A novel strategy for real-time and in situ detection of cytochrome c and caspase-9 in Hela cells during apoptosis.

Cytochrome c (cyt c) and caspase-9 were critical biomarkers in mitochondria-mediated apoptosis. A novel electrochemical immunosensor was developed for in situ analysis of cyt c and caspase-9 in the cytosol. Gold nanoparticle-polydopamine (AuNP/PDA) composites were used to fabricate the interface of the sensor. The anti-cyt c or anti-caspase-9 functionalized-immunosensor provided a biomimetic interface for immunosensing of cyt c or caspase-9 in Hela cells during apoptosis. The changes in the expression level of cyt c and caspase-9 in the cytosol upon curcumin-induced apoptosis were detected by using the proposed method, and also the influence of different concentrations and incubation times of curcumin-induced Hela cells was investigated. This method achieved a linear range (0.1-100 µM) for standard cyt c and caspase-9, with a detection limit of 0.03 ± 0.01 µM for standard cyt c and 0.08 ± 0.02 µM for standard caspase-9. Moreover, this method was used to detect cells which could detect as low as 100 cells which expressed cyt c and caspase-9, and also the results are in good agreement with standard flow cytometry analysis. The developed electrochemical immunosensor offered a simple and rapid approach for sensitive evaluation of apoptosis markers with considerable specificity and reproducibility, and also the developed strategy could be of great importance in clinical diagnosis and therapeutic research.

Synthesis and synergetic anti-tumor activity evaluation of dihydroartemisinin-organogermanium(IV) compound.

Dihydroartemisinin (DHA), a semi-synthetic derivative of the herb artemisinin, has shown commendable bioactivity. In this paper, a novel dihydroartemisinin-organogermanium (DHA-Ge) compound was synthesized, characterized and its potential anti-tumor activity was evaluated by various methods. MTT results demonstrated that DHA-Ge could effectively inhibit the proliferation of HepG2 cells and showed their dose-dependent properties. The IC50 value of inhibition effect on HepG2 cells of DHA-Ge was 10.23 µg/ml which was lower than 39.44 µg/ml of DHA. Flow cytometric results suggested that DHA-Ge could induce apoptosis of HepG2 cells and the apoptosis rate was 20.26% after 24h treatment with 56.8 µg/ml DHA-Ge concentration. Atomic force microscopy images showed that HepG2 cells were collapsed and the cell nucleus were fragmented after 24h treatment. All these results together showed that the DHA-Ge possessed desirable synergetic enhanced anti-tumor effects and could be developed as a suitable tumor therapeutic agent.

Synthesis and synergetic effects of chrysin-organogermanium (IV) complex as potential anti-oxidant.

Organogermanium(IV) (Ge) is considered to play an important role in the anti-oxidative activities of some Chinese medicines. Here, a new chrysin-organogermanium (Chry-Ge) complex was synthesized and investigated for its potential biological activities. The radicals-sensitive Ge-O bond was introduced to Chry-Ge complex to enhance bioactivities of organic Ge or Chry. Results showed that Chry-Ge complex possessed great anti-oxidative activities, showing stronger hydroxyl scavenging effects than their corresponding ligands. We also demonstrated Chry-Ge complex inhibited ROS-dependent oxidative damage in cells. Moreover, the morphological and biophysical recoveries in oxidation-damaged cells induced by Chry-Ge complex were characterized by atomic force microscopy. All these results collectively suggested that Chry-Ge complex has synergetic effect for radicals scavenging and could be served as promising pharmacologically active agent against anti-oxidative treatment.

Synthesis and biological evaluation of Germanium(IV)-polyphenol complexes as potential anti-cancer agents.

Germanium (Ge) is considered to play a key role in the pharmacological effects of some medicinal plants. Here, two new Ge(IV)-polyphenol complexes were synthesized and measured for their potential biological activities. The results indicated that these Ge(IV)-polyphenol complexes possessed great anti-oxidative activities, both showing stronger hydroxyl scavenging effects than their corresponding ligands. We also demonstrated the strong intercalating abilities of Ge(IV)-polyphenol complexes into calf thymus-DNA molecules. In addition, these two Ge(IV)-polyphenol complexes showed strong proliferative inhibition effect on HepG2 cancer cells. Moreover, the morphological changes in HepG2 cells induced by Ge(IV)-polyphenol complexes were detected by atomic force microscopy. All these results collectively suggested that Ge(IV)-polyphenol complexes could be served as promising pharmacologically active substances against cancer treatment.

Anti-tumor activity evaluation of novel chrysin-organogermanium(IV) complex in MCF-7 cells.

Chrysin (5,7-dihydroxylflavone, Chry) is a natural product extracted from plants, honey, and propolis. In this work, a novel chrysin-organogermanium(IV) complex (Chry-Ge) with enhanced anticancer activities was synthesized, and its potential anticancer effects against cancer cells were measured using various methods. MTT results showed that Chry-Ge had significant inhibition effects on the proliferation of MCF-7, HepG2 and Colo205 human cancer cell lines in a dose-dependent manner while had little cytotoxic effects on MCF-10A human normal cells (MCF-10A cells) with the same treatment of Chry-Ge. These results suggested that Chry-Ge possessed enhanced anticancer effects and high selectivity between cancer cells and normal cells. The immuno-staining results showed that the nuclei of MCF-7 cells represented a total fragmented morphology and a disorganized cytoskeletal network in MCF-7 cells after Chry-Ge treatment. Besides, atomic force microscopy (AFM) was applied to detect the changes of ultrastructural and biomechanical properties of MCF-7 cellular membrane induced by Chry-Ge. The AFM data indicated that Chry-Ge treatment directly caused the decrease of cell rigidity and adhesion force of MCF-7 cells, suggesting that membrane toxicity might be one of the targets for Chry-Ge in MCF-7 cells. Moreover, the fluorescence-based flow cytometric analysis demonstrated that Chry-Ge could induce apoptosis in MCF-7 cells in ROS-dependent mitochondrial pathway. All results collectively showed that Chry-Ge could be as a promising anticancer drug for cancer therapy.

Electrochemical activity of holotransferrin and its electrocatalysis-mediated process of artemisinin.

Holotransferrin, the iron (III) transport protein in the blood, can significantly increase the anticancer activity of artemisinin, which is isolated from the Chinese plant qinghaosu. This paper investigates the action process of holotransferrin-induced electrocatalytic reduction of artemisinin by spectroscopic and electrochemical techniques. Results show that holotransferrin(Fe(III)) is the electrochemical sites of holotransferrin, which can catalyze the reduction of artemisinin through lowering the overpotential by about 80 mV. Compared with the different electrochemical behaviors of artemisinin with apotransferrin and holoprotein (apotransferrin in the presence of Fe(III)), respectively, it demonstrates that holotransferrin(Fe(III)) plays an important role in the electrocatalytic reduction of artemisinin, which can catalyze the cleavage of the endoperoxide bridge in artemisinin. A reliable two-step process is proposed to explain that artemisinin is activated by holotransferrin(Fe(III))-mediated electrocatalytic reduction. These results can provide further information for better understanding the anticancer action of holotransferrin-conjugated artemisinin.

[Immobilization of anti-transferrin on nano-gold and its immune recognition of transferrin].

A novel strategy based on antigen-antibody interaction was developed by means of backfilling transferrin on antibody functionalized gold nanoparticles surfaces in the present study. Nano gold particles were immobilized with cysteamine layer by self-assembly, whose surfaces were chemically coupled with anti-transferrin antibodies by using EDCX, forming the anti-transferrin-Au immuno-probes. The particles and the nano-probes were characterized by the integrated tools of resonance Rayleigh scattering (RRS), UV-Vis absorption spectra, transmission electron microscopy (TEM) and laser light scattering. The result showed that the nano-probe with the ability of specific recognition of transferrin had good immune activity. The RRS peak at 470 nm was amplified obviously by using this signal amplification, and the antigen recognition was monitored via the enhancement of 470 nm RRS intensity when this binding event occurred. The result showed that the amplification strategy led to a dramatic improvement of the detection sensitivity of transferrin, and the detection of transferrin featured a linear range of 0.85 to 33.9) x 10(-10) mol x L(-1) with the detection limit of 8.5 x 10(-11) mol x L(-1).

Gold nanoprobes-based resonance Rayleigh scattering assay platform: Sensitive cytosensing of breast cancer cells and facile monitoring of folate receptor expression.

A rapid, facile assay for sensitive cytosensing of breast cancer cells should help to guide potential medical evaluation for breast cancer. Here, we report development of novel resonance Rayleigh scattering (RRS) cytosensor for cell recognitions and folate (FA) receptor expression analyses on living cells. Using FA-conjugated gold nanoparticles (FA-AuNPs) as nanoprobes, the constructed nanoprobes-assembled recognition interface could increase the binding capacity for cell recognition, amplify Au-aggregates-enhanced RRS signal, and then enhance the sensitivity for membrane antibody assay. FA-AuNPs-based RRS measurements enabled a distinct 34-times-enhancement in RRS intensities after incubation with human breast cancer cells, compared with normal cells. Receptor-targeted cytosensor was used to quantitatively detect human breast cancer MCF-7, liver cancer HepG2 and normal cells, which expressing different amount of FA receptor, respectively. The detection limit for MCF-7 cells was 12 cells/mL with good selectivity and reproducibility. Furthermore, the proposed cytosensor allowed for dynamic evaluation of FA receptor expression on different living cells after dihydroartemisinin stimulus. This assay platform shows the good potential for clinical diagnostics and antibody-targeted drug screening.

In-situ detection of resveratrol inhibition effect on epidermal growth factor receptor of living MCF-7 cells by Atomic Force Microscopy.

A new method based on Atomic Force Microscopy (AFM) was developed to real-time and in-situ detect epidermal growth factor receptor (EGFR) expression levels on living MCF-7 cells for evaluating the anticancer activity of resveratrol. Here, the inhibition effect of resveratrol on EGFR expression levels on MCF-7 cells was probed by epidermal growth factor (EGF)-functionalized tips for the first time. Changes in morphology and stiffness of single cell stimulated by resveratrol at different concentrations were detected by AFM. The consequences showed that resveratrol influenced the cellular state and reduced expression of EGFR on the cell surface, which were also interpreted by MTT assay and confocal microscopy assay. AFM, which was used to investigate potential targets for anti-tumor drug on living cells and realize a better understanding of drug action mechanism, was expected to be developed into a promising tool for screening of drugs.

Facile synthesis of multifunctional germanium nanoparticles as a carrier of quercetin to achieve enhanced biological activity.

A simple method for preparing quercetin surface-functionalized germanium nanoparticles (Qu-GeNPs) with enhanced antioxidant and anticancer activity is reported. Spherical germanium nanoparticles (GeNPs) were capped by quercetin (Qu) with a mean particle size of approximately 33 nm and were characterized by TEM, AFM, UV-visible absorption spectroscopy, FTIR, and XRD measurements. The in vitro drug release of Qu from the Qu-GeNPs indicated that Qu could principally be distributed around tumor tissues rather than in the normal section and Qu-GeNPs were internalized by MCF-7 cells. Their biological activity test results indicated that these Qu-GeNPs possessed stronger hydroxyl-scavenging effects and proliferative inhibition effect on MCF-7 cancer cells than quercetin, thus suggesting that the strategy to use GeNPs as a carrier of Qu could be an efficient way to achieve enhanced antioxidant and anticancer activity. In addition, Qu-GeNPs possessed a high apoptotic induction effect in cancer cells, especially in high dosages, and could arrest MCF-7 cells in the S phase.

Highly sensitive detection of chromium (III) ions by resonance Rayleigh scattering enhanced by gold nanoparticles.

Simple and sensitive determination of chromium (III) ions (Cr(3+)) has potential applications for detecting trace contamination in environment. Here, the assay is based on the enhancement of resonance Rayleigh scattering (RRS) by Cr(3+)-induced aggregation of citrate-capped gold nanoparticles (AuNPs). Transmission electron microscopy (TEM) and UV-vis absorption spectroscopy were employed to characterize the nanostructures and spectroscopic properties of the Cr(3+)-AuNP system. The experiment conditions, such as reaction time, pH value, salt concentration and interfering ions, were investigated. The combination of signal amplification of Cr(3+)-citrate chelation with high sensitivity of RRS technique allow a selective assay of Cr(3+) ions with a detection limit of up to 1.0 pM. The overall assay can be carried out at room temperature within only twenty minutes, making it suitable for high-throughput routine applications in environment and food samples.

A novel gold nanoparticle-doped polyaniline nanofibers-based cytosensor confers simple and efficient evaluation of T-cell activation.

A rapid, easy assay for monitoring dynamics of T-cell activation should help to guide potential medical evaluation of immune responses or immunopathogenesis. Here, we report development of novel electrochemical cytosensors for dynamic analyses of T-cell activation markers on living cells. Gold nanoparticles-doped polyaniline nanofiber (Au/PANI-NFs) composite was greenly prepared by in situ one-step chemical inertness of PANI-NFs with gold nanoparticles to fabricate impedance-based electrochemical biosensors. Transmission electron micrographs indicated that the gold nanoparticles were uniformly anchored along with the structure of PANI-NF surface, displaying fibrillar morphology with a ~60 nm diameter. Au/PANI-NFs-based cytosensors coated with anti-CD Ab molecules could provide biomimetic interface for multiple immunosensing of T-cell surface activation markers (CD69, CD25, and CD71). The dual signal amplification of Au nanoparticle and PANI-NFs-based electrochemical impedance spectroscopic (EIS) measurements enabled the cytosensors considerably sensitive, with a detection limit of 1×10(4) cells/ml of activated T-cells. The activation-targeted cytosensors detected early, middle and late stages for expression of activation markers CD69, CD25, and CD71 at 8 h, 24 h, and 36 h, respectively, after concanvalin A stimulation of T cells. The quantitative results consisted with those derived from flow cytometric analysis. Furthermore, activation-targeted cytosensor allowed for dynamic analysis of the immune inhibition of T-cell activation by immune regulatory drug icariin (ICA). Thus, Au/PANI-NFs-based cytosensors offer simple and fast approach for non-destructive, quantitative evaluation of T-cell activation markers, with considerable specificity, reproducibility, and low background noise.

A label-free electrochemiluminescence cytosensors for specific detection of early apoptosis.

A novel electrochemiluminescence (ECL) cytosensors was developed for the detection of early apoptotic cells by the specific interaction between Annexin V and phosphatidylserine(PS) based on ECL signal of CdS-QDs. Immobilization of Annexin V on a L-cysteine-capped CdS-QDs/Polyaniline nanofibers (PANI-NF) resulted in the stable and high loading of Annexin V on the sensor surface and the possibility of sensitivity enhancement. Early apoptotic cells showed an increased exposure of PS on the cell membrane caused by physiological and pathological response reactions, leading to a strong interaction between the apoptotic cells and the sensor surface, which could be probed by the ECL. Using a real of early apoptotic HepG2 cell induced by resveratrol (RVL), the proposed novel strategy has demonstrated its simplicity, high sensitivity, good selectivity and high reproducibility and label-free capability which might hold a great potential for rapid detection of cell apoptosis and drug screening. The results from this approach have showed good agreement with those obtained using inverted microscope, flow cytometry(FCM) and Atomic force microscopy(AFM). The linear range for early apoptotic cells detection ranged from 500 to 1.0 × 10(6) cells mL(-1) with a detection limit of 500 cells mL(-1). The reported strategy has provided a promising platform for highly sensitive cytosensing and convenient screening of some clinically anticancer drugs.

Hydroxyl radical scavenging mechanism of human erythrocytes by quercetin-germanium (IV) complex.

Quercetin is a popular flavonoid in plant foods, herbs, and dietary supplement. Germanium, a kind of trace elements, can enhance the body immunity. This study investigated the hydroxyl-radical-scavenging mechanism of the quercertin-germanium (IV) (Qu-Ge) complex to human erythrocytes, especially the effects on ultrastructure and mechanical properties of cell membrane, plasma membrane potential and intracellular free Ca(2+) concentration. Results showed that QuGe(2), a kind of the Qu-Ge complex, could reduce the oxidative damage of erythrocytes, change the cell-surface morphology, and partly recover the disruption of plasma membrane potential and intracellular free Ca(2+) level. Atomic force microscopy (AFM) was used to characterize the changes of the cell morphology, cell-membrane ultrastructure and biophysical properties at nanoscalar level. QuGe(2) has triggered the antioxidative factor to inhibit cellular damage. These results can improve the understanding of hydroxyl-radical-scavenging mechanism of human erythrocytes induced by the Qu-Ge complex, which can be potentially developed as a new antioxidant for treatment of oxidative damage.

[Study of DNA damage induced by potassium dichromate and glutathione with atomic force microscope].

OBJECTIVE: To observe calf thymus DNA damage induced by potassium dichromate in combination with glutathione (GSH). METHODS: Atomic force microscope and ultraviolet spectrum (UV) were used to observe the alterations of the DNA ultrastructure and absorption spectrum. RESULTS: Atomic force microscopy revealed no breaks of the DNA strand in response to treatment with potassium dichromate alone, but when coupled with GSH at proper concentrations, potassium dichromate induced alterations in the DNA structure and DNA fragmentation. UV examination also confirmed these findings by showing increased absorption intensity of the maximum UV peak following combined treatment of the DNA with potassium dichromate and GSH. CONCLUSION: These morphological and spectrographic evidences verified the important role of GSH in mediating the generation of various tumor-inducing intermediate products of potassium dichromate.

Transferrin-mediated gold nanoparticle cellular uptake.

Targeted drug delivery is an important research area in specific therapy. Transferrin-conjugated nanoparticles are an attractive formulation as a vehicle for specific cellular uptake and targeted drug delivery. In this report, atomic force microscopy imaging was used to visualize the process of cellular uptake of transferrin-coupled gold nanoparticles on the surfaces of live cells for the first time. High-resolution images were captured, showing the endocytosis of transferrin-conjugated nanoparticles taking place during the process of internalization. This specific transferrin-mediated nanoparticle uptake was validated by confocal scanning imaging and transferrin competition experiments.

The stepwise process of chromium-induced DNA breakage: characterization by electrochemistry, atomic force microscopy, and DNA electrophoresis.

DNA conformational change and breakage induced by Cr(VI)-GSH interaction were characterized by the integrated tools of electrochemistry, atomic force microscopy (AFM), and DNA electrophoresis. While electrochemistry confirmed the formation of the active species generated from Cr(VI)-GSH reduction, which causes the DNA conformational changes, AFM imaging vividly demonstrated the stepwise process of the DNA denaturation and breakage for the first time. Our DNA electrophoresis further validated that the DNA breakage occurs unevenly at both of the single strands of the molecule. A scheme was drawn based on the experimental observations to explain the phenomenon of the Cr-induced DNA cleavage.