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WO2006084276A2 - Synthese de nanoparticules metalliques catalysee par uneenzyme - Google Patents

Synthese de nanoparticules metalliques catalysee par uneenzyme Download PDF

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Publication number
WO2006084276A2
WO2006084276A2 PCT/US2006/004307 US2006004307W WO2006084276A2 WO 2006084276 A2 WO2006084276 A2 WO 2006084276A2 US 2006004307 W US2006004307 W US 2006004307W WO 2006084276 A2 WO2006084276 A2 WO 2006084276A2
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enzyme
nadph
nanoparticle
gold
fad
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WO2006084276A3 (fr
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Daniel M. Scott
Michael D. Toney
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University of California Berkeley
University of California San Diego UCSD
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University of California San Diego UCSD
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/04—X-ray contrast preparations
    • A61K49/0409—Physical forms of mixtures of two different X-ray contrast-enhancing agents, containing at least one X-ray contrast-enhancing agent which is not a halogenated organic compound
    • A61K49/0414—Particles, beads, capsules or spheres
    • A61K49/0423—Nanoparticles, nanobeads, nanospheres, nanocapsules, i.e. having a size or diameter smaller than 1 micrometer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B22—CASTING; POWDER METALLURGY
    • B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
    • B22F9/00—Making metallic powder or suspensions thereof
    • B22F9/16—Making metallic powder or suspensions thereof using chemical processes
    • B22F9/18—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds
    • B22F9/24—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds starting from liquid metal compounds, e.g. solutions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B22—CASTING; POWDER METALLURGY
    • B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
    • B22F2998/00—Supplementary information concerning processes or compositions relating to powder metallurgy

Definitions

  • Nanotechnology and biotechnology are two fields of ever-increasing importance that are beginning to merge.
  • the former is focused largely on the synthesis and properties of nanometer-scale structures, while the latter largely exploits the extraordinary properties of biological molecules to solve significant medical, chemical, and engineering problems.
  • Applications in which these two fields overlap include quantum dots in optical imaging of biological samples, biosensor applications, and gold nanoparticles in immunochemistry (J. L. West et al, Annu Rev BiomedEng, 5:285 (2003)), as well as the use of gold nanoparticles to facilitate electron transfer between enzymes and electrodes (Y. Xiao et al, Science, 299:1877 (Mar 21, 2003)).
  • biotechnology has historically been the beneficiary of nanotechnology.
  • a role reversal is described in which biotechnology is applied to the furtherance of nanotechnology, with far-reaching implications for nanoscience.
  • GR is an NADPH-dependent flavoenzyme of known structure (P. R. E. Mittl et al, Protein Science, 3:799 (1994)) that normally catalyzes the reduction of oxidized glutathione via a disulfide exchange reaction involving two active site cysteine residues (Cys42 and Cys47; Figure IA) (P. Rietveld et al, Biochemistry, 33:13888 (1994)).
  • the sulfhydryl groups on the catalytic cysteines provide a good binding site for soft metal ions such as Au 3+ and Pt 4+ . This would poise them for reduction by NADPH via flavin-mediated electron transfer (analogous to the reaction catalyzed by the homologous enzyme mercuric ion reductase), potentially giving rise to nanoparticle formation in the active site. Surprisingly, this invention meets this and other needs.
  • the present invention provides a method of preparing a nanoparticle, comprising the step of providing at least one redox-active enzyme-coenzyme complex.
  • the method further comprises the step of contacting the enzyme-coenzyme complex with a first metal ion and an electron-donor such that the enzyme-coenzyme complex repetitively catalyzes the reduction of the metal ion thereby preparing the nanoparticle bound to the enzyme-coenzyme complex via repetitive metal ion reduction by the enzyme-coenzyme complex.
  • the method also comprises the step of collecting the nanoparticle.
  • the method of the present invention further comprises the step of repeating the contacting step with a second metal ion.
  • the enzyme-coenzyme complex is a member of the pyridine nucleotide-disulfide oxidoreductase family of enzymes.
  • the enzyme-coenzyme complex is a member selected from the group consisting of thioredoxin reductase, lipoamide dehydrogenase, trypanothione reductase, coenzyme A disulfide reductase, alkyl hydroperoxide reductase, NADH oxidase, NADH peroxidase, mercuric ion reductase and glutathione reductase.
  • the enzyme-coenzyme complex is glutathione reductase.
  • the coenzyme of the enzyme-coenzyme complex is flavin adenine dinucleotide.
  • the first metal ion is a member selected from the group consisting of Au (III), Pt (IV), Co (II), Ni (II), Fe (III), Ag (I) and Cd (II).
  • the first metal ion is Au (III).
  • the first metal ion is Pt(IV).
  • the electron-donor is nicotinamide adenine dinucleotide (phosphate).
  • the collecting step comprises releasing said nanoparticle from said enzyme-coenzyme complex using a sulfhydryl-containing reagent.
  • the second metal ion is a member selected from the group consisting of Pt (IV), Co (II), Ni (II) and Fe (III).
  • the enzyme-coenzyme complex is attached to a solid support selected from the group consisting of agarose, cyanogen bromide-activated agarose, Sephadex®, cellulose, dextran, starch, chitin, glass, polystyrene, polyacrylamide, polyethyleneglycol, polyethyleneimine, graphite, silica, silica gel and hydroxyapatite (calcium phosphate).
  • a solid support selected from the group consisting of agarose, cyanogen bromide-activated agarose, Sephadex®, cellulose, dextran, starch, chitin, glass, polystyrene, polyacrylamide, polyethyleneglycol, polyethyleneimine, graphite, silica, silica gel and hydroxyapatite (calcium phosphate).
  • the present invention provides an electron transfer system comprising the following elements in electrical connection: an electron donor or acceptor of the present invention, a redox-active enzyme-coenzyme complex of the present invention, a nanoparticle of the present invention, and an electrode, wherein the metal nanoparticle is grown from the enzyme-coenzyme complex via repetitive metal ion reduction by the enzyme- coenzyme complex.
  • the enzyme-coenzyme complex and the nanoparticle elements of the electron transfer system are electrically connected to said electrode via a nanotube or nanowire.
  • the nanotube is a single- walled carbon nanotube.
  • the nanowire is a LiMo 3 Se 3 nanowire.
  • the present invention provides a method of generating an electrical current using the electron transfer system of the present invention.
  • FIG. 1 The cognate reaction catalyzed by GR, reduction of oxidized glutathione.
  • B Schematic of nanoparticle synthesis catalyzed by GR.
  • FIG. 1 Absorbance spectra of FAD bound to GR.
  • the oxidized form of the enzyme is shown in blue, the reduced form obtained by NADPH addition to the oxidized form is shown in red, and the reduced enzyme to which excess AuCl 4 was added is shown in green.
  • the addition OfAuCl 4 " reoxidizes the reduced enzyme, demonstrating electron transfer from NADPH to AuCl 4 , mediated by the GR-bound FAD.
  • the inset shows the initial rates of oxidation of NADPH or NADH in the presence and absence OfAuCl 4 .
  • the lower rate of NADH oxidation is consistent with the known preference of GR for NADPH (N. S. Scrutton et al, Nature, 343:38 (Jan 4, 1990)).
  • FIG. 1 (A) MALDI-TOF mass spectra of GR reacted with excess NADPH and various stoichiometrics OfAuCl 4 .
  • the higher m/z peaks are for the singly charged dimeric forms of the GR holoenzyme with bound gold, while the lower m/z peaks are for the doubly charged forms.
  • the inset shows the linear correlation between the number of gold atoms added per enzyme active site and the number found by mass spectrometry.
  • FIG. 1 Schematic diagram of the use of resin-immobilized GR to synthesize nanoparticles in a batchwise process. The enzyme was attached to cyanogen bromide activated agarose. NADPH and AuCl 4 " were added, followed by mercaptoethylamine to release the GR-bound AuNPs.
  • B Transmission electron micrograph of the AuNPs released from the resin-bound GR by mercaptoethylamine.
  • Figure 7 Energy dispersive x-ray spectroscopy showing resin-bound metal to be gold.
  • Figure 9 Cyclic voltammetry showing the pH dependence of the redox potential of graphite-bound GR-Au50.
  • FIG. 10 Redox potential vs. pH data for the same E. coli GR in solution, taken from a literature paper (Veine DM, Arscott LD, Williams CH Jr. "Redox potentials for yeast, Escherichia coli and human glutathione reductase relative to the NAD+/NADH redox couple: enzyme forms active in catalysis.” Biochemistry (1998) 37, 15575-82).
  • nanoparticle refers to a defined particle of typically 5 to 500 atoms. Typical dimensions of the nanoparticles of the present invention are on the scale of a few nanometers, and can be tens of nanometers. The nanoparticles of the present invention typically have dimensions of less than 100 nanometers.
  • redox-active enzyme-coenzyme complex refers to an enzyme-coenzyme complex that is capable of participating in a reduction-oxidation reaction.
  • Enzyme-coenzyme complexes useful in the present invention include those that can be oxidized as well as those that can be reduced.
  • the enzyme-coenzyme complexes of the present invention can be catalytic, and include, but are not limited to, the pyridine nucleotide- disulfide oxidoreductase family of enzymes.
  • pyridine nucleotide-disulfide oxidoreductase family of enzymes include, but are not limited to, thioredoxin reductase, lipoamide dehydrogenase, trypanothione reductase, coenzyme A disulfide reductase, alkyl hydroperoxide reductase, NADH oxidase, NADH peroxidase, mercuric ion reductase and glutathione reductase.
  • coenzymes refers to a non-proteinaceous organic or inorganic component that functions by mediating transfer of an electron from the donor to the metal ions thereby reducing the metal ions.
  • the coenzymes of the present invention include, but are not limited to, flavin adenine dinucleotide (FAD).
  • metal ion refers to elements of the periodic table that are metallic and that are negatively or positively charged as a result of having more or fewer electrons in the valence shell than is present for the neutral metallic element.
  • Metals that are useful in the present invention include the earth metals, alkali earth metals, transition metals and post-transition metals.
  • Alkali metals include Li, Na, K, Rb and Cs.
  • Alkaline earth metals include Be, Mg, Ca, Sr and Ba.
  • Transition metals include Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, La, Hf, Ta, W, Re, Os, Ir, Pt, Au, Hg and Ac.
  • Post-transition metals include Al, Ga, In, Tl, Ge, Sn, Pb, Sb, Bi, and Po.
  • Metal ions useful in the present invention include, but are not limited to, Au (III), Pt (IV), Co (II), Ni (II), Fe (III), Ag (I) and Cd (II), Pd (II), Pb (II), Ru (IV), Cr(VI), Mn (VII), Zn (II), Os (IV), Ir (IV), Mo (VI), Cu (II) and Rh (III).
  • the term "electron-donor” refers to a species that is capable of donating an electron to an electron-acceptor in a biological system.
  • electron-donor includes, but is not limited to, the reduced form of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H).
  • NAD(P)H nicotinamide adenine dinucleotide
  • the nicotinamide adenine dinucleotide (phosphate) is in the oxidized form (NAD(P) + ), operating as an electron- acceptor.
  • sulfhydryl-containing reagent refers to a reagent containing a thiol group.
  • exemplary sulfhydryl-containing reagents include, but are not limited to 2-aminoethanethiol and 2-mercaptoethanol.
  • the term “collecting” refers to displacing the nanoparticle from the enzyme-coenzyme complex and concentrating the nanoparticles for analysis.
  • the term “electrically connected” refers to elements that are connected so as to allow the free flow of electrons from one element to another.
  • nanotube refers to a nanometer scale cylindrical structure that is hollow in the center and has one or more walls.
  • Exemplary nanotubes useful in the present invention include, but are not limited to, single-walled carbon nanotubes and multi- walled carbon nanotubes. One of skill in the art will appreciate that other nanotubes are useful in the present invention.
  • nanowire refers to a nanometer scale wire that can be prepared from a variety of materials including, but not limited to, metals, semiconductors, inorganics, or organic materials. Exemplary materials include, but are not limited to, LiMo 3 Se 3 . One of skill in the art will appreciate that other materials are useful as the nanowires of the present invention.
  • Enzyme-coenzyme complexes useful in the present invention include those that can be oxidized as well as those that can be reduced.
  • the enzyme-coenzyme complexes of the present invention can be catalytic, and include, but are not limited to, the pyridine nucleotide- disulfide oxidoreductase family of enzymes.
  • Examples of the pyridine nucleotide-disulfide oxidoreductase family of enzymes include, but are not limited to, thioredoxin reductase, lipoamide dehydrogenase, trypanothione reductase, coenzyme A disulfide reductase, alkyl hydroperoxide reductase, NADH oxidase, NADH peroxidase, mercuric ion reductase and glutathione reductase.
  • the enzymes include glutathione reductase, thioredoxin reductase, and lipoamide dehydrogenase.
  • the enzyme is glutathione reductase (GR).
  • Coenzymes useful in the present invention include non-proteinaceous organic or inorganic components that functions by mediating transfer of an electron from the donor to the metal ions thereby reducing the metal ions.
  • the coenzymes of the present invention include, but are not limited to, flavin adenine dinucleotide (FAD).
  • FAD flavin adenine dinucleotide
  • Metals useful in the present invention include elements of the periodic table that are metallic and that are negatively or positively charged as a result of having more or fewer electrons in the valence shell than is present for the neutral metallic element.
  • Metals that are useful in the present invention include the earth metals, alkali earth metals, transition metals and post-transition metals.
  • Alkali metals include Li, Na, K, Rb and Cs.
  • Alkaline earth metals include Be, Mg, Ca, Sr and Ba.
  • Transition metals include Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, La, Hf, Ta, W, Re, Os, Ir, Pt, Au, Hg and Ac.
  • Post-transition metals include Al, Ga, In, Tl, Ge, Sn, Pb, Sb, Bi, and Po.
  • Metal ions useful in the present invention include, but are not limited to, Au (III), Pt (IV), Co (II), Ni (II), Fe (III), Ag (I) and Cd (II), Pd (II), Pb (II), Ru (IV), Cr(VI), Mn (VII), Zn (II), Os (IV), Ir (IV), Mo (VI), Cu (II) and RIi (III).
  • the first metal ion is a member selected from the group consisting of Au, Pt, Co, Ni, Fe, Ag and Cd. In other embodiments, the first metal ion is a member selected from the group consisting of Au (III), Pt (IV), Co (II), Ni (II), Fe (III), Ag (I) and Cd (II). In another embodiment, the first metal ion is Au (III). In still another embodiment, the first metal ion is Pt(IV).
  • the second metal ion is a member selected from the group consisting of Au, Pt, Co, Ni, Fe, Ag and Cd. In other embodiments, the second metal ion is a member selected from the group consisting of Pt (IV), Co (II), Ni (II) and Fe (III).
  • Combinations of metals useful in the present invention include, but are not limited to, Au and Pt, Au and Ni, Au and Co, Au and Fe, Pt and Ni, Pt and Co, and Pt and Fe. Combinations of three or more metals are also useful in the present invention.
  • core-shell structures result from syntheses in which two different metal ions are initially mixed with reductant. This is due to the relative order of the redox potentials, with the metal of higher potential being deposited first, forming the core, followed by the metal of lower potential depositing to form the shell (M. C. Daniel et al, Chern Rev, 104:293 (Jan, 2004)).
  • the maintenance of the nanoparticle at a negative potential in the active site of GR potentially allows one to circumvent metal deposition based strictly on redox ordering. For example, one could initially deposit a metal of lower potential followed by a metal of higher potential, without any associated oxidation of the first metal deposited (M. C. Daniel et al, Chem Rev, 104:293 (Jan, 2004), Y. Sun et al, Science, 298:2176 (Dec 13, 2002)), since the FAD supplies low potential electrons that are used preferentially in metal reduction.
  • the GR catalyzed synthesis of metallic nanoparticles enables the preparation of nanoparticle structures that are inaccessible by solution techniques, due to the asymmetry of the enzyme structure.
  • the catalytic apparatus rests at the bottom of a cavity (-15x17x13 A) in the protein (P. R. E. Mittl et al, Protein Science, 3:799 (1994)).
  • Nanoparticle nucleation happens at the active site cysteines and growth can only occur outward (toward solvent) from this locus due to the steric bulk of the protein matrix.
  • Mixed-metal nanoparticles are preferred to be layered, rather than the symmetric core-shell structures that are commonly formed in solution reactions.
  • Electron donors useful in the present invention include, but are not limited to, nicotinamide adenine dinucleotide (phosphate).
  • phosphate nicotinamide adenine dinucleotide
  • a variety of solid supports are useful in the present invention including, but not limited to, agarose, cyanogen bromide-activated agarose, Sephadex®, cellulose, dextran, starch, chitin, glass, polystyrene, polyacrylamide, polyethyleneglycol, polyethyleneimine, graphite, silica, silica gel and hydroxyapatite (calcium phosphate).
  • the solid support is agarose.
  • the solid support is cyanogen bromide- activated agarose.
  • One of skill in the art will appreciate that other solid support materials are also useful in the present invention.
  • Nanotubes useful in the present invention include, but are not limited to, carbon nanotubes.
  • SWCNTs single-walled carbon nanotubes
  • multi- walled carbon nanotubes are useful in the present invention.
  • Nanotubes of other material are also useful.
  • Nanowires useful in the present invention can be prepared from a variety of materials including LiMo 3 Se 3 .
  • materials including LiMo 3 Se 3 .
  • One of skill in the art will appreciate that other materials can be used for the nanowires of the present invention.
  • the nanoparticles of the present invention can be collected using a variety of techniques known to one of skill in the art. Collection first requires releasing the nanoparticle from the enzyme-coenzyme complex. This can be accomplished by adding a competitive binding agent that displaces the enzyme-coenzyme complex. Following displacement of the enzyme-coenzyme complex, the nanoparticle can then be collected using a variety of techniques known to one of skill in the art, such as filtration or centrifugation.
  • the present invention provides an electron transfer system comprising the following elements in electrical connection: an electron donor or acceptor, a redox-active enzyme- coenzyme complex, a nanoparticle, and an electrode, wherein the metal nanoparticle is grown from the enzyme-coenzyme complex via repetitive metal ion reduction by the enzyme- coenzyme complex.
  • the electron transfer systems of the invention can be used as electrodes (either anode or cathode) in an enzyme-based fuel cell wherein the NAD(P) 4 VNAD(P)H pair acts as an electron shuttling component.
  • the electron transfer systems can also be used for the purpose of sensitive amperometric detection of compounds that are acted on by NAD(P) + dependent dehydrogenases, including but not limited to glucose, lactate, ethanol, amino acids, and the like.
  • Applications of the latter mode of employment include but are not limited to blood glucose monitoring for diabetics, either discontinuously or continuously, depending on the mode of employment. Additionally, it can be used to regenerate NAD(P)H electrochemically, which is frequently used as a reductant in enzyme-catalyzed reactions employed for stereospecific reductions in commercial organic synthetic applications in the pharmaceutical and other industries.
  • the present invention provides that the enzyme-co enzyme complex and the nanoparticle are electrically connected to the electrode via a nanotube or a nanowire.
  • the nanotube can be made of carbon or other materials.
  • the nanotube can have a single wall or have multiple walls.
  • the nanotube is carbon
  • the nanotube can be a single-walled carbon nanotube.
  • the carbon nanotube can also be a multi-walled carbon nanotube.
  • the electrodes of the present invention are prepared from materials recognized by one of skill in the art.
  • the electron transfer system of the present invention provides that the electron donor or acceptor is nicotinamide adenine dinucleotide phosphate.
  • the enzyme-coenzyme complex is a member of the pyridine nucleotide-disulfide oxidoreductase family of enzymes.
  • the enzyme-coenzyme complex is glutathione reductase.
  • the nanoparticle comprises at least one metal selected from the group consisting of Au, Pt, Co, Ni, Fe, Ag and Cd.
  • the enzyme- coenzyme complex and said nanoparticle are electrically connected to said electrode via a nanotube or nanowire.
  • the nanotube is a single-walled carbon nanotube.
  • the nanowire is a LiMo 3 Se 3 nanowire.
  • the present invention also provides a method of generating an electrical current using the electron transfer system.
  • Glutathione reductase purification The gene for E. coli glutathione reductase (a gift from Professor Charles Williams) as subcloned into plasmid pProExb-Hta and overexpressed in E. coli BL21 DE3 gold. The harvested cells were resuspended in 100 mM potassium phosphate buffer pH 7.6, sonicated at 4 0 C for 30 min, and centrifuged at 20,000 g for 40 min. GR was purified from the supernatant using Ni-NTA Superflow resin (Qiagen) via the 6xHis tag added to the GR gene in the pProExb-Hta-GR construct. GR was eluted with 300 mM imidazole, dialyzed into 100 mM potassium phosphate buffer pH 7.6, and stored frozen at -7O 0 C.
  • GR catalyzed reduction of AuCl 4 " by NADPH NADPH (265 ⁇ M) oxidation was monitored in the presence of GR (14 ⁇ M) and AuCl 4 (72 mM), individually and together, to demonstrate that GR catalyzes the NADPH-dependent reduction OfAuCl 4 . This was performed in 66 mM potassium phosphate buffer at pH 7.6 at room temperature. The reaction mixtures also contained 0.05 mg/mL glucose oxidase from Aspergillus niger, 105 mM glucose and 0.02 mg/mL catalase from bovine liver to remove oxygen from the solution. The sample cuvettes were flushed with Argon.
  • the concentration OfAuCl 4 was varied from 0.043 mM to 3 mM in the presence of a constant concentration of GR (0.64 ⁇ M) and NADPH (253 ⁇ M), in 95 mM potassium phosphate buffer pH 7.6 at room temperature.
  • the initial rates of GR catalyzed NADPH oxidation in the presence OfAuCl 4 showed saturation kinetics ( Figure 5).
  • Figure 3 A presents the MALDI-TOF spectra of GR that was incubated for 30 min in the presence of 4.5 mM NADPH and various stoichiometries OfAuCl 4 .
  • the addition of 20 molecules OfAuCl 4 per enzyme in the presence of excess NADPH produces a spectrum ("GRAu 20 " in Figure 3A) that is shifted by the expected mass, assuming all 20 atoms of gold are retained on the enzyme in a metallic cluster.
  • EXAMPLE 2 PREPARATION OF A BIMETALLIC PARTICLE
  • Pt and Au were found to remain on the enzyme through these manipulations while other metals (e.g., Ag, Cd, and Fe) were not observed. NADPH oxidation in the presence of GR and Ag, Cd, Fe, Co, or Ni salts was not observed.
  • metal ions that are incompatible with potassium phosphate buffer were performed in triethanolamine-HCl buffer pH 7.6. Conversely, NADPH oxidation was observed with these metal ions when their addition was initiated with GR to which either Au or Pt was previously added.
  • FIG. 6 shows the MALDI spectra for a GRPtAu complex, produced as follows. First, 46 ⁇ M GR and 4.6 mM NADPH were mixed with 2.5 mM PtCl 6 2" in 46 mM potassium phosphate pH 7.6 at room temperature, and allowed to incubate for 1 hour. Previous results predicted these conditions to generate GRPt 10 . The observed mass increase was equivalent to 11 atoms of platinum. The remainder of the sample was placed over a gel filtration column equilibrated in 100 mM potassium phosphate pH 7.6 to remove unreacted platinum.
  • GR activity assays were performed in 10 - 100 mM potassium phosphate buffer pH 7.6, 0.76 nM GR, 0.25 mM NADPH, and 5.0 mM GSSG at room temperature. NADPH oxidation was followed at 340 nm.
  • a 0.2 ⁇ M solution of preformed 2 nm AuNPs was incubated with different samples of 0.1 ⁇ M GR. Aliquots of the incubations were tested for their ability to reduce oxidized glutathione (GSSG) in the presence of NADPH. GSSG reduction was inhibited 35% after GR was incubated for 10 min with preformed 2 nm AuNPs in the absence of NADPH. When the incubation was performed in the presence of 0.9 mM NADPH, GR lost >98% of its enzymatic activity after 10 min.
  • GSSG oxidized glutathione
  • GR protects the AuNPs from aggregation in the absence of exogenous surface ligands (e.g. amines or thiols).
  • surface ligands e.g. amines or thiols.
  • GRAu 100 without added surface ligand is stable toward aggregation (as detected colorimetrically by surface plasmon resonance) for hours in buffer at room temperature, with aggregation detected colorimetrically only after prolonged incubation.
  • Inhibition of AuNP aggregation is strong evidence that it is sequestered in the deep active site cleft (as opposed to being on a surface binding site), such that collisional encounter with other AuNPs is prevented by the steric bulk of the enzyme.
  • GR was covalently attached to cyanogen bromide-activated agarose according to the supplier's instructions. GR concentrations on the resin were determined by activity assays with GSSG. The preparation OfAu 200 for imaging was accomplished as follows. GR (50 ⁇ L of CB resin containing 230 nmoles of enzyme in 50 mM phosphate buffer pH 7.6) was allowed to reduce 46 ⁇ moles OfAuCl 4 in the presence of NADPH. The concentrations of AuCl 4 and NADPH were maintained at or below 1.1 mM and 2.0 mM, respectively, by adding aliquots of stock solutions in 10 min intervals.
  • the 230 nmols of GR was allowed to reduce 11.5 ⁇ mols OfAuCl 4 every 10 min.
  • the resin was washed with buffer and then soaked in 100 mM 2-aminoethanethiol for 2 hours. The supernatant was filtered from the resin and analyzed by TEM.
  • the electron micrograph confirms the presence of individual gold nanoparticles of ⁇ 2 nm size (in good agreement with the expected 200 atoms of gold per enzyme in this experiment) that were released from the enzyme.
  • the production of metallic nanoparticles of defined size and composition in large quantities is therefore a matter of scaling up this aqueous, green chemistry solid phase methodology, with large-scale batch nanoparticle growth and elution a potentially highly automatable process.
  • EXAMPLE 5 GENERATION OF AN ELECTRIC CURRENT [0081] Cyclic voltarnrnetry measurements were performed with a Perkin Elmer 263A Potentiostat using an EG&G micro-cell kit model K0264. Working electrodes were 1 x 1.5 cm Toray graphite TGP-060 donated by Ballard Power Systems.
  • the GRAu 50 complex was examined by cyclic voltammetry (CV) to determine if electron transfer between GR-bound FAD and a graphite electrode is facilitated by the presence of the AuNP. This was done by allowing a 244 ⁇ L sample of 50 ⁇ M GR, 4.6 mM NADPH, 56 mM potassium phosphate buffer pH 7.6 and either 0.26 mM or 2.6 mM AuCl 4 " to soak at room temperature for 1 hour onto a 1 x 1.5 cm Toray graphite electrode. Electrodes were then rinsed thoroughly before any CV work was done. A gold clip held the electrodes into solution. Measurements were done in 100 mM potassium phosphate buffer pH 7.6 using a saturated calomel electrode as a reference. Data are reported versus the standard hydrogen electrode (SHE). Platinum wire was used as a counter electrode. Argon gas was used to deoxygenate the cell. The scan rate for the CV measurements in Figure 8 was lOO mV/sec.
  • Figure 9 presents redox potential vs. pH data for the same E. coli GR in solution, taken from a literature paper (Veine DM, Arscott LD, Williams CH Jr. "Redox potentials for yeast, Escherichia coli and human glutathione reductase relative to the NAD+/NADH redox couple: enzyme forms active in catalysis.” Biochemistry (1998) 37, 15575-82).
  • Figure 11 is a plot of the redox potential vs. pH data for graphite-bound GR-Au50 presented in Figure 9.
  • AuNP to graphite is to use a single-walled carbon nanotube (SWCNT) to connect the AuNP to a macroscopic electrode.
  • SWCNT single-walled carbon nanotube
  • Carbodiimide chemistry has previously been applied to the coupling of proteins to SWCNTs. They are just the right size to couple to lysine residues at the edge of the GR active site, once the SWCNTs have been oxidized with nitric acid to form carboxylic acids at their termini. Again, nanoparticle formation would be performed both before and after SWCNT coupling to test for different effects on electrical connection.
  • LiM ⁇ 3 Se 3 nanowires In a different vein, others have demonstrated that inexpensive, water soluble LiMo 3 Se 3 nanowires can be used as electrical connectors between gold nanoparticles and a macroscopic electrode. The LiMo 3 Se 3 nanowires bind to gold nanoparticles via covalent Au-Se interactions. Highly conductive films of the LiMo 3 Se 3 - AuNP composite can be easily fabricated on silicon, gold or glass substrates by drop coating the corresponding solutions. There are at least two approaches for application of the LiMo 3 Se 3 nanowires.
  • the first approach is to form LiMo 3 Se 3 -AuNP composite with ⁇ 2 nm AuNPs (which have been shown to bind to the GR active site), followed by attachment of GR to the AuNPs in the presence of NADPH. Binding of the GR-AuNP to the nanowires will be determined by electron microscopy and/or atomic force microscopy. This nanocomposite will then be adsorbed to either graphite or gold electrodes to test for electrical connection using standard electrochemical experiments (eg cyclic voltammetry).
  • the second approach is to adsorb the GR-AuNP conjugate to the LiMo 3 Se 3 nanowires in a manner similar to that done previously with simple citrate coated AuNPs.
  • low pH and/or high ionic strength facilitates the adhesion process since charge repulsion between the anion-coated surface of the AuNP and the anionic surface of the MoSe nanowires is a barrier to adhesion.
  • the pH of the GR-AuNP solution can be safely lowered to ⁇ 5 without loss of enzyme activity and high ionic strength can be achieved in two ways. The first will be to use 1 M TEA-HCl as a buffer. At the low pH, this will largely be in the salt form.
  • a second way is to use ammonium sulfate, which is commonly used in the purification of enzymes.
  • the GR-AuNP solution will be added to the LiMo 3 Se 3 nanowire solution and then slowly add ammonium sulfate until the enzyme precipitates as an adduct with the nanowires.
  • a second approach for connecting to a gold surface involves forming a self- assembled monolayer of 1,4-benzenedithiol on the surface followed by addition of GR with an active site bound AuNP.
  • the free sulfhydryls on the monolayer will chemiadsorb to the AuNP in the GR active site, providing a it electron pathway for facile conduction of electrons from FAD to the electrode.
  • a variation on this experiment will be attempted in which the 1,4-benzenedithiol is doped with a small amount of 5-thiopentanoic acid, which will allow covalent attachment of GR via carbodiimide chemistry to strengthen the interaction of the enzyme with the electrode.
  • a method of preparing a nanoparticle comprising: a) providing at least one redox-active enzyme-coenzyme complex; b) contacting said enzyme-coenzyme complex with a first metal ion and an electron- donor such that said enzyme-coenzyme complex repetitively catalyzes the reduction of said metal ion thereby preparing said nanoparticle bound to said enzyme-coenzyme complex via repetitive metal ion reduction by said enzyme- coenzyme complex; and c) collecting said nanoparticle.
  • step d) repeating step b) with a second metal ion.
  • said enzyme-coenzyme complex is a member selected from the group consisting of thioredoxin reductase, lipoamide dehydrogenase, trypanothione reductase, coenzyme A disulfide reductase, alkyl hydroperoxide reductase, NADH oxidase, NADH peroxidase, mercuric ion reductase and glutathione reductase.
  • said first metal ion is a member selected from the group consisting of Au (III), Pt (IV), Co (II), Ni (II), Fe (III), Ag (I) and Cd (II).
  • said collecting step comprises releasing said nanoparticle from said enzyme-coenzynie complex using a sulfhydryl- containing reagent.
  • said second metal ion is a member selected from the group consisting of Pt (IV), Co (II), Ni (II) and Fe (III).
  • An electron transfer system comprising the following elements in electrical connection: an electron donor or acceptor, a redox-active enzyme-coenzyme complex, a nanoparticle, and an electrode, wherein said metal nanoparticle is grown from said enzyme-coenzyme complex via repetitive metal ion reduction by said enzyme-coenzyme complex.
  • nanoparticle comprises at least one metal selected from the group consisting of Au (III), Pt (IV), Co (II), Ni (II), Fe (III), Ag (I) and Cd (II).

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Abstract

L'invention concerne l'enzyme glutathione réductase FAD-dépendante qui permet de catalyser la synthèse NADPH-dépendente de nanoparticules d'or, de platine et de métaux mélangés qui sont fortement liées à un site via les résidus cystéine actifs de redox. Ladite enzyme permet de stabiliser de très petites grappes métalliques (?#126, 5 atomes) et empêche l'agrégation de grappes plus importantes en l'absence de ligands de protection. La juxtaposition des nanoparticules avec le cofacteur FAD via les cystéines de site actif permet de maintenir les nanoparticules à un bas potentiel en présence d'un excès de NADPH, ce qui permet de déposer des métaux en couche indépendamment du potentiel redox et de stabiliser des métaux à bas potentiel destinés à être oxydés dans une solution aqueuse. La synthèse des nanoparticules d'or catalysée par la glutathione réductase en phase solide est démontrée et proposée comme moyen de production à grande échelle d'une variété de structures de nanoparticules uniques.
PCT/US2006/004307 2005-02-04 2006-02-03 Synthese de nanoparticules metalliques catalysee par uneenzyme Ceased WO2006084276A2 (fr)

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WO2007146162A3 (fr) * 2006-06-08 2008-03-13 Univ Pittsburgh Dispositifs, systèmes et procédés de réduction de la concentration d'une entité chimique dans des fluides
CN101349667B (zh) * 2007-07-16 2011-11-23 中国科学院化学研究所 一种生理活性物质的电化学检测装置及其专用电化学传感器和制备方法
EP2315837A4 (fr) * 2008-08-21 2012-07-04 Ca Nat Research Council Production de nanoparticules exemptes de soufre par une levure
CN108642035A (zh) * 2018-05-08 2018-10-12 江苏理工学院 一种硅胶固定gdh催化制备nadph的方法
US10322221B2 (en) 2013-01-18 2019-06-18 University of Pittsburgh—of the Commonwealth System of Higher Education Removal of carbon dioxide via dialysis
WO2020036831A3 (fr) * 2018-08-10 2020-05-28 Colorado State University Research Foundation Marqueur enzymatique réducteur de métaux pour microscopie optique et électronique
CN111205852A (zh) * 2020-01-16 2020-05-29 吉林大学 一种谷胱甘肽保护的强荧光发射的金铂合金纳米簇及其可控制备方法
US10801052B2 (en) 2011-10-03 2020-10-13 Oxford University Innovation Limited Cofactor regeneration system

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US6872645B2 (en) * 2002-04-02 2005-03-29 Nanosys, Inc. Methods of positioning and/or orienting nanostructures

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US8323379B2 (en) 2006-06-08 2012-12-04 University of Pittsburgh—of the Commonwealth System of Higher Education Devices, systems and methods for reducing the concentration of a chemical entity in fluids
US7763097B2 (en) 2006-06-08 2010-07-27 University of Pittsburgh—of the Commonwealth System of Higher Education Devices, systems and methods for reducing the concentration of a chemical entity in fluids
US8043411B2 (en) 2006-06-08 2011-10-25 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Devices, systems and methods for reducing the concentration of a chemical entity in fluids
WO2007146162A3 (fr) * 2006-06-08 2008-03-13 Univ Pittsburgh Dispositifs, systèmes et procédés de réduction de la concentration d'une entité chimique dans des fluides
CN101349667B (zh) * 2007-07-16 2011-11-23 中国科学院化学研究所 一种生理活性物质的电化学检测装置及其专用电化学传感器和制备方法
US8986975B2 (en) 2008-08-21 2015-03-24 National Research Council Of Canada Production of sulfur-free nanoparticles by yeast
EP2315837A4 (fr) * 2008-08-21 2012-07-04 Ca Nat Research Council Production de nanoparticules exemptes de soufre par une levure
US10801052B2 (en) 2011-10-03 2020-10-13 Oxford University Innovation Limited Cofactor regeneration system
US10975408B2 (en) 2011-10-03 2021-04-13 Oxford University Innovation Limited Cofactor regeneration system
US10322221B2 (en) 2013-01-18 2019-06-18 University of Pittsburgh—of the Commonwealth System of Higher Education Removal of carbon dioxide via dialysis
CN108642035A (zh) * 2018-05-08 2018-10-12 江苏理工学院 一种硅胶固定gdh催化制备nadph的方法
WO2020036831A3 (fr) * 2018-08-10 2020-05-28 Colorado State University Research Foundation Marqueur enzymatique réducteur de métaux pour microscopie optique et électronique
US12339286B2 (en) 2018-08-10 2025-06-24 Colorado State University Research Foundation Metal-reducing enzymatic tag for optical and electron microscopy
CN111205852A (zh) * 2020-01-16 2020-05-29 吉林大学 一种谷胱甘肽保护的强荧光发射的金铂合金纳米簇及其可控制备方法
CN111205852B (zh) * 2020-01-16 2021-07-09 吉林大学 一种谷胱甘肽保护的强荧光发射的金铂合金纳米簇及其可控制备方法

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