Construction of a Dual-Functional, Reversible, Ratiometric, Golgi-Targeting Fluorescent Probe for Real-time Monitoring of Dynamics of Intracellular Redox Homeostasis.

Intracellular redox homeostasis is highly important for the physiological processes of living organisms. Real-time monitoring of the dynamics of this intracellular redox process is pivotal but challenging because the biological redox reactions involved in the process are reversible and require at least one pair of oxidizing and reducing species. Thus, biosensors for investigating intracellular redox homeostasis need to be dual-functional, reversible, and, ideally, ratiometric in order for them to have real-time monitoring capacity and to provide accurate imaging information. In light of the importance of the redox pair between ClO- and GSH in living organisms, herein, we used the phenoselenazine (PSeZ) moiety as an electron donor and a reaction site to design a coumarin-based fluorescent probe, PSeZ-Cou-Golgi. After successive treatment with ClO- and GSH, the probe PSeZ-Cou-Golgi experienced an oxidation of selenium (Se) to selenoxide (Se═O) by ClO- and a subsequent reduction of Se═O to Se by GSH. The redox reactions alternatively changed the electron-donating strength of the donor in the probe PSeZ-Cou-Golgi, in turn affecting the intramolecular charge transfer process that resulted in the reversible, ratiometric change of fluorescence from red to green. After four cycles of reversible ClO-/GSH detection during in vitro experiments, the probe PSeZ-Cou-Golgi still had good performance. With the Golgi-targeting group, the probe PSeZ-Cou-Golgi was able to monitor the dynamic change of the ClO-/GSH-mediated redox state during Golgi oxidative stress, making it a versatile molecular tool. More importantly, the probe PSeZ-Cou-Golgi could facilitate the imaging of the dynamic redox state during acute lung injury progression.

MoO3-x nanosheets-based platform for single NIR laser induced efficient PDT/PTT of cancer.

Traditional combination therapy of photodynamic therapy (PDT) and photothermal therapy (PTT) is limited in the field of clinical cancer therapy due to activation by light with separate wavelengths, insufficient O2 supply, antioxidant ability of glutathione (GSH) in tumor cell, and low penetration depth of light. Here, a multifunctional nanoplatform composed of MoO3-x nanosheets, Ag nanocubes, and MnO2 nanoparticles was developed to overcome these drawbacks. For this nanoplatform, hyperthermia and reactive oxygen species (ROS) were simultaneously generated under single 808 nm near-infrared (NIR) light irradiation. Once this nanoplatform accumulated in the tumor region, GSH was depleted by MnO2 and intracellular H2O2 was catalyzed by MnO2 to produce O2 to relieve hypoxia. Ultrasound (US) imaging confirmed in-situ O2 generation. Magnetic resonance (MR) imaging, photoacoustic (PA) imaging, and fluorescence imaging were used to monitor in vivo biodistribution of nanomaterials. This provides a paradigm to rationally design a single NIR laser induced multimodal imaging-guided efficient PDT/PTT cancer strategy.

Hyaluronic Acid-Modified Porous Carbon-Coated Fe3O4 Nanoparticles for Magnetic Resonance Imaging-Guided Photothermal/Chemotherapy of Tumors.

Chemotherapy is an effective method for treating cancer, clinically. However, side effects of drug and multidrug resistance restrict its application. In recent years, the combined treatment of chemotherapy and photothermal therapy (PTT) is becoming a promising method for treating cancer. PTT utilizes nanomaterials absorbing near-infrared light and producing heat to acquire advanced hyperthermia strategy for cancer treatment. Carbon nanomaterials with good biocompatibility, high surface area, and excellent photothermal properties are an excellent nanoplatform for drug delivery and PTT. Herein, porous carbon-coated magnetite nanoparticles (PCCMNs) were successfully synthesized by a one-pot solvothermal method. Magnetite, a contrast agent, can be used for magnetic resonance imaging. Hyaluronic acid was used to modify the PCCMNs to achieve targeted therapy. The obtained nanohybrid with a good photothermal effect can realize combined PTT/chemotherapy and will be a promising nanoplatform for high efficacy theranostics.

Surface plasmon resonance biosensors for simultaneous monitoring of amyloid-beta oligomers and fibrils and screening of select modulators.

Oligomeric amyloid-beta (Aß) peptides are considered as the most toxic species in Alzheimer's disease (AD). Monitoring of the Aß aggregation profiles is critical for elucidating the oligomer toxicity and may serve as a therapeutic target for AD. By immobilizing the capture antibodies of A11 and OC that are specific to the oligomers and fibrils, respectively, in separate fluidic channels, a novel surface plasmon resonance (SPR) biosensor was designed for monitoring the oligomeric and fibrillar species of Aß(1-42) simultaneously. The influence of curcumin, Cu(2+) and methylene blue on the amount of toxic oligomers and fibrils was evaluated. The half maximal inhibitory concentration (IC50) of curcumin and methylene blue was determined. The formation of Aß fibrils was also validated by the thioflavin T (ThT) fluorescence assay. The results demonstrate the utility of SPR as an analytical tool for rapid and comprehensive monitoring of Aß aggregation and screening of Aß modulators.

Amplified voltammetric characterization of cleavage of the biotinylated peptide by BACE1 and screening of BACE1 inhibitors.

Cleavage of amyloid precursor protein (APP) by the ß-site APP cleaving enzyme 1 (BACE1) is a key step in the formation of amyloid beta (Aß) peptide, the main component of amyloid plaques in Alzheimer's disease (AD). Suppression of BACE1 activity has thus become an efficient way for the treatment of AD. In this study, BACE1 in the absence or presence of BACE1 inhibitors was exposed to the biotinylated peptide substrate-modified electrode. This step was followed by the attachment of ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates. Due to the blockage of the BACE1 activity by select inhibitors, well-defined voltammetric peaks of high signal intensity were obtained. However, featureless voltammogram was obtained upon initiating the cleavage reaction. The proposed method is simple, sensitive, and suitable for monitoring of BACE1 activity and screening of BACE1 inhibitors.

Sensitive and continuous screening of inhibitors of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) at single SPR chips.

The development of new methods that meet the demand of high-throughput, high-fidelity screening of hit compounds is important to researching modalities of important diseases such as neurological disorders, HIV, and cancer. A surface plasmon resonance- (SPR-) based method capable of continuously screening enzyme inhibitors at a single chip with antibody-amplified signal enhancement has been developed. The proof of concept is demonstrated by monitoring the cleavage of chip-confined peptide substrates [a segment of the amyloid precursor protein (APP) with the Swiss mutation] by ß-site APP-cleaving enzyme 1 (BACE1). In the presence of a noninhibitor, BACE1 clips the peptide substrate at the cleavage site, detaching a fragment that is homologous to the N-terminus of the amyloid beta (Aß) peptide. Consequently, a subsequent injection of the Aß antibody does not lead to any molecular recognition or SPR signal change at the chip. In contrast, suppression of the BACE1 activity by a strong inhibitor leaves the peptide substrate intact, and the subsequent antibody attachment produces an easily detectable SPR signal. Compared to the widely used FRET (fluorescence resonance energy transfer) assay, the method reported here is more cost-effective, as unlabeled peptide is used as the BACE1 substrate. Furthermore, the assay is faster (each screening cycle lasts for ca. 1.5 h) and can be continuously carried out at a single, regenerable SPR chip for more than 30 h. Consequently, excellent reproducibility (RSD < 5%) and throughput can be attained. Two inhibitors were screened, and their half-maximal inhibitory concentrations (IC50) determined by the SPR method were in excellent agreement with values deduced from ELISA and mass spectrometry.

Analysis of fuzhisan and quantitation of baicalin and ginsenoside Rb(1) by HPLC-DAD-ELSD.

Fuzhisan (FZS) is a traditional Chinese medicine composed of Radix Ginseng Rubra (Araliaceae family), Scutellaria baicalensis Georgi (Labiatae family), Angelica sinensis (Oliv) Diels (Umbelliferae family), Anemone altaica Fisch. Ex C.A. Mey (Araceae family) and Glycyrrhiza uralensis (Leguminosae family). To establish the chemical fingerprint of the components of FZS and quantify the components, baicalin and ginsenoside Rb(1), a high performance liquid chromatography method coupled with diode array and evaporative light scattering detectors (DAD-ELSD) was developed. Separation of 36 components from 12 batches of FZS was performed on a C(18) column, with a mobile phase consisting of acetonitrile and 0.1% acetic acid-water, with gradient elution at a column temperature of 30 degrees C. The optimum detection wavelength was set at 335 nm, the drift tube temperature of ELSD was set at 80 degrees C, the carrier gas pressure was 25 psi, and the gain = 10. The similarity among 12 batches of FZS was over 0.95. Five constituents of FZS, namely baicalin, ferulic acid, and ginsenosides Rg(1), Re, and Rb(1), were identified based on their retention times (RT). Calibration curves for baicalin and ginsenoside Rb1 showed good linearity (r (2) > 0.9992); recoveries ranged from 95% to 99%. This quantification method is reproducible and simple, and may provide a tool to assess the quality of FZS.

[Determination of the cysteine residues in the surface-confined biomolecules by using electrochemical desorption and fluorescence detection].

To develop a method for the detection of surface-confined peptides containing cysteine residues or oligodeoxynucleotides (ODNs) whose 3' ends modified with thiol groups, and a thiol-specific fluorescent cross-linker, N-(9-acridinyl) maleimide (NAM) was used. The peptides studied herein include both the oxidized and reduced forms of glutathione, and a hexapeptide (FT). Peptides are first attached onto the activated 11-mercaptoundecanoic acid (MUA)-terminated alkanethiol self-assembled monolayers (SAMs) and then derivatized with NAM. The cysteine residues was determined by using electrochemical desorption and fluorescence detection. GSH concentration as low as 40 pmol x L(-1) can be measured. The fluorescence intensity in the case of FT is about 3 times as high as that for GSH, which is consistent with the molar ratio of cysteine residues in these two molecules. The analytical performance of gene analysis was also evaluated through the analyses of a complementary target and targets with varying numbers of mismatching bases. The method described here is simple, sensitive, reproducible, and does not require sophisticated analytical instrumentation and separation procedures.

[Fingerprinting of Fuzhisan by high-performance liquid chromatography with ultraviolet and evaporative light scattering detector].

OBJECTIVE: To establish a method for fingerprinting of Fuzhisan (FZS, a traditional Chinese medicinal preparation) using high-performance liquid chromatography with ultraviolet and evaporative light scattering detector (HPLC-UV/ELSD) to allow simultaneous determination of 5 major constituents in the preparation. METHODS: HPLC-UV/ELSD analysis was performed on water AlltechC18 column (5 microm, 4.6 mm x 250 mm) with a mixture of acetonitrile (A) and 0.1% acetice acid water (B) as the mobile phase. The solvent A gradient for elution was 0, 12%; 25, 20%; 30, 20%; 75, 30%; 105, 40%; 120, 80%; 130, 12%, with the flow rate of 1.0 ml/min; and the column temperature at 30 degrees . The detective wavelength was 335 nm, drift tube temperature was 80 degrees , pressure of nebulizer gas was 25 psi. The similarities between the HPLC-UV/ELSD fingerprints of the 12 extracts were calculated using similarity evaluation software. RESULTS: The fingerprint of FZS was established and the 5 major constituents were identified. The complementarity between the fingerprints of UV and ELSD was analyzed, showing good correlation between 12 batches of FZS. CONCLUSION: The method for fingerprinting can simultaneously characterize the main chemical constituents in FZS and allows stable, effective and comprehensive quality control and evaluation of FZS for a single sample.