Design,Synthesis And Application Of Novel Fluorescent Probes And Photoacoustic Imaging Probes

Biomarkers can be used for disease diagnosis and disease severity assessment.Due to the abnormal changes of the concentration of specific biomarkers(including enzymes,proteins,metabolites,nucleic acids,etc.)in various diseases,the development of new and efficient detection methods for biomacromolecules and physiological processes has become a research hotspot in the field of probe research.Fluorescence imaging technology has been used in disease diagnosis and real-time monitoring due to its advantages of high sensitivity,simplicity and high efficiency,non-destructive in-situ imaging.However,due to the strong light scattering characteristics of biological tissue,most of the traditional microscopic imaging techniques can not show good spatial resolution.This greatly limits the imaging of deeper tissues in living organisms.New non-invasive and non ionized photoacoustic imaging methods are expected to overcome these difficulties.Photoacoustic imaging collects acoustic signals,while the scattering of biological tissue to acoustic waves is much smaller.Therefore,it can achieve high-resolution imaging of deep tissue.But photoacoustic imaging can't image living cells in real time.Based on the advantages and disadvantages of the two imaging technologies,it is very necessary to design and synthesize a probe that can perform fluorescence photoacoustic dual-mode imaging at the same time.On the one hand,it can realize the real-time imaging ability of fluorescent probe for living cells,and it can also have the advantages of photoacoustic probe for deep tissue imaging.In this paper,we constructed and synthesized a battery of organic small molecule fluorescence and fluorescence/photoacoustic dual-mode imaging probes for detecting biological proteins,active enzymes,novel xanthene photoacoustic contrast agent and their physiological processes,and explored their imaging applications in cells and living tissues.The main works of this paper are as follows:(1)We have developed an environmentally sensitive red fluorescent probe BDI probe that can specifically detect vicinal dithiol-containing proteins(VDPs)in mitochondria.The BDI probe is composed of a positively charged environment sensitive dye and a recognition site of VDPs linked by a chemical bond probe has almost no fluorescence in aqueous solution.When it reacts with VDPs,the probe covalently crosslinks with protein,the dye group is brought into the hydrophobic pocket of protein and the rotation is inhibited,and the fluorescent group emits strong fluorescence,thus realizing the activated detection of VDPs fluorescence signal.After adding ethylenedithiol to the reaction solution,the fluorescence of the reaction solution decreased,which proved the reversibility of the probe.The results showed that the BDI probe had the advantages of high specificity,high sensitivity and fast response to VDPs.The probe has been successfully used to image the VDPs in the mitochondria of cells,which proves that the probe has a good prospect for further study of the physiological function of VDPs.(2)We prepared a kind of near-infrared fluorescence probe NIR-GGT,using Bcy-NH₂as the near-infrared fluorescence group,and then selected Boc-Glu-Ot Bu to protect the amino group in the fluorescence group,and then obtained the near-infrared fluorescence probe of?-glutamyltranspeptidase(GGT).The response mechanism of the probe was confirmed by mass spectrometry and liquid phase detection.After the glutamate was specifically cut off by GGT,the amino near-infrared fluorescence group was dissociated,thus the GGT fluorescence signal could be activated.The results of in vitro experiments show that the probe NIR-GGT has the advantages of high specificity and sensitivity in response to GGT.The enzyme kinetic analysis shows that glutamate is a good substrate for GGT recognition and response,and the probe has been successfully applied to real-time imaging of GGT in cell mitochondria,which proves that the probe has a good application prospect in the physiological function of GGT.(3)We designed and synthesized a fluorescence photoacoustic bimodal imaging probe Rhodol-PA for the detection of human hydroquinone reductase(h NQO1).The probe is based on the rhodol derived near-infrared dye Rhodol-NIR and h NQO1recognition group trimethylquinone acid linked by ester bond.After the hydroxyl in the near-infrared dye Rhodol-NIR is protected by esterification,due to the formation of lactone structure,the conjugated structure of the fluorescent group is interrupted,so the probe Rhodol-PA molecular structure is in a closed-loop state,and there is no absorption in the near-infrared range receiving and fluorescent emission.When h NQO1 specifically reduced quinone to phenol,the ester bond in the probe of intramolecular cyclization was destroyed,which released the fluorescent dye,restored the near-infrared absorption and fluorescence emission,and realized the fluorescence photoacoustic dual-mode activated detection of h NQO1.In vitro data analysis showed that Rhodol-PA probe had the advantages of high signal to multiple ratio,fast response speed and strong specificity in response to h NQO1,and successfully performed real-time fluorescence imaging of h NQO1 in tumor cells,and established a mouse model of human cervical cancer cells with high expression of h NQO1 and human breast cancer cells with low expression of h NQO1 in vivo.The in vivo imaging of two cell lines with this probe system was discussed.(4)We have designed and synthesized a series of new photoacoustic contrast agents PA1-3 based on fluorescein analogues.These photoacoustic contrast agents have excellent photophysical properties,such as high molar absorption coefficient,high photothermal conversion efficiency,low fluorescence quantum yield,good photostability,etc.In vivo tissue imaging,high contrast photoacoustic imaging results can be obtained,and these dyes retain the classic fluorescein dye through lactone unique"on-off"ring structure to regulate the"on-off"characteristics of UV absorption.All in all,the novel oxyxanthene fluorescein analogues have great application prospects in the field of photoacoustic imaging,and provide a new general strategy for the construction of active photoacoustic imaging probes.