Development Of Upconversion Nanoprobe With High Energy Transfer Efficiency And Its Application In MMPs Secretion Detection

Matrix metalloproteinases(MMPs)are a family of zinc-dependent endopeptidase enzymes that play an important role in tumor invasion and metastasis by degrading the extracellular matrix and basement membrane.Therefore,in situ imaging and quantitative analysis of matrix metalloproteinases have important pathological significance in evaluating the invasiveness of tumor cells and the diagnosis of diseases and cancers.Currently,the commonly used matrix metalloproteinase secretion detection methods are almostly based on extraction and detection in homogeneous supernatant,which is difficult to achieve real-time monitoring of MMPs secretion and cannot reflect the cell individual differences.In order to solve this problem,we design the cell membrane targeting ratiometric upconversion nanoprobe and realize in situ imaging and quantitative analysis of MMPs secreted by cancer cells on cell membrane based on luminescence resonance energy transfer(LRET)strategy,which provides a new platform for in situ detection cell secretions.This paper mainly contains the following two parts:1.Development of Upconversion Nanoprobe with High Energy Transfer EfficiencyIn this chapter,oil-phase upconversion nanoparticles were modified with alendronate sodium under acidic conditions for surface hydrophilic modification and amino functionalization.Cy3 was further modified to serve as an energy transfer relay station and achieve two short range LRET processes from UCNP to dye relay station Cy3 to peptide terminal quenching agent for high energy transfer efficiency.Firstly,the oleic acid ligands were replaced by alendronate sodium via ligand change in acidic conditions.Secondly,Cy3 was modified via amide reaction to achieve the first LRET process between the energy donor UCNPs and the energy receptor Cy3.Thirdly,polypeptide chain labeled with quencher QSY7 was further modified through functionalized short PEG chain on the UCNP surface to realize the second LRET process between the energy transfer relay station Cy3 and the energy receptor QSY7,and Cy3 luminescence was quenched.Since MMP2 has specific enzyme digestion on the polypeptide substrate,the Cy3 fluorescence signal is recovered when the MMP2 exists.Thus,the quantitative detection of MMP2 can be achieved by detecting the recovery of the Cy3 fluorescence signal.2.In Situ Imaging and Quantitative Analysis of Matrix Metalloproteinase SecretionBy conjugating of specific antibody that recognize MDA-MB-231 cells via the epidermal growth factor receptor(EGFR)on the cell membrane,the upconversion nanoprobe was connected to the cell membrane to achieve in situ imaging and quantitative analysis of MMP2 secretion from the cells.Firstly,anti-EGFR antibodies were covalently combined through the long chain PEG on the surface of upconversion nanoprobe,which can target and anchor on the cell membrane through specific antigen and antibody interactions.When MMP2 was secreted under lipopolysaccharide stimulation,it was immediately captured by the probe anchored on the cell membrane,leading to the protease hydrolysis of the polypeptide substrate and the detachment of QSY7 labled peptide residual chain,which caused the recovery of Cy3 fluorescence.This method enables rapid in situ monitoring of MMP2 secretion by living cells in cell membrane confined space.We provided a new method for sensitive and rapid detection of other cell secretions,which has potential application values in the study of in situ qualitative and quantitative analysis of other cell secretions.