Design Of Long-wavelength Biological Enzyme Fluorescence Probe And Imaging Application

As a special biological macromolecule with catalytic function,enzymes play an extremely important role in maintaining the homeostasis and normal life activities of biological systems.The abnormal expression of enzyme activity is directly related to the occurrence and development of many major diseases such as cancer,heart disease,nervous system disease and inflammation.To study the distribution level and action mechanism of enzymes in organisms,it is important to develop an effective method to detect enzymes.In recent years,small molecule fluorescent probes have significant advantages in the detection of enzyme activities because of their high sensitivity,good biocompatibility and non-destructive real-time detection ability.In addition,the fluorescence probes are convenient to synthesize and easy to modify,which can be used for imaging analysis of different enzymes in organisms and to explore the physiological role of enzymes in specific diseases,providing important information for personalized therapy and related drug research.However,small molecular fluorescent probes still face the problem of low sensitivity in enzyme detection and imaging.The main reason is that,on the one hand,the short-wavelength enzyme fluorescence probes are easy to be interfered by the background signals of biological tissues and has low tissue penetration depth,resulting in large light damage to biological samples.On the other hand,fluorescent probes with small Stokes shift are prone to be interfered by excitation wavelength when detecting enzymes,resulting in a lower signal-to-noise ratio and reduced imaging sensitivity.In addition,the enzyme fluorescence probes with a single signal are prone to the potential interference of environmental factors,which reduce the sensitivity and accuracy of the detection.To deal with these challenges,this paper takes the accurate detection of enzyme as the starting point,combines the advantages of two-photon and near-infrared fluorescence imaging technology and ratio detection technology,and constructs a series of long-wavelength fluorescence probes with high sensitivity and good accuracy.The probes have successfully realized the detection and imaging study of the target enzymes,providing an effective tool for exploring the mechanism of enzyme in vivo.The specific research content of this paper is as follows:（1）Acetylcholinesterase（ACh E）plays a key role in regulating oxidative stress.Monitoring ACh E level under different oxidative stress conditions is a hot and difficult problem in current research.In chapter 2,two kinds of near infrared fluorescence probes（SNCN-AE and SNC-AE）were designed combining ESIPT and ICT mechanism to explore the change of ACh E level under oxidative stress.The probes had a low fluorescence signal.After reacting with ACh E,the strong electron push-pull effect enhanced the ICT process,and the probes produced a significant near infrared fluorescence signal（SNCN-AE=710 nm;SNC-AE=695 nm）.Meanwhile,due to the synergism of ESIPT effect,the probes were accompanied by a large Stokes shift（SNCN-AE=285 nm;SNC-AE=270 nm）.The performance analysis in vitro showed that SNCN-AE had good stability（fluorescence intensity did not change within 1 h）,fast response（fluorescence intensity rapidly increased within 2 minutes）,and low detection limit（0.13 U/m L）.Importantly,SNCN-AE had been successfully applied to monitor ACh E level in cellular and zebrafish oxidative stress models and was expected to be an important tool to explore the mechanism of ACh E in oxidative stress and related diseases in organisms.（2）β-glucuronidase（GLU）as an important signaling molecule,is closely related to intestinal injury.Currently,the distribution level of GLU in vivo has been extensively studied.However,molecular probes with short wavelength and small Stokes shift limit its application in imaging analysis of GLU in vivo and various organs.To solve the above problems,in Chapter 3,a near-infrared fluorescent probe DP-GLU with large Stokes shift was constructed to detect and image GLU levels in mouse tumor models and intestines.After DP-GLU reacted with GLU,the probe rapidly showed strong fluorescence emission at 676 nm,and the Stokes shift could reach 131 nm.Meanwhile,DP-GLU showed high sensitivity to GLU,and the detection limit was as low as 1.45×10^-2μg/L.Bioimaging results showed that DP-GLU could image GLU in the gut of mice,revealing fluctuations in GLU concentration between different cells,and thus effectively distinguishing tumor cells with overexpression of GLU from normal cells.（3）Colon cancer is the main malignant tumor of digestive tract with high mortality.As a lysozyme glycosidase,the change of GLU level is related to the pathological process of colon cancer.Based on the work in the chapter 3,the distribution of GLU in the intestine was visualized.In chapter 4,the expression level of GLU in colon tumor tissues was further explored.In order to improve the sensitivity and accuracy of GLU detection,a two-photon ratiometric fluorescent probe RN-GLU was developed in this study to detect and image GLU in colon tumor tissues.After RN-GLU activated by GLU,the reaction system showed a significant ratio fluorescence enhancement(I_{553 nm}/I_{441 nm},214-fold).The results of in vitro test showed that RN-GLU had rapid response to GLU（fluorescence intensity rapidly increased within 1 minutes）,low detection limit(1.2×10^-2μg/L),and good stability（fluorescence intensity remained stable within 24 h）.In addition,RN-GLU had good lysosomal targeting ability in cell imaging.Benefited from the two-photon excitation property,RN-GLU had deep tissue penetration,with imaging depths up to 200μm.The probe RN-GLU is expected to be a potential tool to investigate the role of GLU in the pathological process of colon cancer.（4）β-galactosidase（β-gal）,as another important glycoside hydrolase in lysosomes,is closely related to the occurrence and development of tumors.Currently,fluorescence probes with high sensitivity forβ-gal detection are still being explored.In chapter 5,the construction strategy of two-photon ratiometric fluorescence probes described in the previous chapter was further expanded,and a two-photon ratiometric fluorescence probe FTR-βgal based on fluorescence resonance energy transfer was reasonably designed for the detection and imaging ofβ-gal.In probe FTR-βgal,naphthalene derivative was used as energy donors,Rhodamine derivatives as energy acceptors,and galactoside asβ-gal recognition groups.After FTR-βgal reacted withβ-gal,the fluorescence emission of the probe increased at 540 nm and decreased at 450 nm,showing an obvious change in the ratio fluorescence signal,thus achieving an accurate detection ofβ-gal.In addition,the probe FTR-βgal has been successfully used for two-photon fluorescence imaging of endogenousβ-gal in living cells and tissues,and has great potential for development in the study of major diseases related toβ-gal.