Droplet Cell Array Assays

描述（由申请人提供）：在细胞水平上了解癌症的基本遗传和分子标记对于预防、诊断和治疗癌症至关重要。我们实验室最近的工作带来了操纵含有生物分子的小液滴的新技术。我们使用激光诱导表面张力梯度，这使我们能够选择性地移动非常小的单个液滴（μ L-pL），并且我们已经使用这种方法进行了简单的酶分析。由于我们的技术使用激光加热作为液滴控制的基础，我们相信它非常适合基因组分析方法，如依赖热循环的聚合酶链反应（PCR）。我们建议将基于激光的液滴控制技术与基因组分析工具相结合，开发一种用于筛选大量单个细胞的设备。为了测试我们的仪器的能力，我们提出了一个R21项目，其具体目标如下：目标1：优化和自动化基因组分析的液体处理系统。我们将为我们的仪器选择最合适的材料和试剂。我们将自动化我们设备的液滴输送和液滴处理能力。目的2：在我们基于液滴的系统中进行实时PCR。我们将测试总体灵敏度，并在我们的二元液体系统中量化非特异性扩增。目的3：检查小液滴内的单个细胞。该检查将包括测试短期和长期细胞活力和单细胞基因组物质的PCR扩增。本研究与RFA-CA-06-002中概述的目标完全一致。如果这个项目取得成功，我们相信由此产生的技术将证明在帮助医生和科学家更好地了解癌症的分子基础方面是无价的，同时也提供了帮助诊断特定疾病变体、计划和评估治疗以及监测疾病复发的工具。

DESCRIPTION (provided by applicant): Understanding the basic genetic and molecular markers of cancer at the cellular level is vital for preventing, diagnosing, and treating cancer. Recent work in our lab has led to new technologies for manipulating small aqueous drops containing biological molecules. We use laser light to induce surface tension gradients, which allows us to selectively move very small individual droplets (mu L-pL), and we have performed simple enzymatic assays with this approach. Because our technique uses laser heating as a basis for droplet control, we believe that it is well suited to genomic analysis methods, such as polymerase chain reaction (PCR), which rely on thermal cycling. We propose to combine our laser-based droplet control techniques with genomic analysis tools to develop a device for screening large numbers of individual cells. To test the capabilities of our apparatus, we propose an R21 project with the following specific aims: Aim 1: Optimize and automate the liquid handling system for genomic analysis. We will choose the optimal materials and reagents for use in our apparatus. We will automate both the droplet delivery and the droplet handling capabilities of our device. Aim 2: Perform a real-time PCR in our droplet-based system. We will test the overall sensitivity and quantify nonspecific amplification in our binary liquid system. Aim 3: Examine single cells within small droplets. This examination will include testing for short- and long-term cell viability and PCR amplification of single-cell genomic material. This research is fully consistent with the goals outlined in RFA-CA-06-002. If this program is successful, we believe that the resulting technology will prove invaluable in helping doctors and scientists better understand the molecular basis of cancer while also providing tools to help diagnose specific variants of disease, plan and assess therapy, and monitor disease recurrence.