An RNA Sensor for Detection of Circulating Tumor Cells

用于检测循环肿瘤细胞的 RNA 传感器

• 批准号: 7024299
• 负责人: GARY A CLAWSON
• 金额: $ 29.3万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-15 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7024299
• 关键词: RNA; clinical research; library; measurement; neoplasm /cancer

DESCRIPTION (provided by applicant): 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 (R??) will be detected by direct electrical read-out, with voltage (quantitatively) related to binding events (R??) will initially be detected optically). We have successfully measured R? 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.

描述（由申请人提供）： 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”。已经为临床上重要的癌症（前列腺、乳腺和黑素瘤）选择了两种靶RNA，并且将利用文库选择方案来鉴定/优化反义寡核苷酸（阿索）结合的可接近位点。然后将硅纳米线用阿索共价衍生到靶RNA中的文库选择位点（阿索-1）。然后，ASO 1纳米线将通过流体沉积沉积到芯片上，并集成到下面的CMOS电路中。靶RNA将从细胞制备物中纯化，然后将与ASd-纳米线杂交。靶向第二文库选择位点的ASO 2将共价连接至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纳米线中的共振频率偏移，其被金颗粒的质量极大地放大。该共振频率偏移（R？？）将通过直接电读出检测，电压（定量）与结合事件（R？？）最初将被光学地检测）。我们已经成功地测量了R？的300纳米硅纳米线（具有高质量因子）在环境条件下。理论计算预测硅纳米线在H2O中的质量因子非常好，并且应该可以实现单个结合事件的检测。 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”合成和纳米线衍生化，R？。纳米线的测量以及纳米线在芯片上的流体沉积。在基本开发步骤完成后，实验将包括使用检测装置与QPCR扩增相比定量测定靶RNA。本资助期的具体目标旨在开发一种适合随后用于循环肿瘤细胞临床验证研究的RNA传感器。这种RNA传感器的成功开发将提供优于基于PCR的测定的主要优势，并且可以形成用于同时检测许多不同循环肿瘤细胞类型的高通量筛选测试的基础。