Study On Highly Sensitive Biosensors Based On Nuclease-Assisted Signal Amplification Strategy

In this thesis, we constructed three novel and highly sensitive biosensors based on nuclease-assisted cascade amplification and DNAzyme-assisted signal amplification strategy, to detect nucleic acid, protein and enzymes.In chapter â…¡, we constructed a highly sensitive fluorescent biosensor by the coupling of Exo â…¢-assisted cascade target recycling and DNAzyme amplification strategy to detect target DNA. A hairpin DNA probe was designed, which contained the DNA fragment for target recognition and the DNAzyme sequence, the sequence of DNAzyme was caged in the loop region and its activity was suppressed. In the presence of target, the probe can recognize and hybrid with it, trigger Exo â…¢-assisted cascade cleavage recycling process, releasing of target DNA and secondary target. Also, the DNAzyme unit could be released from this hairpin DNA probe. Then, the cyclic cleavage of the activated DNAzyme toward the molecular beacon substrate induced a remarkable amplified fluorescence signal for target detection. A low detection limit of 20 fM with an excellent selectivity toward target DNA could be achieved.In chapter â…¢, the catalytic hairpin DNA assembly-programmed active Mg2+-dependent DNAzyme was proposed for highly sensitive fluoresecent detection toward protein and DNA. The protein detection was implemented with the further combination of an additional terminal protection strategy, with the use of avidin as a model analyte. Exo I fails to catalyze the stepwise hydrolysis of the small-molecule-linked DNA when it is bound to the protein target through the small-molecule moiety, leaving the ssDNA intact. Then, the catalytic hairpin DNA assembly was proposed as a scaffold for the autonomous organization of the active Mg2+-dependent DNAzyme structure from its inactive or caged subunits, ultimately accomplishing the dual-signal amplification toward protein analysis. The detection limit toward avidin could be achieved as 2 pM. Furthermore, this dual-signal amplification strategy based on catalytic hairpin DNA assembly-programmed active Mg2+-dependent DNAzyme could be demonstrated for target DNA detection, and the detection limit could be as low as 0.5pM with an excellent selectivity.In chapter â…£, by using of isothermal strand displacement amplification combind with DNAzyme-assisted signal amplification, we reported a new method for highly sensitive detection of DNA methyltransferase activity. A new hairpin probe DNA and template DNA was designed in this experiment. The probe can be methylated by sepical DNA methyltransferase and prevent the restriction endonuclease cleavage it. Then, with the help of polymerase and nicking endonucleasecan, it can polymerize many DNA fragment, which would induce another isothermal strand displacement amplification producing a lot of functionalized DNAzyme units with the template DNA-assisted. In this chapter, we have proposed a new method for highly sensitive detection of DNA methyltransferase activity with a low detection limit of 1mU ml-1.