Applications Of One-Dimensional Microfluidic Beads Array Chip In Nucleic Acids And Protein Kinase Analysis

One-dimensional microfluidic beads array (1-D chip) is a novel biomolecular microanalysis platform with characteristics of multiplexed and parallel assay. This design combines the merits of microfluidics, microarray and beads heterogeneous binding technologies. Such a platform offer excellent advantages in flexible array design, small sample volumes and potential of high throughput analysis in a single microchannel. Applications extension of this platform can make this system serve as a universal chemical and biological detection platform. In this thesis, focusing on the applications extension and performance improvement of 1-D chip, three parts of works have been demonstrated as follows:(1) Nucleic acids detection using cationic fluorescent polythiophene based on 1-D chipThe cationic water-soluble polythiophene derivatives was introduced into 1-D chip for fluorescence detection of nucleic acids, and demonstrated its capability on rapid detection of p53 target cDNA with different concentration. Furthermore, we have examined simultaneously the transcripts expression changes of p53, c-myc and cyclin-d1 in CNE2 before and after anti-tumor drug treatment. This methodology integrates the advantages of both 1-D chip and the fluorescent polymeric transducer. A more desirable feature is that it offers a label-free, low-cost and sensitive approach for multiple targets detection.(2) Phosphor-specific antibody for protein kinase activity analysis on 1-D chipThe peptide-functional beads were deposited along a microchannel to form an addressable beads microarray. After phosphorylation of the peptides, phosphorylation was detected by using anti-phosphorylated amino acids antibody. Based on 1-D chip, we have successfully determined protein kinase A (PKA) as a model with a linear dynamic range of 5-100 nM. Moreover, the assay was available to investigate the effect of inhobitor H-89 on PKA. This approach extends upon our prior efforts of the 1-D chip, and will find wider application in high-throughput kinase assay and drug screening.(3) Kinase-catalyzed biotinylation for protein kinase assay with 1-D chip.Based on the labeling of a specific phosphorylation event with fluorophores, a simple method for cost-effective kinase activity and inhibitor screening assays has been developed. The phosphorylation reaction is coupled with the biotinylation of the kinase substrate using a biotin-modified ATP as the co-substrate. When the phosphorylated and biotinylated kinase substrate is exposed to streptavidin-coated fluorophores, the high affinity between the streptavidin and biotin resulted in the attachment of fluorophores on the kinase substrate. The fluorescent response enables monitoring the activity of the kinase, as well as the inhibition of small molecule inhibitors. We determined the activity of protein kinase A and Abl protein tyrosine kinase with their substrate peptides, respectively. The detection limits for PKA and Abl were determined as 35 U/ml and 15 U/ml. Subsequently, multiplexed protein kinases and inhibitor screening has been examined simultaneously with 1-D chip. Compared with the former methods, this work has shown many advantages involving low-cost, more practical and convenient.