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

高通量、免纯化、超灵敏跨膜纳米传感器阵列，用于对完整外泌体的 microRNA 生物标志物进行数字计数

• 批准号: 10613253
• 负责人: Rizal Fajar Hariadi
• 金额: $ 22.02万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-15 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10613253
• 关键词: Address; Basic Science; Biological Assay; Biological Markers; Biopsy; Blood; Body Fluids; Cancer Detection; Cancer Diagnostics; Cancer Patient; Cancer Prognosis; Cell Communication; Cell Line; Cells; Clinical; Complement; Consumption; Cytolysis; DNA; Data; Detection; Development; Devices; Diagnosis; Diagnostic; Differential Diagnosis; Disease; Encapsulated; Fluorescence; Heterogeneity; Human Milk; Individual; Investigation; Legal patent; Malignant Neoplasms; Malignant neoplasm of pancreas; Mediating; Membrane; Membrane Lipids; Messenger RNA; Methods; MicroRNAs; Microarray Analysis; Microscopy; Miniaturization; Molecular; Monitor; Nanoarray Analytical Device; Neoplasm Metastasis; Nucleic Acids; Operative Surgical Procedures; Optics; Patients; Pattern; Physicians; Plasma; Positioning Attribute; Primary Neoplasm; Procedures; Process; Prognosis; Prognostic Marker; Proteins; Quantitative Reverse Transcriptase PCR; RNA; Reaction; Reporting; Research; Resources; Role; Saliva; Sample Size; Sampling; Scientist; Sensitivity and Specificity; Serum; Signal Transduction; Solid; Specificity; Surface; Technology; Testing; Time; Tissue Sample; Tissues; Treatment Efficacy; Tumor Tissue; Urine; Vesicle; Work; amplification detection; biomarker selection; cancer biomarkers; cancer diagnosis; cancer therapy; clinical diagnostics; clinical translation; cost effective; density; design; detection sensitivity; diagnostic biomarker; digital; exosome; improved; interest; isothermal amplification; liquid biopsy; microRNA biomarkers; nanoscale; nanosensors; pancreatic cancer patients; rapid detection; screening; sensor; single molecule; skills; stoichiometry; tumor heterogeneity; tumor progression; tumorigenesis

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.

项目概要/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，肿瘤组织活检是常规的。 目前的测试活检需要手术采集的组织来检测和监测癌症进展。 来自可检测的原发性或转移性肿瘤的样本存在一些困难，例如给患者带来不便， 多步骤的复杂程序、部分抽样和非特异性发现使这个过程缓慢， 侵入性、昂贵、不适合筛选大样本且容易出错。 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 最近的研究表明，手术组织活检在癌症患者的诊断和预后中的作用。 外泌体 microRNA（exmiR）作为有前途的液体活检生物标志物来检测癌症进展和 然而，目前的 ex-miR 检测技术， 例如 qRT-PCR 和微阵列筛选测试，需要大量样本，价格昂贵、缓慢、繁琐， 需要高度专业的技能和资源，例如超速离心机、昂贵的 RNA 提取试剂盒等。 单外泌体水平的研究可以显着规避这些问题，但是单分子水平的研究却很少。 ex-miR 定量尝试缺乏扩增策略，因此限制了其在资源密集型领域的应用 为了解决这些问题，我们开发了一种基于分子信标的方法。 跨膜纳米传感器 (TraNS) 将自身插入脂质囊泡膜并发出信号 通过增强荧光来检测 DNA 靶标的存在 我们已经成功证明了这种能力。 TraNS 装置可自发插入脂质膜并感知膜封闭的核酸 在这项研究中，我们建议 (1) 优化 TraNS 设备来检测癌症。 来自生物流体的特定 ex-miR，(2) 利用 TraNS 的跨膜结构重构来开发 等温信号放大方法显着提高检测灵敏度，以及（3）集成 TraNS 设备配备我们获得专利的基于 DNA 折纸的生物标志物检测阵列，可提高通量， 我们将使用低样本量的 ex-miR 化学计量的数字定量的特异性和灵敏度。 该平台对胰腺癌患者临床样本的敏感性、特异性和通量 这项努力的潜在影响可以帮助医生和战士快速、 超灵敏、精确且经济高效的癌症诊断，无需手术组织活检。