Micromagnetic Aptamer PCR System for Ultrasensitive Multiplexed Protein Detection

描述（由申请人提供）：对于许多疾病，有效的诊断和预后取决于定量测量临床样品中低浓度蛋白质生物标志物的能力。例如，细胞因子（如血小板衍生生长因子（PDGF））的血清浓度改变可以指示肿瘤血管生成，而病毒相关蛋白（如血凝素）可以指示病毒病原体感染的程度。虽然血清是诊断信息的丰富来源，但利用血清生物标志物的分析挑战来自于它含有数千种蛋白质的事实，这些蛋白质的浓度范围超过12个数量级。例如，白蛋白构成血清蛋白的大约一半，并且以30 - 50 mg/mL（0.5 - 0.8 mM）存在，而许多重要的生物标志物以1 pg/mL（10 - 100 fM）的浓度存在。由于这种宽的动态范围，在单一测定中特异性和定量检测蛋白质生物标志物特别困难。因此，迫切需要具有以下能力的创新蛋白质检测技术：I）集成样品制备-传感器应具有直接处理临床样品的能力II）传感器应提供多个标志物的定量结果，所述多个标志物提供可操作的诊断价值，3.传感器必须具有高灵敏度IV）检测系统必须产生可再现的结果并且具有低的假阳性率和假阴性率，V）传感器应该具有短的总测定时间，并且最重要的是，VI）诊断信息的总成本必须低。为了应对这些挑战，已经开发了许多最近的免疫学方法，例如免疫PCR、邻近连接和生物条形码测定，以实现超过酶联免疫吸附测定（ELISA）能力的检测性能，酶联免疫吸附测定（ELISA）是三十多年来的金标准。然而，这样的测定存在反应化学被背景血清蛋白和临床样品中的其他污染物阻碍的事实。作为这个问题的解决方案，我们建议将样品制备，扩增和检测集成在一个一次性系统中，这样我们就可以有效地将均相结合检测转化为非均相检测系统。我们建议开发微磁分离-定量聚合酶链反应（MMS-QPCR）系统，其中我们将基于芯片的高梯度微磁分离与基于适体的定量PCR相结合，以直接从未稀释的血清中实现具有飞摩尔检测灵敏度的定量、多重蛋白质检测。该项目将分为三个部分：首先，我们将设计多套抗体-适体对，以高亲和力和特异性捕获和标记靶蛋白。第二，我们将开发微磁分离芯片（MMS），从背景血清中纯化目标蛋白，用于下游检测。磁性颗粒的使用将显著改善反应动力学，减少孵育时间，并消除大量的洗涤步骤。MMS芯片将作为前端直接连接到QPCR检测系统。第三，使用嵌入适体中的引物序列，我们将进行QPCR以实现在飞摩尔浓度下对靶蛋白的定量、多重检测。作为一个模型系统，我们将证明同时检测三种癌症标志物，血小板衍生生长因子（PDGF），肝细胞生长因子（HGF），甲状腺转录因子（TTF1），在飞摩尔浓度水平直接从血清。根据初步结果，我们预计检测限为~低fM（比传统ELISA技术高5个数量级），具有跨越6个数量级（毫微微摩尔至纳摩尔浓度）的宽动态范围。公共卫生相关性：对于许多疾病，有效的诊断和预后取决于定量测量临床样品中以低浓度存在的蛋白质生物标志物的能力。虽然血清是此类信息的丰富来源，但挑战来自于它含有数千种蛋白质的事实，这些蛋白质的浓度范围超过12个数量级（微摩尔至飞摩尔），使得稀有蛋白质标记物的分析极其困难。作为这一重要问题的解决方案，我们提出将基于联合收割机的高梯度微磁分离技术与基于适体的定量PCR相结合，以直接从未稀释的血清样品中获得具有飞摩尔检测灵敏度的定量多重蛋白质检测。

DESCRIPTION (provided by applicant): For many diseases, effective diagnosis and prognosis depends on the ability to quantitatively measure protein biomarkers present at low concentrations in clinical samples. For example, altered serum concentrations of cytokines, such as platelet-derived growth factor (PDGF) can point to tumor angiogenesis, while virus-related proteins, like hemagglutinin can indicate the extent of viral pathogen infection. Although blood serum is a rich source of diagnostic information, the analytical challenge in utilizing serum biomarkers arises from the fact that it contains thousands of proteins whose concentrations range over 12 orders of magnitude. Albumin, for example, constitutes approximately half of the serum proteins and is present at 30-50 mg/mL (0.5-0.8 mM), while many important biomarkers exist at concentrations at 1 pg/mL (10-100 fM). Due to this wide dynamic range, specific and quantitative detection of protein biomarkers in a single assay has been particularly difficult. Therefore, an innovative protein detection technologies with the following capabilities are critically needed: I) Integrated sample preparation - the sensor should have the capability to directly process clinical samples (i.e. whole blood or blood serum), II) the sensor should provide quantitative results of multiple markers that provides actionable diagnostic value, III) the sensor must have high sensitivity (femtomolar detection limits) and broad dynamic range, IV) the detection system must produce reproducible results and have low rates of false positives and negatives, V) the sensor should have a short total assay time and most importantly, VI) the total cost of the diagnostic information must be low. In order to address these challenges, a number of recent immunological methods such as ImmunoPCR, Proximity Ligation, and the Bio-Barcode assays have been developed to achieve detection performances beyond the capabilities of the enzyme-linked immunosorbent assay (ELISA), which as been the gold-standard for over three decades. However, such assays suffer from the fact that the reaction chemistry is hindered by the background serum proteins and other contaminants in the clinical samples. As a solution to this problem, we propose to integrate sample preparation, amplification and detection in a single disposable system such that we effectively transform a homogenous binding assay into a heterogeneous detection system. We propose to develop the the Micro-Magnetic Separation - Quantitative Polymerase Chain Reaction (MMS-QPCR) system wherein we integrate chip-based, high-gradient micro-magnetic separation with aptamer-based quantitative PCR to achieve quantitative, multiplexed protein detection with femtomolar detection sensitivities directly from undiluted serum. The project will be organized in three sections: first, we will design multiple sets of antibody - aptamer pairs to capture and label the target proteins with high affinity and specificity. Second, we will develop the micro-magnetic separation (MMS) chip to purify the target proteins from background serum for downstream detection. The use of magnetic particles will significantly improve the reaction kinetics, reduce incubation times, and eliminate extensive washing steps. The MMS chip will be directly interfaced as a front-end to a QPCR detection system. Thirdly, using the primer sequences imbedded in the aptamers, we will perform QPCR to achieve quantitative, multiplexed detection of target proteins at femtomolar concentrations. As a model system we will demonstrate simultaneous detection of three cancer markers, platelet derived growth factor (PDGF), hepatocyte growth factor (HGF), and thyroid transcription factor (TTF1), at femtomolar concentration levels directly from serum. From the preliminary results, we expect the limit of detection to be ~ low fM (5 orders of magnitude higher than traditional ELISA technique), with a wide dynamic range spanning 6 orders of magnitude (femto molar to nanomolar concentrations). PUBLIC HEALTH RELEVANCE: For many diseases, effective diagnosis and prognosis depends on the ability to quantitatively measure protein biomarkers which are present at low concentrations in clinical samples. Although blood serum is a rich source of such information, the challenge arises from the fact that it contains thousands of proteins whose concentrations range over 12 orders of magnitude (micromolar to femtomolar), making the analysis of rare protein markers extremely difficult. As a solution to this important problem, we propose to combine chip-based, high-gradient micro-magnetic separation technology with aptamer- based quantitative PCR to achieve quantitative, multiplexed protein detection with femtomolar detection sensitivities directly from undiluted serum samples.