A Novel DNA Detection Method Based On Bio-barcode And Gold Nanoparticle Amplification

Cancer is one of the great threats to human health.The occurrence and development of cancer are closely related to genes.The concentrations of relevant DNA markers are very low at the early stage of cancer which are difficult to be detected detect by general methods,resulting in the delay of treatment.Therefore,it is great significant that develop highly sensitive and highly specific DNA detection methods to achieve early diagnosis of the diseases such as cancer and improve the survival of patients.In this thesis,using cancer related gene as the target DNA,two highly sensitive DNA detection methods were designed based on nanotechnology and signal amplification technology.One is gold nanoparticles/bio-barcode amplification ICP-MS gene detection,the other is bio-barcode/gold label silver stain amplification electrochemical gene detection.The main contents of this thesis are as follows:?1?Preparation,modification and characterization of nanoparticles.The sodium citrate reduction method,chemical co-precipitation method and St?ber hydrolysis method were used to synthesize gold nanoparticles?AuNPs?,magnetic nanoparticles?MNPs?and silica nanoparticles?SiO₂?,respectively.The magnetic nanoparticles and silica nanoparticles were amino functionalized with aminosilane.Transmission electron microscopy?TEM?,fourier transform infrared spectrometer?FTIR?,ultraviolet-visible spectrometer?UV-Vis?,X-ray diffractometer?XRD?and vibrating sample magnetometer?VSM?were used to characterize the as-prepared nanoparticles.The results showed that the size of gold nanoparticles and silica nanoparticles were around 13 nm and 95 nm respectively with good dispersibility.The magnetic nanoparticles have a particle size around 31 nm and a saturation magnetization of 58 emu/g with superparamagnetism.The aminosilane was successfully modified on the surface of magnetic nanoparticles and silica nanoparticles.?2?A novel ICP-MS gene detection method based on AuNPs and bio-barcode signal amplification was developed.The SiO₂/AuNPs barcode probes were constructed by combining SiO₂ nanoparticle-labeled DNA with AuNPs based on bio-barcode technology,which hybridized with target DNA and magnetic nanoparticle surface-modified DNA to form a sandwich structure.After magnetic separation,the gold nanoparticles were released using dithiothreitol?DTT?to substitute oligonucleotides on the surface of gold nanoparticles,and then the concentration of gold ions was detected by inductively coupled plasma mass spectrometry?ICP-MS?.The preparation of SiO₂/AuNPs barcode probes,DNA hybridization temperature and hybridization time were optimized.The results showed that the optimal DNA ratio of SiO₂/AuNPs barcode probe was 75:1.The optimal modification amount of AuNPs dispersion was 4 mL,the optimal hybridization temperature and hybridization time were 42°C and 90 min,respectively.The limit of detection of this DNA detection method was 1 fmol/L,and the linear range was 1 to 500 fmol/L.This method can clearly distinguish the complementary and mismatched DNA,which has a good detection specificity.?3?A novel electrochemical gene detection method based on bio-barcode/gold label silver stain amplification was presented.The SiO₂/AuNPs barcode probes were constructed by combining SiO₂nanoparticle-labeled DNA with AuNPs,which hybridized with target DNAs and the electrode surface-modified DNA probes.The silver ions were reduced on the surface of gold nanoparticle to form a silver shell by the catalysis of gold nanoparticles,and then the electrochemical detection of deposited silver was performed by linear sweep voltammetry?LSV?.In this thesis,the effects of electrode blocking agent,blocking time and silver deposition time on electrochemical detection were investigated.The results showed that the background signal can be effectively decreased by using decylhexanol to block the gold electrode.The optimal blocking time was 3 h,and the optimal silver deposition time was 6 min.The electrochemical DNA biosensor had a low detection limit of 0.23fmol/L and a linear range of 1 fmol/L to 10 pmol/L.This method can clearly distinguish the complementary and mismatched DNA,with good repeatability and stability.