A novel AIE fluorescent probe for the detection and imaging of hydrogen peroxide in living tumor cells and in vivo.

Hydrogen peroxide (H2O2), a key reactive oxygen species (ROS), plays crucial roles in redox signaling pathways and immune responses associated with cell proliferation, differentiation, migration, and disease progression. The selective monitoring of overproduced H2O2 is important for understanding the diagnosis and pathogenesis of diseases such as cardiovascular disease, cancers, diabetes, Parkinson's disease, Alzheimer's disease, and inflammation. In this paper, an AIE fluorescent probe BQM-H2O2 was developed by connecting phenyl borate with the fluorophore BQM-PNH for selective detection of H2O2. In the presence of H2O2 at fw = 99% (pH = 7.4, 1% DMSO), the probe BQM-H2O2 could generate strong fluorescent signals due to the oxidation of the borate ester. The probe exhibited high selectivity and a low detection limit toward H2O2 with the calculated LOD of 112.6 nM. Importantly, it was employed in the detection of exogenous and endogenous hydrogen peroxide in 4T1 cells with low cytotoxicity. This probe has also been successfully applied to imaging of H2O2 in Blab/c mice bearing 4T1 graft tumors.

A novel fluorescent probe for rapid detection of sulfatase in vitro and in living cells.

Sulfatase participates in a variety of physiological processes in organisms including hormone regulation, cell signaling, and bacterial pathogenesis. Current sulfatase fluorescent probes can be used to track sulfate esterase overexpression in cancer cells for diagnostic purposes and to understand the pathological activity of sulfate esterase. However, some sulfatase fluorescent probes based on the hydrolysis of the sulfate bond were easily disturbed by the catalytic activity of sulfatase. Herein, we developed the fluorescent probe BQM-NH2 for sulfatase detection, which was based on the quinoline-malononitrile. The probe BQM-NH2 showed a fast response to sulfatase within 1 min and satisfactory sensitivity with a calculated LOD of 1.73 U/L. Importantly, it was successfully used to monitor endogenous sulfate in tumor cells, indicating BQM-NH2 has the potential to monitor sulfatase under physiological and pathological conditions.

Highly selective fluorescent probe based on AIE for identifying cysteine/homocysteine.

Cysteine (Cys) and homocysteine (Hcy) are involved in maintaining homeostasis in the body and are relevant to various diseases. While the level of Cys and Hcy is much lower than GSH in the living system, which leads to a major challenge to selectively identify Cys/Hcy in the presence of large amounts of GSH. In this paper, an AIE fluorescent probe SQM-NBD was obtained by connecting NBD to the hydroxyl group of the fluorophore SQM-OH for selective detection of Cys/Hcy. Probe SQM-NBD had no fluorescence itself. But, under the disturbance of GSH, the fluorescence signal of probe SQM-NBD could be turned on by Cys/Hcy. The study of the response mechanism showed that probe SQM-NBD could release both SQM-OH and Cys/Hcy-NBD after reacting with Cys/Hcy. While Cys/Hcy continued to quench the fluorescence of SQM-OH through the combination of Michael addition and the ion rich environment, resulting in only the fluorescence signal of Cys/Hcy-NBD being observed. SQM-NBD appeared superior sensitivity to Cys/Hcy, with LOD of 54 nM and 72 nM, respectively. Significantly, probe SQM-NBD realized the application of fluorescence imaging of Cys/Hcy in HeLa cells, indicating that probe SQM-NBD has the potential for Cys/Hcy identification under physiological and pathological conditions.

[The Establishment of a Three-color Flow Cytometry Approach for the Scoring of Micronucleated Reticulocytes in Rat Bone Marrow].

OBJECTIVE: To develop and verify a flow cytometric measurement of reticulocytes (RETs) micronucleus in rat bone marrow. METHODS: In our flow cytometric protocol, reticulocytes, leukocytes and DNA were labeled by anti-CD71-fluorescein isothiocyanate (FITC), anti-CD45-phycoerythrin (PE) and DRAQ5, respectively. Sprague-Dawley (SD) rats were assigned to four treatment groups randomly, and were exposed to ethyl methanesulfonate (EMS), cyclophosphamide (CP), ethyl nitrosourea (ENU) and colchicine (COL) respectively. Each treatment group was divided into four subgroups (5 rats per subgroup) according to different exposure dosage. A exposure dose of 0 was used as vehicle control for each group. Rats were administered with testing mutagens by gavage twice with a 24 h interval. Bone marrow from both femurs were collected 24 h after the last administration. The frequency of micronucleated reticulocytes (MN-RETs) and the percentage of reticulocytes (RETs%) were determined by flow cytometric measurement established in this study. And the manual counting method with microscope (by Giemsa staining) was conducted at the same time. RESULTS: A method for detection of reticulocyte micronucleus in bone marrow based on flow cytometry was successfully established. The MN-RETs in rat bone marrow of 20 SD rats treated by vehicle (i.e., background value of MN-RETs) was 0.83‰±0.12‰ by this method. The background value of MN-RETs in manual enumeration method was 1.43‰±0.44‰. It was obvious that the flow cytometric method had lower background value and more stable results. The trend, in which MN-RETs ascended and RETs% descended with increasing dose, can be detected by both methods in rats that exposed to EMS, CP, ENU and COL. Both methods were good to detect the correlation of induced-MN-RETs with four testing mutagens (the correlation coefficients were ranged from 0.834 3 to 0.913 7). CONCLUSION: With its sensitivity, rapidity, easy operation and low background value, the three-color flow cytometric enumerative protocol established in our laboratory can be used as a good substitute for manual micronucleus counting method and used in genotoxicity assessment of chemical substances.