Preparation And Properties Research Of Nitrogen Heterocyclic Two-photon Fluorescence Nucleic Acid Probe

Two-photon fluorescent nucleic acid probes have important application prospects in the field of DNA two-photon fluorescence microscopic imaging field.In this paper,eight quinolinylcarbazole two-photon fluorescent nucleic acid probe materials were designed and synthesized by Vilsmeier formylation reaction and piperidine-catalyzed aldol condensation reaction.Seven of the compounds were not reported.The electron transition absorption spectra and fluorescence emission spectra in DMF were studied,and the quantum yield was calculated.Using a pulsed laser with 120 fs pulse width,800 nm wavelength and 1 kHz repetition rate(Ti: sapphire laser),the two-photon optical properties of the compounds were studied.And two-photon-induced fluorescence spectra,two-photon absorption cross-section and action absorption cross-section were measured.The electron transition absorption spectra,fluorescence emission spectra and two-photon fluorescence emission spectra of the compound as a nucleic acid probe interact with calf thymus DNA in Tris-HCl-NaCl buffer solution were studied.The effects of molecular structure on the optical properties of compounds and the optical switching effect of DNA were discussed.To investigate the changes of electron transition absorption spectra,fluorescence emission spectra and circular dichroism spectra after compounds interacting with bases,nucleotide,and DNA in vitro,and to use Gaussian 09 software and time-dependent density functional theory,the electron transition energy levels of probe molecules in water and non-water environments were calculated.The reason of the optical property changes of the compounds under the interaction with DNA and the mechanisms of compounds binding with DNA were discussed.3T3 cells were stained with compounds,and a series of cells biofluorescence imaging studies were performed using a Leica DMI3000 B inverted microscope and a Carl Zeiss LSM710 laser scanning confocal microscope.The membrane permeation ability and nuclear localization level of compounds as fluorescent nucleic acid probes were studied by co-staining imaging experiments with commercial nucleic acid dye Hochest 33342.The advantages of two-photon imaging were studied by fluorescence imaging experiments under 400 nm and 800 nm light excitation.The three-dimensional imaging ability of the compound was studied by laser layered scanning technique in different irradiation depths.The effect of molecular structure on the photostability of the compounds were discussed by using the delayed laser continuous irradiation test to study the change of two-photon fluorescence intensity with time,fluorescence duration,fluorescence attenuation amplitude and average fluorescence decay rate.It is found that the compounds have lower fluorescence bleaching and higher stability of two-photon fluorescence.The introduction of double-branch quinoline cationic groups at two sides terminal of the molecule,an ortho-vinyl substituted quinolium,and iodemethylation of the molecules can effectively enhance the stability of the two-photon cell imaging of the compounds.Compounds with a large action two-photon absorption cross-section have a lower rate of fluorescence decay.The cytotoxicity level of the compound as a bioprobe was studied by MTT assays.