Electrochemiluminescence aptasensing method for ultrasensitive determination of lipopolysaccharide based on CRISPR-Cas12a accessory cleavage activity

Talanta. 2024 May 15:272:125828. doi: 10.1016/j.talanta.2024.125828. Epub 2024 Feb 24.

Abstract

In this study, an ultrasensitive electrochemiluminescence (ECL) aptasensing method was developed for lipopolysaccharide (LPS) determination based on CRISPR-Cas12a accessory cleavage activity. Tris (2,2'-bipyridine) dichlororuthenium (II) (Ru(bpy)32+) was adsorbed on the surface of a glassy carbon electrode (GCE) coated with a mixture of gold nanoparticles (AuNPs) and Nafion film via electrostatic interaction. The obtained ECL platform (Ru(bpy)32+/AuNP/Nafion/GCE) exhibited strong ECL emission. Thiol-functionalized single-stranded DNA (ssDNA) was modified with a ferrocenyl (Fc) group and autonomously assembled on the ECL platform of Ru(bpy)32+/AuNP/Nafion/GCE via thiol-gold bonding, resulting in the quenching of ECL emission. After hybridization of the LPS aptamer strand (AS) with its partial complementary strand (CS), the formed double-stranded DNA (dsDNA) could activate CRISPR-Cas12a to indiscriminately cleave ssDNA-Fc on the surface of Ru(bpy)32+/AuNP/Nafion/GCE, resulting in recovery of the ECL intensity of Ru(bpy)32+ due to the increasing distance between Fc and the electrode surface. The combination of LPS and AS suppressed the formation of dsDNA, inhibited the activation of CRISPR-Cas12a, and prevented further cleavage of ssDNA-Fc. This mechanism aided in upholding the integrity of ssDNA-Fc on the surface of the electrode and was combined with ECL quenching induced by the target. The ECL intensity decreased linearly as the concentration of LPS increased from 1 to 50,000 pg/mL and followed a logarithmic relationship. This method exhibited a remarkably low detection limit of 0.24 pg/mL, which meets the requirement for low-concentration detection of LPS in the human body. The proposed method demonstrates the capacity of CRISPR-Cas12a to perform non-specific cutting of single-stranded DNA and transform the resultant cutting substances into changes in the ECL signal. By amalgamating this approach with the distinct identification abilities of LPS and its aptamers, a simple, responsive, and discriminatory LPS assay was established that holds immense significance for clinical diagnosis.

Keywords: Aptamer; Aptasensing; CRISPR-Cas12a; Electrochemiluminescence; Lipopolysaccharide.

MeSH terms

  • Biosensing Techniques* / methods
  • CRISPR-Cas Systems
  • DNA, Single-Stranded
  • Electrochemical Techniques / methods
  • Fluorocarbon Polymers*
  • Gold
  • Humans
  • Lipopolysaccharides
  • Luminescent Measurements / methods
  • Metal Nanoparticles*
  • Sulfhydryl Compounds

Substances

  • perfluorosulfonic acid
  • Lipopolysaccharides
  • DNA, Single-Stranded
  • Gold
  • Sulfhydryl Compounds
  • Fluorocarbon Polymers