Highly Sensitive Bioassay Based On Element Tagging And Hybridization Chain Reaction

Nucleic acid amplification strategy has been widely used in the highly sensitive detection of biological molecules,such as nucleic acid,protein,small biomolecules,cell,thanks to its strong ability of signal amplification.Comparing with thermal cycle amplification technology and isothermal amplification based on enzyme,hybridization chain reaction(HCR)has the feature of isothermal,mild conditions and excellent amplification efficiency,and enzyme-free nature.Based on its unique physical and chemical properties,metal nanoparticles have been widely applied in biological signal recognition and signal transduction,its strong load capacity of atoms,especially,inspired people to design the new signal amplification strategy.Inductively coupled plasma mass spectrometry(ICPMS)is an ideal instrument to detect elements,thanks to its high sensitivity,broad dynamic range,low detection limit,multiplexing potential,and absolute quantitation.ICPMS has been widely applied in the sensitive detection of biological molecule,because of the rapid development of metal stable isotope tagging strategy.This paper developed a highly sensitive detection method for nucleic acid and protein,based on metal nanoparticles labeling and hybridization chain reaction.In this thesis,combined with the advantages in signal amplification of metal nanoparticles labeling,hybridization chain reaction and ICPMS technology,a high sensitivity,wide linear range and good specificity method has been established to detect nucleic and protein.In the study of nucleic acid detection,the target DNA was combined with the captured DNA through hybridization,and immobilized on the magnetic beads.Target DNA,as the primer,will triggered HCR by the hybrid DNA.Then SA-AuNPs combined with the double-stranded DNA(dsDNA),which was the product of HCR,by the interaction between streptavidin and biotin.SA-AuNPs were dissolved by aqua regia,and the concentration of gold atoms will be detected by ICPMS.Finally,through the linear relationship between the gold ion signal and the target DNA concentration,the sensitive detection of target DNA was achieved.The detection limit of this method for target DNA was 0.56 amol,and the linear range was 10 amol-10~6 amol,the recovery rate of serum samples was between 89-108%,indicating the potential application in clinical samples.In the study of protein detection,target antigens were captured on the surface of magnetic beads,based on the specific recognition of target antigen and antibody.The antibodies and DNA were facilely functionalized on the surface of AuNPs.The antibody on the AuNPs combined with antigen to form a sandwich structure.The DNA on the AuNPs,as a primer,initiate HCR.Multiple DNAs were localized on the AuNPs at the same time,so a great deal of HCR was initiated.The dsDNA will combine with a lot of SA-AuNPs by the interaction of SA and biotin.AuNPs were dissolved and the gold atoms are detected by ICPMS.Finally,through the linear relationship between the gold ion signal and the target protein concentration,the sensitive detection of target protein was achieved.The detection limit of human IgG was 2.0 pg/mL,linear range was 0.01-1 ng/mL,and the recovery rate was 95-115%in serum,which demonstrated the advantage of this method in the detection of real samples,and provides a potential for the highly sensitive multiplex assay.