| One-dimensional (1-D) microfluidic beads array is a novel biomolecular microanalysis platform with characteristics of multiplxed and parallel assay. This technology combines the characteristics of beads array with the rapid binding kinetics of homogeneous assays, and the liquid handling capability of microfluidics. Such a platform offers 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 chip are an extremely versatile polymer platform with many cabinets along a single microchannel that has been successfully used for the protein profiling detection. To further extend its applications for the mutations detection is obviously a promising work, which can represents an important step in the direction of making this system serve as a universal chemical and biological detection platform. In this thesis, aiming for higher sensitivity and lower cost of per assay, we have combined a series of methods based on the 1-D chip for mutations discrimination and the main details are as follows:(1) The application of a one-dimensional (1-D) microfluidic beads array that composed of individually addressable functionalized SiO2 beads has been demonstrated for detection of single-base mutations based on"sandwich"hybridization assay without additional sample labeling and PCR amplification. According to the coding sequence comprising codon 175 of p53 gene, a series of capture probes were designed and immobilized on surface of microbeads using biotin-streptavidin system. This 1-D microfluidic beads array was sufficiently sensitive to identify single-nucleotide mutations in 40 pM quantities of DNA targets and could discriminate the mutated alleles in an excess of non-mutated alleles at a level of 1 mutant in 100 wild-type sequences. The surface of beads was regenerated and rehybridized up to six times without obvious loss of signal. The entire reaction process was done at room temperature within minutes, and only 2-10μL sample solution was needed to complete the whole detection process. The p53 genotypes of A549 cell line, CNE2 cell line and SKBr-3 cell line also were correctly evaluated by using mRNA extracts as target without need for sample labeling and amplification. Thus, this platform enabled rapid and exact discrimination of gene mutations in the form that is reusable, simple handling of liquid, low cost, and little reagent consumption. (2)In this paper, an on-chip oligonucleotide ligation approach that arrayed a series of functionalized beads in a single microfluidic channel was described for detection of low-abundant point mutations in p53 gene. As a demonstration, it was found that the on-chip beads ligation held high mutation discrimination sensitivity in 1 pM quantities at a SNR (signal-to-noise ratio)>2 using synthesized DNA oligonucleotides in accordance with target fragment. The RT-PCR products of tumor cell line A549, CNE2 and SKBr-3 were further examined to distinguish the point mutation at codon 175 of p53 gene. This approach was capable of detecting a point mutation in a p53 oncogene at a level of 1 mutant in 1000 wild-type sequences using PCR products without the need of LDR amplification. Additionally, this on-chip beads ligation approach also displayed other microfluidic-based advantages of simple handling (one sample injection per test), little reagent quantities, and low potential of contaminations.(3) This study reported the development of a one-dimensional (1-D) microfluidic beads array-based apyrase-mediated allele-specific primer extension approach for typing of single nucleotide polymorphisms (SNP). This chip approach also was utilized to predict risk of weaning steroid after 1-year post-transplantation based on designed extension primers according to two polymorphisms C3435T and G2677T of MDR1 gene. This on-chip beads extension approach could sensitize to 30 pM quantities of synthesized DNA oligonucleotides at a SNR (signal-to-noise ratio)>2, that corresponded to an absolute detection sensitivity of～10-17 mol of target molecules in about 2μl sample. The multiplexing capacity of 1-D microfluidic beads array was demonstrated by simultaneous detection of two polymorphisms C3435T and G2677T and the correctness of on-chip beads extension for SNP typing of practical samples was also further confirmed through the sequencing of PCR products. This method could use the target DNA without labelling for direct discrimination of gene mutations, and decreased the cost of test and avoided complicated labelling process. This on-chip beads extension approach provided a simple, efficient and rapid analysis mode that could be applied in the detection of functional SNP associated with the drugs resistance to predict the therapy efficacy of drugs.(4)This study reports the development of a novel alkaline phosphatase (originated from shrimp)-mediated enzymatic approach for typing of single nucleotide polymorphisms (SNP) based on allele-specific primer extension and its application on fabricated one-dimensional (1-D) microfluidic beads array. This alkaline phosphatase-mediated extension discrimination took advantage of the fact that the reaction kinetics differed between matched and mismatched configurations of allele-specific primers hybridized to DNA template. This difference allowed the incorporation of nucleotides when the reaction kenetics was fast as a result of matched hybridization structure, but degraded the nucleotides before extension by alkaline phosphatase (AP) when the reaction kenetics was slow caused by the mismatches. The capacity of mutation discrimination of this AP-mediated allele-specific primer extension was determined using fabricated one-dimensional (1-D) microfluidic beads array and indicated excellent specificity when alkaline phosphatase was included in the extension mixture despite of the existence of mismatched duplexes. Additionally, this on-chip beads extension approach could sensitize to 0.1 pM quantities of synthesized DNA oligonucleotides at a SNR (signal-to-noise ratio)>2, that corresponded to an absolute detection sensitivity of～10-19 mol of target molecules in about 2μl sample. The multiplexing capacity of on-chip beads extension was demonstrated by the simultaneous detection of two polymorphisms C3435T and G2677T and the correctness of on-chip beads extension for SNP typing of practical samples was also further confirmed through the sequencing of PCR products. In conclusion, this novel enzymatic approach displayed reliable specificity and cost-effectiveness for SNP typing.(5) This study reports the development of a microfluidic rolling circle amplification (RCA) approach for mutation discrimination based on one-dimensional microfluidic beads array chip. This methid used oligonucleotide ligation assay for mutation discrimination and RCA for signal production. This microfluidic RCA process could sensitize to 30 pM quantities of synthesized DNA oligonucleotides at a SNR (signal-to-noise ratio)>2. The RT-PCR products of tumor cell line A549, CNE2 and SKBr-3 were further examined to distinguish the point mutation at codon 175 of p53 gene. The combination of 1-D microfluidic beads array and RCA could make it very sensitive for mutation discrimination. Although actual sensitivity was not satisfactory, the further research will greatly improve it.
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