传统的基于亲和力的比色酶联免疫吸附测定（ELISA）是最广泛使用的生物标志物检测方法之一。然而，通过传统 ELISA 对多种癌症生物标志物进行快速现场护理 (POC) 检测受到长孵育时间、大试剂量、昂贵的仪器以及由于比色方法的性质导致的低灵敏度的限制。在此，我们开发了一种可重复使用且经济高效的聚合物池纸（PiPP）混合微流体微板，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，而无需使用专门的仪器。放置在 PMMA 聚合物池中的一张预先图案化的层析纸有助于快速固定蛋白质，以避免聚合物复杂的表面修饰，并且可以更换新的纸层以重复使用该设备。试剂可以通过流通微孔简单地从顶部 PMMA 层输送到中间 PMMA 层的多个微孔，从而提高洗涤效率并避免重复的手动移液或昂贵的机器人。通过计算办公室扫描仪或智能手机摄像头扫描的图像的亮度来实现定量比色分析。经过多种检测条件的优化后，在PiPP混合装置中进行了夹心式免疫检测。癌胚抗原（CEA）的检出限为0.32 ng·mL-1，前列腺特异性抗原（PSA）的检出限为0.20 ng·mL-1，比市售ELISA试剂盒的检出限提高了约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的环境中的各种生物测定中具有广泛的应用。

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.