Synthesis,Biofunctionalization And Application In Multiplex Detection Of Proteases Of Multicolor Upconversion Nanoparticles

Matrix metalloproteinases(MMPs)are a class of proteases closely associated with tumors.In recent years,the fluorescence method based on fluorescence or luminescence resonance energy transfer(FRET or LRET)has received extensive attention in enzyme detection analysis.Although these methods have highly sensitive detection capabilities,they usually focus on the detection of one protease.On the one hand,conventional fluorescent probes have defects such as photobleaching and autogenic background interference;on the other hand,it is time-conssuning and labor-intensive to obtain multichannel detection signals by simply mixing multiple probes in the sane system.Lanthanide-doped upconversion nanomaterials exhibit outstanding advantages over conventional downconversion fluorescent materials.It is excited by near-infrared light sources and emits ultraviolet and visible light.Its emission peak has a narrow half-width.It is highly resistant to optical blinking and photobleaching and can minimize the background interference of autofluorescence in biological samples.The potential of high-sensitivity single-molecule detection using upconversion nanomaterials has been exploited,but there are few reports on multiplexed detection.Thus,it is highly desirable to develop a novel universal platfomns to simultaneous detection of multiplex proteases.In view of the above problems,We have synthesized a multicolor upconverting nanosensor for multiplex detection of matrix metalloproteinases.This thesis mainly studies fiom two aspects.(1)Synthesis of a novel multicolor tuning of upconverting nanoparticles.In order to synthesize upconverting nanoparticles capable of multiple emission and high fluorescence intensity and satisfy LRET conditions,we designed a structure of active-core/luninescent-shell.We doped gadolinium ions(Gd3+)in the core to adjust the size of the core to obtain a monodisperse core-shell structure and doped 60%ytterbium(Yb3+)to eliminate the internal quenching effect to obtain a suitable intensity emission spectrum.In additon,the ratio of emission peak is controlled by changing the doping concentration of Er3+and Tm3+in the shell.(2)Peptide functionalized upconversion nanoprobe for multiplex detection of MMPs.The probe was assembled by coupling two designed fluorescent molecule-modified polypeptides which was comprised from a matrix metalloproteinase-2 4NIP-2)substrate and a MMP-7 substrate,respectively.Once the targeted proteases were added,the corresponding polypeptide sites were specifically cleaved and luminescence resonance energy transfer(LRET)was impeded between UCNPs and fluorescent molecule,resulting in upconversion luminescence recovery.