[Quercetin induces apoptosis of human breast cancer cells by activiting PTEN and inhibiting PI3K/AKT and JNK signaling pathways].

Objective To investigate the effect of quercetin on cell proliferation and apoptosis of MCF-7 human breast cancer cells, and effects of signaling pathways of PTEN/PI3K/AKT and c-Jun N-terminal kinase (JNK) signaling pathway. Methods MCF-7 cells were treated with quercetin (0, 20, 40, 60, 80, 100 µmol/L) CCK-8 assay was used to detect the cell proliferation, and cell apoptosis was assayed by flow cytometry. Hochesst33342 staining was used to observe the changes of nuclear number and karyotype, and flow cytometry was employed to detect cell apoptosis. The expression of PTEN and phosphorylated JNK (p-JNK) were detected by immunofluorescence cytochemistry, and the protein levels of PTEN, phosphorylated PI3K (p-PI3K), p-JNK and phosphorylated AKT (p-AKT) were detected by Western blot analysis. After treated with PI3K/AKT pathway inhibitor LY294002, the cell apoptosis and related protein expression were detected by the above methods again. Results With increased quercetin concentration, cell activity decreased and cell growth was inhibited in a concentration dependent manner; the nuclear concentration and apoptosis also increased. High concentration of quercetin significantly enhanced the expression and distribution of PTEN protein, decreased the levels of p-PI3K and p-AKT protein, and inhibited the expression of p-JNK protein. After adding LY294002 to inhibit PI3K/AKT, the apoptosis rate increased for cells treated with both LY294002 and quercetin; the expression of PTEN protein also showed an increase. Quercetin could significantly enhance the effect of LY294002 on p-PI3K/AKT/PTEN protein. Conclusion Quercetin can signeristically inhibit cell viability and induce cell apoptosis on MCF-7 cells, by up-regulating the expression of PTEN and down-regulating PI3K/AKT and JNK pathways.

Electrochemical mixed aptamer-antibody sandwich assay for mucin protein 16 detection through hybridization chain reaction amplification.

A mixed aptamer-antibody sandwich assay for the determination of mucin protein 16 (MUC16) was developed based on hybridization chain reaction (HCR) with methylene blue (MB) as an electrochemical indicator. First, MUC16 antibody was adsorbed onto the surface of the Au nanoparticle (AuNP)-modified indium tin oxide (ITO) electrode to effectively capture the target MUC16. After MUC16 was captured by the MUC16 aptamer, an antibody/MUC16/aptamer sandwich structure formed for the highly selective detection of MUC16. The 3' end of the aptamer was then subjected to HCR with the assistance of auxiliary probes to obtain DNA concatemers. Numerous MB molecules bonded with G bases in the DNA concatemers by immersing the modified ITO electrode into a stirred solution containing MB with KCl. Stepwise changes in the microscopic features of the electrode surface were studied by scanning electron microscopy (SEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the electrochemical behavior of the different modified electrodes. The oxidation current of MB was detected by differential pulse voltammetry (DPV). Under the optimum conditions, the proposed mixed aptamer-antibody sandwich assay showed wide dynamic range from 0.39 to 200 unit mL-1 with a low detection limit of 0.02 unit mL-1 (S/N ratio = 3). The proposed method showed good accuracy, selectivity, and acceptable reproducibility. Graphical abstract An electrochemical mixed aptamer-antibody sandwich assay based on the aptamer-induced HCR amplification strategy was fabricated for the highly sensitive detection of MUC16. The mixed aptamer-antibody sandwich assay showed acceptable performance of detection range, detection limit, reproducibility, and selectivity.

Amperometric sandwich immunoassay for determination of myeloperoxidase by using gold nanoparticles encapsulated in graphitized mesoporous carbon.

An ultrasensitive sandwich-type electrochemical immunosensor was developed for the amperometric determination of serum myeloperoxidase (MPO). The method is making use of (a) gold nanoparticles encapsulated in graphitized mesoporous carbons (AuNP@GMC); and (b) horseradish peroxidase (HRP) labeled secondary antibody (HRP@Ab2) immobilized on AuNP@GMC. MPO capture antibody (Ab1) was immobilized on the electrode modified with an AuNP-graphene oxide nanocomposite. The sandwich immunoreaction leads to the formation of the complex composed of Ab1, MPO, and HRP@Ab2. An amplified electrochemical signal is produced by electrocatalytic reduction of H2O2 (at a typical voltage of -0.18 V vs. Ag/AgCl) in the presence of enzymatically oxidized thionine. The peak current of thionine was measured using differential pulse voltammetry. Under optimized steady-state conditions, the reduction peak increases in the 1 to 300 pg.mL-1 MPO concentration range, and the detection limit is 0.1 pg.mL-1 (at S/N = 3). Graphical abstract Schematic presentation of AuNP-GO based sandwich-type electrochemical immunoassay for the determination of myeloperoxidase by using gold nanoparticles encapsulated in graphitized mesoporous carbons (AuNP@GMC) as a carrier for horseradish peroxidase (HRP) labeled secondary antibody (HRP@Ab2).

Electrochemical determination of the activity of DNA methyltransferase based on the methyl binding domain protein and a customized modular detector.

An electrochemical method is described for the determination of the activity of the DNA methyltransferase (MTase). The assay was based on the use of a commercially available customized electromagnetic modular detector, which consisted of a magnetic switch, electrical connectors and a screen-printed electrode modified with graphene oxide. The biotinylated single-strand DNA (ss-DNA) S1 was absorbed by streptavidin-modified magnetic beads (MBs) via streptavidin-biotin interaction. The biotinylated ss-DNA S1 was hybridized with the complementary ss-DNA S2. After the symmetrical sequences 5'-CCGG-3' of the duplex DNA (ds-DNA) were methylated by M. SssI CpG methyltransferase (M. SssI MTase), the symmetrical sequences 5'-CCGG-3' in the ds-DNA were recognized by glutathione S-transferase (GST) tagged methyl CpG binding protein 2 (MeCP2). The unmethylated 5'-CCGG-3' sequences were specifically cleaved by HpaII restriction endonuclease. After magnetic separation and washing, HRP-labeled GST tag monoclonal antibody and H2O2 were used as a tracer label and enzyme substrate, respectively. Electrochemical measurement was carried out at pH 7.4 in the presence of 50 µM thionine and 0.5 mM H2O2. Stepwise changes in the microscopic features of the SPE surface upon the formation of each layer were studied by scanning electron microscopy. Cyclic voltammetry and differential pulse voltammetry were used to characterize the electrochemical behavior of the different modified electrodes. Under the optimal conditions, the activity of M. SssI MTase can be determined in the activity range of 0.5-125 unit·mL-1 with a detection limit of 0.2 unit·mL-1 (at an S/N ratio of 3). The sensitivity of the immunoassay is 0.489 µA·µM-1·cm-2. Graphical abstract Schematic presentation of the electrochemical immunosensor for the determination of the activity of M. SssI CpG methyltransferase (M. SssI MTase). It is based on an electromagnetic modular detector and the use of glutathione S-transferase tagged methyl CpG binding protein 2 (GST-MeCP2).

Electrochemical determination of BCR/ABL fusion gene based on in situ synthesized gold nanoparticles and cerium dioxide nanoparticles.

An efficient DNA electrochemical biosensor, based on the gold nanoparticles (GNPs) in situ synthesized at the surface of multiwalled carbon nanotubes (MWCNTs), cerium dioxide (CeO2) and chitosan (Chits) composite membrane, was developed for the detection of BCR/ABL fusion gene in chronic myelogenous leukemia (CML). The capture probe was attached onto the nanocomposite membrane modified glassy carbon electrode (GCE) through the conjugated structure. Owing to the synergistic effects of CeO2 nanoparticles with a strong adsorption ability and MWCNTs with a large surface area and excellent electron transfer ability, the prepared composite membrane was demonstrated an efficient electron transfer ability. The biosensor was electrochemically characterized by cyclic voltammogram (CV) and differential pulse voltammetry (DPV), and the decrease of the peak currents upon hybridization was observed using methylene blue (MB) as the electroactive indicator. Under the optimized conditions, peak currents were linear over the range from 1 × 10(-9) M to 1 × 10(-)(12) M, with a detection limit of 5 × 10(-)(13) M (based on the 3σ). And the proposed method was successfully applied for the detection of PCR real samples with satisfactory results. Furthermore, the developed DNA biosensor was demonstrated a good selectivity, a reasonable stability and a favorable reproducibility, which could be regenerated easily.

Ultrasensitive electrochemical immunosensor for HE4 based on rolling circle amplification.

A novel electrochemical immunoassay system for the detection of human epididymis-specific protein 4 (HE4) was developed. A chitosan-titanium carbide (TiC) nanocomposition film was first electrodeposited onto a tin-doped indium oxide (ITO) electrode at a constant potential. Gold (Au) nanoparticles were then electrodeposited on the surface of the chitosan-TiC film by cyclic voltammetry (CV). The capture antibody (anti-HE4) was adsorbed onto the Au and TiC nanoparticles. After a specific sandwich immunoreaction among the capture antibody, HE4, and biotinylated secondary antibody, biotinylated primer DNA was immobilized on the secondary antibody by biotin-streptavidin system. Appropriate amounts of circular template DNA and biotinylated primer DNA were used for rolling circle amplification (RCA) under optimal conditions. The RCA products provided a large number of sites to link DNA detection probes. Doxorubicin hydrochloride intercalated the CG-GC steps between the RCA products and the DNA detection probes, which was monitored by differential pulse voltammetry (DPV) based on the current signal of doxorubicin hydrochloride. With the above-mentioned amplification factors, the current responded to HE4 linearly in the concentration range of 3-300 pM under optimal detection conditions, with a detection limit of 0.06 pM. Stepwise changes in the microscopic features of the surfaces and electrochemical properties upon the formation of each layer were confirmed by scanning electron microscopy (SEM), atomic force microscopy (AFM), and electrochemical impedance spectroscopy (EIS). This system was successfully employed for the detection of HE4 with good accuracy and renewable ability.

Improved electrochemical immunosensor for myeloperoxidase in human serum based on nanogold/cerium dioxide-BMIMPF6/L-Cysteine composite film.

An electrochemical immunosensing assay for myeloperoxidase (MPO) determination in human serum has been developed. Firstly, L-Cysteine was initially electropolymerized on an Au electrode to form L-Cysteine film. After that cerium dioxide (CeO2) dispersed in 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6) were immobilized on the L-Cysteine film. Then the negatively charged nanogold particles were adsorbed onto the membrane via the positive charge of CeO2, which aimed at assembling more antibody of MPO (anti-MPO). The resulting immunosensor showed a high sensitivity, broad linear response to the MPO concentration comprised between 10 ng/mL and 400 ng/mL with a detection limit of 0.06 ng/mL. Moreover, the surface morphology of the electrode was studied by means of a scanning electron microscope and the electrochemical properties of the fabricated immunosensor were further characterized by cyclic voltammetry. Also, factors influencing the performance of the resulting immunosensors were studied in detail.