Development Of Chemical Fluorescent Probe And Its Application In Cancer Detection

Recent advance in bioanalytical techniques,especially fluorescent imaging technology has accelerated the deep understanding of cellular states and life science.Compared with traditional sensing technology,fluorescence imaging technology has many benefits like high sensitivity,rapid response and excellent selectivity.On one hand,fluorescent probe has been used for the sensing of ions and bioactive small molecules which involves in pathological process,to diagnose the disease.On the other hand,remarkable advance has been achieved in protein localization and dynamics,and protein-protein interactions.Also,cancer is still the leading cause of the cancer death worldwide,the diagnosis and treatment for cancer is still the biggest challenging for scientists.Currently,hundreds of chemical small molecule probe for the sensing of ions and bioactive small molecules has been reported.However,for the detection of enzymatic activity,traditional method like high-performance liquid chromatography,mass spectra,enzyme-coupled fluorescence assay using fluorogenic peptides and western blotting using antibody are laborious and involve the handling of radioactive species.So,we want to design chemical probe for the detection of key biomarker in cell biological process.The details are described as follow:The first work is about the fluorescent light-up aggregation-induced emission probe for screening non–small cell lung carcinoma(NSCLC).The fluorescent probe DEVD-TPE,which was composed by a substrate peptide DEVD and an AIE reporter group TPE,was developed for detecting caspase-3 in living cells.In alkalescent solution,the DEVD-TPE probe was almost nonfluorescent.However,it exhibited significant fluorescence while DEVD-TPE was cleaved by Caspase-3 as well as reporter group TPE aggregating.EGFR inhibitor Gefitinib was used for confirming the efficacy to different NSCLC cell lines such as HCC827 cells,HeLa cells,A549 cells and H1650 cells in this work.Cell proliferation assay and apoptosis assay indicated the different NSCLC cells had different sensitivity to Gefitinib.The results of living cell fluorescent imaging and flow cytometry analysis were consistent with cell proliferation assay and apoptosis assay.It demonstrated Gefitinib was more efficient to HCC827 cells than H1650 cells and A549 cells.This work proved that the fluorescent probe DEVD-TPE was highly sensitive to caspase-3,and was of potential prospect in rapidly screening NSCLC.The second work is a fluorescent probe for the sensing for histone deacetylase(HDAC)in living-cells.Breast cancer is a pominnent cause of cancer-related deaths in women and account for 6.1% of all female death.In addition to genetic mutations,epigenetics also play an important role in breast cancer accordding to increasing number of researches.HDACs are hydrolytic enzymes that regulate the acetylation status of histones,which effects the regulation of gene expression.Inhibitors of HDACs have been found to cause growth arrest,differentiation and apoptosis of many tumours cells and proving to be an exciting therapeutic approach to cancer.Here,we developed a chemical fluorescent probe,R7K(Ac)-CCB,which consists of a peptide substrate containing acetyl-Lys and a coumarin fluorophore with a carbonate ester.The peptide substrate contains a cell-penetrating peptide and nuclear localization sequence as well,which can help the probe to enter the nucleus efficiently.HDAC inhibitor SAHA was used for confirming the efficacy to breast cancer cell line MCF-7 and normal cell line NIH 3T3,and the results indicated SAHA has higher therapeutic efficacy to cancer cells than normal cells.Also,the results of living cell fluorescent imaging demonstrated that the probe has a good cell membrane permeability and be used for rapid live-cell imaging.However,the selectivity for HDAC in cells of R7K(Ac)-CCB still needs to be improved.