| As a novel chemical/biological microanalysis platform, One-dimensional microfluidic beads array chip (1-D chip) combines the merits of microfluidics, microarray and beads heterogeneous binding technologies. The primary applications in protein profiling and nucleic acids detection have demonstrated its predominance in the field of biological research. Applications extension of this platform and further performance improvements in proteins and nucleic acids analysis should make it a practical and high sensitive analytical system. In this thesis, focusing on the applications extension and sensitivity improvement of the 1-D chip, three parts of works have been demonstrated and the details are shown as follows.Firstly, the applications of 1-D chip have been extended to disease-associated genes and virus genes analysis:(1) A sandwich hybridization assay for multiple transcripts detection based on 1-D chip has been developed. Three sandwich probes for p53, c-myc and NME1 have been designed and synthesized according to the specific fractions of the three genes respectively. The microbeads modified with capture probes were arrayed into the microchannel to form one dimensional nucleic acids beads array for multiple transcripts detection. The array based sandwich assay performance for DNA detection was first tested and simultaneous multiple DNA analysis in the mixed sample has also been achieved. Three transcripts expressions in tumor cell line samples before and after 5-flouorouracil stimuli were then demonstrated. The LOD of this platform for DNA analysis was found to be 0.02 nM and a linear range of 0.02-1.0 nM was obtained.(2) Tumor metastasis is the main cause for mortality and has close relationships with tumor metastasis associated genes. Based on the previous work, 1-D chip was utilized to perform tumor metastasis associated genes expression research. In the experiments, by choosing three genes tightly associated with tumor metastasis, i.e. E-cadherin, mts1 and NME1 as target genes, using two colon cancer cell lines (SW480 and LoVo) and two prostate cancer cell lines (PC-3M-2B4 and PC-3M-1E8) with different metastatic abilities as two models, we demonstrated the three tumor metastasis associated genes expression differentiation between the cancer cells with different metastatic abilities. The results show that the 1-D chip may hold potential in tumor early diagnosis, forecast and therapy. (3) Hepatitis B virus (HBV) has 8 genotypes and various subtypes HBV show different nosogenesis and drug-fast characteristics. One dimensional microfluidic beads array was exploited to find its application in HBV genotyping. According to the gene sequences of chinese most prevalent B, C and D subtypes published in GenBank, three sandwich genotyping probes were designed and utilized to build B, C, D genotyping 1-D chip. By optimizing the hybridization and elution conditions, we realized three HBV subtypes genotyping based on the 1-D chip. This method is simple, fast and accurate. It holds the potential for clinical HBV diagnosis.Secondly, sensitive methods for nucleic acids and proteins analysis based on 1-D chip have been developed:(4) Based on 1-D chip, combining the gold nanoparticle probes and polymerase catalyzed fluorescence labeling technology, an in situ signal amplification method for sensitive and high throughput nucleic acids detection has been developed. By employing a novel agarose beads and optimizing experimental conditions, the LOD of this method for DNA analysis was 7.0 fM, capable of detecting p53 mRNA in cell lysate containing as low as 140 CNE2 cells per microlitre sample. Compared with previous sandwich hybridization assay, a 3 orders higher sensitivity was obtained by this method. Furthermore, this in situ signal amplification technology was readily suitable for parallel multi-target detection. This work dramatically improves the performance of the 1-D chip and makes this platform more competent in nucleic acids detection.(5) In order to further improve the sensitivity for protein detection on the basis of our previous"two-site"sandwich immunoassay, a telomerase catalyzed fluorescent probes for sensitive proteins detection based on 1-D chip has been developed. Telomerase can synthesize repeated TTAGGG units by its RNA templates and add these units to the 3'of telomere. Utilizing this characteristics, a biotin-labeled fluorescent probes containing numerous fluorescein was synthesized. The generated fluorescent probes were specifically captured by biotin-labeled primary antibody through biotin-streptavidin linkage and thus to realize high sensitive antigen detection. The LOD of this method for purified p53 protein analysis was 1.0 pM, nearly 2 orders higher than our previous"two-site"sandwich immunoassay. It can perform p53 protein detection in CNE2 cell lysate containing as low as 85 cells per microlitre sample. Three genes expression variations in CNE2 cells before and after 5-flouorouracil stimuli were also assessed using this method. Compared with immuno-PCR and immuno-RCA, this technology is simple, fast and able to perform high throughput protein profiling. It may play an important role in low-abundant cellular proteins detection.At last, the 1-D chip has been improved by fully integration of micropump based on evaporation and capillary effects:(6) To solve the limitations of the liquid driven models we have adopted in 1-D chip such as pressure drive, electroosmosis drive and gravity drive, the micropump based on evaporation and capillary effect was fully integrated into the 1-D chip. Such an integrated beads array was firstly designed and fabricated, and then the effects of temperature, evaporation area and humidity on the flow rate were tested respectively. The micropump performance in long time running under certain conditions was also assessed. The results show that the integrated micropump can provide steady flow rate under the general conditions and the flow rate can be readily adjusted by changing temperature and evaporation area, make the experiment results more accurate and reproducible.
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