| Because of its excellent characteristics such as high sensitivity,high selectivity and low biological toxicity,fluorescent dyes are often used as a new,efficient and fast real time in situ detection tool in food science,material science,analytical chemistry the research of modern biological science.and clinical medical diagnosis.Active substance in oranisms participate in various life activities of organisms and play a pivotal role in maintaining normal metabolism and physiological activities of cells.Proteins and enzymes are important active substances in the organism,and in the cell,the abnormal of some proteins and enzymes are closely related to the occurrence and development of many diseases.For example,the abnormal content of human serum albumin（HSA）is the precursor of diseases such as kidney,and the concentration and activity of nitro reductase are significantly increased in hypoxic tumors.Therefore,the development of simple and efficient detection tools for monitoring the content and activity of bioactive substances is of great significance for understanding the pathogenesis and early diagnosis of many diseases.At present,many fluorescent dyes used in the detection of bioactive substances have been reported,but few fluorescent dyes that can simultaneously detect and treat related diseases through photodynamic therapy are available at the same time.Based on the above research status,two cases of triphenylamine based fluorescent dyes were designed and constructed in this work,and their response ability to different bioactive substances in vivo and their ability to realize the treatment of hypoxic tumors through light stimulation were studied.As an important bioactive substance,serum albumin（SA）accounts for more than 50%of plasma protein content and is one of the main components of plasma.Serum albumin plays an important role in physiological activities such as maintaining blood osmotic pressure,stabilizing p H value,and carrying various drugs and metabolites.When the serum albumin content is lower than the normal physiological level,it will cause cirrhosis,liver failure and other diseases.Higher than normal levels can lead to conditions such as diabetes,coronary hart disease and cerebal infarction.Therefore,as an important indicator of life function,the accurate detection of serum albumin is conducive to the assessment of the health status of organisms and the clinical diagnosis and treatment of diseases.Therefore,in this work,a triphenylamine based fluorescent dye TPA-BM-NO2 was developed and synthesized.The dye achieved a specific response to bovine serum albumin（BSA）in vitro and in vivo.The dye fluorescence emission signal showed an"OFF-ON"change at 516 nm through protein activation.The fluorescent dye has good p H stability,excellent selectivity and remarkable optical properties,and can achieve a specific response to bovine serum albumin in the presence of various interference substances.The dye TPA-BM-NO2 in the free state almost had no fluorescence,but the fluorescence intensity of the fluorescent dye TPA-BM-NO2 was significantly enhanced after the addition of bovine serum albumin,and the fluorescence signal changed off-on.At the same time,the dye TPA-BM-NO2also has good biocompatibility,as well as the ability of specific biological targeting,and has been successfully located in the cell liposome,realizing the analysis and application of dye molecule TPA-BM-NO2 in living organisms.Hypoxia is the result of insufficient oxygen supply in cells or tissues,often leading to a variety of diseases（such as vascular disease,lung disease,etc.）.It is also an important feature of solid tumors.Nitroreductase,as an important bioactive substance,is also an important marker for hypoxia detection.It is very important for early detection of cancer cells to detect the oxygen status in cancer cells by the method that dye can be specifically activated by nitroreductase.In addition,NAD+,an oxidation-type coenzyme used in tumor hypoxia microenvironment detection,also has changes in its content under hypoxia conditions.In this work,a case of fluorescent dye TPA-NO2,which can be activated by nitro reductase and coenzyme under hypoxia state,was designed and constructed.The fluorescence signal of the dye can be changed in a ratio type under the combined action of coenzyme and nitro reductase,and the fluorescence intensity has a good linear relationship with the concentration of the two enzymes.The detection limits are 0.11μg/m L and 27μmol/L.Based on the good water solubility,excellent optical properties and negligible cytotoxicity of fluorescent dyes,the specific identification and detection of nitro reductase and coenzyme in vitro and in vivo were realized.At the same time,after TPA-NO2 is reduced by nitro reductase,the product TPA-NH2 has amazing cell phototoxicity and can specifically kill cancer cells under light conditions.This ability to specifically kill cancer cells in hypoxia microenvironment has excellent application potential in the clinical treatment of cancer. |
