A Burkholderia Pseudomallei Detection Strategy Based On MOF Nanocubes Combined With Three-dimensional Magnetic Walking Nano-machines

Objective:To establish a new electrochemiluminescence sensor based on multi-functional MOF nano-cube and three-dimensional magnetic walking nano-machine for the ultra-sensitive detection of Burkholderia pseudomallei,and to realize the rapid,simple and sensitive detection of nucleic acid.Methods:In this study,we developed a highly sensitive electrochemiluminescence biosensor with"off-on"structure,which is based on Au@Co-MOF@ABEI nano-cube and three-dimensional magnetic walking nano-machine to detect Burkholderia mallei nucleic acid.In the presence of the target DNA chain,the target chain reacts with the protective probe（PP）on the DNA Walker by strand replacement reaction（SDA）to release the walking probe（DWT）.Subsequently,DWT can replace S3 to form the blunt 3’end of DWT and DWT1.Then,the Exo III is used to cut the orbit probe（DWT1）,and the walking DWT is driven to move independently along the designed 3D orbit,and the SMB is separated by magnets through magnetic separation,thus obtaining a large number of intermediate DNA S3.The released S3 chain can replace the DA-S2 chain on the electrode to restore the ECL signal,and the process is verified by detecting the electrochemiluminescence signal.Results:（1）Through the detection of fluorescence signal,we can see that the sample with target has a strong fluorescence signal,while the sample without target has only a very small fluorescence signal.It is proved that some of the experiments of DNA Walker are feasible.Through the detection of the electrochemical signal of ECL,we can see that the electrochemiluminescence signal of Au@Co-MOF@ABEI is much higher than that of Co-MOF@ABEI,Au@ABEI and ABEI.At the same time,when we added intermediate DNA S3 to the sensor modified with DA-S2 chain,the electrochemiluminescence signal of ECL was significantly enhanced,indicating that the intermediate DNA S3 produced by the part of DNA Walker could react with the modified DA-S2 chain on the electrode.These results verify the catalytic performance of Au@Co-MOF and the feasibility of the whole experimental scheme.（2）the experimental conditions of the scheme were optimized,and the optimal conditions were determined as follows:（1）the optimal ratio between walking chain（PWT）and orbital chain（SDs）was 1:20;（2）the concentration of Exo III exonuclease was 250U m L^-1,（3）the concentration of quencher dopamine was 0.31μM;（4）the time of chain replacement reaction was 1.5h.（3）the experimental performance analysis is carried out under the optimal experimental conditions,and a linear equation is obtained in 100a M～100p M,which is I=1362.9lgc-627.9,and the correlation coefficient is 0.996.（4）We also analyzed the specificity and stability of the experimental method,and the results showed that this method had good specificity and stability.Conclusion:In this study,a highly sensitive Au@Co-MOF@ABEI biosensor with"off-on"structure was successfully constructed by combining multi-functional ECL nanotubes and three-dimensional magnetic walking nanomachines,and a rapid,simple and sensitive method for the detection of Burkholderia mallei nucleic acid was realized.This method has good specificity and stability.In addition,it also has the following advantages:（1）nanotubes can not only fix a large amount of ABEI,but also shorten the distance between catalysis and luminescence system,so that the ECL signal is enhanced;（2）Exo III avoids the interference of non-specific DNA chains,and Exo III effectively drives the multi-functional walking nano-machine by amplifying the signal-to-noise ratio（SNR）.（3）The ECL biosensor developed for nucleic acid detection of Burkholderia pseudomallei provides a new detection method for the detection of Burkholderia pseudomallei,and has great application potential in the early diagnosis and treatment of gangrene-related diseases.