2-D Diffusion Assay on Polymer Brush for POC Cardiac Infarction Diagnosis

描述(由申请人提供)：本R21提案的目标是开发一种用于心肌梗死的多分析二维扩散点检测(POC)。2-D扩散法由一个“无污染”聚甲基丙烯酸寡乙二醇酯(POEGMA)刷涂玻璃芯片组成，其中包含两种类型的印刷点：捕获抗体(ABC)的“稳定”点和检测试剂的“可溶”点，这些点印有辅料，使它们与血液接触时溶解。将一根手指的血液涂抹在表面，血液中的蛋白质分析物在无污染的POEGMA刷子上扩散，并与表面上个人捕获抗体(ABC)的固定斑点结合。同时，血液从不稳定的斑点溶解荧光标记的检测抗体(ABD)，然后结合到各自的分析物-ABC斑点，完成三明治。使用缓冲液的最后置换步骤从芯片中移除血液成分，揭示在打印未标记的检测剂的位置形成的荧光点。该分析不需要样品前处理，将在同一芯片上多路复用以检测心肌肌钙蛋白(CTnI)和脑钠素前体多肽(Pro-BNP)，并将提供分析物浓度的定量读出，从而提供优于当前金标准-侧向流动分析的POC测试能力的新维度。 与公共卫生相关：这项拟议的研究将为心肌梗死开发一种新的临床点检测方法--二维扩散法，不需要样品纯化 测试中的最小步骤。在这种化验方法中，在一层薄的聚合物膜表面滴入一滴血，就可以使患者血液中的一种标记物与捕获抗体的“稳定”捕获斑点结合，同时，血液溶解打印成“可溶”斑点的光学标记检测抗体，从而使它们迁移到 溶液将稳定的斑点捕获分子结合到标记物上，并完成检测，产生可见信号。

DESCRIPTION (provided by applicant): The objective of this R21 proposal is to develop a multianalyte 2-D diffusion point-of-care assay (POC) for cardiac infarction. The 2-D diffusion assay consists of a "nonfouling" poly(oligoethyene glycol methacrylate (POEGMA) brush coated glass chip that contains two types of printed spots: "stable" spots of capture antibodies (Abc) and "soluble" spots of detection reagents that are printed with excipients so that they dissolve upon contact with blood. A finger stick of blood is applied to the surface, and the protein analytes in the blood diffuse across the nonfouling POEGMA brush and bind to immobile spots of the individual capture antibodies (Abc's) on the surface. Simultaneously, the blood dissolves the fluorescently labeled detection Ab's (Abd) from the labile spots, which then bind to their respective analyte-Abc spots, completing the sandwich. A final displacement step with buffer removes the blood components from the chip, revealing the formation of a fluorescent spot at the position where the unlabeled detection agent had been printed. This assay will require no sample preprocessing, will be multiplexed to detect cardiac troponin (cTnI) and the brain natriuretic pro-polypeptide (pro-BNP) on the same chip, and will provide quantitative read-out of analyte concentration, thereby providing a new dimension of capabilities to POC tests that are superior to the current gold standard - lateral-flow assays. PUBLIC HEALTH RELEVANCE: The proposed research will develop a new point-of-care clinical test -the 2-D diffusion assay- for cardiac infarction that requires no sample purification and minimal steps in testing. In this assay, adding a drop of blood to the surface of a thin polymer film allows a marker that is present in the patient's blood -and diagnostic of a disease- to bind to "stable" capture spots of capture antibodies, while simultaneously, the blood dissolves optically labeled detection antibodies that are printed as "soluble" spots, so that they migrate in solution to the stable spots of capture molecules bound to the marker, and complete the assay, resulting in a visible signal.