Enzyme-Channeling Based Electrochemical Biosensors
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Abstract
Low-cost, non-toxic and fast immunoassay systems (immunosensors) and uses thereof as analytical and diagnostic tools for detecting an immune response in a subject are disclosed. The systems and methods disclosed are based on recording an electrochemical signal which is generated proportionally to an enzymatic cascade reaction (enzyme-channeling) upon detecting an analyte, and therefore can be used to determine the titer level of an antibody analyte in a liquid sample such as artificial media, serum or blood both qualitatively and quantitatively, in a one-step and separation free immunoassay. Systems and methods based on recording an electrochemical signal which is generated proportionally to an enzymatic cascade reaction (enzyme-channeling) upon detecting an analyte, which utilize a non-toxic secondary substrate such as acetaminophen are also disclosed.
42 Citations
93 Claims
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1-68. -68. (canceled)
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69. A system for detecting an antibody in a liquid sample, the system comprising:
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an electrochemical cell which comprises; a reference electrode; a counter electrode; an electrolytic solution; a current detecting unit; and a working electrode having immobilized thereon an antigen and a first enzyme of an enzymatic cascade; a conjugate which comprises an agent capable of specifically binding to the antibody and a second enzyme of said enzymatic cascade being conjugated to said agent; and a substrate of said first enzyme of said enzymatic cascade; wherein; said antigen is capable of specifically binding to the antibody; and said first enzyme is capable of catalyzing a formation of a substrate of said second enzyme, and further wherein said second enzyme generates an electrochemically detectable moiety upon binding of said conjugate to the antibody and binding of the antibody to the antigen, whereas a presence and/or amount of said electrochemically detectable moiety is detectable by said detecting unit. - View Dependent Claims (70, 71, 72, 73, 74, 80, 81, 82)
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75. A kit for detecting an antibody in a liquid sample, the kit comprising:
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a working electrode having immobilized thereon an antigen and a first enzyme of an enzymatic cascade; wherein; said antigen is capable of specifically binding to the antibody; said first enzyme is capable of catalyzing a formation of a substrate of a second enzyme; and said second enzyme is capable of catalyzing a formation an electrochemically detectable moiety. - View Dependent Claims (76, 77, 78, 79)
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83. A system for detecting a first member of a binding pair in a liquid sample, the system comprising:
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an electrochemical cell which comprises; a reference electrode; a counter electrode; an electrolytic solution; a current detecting unit; and a working electrode having immobilized thereon a second member of the binding pair and a first enzyme of an enzymatic cascade; a conjugate which comprises an agent capable of specifically binding to the first member of the binding pair and a second enzyme of said enzymatic cascade conjugated to said agent; a substrate of said first enzyme of said enzymatic cascade; and a secondary substrate of said second enzyme of said enzymatic cascade, wherein; said first enzyme of said enzymatic cascade is a hydrogen peroxide-producing enzyme; said second enzyme of said enzymatic cascade is a peroxidase; and said secondary substrate is acetaminophen, and further wherein; said second enzyme generates a detectable form of said acetaminophen upon binding of said conjugate to the first member of the binding pair and binding of the first member to said second member of the binding pair, whereas a presence and/or amount of said detectable form of said acetaminophen is detectable by said detecting unit. - View Dependent Claims (84, 85, 86)
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- 87. An electrode for detecting an antibody in a liquid sample, the electrode comprising a body and a surface having immobilized thereon an antigen and a first enzyme of an enzymatic cascade, said antigen is capable of specifically binding to the antibody, said first enzyme is capable of catalyzing a formation of a substrate of a second enzyme in said enzymatic cascade, said second enzyme capable of generating an electrochemically detectable moiety upon binding of a conjugate to the antibody and binding of the antibody to the antigen, whereby said conjugate comprises an agent capable of specifically binding to the antibody and said second enzyme of said enzymatic cascade being conjugated to said agent.
Specification