Single Molecule Fiber Arrays for the Detection of Low Abundance Proteins

描述（由申请人提供）：该项目的目的是开发一种新技术，可用于检测极低浓度（飞摩尔及以下）的蛋白质，用于诊断癌症。目前，癌症的早期诊断受到现有技术（如ELISA）的检测限（LOD）高于可指示疾病发病的低丰度蛋白质的循环浓度这一事实的限制。该项目将扩大和加强新型光纤阵列单分子检测技术的使用，用于极高灵敏度的癌症标志物测量。这项技术本质上是数字化的，也就是说，单个分子通过它们的存在（“开”信号）或不存在（“关”信号）来量化。通过聚焦检测在单分子水平，显着提高了灵敏度，实现了蛋白质。该项目的第一阶段表明，单分子检测可以检测到比ELISA低一千倍的蛋白质浓度。采用单分子检测，检测血清中前列腺特异性抗原（PSA）浓度小于1飞摩尔；目前的免疫分析仪检测PSA的LOD约为3皮摩尔。第二阶段项目有三个具体目标，将以这些主要证明实验为基础，扩大和加强该技术在检测癌症蛋白质标记物方面的应用，并使该技术更接近商业化，供医学研究人员和临床医生使用。首先，超灵敏的PSA检测将用于检测人类临床样本中的标记物，特别是用于测量接受根治性前列腺切除术的前列腺癌患者的PSA水平。目前的技术无法检测这些样品中的PSA；该技术可以早期发现血清中PSA的回归，从而提示残留疾病。其次，该技术将扩展到更多种类的蛋白质，如融合蛋白和翻译后修饰，以使其更广泛的应用。第三，将开发使用小样本量在低浓度下测量蛋白质面板的方法。这些小组将使诊断难以发现的疾病，如卵巢癌，具有更高的灵敏度和特异性。这项研究的长期目标是建立一个新的标准，在高灵敏度检测生物分子（蛋白质，多肽，RNA，细胞）使用仪器，将广泛应用于学术和临床实验室。这种创新将使研究人员能够以前所未有的水平检测癌症标志物，用于早期诊断应用，并将促进发现新的低丰度癌症标志物。这一长期目标满足了NIH若干方面的使命，特别是为改进人类疾病的诊断、预防和治疗制定创新的研究战略。癌症的早期诊断是由临床肿瘤学家检测疾病的能力决定的。Quanterix正提议开发一种技术，使临床医生能够检测血液中非常低浓度的蛋白质，从而实现疾病的早期诊断。这项技术有可能帮助医学研究人员和医生识别和检测更多的早期癌症诊断标志物，并改善患者的预后。

DESCRIPTION (provided by applicant): The aim of this project is to develop a new technology that can be used to detect very low concentrations (femtomolar and below) of proteins that are diagnostic for cancer. Presently, early diagnosis of cancer is limited by the fact that the limits of detections (LOD) of available technologies, such as ELISA, are higher than the circulating concentrations of low abundance proteins that could indicate the onset of disease. This program would broaden and strengthen the use a novel fiber optic array single molecule detection technology for extremely high sensitivity measurement of cancer markers. This technology is digital in nature, i.e., individual molecules are quantified by their presence ("on" signal) or their absence ("off" signal). By focusing detection at the single molecule level, dramatic improvements in sensitivity are achieved for proteins. Phase I of this project demonstrated that single molecule detection makes it possible to detect concentrations of proteins at levels a thousand times lower than ELISA. Using single molecule detection, prostate specific antigen (PSA) was detected in serum at concentrations less than 1 femtomolar; current immunoanalyzers have an LOD of about 3 picomolar for PSA. The Phase II project has three specific aims that would build on these proof-of-principal experiments to broaden and strengthen the use of the technique for detection of cancer protein markers, and to move the technology closer to commercialization for use by medical researchers and clinicians. First, the ultra-sensitive PSA assay will be used to detect the marker in human clinical samples, specifically to measure the levels of PSA in prostate cancer patients who have undergone radical prostatectomy. Current technologies cannot detect PSA in these samples; this technology could enable the early detection of the return of PSA in serum, which would indicate residual disease. Second, the technology will be expanded to cover more classes of proteins, such as fusion proteins and post-translational modifications, to enable its broader implementation. Third, methods will be developed to measure panels of proteins at low concentrations using small sample volumes. These panels would enable the diagnosis of difficulty to detect diseases, such as ovarian cancer, with greater sensitivity and specificity. The long term objective of this research is to establish a new standard in high sensitivity detection of biological molecules (proteins, peptides, RNA, cells) using instrumentation that would be widely employed in both academic and clinical laboratories. Such an innovation would enable researchers to detect cancer markers at unprecedented levels for early diagnostic applications, and would facilitate the discovery of new low abundance cancer markers. This long term goal satisfies several aspects of the mission of the NIH, in particular, to develop innovative research strategies towards improving the diagnosis, prevention and cure of human diseases. The early diagnosis of cancer is determined by the ability of the clinical oncologist to detect the disease. Quanterix is proposing to develop a technology that would enable clinicians to detect very low concentrations of proteins in blood and, therefore, enable the early diagnosis of disease. This technology has the potential to help medical researchers and doctors to identify and detect many more early cancer diagnostic markers and improve outcomes for patients.