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志物对于预防、诊断和治疗癌症至关重要。我们实验室最近的工作导致了操纵含有生物分子的小水滴的新技术。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），我们已经用这种方法进行了简单的酶测定。由于我们的技术使用激光加热作为液滴控制的基础，我们认为它非常适合基因组分析方法，如依赖于热循环的聚合酶链反应（PCR）。我们建议联合收割机结合我们的基于激光的液滴控制技术与基因组分析工具，以开发用于筛选大量单个细胞的设备。为了测试我们的设备的能力，我们提出了一个R21项目，具有以下具体目标：目标1：优化和自动化用于基因组分析的液体处理系统。我们将选择最佳的材料和试剂用于我们的仪器。我们将自动化我们设备的液滴输送和液滴处理功能。目的2：在我们的基于液滴的系统中进行实时PCR。我们将测试整体灵敏度，并在我们的二元液体系统中定量非特异性扩增。目的3：检查小液滴中的单个细胞。该检查将包括短期和长期细胞活力测试以及单细胞基因组材料的PCR扩增。本研究完全符合RFA-CA-06-002中概述的目标。如果该计划成功，我们相信所产生的技术将证明是非常宝贵的，可以帮助医生和科学家更好地了解癌症的分子基础，同时还可以提供工具来帮助诊断疾病的特定变体，计划和评估治疗，并监测疾病复发。

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.