Methods for measuring an amount of an analyte in a complex sample
A method for detecting an amount of an analyte in a solution includes providing an assay chamber including an electrode positioned at a first end of the assay chamber and a capture molecule attached to the electrode via a linker. A solution including an analyte, a binding partner of the analyte, at least one electrochemically active agent, and a detecting probe having a signaling tag attached thereto may be provided to the assay chamber. An electrical signal may be applied to the electrode to change the pH of the solution in the area near the electrode. The analyte may bind to the capture molecule and to the detecting probe at the first end of the assay chamber at the new pH. A signal produced by the signaling tag at the first end of the assay chamber may be measured to calculate the amount of the analyte in the solution.
1 . A method for detecting an amount of an analyte in a solution comprising:
providing an assay chamber including an electrode positioned at the assay chamber and a capture molecule attached to the electrode via a linker;
providing to the assay chamber a solution including an analyte, a binding partner of the analyte, at least one electrochemically active agent, and a detecting probe having a pH-dependent signaling tag attached thereto, the solution is in contact with the electrode and has a first pH value at which the pH-dependent signaling tag does not produce a signal and the analyte is not bound to the binding partner of the analyte;
applying a first electrical signal to the electrode to change the pH of the solution near the surface of the electrode from the first pH value to a second pH value, while the pH of other areas of the solution remains at the first pH value;
binding the analyte to the capture molecule and to the detecting probe at the first end of the assay chamber; and
measuring a signal produced by the pH-dependent signaling tag at the second pH value near the surface of the electrode, wherein the pH-dependent signaling tag in the other areas of the solution at the first pH value does not produce a signal.
2 . The method of claim 1 , wherein the second pH value is higher than the first pH value.
3 . The method of claim 1 , wherein the analyte is an anti-drug antibody (ADA).
4 . The method of claim 1 , wherein the electrochemically active agent is selected from the group consisting of quinone, catechol, aminophenol, hydrazine, hydroquinone, benzoquinone, naphthoquinone, derivatives thereof, and combinations thereof.
5 . The method of claim 1 , wherein the signaling tag is selected from the group consisting of a fluorescent tag, a fluorescent dye, a fluorescent protein, an electroluminescent dye, a chemiluminescent dye, and an enzyme.
6 . A method for detecting an amount of an analyte in a solution comprising:
providing an assay chamber including an electrode positioned at the assay chamber and a capture molecule attached to the electrode via a linker;
providing to the assay chamber a solution including an analyte, a binding partner of the analyte, at least one electrochemically active agent, and a detecting probe having a pH-dependent signaling tag attached thereto, the solution is in contact with the electrode and has a first pH value at which the pH-dependent signaling tag does not produce a signal and the analyte is not bound to the binding partner of the analyte;
applying a first electrical signal to the electrode to change the pH of the solution near the surface of the electrode from the first pH value to a second pH value, while the pH of other areas of the solution remains at the first pH value;
binding the analyte to the capture molecule and to the detecting probe at the first end of the assay chamber, wherein the pH-dependent signaling tag near the surface of the electrode produces a first signal at the second pH value and wherein the pH-dependent signaling tag in the other areas of the solution at the first pH value does not produce a signal;
applying a second electrical signal to the electrode to change the pH of the solution near the surface of the electrode from the second pH value to a third pH value, while the pH of other areas of the solution remains at the first pH value; and
measuring a second signal produced by the pH-dependent signaling tag at the third pH value near the surface of the electrode, wherein the pH-dependent signaling tag in the other areas of the solution at the first pH value does not produce a signal.
7 . The method of claim 6 , wherein the second pH value is higher than the first pH value.
8 . The method of claim 6 , wherein the third pH value is higher than the second pH value.
9 . The method of claim 6 , wherein the analyte is an anti-drug antibody (ADA).
10 . The method of claim 6 , wherein the electrochemically active agent is selected from the group consisting of quinone, catechol, aminophenol, hydrazine, hydroquinone, benzoquinone, naphthoquinone, derivatives thereof, and combinations thereof.
11 . The method of claim 6 , wherein the signaling tag is selected from the group consisting of a fluorescent tag, a fluorescent dye, a fluorescent protein, an electroluminescent dye, a chemiluminescent dye, and an enzyme.
12 . A method for detecting an amount of an analyte in a solution comprising:
providing an assay chamber including an electrode positioned at the assay chamber and a capture molecule attached to the electrode via a linker;
providing to the assay chamber a solution including an analyte, a binding partner of the analyte, at least one electrochemically active agent, and a detecting probe having a pH-dependent signaling tag attached thereto, the solution is in contact with the electrode and has a first pH value at which the pH-dependent signaling tag does not produce a signal and the analyte is bound to the binding partner of the analyte;
applying a first electrical signal to the electrode to change the pH of the solution near the surface of the electrode from the first pH value to a second pH value, while the pH of other areas of the solution remains at the first pH value;
dissociating the binding partner of the analyte from the analyte at the first end of the assay chamber at the second pH value;
applying a second electrical signal to the electrode to change the pH of the solution near the surface of the electrode from the second pH value to the first pH value, while the pH of other areas of the solution are at the first pH value;
binding the analyte to the capture molecule and to the detecting probe at the first end of the assay chamber;
applying a third electrical signal to the electrode to change the pH of the solution near the surface of the electrode from the first pH value to the second pH value, while the pH of other areas of the solution remains at the first pH value;
dissociating the binding partner of the analyte from the analyte at the first end of the assay chamber at the second pH value;
applying a fourth electrical signal to the electrode to change the pH of the solution near the surface of the electrode from the second pH value to the first pH value, while the pH of other areas of the solution are at the first pH value;
binding the analyte to the capture molecule and to the detecting probe at the first end of the assay chamber;
applying a next electrical signal to the electrode to change the pH of the solution at the first end of the assay chamber from the second pH value to a third pH value, the pH-dependent signaling tag is in an on state at the third pH value; and
measuring a signal produced by the pH-dependent signaling tag at the third pH value.
13 . The method of claim 12 , further comprising shifting the pH of the solution in the first end of the assay chamber between the first and second pH values prior to applying the next electrical signal to the electrode to change the pH of the solution at the first end of the assay chamber from the second pH value to the third pH value.
14 . The method of claim 12 , wherein the second pH value is lower than the first pH value.
15 . The method of claim 12 , wherein the third pH value is higher than the second pH value.
16 . The method of claim 12 , wherein the analyte is an anti-drug antibody (ADA).
17 . The method of claim 12 , wherein the electrochemically active agent is selected from the group consisting of quinone, catechol, aminophenol, hydrazine, hydroquinone, benzoquinone, naphthoquinone, derivatives thereof, and combinations thereof.
18 . The method of claim 12 , wherein the signaling tag is selected from the group consisting of a fluorescent tag, a fluorescent dye, a fluorescent protein, an electroluminescent dye, a chemiluminescent dye, and an enzyme.
19 . The method of claim 12 , wherein the linker is configured to immobilize the capture molecule through adsorption.
20 . The method of claim 12 , wherein the linker is configured to immobilize the capture molecule through covalent bonding.