T4 DNA Polymerase-Proofread DNA Binding Identifier for Sensitive Homogeneous Immunoassays

Abstract

Aptamer-based homogeneous immunoassays exhibit considerable potential in the domains of bioanalysis and biodiagnosis owing to their universality in analyzing both proteins and small molecules as well as their compatibility with nucleic acid amplification technologies. Nevertheless, the substantial signal leakage by nonspecific aptamer allostery poses a challenge to enhancing sensitivity further. Herein, we reported a T4 DNA polymerase-proofread DNA binding identifier (ReID). This strategy could harness the dual-enzymatic activity of T4 DNA polymerase to eliminate the leaked signal, thereby efficiently integrating target-induced aptamer allostery with subsequent polymerase chain reaction signal amplification. Moreover, we explored the regulation mechanism of dNTPs concentration on the dual-enzymatic activity of the T4 DNA polymerase. As a result, this strategy achieved an ultrasensitive protein detection limit of 8 fg/mL, validating the effectiveness of this proofreading approach. The universality was further confirmed by highly sensitive detection of small molecules. The exploration of ReID represents a significant advancement in the sensitivity and universality of immunoassays, even demonstrating the potential for multiple proteomic assays, offering a novel perspective for the development of high-performance homogeneous immunoassays.