School of Chemical and Biomedical Engineering, Center for Advanced Bionanosystems, Nanyang Technological University, Singapore 637457, Singapore.
Biomed Microdevices. 2011 Aug;13(4):769-77. doi: 10.1007/s10544-011-9547-1.
Flow-through immunoassay is an attractive method for fast, inexpensive and high-throughput protein analyses. However, its practical application is limited by low sensitivity. In this work, a highly sensitive flow-through microarray immunoassay device is developed, in which a poly[glycidyl methacrylate-co-poly(ethylene glycol) methacrylate] (P(GMA-co-PEGMA)) brush as a flexible matrix is uniformly coated on a glass slide through a purge-free surface-initiated atom transfer radical polymerization (SI-ATRP) to immobilize capture proteins for a larger loading capacity and higher bioactivity while allowing easy target access to the brush-attached probes for efficient antibody-antigen (Ab-Ag) bindings. The integrated device is then constructed by simply laminating the protein-arrayed slide onto a ready-for-bonding double-sided adhesive tape-attached poly(methyl methacrylate) (PMMA) microfluidic structure. As a demonstration, a parallel microarray panel is designed to perform flow-through immunoassays for good detection flexibility and simultaneous analysis of various samples. The limit of detection (LOD) of 1-10 pg/mL for detected target proteins is achieved, which is one to two orders better than those of reported flow-through immunoassays. The device also demonstrates significantly reduced total assay time over the static microarray immunoassay. The rapid and sensitive detection can be mainly ascribed to the P(GMA-co-PEGMA) brushed substrate, of which both the hydrophilicity from its PEG component and the binding capability from its GMA moiety result in higher protein loading capacity, lower nonspecific adsorption, and higher Ab-Ag binding efficiency. The integrated microfluidic device was further used to detect an important cancer biomarker carcinoembryonic antigen (CEA) in serum and achieved a LOD of 10 pg/mL, demonstrating its great potential for clinical applications.
流变法免疫分析是一种快速、廉价、高通量的蛋白质分析方法。然而，其实际应用受到灵敏度低的限制。在这项工作中，开发了一种高灵敏度的流变法微阵列免疫分析装置，其中聚[甲基丙烯酸缩水甘油酯-co-聚乙二醇甲基丙烯酸酯]（P（GMA-co-PEGMA））刷作为柔性基质通过无吹扫表面引发原子转移自由基聚合（SI-ATRP）均匀涂覆在玻璃载玻片上，以固定捕获蛋白，从而具有更大的载量和更高的生物活性，同时允许目标物轻松进入刷状附着的探针，以实现有效的抗体-抗原（Ab-Ag）结合。然后，通过简单地将蛋白阵列玻片层压到附有双面胶的聚甲基丙烯酸甲酯（PMMA）微流控结构上，即可构建集成装置。作为一个演示，设计了一个平行微阵列面板，用于进行流变法免疫分析，具有良好的检测灵活性和同时分析各种样品的能力。检测目标蛋白的检测限（LOD）为 1-10 pg/mL，比报道的流变法免疫分析提高了一到两个数量级。该装置还显著减少了总分析时间。快速灵敏的检测主要归因于 P（GMA-co-PEGMA）刷状基底，其亲水性来自其 PEG 部分，结合能力来自其 GMA 部分，从而具有更高的蛋白载量、更低的非特异性吸附和更高的 Ab-Ag 结合效率。集成微流控装置进一步用于检测血清中的重要癌症生物标志物癌胚抗原（CEA），检测限为 10 pg/mL，显示出其在临床应用中的巨大潜力。
