Preparation Of Au(Ag)/DNA Conjugate-based Two-photon Nanoprobes And Its Imaging Application In Live Cells

In recent years,two-photon microscopy based on two near-infrared excitation has been the focus of attention in the field of biological detection and biomedical researches.As compared with one-photon microscopy(OPM),two-photon microscopy(TPM)has several advantages.TPM allows to image of deeper tissue,offers higher spatial resolution and longer observation time for the light source it uses.However,most of the fluorescent molecules employed in TPM have low two-photon absorption,which means that their applications have been limited.What is more,because of the intrinsic characters of organic molecules,there may exist some disadvantages in practical applications.For a variety of organic molecules,their ability of penetrating the cell membrane is poor,and they are not capable of resisting to photobleaching.So,it is still the most important to design and synthesize TP molecules with excellent characteristics.Nanomaterials has triggered more and more attention in biomedical researches.There are many kinds of nanomaterials available in application,such as metal nanoparticles,meso-porous silicon,and metal nanorodes.They are not only carriers of best choice,but also have a tunable circulation lifetime,enhanced permeability and retention(EPR)effect.Given the advantages of TP fluorescent probes in bioimaging and the features of nanomaterials in applications,several works were involved in this thesis:1.The TP molecule with excellent properties was designed and prepared which was named BMVC-Bu.In order to have a better understanding of the BMVC-Bu,the interaction between BMVC-Bu and different DNAs was studied.There were two aspects to be considered,one is the fluorescence enhancement of DNAs to BMVC-Bu,the other is association constant and binding ratio of BMVC-Bu with HPDNA,which increase the fluorescence of BMVC-Bu the most obviously.At the same time,the two photon properties of BMVC-Bu and DNA/BMVC-Bu was also studied for providing the following applications with theoretical foundation.2.Considering the reaction of GSH and AgNPs,the AgNPs/DNA/BMVC-Bu nanoflare was designed for the detection of GSH.Firstly,the HPDNA/BMVC-Bu was used as the template of preparing AgNPs.With the formation of AgNPs,the narrow distance between AgNPs and BMVC-Bu led to the reduced fluorescence because of the energy transfer.GSH can react with AgNPs for the formation of Ag-S bond,and the competitive interaction may result in AgNPs falling away from HPDNA/BMVC-Bu.As a result,the fluorescence of BMVC-Bu recovered.The AgNPs/DNA/BMVC-Bu nanoflare has been successfully applied in imaging cellular GSH,and further used in monitoring GSH variation in the process of apoptosis.3.Given the interaction between Dnase I and DNAs,the AuNPs based nanoprobe AuNPs/DNA/BMVC-Bu was designed to detect Dnase I.The formation of BMVC-Bu/DNA induced fluorescence enhancement of BMVC-Bu.The BMVC-Bu/DNA can be attached to the surface of AuNPs via Au-S bond,which lowed down the fluorescence of BMVC-Bu for a process of energy transfer.The DNA can be degraded into little pieces by Dnase I.As a result,BMVC-Bu was away from AuNPs and the energy transfer process was broken,which led to f luorescence enhancement of BMVC-Bu.Detection of Dnase I activity through TPM can be achieved.