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

项目总结/摘要 快速和特异性的组织病理学诊断对于癌症治疗至关重要。肿瘤组织活检常规 用于检测和监测癌症进展。目前的测试活组织检查需要解剖收集的组织 来自可检测的原发性或转移性肿瘤的样本。几个困难，如患者不便， 多步骤的复杂程序、部分取样和非特异性发现，使这一过程缓慢， 侵入性、昂贵、不适合筛选大样本量且容易出错。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很大的希望。 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 外泌体微小RNA（exmiRs）作为检测癌症进展的有前景的液体活检生物标志物， 具有较高的敏感性和特异性。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，需要高样品体积，昂贵、缓慢、繁琐， 需要高度专业化的技能和资源，例如超浓缩技术、昂贵的RNA提取试剂盒等。 单外泌体水平的研究可以显著地规避这些问题。然而，少数单分子 ex-miR定量尝试缺乏扩增策略，因此将其应用限制于资源密集型 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），将其插入脂质囊泡的膜中，并发出信号， 通过荧光的增加来检测DNA靶的存在。我们已经成功地证明了 自发插入脂质膜并检测膜封闭核酸的TraNS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TraNS设备以检测癌症- （2）利用TraNS的跨膜结构重构， 等温信号放大法，显著提高检测灵敏度，（3）积分 TraNS设备与我们的专利DNA折纸为基础的生物标志物检测阵列，以提高吞吐量， 特异性和灵敏度，以低样品体积进行ex-miR化学计量的数字定量。我们将使用 该平台对胰腺癌患者临床样本的灵敏度、特异性和通量 与健康对照组相比。这项工作的潜在影响可以帮助医生和临床医生快速， 超灵敏、精确和经济高效的癌症诊断，无需手术组织活检。

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.