Development and application of highly integrated protein and cell chip devices

虽然人类基因组计划已报道了约3万个人类基因，但大多数基因的功能和表达机制仍不清楚。因为基因功能的命运是由蛋白质的表达决定的，而蛋白质的表达是由细胞功能控制的。因此，有必要开发能够在单细胞和单分子水平上对蛋白质和细胞进行高通量筛选和分析的微阵列芯片设备。为了实现单细胞或单分子分析，可以在微微升和纳升体积水平进行分析的高度集成的微阵列系统是实现后基因组研究的迫切需要，例如蛋白质组学和细胞组学。在我们的项目研究中，为了实现对多个目标DNA的同时检测，利用TaqMan聚合酶链式反应提高了我们的微室阵列的可行性。从三种不同的D-…中扩增出三种不同的DNA序列在同一微室阵列中同时检测到更多的NA模板。此外，不仅通过实时监测荧光强度，还通过观察终点荧光信号，实现了对存在于微室中的初始DNA浓度的量化，每个微室从0到12个拷贝。因此，该系统被证明是一种低成本、高通量的DNA扩增和检测设备，用于临床诊断，也可以由非专家用户操作。此外，我们报告了改进的微室阵列，可以在单细胞水平上同时监测超过25,000个细胞的钙动员。我们利用微阵列开发了一种新的高通量的抗原特异性单个B细胞筛选和分析系统，该系统是通过检测针对感兴趣抗原的抗原特异性单个B细胞来进行的。单细胞芯片系统不需要像传统杂交瘤技术那样使用骨髓瘤，可以直接从细胞悬液中筛选抗原特异性单个B细胞，并在单细胞水平上分析抗原特异性抗体DNA。该系统操作简单，与传统技术相比，制备单抗的速度足够快。此外，该系统还可以利用芯片器件进行高通量的单细胞分析。因此，我们在本项目中使用微微升或纳米升的小室阵列系统来进行DNA、蛋白质和细胞的分析。它们还可能应用于DNA和细胞的检测，这将导致未来的免疫治疗或基因治疗。较少

Though the number of human genes was reported to be approximately 30,000 from human genomic project, the functions and expression mechanism for most of the genes are remain to be unknown. Because the fate of the genes functionality is determined after protein expression, but proteins expression is controlled by cellular function. Therefore, it is necessary to develop the microarray chip devices that can perform high-throughput screening and analysis of proteins and cells at single-cell and single-molecule level. For achieving single-cell or single-molecule analysis, highly integrated microarray systems that can perform assays at pico- and nano-liter volume level are greatly desirable to realize post-genomic research, such as proteomics and cellomics. In our project research, for achieving simultaneous detection of several numbers of target DNA, the feasibility of our microchamber array was improved by using TaqMan PCR. Three different DNA sequences were amplified from three different D … More NA templates and detected in the same microchamber array simultaneously. In addition, the quantification of initial DNA concentration present in a microchamber was achieved from 0 to 12 copies per chamber, not only by monitoring the real-time fluorescence intensity but also by observing the end point fluorescence signal. Therefore, this system proves to be a promising device for the low-cost, high-throughput DNA amplification and detection for point-of-care clinical diagnosis, which can also be handled by non-specialist users.Further, we report improved microchamber array to monitor Ca^<2+> mobilization of over 25,000 cells simultaneously at a single-cell level. And we have developed a novel high-throughput screening and analysis system for antigen-specific single B-cells using the microarray, which was carried out by detecting antigen-specific single B-cells against an antigen of interest. The single-cell microarray system does not need to use myeloma as in the case of conventional hybridoma technique, and can screen the antigen-specific single B-cells directly from cell suspension and analyze antigen-specific antibody DNA at a single-cell level. This system is simple and easy in its operation, and quick enough for making monoclonal antibodies when compared to conventional techniques. Moreover this system can perform high-throughput single-cells analysis using chip devices.Therefore, we have addressed the analysis of DNA, protein and cell using pico- or nano-liter chamber array system in this project. They might also be applicable for detection of DNA and cells, which lead to immune therapy or gene therapy in the future. Less