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（亚皮摩尔）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。该项目将建立一种新型光纤阵列单分子检测技术的可行性，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，即单个分子通过它们的存在（信号）或不存在（信号）来量化。通过在单分子水平上聚焦检测，可以实现对蛋白质的3-7 log量级的灵敏度的显着提高。该项目有两个具体目标，即建立检测癌症蛋白标记技术的可行性。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决定的，因此表面化学将被开发用于最小化或消除来自复杂基质（如人类血清）的生物分子的非特异性结合。这些表面化学物质将包含用于特异性捕获和检测低丰度靶蛋白的固定化抗体。其次，将实施表面化学技术创新，开发前列腺特异性抗原（PSA）的高灵敏度检测方法，PSA是前列腺癌的标志。这个特定的目的是为了证明纤维阵列技术可以用来跟踪PSA水平，比目前的技术低几个对数，从而使前列腺切除术患者能够确保没有残留的疾病。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，多肽，RNA，细胞）使用仪器，将广泛应用于学术和临床实验室。这种创新将使癌症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。

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.