One-Dimensional Microfluidic Beads Array For Nucleic Acids Detection

One-dimensional microfluidic beads array (1-D chip) is a novel biomolecularmicroanalysis platform that combines the characteristics of beads array with the rapidbinding kinetics of homogeneous assays, and the liquid handling functions ofmicrofluidics. Such a platform offer excellent advantages in flexible array design,small sample volumes, short assay times, simple assay protocols, reduced cost per test,and potential of high throughput analysis. The central components of this 1-D chipare an extremely versatile polymer platform with many cabinets along a singlemicrochannel that has been successfully used for the protein profiling detection. Tofurther extend its applications for the nucleic acids detection is obviously a promisingwork, which can represents an important step in the direction of making this systemserve as a universal chemical and biological detection platform. In this thesis, aimingfor higher sensitivity and lower cost of per assay, we have combined a series ofmethods with the 1-D chip for nucleic acids detection and the main details are asfollows:(1) According to the special sections of human gene p53, c-myc and cyclin d1mRNA sequences respectively, three biotinylated molecular beacons have beeddesigned and then modified on the surface of the microbeads in the 1-D chip channelto form a one dimensional microfluidic molecular beacon beads array for multiplenucleic acids detection. In the experiment, as a demonstration, the synthesized DNAtargets according to the same sequence with a fraction of human gene p53 mRNAwere investigated with a limit of detection 0.5 nM. We have further examined theasymmetry PCR products of human gene p53, c-myc and cyclin-d1 to detect themRNA expression changes before and after anticancer drug 5-flouorouracil treatment.(2) Surface-immobilized molecular beacons usually have relatively lowfluorescence enhancement after hybridization, which causes significant limitations totheir applications in 1-D chip. To solve this problem, we have designed a molecularbeacon with a restriction endonuclease recognition site at its loop structure, to studythe behavior of the molecular beacon on solid matrix and to improve its performance.The maximum fluorescence enhancement of the molecular beacon caused byrestricting cleavage after hybridization was 5.2, close to its correspondingenhancement 5.9 in solution, higher than its corresponding enhancement 2.7 that justcaused by hybridization. Moreover, as a demonstration, such a immobilized molecular...