Daily Quantification of Cancer-Associated Exosomal miRNA in Patient Blood by Photonic Crystal-Enhanced Quantum Dot Emission

摘要 这项提案的主要目标是开发一种新技术，使患者能够重复测量 基于核酸的癌症生物标记物，每天从一滴血中提取。这种化验方法正在研制中 用于特定的临床应用，以确定药物治疗的效果，预测生存，并监测术后 通过评估从肿瘤脱落到癌症患者血液中的候选核酸进行治疗干预。 我们专注于检测microRNA，因为我们的团队最近发现了与生存密切相关的因素 被确认为转移性前列腺癌患者。我们假设通过测量这些物质的浓度 这些标志物在患者治疗过程中频繁出现，可以准确调整治疗方案 针对个别患者的治疗方案。然而，准确测量血液中的microRNA需要极高的 低浓度的检测极限，在宽浓度范围内的检测，以及高序列- 特异性，目前不可能用于指纹血样常规筛查的属性 标准的检测方法，如聚合酶链式反应。为此，我们的临床需求激发了一种新的形式的 在显微镜下通过直接分子计数来测量血液中的核酸的方法。现在，这已成为可能 因为我们已经开发出新的方法来放大来自单个分子的信号，通过一系列 协同技术，包括发光量子点、电场增强光子晶体和 一步法序列特异性酶促生长microRNA。我们现在将结合这些技术来设置 使用已在临床诊断中可用的低成本设备测量microRNA的阶段 实验室将临床采用的翻译障碍降至最低。为了实现这些目标，我们的多名调查员 团队在单分子成像的探测器方面拥有广泛的专业知识(安德鲁·史密斯)，在低分子成像的光学检测方面拥有丰富的专业知识 成本设备(Brian Cunningham)、临床肿瘤学(Manish Kohli)、生物标记物发现(王亮)，以及 流行病学/生物统计学(丽贝卡·史密斯)。我们将结合使用合成和优化我们的平台 临床血液样本，并彻底分析我们的分析的序列选择性，特别是关注 MicroRNA变种，并将我们的结果与定量PCR分析的结果进行密切比较。到这个结束的时候 在获奖期间，我们希望开发出第一个用于人类样本的直接读出microRNA分析 与低成本设备兼容，优化了量子点和 光子晶体，并首次测量了血液中microRNA的精确(数字)浓度 在100名受试者中，在标准护理过程中，每名受试者前瞻性登记并随访超过6天 目前尚无预测或预后生物标记物的治疗转移性前列腺癌 癌症。如果成功，这项工作的结果将填补在如何匹配和匹配的知识的一个重大临床空白 根据个别分子图谱微调治疗方法。

ABSTRACT The primary goal of this proposal is to develop a new technology that can allow patients to repeatedly measure nucleic acid-based cancer biomarkers from a single drop of blood on a daily basis. This assay is being developed for specific clinical applications to determine drug treatment efficacies, prognosticate survival, and monitor post- treatment intervention by evaluating candidate nucleic acids shed from the tumor into blood of a cancer patient. We are focusing on detecting microRNA due to a very strong correlation with survival that our team has recently identified for patients with metastatic prostate cancer. We hypothesize that by measuring the concentrations of these markers in patients on a frequent basis during the course of therapy, we can precisely adjust therapeutic regimens for individual patients. However, accurately measuring microRNA in blood requires an extremely high limit of detection due to low concentrations, detection over a broad range of concentrations, and high sequence- specificity, attributes that are not currently possible for routine screening of fingerstick blood samples using standard methods of detection, such as PCR. Toward this end, our clinical needs have inspired a new form of assay to measure nucleic acids in blood through direct molecular counting in a microscope. This is now possible because we have developed novel ways to amplify the signals from individual molecules through a series of synergistic technologies, including light-emitting quantum dots, electric field-enhancing photonic crystals, and single-step sequence-specific enzymatic growth of microRNA. We now will combine these technologies to set the stage for measurement of microRNA using low-cost equipment that is already available in clinical diagnostic laboratories to minimize translational barriers to clinical adoption. To achieve these goals, our multi-investigator team has extensive expertise in probes for single-molecule imaging (Andrew Smith), optical detection in low- cost devices (Brian Cunningham), clinical oncology (Manish Kohli), biomarker discovery (Liang Wang), and epidemiology/biostatistics (Rebecca Smith). We will optimize our platform using a combination of synthetic and clinical blood specimens and thoroughly analyze the sequence selectivity of our assay, particularly focusing on microRNA variants, and closely compare our results with those from quantitative PCR assays. By the end of this award period, we expect to have developed the first direct-readout microRNA assay for use in human samples that is compatible with low-cost equipment, optimized the synergistic integration between quantum dots and photonic crystals, and measured, for the first time, the precise (digital) concentrations of microRNA in the blood of 100 subjects, prospectively enrolled and followed over 6 days each during the course of standard of care treatments for which no predictive or prognostic biomarkers currently exist in the treatment of metastatic prostate cancer. If successful, the outcome of this work will fill a major clinical gap in knowledge of how to match and finely tune treatments to individual molecular profiles.