Enhancing peroxidase activity of NiCo₂O₄ nanoenzyme by Mn doping for catalysis of CRISPR/Cas13a-mediated non-coding RNA detection

CRISPR/Cas13a with precise and controllable programming of endonuclease activity has been served as powerful tool for RNA sensing. Although with high sensitivity, existing CRISPR/Cas13a-based biosensors need complex amplification procedure or special equipment that limited quantification capability. Here, Mn-doped NiCo₂O₄ (Mn/NiCo₂O₄) nanozyme with enhanced peroxidase activity was synthesized and combined with CRISPR/Cas13a-based reaction to develop a simple, sensitive and universal biosensor for RNA detection, which is achieved through target recognition that activates Cas enzymes to cleave RNA reporter for inhibiting Mn/NiCo₂O₄ nanozyme to assemble on microplate. The Mn/NiCo₂O₄ nanozyme assembled on microplate can be monitored through colorimetric and fluorometric approaches. On one hand, Mn/NiCo₂O₄ nanozyme offers ideal peroxidase activity to catalyze colorimetric reaction, and as low as dozens of amol level of RNA target can be sensitively detected by naked eyes without any amplification procedures. On the other hand, Mn/NiCo₂O₄ can be also served as a signal amplifier to produce large amount of Co²⁺, Mn²⁺and Ni²⁺ to quench the fluorescence of calcein. The fluorescent approach can achieve higher sensitivity (about 40-fold) than colorimetric method. More importantly, the proposed biosensor can work well for multiple RNA detection in real biological samples, showing a great potential for monitoring non-coding RNA-related diseases.