sepharose and cyanine-dye-3

To improve the sensitivity of fluorescence detection in biochip, a new kind of substrates was developed by agarose coating on silica opal film. In this study, silica opal film was fabricated on glass substrate using the vertical deposition technique. It can provide stronger fluorescence signals and thus improve the detection sensitivity. After coating with agarose, the hybrid film could provide a 3D support for immobilizing sample. Comparing with agarose-coated glass substrate, the agarose-coated opal substrates could selectively enhance particular fluorescence signals with high sensitivity when the stop band of the silica opal film in the agarose-coated opal substrate overlapped the fluorescence emission wavelength. A DNA hybridization experiment demonstrated that fluorescence intensity of special type of agarose-coated opal substrates was about four times that of agarose-coated glass substrate. These results indicate that the optimized agarose-coated opal substrate can be used for improving the sensitivity of fluorescence detection with high quality and selectivity.

Topics: Carbocyanines; Drug Stability; Glass; Materials Testing; Minerals; Oligonucleotide Array Sequence Analysis; Sensitivity and Specificity; Sepharose; Silicon Dioxide; Spectrometry, Fluorescence

The antibody microarray, a high-throughput multiplex immunoassay method, has become a significant tool for quantitative proteomics studies. We describe here the strategies for optimizing the condition of antibody microarray building based on agarose-coated slides. In this study, modified glass slides were robotically printed with capture antibodies against monocyte chemoattractant protein 1 (MCP-1), then dilutions of the cytokine were applied to the arrays, and the protein was detected with biotin-labeled antibody coupled with Cy3-conjugated streptavidin. Thus a protein profiling microarray based on sandwich immunoassay has been established. Various factors in the production of antibody microarrays were analyzed: the capture antibody concentrations, shelf life of the postprinting slides, blocking buffers, and reproducibility of the system. A calibration curve with a correlation coefficient of 0.9995 was established which suggested that the matrix can retain arrayed proteins in near-quantitative fashion. The results revealed high signal uniformity and reproducibility with regard to intra-array (1.3%) and the interarray (8.7%) variation at the capture antibody concentration of 125 microg/mL. Besides, the printed arrays could be stored for at least two months without any apparent change of the performance parameters.

Topics: Antibodies; Biotin; Carbocyanines; Chemokine CCL2; Immunoassay; Protein Array Analysis; Proteomics; Sepharose; Streptavidin