Microbial biosensor for detection of methyl parathion using screen printed carbon electrode and cyclic voltammetry

Whole cells of recombinant Escherichia coli were immobilized on the screen printed carbon electrode (SPCE) using glutaraldehyde. Recombinant E. coli was having high periplasmic expression of organophosphorus hydrolase enzyme, which hydrolyzes the methyl parathion into two products, p-nitrophenol and dimethyl thiophosphoric acid. Cells immobilized SPCE was studied under SEM. Cells immobilized SPCE was associated with cyclic voltammetry and cyclic voltammograms were recorded before and after hydrolysis of methyl parathion. Detection was calibrated based on the relationship between the changes in the current observed at +0.1 V potential, because of redox behavior of the hydrolyzed product p-nitrophenol. As concentration of methyl parathion was increased the oxidation current also increased. Only 20 μl volume of the sample was required for analysis. Detection range of biosensor was calibrated between 2 and 80 μM of methyl parathion from the linear range of calibration plot. A single immobilized SPCE was reused for 32 reactions with retention of 80% of its initial enzyme activity.     

Novel immunoassay for carcinoembryonic antigen based on protein A-conjugated immunosensor chip by surface plasmon resonance and cyclic voltammetry

In this study, an immunosensor chip utilizing surface plasmon resonance (SPR) and cyclic voltammetry (CV) was fabricated for detecting carcinoembryonic antigen (CEA). Specifically, we applied in parallel an SPR instrument and a CV device to monitor the assembly of carcinoembryonic antibody (anti-CEA) on a protein A-conjugated surface and the subsequent ligand reaction. The immunosensor chips were constructed by various concentrations of protein A. To determine the surface characteristics of different self-assembly monolayers (SAMs), several quantitative and kinetic measurements were carried out. The extent of immobilization of anti-CEA and the immune response of anti-CEA antibody against CEA were measured using the SPR instrument and CV device. The terminal functional groups of protein A have different effects on the adsorption and covalent binding of immunoprotein depending on the steric hindrance. Through the parallel measurements, we demonstrate that SPR and CV are sensitive to measure the antigen–antibody binding capacity.     

The effect of proflavine on HeLa cells

1. The effect of proflavine on the metabolism of RNA, DNA and protein of HeLa cells was studied. 2. The synthesis of RNA, DNA and protein was progressively inhibited by concentrations of proflavine up to 43mum. 3. There was no simple relationship between the degrees of inhibition of synthesis of RNA, DNA and protein by increasing concentrations of proflavine: the synthesis of RNA was most readily inhibited, and the synthesis of protein was relatively insensitive. 4. A concentration of 22mum-proflavine inhibited synthesis of RNA and DNA and caused a progressive loss of RNA from both nucleus and cytoplasm without any accompanying loss of DNA or dry weight from the cells. 5. The rapidly labelled RNA in the nucleus was preferentially degraded and was not transferred in a stable form to the cytoplasm.     

Protein-film voltammetry: a theoretical study of the temperature effect using square-wave voltammetry

Square-wave voltammetry of surface redox reactions is considered as an adequate model for a protein-film voltammetric setup. Here we develop a theoretical approach to analyze the effects of temperature on square-wave voltammograms. The performed simulations address the surface redox reactions featuring slow, modest and fast electron transfer. The theoretical calculations show that the temperature affects the square-wave voltammetric responses in a complex way resulting in a variety of peak shapes. Temperature effects on the phenomena known as "quasireversible maximum" and "split SW peaks" are also analyzed. The simulated results can be used to analyze the redox mechanisms and kinetic parameters of electron transfer reactions in protein-film criovoltammetry and other surface-confined redox systems. Our analysis also shows how "abnormal" features present in some square-wave voltammetric studies can easily be misinterpreted by postulating "multiple species", "stable radicals", or additional processes. Finally we provide a simple algorithm to use the "quasireversible maximum" to determine the activation energy of electron transfer reactions by surface redox systems.     

Electrochemical cell-based chip for the detection of toxic effects of bisphenol-A on neuroblastoma cells

A cell-based chip was fabricated for the electrochemical detection of the dose-dependent effects of bisphenol-A (BPA) on neuroblastoma cells (SH-SY5Y), which showed dual-mode correlation as a standard curve. Toxicity assessment of BPA became very important in environmental toxicants detection since BPA can be reached out easily from various common plastic-based product and give negative cellular effects on living organism. Cell chip was fabricated by immobilizing cells on C(RGD)(4) peptide coated electrode to detect the cytotoxicity of BPA electrochemically. Redox properties in living cells were determined by cyclic voltammetry using a home-made three-electrode system, and the cathodic peak current (I(pc)) was used as a parameter for measurement of the effect of BPA on cell viability. The peak current, I(pc) value increased with the concentration of BPA up to 300 nM and then decreased because of the stimulation of cancer cell activity at the concentration of BPA below 300nM and cytotoxicity at the concentration of BPA above 300 nM, respectively. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and optical microscopy-based morphological analysis confirmed the results of electrochemical study. This dual-mode correlation between the concentration of BPA and voltammetric signal intensity should be firstly considered to analyze its dose-dependent stimulus and cytotoxic effects on neuroblastoma cells by cell chip.     

Occurrence of the Crabtree effect in HeLa cells

The occurrence of a Crabtree effect in HeLa cells was detected. Some properties of pyruvate kinase (PK) were also evaluated. Hexose phosphate, triose-phosphate and phosphoenolpyruvate (PEP) significantly decreased the oxygen consumption of digitonin-permeabilized HeLa cells, which were oxidizing succinate. The Crabtree effect promoted by PEP was concentration-dependent and was lowered by an increase of ADP concentration, suggesting a participation of PK. The dependence of fructose-1,6-bisphosphate (FDP) by HeLa cell PK was observed. The PK of HeLa cells was inhibited by L -alanine only in the absence of FDP, while in the presence of the metabolite, an increase in the activity was observed. PK was also inhibited in the presence of L -histidine and L -leucine, while L -serine promoted activation. L -Cysteine and L -phenylalanine also inhibited the PK of HeLa cells. This, together with the sigmoidal character in relation to substrate concentration, suggests the presence of the K-type of PK in HeLa cells. © 1998 John Wiley & Sons, Ltd.     

Cell-based multiwell assays for the detection of substrate accumulation and oxidation

We describe multiwell assays for detecting the accumulation as well as the subsequent oxidation of (14)C-labeled substrates in cultured cells. Accumulation is monitored in real time by an established scintillation proximity assay in which the scintillator is embedded in the plate base primarily detecting cell-associated radiolabel. The substrate oxidation assay is a novel variant of previously described experimental approaches aimed at trapping (14)CO(2) produced by isolated enzymes, organelles, or intact cells. This method uses a standard 96-well tissue culture plate and, on top, an inverted filter plate immersed with NaOH that are clamped into a sandwich sealed with a silicon gasket to obtain gas-tight compartments. (14)CO(2) is captured in the filter and quantified by conventional scintillation. We demonstrate both the accumulation and subsequent oxidation of (14)C-labeled substrates in cultured human myotubes, adipocytes, and hepatocytes. Both methods are adaptable for compound screening; at the same time, these protocols provide easy-to-use and time- saving methods for in vitro studies of cellular fuel handling.     

蛋白激酶C抑制剂staurosporine对HeLa细胞周期的影响

蛋白激酶Ｃ抑制剂ｓｔａｕｒｏｓｐｏｒｉｎｅ对ＨｅＬａ细胞周期的影响石法武＊任洪波张兆山＊＊王会信周廷冲（军事医学科学院基础医学研究所,＊＊生物工程研究所,北京１００８５０ＴｈｅｅｆｅｃｔｏｆｓｔａｕｒｏｓｐｏｒｉｎｅｏｎｔｈｅｃｅｌｃｙｃｌｅｏｆＨｅ...     

Pleiotropic effect of anticapsin on HeLa S3 cells

Anticapsin, the terminal epoxyaminoacid moiety of tetaine, inhibits irreversibly growth of HeLa S3 cells. The antibiotic decreases to a similar extent incorporation of 3H-labelled precursors into nucleic acids and protein in intact cells: inhibition of protein synthesis prevails on prolonged incubation. Also incorporation of [3H]dTTP and [3H]UTP is inhibited in the presence of anticapsin into permeabilized cells. These effects, however, are not due to the interference with DNA or RNA polymerases since anticapsin only slightly suppresses RNA polymerase activity and has no effect on DNA polymerase in the cell-free systems. The results indicate that the mechanism of antiproliferative action of anticapsin in HeLa S3 cells differs from that of tetaine and imply that inhibition of protein synthesis might be the primary effect of anticapsin.     

The use of cyclic voltammetry for the evaluation of antioxidant capacity

Low-molecular weight antioxidants (LMWAs) play a major role in protecting biological systems against reactive oxygen-derived species and reflect the antioxidant capacity of the system. Cyclic voltammetry (CV), shown to be convenient methodology, has been validated for quantitation of the LMWA capacity of blood plasma, tissue homogenates, and plant extracts. Analysis of the CV tracing yields the values of (i) the biological oxidation potential, E and E(1/2), which relate to the nature of the specific molecule(s); (ii) the intensity (Ia) of the anodic current; and (iii) the area of the anodic wave (S). Both Ia and S relate to the concentration of the molecule(s). LMWA components of human plasma and animal tissues were identified and further validated by reconstruction of the CV tracing and by high-performance liquid chromatography-electrochemical detection. To reflect the oxidative stress status, the use of an additional parameter, R, has been proposed. R represents the level (%) of oxidized ascorbate (compared with total ascorbate) and is measured by high-performance liquid chromatography-electrochemical detection. All these parameters were monitored in healthy human subjects as well as in chronic (diabetes mellitus) and acute care patients (subjected to total body irradiation before bone marrow transplantation). The electroanalytical methodologies presented here could be widely employed for rapid evaluation of the status of subjects (in health and disease) for monitoring of their response to treatment and/or nutritional supplementation as well as for screening of specific populations.     

Synthesis of novel spirobibenzopyrans as potent anticancer leads inducing apoptosis in HeLa cells

Spirobibenzopyrans are an unexplored class of therapeutics. We report the anticancer activity of novel spirobibenzopyrans, synthesized by a one-pot reaction and extensively characterized. Structure of one of the spirobibenzopyran has been determined by the single crystal XRD technique. The in vitro anticancer activity of these derivatives across the NCI 60-cell line panel was evaluated and for the first time their mechanism of action against HeLa cells was probed via cell morphology analysis and cell cycle analysis. They were determined to be apoptosis inducers with cell cycle arrest in G₀/G₁ and S phase suggesting CDK-4 protein inhibition and the inhibition of DNA replication. The DNA inhibition was studied and confirmed using the alkaline comet assay for the compound CHX-4MO-SAL showing S phase inhibition. Further, conformity with the in silico Lipinski’s score signify the potential of spirobibenzopyrans as anticancer leads.     

The effect of biotin on cellular functions in HeLa cells

HeLa cells cultured in a biotin-deficient medium show reduced rate of protein synthesis, DNA synthesis and growth. Addition of exogenous biotin to the cells cultured in biotin-deficient medium results in enhanced protein synthesis, DNA synthesis and cell growth. Continuous protein synthesis is required for the increase in DNA synthesis observed upon the addition of exogenous biotin to the cells cultured in biotin-deficient medium. These results suggest that cells cultured in biotin-deficient medium are arrested in the G1 stage of cell cycle and this block is removed upon the addition of biotin to the deficient medium.     

A prototype microfluidic chip using fluorescent yeast for detection of toxic compounds

A microfluidic chip has been developed to enable the screening of chemicals for environmental toxicity. The microfluidic approach offers several advantages over macro-scale systems for toxicity screening, including low cost and flexibility in design. This design flexibility means the chips can be produced with multiple channels or chambers which can be used to screen for different toxic compounds, or the same toxicant at different concentrations. Saccharomyces cerevisiae containing fluorescent markers are ideal candidates for the microfluidic screening system as fluorescence is emitted without the need of additional reagents. Microfluidic chips containing eight multi-parallel channels have been developed to retain yeast within the chip and allow exposure of them to toxic compounds. The recombinant yeast used was GreenScreentrade mark which expresses green fluorescent proteins when is exposed to genotoxins. After exposure of the yeast to target compounds, the fluorescence emission was detected using an inverted microscope. Qualitative and quantitative comparisons of the fluorescent emission were performed. Results indicated that fluorescent intensity per area significantly increases upon exposure to methyl-methanesulfonate, a well known genotoxic compound. The microfluidic approach reported here is an excellent tool for cell-based screening and detection of different toxicities. The device has the potential for use by industrial manufacturers to detect and reduce the production and discharge of toxic compounds, as well as to characterise already polluted environments.