Methods for generating pH/ionic concentration gradient near electrode surfaces for modulating biomolecular interactions
First Claim
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1. A method comprising:
- a) providing a biosensor comprising a multisite array of test sites, each test site comprising a support in an aqueous solution, the support comprising one or more electrodes or an electromagnet, and a biomolecule interface layer having one or more immobilized probes thereon;
b) adding an electrochemically active agent, an enzyme, an enzyme substrate, a buffer inhibitor, or a combination thereof to the aqueous solution;
c) during a test, independently varying for each of the test sites a degree of biological interaction that can occur with a biomolecule analyte at the respective test site, to obtain a collection of varied signals, wherein the varying includes reacting the electrochemically active agent, the enzyme, the enzyme substrate, or combination thereof in the aqueous solution to produce H+ ion or OH−
ions, or increasing the diffusion of H+ ions or OH−
ions with the buffer inhibitor, or inhibiting the interaction between H+ ions or OH−
ions and buffering salts with the buffer inhibitor; and
d) analyzing the collection of varied signals to obtain a measurement.
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Abstract
Methods of modulating the binding interactions between a (biomolecular) probe or detection agent and an analyte of interest from a biological sample in a biosensor having a multisite array of test sites. In particular, the methods modulate the pH or ionic concentration gradient near the electrodes in such biosensor. The methods of modulating the binding interactions provide a biosensor and analytic methods for more accurately measuring an analyte of interest in a biological sample.
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Citations
35 Claims
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1. A method comprising:
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a) providing a biosensor comprising a multisite array of test sites, each test site comprising a support in an aqueous solution, the support comprising one or more electrodes or an electromagnet, and a biomolecule interface layer having one or more immobilized probes thereon; b) adding an electrochemically active agent, an enzyme, an enzyme substrate, a buffer inhibitor, or a combination thereof to the aqueous solution; c) during a test, independently varying for each of the test sites a degree of biological interaction that can occur with a biomolecule analyte at the respective test site, to obtain a collection of varied signals, wherein the varying includes reacting the electrochemically active agent, the enzyme, the enzyme substrate, or combination thereof in the aqueous solution to produce H+ ion or OH−
ions, or increasing the diffusion of H+ ions or OH−
ions with the buffer inhibitor, or inhibiting the interaction between H+ ions or OH−
ions and buffering salts with the buffer inhibitor; andd) analyzing the collection of varied signals to obtain a measurement. - View Dependent Claims (3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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2. A method of modulating the pH or ionic concentration in a biosensor, the method comprising:
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a) providing a biosensor comprising a multisite array of test sites in which the conditions for interacting with a biomolecule analyte can be varied independently, each test site comprising a support in an aqueous solution, the support comprising one or more electrodes or an electromagnet, and a biomolecule interface layer having one or more immobilized probes thereon; b) adding an electrochemically active agent, an enzyme, an enzyme substrate, a buffer inhibitor, or a combination thereof to the aqueous solution; and c) reacting the electrochemically active agent, the enzyme, the enzyme substrate, or combination thereof in the aqueous solution to produce H+ ion or OH−
ions, or increasing the diffusion of H+ ions or OH−
ions with the buffer inhibitor, or inhibiting the interaction between H+ ions or OH−
ions and buffering salts with the buffer inhibitor;wherein; the aqueous solution comprises a water-miscible organic co-solvent selected from the groups consisting of acetonitrile, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), N,N-dimethyl acetamide (DMAc), and mixtures thereof; the biomolecular interface layer comprises a silane immobilized polyethylene glycol (PEG), and at least a portion of the surface-immobilized PEG comprises terminal functional groups selected from the group consisting N-hydroxysuccinimide (NHS) ester, maleimide, alkynes, azides, streptavidin and biotin; the biosensor comprises a support in an aqueous solution, the support comprising one or more electrodes, and a biomolecule interface layer having one or more immobilized probes and one or more immobilized enzymes, in step b) an enzyme substrate is added to the aqueous solution, and in step c) the enzyme substrate is enzymatically oxidized or reduced;
orthe support comprises an electromagnet and the biomolecule interface layer has one or more immobilized probes, in step b) one or more enzymes immobilized onto magnetic micro- or nano-particles are added prior to or simultaneously with an enzyme substrate, and in step c) the enzyme substrate is enzymatically oxidized or reduced. - View Dependent Claims (4, 16, 17, 18, 19, 20, 21, 22)
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23. A method of modulating the pH or ionic concentration in a biosensor, the method comprising:
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a) providing a biosensor comprising a multisite array of test sites in which the conditions for interacting with a biomolecule analyte can be varied independently, each test site comprising a support in an aqueous solution, the support comprising one or more electrodes or an electromagnet, and a biomolecule interface layer having one or more immobilized probes thereon; b) adding an enzyme to the aqueous solution; and c) reacting the enzyme at the electrode surface. - View Dependent Claims (24)
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25. A method of modulating the pH or ionic concentration in a biosensor, the method comprising:
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a) providing a biosensor comprising a multisite array of test sites in which the conditions for interacting with a biomolecule analyte can be varied independently, each test site comprising a support in an aqueous solution, the support comprising one or more electrodes or an electromagnet, and a biomolecule interface layer having one or more immobilized probes thereon; b) adding a buffer inhibitor to the aqueous solution; and c) reacting the electrochemically active agent, the enzyme, the enzyme substrate, or combination thereof in the aqueous solution to produce H+ ion or OH−
ions, or increasing diffusion of H+ ions or OH−
ions with the buffer inhibitor, or inhibiting the interaction between H+ ions or OH−
ions and buffering salts is inhibited with the buffer inhibitor.
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26. A method of modulating the pH or ionic concentration in a biosensor, the method comprising:
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a) providing a biosensor comprising a multisite array of test sites in which the conditions for interacting with a biomolecule analyte can be varied independently, each test site comprising a support in an aqueous solution, the support comprising one or more electrodes or an electromagnet, and a biomolecule interface layer having one or more immobilized probes thereon, wherein the biomolecule interface layer is covering the one or more electrodes and has a thickness of 5-150 nm; b) adding an electrochemically active agent, an enzyme, an enzyme substrate, a buffer inhibitor, or a combination thereof to the aqueous solution; and c) reacting the electrochemically active agent, the enzyme, the enzyme substrate, or combination thereof in the aqueous solution to produce H+ ion or OH−
ions, or increasing the diffusion of H+ ions or OH−
ions with the buffer inhibitor, or inhibiting the interaction between H+ ions or OH−
ions and buffering salts with the buffer inhibitor.
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27. A biosensor for use in detecting a biomolecule analyte comprising:
a multisite array of test sites, each test site comprising;
a support in an aqueous solution;
whereinthe aqueous solution has added thereto an electrochemically active agent, an enzyme, an enzyme substrate, a buffer inhibitor, or a combination thereof; the support comprises one or more electrodes or an electromagnet, and a biomolecule interface layer having one or more immobilized probes thereon; and the biosensor is configured for; during a test, independently varying for each of the test sites a degree of biological interaction that can occur with a biomolecule analyte at the respective test site, to obtain a collection of varied signals, the varying including reacting the electrochemically active agent, the enzyme, the enzyme substrate, or combination thereof in the aqueous solution to produce H+ ion or OH−
ions, or increasing the diffusion of H+ ions or OH−
ions with the buffer inhibitor, or inhibiting the interaction between H+ ions or OH−
ions and buffering salts with the buffer inhibitor; andanalyzing the collection of varied signals to obtain a measurement. - View Dependent Claims (28, 29, 30, 31, 32)
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33. A method for detecting a biomolecule analyte in a biological sample, the method comprising:
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a) providing a biosensor comprising a multisite array of test sites in which the conditions for interacting with a biomolecule analyte can be varied independently, each test site comprising a support in an aqueous solution, the support comprising one or more electrodes or an electromagnet, and a biomolecule interface layer having one or more immobilized probes thereon; b) adding in each test site an electrochemically active agent, an enzyme, an enzyme substrate, a buffer inhibitor, or a combination thereof to the aqueous solution; c) reacting the electrochemically active agent, the enzyme, the enzyme substrate, or a combination thereof in the aqueous solution to produce H+ ion or OH−
ions, or increasing the diffusion of H+ ions or OH−
ions with a buffer inhibitor, or inhibiting the interaction between H+ ions or OH−
ions and buffering salts with the buffer inhibitor;d) adding a biological sample to each test site; and e) detecting the biomolecule analyte in each test site, wherein the amounts added in step b) and the reaction in step c) are varied between test sites in a subset array of test sites in order to obtain sets of test sites in which the pH or ionic concentration near electrode surfaces in the test sites varies. - View Dependent Claims (34, 35)
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Specification