Exosome separation and digital resolution detection of blood-based nucleic acid biomarkers for noninvasive therapeutic diagnostics in cancer

摘要 多种药物疗法已被批准用于治疗晚期癌症。然而， 每一个都是可变的，并且监测或预测个体患者的功效的能力还不发达。我们的团队 最近证明（使用传统的基于测序的方法），特异性的表达水平， 血液中的microRNAs（miRNAs）可以有效预测治疗结果。该提案的目标是发展 创新的技术将使我们能够频繁地测量来自患者的miRNA， 方便快捷，可精确调整治疗。这是目前使用RT-PCR无法实现的 或基于测序的检测。所有癌症都与异质性体细胞遗传改变有关， 开创了新一代基于核酸的靶向治疗。体细胞基因组的测定 需要基于生物标志物来评估、监测和改变治疗。循环外泌体miRNAs 代表了一类高度特异性的癌症相关基因突变标记物， 从血液中取样，其定量可以为临床医生提供以前无法获得的信息， 在广泛的选择方案中选择有效的治疗方法，从而做出明智的决定。为了 为了有效地常规使用miRNA癌症生物标志物，需要开发新的技术方法 其可以提供高度多路复用、定量、超灵敏度、低成本、简单、集成样品 处理和适用于护理点（POC）设置的稳健仪器。 我们将一种新展示的显微镜形式，称为纳米粒子光子谐振器吸收， 使用简单有效的外泌体分离方法进行样品制备的NP-PRAM显微镜 产生用于检测的外泌体miRNA。使用其共振波长匹配一个共振波长的等离子体纳米粒子， 光子晶体表面，NP-PRAM展示了高对比度的“数字分辨率”精确感测外泌体 miRNAs。我们计划开发同时检测从单个细胞中提取的5个miRNA序列的检测方法。 使用不需要荧光发射器或酶促扩增的快速测定方案测量血液微滴。 我们利用模拟引导的miRNA探针设计进行超特异性杂交。我们将NP-PRAM应用于 一组经临床验证的用于晚期前列腺癌的miRNA生物标志物的背景。 与现有的循环核酸检测方法相比，我们的方法具有重要的优势。 酸性生物标志物：与基于RT-PCR的10-20 ml血液不同，它只需要约50 µl的测试样品液滴 检测方法NP-PRAM检测产生高度定量的结果，因为纳米颗粒标签不被检测到。 受到荧光染料常见的淬灭或背景荧光的影响。测定 是等温的，在室温下进行，并且具有高度选择性，而不需要酶 扩增或洗涤步骤。该方法可应用于miRNA生物标志物的定量表征 对于所有癌症类型，尽管在这里我们特别关注临床验证的前列腺癌miR集。

Abstract Multiple drug therapies have been approved for treating advanced cancer. However, the effectiveness of each is variable and the ability to monitor or predict efficacy in individual patients is underdeveloped. Our team recently demonstrated (using traditional sequencing-based methods) that expression levels of specific microRNAs (miRNAs) in blood can effectively predict treatment outcomes. The goal of this proposal is to develop innovative technologies that will allow us to measure miRNAs from a patient on a frequent basis, in a way that is convenient and rapid, to enable precise adjustment of therapy. This is currently not achievable using RT-PCR or sequencing-based detection. All cancers are associated with heterogeneous somatic genetic alterations, ushering in a new generation of nucleic-acid-based targeted treatments. The measurement of somatic genome based biomarkers to assess, monitor, and change treatments is needed. Circulating exosomal miRNAs represent one class of highly specific markers of cancer-associated genetic mutations that can be noninvasively sampled from blood, whose quantitation can provide previously-unavailable information to clinicians for generating informed decisions on selection of effective treatments among the wide array of options. In order to make effective routine use of miRNA cancer biomarkers, novel technical approaches will need to be developed that can offer a high degree of multiplexing, quantitation, ultrasensitivity, low cost, simplicity, integrated sample processing, and robust instrumentation suitable for point of care (POC) settings. We link a newly demonstrated form of microscopy, called NanoParticle Photonic Resonator Absorption Microscopy (NP-PRAM) with a simple and effective exosome isolation approach to perform sample preparation that yields exosomal miRNA for detection. Using plasmonic NPs whose resonant wavelength matches a photonic crystal surface, NP-PRAM demonstrates high contrast “digital resolution” precision sensing of exosomal miRNAs. We plan to develop assays for simultaneous detection of 5 miRNA sequences extracted from a single droplet of blood with a rapid assay protocol that does not require fluorescent emitters or enzymatic amplification. We utilize simulation-guided miRNA probe design for ultraspecific hybridization. We will apply NP-PRAM in the context of a panel of clinically validated miRNA biomarkers for advanced prostate cancer. Our approach offers important advantages compared to existing methods for detection of circulating nucleic acid biomarkers: It requires only a ~50 µl droplet of test sample unlike 10-20 ml of blood for RT-PCR based detection methods. NP-PRAM detection produces highly quantified results because nanoparticle tags are not subject to the effects of quenching or background fluorescence that are common to fluorescent dyes. The assay is isothermal, conducted at room temperature, and highly selective, while it does not require enzyme amplification or wash steps. The approach can be applied to quantitative characterization of miRNA biomarkers for all cancer types, although here we specifically focus on a clinically validated set of miRs for prostate cancer.