A Novel Ratiometric Two-Photon Fluorescent Probe for the Detection of Biothiols in Solution and Imaging of Living Cells

摘要

Mercaptan biomolecules, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), are abundant in the organism and play important roles in many biological processes. Abnormal levels of cellular thiols are associated with many human diseases, such as cardiovascular disease, Alzheimer's disease, leucocyte loss, liver damage, and cancer. For this reason, the development of effective methods for the detection of biothiols is urgently needed. Optical approaches based on synthetic colorimetric and fluorescent molecular probes have attracted increasing interest due to their simplicity, low cost, and high sensitivity and selectivity. So far, a large number of fluorescent molecular probes have been exploited that mainly function through various chemical reactions with thiols to trigger the corresponding spectral alterations; examples are the Michael reaction, cyclization reaction with an aldehyde, and cleavage reaction by thiols, among others. The majority of these probes are based on a turn-on fluorescence response in which the fluorescence is measured at a single wavelength and may be influenced by variations in probe concentration and the environment. By contrast, ratiometric fluorescent probes allow for the measurement of the emission intensity at two wavelengths, which should provide a built-in correction for environmental effects. Although ratiometric fluorescent variation can be achieved through Forster resonance energy transfer (FRET), FRET-based probes are normally composed of two chromophores; hence, their synthesis is more complicated compared to that of single-chromophore probes. In addition, under one-photon excitation, short excitation wavelengths (<500 nm) limit the applications of the probes with regard to deep-tissue imaging due to the shallow penetration depth (<100 μm). An attractive approach to the detection of organelles deep inside the tissue is through the use of two-photon microscopy (TPM), which employs near-infrared photons for excitation. TPM can obtain images deep inside intact tissues for a long period of time with high spatial resolution when combined with appropriate two-photon probes. However, so far only a few two-photon fluorescent probes have been reported.