Design And Synthesis Of Novel Two-Photon Fluorescent Probes And Their Application In Bioimaging

Developing organic molecular fluorescent probe to image the vital movement is one of important research fields in life science. Usually, the traditional xanthene dyes(fluorescein and rhodamine, etc.) are all single-photon excited fluorescent dyes, and easily suffer from photobleaching, autofluorescence interference of matrix and small penetration depth(<100 μm), which impede their application in fluorescence imaging. Since two-photon fluorescence dyes are excited by near infrared light, fluorescence based two-photo fluorescence dyes can penetrate deep tissue(> 500 μm), and reduce fluorescence background, Rayleigh scattering and tissue damage. Thus, their applications in biological imaging have attracted more and more attentions. However, the emission wavelengths of these two-photon fluorescent probes reported so far are general in the visible region. Therefore, these probes are susceptible to interferences from other factors, such as probe concentration, environmental conditions and detection devices. To address the question mentioned above, a series of novel ratiometric fluorescence probe based on two-photo fluorescence dyes have been designed for imaging metal ions, p H, organelle and signal molecules in cell and tissue in this thesis. The main research contents of the thesis are summarized as follows:1. In Chapter 2, a two-photon fluorophore and a rhodamine B derivative was conjugated by using through bond energy transfer(TBET) strategy to construct a novel two-photon ratiomertic fluorescence probe platform. This design could address the problem of small stokes shift caused by single fluorophore and broadens the types of two-photon dyes. In addition, the platform had a large two-photon active absorption cross section, high resolution and deep penetration depth. By integrating the platform and a copper ion-responsive element, copper ions could be detected as low as 3.0 × 10-7 M in tube. Experimental results also showed that the probe had good photo-stability in the range of p H 2.0-10.0, and also had no response to other metal ions. Moreover, Cu2+ ions in cells and tissues imaging can be also achieved with high resolution and large penetration depth(70-180 μm).2. In chapter 3, we have firstly designed and synthesized the palladium ions-responsive two-photon fluorescence probes by integrating the two-photon ratiomertic platform reported in Chapter 2 and amide Schiff base unit. This probe was based on the through bond energy transfer(TBET) mechanism to achieve a single excitation, dual emission detection Pd(II). In the presence of Pd(II) ions, amide Schiff base unit could form 1:1 coordination compounds with Pd(II), resulting two emission peaks of the fluorescence intensity changes. The distance between the two emission peaks of the probe was 100 nm, which could avoid the cross-talking between the two channels with high-resolution imaging pictures. Experimental results showed that this probe had a good signal to noise ratio 31.2, low detection limit of 2.3×10-7 M, good p H working range, good selectivity, and high penetration depth 90-270 μm.3. In chapter 4, a lysosome-localized p H-responsive two-photon ratiomertic fluorescence probe was synthesized by conjugating 4-substituted-naphthalimide, a rhodamine B derivative and a p H-responsive element. This probe has a strong ability of lysosomal localization with high resolution, and large Stokes shift. Moreover, the probe had little cytotoxicity. The probe was successfully used to image the change of p H in mouse tissue and onion tissue, and its penetration depth could reach 180 μm.4. In Chapter 5, HPQ, a small organic dye known for its classic luminescence meachanism through excited-state intramolecular proton transfer, was used as a precursor to develop a localizable, photoactivatable two-photon probe(PHPQ) for spatiotemporal bioimaging applications. PHPQ released a precipitating HPQ fluorophore with both one-photon and two-photon excited yellow-green fluorescence after photocleavage, thereby producing a mitochondrion-localized fluorescence signal that affords high spatial resolution for imaging mitoch ondrion, with more than 200-fold one-photo and 150-fold two-photon fluorescence enhancement.5. In chapter 6, a novel two-photon fluorescent dye TP-NI3 with near infrared excitation wavelength and emission wavelength was designed and synthesized. The fluorescent dye had good photostability and large Stokes shift, its fluorescence signal was almost unaffected in the range of p H 2-8. Colocalization experiments demonstrated that the dye was mitochondrion-localizing. After intravenous injection, it could quickly penetrate the blood-brain barrier, and was metabolized through kidney, which was similar to metabolic probe ICG. By changing azido group into amino group, a H2S-specific fluorescence probe was successfully constructed. The probe could detect exogenous and endogenous hydrogen sulfide in cancer cells.