Strand displacement amplification triggered 3D DNA roller assisted CRISPR/Cas12a electrochemiluminescence cascaded signal amplification for sensitive detection of Ec-16S rDNA

Abstract

Background: Escherichia coli can cause gastrointestinal infection, urinary tract infection and other infectious diseases. Accurate detection of Escherichia coli 16S rDNA (Ec-16S rDNA) in clinical practice is of great significance for the identification and treatment of related diseases. At present, there are various types of sensors that can achieve accurate detection of Ec-16S rDNA. Electrochemiluminescence (ECL) has attracted considerable attention from researchers, which causes excellent performance in bioanalysis. Based on the previous research, it is significance to develop a novel, sensitive and efficient ECL biosensor.

Results: In this work, an ECL biosensor for the detection of Ec-16S rDNA was constructed by integrating CRISPR/Cas12a technology with the cascade signal amplification strategy consisting of strand displacement amplification (SDA) and dual-particle three-dimensional (3D) DNA rollers. The amplification products of SDA triggered the operation of the DNA rollers, and the products generated by the DNA rollers activated CRISPR/Cas12a to cleave the signal probe, thereby realizing the change of the ECL signal. The cascade amplification strategy realized the exponential amplification of the target signal and greatly improved the sensitivity. Manganese dioxide nanoflowers (MnO₂ NFs) as a co-reaction promoter effectively enhanced the ECL intensity of tin disulfide quantum dots (SnS₂ QDs). A new ternary ECL system (SnS₂ QDs/S₂O₈^2-/MnO₂ NFs) was prepared, which made the change of ECL intensity of biosensor more significant. The proposed biosensor had a response range of 100 aM-10 nM and a detection limit of 27.29 aM (S/N = 3).

Significance and novelty: Herein, the cascade signal amplification strategy formed by SDA and dual-particle 3D DNA rollers enabled the ECL biosensor to have high sensitivity and low detection limit. At the same time, the cascade signal amplification strategy was integrated with CRISPR/Cas12a to enable the biosensor to efficiently detect the target. It can provide a new idea for the detection of Ec-16S rDNA in disease diagnosis and clinical analysis.

Keywords: CRISPR/Cas12a; Ec-16S rDNA; Electrochemiluminescence; Strand displacement amplification; Three-dimensional DNA roller.