Determination Of Nucleic Acid And Thrombin Based On Hybridization Chain Reaction And Nano-particle Labelling

With the development of nanotechnology,nanomaterials can be designed as sensitive chemical and biological sensors.As a zero-dimension nanomaterial,nanoparticle become more and more popular in biosensors due to their size-dependent optical,electronic properties and their dimensional similarities with biomacromolecules.As a common amplification technique,the hybridization chain reaction（HCR）has many advantages,such as simple protocols and high amplification efficiency.In a typical HCR process,the primer triggers a cascade of hybridization events to form a long double-strand DNA.As a highly efficient signal amplification technology,HCR has been applied to detect a variety of analytes,including nucleic acids,proteins,small molecules and cells.In this paper,two universal and sensitive optical analysis methods were established based on the combination of nanomaterials（gold nanoparticles,quantum dots）with the HCR amplification technology,which realized the detection of nucleic acid and thrombin.The main research contents are as follows:（1）DNA functionalized gold nanoparticles（DNA-AuNPs）were prepared by freezing method.And a universal and sensitive colorimetric analysis method was established combining HCR amplification with DNA-AuNPs,which realized the detection of target DNA and thrombin.For the detection of target DNA,the presence of the target DNA triggered HCR,then the formed HCR product hybridized with DNA-AuNPs to induce the aggregation of DNA-AuNPs,as a result,the absorbance of the detection system decreased.According to the relationship between the absorbance of solution and the concentration of target DNA,the quantitative analysis method of target DNA was established.The linear range of this method for target DNA detection was 10-200 p M and the detection limit was 4.47 p M.For the detection of thrombin,the primer could be designed according to the thrombin aptamer.In the absence of thrombin,the primer hybridized to the thrombin aptamer to prevent HCR.Once upon the addition of thrombin,the thrombin bound to the aptamer so the primer could be released to trigger HCR.Afterwards,the HCR product was hybridized with DNA-AuNPs to induce the aggregation of DNA-AuNPs,as a result,the absorbance of the detection system decreased.Thus,thrombin concentration was quantified according to the change in absorbance.Hence,the concentration of thrombin was quantified by the determination of absorbance.The linear range of this method for thrombin detection was 20 p M-1 n M and the detection limit was 1.26 p M.The method was used to detect thrombin in human serum,and the recovery was in the range of 90.1-104%.（2）DNA functionalized quantum dots（DNA-QDs）were synthesized by hydrothermal method.And a universal and sensitive fluorescence analysis method was executive combining HCR amplification with DNA-QDs,which realized the detection of thrombin.In the absence of thrombin,the primer bound to the thrombin aptamer to prevent HCR.Once upon the addition of thrombin,the thrombin interacted with the aptamer so the primer was released to trigger HCR.Subsequently,the HCR product hybridized with DNA-QDs,then the DNA-QDs labeled HCR products were separated by the streptavidin-modified magnetic beads.According to the relationship between the fluorescence intensity of supernatant and the concentration of thrombin,the quantitative analysis method of thrombin was established.Under optimized experimental conditions,the linear range of this method for thrombin detection was30 p M-2.5 n M and the detection limit was 0.15 p M.The method was used to detect thrombin in human serum,and the recovery was in the range of 97-108%.