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，这一政策的有效性 每一种都是可变的，监测或预测个别患者的疗效的能力还不发达。我们的团队 最近证明(使用传统的基于测序的方法)特定的表达水平 血液中的microRNAs(MiRNAs)可以有效地预测治疗结果。这项提议的目标是发展 创新技术，使我们能够频繁地测量患者的miRNA，以一种 方便快捷，使治疗能够精确调整。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基因改变有关， 迎来了新一代以核酸为基础的靶向治疗。体细胞基因组的测量 需要基于生物标志物来评估、监测和改变治疗方法。循环外体miRNAs 代表了一类高度特异的癌症相关基因突变的标记，可以是非侵入性的 从血液中采样，其定量可以为临床医生提供以前无法获得的信息 就在众多选项中选择有效的治疗方案做出明智的决定。为了 要有效地常规使用miRNA癌症生物标记物，需要开发新的技术方法 它可以提供高度的多重、定量、超灵敏、低成本、简单、集成的样品 处理，以及适用于护理点(POC)设置的坚固的器械。 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 显微镜(NP-PRAM)，采用简单有效的外切体分离方法进行样品制备 这将产生用于检测的外体miRNA。使用共振波长匹配的等离子体纳米粒子 光子晶体表面，NP-PRAM展示了对外体的高对比度“数字分辨率”精确传感 MiRNAs。我们计划开发一种同时检测从一个单一的 不需要荧光发射器或酶扩增的快速检测方法可以检测血液中的液滴。 我们利用模拟引导的miRNA探针设计进行超特异性杂交。我们将把NP-PRAM应用于 一组经临床验证的晚期前列腺癌miRNA生物标记物的背景。 与现有的检测循环核的方法相比，我们的方法具有重要的优势。 酸性生物标志物：只需要一滴~50微米的L样本，而不是10-20毫升的血液，用于RT-PCR法 检测方法。NP-PRAM检测产生高度量化的结果，因为纳米颗粒标签不是 受到荧光染料常见的猝灭或背景荧光的影响。化验结果 是恒温的，在室温下进行，并且具有高度的选择性，而它不需要酶 放大或洗涤步骤。该方法可用于miRNA生物标志物的定量表征 对于所有类型的癌症，尽管在这里我们特别关注一组经过临床验证的前列腺癌MIR。

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.