An electrochemical method based on CRISPR-Cas12a and enzymatic reaction for highly sensitive detection of tumor marker MUC1 mucin

Abstract

The early warning of cancer is crucial in cancer prevention and anti-cancer, and the highly sensitive methods for detecting cancer biomarkers are essential for cancer early diagnostic. Herein, an electrochemical aptamer biosensor based on CRISPR-Cas12a system was constructed for the detection of cancer tumor biomarker MUC1 mucin. The sensitivity was significantly prompted by enzyme-catalyzed signal amplification, and the selectivity was improved by dual recognition of the aptamer to MUC1 and the crRNA-Cas12a system to aptamer. Glucose oxidase (GOD) was loaded on the surface of magnetic Fe3O4@Au (MGNP) via probe single-stranded DNA (pDNA) with terminal modification of mercapto (-SH) to form GOD-pDNA/MGNP. The corresponding aptamer of MUC1 (MUC1 Apt) binds to its complementary ssDNA (cDNA) to form the activator Apt/cDNA, which is specifically recognized by crRNA-Cas12a and excites the trans-cleavage function of Cas12a, thus in turn trans-cleave pDNA and detached GOD from the magnetic particles. The magnetic beads were separated and transferred into a glucose solution, and the oxidation current of H2O2 produced by the catalytic reaction of GOD was measured on a Pt modified magnetically controlled glassy carbon electrode, resulting in an indirect determination of MUC1. The current change was linear with the logarithm of MUC1 concentration in the range of 1.0×10-17 g/mL to 1.0×10-10 g/mL. The detection limit was as low as 7.01×10-18 g/mL. The method has been applied to the detection of MUC1 in medical samples.