Development Of Paper Sensor Based On Programmable Cas12a/3D DNAzyme For Rapid Genotypic Antibiotic Susceptibility Test

Antibiotic resistance of clinical pathogens has become a major problem that needs to be solved urgently in the world.Rapid and accurate assessment of the susceptibility of bacteria to antibiotics is the key to solve the rapid development of antibiotic resistance.Therefore,there is an urgent need to develop new rapid analysis methods for antibiotic resistance of pathogens.In recent years,the development of analysis methods for drug-resistant genotypes can quickly screen super-resistant bacteria,which is of great guiding significance for clinical antibiotics.However,the commonly used polymerase chain reaction(PCR)method to analyze the genotype of bacteria requires professional laboratories and operators,and is difficult to use for rapid detection in resource-limited areas.Herein,we report a paper-based sensor constructed with CRISPR/Cas12 a and 3D DNAzyme for rapid detection of specific antibiotic-resistant gene(NDM-1)with high sensitivity.The DNA nanostructures prepared by Rolling Circle Amplification(RCA)make the paper sensor both programmability and high stability.By designing a DNAzyme domain containing a G-quadruplex on the DNA circular template(DNA Circle),the nanometer-sized 3D DNAzyme is able to adhere strongly to paper surface,producing highly bioactive paper sensor with high density of functional DNAzyme sequences.We design a cr RNA that can specifically target the NDM-1 gene.After Cas12 a and cr RNA form a complex,Cas12a/cr RNA can be activated by NDM-1 gene and subsequent collateral cleavage of nearby single-stranded nontargeted nucleic acids.In the absence of NDM-1 gene,3D DNAzyme is able to catalyze a colorimetric reaction for generating blue color signal.When there is NDM-1 gene in sample,the collateral cleavage activity of Cas12 a would be activated for cleaving 3D DNAzyme,resulting in no obvious colorimetric signal.We investigated the sensitivity and specificity of the sensor and its potential to identify bacterial species.This paper sensor provides rapid and low-cost detection of antibiotic-resistant genes carried by various pathogenic microorganisms with femtomolar level sensitivity and the detection results can be identified by naked eye or smartphone camera.The whole analysis approach only requires < 1 h assay time.Futhermore,based on the programmable CRISPR probe design of this paper sensor,we can develop detection probes for different genes,which has great potential to deal with new global epidemics.