An RNA Sensor for Detection of Circulating Tumor Cells

该提案旨在开发用于检测循环肿瘤细胞的RNA传感器。 RNA检测基于杂交“夹心”。已经选择了两种靶RNA用于临床 重要的癌症（前列腺、乳腺和黑色素瘤），并且文库选择方案将用于 鉴定/优化反义寡核苷酸（阿索）结合可及位点。硅纳米线将被 用阿索共价衍生至靶RNA中的文库选择位点（阿索-1）。ASOi纳米线将 然后通过流体沉积被沉积到芯片上，并被集成到下面的CMOS电路中。目标 RNA将从细胞制备物中纯化，然后与ASd纳米线杂交。一种抗氧化剂， 靶向第二文库选择的位点，将共价连接到12 nm金颗粒（ASO 2-纳米探针）。 ASO 2-纳米探针与靶RNA-ASO 1-纳米线复合物的结合将诱导共振 纳米线中的频率偏移，其被金颗粒的质量极大地放大。该谐振 频率偏移（RXA）将通过直接电读出检测，电压（定量）与 结合事件（RX，A）最初将被光学检测）。我们已经成功地测量了300 nm的RX 硅纳米线（具有高质量因子）在环境条件下。理论计算预测， 在H2O中的硅纳米线的良好质量因子，以及单一结合事件的检测应该是 可实现的。 已经获得了与RNA传感器开发的各个方面相关的初步数据。其中包括： 前列腺DD 3 RNA中靶位点的文库选择，夹心杂交特异性“芯片外”合成 和纳米线的衍生化，R>。纳米线的测量，以及纳米线在 芯片在基本的开发步骤完成后，实验将包括定量测定 与QPCR扩增相比，使用检测装置检测靶RNA。这笔资金的具体目标 期间设计用于开发适合随后用于临床验证研究的RNA传感器 循环肿瘤细胞。这种RNA传感器的成功开发将提供一个主要的优势， 基于PCR的检测，并可以形成高通量筛选测试的基础，同时 检测许多不同的循环肿瘤细胞类型。

This proposal seeks to develop an RNA Sensor to be employed for detection of circulating tumor cells. RNA detection is based upon an hybridization "sandwich". Two target RNAs have been chosen for clinically important cancers (prostate, breast, and melanoma), and library selection protocols will be utilized to identify/optimize accessible sites for antisense oligonucleotide (ASO) binding. Silicon nanowires will then be covalently derivatized with ASO to a library-selected site (ASO-,) in the target RNA. The ASOi nanowires will then be deposited by fluidic deposition onto chips, and integrated into the underlying CMOS circuitry. Target RNA will be purified from cellular preparations, and will then be hybridized to the ASd-nanowires. An ASO2, targeted to a 2nd library-selected site, will be covalently attached to 12 nm gold particles (ASO2-nanoprobe). Binding of the ASO2-nanoprobe to the target RNA-ASOi-nanowire complexes will induce a resonance frequency shift in the nanowires, which is greatly amplified by the mass of the gold particle. This resonance frequency shift (RXA)will be detected by direct electrical read-out, with voltage (quantitatively) related to binding events (RX,A) will initially be detected optically). We have successfully measured RX of 300 nm silicon nanowires (with high Quality-Factors) under ambient conditions. Theoretical calculations predict very good Quality-Factors for silicon nanowires in H20, and detection of single binding events should be achievable. Preliminary data related to all aspects of RNA Sensor development have been obtained. These include: library selection of target sites in prostatic DD3 RNA, sandwich hybridization specificity "off-chip" synthesis and derivatization of nanowires, R>. measurements with nanowires, and fluidic deposition of nanowires on chips. After basic developmental steps are completed, experiments will include quantitative determination of target RNAs using the detection device compared to QPCR amplification. The Specific Aims for this funding period are designed to develop an RNA Sensor appropriate for subsequent use in clinical validation studies for circulating tumor cells. Successful development of this RNA Sensor would provide a major advantage over PCR-based assays, and could form the basis for high-throughput screening tests for simultaneous detection of many different circulating tumor cell types.