Electrochemical and photochemical electrodes and their use
First Claim
1. A process for preparing an electrode having FAD-dependent redox enzymes immobilized thereon, the process comprising:
- (a) preparing apo-enzymes by treating an FAD-dependent enzyme so as to remove the FAD-cofactor therefrom;
(b) preparing a functionalized FAD by covalent binding to a binding moiety capable of chemical association with, attachment to or a chemical sorption to the surface of the electrode;
(c) reacting the functionalized FAD with an electrode under conditions such that the modified FAD becomes immobilized onto the electrode through chemical association, attachment or sorption of the binding moiety onto the surface of the electrode;
(d) reacting the electrode obtained in (c) with the apo-enzyme under conditions in which the apo-enzyme combines with the modified FAD to yield functional immobilized enzymes; and
(e) reacting the electrode obtained in (d) with an enzyme substrate in the presence of a diffusional electron mediator.
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Accused Products
Abstract
Electrodes carrying FAD-dependent enzymes on their surface are enclosed. The FAD is modified to include a functional group or moiety which affects the properties of the enzyme. The functional group or moiety can be a binding moiety through which the FAD-enzyme complex is immobilized onto the electrode; it can be an electron mediator group for transferring electrons between the electrode and the FAD; or it can be a photoisomerizable group which can undergo photoisomerization which yields a change in the rate of the electrically induced catalytic activity of the enzyme. The electrodes can be used in electrochemical systems for deforming analytes in liquid medium or for recordal of optical signals.
13 Citations
14 Claims
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1. A process for preparing an electrode having FAD-dependent redox enzymes immobilized thereon, the process comprising:
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(a) preparing apo-enzymes by treating an FAD-dependent enzyme so as to remove the FAD-cofactor therefrom;
(b) preparing a functionalized FAD by covalent binding to a binding moiety capable of chemical association with, attachment to or a chemical sorption to the surface of the electrode;
(c) reacting the functionalized FAD with an electrode under conditions such that the modified FAD becomes immobilized onto the electrode through chemical association, attachment or sorption of the binding moiety onto the surface of the electrode;
(d) reacting the electrode obtained in (c) with the apo-enzyme under conditions in which the apo-enzyme combines with the modified FAD to yield functional immobilized enzymes; and
(e) reacting the electrode obtained in (d) with an enzyme substrate in the presence of a diffusional electron mediator.
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2. A process for preparing an electrode having FAD-dependent redox enzymes immobilized thereon, the process comprising:
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(a) treating an electrode to obtain a monolayer comprising an electron mediator group, the electron mediator group having a binding moiety which is capable of chemical association with, attachment to or chemical sorption to the surface of the electrode, the treatment comprising binding of the binding moiety onto the surface of the electrode;
(b) reacting the electrode obtained in (a) with an FAD such that the FAD becomes immobilized onto the electrode through chemical attachment to the electron mediator group;
(c) reacting the electrode obtained in (b) with apo-enzyme of an FAD-dependent enzyme under conditions in which the apo-enzyme combines with the FAD component of the immobilized FAD.
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3. A process for preparing electrodes comprising immobilized enzymes with an associated photoisomerizable group which can change its isomerization state by exposure to light at a certain wavelength, thereby changing the rate of electrically induced catalytic activity of the enzyme, the process comprising:
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(a) preparing an apo-enzyme by treating a FAD-dependent enzyme so as to remove the FAD therefrom;
(b) preparing a modified FAD by covalent binding of a group capable of attachment or binding to a photoisomerizable group;
(c) reacting the modified FAD with the photoisomerizable group to yield a photoisomerizable FAD;
(d) combining the apo-enzyme with the photoisomerizable FAD to yield a reconstituted photoisomerizable redox enzyme; and
(e) providing an electrode carrying linking groups immobilized thereon and reacting the reconstituted enzymes with the electrodes such that the enzymes become covalently bound to the linking group.
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4. An electrochemical system for determining the presence of an analyte liquid medium, the system comprising:
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(a) an electrode carrying on its surface FAD-dependent enzymes, the enzymes being capable of catalyzing a redox reaction in which an analyte is converted into a product, the enzymes comprising a functionalized FAD having a binding moiety which is chemically associated with, attached to or chemically sorbed onto the surface of the electrode;
(b) an electron mediator group which can transfer electrons between the surface of the electrode and the FAD;
(c) an electrical circuitry for charging the electrode and measuring the electrical response. - View Dependent Claims (5, 6, 7, 8, 9)
(a) providing a system according to claim 4, (b) introducing a sample of said liquid medium into the electrochemical cell of the system;
(c) charging the electrode and measuring the electrical response, a change in the electrical response as compared to an electrical response under the same condition in a control medium which does not comprise the analyte, indicating the presence of the analyte in the system.
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9. A method according to claim 8, wherein the analyte determination is performed in vivo.
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10. An electro chemical system for the recording of optical signals having a first wavelength and electrical transduction of the recorded signals, the system having an electrochemical cell comprising:
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(a) an electrode carrying immobilized FAD-dependent redox enzymes, the enzyme having a functionalized FAD comprising a photoisomerizable group which changes its isomerization state from a first to a second state upon photostimulation of light of the first wavelength, a change in the isomerization state giving rise to a change in the rate of catalytic activity of the redox enzyme;
(b) an electron mediator group which can transfer electrons between the electrode and the FAD;
(c) a substrate for the catalytic activity of the enzyme; and
(d) an electric circuitry for charging the electrode and measuring the electrical response. - View Dependent Claims (11, 12, 13, 14)
(a) providing a system according to claim 10;
(b) exposing the electrode to a light source;
(c) charging the electrode and measuring the electrical response, changing the electrical response indicating exposure to light having said first wavelength.
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Specification