Construction Of Two-photon Fluorescent Nanoprobes And Their Application In Biological Analysis

There are a variety of biological molecules in living organisms.They have special biological functions and play a vital role in physiological and pathological processes.Therefore,real-time monitoring of the change of concentration and distribution of biomolecules in living organisms is of great significance for the accurate diagnosis of diseases and the study of related pathologies.Fluorescent probes have become important tools for monitoring disease markers due to their advantages such as high sensitivity,good specificity,and in-situ imaging.However,traditional small-molecule based fluorescent probes have disadvantages of low cell entry efficiency,poor water solubility,and easy photobleaching,which limit their further application in vivo.Therefore,developing a fluorescent probe with strong membrane penetrability,good water solubility,and excellent cell imaging effect is of great significance for accurate diagnosis of diseases.Nanomaterials(such as graphene quantum dots,silica nanoparticles,and manganese dioxide nanosheets,etc.)have shown great application prospects in the field of fluorescent probe due to their higher cell membrane penetrability,excellent biocompatibility,and higher loading capability.In addition,compared with single-photon imaging technology,two-photon imaging technology uses two-photons with lower frequency and energy(longer wavelengths)as the excitation light source,which can reduce interference of biological background fluorescence and bioligcal seample damage as well as light scattering.Moreover,deeper tissues imaging and higher imaging resolution also can be achieved.These advantages make two-photon fluorescent probes more suitable for the study of biological samples.Therefore,the two-photon fluorescent nanoprobe constructed by combining nanomaterials and two-photon imaging technology possess broad application potentialin the research of living cells and tissues..To address the above problems,combining with the advantages of nanomaterials and two-photon imaging technology,we used graphene quantum dots and two-photon silicon dioxide as signal units,and manganese dioxide nanosheets and divalent iron ions as identification units to construct two kinds of two-photon nanoprobe for accurate diagnosis of diseases.The details are as follows:(1)In Chapter 2,based on the special optical properties of two-photon silicon nanoparticles and the Fenton reaction between divalent iron ions and hydrogen peroxide,we have developed a two-photon fluorescent nanoprobe for two-photon imaging of hydrogen peroxide in cells.In the presence of hydrogen peroxide,the ferrous iron ions generate hydroxyl radicals through a Fenton reaction,which can effectively quench the fluorescence of silicon nanoparticles.With the increase of hydrogen peroxide concentration,the fluorescence intensity in the system was gradually quchened.The detection limit of this probe is 336 n M,and it shows good selectivity.Finally,the probe was successfully used imaging of hydrogen peroxide in living cells,and real-time monitoring the change of hydrogen peroxide in living cells.(2)In Chapter 3,to overcome the shortcomings of the short emission wavelength and single function of the traditional two-photon fluorescent probes,we designed and synthesized a graphene quantum dot with two-photon properties whose excitation and emission wavelengths are in the near-infrared region.The nanomaterial also has the property of producing singlet oxygen.In this chapter,the quantum dots and manganese dioxide nanosheets are combined to construct a multifunctional two-photon fluorescent nanoprobe.The detection limit of this probe was 83 n M in a buffer solution,and showed higher selectivity.Combined with two-photon imaging technology,the probe was successfully used to monitor the changes of target concentration in living cells and deep tissues.Finally,we also preliminary explored the photodynamic therapy effect of the probe.The experimental results suggested that the manganese dioxide nanosheets in this probe can consume glutathione in the cell and greatly enhance the photodynamic therapy effect on tumors.