Conventional affinity-based colorimetric enzyme-linked immunosorbent assay (ELISA) is one of the most widely used methods for the detection of biomarkers. However, rapid point-of-care (POC) detection of multiple cancer biomarkers by conventional ELISA is limited by long incubation time, large reagent volume, and costly instrumentation along with low sensitivity due to the nature of colorimetric methods. Herein, we have developed a reusable and cost-effective paper-in-polymer-pond (PiPP) hybrid microfluidic microplate for ultrasensitive and high-throughput multiplexed detection of disease biomarkers within an hour without using specialized instruments. A piece of pre-patterned chromatography paper placed in the PMMA polymer pond facilitates rapid protein immobilization to avoid intricate surface modifications of polymer and can be changed with a fresh paper layer to reuse the device. Reagents can be simply delivered from the top PMMA layer to multiple microwells in the middle PMMA layer via flow-through microwells, thereby increasing the efficiency of washing and avoiding repeated manual pipetting or costly robots. Quantitative colorimetric analysis was achieved by calculating the brightness of images scanned by an office scanner or a smartphone camera. Sandwich-type immunoassay was performed in the PiPP hybrid device after the optimization of multiple assay conditions. Limits of detection of 0.32 ng mL⁻¹ for carcinoembryonic antigen (CEA) and 0.20 ng mL⁻¹ for prostate-specific antigen (PSA) were obtained, which were about 10-fold better than those of commercial ELISA kits. We envisage that this simple but versatile hybrid device can have broad applications in various bioassays in resource-limited settings.

中文翻译：

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用于癌症生物标志物多路超灵敏检测的聚合物池纸 （PiPP） 混合微流控微孔板


常规的基于亲和力的比色酶联免疫吸附测定 （ELISA） 是检测生物标志物的最广泛使用的方法之一。然而，由于比色法的性质，传统 ELISA 对多种癌症生物标志物的快速即时 （POC） 检测受到孵育时间长、试剂量大、仪器费用昂贵以及灵敏度低的限制。在此，我们开发了一种可重复使用且具有成本效益的聚合物纸池 （PiPP） 混合微流控微孔板，无需使用专用仪器即可在一小时内对疾病生物标志物进行超灵敏和高通量多重检测。放置在 PMMA 聚合物池中的一张预先图案化的色谱纸有助于快速固定蛋白质，以避免聚合物的复杂表面修饰，并且可以更换新纸层以重复使用设备。试剂可以通过流通式微孔从顶部 PMMA 层简单地输送到中间 PMMA 层的多个微孔，从而提高清洗效率，避免重复手动移液或昂贵的机器人操作。通过计算办公室扫描仪或智能手机相机扫描的图像的亮度来实现定量比色分析。优化多个检测条件后，在 PiPP 杂交装置中进行夹心型免疫测定。获得了 0.32 ng ^mL-1 的癌胚抗原 （CEA） 和 0.20 ng ^mL-1 的前列腺特异性抗原 （PSA） 的检测限，比商业 ELISA 试剂盒的检测限好约 10 倍。我们设想这种简单但多功能的混合设备可以在资源有限环境中的各种生物测定中具有广泛的应用。