Design, Construction And Application Of Two-photon Fluorescence Functional Nanoprobes

Due to their advantages of less tissue optical damage and phototoxicity,deep tissue penetration,reduced photobleaching and avoidance of auto-fluorescence,two-photon fluorescence imaging technology has been widely used in the detection and imaging of biological analytes in cells,tissues and in vivo,which provides an effective technical method for further understanding the function of biomolecules and their connection with the organism.Through the analysis of specific biomarkers for a certain disease,early diagnosis of diseases can be achieved.Furthermore,real-time monitoring of disease processes provides a theoretical basis for the design of new drugs.Therefore,how to design the new two-photon fluorescence bioprobes,which provide the solutions for many problems in biosensing and biomedical research,is the starting point and the foothold of this paper.Aiming at the key and difficult problems faced by two-photon fluorescence probes in biosensing and biomedical applications,this paper designs and constructs a series of new two-photon fluorescence probes with excellent performance and applies them to detect and image the target biological analytes in living cells,tissues and the mouse disease model,which could further investigate the important role of biomolecules in the physiological functions of the body,disease development or disease treatment.The details are as follows:1)In Chapter 2,by analyzing the important role of?-glutamyltranspeptidase?GGT?in the production of oxidative stress,we developed a novel two-photon fluorescence probe Np-Glu for detection and imaging of GGT activity.Np-Glu has high sensitivity,high selectivity and fast response to GGT.We first constructed the oxidative stress model in HepG2 cells by menaquinone as an oxidative stress stimulator,revealing an up-regulation of GGT levels caused by menadione-mediated oxidative stress in HepG2 cells.In addition,we have demonstrated the high expression levels of GGT in the development process of cancer by analyzing the changes of GGT content in normal tissues and tumor tissues,which means that Np-Glu is expected to be applied in early diagnosis and monitoring treatment of oxidative stress related diseases.2)In Chapter 3,in order to solve the problem that the traditional two-photon fluorescence probe is difficult to quantitatively detect biological analytes,we developed a ratiometric two-photon fluorescence nanoplatform TPSNP based on supramolecular assembly.TPSNP consists of three parts by host-guest supramolecular recognition:the host molecule poly-?-CD,the potential two-photon guest probe molecule Np-Ad and the two-photon guest internal reference molecule NpRh-Ad.TPSNP has excellent physicochemical properties,excellent biocompatibility and cell internalization.We constructed a two-photon fluorescence nanoprobe TPSNP-1 by modifying the potential two-photon guest probe molecule Np-Ad,for the quantification detection and imaging of gas signal molecules hydrogen sulfide?H₂S?in living cells,deep tissues and important organs of mice,which obtained satisfactory results.TPSNP will help to explore more functions and applications of supramolecular chemistry,and will also open the way for the design and fabrication of advanced supramolecular bioprobes for biosensing and biomedical applications.3)In Chapter 4,due to the interference of biological macromolecules including enzymes on the detection performance of fluorescent probes,we developed the first covalent organic frameworks?COFs?based two-photon fluorescence nanoprobe TpASH-NPHS.TpASH-NPHS has the excellent chemical stability and photostability,convenient synthesis,versatile post-modification strategys and the ideal nanoscale size.TpASH-NPHS can perform high-sensitivity and high-selectivity fluorescence detection on the gas signal molecule H₂S,and realize two-photon imaging of H₂S at the living cells and deep tissues.More importantly,we have demonstrated that TpASH-NPHS can avoid the influence of endogenous biological enzymes on the detection process and achieve the interference-resistant fluorescence imaging.In addition,we constructed a mouse cirrhosis model and studied the important physiological roles of H₂S and related enzymes in the process of cirrhosis by using TpASH-NPHS for accurately detecting H₂S.4)In Chapter 5,in view of the therapeutic effect of tumor treatment in hindsight and the untimely treatment scheme changes,we have constructed a new two-dimensional black phosphorus nanosheets,BP-MNAH,for two-photon fluorescence self-reporting photodynamic therapy.BP-MNAH consists of three parts:black phosphorus nanosheets,NH₂-PEG₂₀₀₀-NH₂ and the two-photon fluorescence probe MNAH-COOH.And each part plays a different role:under 660 nm laser irradiation,black phosphorus nanosheets can effectively produce ¹O₂ for photodynamic therapy;NH₂-PEG₂₀₀₀-NH₂ functions as a linker for black phosphorus nanosheet and MNAH-COOH,and can effectively enhance the stability of black phosphorus nanosheets in solution;MNAH-COOH has the capability of ¹O₂-activated two-photon fluorescence signal changes and enables real-time monitoring ¹O₂ generation.In live cells and mouse tumor model,the photodynamic therapy effect has a good linear relationship with the intensity of the two-photon fluorescence signal of BP-MNAH,which can realize the real-time prediction of the therapeutic effect and guide the timely adjustment of the treatment schemes.We believe that the two-photon fluorescence self-reporting photodynamic therapy of BP-MNAH has broad application prospects and provides the novel ideas and methods for personalized medicine.