Single DNA Detection With Magnetic Microsphere Probe Based On Volume Amplification And Electrochemical Technique

In chapter one of this thesis,we provided a novel method to detect single DNA molecules.A sandwich-type hybridization assay is performed on a streptavidin-coated substrate.A large number of biotinylated capture DNAs(c-DNAs)are first covalently bound to the substrate through the interaction between streptavidin and biotin.Then, the complementary target DNAs(t-DNAs)are hybridized with the c-DNAs attached to the substrate,followed by hybridization between t-DNAs and biotinylated first probe DNAs(p-DNAs)conjugated to streptavidin-coated magnetic microparticles (MMPs).In this case,one t-DNA binds one MMP which could be observed under an optical microscope.To quantitatively determine t-DNAs,The images of all MMPs were taken by DP70,then the number of MMPs on the images were obtained by using a MateMorph software.The number of the MMPs is linearly proportional to the concentration of t-DNA in a range of 5×10^-16-1×10^-14mol/L。Additionally,we disscuss the influence of hybridization time,hybridization form,hybridization buffered solution and the times of the cleaning to hybridization effect.This size amplification method was simple and could be applied to other biomolecules.In chapter two,we describe a magnetic nanobead-based single-molecule electrochemical detection method of DNA by combining DNA amplification and enzyme-catalysis amplification.A sandwich-type hybridization assay is performed on a streptavidin-coated substrate.A large number of biotinylated capture DNAs (c-DNAs)are first covalently bound to the substrate through the interaction between streptavidin and biotin.Then,the complementary target DNAs(t-DNAs)are hybridized with the c-DNAs attached to the substrate,followed by hybridization between t-DNAs and biotinylated first probe DNAs(p-DNA1s)conjugated to streptavidin-coated magnetic nanobeads(MNBs).In this case,one t-DNA binds one MNB.Subsequently,the MNBs are released from the substrate and transferred to another vessel.Then,biotinylated second probe DNAs(p-DNA2s)are hybridized to the p-DNA1s on the surface of the MNBs.After magnetic separation from the reaction media,the biotinylated p-DNA2 on the MNBs are labeled with alkaline phosphatase(AP)conjugated by streptavidin through the interaction between streptavidin and biotin.The MNBs with DNA hybrid labeled by AP(AP-DNA-MNBs) with enzyme substrate disodium phenyl phosphate(DPP)are continuously introduced by pressure through a capillary as the microsampler and microreactor by means of a microsyringe pump.AP on the AP-DNA-MNBs converts a huge number of DPP into its product phenol around each AP-DNA-MNB during AP-DNA-MNB movement in the capillary.The phenol zones with the moving AP-DNA-MNBs are continuously delivered to the capillary outlet and detected by electrochemical detection.One phenol zone corresponding to one t-DNA produces one peak on elution curve.The number of the peaks is linearly proportional to the concentration of t-DNA in a range of 5.0×10^-16-1.0×10^-13.The high sensitivity is because that in this method,phenol is detected rather than t-DNA.The phenol is generated through double amplification, one t-DNA molecule produces 7000 AP molecules on the surface of one AP-DNA-MNB(DNA amplification)and one AP molecule produces 5×10⁴ phenol molecules(enzyme amplification).Therefore,an amplification factor of 10⁸ is obtained in the method,which leads to that electrochemical technique can detect single DNA molecules.The quantitative method relies on counting the number of the peaks corresponding to single t-DNA molecules rather than signal intensity of the peaks.Thus,the detected signal intensity is not important.This feature guarantees the reliability of the electrochemically quantitative assay for DNA.Additionally,the selectivity of the method is high.