High-throughput, purification-free, and ultrasensitive transmembrane nanosensor arrays for digital counting of microRNA biomarkers of intact exosomes

项目摘要/摘要 快速和特异的组织病理学诊断是癌症治疗的关键。肿瘤组织活检是常规的 进行检查以检测和监测癌症进展。目前的测试活组织检查需要手术收集的组织 来自可检测的原发或转移肿瘤的样本。几个困难，如病人不便， 多步骤复杂的程序、部分采样和非特定的发现，使这一过程变得缓慢， 有创、昂贵、不适合筛查大样本量，而且容易出错。非侵入性选择 体液中的生物标志物，称为液体活检，在补充甚至替代方面提供了巨大的希望。 手术组织活检在癌症患者诊断和预后中的作用。最近的研究表明 Exosomal microRNAs(ExmiRs)作为液体活检生物标记物在检测肿瘤进展和转移中的应用 治疗效果敏感度高，特异度高。然而，目前用于ex-miR检测的技术， 例如qRT-PCR和微阵列筛选测试，需要高样本量，昂贵、缓慢、繁琐， 需要高度专业化的技能和资源，如超速离心机、昂贵的RNA提取试剂盒等。 单外切体水平的研究可以显著地绕过这些问题。然而，为数不多的单分子 Ex-miR量化尝试缺乏放大策略，因此其应用仅限于资源密集型 研究设置。为了解决这些问题，我们开发了一种基于分子信标的 跨膜纳米传感器(TRANS)，它插入脂泡的膜中，并发出信号 由于荧光增强而导致DNA靶标的存在。我们已经成功地证明了 自发插入脂膜并感测膜包被核酸的反式装置 具有高度特异性的生物标志物。在这项研究中，我们建议(1)优化TRAN设备以检测癌症- 来自生物体液的特异性exmiRs，(2)利用反式的跨膜结构重组来开发 采用等温信号放大方法，显著提高了检测灵敏度；(3)积分 TRANS设备与我们基于DNA折纸的专利生物标记物检测阵列相结合，以提高吞吐量， 低样本量的ex-miR化学计量学数字定量的特异度和灵敏度。我们将使用 该平台对胰腺癌患者临床样本的敏感性、特异性和吞吐量 来对抗他们的健康对照。这一努力的潜在影响可以帮助医生和临床医生快速、 无需手术组织活检，即可实现超灵敏、精确、经济的癌症诊断。

Project Summary/Abstract Rapid and specific histopathologic diagnoses are critical for cancer treatment. Tumor tissue biopsy is routinely performed to detect and monitor cancer progression. Current test biopsies require surgically-collected tissue samples from detectable primary or metastatic tumors. Several difficulties, such as patient inconvenience, multistep complicated procedure, partial samplings, and non-specific findings, make this process slow, invasive, expensive, unfit for screening large sample sizes, and error-prone. Non-invasive selections of biomarkers in body fluids, known as liquid biopsy, offer great promise in complementing or even substituting surgical tissue biopsy in the diagnosis and prognosis of cancer patients. Recent studies have indicated exosomal microRNAs (exmiRs) as promising liquid biopsy biomarkers in detecting cancer progression and efficacy of therapy with high sensitivity and specificity. However, current technologies for ex-miR detection, such as qRT-PCR, and microarray screening tests, require high sample volume, are expensive, slow, tedious, requiring highly specialized skills and resources such as ultracentrifuge, expensive RNA extraction kits, etc. Single-exosome level studies can significantly circumvent these problems. However, the few single-molecule ex-miR quantification attempts lack amplification strategy, thus limiting their applications to resource-heavy research settings. To address these problems, we have developed a molecular beacon-based Transmembrane Nano-Sensor (TraNS) that inserts itself into the membrane of lipid vesicles and signals the presence of a DNA target by an increase in fluorescence. We have successfully demonstrated the ability of the TraNS device to spontaneously insert into the lipid membrane and sense membrane-enclosed nucleic acid biomarkers with high specificity. In this study, we propose to (1) optimize the TraNS device to sense cancer- specific ex-miRs from biofluids, (2) harness the transmembrane structural reconfiguration of TraNS to develop an isothermal signal amplification method to improve the sensitivity of detection significantly, and (3) integrate the TraNS device with our patented DNA origami-based biomarker detection array to improve the throughput, specificity, and sensitivity of digital quantification of ex-miR stoichiometry with low sample volume. We will use the platform’s sensitivity, specificity, and throughput on clinical samples from pancreatic cancer patients against their healthy controls. This effort’s potential impact can help physicians and clinicians with rapid, ultrasensitive, precise, and cost-effective cancer diagnostics without a surgical tissue biopsy.