Preparation, Optimization And Application Of One-Dimensional Microfluidic Microbead Arrays For Protein Analysis

Microchip technology has become one of the most important areas in the field of chemistry and life sciences. One-dimensional (1-D) microfluidic bead arrays are kinds of novel biomolecular microanalysis platform that we proposed. These microchip platforms have excellent advantages in flexible array design, easy fluidic control and low consumption of the samples and reagents. In this thesis, based on this platform, modification of the antibodies on the bead surface and experimental optimization have been further investigated and have been applied for protein analysis successfully. This thesis includes three major parts as follows:1. Denaturalization of the inner wall of the microfluidic channels and preparation of the antibody-modified microbeads. In our investigation, non-specific protein adsorption on the inner wall of the PDMS micro-channels can be successfully restrained via proper modification of the inner wall. In antibody-beads preparation, the antibody immobilization was influenced greatly by protective proteins such as bovine serum albumin (BSA), which are commonly added at a high concentration to prevent the commercial antibody products from titer decrease. A simple pretreatment protocol based on ultra-filtration was carried out and was proved to be efficient for the BSA removal. Approach of protein A linkage for the improvement of antibody immobilization was also investigated.2. Optimization of experimental conditions. This part includes preparation of total protein samples from the culture cells, on-bead immuno-binding reaction and wash step, and choice of sampling. The optimized conditions were investigated and the results show that the performance of the 1-D protein arrays has been improved.3. Quantitative analysis of protein expression. The sensitivity and limit of detection was gained for the detection of P53 protein using the 1-D protein arrays. Also, we determined P53 copy number in average single A549 cell. In the illustration of differential protein expression analysis, a three-protein profiling of P53, c-Myc andβ-Actin was demonstrated. Pharmaceutical-mediated differential expressions of P53 and c-Myc in several kinds of cell lines were investigated and the reliability of the 1-D protein arrays were verified by Western blotting. In addition, preliminary work on neck malignant tumor typing has been carried out.