Nanodroplets Based High Throughput Single-Cell PCR System

描述(由申请人提供)：聚合酶链式反应系统对细胞内容物的化学分析，如核酸和蛋白质，对生物、医学和制药界具有重要意义。细胞内物质中异常基因和蛋白的检测为疾病的早期诊断提供了重要线索。对逐渐变小的纯细胞群体进行精确分子分析的愿望已经发展成为微基因组学的新兴领域。不幸的是，由于缺乏确保此类研究可靠性所需的过程控制，研究人员倾向于放弃使用微基因组技术。微基因组技术中的主要困难是处理少量样本的困难，无法制备和操纵单细胞，以及无法具有高通量能力。更重要的是，这是极其劳动密集型的，需要昂贵的设备来分离单个细胞并对每个细胞进行扩增，所以每次可以检测的细胞数量非常有限。因此，Maxwell Sensors Inc.建议开发一种基于纳米液滴的高通量单细胞聚合酶链式反应(HTSC)芯片/系统，该芯片/系统将提供对所需目标细胞的病理学、肿瘤发生和其他过程的发展至关重要的前所未有的信息。HTSC聚合酶链式反应系统结合了纳米液滴、微流控和荧光逆转录(RT)聚合酶链式反应技术，以快速、高通量和经济高效的方式进行单细胞(SC)逆转录聚合酶链式反应。这一高度集成的平台被配置为精确地将单个细胞制备和封装在纳米液滴中，同时执行数百或数千个纳升体积的单细胞聚合酶链式反应，快速分析芯片上的聚合酶链式反应结果，并为用于临床诊断或生物医学研究目的的大规模人群的单细胞分析开辟道路。纳米液滴的高保真操作和执行核酸放大的能力相结合，为开发强大的自动化仪器提供了可能性。

DESCRIPTION (provided by applicant): PCR System Chemical analysis of cellular contents, such as nucleic acids and proteins, is of significant interest to the biological, medical, and pharmaceutical communities. Detection of abnormal gene and protein in the intracellular materials provide important clues for early disease diagnosis. The desire to perform precise molecular analysis on progressively smaller, pure cell populations has developed into the bourgeoning field of microgenomics. Unfortunately, investigators have tended to forgo the use of microgenomic technologies due to a lack of process control needed to ensure reliability of such studies. Key difficulties in microgenomic technologies are the difficulty in handling a minute amount of sample, inability to prepare and manipulate single cell, and inability to have high throughput capability. More importantly, it is extremely labor intensive and requires expensive equipment to isolate single cells and to perform amplification on each cell, so that the cell number that can be tested each time is very limited. Therefore, Maxwell Sensors Inc. proposes to develop a nanodrop based High Throughput Single Cell PCR (HTSC PCR) chip/system that will provide unprecedented information critical to the development of pathology, oncogenesis, and other processes of a desired target cell. The HTSC PCR system combines nanodroplet, microfluidics, and fluorescent reverse transcription (RT) PCR technologies for performing single cell (SC) RT PCR in a quick, high throughput and cost effective fashion. This highly integrated platform is configured to precisely prepare and encapsulate a single cell in a nanodroplet, simultaneously perform hundreds or thousands of single cell PCR in nanoliter volumes, rapidly analyze the PCR results on chip, and open the road to single cell analyses of large populations for clinical diagnostics or biomedical research purpose. The combination of high fidelity manipulation of nanodroplets and the ability to perform nucleic acid amplification offers the possibility of developing powerful automated instruments.