Electrochemical DNA Sensing Based On Biological Enzymes And Nanomaterials

Recently, with various diseases reported, the detection of specific DNA sequence has attracted considerable interest due to its extensive applications in molecular, genetics therapy, and early diagnosis of cancer. Therefore, developing a novel, sensitive and fast method to detect DNA is of great importance. Compared with traditional DNA detection methods, the electrochemical method has various advantages, such as rapid response, high sensitivity, simple design, miniaturization of the instruments, low cost and so on. In this thesis, Mspl endonuclease could recognize its specific sequence in double-stranded (dsDNA) DNA and cleave the dsDNA fragment off the electrode. The specificity of the method can also be improved owe to Mspl endonuclease specific interaction with dsDNA. Mspl endonuclease combining with horseradish peroxidase and CdSe quantum dots was applied in DNA detection field. Once probe DNA hybridized with target DNA to form dsDNA, Mspl endonuclease could recognize the specific sequence in the dsDNA and cleave the fragment linked with signal label off the electrode, he remaining electrochemical signal decreased with increase of target DNA concentrations. Based on this, we can detect Mtb DNA sensitively. Acetylcholinesterase (AChE) can be immobilized on SnSe2hollow spheres and its biological activity retained well, based on the inhibition toward the enzymatic activity of AChE, the biosensor for detection of phoxim was constructed. Undecylic acid coated Fe3O4nanoparticles affect on the electrochemical signal of surface-confined methylene blue when they were attracted to the electrode surface by the external magnet, in this case, the property of electrode surface was changed, and the electron transfer of methylene blue was confined. The details are listed as follows:1. A novel electrochemical strategy for monitoring DNA was developed based on specific cleavage of Mspl endonuclease combining with streptavidin-horseradish peroxidase (SA-HRP) conjugate. By introducing the strategy to mycobacterium tuberculosis (Mtb) DNA detection, the probe DNA (its5’end is labeled with SH and3’end is labeled with biotin) was immobilized on Au electrode via Au-S bond and SA-HRP conjugate was linked to probe DNA by biotin-SA specific interaction. Once recognition of the probe DNA assembled on the electrode to target DNA, probe DNA hybridized with target DNA to form double-stranded DNA (dsDNA). MspI endonuclease could recognize the specific sequence in the dsDNA and cleave the fragment linked with HRP-SA off the electrode, which led to the electrochemical signal decreases. And the value decreased linearly with the increase of target DNA concentrations. This proposed electrochemical method exhibited satisfactory performance which could detect Mtb DNA linearly ranging from10pM to100nM with a detection limit of2.3pM. The novel strategy of DNA analysis showed a promising application in clinic diagnostics.2. A novel electrochemical strategy for the detection and identification of Mtb DNA based on the site-specific cleavage of MspI endonuclease and signal amplification with gold nanoparticles (AuNPs) was designed. The probe DNA (its5’ end was labeled with SH and3’end was labeled with biotin) was self-assembled on a gold nanoparticles coated glass carbon electrode (GCE), CdSe quantum dots labeled streptavidin (QDs-SA) was linked to the probe DNA via streptavidin (SA)-biotin interaction. Once recognition of the probe DNA assembled on the electrode to target DNA, probe DNA hybridizes with target DNA to form dsDNA. Mspl endonuclease could recognize the specific sequence in the dsDNA and cleave the dsDNA fragment linked with QDs-SA off the electrode, and the remaining attached signal tags can be easily read out by square-wave voltammetry using electrodeposited bismuth film modified glass carbon electrode after dissolving with acid. This newly designed protocol provided an ultrasensitive electrochemical detection of DNA down to8.7×10-15M with a linear range of5orders of magnitude (from1×10-14to1×10-9M). The developed strategy also showed high selectivity against single-base mismatch sequences. These satisfactory performances made this method a great potential for early diagnosis in gene-related disease.3. A simple method for immobilization of acetylcholinesterase (AChE) on SnSe2hollow spheres for detection of phoxim was described. SnSe2hollow spheres were prepared by hydrothermal methods and were characterized by transmission electron microscope (TEM). The immobilized AChE retained its biological activity well and could catalyze the hydrolysis of acetylthiocholine to form thiocholine, which was then oxidized to produce the detectable signal. Based on the inhibition toward the enzymatic activity of AChE by phoxim, under optimal conditions, the sensors could be used for the determination of phoxim ranging from0.008to56ug/mL with the detection limit of0.004μg/mL. The developed phoxim biosensors exhibited good stability and reproducibility. This work demonstrated that SnSe2hollow sphere could be served as an ideal carrier for immobilization of AChE to fabricate the corresponding biosensor.4. Undecylic acid coated Fe3O4nanoparticles were prepared, and the effect on the signal of surface-confined methylene blue of Fe3O4nanoparticles when the nanoparticles were retracted from or attracted to the electrode surface by the external magnet was investigated. This work demonstrated that the signal of methylene blue in phosphate buffered saline system woule be decreased with reversible attachment to and away from the electrode surface of the Fe3O4nanoparticles. All this may be illustrated that when the Fe3O4nanoparticles were attracted to the electrode surface, properties of electrode surface were changed, and the electron transfer of methylene blue was confined.