Electronically modulated biological functions of molecular interfaced enzymes and living cells.

Conducting polymer molecular interfaces have been implemented to modulate biological functions of fructose dehydrogenase, pyruvate oxidase and Saccharomyces cerevisiae at the electrode surface by adjustment of electrode potential. The enzyme activity of the polypyrrole-interfaced fructose dehydrogenase was electronically modulated by means of electron transfer between the enzyme and the electrode surface. The enzyme activity of polypyrrole-interfaced pyruvate oxidase was modulated by an electronically driven change of substrate concentration. The gene expression in polypyrrole-interfaced Saccharomyces cerevisiae was electronically induced by a change in the phosphate concentration.

Biosensing of benzene derivatives in the environment by luminescent Escherichia coli.

Sensitive and convenient biosensing of environmental pollutants has been developed by fusing a gene of firefly luciferase to the TOL plasmid. TOL plasmid of Pseudomonas putida encodes a series of enzymes for degradation of benzene and its derivatives. The expression of these enzymes is controlled with the regulating proteins xylR and xylS, whose promoters are activated in the presence of aromatic compounds. The structural gene of firefly luciferase, as a reporter enzyme, was inserted under the control of the promoter of xylS protein, and gene fusion plasmid pTSN316 was constructed. The recombinant Escherichia coli transformed with this plasmid was applied to the environmental biosensing of benzene derivatives. The expression of luciferase was induced in the presence of aromatic compounds and the lower detection limit for m-xylene was 5 microM.

Electrical effects on the proliferation of living HeLa cells cultured on optically transparent electrode surface.

Low d.c. potential application induced changes of cellular morphology and growth of living cells on a potential-controlled electrode. At a potential range higher than +0.7 V (vs. Ag/AgCl), serious electric effects on cell viability, membrane permeability, and cytoskeletal morphology of HeLa cells were observed. On the other hand, at lower than +0.5 V no effect was observed. At the boundary potential range between +0.5 V to +0.7 V, where HeLa cells were cultured on the potential-controlled optically transparent In2O3 electrode (OTE) surface, intriguing effects on HeLa cells appeared. At this potential range, where HeLa cells cultured on a potential-applied OTE, all the cells were alive accompanying morphological change. The morphology of HeLa cells returned to their normal spindle shape, when potential application to the electrode was cut off. At a potential of +0.65 V, cell proliferation ratio of cultured cell on an electrode was about one-fifth of that on a non-controlled electrode. These results suggest that low d.c. electrical effects induce significant change in cellular morphology and function.

Production of the chimeric-binding protein, maltose-binding protein-protein A, by gene fusion.

A fusion protein between maltose-binding protein (MBP) and staphylococcal protein A (SpA) was genetically produced. The gene fusion plasmid, pMALPA2, was constructed by inserting the protein A gene into an expression vector of maltose-binding protein in frame, and was expressed efficiently in Escherichia coli. The resulting fusion protein of molecular mass 65 kDa, retained the activity of both MBP and SpA (binding capability to amylose and immunoglobulin G). This chimeric-binding protein was used as an adhesive molecule for immobilization of antibodies to a solid-phase surface for enzyme immunoassay. An enzyme immunoassay was performed with the fusion protein, and human IgG was determined in the concentration range from 10(-4) to 10(-6) g ml-1.

Hyperproduction of a bifunctional hybrid protein, metapyrocatechase-protein A, by gene fusion.

A hybrid protein between metapyrocatechase and Staphylococcal protein A was produced by recombinant DNA techniques. A plasmid carrying the fusion gene that encodes the hybrid protein was constructed and expressed in E. coli. Over 70% of soluble proteins of the cell extracts was estimated to be the hybrid protein. This fusion protein is about 65,000. Both the IgG-binding activity of protein A and the metapyrocatechase activity were found in the hybrid protein. The optimum pH of metapyrocatechase in the fusion protein was at around 6.5 and Km was 1.3 X 10(-5) M. A simple immuno-enzymometric assay was developed for anti-BSA antibody using the fusion protein.

Enzymatic synthesis of polyaniline film using a copper-containing oxidoreductase: bilirubin oxidase.

Electroactive polyaniline films have been synthesized by using a copper-containing oxidoreductase, bilirubin oxidase (BOD). Enzymatic polymerization took place on the surface of BOD-adsorbed solid matrix which was in contact with a buffer solution containing aniline. Optimum conditions for enzymatic polymerization of aniline were investigated. Elemental analysis and IR spectroscopy indicated that the enzymatically synthesized film was polyaniline. The cyclic voltammetric studies demonstrated that the polyaniline film was electrochemically reversible in the redox properties in acidic aqueous solutions. Since the film retained enzymatic activity of BOD which was employed as a catalyst for polymerization, enzymatic polymerization seems promising in preparation of immobilized enzyme membranes.

Electrically controlled proliferation of human carcinoma cells cultured on the surface of an electrode.

Human carcinoma cells, MKN45, were cultured on the surface of a metal-coated plastic plate electrode the potential of which was controlled. The proliferation rate and cell morphology were altered depending on the applied potential. Cell proliferation was halted in the potential range above 0.4 V vs. Ag/AgCl, although cells started to proliferate again when the applied potential was shifted from 0.4 V to 0.1 V vs. Ag/AgCl. Fluorescence probe studies indicated that the fluidity of plasma membrane decreased in association with halting of cell proliferation. These results suggest that electrical stimulation causes cells to temporarily halt proliferation, and that cell proliferation was reversibly controlled by electrode potential. The mechanism is interpreted in relation to the change of plasma membrane structure represented by membrane fluidity.

Delayed luminescence of luminol initiated by a membrane-bound peroxidase.

The luminescense of the luminol-H2O2 system was initiated by either free or membrane-bound horseradish peroxideae (HRP). The instantaneous luminescene decayed rapidly and was followed by the delayed luminescence in the presence of excess luminol. The delayed luminescence was characterized by a chain reaction, in which luminescence intensity increased exponentially. Membrane-bound HRP demonstrated that the delayed luminescence took place even in the absence of HRP if the instantaneous luminescence was initiated by HRP. A mechanism for the nonenzymatic luminescence is proposed and discussed.

Stabilization and translation of immobilized mRNA on latex beads for cell-free protein synthesis system.

The stability of immobilized mRNA against ribonucleases was investigated in a cell-free protein synthesis system. The plasmid-encoding protein A with the 20-mer poly(A) tail under the control of T7 promoter was constructed, and the corresponding mRNA was synthesized by T7 RNA polymerase reaction. The resulting mRNA was immobilized on oligo(dT)-immobilized latex beads by hybridization utilizing the poly(A) tail of mRNA at the 3'-terminus. The mRNA was stabilized against three types of nucleases (3'-OH exonuclease, 5'-OH exonuclease, and endonuclease) by immobilization. Translation of immobilized mRNA with a continuous-flow cell-free protein-synthesizing system from Saccharomyces cerevisiae was ascertained. Reusability of the immobilized mRNA as genetic information was also examined.

Translation of immobilized genetic information by yeast cell-free protein synthesizing system.

A cell-free protein-synthesizing system for the purpose of specific genetic information translation was constructed: ribosome, t-RNA, and enzymes extracted from yeast cells were combined with an immobilized mRNA. The soluble fraction mixed with energy sources and amino acids was incubated with the immobilized mRNA such as poly(U), yeast mRNA, and myeloma mRNA to incorporate [(3)H]phenylalanine into polypeptides of de novo synthesis. By supplying amino acids to these protein-synthesizing systems, amino acid incorporation was ascertained. Lower efficiency of the incorporation in the immobilized system than that of the homogeneous system was mainly attributed to the heterogeneous reaction where the mass-transfer process is diffusion limited. Results obtained show the possibility of a system for specific translation of a desired genetic code.

Electrochemical luminescence-based homogeneous immunoassay.

Aromatic hydrocarbon such as pyrene capable of generating electrochemical luminescence was employed as a label of immunoassay. Pyrene labeled antigen generated luminescence upon electrolytic reduction, while the luminescence decreased remarkably in the presence of antibody. The labeled antigen (constant) and free antigen were competitively reacted to the constant amount of antibody. The luminescence was correlated to the antigen concentration as little as 10(-6)M antigen. The proposed method is a very unique immunochemical technique which requires no BF separation.

Multilabeling of ferrocenes to a glucose oxidase-digoxin conjugate for the development of a homogeneous electroenzymatic immunoassay.

A new homogeneous electroenzymatic immunoassay was developed to determine antibody concentration using glucose oxidase and ferrocene as enzymatic and electrochemical amplifier, respectively. Digoxin (Dig)-conjugated glucose oxidase (GOx) was modified with ferrocene (Fec) to form Fec-GOx-Dig conjugate. After immunocomplex formation between the Fec-GOx-Dig conjugate and the anti-digoxin antibody, the complex underwent less electrochemical reaction due to the steric hindrance of the antibody. The ferrocene multilabeled conjugate was provided for the determination of anti-digoxin antibody. Since the strategy taken here is based on a combined effect of GOx and ferrocene, i.e., enzymatic amplification by GOx and electrochemical amplification by multilabeled ferrocenes, the antibody concentration was determined in the range from 1/50 to 1/500 dilution.

Electrically regulated cellular morphological and cytoskeletal changes on an optically transparent electrode.

Electrically regulated morphological and cytoskeletal changes of HeLa cells were studied on an optically transparent electrode (OTE), on which potential-applied surface cellular behavior and morphogenesis were easily observed. Upon application of a potential, HeLa cells in an OTE exhibited remarkable morphological changes above +0.5 V (vs. Ag/AgCl) and below 0 V. At each potential in this potential range, change in F-actin distribution was observed using a fluorescent probe (rhodamine phalloidin). These results suggest that an electrical field induces a subcellular cytoskeletal change. Electrostimulation of cells with OTE can be a valuable strategy for the manipulation of cultured human cells.

Ca(2+)-responsive extensible monolayer membrane of calmodulin-albumin conjugate.

A Ca(2+)-responsive monolayer protein membrane was prepared by developing calmodulin and bovine serum albumin at the air-water interface and by conjugating them with a bifunctional agent. In the case of the BSA monolayer, complex formation with Mg2+ generated a larger change in surface pressure than that with Ca2+. On the other hand, a drastic change in surface pressure was observed for the conjugated thin membrane associated with Ca2+ than that associated with Mg2+. Due to a drastic change in the conformation of calmodulin, the conjugated protein film changes its morphology (STM image), depending on Ca2+ concentration: the extended structure in the presence of Ca2+ transforms to a shrinked structure in the absence of Ca2+. The largest surface pressure change was detected when calmodulin was mixed with an equimolar amount of bovine serum albumin.

Stimulation of acid phosphatase induction in Saccharomyces cerevisiae by electrochemical modulation of effector concentration.

A novel modulating method of the expression of Saccharomyces PHO 5 gene, responsible for acid phosphatase (APase), is proposed. The method is based on electrochemical modulation of an effector (inorganic phosphate) concentration, as the gene expression is initiated below a threshold concentration of phosphate and is terminated above the threshold value. By positioning the yeast in the close neighborhood of a conducting polymer, the authors show the effectiveness of the electrochemical approach toward PHO 5 induction. Based on the approach, phosphate concentration is easily modulated at the boundary concentration by taking advantage of anion doping-undoping at a conducting polymer and the resulting anion localization-delocalization in the polymer, as the local enrichment of phosphate in the polymer results in the lowering of phosphate in the vicinity of polypyrrole. External phosphate concentration is thus electrochemically modulated when the conducting polymer is positioned in the close neighborhood of the yeast cells; thereby the PHO gene is induced. Here an electrochemical approach for the APase expression as a strategy of selective induction of specific genetic information is described. (c) 1993 John Wiley & Sons, Inc.

Luminescence biosensors.

A novel optical biosensor for homogeneous immunoassay has been developed on the basis of the finding that electrochemical luminescence of pyrene-labelled antigen is extremely inhibited by immunochemical complexation. Electrochemical luminescence homogeneous immunoassay for human serum albumin (HSA), as a model analyte, was performed with a platinum plate electrode which was located in the vicinity of an optical fibre tip. HSA was determined in the concentration range of 3-25 X 10(-6) mol/l. To improve electrochemical luminescence measurement an optical fibre electrode has been developed by fabricating a transparent platinum film on the top of an optical fibre. The minimum detectable limit of luminol was 10(-11) mol/l with the optical fibre electrode. Luminol was applied as a label for homogeneous immunoassay.

Bioluminescent immunoassay with a protein A-luciferase fusion protein.

Protein A and firefly luciferase were genetically fused and the resulting fusion protein was applied to a bioluminescent immunoassay. The gene fusion plasmid, pMALU2, was constructed by inserting the structural gene of luciferase into a protein A expression vector, and was expressed in Escherichia coli. The resulting fusion protein of molecular weight 91 kDa retained not only the enzymatic activity of luciferase but also the binding capability of protein A to the Fc region of immunoglobulin G (IgG). The bioluminescent immunoassay was performed with the fusion protein and human IgG was determined in the concentration range from 10(-3) to 10(-7) g/ml.

Application of a fusion protein, metapyrocatechase/protein A, to an enzyme immunoassay.

A fusion protein of metapyrocatechase and protein A was genetically produced for demonstration of effective conjugation of an enzyme with a binding protein employed in enzyme immunoassay. Plasmid pMPRA3, constructed by inserting the protein A gene into a plasmid pMK12 vector derived directly from the structural gene of metapyrocatechase, was expressed in Escherichia coli. The resulting fusion protein was shown to have promising properties for use in enzyme immunoassays due to the specific binding of the protein A moiety to the Fc portion of immunoglobulin G and to the high amplification of enzyme. Bovine serum albumin, a model antigen, was successfully determined in the concentration range from 1 x 10(-3) to 1 x 10(-7) g/ml.

Electrically promoted protein production by mammalian cells cultured on the electrode surface.

Protein production of mammalian cells has been promoted by applying a small constant potential to the surface of an electrode on which cells are cultured. Human carcinoma line of MKN45 cells were cultured on the surface of a platinum-coated plastic plate electrode. Low d.c. voltage of constant potential was applied to the electrode during 4-day culture to modulate the production of carcinoembryonic antigen (CEA). The amounts of both secreted and membrane-bound CEA were dependent on the applied potential during culture. Secreted CEA was more than twice in amount in the potential range from 0.2 V to 0.6 V vs. Ag/Agcl as compared with that of normal culture. In the potential range, CEA was also increased in membrane-bound form. The potential-controlled cell culture may have an enhanced effect on protein production.