Real-time Detection Of DNA Based On Optical Tweezers And Upconversion Luminescence Nanolabels

Optical tweezers,also called single beam gradient force optical trap,which can be used to trap and manipulate objects with a size ranging from several nanometers to tens of micrometers,is generated by a strongly focused laser beam.Since its invention in 1986,optical tweezers has been widely applied in research fields such as biophysics,cell biology and nanoscience.In the field of biophysics,optical tweezers has been used to study the mechanics of biological molecules,which has greatly enhanced our knowledge of biological molecules interactions.In the field of cell biology,contact-free manipulation by optical tweezers is well suited for cell manipulation,cell sorting and biological function research.In the field of nanoscience,optical trapping of individual nanoparticles has been used to study photophysics of various nanoparticles.Currently,optical trapping of nanoparticles for biomedical applications such as gold nanoparticles(Au NPs),quantum dots(QDs)and upconversion nanoparticles(UCNPs)has been realized.UCNPs is capable of emitting ultraviolet and visible light when exicted with a near-infrared(NIR)light source,which shows excellent ability of anti-photobleaching,thus has advantages in the fields of biomedical analysis and in vivo imaging.The upconversion luminescence of UCNPs can be simultaneously excited when trapped with the same NIR laser beam.Recently,our group has established a method combining 980 nm NIR optical tweezers and bead suspension assay for sensitive detection of tumor biomarkers.Then we are eager to extend this to establish a method for in situ real-time detection of biological molecules in complex biological system.Towards this goal,this thesis shows some initial work,which includes the following:(1)Water-dispersible poly(maleic anhydride-alt-1-octadecene)coated upconversion nanoparticles(PMAO-UCNPs)has been prepared by a method of amphiphilic polymer coating and polymer cross-linking.Its colloidal stability in phosphate buffer saline(PBS)was studied by measuring hydrodynamic sizes.The result shows that PMAO-UCNPs kept good dispersity even after stored in PBS for 30 days.Then biotin-PEG-UCNPs for streptavidin(SA)specific labeling was prepared by conjugating biotin-PEG-NH₂ to the surface of PMAO-UCNPs.Finally,specific labeling of SA conjugated microspheres using biotin-PEG-UCNPs was realized in PBS.(2)SA was further linked to the surface of biotin-PEG-UCNPs and the obtained SA-UCNPs was used to specific labeling of biotin molecules.A kind of micro-nano composite particles(MNCs)for DNA sensing was constructed by using Na YF₄ or silica particles as substrates,biotin-duplex as linkers,and SA-UCNPs as luminescence labels.Strand displacement reaction was initiated in the presence of invader strand,resulting in the disassembly of MNCs and disassociation of UCNPs.Disassembly and disassociation process occurs in the optical trap when single MNCs was trapped by a 980 nm optical tweezers,which could be real-time detected by continuously measuring the intensity of upconversion luminescence.Real-time detection of 1 n M DNA in PBS was realized.Potentially,this proposed method could be applied in the field of dynamic DNA nanotechnology and might be extended for real-time detection of other biomolecules.