Construction Of CRISPR/Cas14a Combined With Novel MOF Materials For Biosensor Detection Of Burkholderia Pseudomallei

Objective:To develop an electrochemical biosensor based on CRISPR/Cas14a combined with novel metal-organic framework nanomaterials with high sensitivity and specificity for the identification of genomic fragments of Burkholderia pseudomallei for rapid,simple and ultra-sensitive detection of Burkholderia pseudomallei.Methods:In this study,a novel electrochemical biosensor for nucleic acid detection of Burkholderia pseudomallei with high sensitivity and specificity was constructed by combining target-activated CRISPR/Cas14a and PCN-224 nanocomposites functionalized with PtPd nanoparticles as a dual signal amplification strategy.First,PtPd@PCN-224 nanoparticles were prepared by in situ generation of PtPd nanoparticles on the surface of PCN-224 nanomaterials,which exhibited excellent peroxidase-like activity towards H₂O₂ catalysis.In the absence of target DNA,CRISPR/Cas14a was not activated,which Left the phosphorylated ss DNA（P-ss DNA）pre-assembled at the electrode interface uncut.Subsequently,the PtPd@PCN-224nanoenzyme was immobilized on the P-ss DNA modified electrode based on the Zr-O-P coordination bond,and under the addition of H₂O₂ to the reaction system,the PtPd@PCN-224 nanoenzyme exhibited a strong reduction peak current generated by catalytic H₂O₂.In contrast,in the presence of target DNA,which activates CRISPR/Cas14a for trans cleavage activity,the P-ss DNA at the electrode interface is cleaved and the PtPd@PCN-224 nanoenzyme is unable to assemble at the electrode interface through the Zr-O-P bond,resulting in a weaker reduction peak current for H₂O₂.In the“on-off”mode,the electrochemical signal decreases with increasing target DNA concentration,resulting in an ultra-sensitive detection of Burkholderia pseudomallei.Results:（1）Firstly,characterisation by transmission electron microscopy（TEM）and elemental energy spectral surfaces（Mapping）showed that the nanomaterials in this study were synthesised successfully,while X-ray photoelectron spectroscopy（XPS）verified that phosphorylated ss DNA（P-ss DNA）could be stably bound to PtPd@PCN-224 nanoenzyme via Zr-O-P bonds.The Michaelis constant（K_m value）of PtPd@PCN-224 nanase was calculated by applying UV spectrophotometry-kinetics.With the reaction substrate TMB concentration kept constant and the addition of an equal gradient concentration of H₂O₂,the K_m value of PtPd@PCN-224nanase is 0.127×10^-3 M,the maximum reaction rate(V_max)was 4.237×10^-8 M/s,showing great performance advantages compared to other nanoenzymes reported in the literature.The trans cleavage of non-specific ss DNA by CRISPR/Cas14a was verified by non-denaturing polyacrylamide gel electrophoresis experiments.（2）Secondly,by testing the electrochemical differential pulse voltammetry（DPV）signal,we can see that the electrochemical reduction peak currents show great differences in the presence and absence of a target.When no target is present,as there is no activation of the non-specific ss DNA cleavage activity of CRISPR/Cas14a,the PtPd@PCN-224 nanoenzyme can be assembled to the electrode via the Zr-O-P bond,while the electrochemical DPV signal exhibits a strong reduction peak current in the presence of H₂O₂,indicating that PtPd@PCN-224 is attached to the electrode via the Zr-O-P bond attached to P-ss DNA and can catalyse H₂O₂ to generate an electrochemical signal;in contrast,the electrochemical DPV signal exhibited a weak reduction peak current when the target was present,suggesting that the target activated the trans cleavage activity of CRISPR/Cas14a.This resulted in a decrease in signal.These results validate the catalytic performance of the PtPd@PCN-224nanoenzyme and the feasibility of the overall experimental protocol.（3）Finally,we optimised the experimental conditions of this experimental protocol and the optimal experimental conditions were determined as follows:（1）the optimal ratio of CRISPR/Cas14a to sg RNA was 3:2,（2）the optimal p H value of the reaction was 7.4,（3）the optimal concentration of non-specific single-stranded DNA（P-ss DNA）on the electrode was 1μM,and（4）the optimal reaction time was 45 min.（4）After optimising the experimental conditions,we also analysed the performance of the biosensor.The Linear regression equation for this experimental protocol wasΔI%=0.7977+0.0977Lg C_{B.pseudomallei DNA},with a correlation coefficient（R²）of 0.9916,a detection range of 100 a M to 10 n M,and a minimum Limit of detection（LOD value）of 12.8 a M.（5）In addition,we investigated the specificity and stability of the biosensor and comparison with conventional methodology（q-PCR）,and the results showed that the method has good specificity and stability as well as consistency with conventional methodology.Conclusion:In this study,a novel electrochemical biosensor for DNA detection of Burkholderia pseudomallei was successfully established by combining target-activated CRISPR/Cas14a and PtPd nanoparticles functionalized with PCN-224nanocomposite as a dual signal amplification strategy,and with high sensitivity and specificity.An ultra-sensitive detection of Burkholderia pseudomallei was achieved.Furthermore,it is noteworthy that this study proposes a general electrochemical biosensing strategy for the identification of genomic fragments of pathogenic bacteria by well-designed sg RNA in CRISPR/Cas14a,which has great potential in clinical diagnostic applications.