Preparation And Application Of Near Infrared Fluorescent Probe Based On Cyanine Skeleton

Biological imaging technology has been developed rapidly in recent years.Fluorescent imaging technology has been widely utilized in the field of disease diagnosis because of its advantages of low biological damage,simple operation,high sensitivity and low cost.Fluorescent probes are often chosen as the carrier of the imaging technology for the detection of small molecular gas,biological enzymes and neurotransmitters in organisms.Considering that the biomolecules in the organism will absorb and scatter the excitation light and fluorescence from the probe molecules,leading to the reduction of imaging effect.However,the near-infrared fluorescence probe with stronger tissue penetration ability greatly reduces the interference of tissue auto-fluorescence,resulting in high signal-to-noise ratio imaging effect.Thus the near-infrared probes perfectly solved the above problems.In addition,near-infrared light as excitation source can reduce the optical damage to biological cells or tissues.Therefore,the development of near-infrared fluorescent probes has become a research hotspot in the field of analytical detection.Up to now,there are only a few near-infrared fluorescent dyes which have been widely used.And cyanine dyes have the characteristics of adjustable wavelength and easy modification,which are pursued by researchers.As far as enzyme detection is concerned,it faces the problems of probe design and real-time imaging of living cells.The near-infrared fluorescent probe for monitoring of Carboxypeptidase A in living cells has not been developed due to the difficulty in designing carboxypeptidase substrates.And in terms of neurotransmitters norepinephrine detection,it is usually interfered by catecholamines,such as dopamine and epinephrine,causing the near-infrared fluorescent probe to detect it has not been constructed.In this paper,we attempt to solve the above problems by modifying hemicyanine dyes with different functional groups to prepare near-infrared fluorescent probes for carboxypeptidase A and norepinephrine detection,and explore the detection ability and imaging effect in vitro and in vivo.The research works of this paper mainly includes the following aspects:(1)The near-infrared fluorescent probe Cy-NH₂-CPA was prepared by coupling amino hemicyanine Cy-NH₂ with L-phenylalanine salt via urea bond.Then the selection,specificity,sensitivity,response time and enzyme kinetics between Cy-NH₂-CPA and carboxypeptidase A were explored based on the fact that carboxypeptidase A can recognize the L-phenylalanine salt site of probe Cy-NH₂-CPA.The results showed that the probe had the characteristics of good specificity,high sensitivity and fast response time.In addition,the detection limit of0.09 U/m L was calculated by studying the spectral response of the probe at different enzyme concentrations.Finally,after determining the low toxicity of the probe to cells,the ability of Cy-NH₂-CPA to trace the activity of endogenous carboxypeptidase A in live cells and pancreatic cancer bearing mice was studied,which indicated Cy-NH₂-CPA had the ability to monitor norepinephrine in living cells and in vivo.(2)The near-infrared fluorescence probe Cy-OH-NE was synthesized by linking hydroxyl hemicyanine Cy-OH with p-methylthiophenol through thiocarbonate moiety.The selectivity,time response,sensitivity and visualization function between the probe Cy-OH-NE and norepinephrine were studied based on the principle of nucleophilic reaction.Through the above experiments,Cy-OH-NE had the characteristics of good selectivity,fast response time and visualization.In addition,the detection limit and response mechanism of Cy-OH-NE for norepinephrine were explored.Finally,the studies had shown that the probe Cy-OH-NE had the ability to monitor norepinephrine in living cells and in vivo.