Membrane-based microfluidic chip constructed and applied in magnetic microparticle anchoring-and-release digital immunoassay

Digital immunoassay is a valuable method for quantifying protein at the single-molecule level. Traditional digital immunoassay (the single-molecule arrays, SiMoAs) involves distributing the antibody-antigen-antibody sandwich immune complex into droplets, but some targets are lost during this process. To address this issue, we developed a magnetic microparticle anchoring-and-release digital immunoassay method for protein detection. We embedded a commercial track-etched polycarbonate (PCTE) membrane into the microfluidic chip for droplet generation, and modified the magnetic microparticles (MMPs) with enzymes for signal production. This method involves two processes: first, the enzymes modified MMPs are anchored in 96-well plates through the target protein. Second, the enzymes modified MMPs were released, and digitally quantified in the membrane-based microfluidic chip. This magnetic microparticle anchoring-and-released digital immunoassay method was successfully applied to the detection of p24 protein. Results showed that p24 could be digitally detected within the range of 1–100 pg/mL with good selectivity and accuracy using this digital method. By reducing signal loss during the digital immunoassay process, this method represents a significant advancement in digital technology.