Two-Photon Small-Molecule Fluorogenic Probes for Visualizing Endogenous Nitroreductase Activities from Tumor Tissues of a Cancer Patient.

Nitroreductase (NTR), a common enzymatic biomarker of hypoxia, is widely used to evaluate tumor microenvironments. To date, numerous optical probes have been reported for NTRs detection. Approaches capable of concisely guiding the probe design of NTRs suitable for deep-tissue imaging, however, are still lacking. As such, direct optical imaging of endogenous NTR activities from tumors derived from cancer patients is thus far not possible. Herein, aided by computational calculations, the authors have successfully developed a series of two-photon (TP) small-molecule fluorogenic probes capable of sensitively detecting general NTR activities from various biological samples; by optimizing the distance between the recognition moiety and the reactive site of NTRs from different sources, the authors have discovered and experimentally proven that X4 displays the best performance in both sensitivity and selectivity. Furthermore, X4 shows excellent TP excited fluorescence properties capable of directly monitoring/imaging endogenous NTR activities from live mammalian cells, growing zebrafish, and tumor-bearing mice. Finally, with an outstanding TP tissue-penetrating imaging property, X4 is used, for the first time, to successfully detect endogenous NTR activities from the liver lysates and cardia tissues of a cancer patient. The work may provide a universal strategy to design novel TP small-molecule enzymatic probes in future clinical applications.

Molecular Packing-Controlled Mechanical-Induced Emission Enhancement of Tetraphenylethene-Functionalised Pyrazoline Derivatives.

Organic mechanofluorochromic (MFC) materials with mechanical-induced emission enhancement (MIEE) are scarce. Herein, Aggregation-induced emission (AIE)-active tetraphenylethene-functionalised pyrazoline derivatives with various non-conjugated substituent groups (Br, F, N,N-dimethylamino and cyano groups for TPEB, TPEF, TPEN and TPECN, respectively) were designed and synthesised. The fluorescence spectra of the compounds in the solid state were sensitive to mechanical stimuli and exhibited unique MFC properties. The solid fluorescence peaks of all compounds were red shifted, whereas the quantum yields of TPEB, TPEF and TPEN were increased and that of TPECN was slightly reduced after grinding. The MIEE mechanism was systematically investigated through structure-property relationship studies. The results showed that the pyrazoline ring of TPEB with MIEE property formed H-dimer aggregates with weak π-π stacking in the solid state. After grinding, the synergistic effects of conformational planarization and destructive π-π interactions induced the red shift of the fluorescence peak and the intensity enhanced. TPECN formed incompact J-aggregates with weak intermolecular interactions instead. The conformational planarization and increased intermolecular interactions induced by grinding led to the red-shifted peak and the slightly reduced intensity. These results clearly demonstrated that the translation of packing arrangements is an efficient method to design MIEE materials, which opens a new scope for designing unique MFC materials.

Internal standard fluorogenic probe based on vibration-induced emission for visualizing PTP1B in living cells.

Herein, as a proof of concept, we developed the first enzymatic VIE fluorogenic probe for protein tyrosine phosphatase 1B (PTP1B). The detection and imaging of PTP1B using VIE in living cells were both realized. Particularly importantly, the designed probe herein provides a guideline and platform for the development of new VIE-based enzymatic probes.

Two-component ratiometric sensor for Cu2+ detection on paper-based device.

The copper(II) ion (Cu2+) has played an indispensable role in diverse kinds of functional physiological processes of organisms, which has become of growing interest. Despite the fact that numerous Cu2+ test papers using fluorescent probes have been fabricated, sensors featuring the ratiometric property that integrates quenched probes and an inner standard dye are rarely reported. Herein, a two-component ratiometric sensor in a paper-based device is proposed to realize highly selective Cu2+ detection. To overcome shortcomings such as low signal-to-noise ratio and incorrect response of the quenching probe, a novel BODIPY-based turn-off probe (P2017) is designed and introduced into the paper-based device with better water solubility and selectivity for Cu2+ detection. Furthermore, a reference dye (B001), exhibiting an emission at 690 nm when the excitation wavelength is 480 nm, is also introduced into the paper-based device. These two components can enhance the quality of the signal as P2017 is sensitively quenched by Cu2+, while B001 with a photostable property, serving as an internal benchmark, is unable to react with Cu2+. The results indicated that the two components provided a new concept for optimizing paper-based device fabrication and developing accurate, simple, and inexpensive Cu2+ detection methods, which could be potentially applied to monitor human health and the environment in remote areas. Graphical abstract.

A novel pyrimidine based deep-red fluorogenic probe for detecting hydrogen peroxide in Parkinson's disease models.

Parkinson's disease (PD) severely affects life quality of patients and has brought huge economic burden to health system worldwide. Previous studies have shown that the abnormal expression of hydrogen peroxide (H2O2) in the brain is closely related to the development of neurodegenerative diseases such as PD. Herein, we designed a novel deep-red H2O2 fluorogenic probe PB1 to detect the level of H2O2in vivo. PB1 showed a highly selectivity response to H2O2 over other reactants such as reactive oxygen/nitrogen species, biothiols and various ions in aqueous solution at physiological pH. We have demonstrated that PB1 possesses an excellent response to H2O2 in the cells and in the brain tissue of drosophila from confocal fluorescence imaging. These results suggested that PB1 holds great potential in the study of the relationship between H2O2 overexpression and PD.

Rational design of NIR fluorescence probes for sensitive detection of viscosity in living cells.

Developing near-infrared (NIR) fluorescence probes for detection of intracellular viscosity is still sufficiently challenging. In this work, three kinds of D-A-D type naphthyl and 2,1,3­benzoxadiazol hybrid NIR dyes functionalized with amino (NY1), N­methylamino (NY2) and N,N­dimethylamino (NY3) groups for intracellular micro-viscosity detection were designed and synthesized. All the probes exhibited very weak NIR emission in low viscosity environment and obvious fluorescence enhancement with the increased viscosity. Different substituent groups had a high impact on the photophysical properties and response sensitive of the probes to viscosity. The structure-property relationships were systematic investigated. The results showed that stronger electron-donating ability and larger steric effect of N,N­dimethylamino led to a narrower energy gap and more sensitive to viscosity environment. Therefore, NY3 exhibited higher signal noise ratio for viscosity detection and was successfully applied for imaging the changes of intracellular micro-viscosity. This work provides an efficient way to design powerful NIR fluorescence probes for viscosity detection.

A fluorogenic probe based on chelation-hydrolysis-enhancement mechanism for visualizing Zn2+ in Parkinson's disease models.

Developing efficient methods for the real-time detection of Zn2+ levels in biological systems is highly relevant to improving our understanding of the role of Zn2+ in the progression of Parkinson's disease (PD). In this work, a novel Schiff base based Zn2+ fluorescent probe (ZP) was designed, synthesized and systematically investigated. A significant turn-on effect on ZP upon the addition of Zn2+ was observed, accompanied by a blue-shift of the fluorescence spectra. ZP is sensitive to Zn2+ and has excellent selectivity against various biologically relevant cations, anions and amino acids. The sensing mechanism of ZP was studied by 1H NMR, MS, single crystal X-ray diffraction and theoretical calculations. The results showed that the response of ZP to Zn2+ was based on the chelation-hydrolysis-enhancement process. Upon bonding, Zn2+ hydrolyzes the Schiff base to an aldehyde precursor, the resulting aldehyde further coordinates to Zn2+ to form a more stable heterobimetallic complex leading to the emission enhancement and blue-shift. ZP was applied to imaging exogenous/endogenous Zn2+ in live HeLa cells. Furthermore, we successfully measured the Zn2+ levels using in vitro PD models, which provided a visualization method to better understand the relationship between Zn2+ levels and PD development.

Ligand-displacement-based two-photon fluorogenic probe for visualizing mercapto biomolecules in live cells, Drosophila brains and zebrafish.

Investigating the change in expression level of mercapto biomolecules (GSH/Cys/Hcy) necessitates a rapid detection method for a series of physiological and pathological processes. Herein, we present a ligand-displacement-based two-photon fluorogenic probe based on an Fe(iii) complex, TPFeS, which is a GSH/Cys/Hcy rapid detection fluorogenic probe for in vitro analysis and live cell/tissue/in vivo imaging. The "in situ" probe is non-fluorescent and was prepared from a 1 : 2 ratio of Fe(iii) and TPS, a novel two-photon (TP) fluorophore with excellent one-photon (OP) and TP properties under physiological conditions, as a fluorescent ligand. This probe shows a rapid and remarkable fluorescence restoration (OFF-ON) property due to the ligand-displacement reaction of mercapto biomolecules in a recyclable manner in vitro. A significant two-photon action cross-section, good selectivity for biothiols, low cytotoxicity, and insensitivity to pH over the biologically relevant pH range allowed the direct visualization of mercapto biomolecules at different levels between normal/drug-treated live cells, as well as in Drosophila brain tissues/zebrafish based on the use of two-photon fluorescence microscopy.

Anion-controlled dimer distance induced unique solid-state fluorescence of cyano substituted styrene pyridinium.

Molecular packing arrangements play a key role in dominating the photophysical properties of luminophores in aggregated state but fine control of the molecular packing is a great challenge. This article describes a unique cyano substituted styrene pyridinium with interesting solid-state fluorescence that can be finely tuned by simple change of counteranions. The dilute solutions of the organic salts (PyCl, PyNO3, PyOTs and PyPh4B) exhibit very weak fluorescence. The crystals of the organic salts (PyCl, PyNO3, and PyOTs) show much enhanced fluorescence compared with their dilute solutions. It is interesting that the emissions changed from bluish-green to deep-blue and fluorescence quantum yields increase from 2.5% to 13.1% with the increasing of steric hindrance of the anions from chloridion, nitrate, to p-toluenesulfonate. Crystal and DFT studies reveal that the enhanced fluorescence is ascribed to the formation of dimers and bigger anions induce larger molecular separation in dimers. Tetraphenylboron anion with very large steric hindrance impedes the formation of dimers and thus results in non-fluorescent salt (PyPh4B). Meanwhile, this unique dimeric packing endows the crystal of PyNO3 with anisotropic fluorescence.

Complex-formation-enhanced fluorescence quenching effect for efficient detection of picric acid.

Amine-functionalized α-cyanostilbene derivatives (Z)-2-(4-aminophenyl)-3-(4-butoxyphenyl)acrylonitrile (ABA) and (Z)-3-(4-butoxyphenyl)-2-[4-(butylamino)phenyl]acrylonitrile (BBA) were designed for specific recognition of picric acid (PA), an environmental and biological pollutant. The 1:1 host-guest complexes formed between the chemosensors and PA enhanced fluorescence quenching, thus leading to sensitive and selective detection in aqueous media and the solid phase.