Label-Free Colorimetric Aptasensor Based on Nicking Enzyme Assisted Signal Amplification and DNAzyme Amplification for Highly Sensitive Detection of Protein

Abstract

Highly sensitive detection of proteins is essential to biomedical research as well as clinical diagnosis. Here, we develped a novel label-free colorimetric aptasensor based on nicking enzyme assisted signal amplification and DNAzyme amplification for highly sensitive detection of protein. The system consists of a hairpin DNA probe carrying an aptamer sequence for target, a G-riched DNA probe containing two G-riched DNAzyme segments and the recognition sequence as well as cleavage site for nicking enzyme, a blocker DNA, and the nicking enzyme. The hybridization of the G-riched DNA with the blocker DNA prohibits the formation of the activated DNAzymes in the absence of target. Upon addition of target to the system, the hairpin probe is opened by the specific recognition of the target to its aptamer. The open hairpin probe hybridizes with a G-riched DNA and forms a DNA duplex, which triggers the selective cleavage of the G-riched DNA probe by nicking enzyme, leading to the release of the aptamer–target complex and the G-riched DNAzyme segments. The released open hairpin probe then hybridizes with another G-riched DNA probe, and the cycle starts anew, resulting in the continuous cleavage of the G-riched DNA probes, generating a much of G-riched DNAzyme segments. The G-riched DNAzyme segments interact with hemin and generates the activated DNAzyme that can catalyze the H₂O₂-mediated oxidation of 2,2′-azino-bis­(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^2–) to the colored ABTS^•–, thus providing the amplified colorimetric detection of target. With the use of thrombin (Tb) as a proof-of-principle analyte, this sensing platform can detect Tb specifically with a detection limit as low as 1.5 pM, which is at least 4 orders of magnitude lower over the unamplified colorimetric assay. Moreover, the assay does not involve any chemical modification of DNA, which is simple and low-cost. This sensing platform provides a promising approach for the amplified analysis of target molecules