An electrochemical RNA aptasensor based on sheet-like α-Fe2O3/Fe3O4 magnetic nanocomposites and CRISPR/Cas13a system for supersensitive detection of osteopontin

Abstract

Osteopontin (OPN) exhibits markedly elevated expression in malignant tumor tissues, rendering it a crucial tumor marker for cancer prevention and monitoring—underscoring the significance of its detection. This work proposed an electrochemical RNA aptasensor based on a novel sheet-like α-Fe₂O₃/Fe₃O₄ magnetic nanocomposites (MNCs) and CRISPR/Cas13a system to effectively detect OPN. The proposed aptasensor used the sheet-like α-Fe₂O₃/Fe₃O₄ MNCs as the conduction matrix and applied their magnetic property to accomplish self-assembly of the sensing element onto the electrode. The current change of this aptasensor depended on the activation degree of the CRISPR/Cas13a system, which correlated with the amount of OPN expression. Double-stranded RNA (Apt/Activator) was compelled disassembly due to the OPN's strong affinity of Apt, while single-stranded RNA (Activator) could be guided by crRNA to combine with Cas13a/crRNA and activated the enzymatic activity of Cas13a. Cas13a enzyme affected the current and electrical resistance by shearing the nucleic acid strands (Reporter) on the electrode, ultimately enabling the quantitative detection of OPN. The aptasensor demonstrated excellent selectivity, reproducibility, and stability, with the detection limit (LOD) of 0.33 pg·mL⁻¹ and the wide linear detection range of 1 pg·mL⁻¹ − 10 ng·mL⁻¹. These results offer a novel idea for advancing tumor marker electrochemical biosensors.