Highly Selective Two-Photon Fluorescent Probe for Ratiometric Sensing and Imaging Cysteine in Mitochondria

Abstract

A novel ratiometric mitochondrial cysteine (Cys)-selective two-photon fluorescence probe has been developed on the basis of a merocyanine as the fluorophore and an acrylate moiety as the biothiol reaction site. The biocompatible and photostable acrylate-functionalized merocyanine probe shows not only a mitochondria-targeting property but also highly selective detection and monitoring of Cys over other biothiols such as homocysteine (Hcy) and glutathione (GSH) and hydrogen sulfide (H2S) in live cells. In addition, this probe exhibits ratiometric fluorescence emission characteristics (F518/F452), which are linearly proportional to Cys concentrations in the range of 0.5-40 μM. More importantly, the probe and its released fluorophore, merocyanine, exhibit strong two-photon excited fluorescence (TPEF) with two-photon action cross-section (Φσmax) of 65.2 GM at 740 nm and 72.6 GM at 760 nm in aqueous medium, respectively, which is highly desirable for high contrast and brightness ratiometric two-photon fluorescence imaging of the living samples. The probe has been successfully applied to ratiometrically image and detect mitochondrial Cys in live cells and intact tissues down to a depth of 150 μm by two-photon fluorescence microscopy. Thus, this ratiometric two-photon fluorescent probe is practically useful for an investigation of Cys in living biological systems.

A Simple and Rapid Turn On ESIPT Fluorescent Probe for Colorimetric and Ratiometric Detection of Biothiols in Living Cells

“…Introduction of the acrylate group, which has a strong electron-withdrawing ability and is typically used as a functional trigger moiety to detect biological thiols 3, 32–37 , could result in a shift in fluorescence and cause ratiometric fluorescence changes. The cyclization and cleavage reaction with the targets changes the fluorescence as the electron-withdrawing groups depart.…”

A multi-emissive fluorescent probe for the discrimination of glutathione and cysteine

“…For instance, the selective detections of Cys/Hcy were usually performed based on the strategy of cyclization with aldehydes (Rusin et al, 2004;Wang et al, 2012;Barve et al, 2014) and conjugate addition-cyclization with acrylates (Yang et al, 2011;Niu et al, 2016;Han et al, 2015;Guo et al, 2012). On the other hand, the discriminations of GSH from Cys were achieved using aromatic substitution-rearrangement reactions, etc (Niu et al, 2012;Wang et al, 2014c;Lim, 2014;Niu et al, 2015c;Işık et al, 2014).…”

A new fluorescent probe for colorimetric and ratiometric detection of sulfur dioxide derivatives in liver cancer cells

“…However, these methods usually suffered long operation time, low sensitivity, tedious operation and no applications inside living cells. Alternatively, fluorometric analysis has emerged in recent years, which could be used to detect analytes with high sensitivity and high accuracy910. More importantly, in situ and real-time imaging of analytes could be carried out by non-destructive fluorescent probes1112.…”

“…By now, two detection mechanisms have been reported in the design of fluorescent probes for HSO 3 − /SO 3 2− : reactions with HSO 3 − /SO 3 2− and hydrogen-bond formation with HSO 3 − /SO 3 2345678910111213141516. The reactions with HSO 3 − /SO 3 2− include cleavage of levulinate group1718, nucleophilic addition to aldehyde group1920 or “C=C” double bond2122232425262728293031.…”