Y-shaped DNA nanostructures assembled-spherical nucleic acids as target converters to activate CRISPR-Cas12a enabling sensitive ECL biosensing

Abstract

Considering the trans-cleavage capabilities, high-specificity and programmability, the CRISPR-Cas system has been recognized as a valuable platform to develop the next-generation diagnostic biosensors. However, due to the natural interaction with nucleic acids, current CRISPR-Cas-based detection mostly applies in nucleic acid analysis rather than non-nucleic acid analysis. By virtue of spherical nucleic acids (SNAs) with programmability and specificity, the Y-shaped DNA nanostructures assembled-SNAs (Y-SNAs) were rationally designed as target converters to achieve the quantitative activation of CRISPR-Cas12a, enabling a highly specific and sensitive electrochemiluminescence (ECL) determination of alpha-methylacyl-CoA racemase (AMACR), a high specific protein biomarker of prostate cancer. Significantly, the Y-shaped DNA nanostructures comprised of assisted DNA (A1), AMACR aptamer and DNA activator of CRISPR-Cas12a were loaded on Au nanoparticles modified Fe₃O₄ magnetic beads (Au@Fe₃O₄ MBs) to construct the robust Y-SNAs. In the presence of the target AMACR, the Y-SNAs as target converters could achieve quantitative activation of CRISPR-Cas12a by outputting the DNA activators with a linear relationship to the target. The amplified ECL signals were triggered by the release of the ferrocene-labeled quenching probes (QPs) on the electrode surface due to the trans-cleavage activity of CRISPR-Cas12a, thereby realizing the sensitive ECL determination of AMACR from 10 ng/mL to 100 μg/mL with the detection limit of 1.25 ng/mL. In general, this approach provides novel perspectives on how to design a universal ECL platform of the CRISPR-Cas system to detect the non-nucleic acid targets beyond the traditional methods.