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，可用于检测非常低浓度（亚皮摩尔）的诊断癌症的蛋白质。目前，癌症的早期诊断受到以下事实的限制：可用技术（例如ELISA）的检测限（LOD）高于可指示疾病发作的低丰度蛋白质的循环浓度。该计划将建立使用新型光纤阵列单分子检测技术进行癌症标志物极高灵敏度测量的可行性。该技术本质上是数字的，即，单个分子通过它们的存在（开信号）或不存在（关信号）来定量。通过将检测集中在单分子水平，对于蛋白质可以实现3-7个对数级的灵敏度的显著提高。该项目有两个具体目标，即建立检测癌症蛋白标记物技术的可行性。首先，由于该技术的LOD是由来自非特异性事件的假信号的数量确定的，因此将开发表面化学以最小化或消除来自复杂基质如人血清的生物分子的非特异性结合。这些表面化学物质将结合用于特异性捕获和检测低丰度靶蛋白的固定化抗体。其次，将实施表面化学方面的技术创新，以开发一种高灵敏度的前列腺特异性抗原（PSA）检测方法，PSA是前列腺癌的标志物。该特定目的旨在证明纤维阵列技术可用于跟踪比现有技术低几个对数的PSA水平，从而使前列腺切除术患者能够确保没有残留疾病。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，肽，RNA，细胞）使用仪器，将广泛应用于学术和临床实验室。这种创新将使癌症研究人员能够在前所未有的水平上检测癌症标记物，用于早期诊断应用，并将促进新的低丰度癌症标记物的发现。这一长期目标满足了NIH使命的几个方面，特别是开发创新的研究策略，以改善人类疾病的诊断、预防和治疗。

DESCRIPTION (provided by applicant): The aim of this project is to develop a new technology that can be used to detect very low concentrations (sub- picomolar) 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 establish the feasibility of using 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 on the order of 3-7 logs could be achieved for proteins. The project has two specific aims that would establish feasibility of the technique for detection of cancer protein markers. First, as the LOD of the technique is determined by the number of false }on} signals from non- specific events, surface chemistries will be developed to minimize or eliminate non-specific binding of biological molecules from a complex matrix such as human serum. These surface chemistries will incorporate immobilized antibodies that are used to specifically capture and detect low abundance target proteins. Second, the technological innovations in surface chemistry will be implemented to develop a high-sensitivity assay for prostate specific antigen (PSA), which is a marker for prostate cancer. This specific aim is intended to demonstrate that the fiber arrays technology can be used to follow levels of PSA several logs lower than present technology, thereby enabling prostatectomy patients to be assured that there is no residual disease. 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 cancer 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.