Inhibition of Polymerase Chain Reaction by Hydrogel Monomers

Abstract

Hydrogels are increasingly being integrated into polymerase chain reaction (PCR)-based diagnostic platforms because of their biocompatibility and ability to be compartmentalized at the microscale. However, the direct effect of hydrogel monomers on PCR performance is not fully understood. As such, we systematically evaluated the inhibitory effects of commonly used hydrogel monomers, including acrylamide, poly(ethylene glycol) dimethacrylate (PEGDMA), ethylene glycol diacrylate, ethylene glycol dimethacrylate (EGDMA), and gelatin methacryloyl (GelMA), on Taq polymerase activity and amplification efficiency. Our results revealed that even low concentrations of PEGDMA and acrylamide strongly inhibited the PCR, whereas GelMA and EGDMA minimally interfered with PCR. The results of mechanistic studies suggested that the α,β-unsaturated carbonyl groups in monomers inactivate the polymerase through covalent interactions with nucleophilic amino acids. Various PCR enhancers were evaluated to address this issue. Nonionic surfactants with low critical micelle concentrations, such as Tween 20, Tween 80, and NP-40, successfully restored PCR amplification under PEGDMA-rich conditions. In contrast, additives such as dimethyl sulfoxide and Triton X-100 were ineffective. Using excess Taq polymerase mitigated the acrylamide-induced inhibition, supporting direct monomer–enzyme interactions. These findings provide molecular insights into hydrogel–PCR compatibility and can be used to guide the development of strategies for developing robust hydrogel-integrated PCR systems for diagnostics and genomics.

Keywords: PCR enhancers, DNA analysis, protein, diagnostics, genomics