A Miniaturized, Multilayer Structured Vertical Flow Paper Device Enabled One-Step, Multiplex, Spontaneous Detection of Biomarkers in Microliter Blood for Disease Screening.

Developing a miniaturized, portable device for multiplexed detection of a library of protein biomarkers from microliter blood sample is critical for early disease screening in many scenarios, such as home healthcare and emergency care. However, major challenges remain, including multiplexed detection without cross-interference, system-level miniaturization, reliable flow manipulation, and simple user operation. Here, we present a miniaturized, multilayer structured vertical flow paper device that enables one-step, multiplexed, spontaneous detection of biomarkers in microliter blood for screening of heart disease, kidney disease, and inflammation. The device is designed with a multilayer membrane structure that integrates 3D-printed glass fiber membranes, wax-printed nitrocellulose membranes, and cellulose absorption membranes, enabling autonomous wicking of the sample through the device while concurrently distributing the sample into multiple equal parts and executing the sandwich immunoassay and washing steps. Simultaneous quantification of 12 biomarkers is achieved, and clinical performance is validated using clinical samples from patients with acute myocardial infarction (AMI), acute kidney disease (AKD), or sepsis. The device requires about 60 μL of sample, delivers results in 15 min, and is also compact (12.5 cm3). Overall, this platform provides a novel and universal strategy for miniaturized multiplexed immunoassays for rapid clinical screening.