System and Method for Single-Step ELISA via Local pH Modulation

Abstract

A method for detecting a presence and/or a concentration of a target substance in a reagent solution using enzyme-linked immunosorbent assay (ELISA) includes binding the target substance directly or indirectly to an electrode, and binding a detection agent directly or indirectly to the bound target substance. The method further includes modulating a pH of only a portion of the reagent solution in which the bound target substance and the bound detection agent are located using the electrode, the modulated pH of the portion of the reagent solution causing the bound detection agent to undergo a change, and detecting the change in the bound detection agent. The detected change corresponds to the presence of the target substance in the reagent solution and/or the concentration of the target substance in the reagent solution.

What is claimed is: 
     
         1 . A method for detecting a presence and/or a concentration of a target substance in a reagent solution using enzyme-linked immunosorbent assay (ELISA) comprising:
 binding the target substance directly or indirectly to an electrode;   binding a detection agent directly or indirectly to the bound target substance;   modulating a pH of only a portion of the reagent solution in which the bound detection agent is located using the electrode, the modulated pH of the portion of the reagent solution causing the bound detection agent to undergo a change; and   detecting the change in the bound detection agent, the detected change corresponding to the presence of the target substance in the reagent solution and/or the concentration of the target substance in the reagent solution.   
     
     
         2 . The method of  claim 1 , further comprising:
 binding a capture agent to the electrode; and   binding the target substance directly or indirectly to the capture agent.   
     
     
         3 . The method of  claim 1 , wherein the detection agent includes a pH-sensitive reporter, the method further comprising:
 energizing the electrode with a direct current electrical signal for a predetermined time period to modulate the pH of only the portion of the reagent solution from a first value to a second value, the second value different from the first value,   wherein the pH-sensitive reporter undergoes the change in response to the portion of the reagent solution having the second value of pH.   
     
     
         4 . The method of  claim 1 , wherein the portion of the reagent solution is a first portion of the reagent solution and the first portion of the reagent solution is located in a modulation zone, the method further comprising:
 preventing modulation of a pH of a second portion of the reagent solution, the second portion of the reagent solution located in a non-modulation zone,   wherein unbound molecules of the detection agent are located in the non-modulation zone, and   wherein the unbound molecules of the detection agent do not undergo the change.   
     
     
         5 . The method of  claim 1 , further comprising:
 binding a capture agent to an occupied area including an area of the electrode;   blocking an unoccupied area adjacent to the occupied area to prevent binding of the capture agent to the unoccupied area;   removing unbound capture agent from the occupied area and the unoccupied area in a washing step;   binding the target substance directly or indirectly to the bound capture agent; and   detecting the change in the bound detection agent without any further washing steps.   
     
     
         6 . The method of  claim 1 , wherein the detection agent includes a pH-sensitive reporter, the method further comprising:
 detecting the change in the bound detection agent as an intensity of light emitted by the pH-sensitive reporter.   
     
     
         7 . A method for detecting a target substance using enzyme-linked immunosorbent assay (ELISA) comprising:
 binding the target substance directly or indirectly to a first electrode located in a test well including a reagent solution;   adding a detection agent to the test well, a bound portion of the detection agent bound directly or indirectly to the bound target substance and an unbound portion of the detection agent unbound to the bound target substance;   adding additional detection agent and additional reagent solution to a control well that does not include the target substance, a second electrode located in the control well;   modulating a pH of only a portion of the reagent solution in the test well in which the bound portion of the detection agent is located to cause the bound portion of the detection agent located in the portion of the reagent solution and the unbound portion of the detection agent located in the portion of the reagent solution to undergo a first change using the first electrode;   modulating a pH of only a corresponding portion of the additional reagent solution located in the control well to cause a corresponding portion of the additional detection agent located in the corresponding portion of the additional reagent solution to undergo a second change using the second electrode; and   detecting the first change as a first detected value;   detecting the second change as a second detected value; and   generating a test value as a comparison of the first detected value to the second detected value, the generated test value corresponding to a presence of the target substance in the test well and/or a concentration of the target substance in the test well.   
     
     
         8 . The method of  claim 7 , further comprising:
 detecting the first detected value as a value representing a sum of the bound portion of the detection agent located in the portion of the reagent solution having the modulated pH and the unbound portion of the detection agent located in the portion of the reagent solution having the modulated pH; and   detecting the second detected value as a value representing unbound detection agent located in the portion of the other reagent solution having a modulated pH.   
     
     
         9 . The method of  claim 8 , further comprising:
 subtracting the second detected value from the first detected value to generate the test value to isolate an output of the bound detection agent located in the portion of the reagent solution having the modulated pH.   
     
     
         10 . The method of  claim 7 , further comprising:
 adding the reagent solution and the other reagent solution to the same space; and   maintaining a physical separation between the reagent solution and the other reagent solution.   
     
     
         11 . The method of  claim 7 , wherein the detection agent and the other detection agent each include a pH-sensitive reporter and the method further comprises:
 detecting the first change as an intensity of light emitted by the pH-sensitive reporter of the bound detection agent; and   detecting the second change as another intensity of light emitted by the pH-sensitive reporter of the other detection agent located in the corresponding portion of the additional reagent solution.   
     
     
         12 . A system for detecting a presence and/or a concentration of a target substance using enzyme-linked immunosorbent assay (ELISA) comprising:
 a test well;   a reagent solution located in the test well and including the target substance;   at least one electrode located in the test well in direct contact with the reagent solution;   a capture agent including one of a pH-sensitive donor molecule and an acceptor molecule, the capture agent bound to the at least one electrode, the target substance binding directly or indirectly to the capture agent;   a detection agent including the other of the pH-sensitive donor molecule and the acceptor molecule, the detection agent configured to bind directly or indirectly to the bound target substance; and   a controller electrically connected to the at least one electrode and configured to
 modulate a pH of only a portion of the reagent solution in which the bound detection agent is located using the electrode, the modulated pH of the portion of the reagent solution causing the pH-sensitive donor to undergo a first change, and the first change causing the acceptor molecule to undergo a second change; and 
 detect the second change in the acceptor molecule, the detected second change corresponding to the presence of the target substance in the reagent solution and/or the concentration of the target substance in the reagent solution. 
   
     
     
         13 . The system of  claim 12 , further comprising:
 a power source configured to generate and to supply the at least one electrode with an electrical signal that modulates the pH of only the portion of the reagent solution.   
     
     
         14 . The system of  claim 13 , wherein the electrical signal is a direct current electrical signal of a predetermined time period. 
     
     
         15 . The system of  claim 13 , further comprising:
 a memory electrically connected to the controller and configured to store at least one program for generating the electrical signal.   
     
     
         16 . The system of  claim 13 , wherein the reagent solution includes electroactive molecules capable of electrochemical oxidation and/or electrochemical reduction, which results in generation or consumption of protons to modulate the pH of the portion of the reagent solution in response to the electrical signal. 
     
     
         17 . The system of  claim 16 , wherein the reagent solution includes at least one of a quinone derivative, a hydrazine derivative, and phenol-Ru (2,2′-bipyridine) 3   2+ . 
     
     
         18 . The system of  claim 12 , wherein the at least one electrode is formed from at least one of platinum, gold, silver, indium tin oxide, fluorine doped tin oxide, glassy carbon, and graphite. 
     
     
         19 . The system of  claim 12 , wherein:
 the donor molecule is bound to the capture agent,   the acceptor molecule is bound to the detection agent, and   at least one of the donor molecule and the acceptor molecule is pH-sensitive.   
     
     
         20 . The system of  claim 19 , wherein:
 the first change includes the donor molecules generating a donor region,   only the acceptor molecules located in the donor region undergo the second change, and   the second change is an emission of light or a quenching of light.