Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, NC, USA.
Department of Chemical and Biomolecular Engineering, College of Engineering, NC State University, Raleigh, NC, USA.
Methods Mol Biol. 2022;2393:343-365. doi: 10.1007/978-1-0716-1803-5_19.
Sandwich immunoassays are the gold standard for detection of protein analytes. Here, we describe an ultrasensitive point-of-care sandwich immunoassay platform for the detection of biomarkers directly from blood or serum using a custom-built smartphone detector. Testing undiluted blood or serum is challenging due to the complexity of the matrix. Proteins nonspecifically adsorb to and cells often adhere to the assay surface, which can drastically impact the analytical sensitivity of the assay. To address this problem, our assay is built upon a "nonfouling" polymer brush "grafted from" a glass slide, which eliminates nearly all nonspecific binding and therefore increases the signal-to-noise ratio and greatly improves the analytical performance of the test. The two components required to perform a sandwich immunoassay are inkjet-printed directly onto the surface: (1) "stable" capture antibodies that remain entrapped in the brush even after exposure to a liquid sample and (2) fluorescently labeled "soluble" detection antibodies that dissolve upon exposure to a liquid sample. The polymer brush provides hydration to the antibodies, allowing them to remain stable and active over prolonged periods of time. When a liquid sample containing a biomarker of interest is dispensed onto the chip, the detection antibodies dissolve and diffuse to the stable capture spots forming a complex that sandwiches the analyte and that has a fluorescence intensity proportional to the concentration of the biomarker in solution, which can be measured using a custom-built smartphone detector. As multiple capture antibodies can be printed as discrete capture spots, the assay can be easily multiplexed without the need for multiple fluorophores. This chip and detector platform can be utilized for the point-of-care detection of low-abundance biomarkers directly from blood or serum in low-resource settings.
三明治免疫分析是检测蛋白质分析物的金标准。在这里，我们描述了一种超灵敏的即时护理三明治免疫分析平台，用于使用定制的智能手机检测器直接从血液或血清中检测生物标志物。由于基质的复杂性，未经稀释的血液或血清的测试具有挑战性。蛋白质非特异性地吸附到细胞上，并且经常附着在分析物表面上，这会极大地影响分析物的分析灵敏度。为了解决这个问题，我们的分析物建立在“无缺陷”聚合物刷的基础上，聚合物刷“从”玻璃载玻片“接枝”，几乎消除了所有非特异性结合，从而提高了信号与噪声的比率，大大提高了测试的分析性能。执行三明治免疫分析所需的两个组件直接喷墨打印到表面上：（1）“稳定”的捕获抗体，即使在暴露于液体样品后仍保持困在刷中，（2）荧光标记的“可溶性”检测抗体，在暴露于液体样品后溶解。聚合物刷为抗体提供水合作用，使它们在长时间内保持稳定和活性。当含有感兴趣的生物标志物的液体样品分配到芯片上时，检测抗体溶解并扩散到稳定的捕获点，形成一个夹在分析物之间的复合物，其荧光强度与溶液中生物标志物的浓度成正比，可使用定制的智能手机检测器进行测量。由于可以将多个捕获抗体打印为离散的捕获点，因此无需多个荧光团即可轻松实现多重检测。该芯片和检测器平台可用于在低资源环境中直接从血液或血清中进行即时护理的低丰度生物标志物的检测。
