA) was found to be the best substrate for VanX enzyme (KM=8.9+/-1.2 mM, kcat=0.0102+/-0.0016 s(-1), kcat/KM=0.0012 mM(-1)s(-1)). Although it is catalytically inefficient, this new VanX substrate needs essentially no sophisticated synthetic chemistry for preparation and therefore offers a convenient means for routine analysis of enzyme catalysis and the screening of potential inhibitors. Moreover, because it is the uncommon leucine in its D form in D-Leu-pNA, enzymatic activities due to other contaminated species in Escherichia coli used for VanX overproduction should be greatly reduced.