Digital microfluidic chip with photopatterned reactive sites for direct biomolecules immobilization and magnetic beads-free immunoassay

Digital microfluidics (DMF), as an emerging liquid-handling technology, is widely recognized as an ideal platform for enzyme-linked immunosorbent assay (ELISA). However, current DMF-based ELISA methods are highly dependent on magnetic beads (MBs), which usually involves tedious modification of MBs, complex peripherals for MBs control, and impairs the parallel processing capability of DMF. To address these issues, we herein developed a photopatterned reactive sites-integrated DMF chip (PRS-DMF) for MBs-free immunoassay. By taking advantage of click chemistry between N-(allyloxycarbonyloxy) succinimide and thiol-ene substrate, the preparation of PRS is very simple, and leaves the reactive N-hydroxysuccinimide (NHS) ester groups on the surface. This enables direct biomolecules immobilization without any surface modification/activation processes. Moreover, the patterned micropillar array can notably increase the specific surface area, and promote the liquid mixing and immunoreaction. As a consequence, highly sensitive and MBs-free ELISA can be readily achieved on PRS-DMF, with a proof-of-concept H5N1 assay having been successfully demonstrated herein, which offers a low limit of detection (LOD) of 147 pg/mL. This also avoids the use of complex magnetic peripherals, and thus, contributing to the instrument miniaturization into a very compact form. It is therefore reasonably expected that the proposed PRS-DMF may serve as a promising platform for a variety of biological and biomedical applications, especially in the field of point-of-care testing (POCT).