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

描述（由申请方提供）：本R21提案的目的是开发一种用于心肌梗死的多分析物二维扩散床旁检测（POC）。2-D扩散试验由“无污染”聚（低聚乙二醇甲基丙烯酸酯（POEGMA）刷涂的玻璃芯片组成，该芯片含有两种类型的打印点：捕获抗体（Abc）的“稳定”点和检测试剂的“可溶”点，这些点打印有赋形剂，以便它们在与血液接触时溶解。将手指针刺的血液施加到表面上，血液中的蛋白质分析物扩散穿过无污染的POEGMA刷，并结合到表面上的单个捕获抗体（Abc）的固定点。同时，血液溶解来自不稳定斑点的荧光标记的检测Ab（Abd），其然后结合到其各自的分析物-Abc斑点，完成夹心。用缓冲液进行的最终置换步骤从芯片上去除血液成分，揭示了在未标记的检测剂已被打印的位置处形成的荧光点。该检测试剂盒无需样本预处理，可在同一芯片上多重检测心肌肌钙蛋白（cTnI）和脑利钠肽原（pro-BNP），并可提供分析物浓度的定量读数，从而为POC检测提供上级当前金标准-侧流检测试剂盒的新功能。 公共卫生相关性：这项拟议中的研究将开发一种新的即时临床测试-二维扩散测定-用于心肌梗死，不需要样品纯化 和最少的测试步骤。在该测定中，将一滴血添加到薄聚合物膜的表面允许存在于患者血液中的标记物-以及疾病的诊断-结合到捕获抗体的“稳定”捕获点，而同时，血液溶解被打印为“可溶性”点的光学标记的检测抗体，使得它们迁移到标记物中。 将溶液转移到与标记物结合的捕获分子的稳定斑点，并完成测定，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.