Affinity chromatography column and electrochemical detection methods for quantitative and semi-quantitative analysis of biomarkers

Abstract

A detection method for quantitative or semi-quantitative analysis of one or more target analytes includes a step of introducing a sample specimen to an affinity chromatography column through a port of the affinity chromatography column. The affinity chromatography column includes detection antibodies and nanoparticles in an interior of the affinity chromatography column that bind to the one or more target analytes. The method further includes separating the one or more target analytes that are bound to the detection antibodies and nanoparticles from unbound materials; washing the affinity chromatography column in order to remove the unbound materials from the affinity chromatography column; and detecting electric current signal(s) representative of a quantity of the one or more target analytes bound to the detection antibodies and nanoparticles within the affinity chromatography column with at least one electrochemical sensor.

Description

Attorney Reference No. 10697-2506324AFFINITY CHROMATOGRAPHY COLUMN AND ELECTROCHEMICAL DETECTION METHODS FOR QUANTITATIVE AND SEMI-QUANTITATIVE ANALYSIS OF BIOMARKERSCROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of United States Provisional Patent Application No. 63 / 761,367, filed February 21, 2025, the disclosure of which is hereby incorporated by reference in its entirety.BACKGROUNDField of the Invention

[0002] The present disclosure relates to detection devices, systems, and methods for quantitative and semi-quantitative analysis of target analytes and, in particular, to an affinity chromatography column along with methods for using the affinity chromatography column in tandem with one or more electrochemical sensors for detection of target analytes in biological samples.Description of Related Art

[0003] Affinity chromatography is characterized by the specific coupling or binding between selective binding units (SBU), such as antibody-antigen, antibody -biomarkers, avidinbiotin or oligonucleotides, and their complementary strands. Antibodies for many clinically relevant biomarkers have been isolated and are readily available. Existing electrochemical detection of affinity based assays on lab-on-a-chip systems are discussed in the following references.

[0004] International Patent Appl. Pub. No. WO 2000 / 011474A1, entitled “Electrochemical affinity assay,” describes an electrochemical affinity assay system for detection of ligandligand receptor binding. The ligand-ligand receptor binding occurs as follows. Initially, a first member of a ligand-and-ligand receptor pair is immobilized in a redox polymer. Next, a second member is bound to the first member. A third member is then labeled with a detection marker, such as horseradish peroxidase or soybean peroxidase. Associating the labeled third member with the electrode via the second member results in electrical contact between the peroxidase and the redox polymer, which causes an electrical connection between reaction centers of the peroxidase label and the electrode through the conducting redox polymer. Such electrical connection converts a film to a catalyst for the electro-reduction of the hydrogen peroxide produced within the film by the immobilized substrate-generating enzyme.16B33215.DOCXAttorney Reference No. 10697-2506324

[0005] European Patent Appl. Pub. No. EP 2390664A1, entitled “Method for electrochemical detection of binding reactions,” discloses that a first conjugate for a binding reaction can comprise a redox marker and an analyte molecule. The second conjugate can comprise an anti-redox marker antibody or its specific binding fragment, and a molecule binding specifically to the analyte.

[0006] Purification methods for the affinity system matrices are also known. For example, International Patent Appl. Pub. No. WO 2016 / 139128A1, entitled “Sanitization method for affinity chromatography matrices,” discloses a method for cleaning or sanitization for an affinity chromatography matrix.SUMMARY

[0007] According to an aspect of the disclosure, a detection method for quantitative or semi-quantitative analysis of one or more target analytes includes a step of introducing a sample specimen to an affinity chromatography column through a port of the affinity chromatography column. The affinity chromatography column includes detection antibodies and nanoparticles in an interior of the affinity chromatography column that bind to the one or more target analytes. The method further includes separating the one or more target analytes that are bound to the detection antibodies and nanoparticles from unbound materials; washing the affinity chromatography column in order to remove the unbound materials from the affinity chromatography column; and detecting electric current signal(s) representative of a quantity of the one or more target analytes bound to the detection antibodies and nanoparticles within the affinity chromatography column with at least one electrochemical sensor.

[0008] According to another aspect of the disclosure, an affinity chromatography column for quantitative or semi-quantitative analysis of one or more target analytes, the affinity chromatography column includes: a column container having a first end, a second end, and a sidewall extending therebetween; at least one first connector over the first end of the column container configured for introducing fluid through the at least one first connector into an interior of the column container; and at least one second connector over the second end of the column container configured for removing fluid from the interior of the column container through the at least one connector. The affinity chromatography column further includes at least one conductive layer having at least one intrinsically conducting polymer (ICP) over at least portions of an interior surface of the sidewall of the column container providing contact regions for the one or more target analytes and metal nanoparticles that are bound to the target analytes.26B33215.DOCXAttorney Reference No. 10697-2506324

[0009] According to another aspect of the disclosure, a detection method for quantitative or semi -quantitative analysis of one or more target analytes includes a step of introducing a sample specimen to the interior of one of the previously described affinity chromatography columns. The affinity chromatography column includes detection antibodies that bind to the one or more target analytes through the sample injection port of the affinity chromatography column. The method further includes a step of detecting electric current signal(s) representative of a quantity of the one or more target analytes bound to the detection antibodies within the affinity chromatography column with at least one electrochemical sensor.

[0010] According to another aspect of the disclosure, a system for quantitative or semi-quantitative analysis of one or more target analytes includes: any of the affinity chromatography columns as previously described; and at least one electrical sensor electrically connected to the at least one conductive layer of the affinity chromatography column configured to detect electrical signals representative of the one or more target analytes and detection antibodies immobilized on the at least one conductive layer.

[0011] According to another aspect of the disclosure, an affinity chromatography column for quantitative or semi-quantitative analysis of one or more target analytes includes a column container having a first end, a second end, and a sidewall extending therebetween. At least a portion(s) of an inner surface of the sidewall have grooves configured to provide contact regions of increased surface area for immobilizing the one or more target analytes that are bound to detection antibodies against the sidewall. The affinity chromatography column also includes: at least one first connector over the first end of the column container configured for introducing fluid through the at least one first connector into an interior of the column container; and at least one second connector over the second end of the column container configured for removing fluid from the interior of the column container through the at least one connector.

[0012] According to another aspect of the disclosure, a system for quantitative or semi-quantitative analysis of one or more target analytes includes: an affinity chromatography column having an inlet configured to receive a sample specimen containing the one or more target analytes and detection antibodies immobilized on metal nanoparticles contained within the affinity chromatography column that bind to the one or more target analytes in the sample specimen and at least one electrochemical sensor configured to detect electric current signal(s) representative of a quantity of the one or more target analytes bound to the detection antibodies within the affinity chromatography column. The system also includes at least one controller in communication with the at least one electrochemical sensor. The controller is configured to 36B33215.DOCXAttorney Reference No. 10697-2506324receive and process the electric current signal(s) detected by the at least one electrochemical sensor and determine a concentration or level of the one or more target analytes bound to the detection antibodies within the affinity chromatography column based on analysis of the received and processed electric current signal(s).

[0013] Preferred and non-limiting examples of the present disclosure will now be described in the following numbered clauses:

[0014] Clause 1: A detection method for quantitative or semi-quantitative analysis of one or more target analytes, comprising: introducing a sample specimen to an affinity chromatography column through a port of the affinity chromatography column, the affinity chromatography column comprising detection antibodies and nanoparticles in an interior of the affinity chromatography column that bind to the one or more target analytes; separating the one or more target analytes that are bound to the detection antibodies and nanoparticles from unbound materials; washing the affinity chromatography column in order to remove the unbound materials from the affinity chromatography column; and detecting electric current signal(s) representative of a quantity of the one or more target analytes bound to the detection antibodies and nanoparticles within the affinity chromatography column with at least one electrochemical sensor.

[0015] Clause 2: The method of clause 1, further comprising analyzing the detected electric current signal(s) to determine a concentration or level of the one or more target analytes bound to the detection antibodies and nanoparticles within the affinity chromatography column to make diagnostic conclusions from the sample specimen.

[0016] Clause 3 : The method of clause 1 or clause 2, wherein the detection antibodies and nanoparticles in the affinity chromatography column comprise antibody-conjugated-nanoparticles.

[0017] Clause 4: The method of any of clauses 1-3, wherein the one or more target analytes comprises at least one of a clinically relevant biomarker, antigen, pathogen, or protein (e.g., CA125, HE4, corona virus, albumin).

[0018] Clause 5: The method of any of clauses 1-4, wherein the one or more target analytes comprises a clinically relevant biomarker for detection of ovarian cancer or breast cancer.

[0019] Clause 6: The method of any of clauses 1-5, wherein the one or more target analytes comprises one or more of the following clinically relevant biomarkers suitable for cancer detection: CA125, HE4, VEGF, APO-A1, Prostatin, Transthyretin, or Transferrin.

[0020] Clause 7: The method of any of clauses 1-6, wherein the sample specimen comprises at least one bodily fluid (e.g., nasal mucosa, urine, saliva, blood, plasma, or serum).46B33215.DOCXAttorney Reference No. 10697-2506324

[0021] Clause 8: The method of any of clauses 1-7, wherein the affinity chromatography column comprises a plurality of beads disposed within the affinity chromatography column comprising the detection antibodies and nanoparticles.

[0022] Clause 9: The method of clause 8, wherein the plurality of beads comprises at least one of magnetic beads, silica beads, or beads comprising gold nanoparticles.

[0023] Clause 10: The method of any of clauses 1-9, wherein washing the affinity chromatography column comprises introducing a buffer solution through the affinity chromatography column.

[0024] Clause 11 : The method of clause 10, wherein the buffer solution comprises at least one of the following types of buffer solutions: a neutral buffer (e.g., PBS or TBS), a mild detergent (e.g., Tween-20 or Triton X-100), a regeneration buffer (e.g., glycine-HCl), or an electrochemical mediator.

[0025] Clause 12: The method of any of clauses 1-11, wherein the at least one electrochemical sensor comprises a working electrode comprising at least one of gold nanoparticles, graphene nanotubes, or carbon nanotubes.

[0026] Clause 13: The method of clause 12, wherein the working electrode is configured to be connected to nanoparticles in the affinity chromatography column for detection of the electric current signal(s).

[0027] Clause 14: The method of clause 13, wherein the nanoparticles in the affinity chromatography column are in a stationary phase of the affinity chromatography column and do not carry the sample specimen through the affinity chromatography column.

[0028] Clause 15: The method of any of clauses 1-14, wherein the unbound materials comprise one or more of analytes, antibodies, or impurities contained in the sample specimen.

[0029] Clause 16: The method of any of clauses 1-15, further comprising periodically inverting or rolling the affinity chromatography column to introduce periodic laminar flow through the column in order to oscillate the one or more target analytes bound to the detection antibodies within the affinity chromatography column.

[0030] Clause 17: The method of any of clauses 1-16, wherein the affinity chromatography column comprises: a column container comprising a first end, a second end, and a sidewall extending therebetween; at least one first connector over the first end of the column container configured for introducing a buffer solution through the at least one first connector into an interior of the column container; at least one second connector over the second end of the column container configured for removing the buffer solution from the interior of the column container through the at least one second connector; and at least one conductive layer 56B33215.DOCXAttorney Reference No. 10697-2506324comprising at least one intrinsically conducting polymer (ICP) over at least portions of an inner surface of the sidewall providing contact regions for the one or more target analytes and metal nanoparticles that are bound to the target analytes.

[0031] Clause 18: The method of clause 17, wherein detecting the electric current signal(s) is performed using electrodes that are electrically connected to the at least one conductive layer of the affinity chromatography column.

[0032] Clause 19: The method of clause 17 or clause 18, wherein the affinity chromatography column further comprises a degassing chamber and vent membrane positioned between the degassing chamber and the interior of the column container, the method further comprising allowing gasses to escape from the interior of the column container to the degassing chamber while washing the affinity chromatography chamber.

[0033] Clause 20: The method of clause 19, wherein the method is performed in a zero gravity or low gravity environment in which gravity is not used for separating bound analytes from the unbound materials.

[0034] Clause 21 : The method of any of clauses 17-20, wherein the at least one ICP at least partially covers a bottom side of the inner surface of the sidewall of the column container when the column is in a horizontal orientation.

[0035] Clause 22: The method of any of clauses 17-21, wherein the at least one ICP comprises at least one of polyallanine, polypyrrole or Poly(3,4-ethylenedioxythiophene).

[0036] Clause 23: The method of any of clauses 17-22, wherein the contact regions on the inner surface of the sidewall comprise grooves for increasing surface area of the contact regions.

[0037] Clause 24: The method of any of clauses 17-23, further comprising oscillating the one or more target analytes and detection antibodies that are bound to the target analytes through the interior of the column container by applying electric current to at least two conductive members repeatedly and one after another to introduce laminar flow and oscillating movement of the one or more analytes and bound antibodies between the at least two conductive members.

[0038] Clause 25: The method of clause 24, wherein the at least two conductive members of the affinity chromatography column comprise: a first conductive member extending axially over a first portion of an exterior surface of the sidewall; and a second conductive member extending axially over a second portion of the exterior surface of the sidewall.

[0039] Clause 26: The method of any of clauses 17-25, wherein the port of the affinity chromatography column comprises a sample injection port extending through the sidewall of 66B33215.DOCXAttorney Reference No. 10697-2506324the column container, the sample injection port comprising a one-way valve for permitting fluid flow into an interior of the column container and restricting fluid flow out of the column container, and wherein introducing the sample specimen to the affinity chromatography column comprises introducing the sample specimen through the one-way valve.

[0040] Clause 27 : The method of clause 26, wherein the one-way valve comprises a duckbill valve.

[0041] Clause 28: The method of any of clauses 17-27, wherein washing the affinity chromatography column in order to remove the unbound materials from the affinity chromatography column is performed by a buffer solution circulation system comprising a pump fluidly connected between the at least one first connector and the at least one second connector, which introduces the buffer solution into the interior of the column container through the at least one first connector and removes the buffer solution from the column container through the at least one second connector.

[0042] Clause 29: The method of any of clauses 17-28, wherein the column container is hydrophobic and / or charge neutral and comprises at least one of polycarbonate, poly(carboxybetaine) (PCB), polypropylene, polyethylene, or polyester.

[0043] Clause 30: An affinity chromatography column for quantitative or semi-quantitative analysis of one or more target analytes, the affinity chromatography column comprising: a column container comprising a first end, a second end, and a sidewall extending therebetween; at least one first connector over the first end of the column container configured for introducing fluid through the at least one first connector into an interior of the column container; at least one second connector over the second end of the column container configured for removing fluid from the interior of the column container through the at least one second connector; and at least one conductive layer comprising at least one intrinsically conducting polymer (ICP) over at least portions of an interior surface of the sidewall of the column container providing contact regions for the one or more target analytes and metal nanoparticles that are bound to the target analytes.

[0044] Clause 31 : The affinity chromatography column of clause 30, further comprising the detection antibodies in the interior of the column container configured to bind to the one or more target analytes configured to be detected by the affinity chromatography column.

[0045] Clause 32: The affinity chromatography column of clause 30 or clause 31, wherein the at least one first connector and / or the at least one second connector comprise an end cap sealed to the first end or the second end of the container column and comprising a luer lock for connecting the affinity chromatography column to a fluid circulation system.76B33215.DOCXAttorney Reference No. 10697-2506324

[0046] Clause 33: The affinity chromatography column of any of clauses 30-32, further comprising a degassing chamber and vent membrane, which is positioned between the degassing chamber and the interior of the column container, configured to allow gasses to escape from the interior of the column container to the degassing chamber.

[0047] Clause 34: The affinity chromatography column of clause 33, wherein the vent membrane covers an opening in the sidewall of the column container, and where the degassing chamber comprises a gas dome over the vent membrane.

[0048] Clause 35: The affinity chromatography column of clause 33 or clause 34, wherein the degassing chamber and vent membrane are configured to permit use in zero gravity and low gravity environments.

[0049] Clause 36: The affinity chromatography column of clause 30, wherein the ICP at least partially covers a bottom side of the inner surface of the sidewall of the column container when the column is in a horizontal orientation.

[0050] Clause 37: The affinity chromatography column of any of clauses 30-36, wherein the at least one ICP comprises at least one of polyallanine, polypyrrole or Poly(3,4-ethy 1 enedi oxy thi ophene) .

[0051] Clause 38: The affinity chromatography column of any of clauses 30-37, wherein the contact regions on the inner surface of the sidewall comprise grooves for increasing surface area of the contact regions.

[0052] Clause 39: The affinity chromatography column of clause 38, wherein the at least one conductive layer is deposited over the portion(s) of the inner surface comprising the grooves.

[0053] Clause 40: The affinity chromatography column of clause 38 or clause 39, wherein the grooves extend at least partially about an axis of the column in planes that are transverse or substantially transverse to an axis of the column container.

[0054] Clause 41: The affinity chromatography column of any of clauses 30-40, further comprising: a first conductive member extending axially over a first portion of an exterior surface of the sidewall; and a second conductive member extending axially over a second portion of the exterior surface of the sidewall.

[0055] Clause 42: The affinity chromatography column of clause 41, wherein the first conductive member or the second conductive member comprise metallic strips or coils extending axially over the exterior surface of the sidewall and configured to form electromagnetic fields for attracting the one or more target analytes and bound antibodies.86B33215.DOCXAttorney Reference No. 10697-2506324

[0056] Clause 43 : The affinity chromatography column of clause 41 or clause 42, where the first conductive member and the second conductive member are on opposite sides of the container column.

[0057] Clause 44: The affinity chromatography column of any of clauses 41-43, wherein the first conductive member and the second conductive member are separated by about 180 degrees around the exterior surface of the column container, when viewed through a cross-sectional view.

[0058] Clause 45: The affinity chromatography column of any of clauses 41-44, wherein the first conductive member and the second conductive member are configured to be energized repeatedly and one after another to introduce laminar flow and oscillating movement of the one or more analytes and bound antibodies between the first conductive member and the second conductive member.

[0059] Clause 46: The affinity chromatography column of any of clauses 30-45, further comprising a sample injection port extending through the sidewall of the column container, the sample injection port comprising a one-way valve for permitting fluid flow into an interior of the column container and restricting fluid flow out of the column container.

[0060] Clause 47: The affinity chromatography column of clause 46, wherein the one-way valve comprises a duckbill valve.

[0061] Clause 48: The affinity chromatography column of any of clauses 30-47, wherein the first connector and the second connector are configured to be connected to tubing connected to a buffer pump for circulating buffer solution through the interior of the column container through the tubing and first and second connectors.

[0062] Clause 49: The affinity chromatography column of any of clauses 30-48, wherein the column container is hydrophobic and / or charge neutral.

[0063] Clause 50: The affinity chromatography column of any of clauses 30-49, wherein the column container comprises a polymer material that is hydrophobic and / or charge neutral.

[0064] Clause 51: The affinity chromatography column of clause 50, wherein the column container is hydrophobic and / or charge neutral and comprises at least one of polycarbonate, poly(carboxybetaine) (PCB), polypropylene, polyethylene, or polyester.

[0065] Clause 52: A detection method for quantitative or semi-quantitative analysis of one or more target analytes, the method comprising: introducing a sample specimen to the interior of the affinity chromatography column of any of clauses 30-51, wherein the affinity chromatography column comprises detection antibodies that bind to the one or more target analytes through the sample injection port of the affinity chromatography column; and 96B33215.DOCXAttorney Reference No. 10697-2506324detecting electric current signal(s) representative of a quantity of the one or more target analytes bound to the detection antibodies within the affinity chromatography column with at least one electrochemical sensor.

[0066] Clause 53: The method of clause 52, wherein the sample specimen is introduced to the affinity chromatography column through a sample injection port extending through the sidewall of the column container, the sample injection port comprising a one-way valve for permitting fluid flow into an interior of the column container and restricting fluid flow out of the column container, and wherein introducing the sample specimen to an affinity chromatography column comprises introducing the sample specimen through the one-way valve.

[0067] Clause 54: The method of clause 52 or clause 53, further comprising washing the affinity chromatography column in order to remove unbound materials from the affinity chromatography column, wherein washing the affinity chromatography column is performed by a buffer solution circulation system comprising a pump fluidly connected between the at least one first connector and the at least one second connector, which introduces the buffer solution into the interior of the column container through the first connector and removes the buffer solution from the column container through the second connector.

[0068] Clause 55: The method of any of clauses 52-54, wherein detecting the electric current signal(s) comprises detecting the one or more target analytes bound to the detection antibodies that are immobilized on the at least one conductive layer comprising the ICP.

[0069] Clause 56: The method of any of clauses 52-55, further comprising oscillating the one or more target analytes and detection antibodies that are bound to the target analytes through the interior of the column chamber by applying electric current to at least two conductive members repeatedly and one after the other thereby causing the one or more target analytes and bound detection antibodies to oscillate from one of the at least two conductive members to the other conductive member.

[0070] Clause 57: A system for quantitative or semi-quantitative analysis of one or more target analytes, comprising: the affinity chromatography column of any of clauses 30-51; and at least one electrical sensor electrically connected to the at least one conductive layer of the affinity chromatography column configured to detect electrical signals representative of the one or more target analytes and detection antibodies immobilized on the at least one conductive layer.

[0071] Clause 58: The system of clause 57, further comprising a buffer pump fluidly connected between the at least one first connector and the at least one second connector of the 106B33215.DOCXAttorney Reference No. 10697-2506324affinity chromatography column, wherein the buffer pump is configured to circulate buffer solution through the interior of the column container and between the first connector and the second connector.

[0072] Clause 59: The system of clause 57 or clause 58, wherein the affinity chromatography column further comprises: a first conductive member extending axially over a first portion of an exterior surface of the sidewall; and a second conductive member extending axially over a second portion of the exterior surface of the sidewall.

[0073] Clause 60: The system of clause 59, further comprising a controller electrically connected to the first conductive member and the second conductive member, the controller configured to selectively apply electric current to the first conductive member and the second conductive member causing the one or more target analytes and detection antibodies to oscillate back and forth within the interior of the column container.

[0074] Clause 61: The system of clause 60, wherein the controller is configured to apply the electric current to the conductive member that is closest to the at least one conductive layer as a final oscillation step before ceasing applying the electrical current to the conductive members, thereby causing the one or more target analytes and detection antibodies to become immobilized against the at least one conductive layer of the affinity chromatography column.

[0075] Clause 62: The system of any of clauses 57-61, further comprising a controller electrically connected to the sensor configured to: receive and process the electrical signal(s) detected by the at least one electrical sensor; and determine a concentration or level of the one or more target analytes bound to the detection antibodies within the affinity chromatography column based on analysis of the received and processed electric current signal(s).

[0076] Clause 63: The system of clause 62, wherein the at least one controller is further configured to analyze the determined concentration or level of the one or more target analytes bound to the detection antibodies within the affinity chromatography column to make diagnostic conclusions from the sample specimen.

[0077] Clause 64: An affinity chromatography column for quantitative or semi-quantitative analysis of one or more target analytes, the affinity chromatography column comprising: a column container comprising a first end, a second end, and a sidewall extending therebetween, wherein at least a portion(s) of an inner surface of the sidewall comprises grooves configured to provide contact regions of increased surface area for immobilizing the one or more target analytes that are bound to detection antibodies against the sidewall; at least one first connector over the first end of the column container configured for introducing fluid through the at least one first connector into an interior of the column container; and at least one second connector 116B33215.DOCXAttorney Reference No. 10697-2506324over the second end of the column container configured for removing fluid from the interior of the column container through the at least one connector.

[0078] Clause 65: The affinity chromatography column of clause 64, wherein the portion(s) of the inner surface comprising the grooves are on a bottom portion of the inner surface of the sidewall and wherein a top portion of the sidewall is free from grooves.

[0079] Clause 66: The column of clause 64 or clause 65, wherein the portion(s) of the inner surface of the sidewall comprising the grooves are disposed at about 90 degrees to 270 degrees of the inner surface, when viewed from a cross-sectional view.

[0080] Clause 67 : The column of any of clauses 64-66, wherein the portion(s) of the inner surface of the sidewall comprising the grooves are disposed at about 135 degrees to 225 degrees of the inner surface, when viewed from a cross-sectional view.

[0081] Clause 68: The column of any of clauses 64-67, wherein the grooves extend at least partially about an axis of the column in planes that are transverse or substantially transverse to an axis of the column container.

[0082] Clause 69: The column of any of clauses 64-68, wherein the portion(s) of the inner surface comprising the grooves are conductive and configured to be electrically connected to electrodes in order to detect electrical signals influenced by the one or more target analytes that are bound to the detection antibodies and immobilized on the contact areas of the column container.

[0083] Clause 70: A system for quantitative or semi-quantitative analysis of one or more target analytes, comprising: an affinity chromatography column comprising an inlet configured to receive a sample specimen containing the one or more target analytes and detection antibodies immobilized on metal nanoparticles contained within the affinity chromatography column that bind to the one or more target analytes in the sample specimen; at least one electrochemical sensor configured to detect electric current signal(s) representative of a quantity of the one or more target analytes bound to the detection antibodies within the affinity chromatography column; and at least one controller in communication with the at least one electrochemical sensor configured to receive and process the electric current signal(s) detected by the at least one electrochemical sensor and determine a concentration or level of the one or more target analytes bound to the detection antibodies within the affinity chromatography column based on analysis of the received and processed electric current signal(s).126B33215.DOCXAttorney Reference No. 10697-2506324BRIEF DESCRIPTION OF THE DRAWINGS

[0084] The disclosure will be described with reference to the following drawing figures wherein like reference numbers identify like parts throughout.

[0085] FIGS. 1 A and IB are front views of examples of affinity chromatography columns, according to aspects of the present disclosure;

[0086] FIG. 1C is an exploded view of an affinity chromatography column, according to an aspect of the present disclosure;

[0087] FIG. 2 is a schematic drawing showing an affinity chromatography column and electrochemical sensor attached in tandem for carrying out detection, according to an aspect of the present disclosure;

[0088] FIG. 3 is a schematic drawing illustrating a functionalization process of nanoparticles and an antibody-antigen binding event that occurs during a biomarker detection method, according to an aspect of the present disclosure;

[0089] FIG. 4A is a schematic drawing of another example of an affinity chromatography column, according to an aspect of the present disclosure;

[0090] FIG. 4B is a schematic drawing of an analyte detection system including the affinity chromatography column of FIG. 4A, according to an aspect of the present disclosure;

[0091] FIGS. 5 A and 5B are schematic drawings showing analytes and bound nanoparticles oscillating through an affinity chromatography column, according to aspects of the present disclosure; and

[0092] FIG. 6 is a flow chart showing a detection method for quantitative or semi-quantitative analysis of one or more target analytes, according to an aspect of the present disclosure.DETAILED DESCRIPTION

[0093] As used herein, the singular form of “a”, “an”, and “the” include plural referents unless the context clearly states otherwise.

[0094] As used herein, the terms “right”, “left”, “top”, “bottom”, and derivatives thereof shall relate to the invention as it is oriented in the drawing figures. However, it is to be understood that the invention can assume various alternative orientations and, accordingly, such terms are not to be considered as limiting. Also, it is to be understood that the invention can assume various alternative variations and stage sequences, except where expressly specified to the contrary. It is also to be understood that the specific devices and processes 136B33215.DOCXAttorney Reference No. 10697-2506324illustrated in the attached drawings, and described in the following specification, are examples. Hence, specific dimensions and other physical characteristics related to the embodiments disclosed herein are not to be considered as limiting.

[0095] For the purposes of this specification, unless otherwise indicated, all numbers expressing, for example, dimensions, physical characteristics, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the present invention. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any measured numerical value, however, may inherently contain certain errors resulting from the standard deviation found in their respective testing measurements.

[0096] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, are meant to be open ended.

[0097] Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of “1 to 10” is intended to include any and all sub-ranges between and including the recited minimum value of 1 and the recited maximum value of 10, that is, all subranges beginning with a minimum value equal to or greater than 1 and ending with a maximum value equal to or less than 10, and all subranges in between, e.g., 1 to 6.3, or 5.5 to 10, or 2.7 to 6.1.

[0098] The present disclosure is directed to electrochemical devices for analyte detection and, in particular, to an affinity chromatography column 10 configured to be used in tandem with an electrochemical sensor for analyte detection, as well as to detection methods that use the affinity chromatography column 10 and electrochemical sensor for detecting analyte concentrations in biological samples. In examples, target portions or contact regions of the affinity chromatography column 10 can be conductive and / or configured so that particles are connected to or immobilized against the conductive surfaces. As described in further detail herein, the target portions or contact regions can include conductive materials, such as conductive polymers, for detecting, for example, metallic nanoparticles bound to target analytes. The target portions or contact regions can also include textured areas or grooves, which increase available surface areas on which the analytes and bound antibodies can be attached, which can increase signal magnitude or sensitivity compared to chromatography columns with less surface area.146B33215.DOCXAttorney Reference No. 10697-2506324

[0099] The following terms and definitions are used throughout the present disclosure. As used herein, the term “plasma” or “serum” can refer to a liquid portion of whole blood. Plasma is derived by blood cell removal from whole blood in the presence of anticoagulants, such as Ethylenediaminetetraacetic acid (EDTA), Heparin, Sodium Citrate. Blood cells are red blood cells, white blood cells, and platelets. Serum is derived by blood cell removal from whole blood without the presence of anticoagulants. Plasma is obtained by centrifuging whole blood and anticoagulants. Serum is obtained by allowing whole blood to clot without the presence of anticoagulants.

[0100] As used herein, “affinity chromatography” can refer to detection / separation driven by specific binding properties of antibodies to their specific biomarkers. In affinity chromatography, specific antibodies required for the detection of biomarkers or antigens are immobilized onto the stationary phase utilizing any chemical coupling method.

[0101] As used herein, “chromatography” can refer to a common separation technique used in laboratories, mainly for the separation of components present within a sample or mixture. Chromatography works by passing the mixture containing the analyte of interest through a system that consists of a stationary phase and a mobile phase. The components of the mixture interact differently with the stationary and mobile phases, causing them to move at different speeds and thus separating them.

[0102] The “stationary phase” can refer to the immobile material or surface within a chromatography system that the sample components interact with. This phase does not move, and its interaction with the analytes causes them to separate based on their affinity. Different substances in the sample will adhere to the stationary phase with varying strengths, which causes them to move at different speeds when exposed to the mobile phase, resulting in separation.

[0103] The “mobile phase” can refer to a fluid that carries a sample mixture through the chromatography system. It can be either a liquid (in liquid chromatography) or a gas (in gas chromatography). The mobile phase is responsible for moving the analytes through or over the stationary phase, and the differential interaction of the analytes with the mobile phase and stationary phase leads to their separation. Solvents, such as water, salt buffers, methanol, or acetonitrile, are examples of mobile phases.

[0104] The terms “antigens” or “biomarkers” or “target analyte” or “analyte” may be used interchangeably and can refer to the specific biomolecule, which will bind at the binding site of the antibody specific to them. The antigen (or the analyte, or the target analyte) is the156B33215.DOCXAttorney Reference No. 10697-2506324molecule of interest within the sample specimen. In a diagnostic context, the antigen could be a biomarker, pathogen, or protein (e.g., CA125, HE4, corona virus, albumin).

[0105] The term “antibodies” can refer to immunoglobulins in the form of monoclonal, polyclonal, Fab fragments or Fab derivatives. Examples of immunoglobulins are IgG, IgA, IgM.

[0106] The term “antibody-antigen binding” can refer to the binding activity which occurs specifically between the antibody and its antigen. This specific binding occurs due to the high affinity between an antibody and its corresponding antigen, typically at the epitope (i.e., the part of the antigen recognized by the antibody). The binding is specific and follows a lock-and-key mechanism, where the antibody’s paratope (binding site) fits precisely with the antigen’s epitope.

[0107] The term “capture antibodies” may be used interchangeably with “antibodies.” The primary function of the antibody or the capture antibody is to capture or bind specifically to the antigen, biomarker, or target analyte. The capture antibody is the first antibody used in certain Enzyme-Linked Immunosorbent Assay (ELISA) formats (such as the sandwich ELISA). It is immobilized on a solid surface (such as a microplate) and is responsible for specifically binding to the antigen (or target analyte). The capture antibody’s role is to “capture” the antigen from the sample specimen for subsequent detection.

[0108] The “secondary antibody” or “detection antibody” can refer to the antibody used to bind to the antigen already captured by the capture antibody. In sandwich ELISA, the detection antibody is generally linked to an enzyme. This “enzyme-conjugate” generates a signal after binding the antigen, indicating the presence of the target analyte. For example, the secondary antibody could be “enzyme-conjugated” where the antibody has been chemically linked to an enzyme. This enzyme, when provided with a suitable substrate, produces a measurable signal (typically a color change or fluorescence). Common enzymes used in ELISA include horseradish peroxidase (HRP) and alkaline phosphatase (AP).

[0109] The term “label” can refer to any marker (e.g., an enzyme, fluorescent dye, or radioactive isotope) attached to an antibody or antigen that allows for the detection of the antigen-antibody binding event. In ELISA, the label is typically an enzyme, and the signal produced when the substrate undergoes a “redox reaction” (color change or fluorescence) is measured to determine the amount of antigen present in the sample.

[0110] The term “redox reaction” (reduction-oxidation) can refer to a chemical reaction in which electrons are transferred between molecules. In the context of ELISA, enzymatic redox reactions are often involved, particularly when using HRP, which catalyzes the oxidation of a 166B33215.DOCXAttorney Reference No. 10697-2506324substrate such as tetramethylbenzidine (TMB), leading to a color change that can be measured spectrophotometrically.

[0111] The term “immunosorbent assay” can refer to a biochemical technique used to detect the presence of specific proteins (e.g., antibodies, antigens) in a liquid sample. The assay relies on the specific binding between an antigen and its corresponding antibody. The most common format is the Enzyme-Linked Immunosorbent Assay (ELISA), where an enzyme-linked antibody generates a detectable signal (e.g., a color change). For example, there are several types of immunosorbent assays. The most common types are sandwich assays, indirect assays, direct assays, and competitive assays.

[0112] In a sandwich ELISA, the target antigen can be “sandwiched” between two antibodies: capture antibody and detection antibody. First, the capture antibody can be immobilized on a solid surface. Next, when the sample specimen is introduced to the immobilized antibody, the specific antigens present in the sample specimen agglutinates with the antibody. A second detection antibody, conjugated with an enzyme, binds to another site on the antigen. The detection antibody often carries a “label” which allows for detection based on colorimetric or electrochemical changes. This format is highly specific and sensitive.

[0113] In competitive ELISA, the antigens present within the sample specimen compete with a labeled antigen for a limited number of antibody binding sites. The more antigen in the sample, the fewer labeled antigens can bind to the antibodies, resulting in a reduced signal. This format is useful for small molecules or when antigen levels present in the sample specimen are very low.

[0114] In direct ELISA, the antigen is directly immobilized on the solid phase. Next, an enzyme-linked detection antibody binds directly to the antigen. This format is faster and involves fewer steps but may be less sensitive due to fewer amplification steps.

[0115] In indirect ELISA, the antigen is immobilized, and a primary antibody binds to the antigen. A secondary antibody conjugated to an enzyme is then used to bind to the primary antibody, amplifying the signal. This format is often used for detecting antibodies in samples (e.g., in HIV tests, CO VID-19 serology tests).

[0116] In examples, an affinity chromatography column 10 can comprise antibody -conjugated-nanoparticles configured to bind with target analytes. During a biomarker detection method, the affinity chromatography column 10 can receive a sample specimen, allowing the specimen to pass through a stationary phase (e.g., antibody-conjugated-nanoparticles) and a mobile phase (e.g., a system of liquids). The sample specimen carrying the analyte of interest, generally comprises bodily fluids such as nasal mucosa, urine, saliva,176B33215.DOCXAttorney Reference No. 10697-2506324blood, plasma, or serum. In examples, mucosa would need to be mixed with a carrier solution or buffer to permit the mucosa to pass into and flow through an interior of the affinity chromatography column 10.

[0117] In examples, the analytes of interest detected by the devices and method disclosed herein may be any clinically relevant biomarker, antigen, or protein. Once the analytes bind to the detection antibodies within the antibody-conjugated-nanoparticles, several washing steps can be performed to ensure removal of unbound analytes, antibodies and other agents and impurities present in the sample specimen and buffers.

[0118] The present disclosure also describes a diagnostic tool comprising an affinity chromatography column 10 coupled with electrochemical sensing to detect the presence of biomarkers. More particularly, as discussed above, the outlined diagnostic tool can be used to determine the concentration or levels of biomarkers present in plasma or serum samples, such as CA 125, HE4, VEGF, TTR, Transferrin, PSN, and APO-A1. These are clinically relevant biomarkers to detect ovarian and breast cancer; however, the present disclosure is not limited to detecting tumor or cancer biomarkers.

[0119] In some examples, the outlined diagnostic tool, which uses affinity chromatography coupled with electrochemical detection, focuses on providing or optimizing both biomarker capture efficiency and electron conductivity for accurate current measurement. Furthermore, in some examples, the affinity chromatography column 10 comprises the antibody-conjugated-nanoparticles. The affinity chromatography column 10 can be used to receive the sample specimen and pass it through the stationary phase, e.g., antibody-conjugated-nanoparticles with the aid of a mobile phase, such as a system of liquids. The sample specimen carrying the analyte of interest, can be bodily fluids, such as nasal mucosa, urine, saliva, blood, plasma, or serum. The analytes of interest can be any clinically relevant biomarker, antigen, or protein.

[0120] In examples, the present disclosure is also directed to a diagnostic tool which can be used to quantitate the amounts of both redox active and non-redox active biomarkers in a given specimen sample, such as plasma, serum, saliva, nasal mucosa.

[0121] FIGS. 1A and IB are drawings showing examples of liquid or affinity chromatography columns 10 including features of the present disclosure. As shown in FIGS.1A and IB the affinity chromatography column 10 comprises a top or first end 12, a second end 14, and a sidewall 16 extending therebetween. The column 10 can be, for example, about 50.0 mm in length L and can have an outer diameter OD of about 12.0 mm and an inner diameter ID of about 10.0 mm. The column 10 further comprises a branched channel, referred to herein as a sample injection port 18, for introducing a sample specimen into an interior of 186B33215.DOCXAttorney Reference No. 10697-2506324the column 10. FIG. 1A shows the affinity chromatography column 10 in a vertical configuration, in which the sample passes from injection port 18, located near the top end 12 of the column 10, to an outlet at the bottom end 14 of the column 10 by gravity. FIG. IB shows an affinity chromatography column 10 in a horizontal orientation. As described in further detail herein, when in the horizontal orientation, a pump can be used to circulate fluid (e.g., buffer solution) through the affinity chromatography column 10 for separating and / or washing analytes and detection antigens bound thereto.

[0122] FIG. 1C shows an exploded view of an affinity chromatography column 10. In examples, as shown in FIG. 1C, ends caps 40, 42 can be mounted or adhered to the ends 12, 14 of the column 10 in order to form a sealed enclosure configured to contain the sample specimen and buffer solution within the interior of the column 10. In examples, as described in further detail herein, electrodes, such as a conductive member (e.g., a copper wire) can be attached to the column through openings in the end caps 40, 42, in order to form an electrochemical sensor that detects electrical signals from the interior of the column 10.

[0123] The following portions of the disclosure describe suitable materials and components, which can be used in order to perform different steps of the methods disclosed herein. In some examples, the method(s) can utilize binding events between biomarkers and their specific antibody to determine the presence of a biomarker, or vice versa. The general approach to detecting such antibody-biomarkers binding events is to use a label. Some detection methods, such as electrochemical methods (e.g., voltometric, amperometric, etc.) or surface plasmon resonance, allow for detection without labeling. In contrast to these labeled and electrochemical methods, the methods of the present disclosure use a label-free detection method, such as amperometry, to detect the antibody-biomarkers binding event, enabling an integrated diagnostic system to output a quantitative signal directly proportional to concentration of the biomarkers present.

[0124] In some examples, the affinity chromatography column 10 can comprise a cylindrical housing or container formed from an inert, chemically stable, electrically non-conductive, and thermally non-conductive material, such as polypropylene (PP) polyethylene (PE), plexiglass, polycarbonate, borosilicate glass or glass. These example materials are widely used in affinity chromatography because they do not interfere with the biochemical interactions within the affinity chromatography column 10.

[0125] The affinity chromatography column 10 can also include surfaces or structures, such as beads 20 (shown in FIG. 2) or a matrix, composed of a material that support high surface area and efficient antibody immobilization onto the beads for biomarker capture. The 20 beads 196B33215.DOCXAttorney Reference No. 10697-2506324or matrix in the affinity chromatography column 10 may be composed of a material that supports electric conductivity. The pore size of the beads 20 should be tailored to accommodate large protein biomarkers, such as CA 125 (-200,000 Dalton molecular weight), while allowing for efficient flow-through of plasma or serum samples. Alternatively or in addition, as described in further detail herein, inner surface(s) of the affinity chromatography column 10 can be coated with or comprise a conductive layer (e.g., a conductive polymer layer) for immobilizing the target analyte, along with an antigen and / or nanoparticles (e.g., metal nanoparticles) that are bound to the analyte.

[0126] In examples, the beads 20 or matrix of the affinity chromatography column can comprise any of the following materials. Magnetic Beads: Superparamagnetic iron oxide nanoparticles (SPIONs) coated with agarose or dextran. Magnetic beads provide the added advantage of rapid separation and washing steps, and they can also be coupled with electrochemical readouts by altering the surface chemistry. Silica Beads: Silica offers a rigid matrix for affinity chromatography and can be functionalized with silane linkers for covalent binding of antibodies to the stationary phase, stationary matrix, or beads. Gold (Au) nanoparticles: Gold nanoparticles allow for high conductivity of electricity which would enable the detection of the antibody-biomarkers binding event.

[0127] In examples, the beads 20 can also comprise sepharose (Agarose). Agarose-based beads like Sepharose can be a popular choice for protein purification due to their high binding capacity for antibodies and biomolecules. They are chemically stable, hydrophilic, and suitable for large proteins like CA125 and HE4. However, because agarose is not conductive, agarose beads 20 would not be used to form an electrochemical sensor for detecting electrical signals representative of analyte concentration using the devices and methods disclosed herein.

[0128] In examples, a variety of crosslinking chemistries such as carbodiimide / NHS (EDC / NHS), carbodiimide / imidazole (EDC / Im), carbodiimide / sulfo-NHS (EDC / sulfo-NHS), BS3, pegylation, ligation, click chemistry may be used to covalently link detection antibodies (CA125, HE4, etc.) to the stationary phase (i.e., bead’s surface). This ensures strong binding and minimizes leaching during washing steps.

[0129] In examples, surface(s) of the beads 20 may or may not need to be functionalized with carboxyl (e.g., hydroxyl or PEG groups) prior to carrying out the coupling reaction between the bead and the antibody. FIG. 3 is a schematic drawing illustrating a functionalization process of nanoparticles and the antibody-antigen binding event, which occurs in a final step of a biomarker detection method. Steps of functionalization of a nanoparticle, which can be used in order to provide antibody-antigen binding event(s). In 206B33215.DOCXAttorney Reference No. 10697-2506324particular, as shown in FIG. 3, a nanoparticle 22 may be functionalized and then bound to antibodies, forming an antigen-conjugated nanoparticle 24, which can then be used during an antibody-antigen binding event assay 26. Any functional group readily available on the surface of the native antibody may be utilized for the coupling reaction. Non-covalent bonding techniques, such as avidin-biotin, may also be used to immobilize the antibodies to the bead surface. The antibody immobilized stationary phase may be referred to as “antibody-bead conjugates” or “antibody-matrix conjugates” hereafter.

[0130] The column 10 can also contain a buffer solution that allows the sample specimen to move through the column 10, so that target analytes can bind with antigens and / or nanoparticles. In examples, buffers used during the binding, washing, and elution steps of the detection methods should support both biomarker-antibody interactions and stable current measurement. Desirably, the composition, salt concentrations, and the pH of the buffer systems, should be optimized to ensure sensitivity, accuracy, limit of detection (LOD) and specificity of the diagnostic system. Furthermore, the buffers should maintain optimal pH and ionic strength to ensure biomarker or antigen binding and maintain the integrity of the beadantibody conjugates. The buffer systems may also be selected to minimize interference with amperometric readings. Potential buffers can include any of the following.

[0131] Binding Buffer: A neutral buffer, such as PBS (Phosphate-Buffered Saline) or TBS (Tris-Buffered Saline) at pH 7.4, may be used to maintain protein stability and support antibody-antigen or antibody -biomarker binding.

[0132] Washing Buffer: Mild detergents like Tween-20 or Triton X-100 or PBS may be used for washing unbound material, while ensuring that bound biomarkers remain attached. Other detergents, surfactants and polysorbates could also be used interchangeably.

[0133] Regeneration Buffer: After detection, the affinity chromatography column could be regenerated for reuse using buffers such as glycine-HCl (pH 2.5) to dissociate the bound biomarkers from the antibodies, followed by neutralization.

[0134] Electrochemical Mediator: In electrochemical detection, a redox mediator such as ferrocyanide / ferricyanide may be needed to facilitate electron transfer from the immobilized antibody-antigen complex to the working electrode.

[0135] As previously discussed, FIG. 2 shows a schematic illustration of the affinity chromatography column 10 and an electrochemical sensor 28 attached in tandem to provide amperometric detection. More particularly, as shown in FIG. 2, the column 10 comprises an inlet 30 for introducing a sample specimen into the interior of the column, an outlet 32 for removing the sample specimen and / or buffer solution from the column 10, and the beads 20 in 216B33215.DOCXAttorney Reference No. 10697-2506324the column 10 that bind with the target analytes. The beads 20 can be electrically connected to an amperometry voltmeter 34 forming an electrochemical sensor 28 with an anode and a cathode. For example, the sensor 28 can comprise leads 70 extending between the voltmeter 34 and the end caps 40, 42 of the column 10. The leads 70 can be electrically connected to a working electrode that extends, for example, through the endcaps 40, 42 and into the column 10, thereby providing the electrical connection between the interior of the column 10 and the voltmeter 34. In examples, the electrodes can comprise conductive members, such as a length of copper wire. More particularly, as shown in FIG. 2, leads 70 extending from the voltmeter 34 to the top or first end cap 40 can form an anode of the electrochemical sensor 28 and leads 70 extend from the volumeter to the bottom or second end cap 42 form the cathode of the electrochemical sensor 28, though polarity can be reversed if desired. In examples, the electrochemical sensor 28 can comprise one or more of the following electrode materials.

[0136] Working Electrode Material: Gold, platinum, or carbon electrodes are commonly used in amperometry. In some examples, a small section of the affinity chromatography column may be coated with gold nanoparticles or graphene for optimal signal transduction.

[0137] Counter and Reference Electrodes: Platinum wires or silver / silver chloride (Ag / AgCl) electrodes may be utilized for stable current measurement and accurate potentiometric control during amperometry.

[0138] The working electrode and the materials within the affinity chromatography column 10 desirably promote efficient electron transfer to facilitate amperometric measurements after biomarker binding. Potential materials can include any of the following.

[0139] Gold-Coated Beads: Incorporating gold nanoparticles or gold-coated beads 20 in the affinity chromatography column can enhance conductivity and allow for the direct coupling of redox-active mediators. Gold is widely used in electrochemical biosensors due to its excellent conductivity and biocompatibility.

[0140] Graphene or Carbon Nanotube Coating: Graphene-coated or carbon nanotube-modified beads can provide a high surface area for antibody binding while improving the conductivity of the system. These materials support the direct immobilization of antibodies and can boost the sensitivity of the amperometric detection.

[0141] In examples, the methods disclosed herein differ from other detection methods because the methods disclosed herein may not utilize labeling techniques such as Enzyme Linked Immunosorbent Assays (ELISA). The use of ELISA techniques (e.g. sandwich ELISA, direct ELISA, indirect ELISA, competitive ELISA) requires secondary antibodies and redox226B33215.DOCXAttorney Reference No. 10697-2506324reactions. They increase sample testing costs and may reduce the sensitivity of the diagnostic assay due to poor binding activities.

[0142] Furthermore, ELISA techniques generally produce chromogenic responses that are based on colorimetric analyses. Such colorimetric analyses generate qualitative results which output binary answers. For example, in the case of a pregnancy test, the development of a colored line in the “test line” section of the test indicates the presence of hcg hormone in the urine sample specimen. Such binary testing, however, does not indicate how much hcg hormone is present in the urine sample specimen. In order to turn a qualitative test as such into a quantitative test, an “electronic reader” must be utilized. These readers are often expensive and yield inconclusive results. Redox reactions used in such ELISA techniques produce species which could be utilized for electrochemical sensing. However, radicals and free-floating ions may disrupt the electrical signal, making the assay non-reliable.

[0143] By contrast, the methods disclosed herein use biosensors for detecting an analyte of interest in the affinity chromatography column. In presently available electrochemical biosensors, a redox-active mediator has been used to facilitate electron transfer between the analyte and the electrode. The mediator reacts with the analyte upon binding to the antibody, resulting in an electron transfer that generates a measurable current. For example, in glucose biosensors, glucose oxidase catalyzes the oxidation of glucose, producing hydrogen peroxide (H2O2), which is then oxidized at the electrode, generating a current proportional to the glucose concentration.

[0144] Similarly, the present inventor has recognized that if the antibody-antigen binding induces a redox reaction either through direct electron transfer or via a mediator, the current measured by the electrochemical sensor 28 (e.g., by an electrochemical sensor) will be proportional to the concentration of the bound target analyte. In some cases, the target analyte itself may be redox-active or capable of facilitating direct electron transfer when bound to the antibody on the electrode surface. For example, certain biomarkers or small molecules (e.g., hormones, neurotransmitters) may undergo oxidation or reduction upon binding, which can be directly detected via amperometry.

[0145] If the analyte is non-electroactive, it may not directly contribute to the detectable electric current. In such cases, sandwich ELISA can be and has been used to generate redox species which will facilitate electric current flow. Operating principles of sandwich ELISA were previously described. However, modifications to the electrode surface or the use of nanomaterials (e.g., gold nanoparticles, carbon nanotubes) may be needed in order to enhance electron transfer and make the binding event detectable.236B33215.DOCXAttorney Reference No. 10697-2506324

[0146] In some examples, conductive nanoparticles (e.g., gold, silver) can be conjugated to the target analyte or antibody. When binding occurs, these nanoparticles can enhance or impede the electron transfer at the electrode, leading to a measurable increase in current.

[0147] In some cases, the binding of the analyte to the antibody may not inherently cause a significant change in electric current. However, in such instances, signal amplification strategies may be used to enhance the detection sensitivity. For example, similar to ELISA, enzymes such as horseradish peroxidase or alkaline phosphatase, can be attached to the antigen or to the secondary antibody. When the target binds, the enzyme catalyzes a reaction that generates an electroactive product, which can be detected amperometrically. Upon binding of the target analyte to the antibody, the electric current measured at the working electrode changes, typically reflecting either an increase or decrease in electron transfer depending on the system’s design. The magnitude of the electric current is directly proportional to the concentration of the target analyte, allowing for quantitative analysis of biomarker levels in the sample. In the present disclosure, when a specific detection antibody binds to its target analyte (such as an antigen or biomarker), the binding event may be measured via amperometry (e.g., current flow), potential difference (e.g., voltage) or impedance (e.g., resistance to current flow).

[0148] The electric current flow, potential difference, or impedance change may occur due to the specific configuration of the electrodes and the nanoparticles (i.e., stationary phase of the affinity chromatography column). This will hold true if a redox mediator, enzyme label, or conductive nanoparticles are incorporated to facilitate electron transfer. The measured current would reflect the concentration of the analyte and provide a means for quantitative detection. The methods developed as described above may be validated by developing calibration curves and validation graphs prior to deploying in real life applications.

[0149] FIGS. 4 A and 4B show another example of an affinity chromatography column 110 with additional features for improving signal quality for detected electrical signals that are representative of analyte concentration. As in previous examples, the affinity chromatography column 110 is configured to perform quantitative or semi-quantitative analysis of one or more target analytes and can contain detection antibodies and / or nanoparticles that bind to target analytes. The affinity chromatography column 110 shown in FIGS. 4A and 4B can be used with any of the devices, systems, electrochemical sensors, and method, as previously described.

[0150] As shown in FIGS. 4A and 4B, the affinity chromatography column 110 comprises a column container 136 comprising a first end 112, a second end 114, and a sidewall 116 extending between the first end 112 and the second end 114. The column container 136 can be generally cylindrical in shape and, as described in further detail herein, can be configured 246B33215.DOCXAttorney Reference No. 10697-2506324to be placed on its side in a horizontal orientation during use. The column container 136 can comprise and / or can be formed from a hydrophobic and / or charge neutral material, such as a hydrophobic and / or charge neutral polymer (e.g., polycarbonate, poly(carboxybetaine) (PCB), polypropylene, polyethylene, or polyester).

[0151] The affinity chromatography column 110 further comprises a sample inlet or sample injection port 118 for introducing the sample specimen comprising a body fluid (e.g., nasal mucosa, urine, saliva, blood, plasma, or serum) into an interior of the container column 136. For example, the sample specimen may be injected through the port 118 with a fluid delivery device, as a syringe, pipette, or another injector. In examples, as shown in FIGS. 4A and 4B, the injection port 118 can extend through the sidewall 116 of the column container 136 and can be positioned to introduce the specimen sample near the top side of the column container 136, when the column container 136 is oriented horizontally, in order to reduce a likelihood that air bubbles will be introduced to the column container 136.

[0152] In examples, the sample injection port 118 can comprise a valve 138, such as a oneway valve, for permitting fluid flow into an interior of the column container 136 and restricting fluid flow out of the column container 136. In examples, the one-way valve 138 can be a duckbill valve comprising a flap that opens to permit fluid flow into the container 136 and closes preventing fluid flow out of the container 136.

[0153] The affinity chromatography column 110 further comprises a first end cap 140 over or sealed to the first end 112 of the column container 136 and a second end cap 142 over and / or sealed to the second end 114 of the column container 136. The first end cap 140 can comprise a first connector 144, such as a luer connector, configured for introducing fluid into an interior of the column container 136. Similarly, the second end cap 142 can comprise a second connector 146, such as a luer connector, configured for removing fluid from the interior of the column container 136 through the second connector 146. Beneficially, the luer connectors or luer locks can create a leak-proof seal for the connectors 144, 146 preventing leakage from ends of the connectors 144, 146 during use. In examples, as described in further detail herein, the first connector 144 and / or the second connector 146 can be configured to be connected to tubing 148 connected to a buffer pump 150 (shown in FIG. 4B) for circulating buffer solution through the interior of the column container 136 through the tubing 148 and first and second connectors 144, 146.

[0154] As shown in FIGS. 4A and 4B, the column container 136 further comprises a conductive layer 152 comprising, for example, layers of a conductive material, such as a conductive polymer, deposited over portions of an inner surface of the sidewall 116 of the 256B33215.DOCXAttorney Reference No. 10697-2506324column container 136. As described in further detail herein, the conductive layer 152 can be electrically connected to an electrical sensor, such as a voltmeter, by leads or electrodes to detect electrical signals influenced by the analyte concentration in the column 110. The conductive layer 152 can be provided to enhance detection of electrical signals compared to other electrode configurations (e.g., compared to conductive members that merely extend into the container 136 through end caps 140, 142) allowing for detection of electrical signals even at very low values.

[0155] In examples, the conductive polymer of the conductive layer 152 can comprise an intrinsically conducting polymer (ICP), such as polyallanine, polypyrrole or Poly (3,4-ethylenedi oxythiophene), over at least portions of the inner surface of the sidewall 116. The ICP provides points, spaces, or regions where the one or more target analytes and detection antibodies bound to the target analytes can be immobilized against and / or in contact with the ICP. As previously described, in examples, the conductive layer 152 can be electrically connected to and / or in communication with an electrical or electrochemical sensors, such as a voltmeter, for detecting electrical signals from the conductive layer 152.

[0156] In examples, the ICP at least partially covers a bottom side of the inner surface of the sidewall 116 when the column 110 is in a horizontal orientation. For example, when the column is viewed in a cross-sectional view, the ICP may cover an area extending from 90 degrees to 270 degrees or, preferably about 135 degrees to 225 degrees, near the bottom side of the sidewall 116.

[0157] In examples, the contact regions on the inner surface of the sidewall 116 can also comprise grooves 154, which are formed in the inner surface of the sidewall 116, in order to increase surface area available for analytes and other bound particles to connect to the ICP, compared to a flat surface without grooves 154. In examples, the crests, ridges, and / or troughs of the grooves 154 can be covered by the conductive layer 152 comprising the conductive polymer. As shown in FIGS. 4A and 4B, the grooves 154 can be arcuate grooves that extend at least partially about an axis of the column container 136. In other instances, the grooves 154 can be helical or can extend axially through the contact regions of the inner surface of the sidewall 116.

[0158] With continued reference to FIGS. 4A and 4B, the affinity chromatography column 110 can further comprise structures for releasing air bubbles from and / or venting the interior of the column container 136. In particular, the inventor has recognized that pipetting solution / fluid into the column 110 through the sample injection port 118 may cause a gas bubble(s) to form, which would lead to poor volumetric control. In order to avoid forming gas 266B33215.DOCXAttorney Reference No. 10697-2506324bubbles making it difficult to control volume of a buffer or reaction solution in the container 136, as shown in FIGS. 4 A and 4B, the affinity chromatography column 110 further comprises a degassing chamber 156 and a vent membrane 158 (e.g., a hydrophobic membrane, such as PTFE, forming a gas-liquid barrier that allows gas to escape while retaining fluid within the column container 136). The vent membrane 158 is positioned between the degassing chamber 156 and the interior of the column container 136 and is configured to allow gasses to escape from the interior of the column container 136 to the degassing chamber 156 through the vent membrane 158. More particularly, in examples, the vent membrane 158 covers an opening in the sidewall 116 of the column container 136 and is formed from a material that permits gas flow through the membrane 158. The degassing chamber 156 can comprise a gas dome, such as a flexible or elastomeric dome, over the vent membrane, which can be compressed or pushed and released to draw gas through the vent membrane 158 into the degassing chamber 156.

[0159] Rather than waiting for bubbles to pass through the column container to the outlet of the column, the degassing chamber 156 provides a port or opening in the sidewall 116 creating another location for removing bubbles from the interior of the container column 136. Beneficially, including the degassing chamber 156 can allow the affinity chromatography column 110 to be used in zero gravity and low gravity environments where gas bubbles would not move through the column container 136 by gravity.

[0160] FIG. 4B is a schematic drawing showing a system 102 for quantitative or semi-quantitative analysis of one or more target analytes using one or more of the affinity chromatography columns 110 of the present disclosure. In examples, as previously described, the system 102 can comprise a fluid or buffer solution circulation system for circulating fluid between the second connector and the first connector, as shown by the arrows Al in FIG. 4B. More particularly, in examples, the circulation system comprises the buffer pump 150 that is fluidly connected to the first connector 144 and the second connector 146 by a fluid conduit, such as the flexible plastic tubing 148. The pump 150 is configured to periodically draw fluid (e.g., buffer solution) from the second connector 146, through the pump 150, and to the first connector 144 in order to circulate the buffer solution through the column container 136.

[0161] The system 102 further comprises an oscillation system for causing the target analytes and detection antibodies or nanoparticles (e.g., metal nanoparticles) bound to the analytes to oscillate within the interior of the column container 136, for example, between a bottom side and a top side of the column container. In examples, the oscillation system can use conductive members, such as electromagnets, that are operated intermittently to move the analytes and bound antibodies through the column container (e.g., to cause the analytes and 276B33215.DOCXAttorney Reference No. 10697-2506324bound particles to alternate between levitating towards a top side of the container and gravitating towards a bottom side of the container). For example, as shown in FIG. 4B, the affinity chromatography column 110 can comprise a first conductive member 160, such as a metal coil forming an electromagnet, extending axially over a first portion of an exterior surface of the sidewall 116 of the column container 136 and a second conductive member 162, such as a second metal coil forming a second electromagnet, extending axially over a second portion of the exterior surface of the sidewall 116. In some instances, the first and / or second conductive members 160, 162 can be connected or adhered to the exterior surface of the column container 136. In other cases, the conductive members 160, 162 can be suspended above and below the column container leaving a gap between the conductive member(s) and the exterior surface of the sidewall 116. As shown in FIG. 4B, the conductive members 160, 162 can be positioned on opposing sides of the column container 136 spaced apart by, for example, about 180 degrees around the container 136.

[0162] As shown in FIG. 4B, the first conductive members 160 and the second conductive member 162 can be connected to a power source 164 for energizing the conductive members 160, 162 at desired times. In particular, in examples, the system 102 can be configured to intermittently and repeatedly energize the conductive members 160, 162 one after the other causing the analytes and bound antibodies to move through the column container 136. Intermittently energizing the first and second conductive members 160, 162 produces laminar flow of the analytes and bound antibodies through the container 136 between the bottom side of the container and the top side of the container as shown schematically in FIGS. 5A and 5B. More particular, as shown in FIG. 5 A, when the top or first conductive member 160 is energized (e.g., turned ON) and the bottom or second conductive member 162 is deenergized (e.g., turned OFF), particles 166 (e.g., the analytes and bound detection antigens) move upwards towards the first conductive member 160, as shown by arrow A2. By contrast, as shown in FIG. 5B, when the first conductive member 160 is deenergized (e.g., turned OFF) and the second conductive member 162 is energized (e.g., turned on) the particles 166 move downward as shown by arrow A3 towards the bottom of the second conductive member 162 and the bottom side of the column container 136. In examples, the system 102 can be configured to cause the bottom or second conductive member 162 proximate to the conductive (e.g., ICP) layer 152 to be energized last (as shown in FIG. 5B) causing the analytes and particles 166 to flow towards and become immobilized on the conductive layer 152 comprising the ICP.286B33215.DOCXAttorney Reference No. 10697-2506324

[0163] With reference again to FIG. 4B, in examples, the system 102 further comprises a controller 168, such as a computer processor or computing device, electrically connected to the buffer pump 150 and to the power source 164 that energizes the conductive members 160, 162. The controller 168 can be configured to periodically, intermittently, or continually cause the pump 150 to circulate the buffer solution through the column container 136. The controller 168 can also be configured to cause the power source 164 to periodically energize the conductive members 160, 162 according, for example, to a predetermined pattern or sequence. In particular, as previously described, the conductive members 160, 162 can be energized intermittently (e.g., one after the other) and repeatedly causing the analytes and bound particles to move through the column container 136. In examples, the controller 168 can be configured to apply the electric current to the second conductive member 162 (e.g., the conductive member 162 closest to the conductive layer 152) as a final oscillation step before ceasing applying electrical current to the conductive members 160, 162, thereby causing target analytes and detection antibodies to become immobilized against the conductive layer 152.

[0164] In examples, the system 102 further comprises a voltmeter 134 or multimeter that is electrically connected to the conductive layer 152, such as the ICP layer by, for example, leads or electrodes 170, forming an electrical sensor. The electrical sensor can be configured to detect electrical signals representative of the target analytes and detection antibodies immobilized on the conductive layer 152. As shown in FIG. 4B, the voltmeter 134 can be in communication with the system controller 168 and configured to transmit or provide detected electrical signals and / or detected voltage or current values to the controller 168 for processing and analysis. In such cases, the controller 168 can be configured to receive and process the electrical signal(s) detected by the voltmeter 134 and determine, for example, a concentration or level of the target analytes bound to the detection antibodies within the affinity chromatography column 136 based on analysis of the received and processed electric current signal(s). The controller 168 can also be configured to analyze the determined concentration or level of the target analytes bound to the detection antibodies within the affinity chromatography column 136 to make diagnostic conclusions from the sample specimen.

[0165] FIG. 6 is a flow chart showing steps of a detection method for quantitative or semi-quantitative analysis of one or more target analytes using one or more of the affinity chromatography columns 110 of the present disclosure. As shown in FIG. 6, at step 210, the method comprises introducing a sample specimen to the affinity chromatography column 110 through the sample injection port 118 of the affinity chromatography column 110. The sample specimen can be a biological or body fluid, such as nasal mucosa, urine, saliva, blood, plasma,296B33215.DOCXAttorney Reference No. 10697-2506324or serum. In examples, mucus can be mixed with a buffer solution to improve flowability prior to injection into the column container 136. As previously described, the affinity chromatography column 110 can comprise detection antibodies and markers, such as metallic nanoparticles, in the interior of the affinity chromatography column 110 that bind to the target analytes in the sample specimen. For example, the target analytes can comprise clinically relevant antigens, pathogens, or proteins (e.g., CA125, HE4, corona virus, albumin). In particular examples, the target analytes comprise a clinically relevant biomarker for detection of ovarian cancer or breast cancer. Alternatively or in addition, the target analytes can comprise one or more of the following clinically relevant biomarkers suitable for cancer detection: CA125, HE4, VEGF, APO-A1, Prostatin, Transthyretin, or Transferrin. In other examples, the detection antibodies in the affinity chromatography column 110 comprise antibody -conjugated-nanoparticles (e.g., metallic nanoparticles).

[0166] As previously described, the affinity chromatography column 110 can be used with a circulation system for circulating buffer solution through the column 110, which causes the analytes and bound particles to move through the column 136. For example, the first and second connectors 144, 146 of the affinity chromatography column 110 can be connected to the buffer pump 150 for circulating the buffer solution through the column 110. The affinity chromatography column 110 can also comprise the conductive members 160, 162 on the exterior surface of the column 110 that are intermittently energized causing the analytes and particles to oscillate through the column 110. Also, a sensing electrode can be electrically connected to the conductive layer 152 by, for example, leads or electrodes 170 for detecting electrical signals representative of analytes and bound particles immobilized on the conductive layer 152.

[0167] At step 212, once the specimen sample is in the container column 110, the method further comprises separating target analytes that are bound to the detection antibodies from unbound materials (e.g., unbound analytes or antibodies, as well as impurities in the sample specimen). In examples, as previously described, separation can be performed using beads 20 disposed within the affinity chromatography column 110 that comprise detection antibodies. For example, beads 20 can include magnetic beads, silica beads, or gold nanoparticles that bind with the target analytes. In other examples, the beads 20 can be agarose-based beads (e.g., Sepharose) that interact or engage the analytes and bound antigens. In these instances, the antigens or nanoparticles in the affinity chromatography column 110 are in a stationary phase of the affinity chromatography column and do not carry the sample specimen through the affinity chromatography column.306B33215.DOCXAttorney Reference No. 10697-2506324

[0168] In other examples, as previously described, separation can be performed by oscillating the target analytes and bound antigens within the column 110 causing the target analytes and bound antigens to be immobilized on the conductive layer 152 on the inner surface of the sidewall 116 of the column container 136. As previously described, the oscillation can be controlled so that the analyte and bound antigen particles flow towards the conductive layer 152 immediately prior to ceasing oscillation. More particularly, in examples, the conductive member 162 proximate to the conductive layer 152 can be energized last, causing the analytes and bound antigens to flow towards and become immobilized on the conductive layer 152. When it is believed that a reasonable number of analytes and bound antigen particles are on the conductive layer 152, the system 102 can be configured to cease applying the electric current to energize the conductive members 160, 162.

[0169] At step 214, along with separating the bound particles from unbound material, the method further comprises washing the affinity chromatography column 110 in order to remove the unbound materials from the affinity chromatography column 110. For example, washing the affinity chromatography column 110 can comprise circulating buffer solution through the affinity chromatography column 110 using, for example, the circulation system comprising the buffer pump 150. In examples, the buffer solution can comprise at least one of the following types of buffer solutions: a neutral buffer (e.g., PBS or TBS), a mild detergent (e.g., Tween-20 or Triton X-100), a regeneration buffer (e.g., glycine-HCl), or an electrochemical mediator. In examples, washing the affinity chromatography column 110 can also comprise periodically inverting or rolling the affinity chromatography column 110 in order to oscillate the one or more target analytes bound to the detection antibodies within the affinity chromatography column and / or to circulate buffer solution through the interior of the column 110.

[0170] At step 216, once it is believed that the reasonable number of analytes and particles are immobilized on the conductive layer 152, the method further comprises detecting electric current signal(s) representative of a quantity of the target analytes bound to the detection antibodies within the affinity chromatography column 110 with an electrical or electrochemical sensor. For example, the sensor can comprise an electrical voltage sensor (e.g., a voltmeter) that is electrically connected to the conductive layer 152 on the inner surface of the container 136 by the electrodes or leads 170. The voltmeter is configured to detect changes in voltage representative of a level or concentration of analyte and bound antigen particles that are immobilized on the conductive layer 152. In other examples, electrochemical sensors configured to detect the target analyte and bound detection antigens in the column container 136 can comprise beads 20 comprising, for example, nanoparticles (e.g., gold nanoparticles,316B33215.DOCXAttorney Reference No. 10697-2506324graphene nanotubes, or carbon nanotubes) dispersed in the column container 136. In such instances, working electrode(s) or leads can be used for electrically connecting the beads 20 to the voltmeter for detecting electric current and signals from the nanoparticles.

[0171] At step 218, the method further comprises analyzing the detected electric current signal(s) to determine a concentration or level of the target analytes bound to the detection antibodies within the affinity chromatography column 110 to make diagnostic conclusions from the sample specimen.

[0172] Although the invention has been described in detail for the purpose of illustration based on what is currently considered to be the most practical and preferred embodiments, it is to be understood that such detail is solely for that purpose and that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover modifications and equivalent arrangements. Furthermore, it is to be understood that the present invention contemplates that, to the extent possible, one or more features of any embodiment can be combined with one or more features of any other embodiment.EXAMPLES

[0173] To evaluate the methods disclosed herein, the present inventor performed the following experimental example showing that target particles can be detected using the affinity chromatography column and electrochemical sensor of the present disclosure. An explanation of testing performed using a prototype device follows.

[0174] An affinity chromatography column similar in structure to the affinity chromatography columns shown in FIGS. 1A-1C was made by 3-D printing with Polylactic acid (PLA). The column had a length of 50 mm, an outer diameter of 12 mm, and an inner diameter of 10 mm. End caps were placed over open ends of the column. As shown in FIGS.1A-1C, the column also included the sample injection port near the top of the column and extending from the sidewall of the column at an angle of about 45 degree angle.

[0175] The column was assembled by attaching the end caps to the open ends of the column and inserting copper wires through the end caps into the interior of the column, thereby forming a cathode and an anode portion of an electrochemical sensor. The column was filled with a HEPES buffer solution (HEPES buffer, IM, pH 7+ / -0.1 (Biologix, Camarillo, CA)). A multimeter was used as a measuring device to detect an electrochemical signal of the column.

[0176] The aim of testing was to determine if there was a difference between the potential difference (voltage) between polyvinylpyrrolidone (“PVP”, 4kDa molecular weight, 0.35g / mL bulk density (RND Center Inc., La Jolla, CA)) bound vs. unbound silver nanoparticles. PVP 326B33215.DOCXAttorney Reference No. 10697-2506324was used as an analog for a biomarker. Beneficially, PVP is a water soluble and non-toxic synthetic polymer, which is used widely in pharmaceutical products. These characteristics combined with the molecular weight, hydrophilicity and ring structure of the molecule makes it a suitable alternative molecule as a stand-in for experimental calibration, especially for the purposes of testing signal changes in the present electrochemical prototype due to macromolecular presence, viscosity shifts, or non-specific interactions. However, the PVP was not intended to mimic binding affinity, redox activity, or biological specificity of the real biomarkers.Method

[0177] This experimental example involved the following method. Approximately 6% w / v (60g / L) PVP solution was made by using 1.75g of PVP and adding HEPES buffer to make a final volume of 30mL. ImL of this PVP solution was incubated with ImL of 0.02mg / mL silver nanospheres (Silver nanosphere - 4nm, citrate buffer, 0.02mg / mL (Nanocomposix)) suspended in citrate buffer. The final solution was injected to the affinity chromatography column and left to incubate at room temperature for approximately 10 hours. To prevent agglomeration of silver nanospheres, the affinity chromatography column laying horizontally was inverted several times in approximately 5 hour intervals.

[0178] After the incubation period, this constituted the “Test Sample” listed below and discussed hereafter. Each sample listed below under “Controls and Test Samples” were injected into the affinity chromatography column using a ImL pipette tip and pipettor, through the sample injection port extending from the sidewall of the column. The voltage across the affinity chromatography column was measured by connecting the cathode and an anode of the Multimeter (Fluke 600V, CAT III) to the copper wires extending from the end caps of the column. Each sample listed below under “Controls and Test Samples” were tested in triplicates. The average is reported herein along with 1 standard deviation (1SD). In between measuring the voltage across the affinity chromatography column containing the Test Sample and the controls, the affinity chromatography column was washed with at least 5 mL of deionized (DI) water in order to prevent cross contamination.Controls and Test Samples

[0179] Test sample: 2mL PVP solution and silver nanospheres prepared as described above.

[0180] Negative Control 1: 2mL of HEPES buffer.

[0181] Positive control: 2mL of 0.02mg / mL silver nanospheres suspended in citrate buffer.336B33215.DOCXAttorney Reference No. 10697-2506324Results and Discussion

[0182] Multimeter Calibration: Prior to conducting tests, a known electronic, such as an AA battery, was used to check if the voltmeter (Fluke 600V CAT III Multimeter) used for detection, was functioning as intended. The voltmeter measured voltage ranging from 1.5 V to 1 ,7V for the AA battery.

[0183] Test Results: When testing was carried out as described in the methods section, there was a practical difference in the voltage values measured across the affinity chromatography column containing the Test Sample (0.012 ± 0.001V), Negative Control (0.006 ± 0.002V) and the Positive Control (0.021 ± 0.004V).

[0184] Individual voltage measurements across the affinity chromatography column containing 3 different types of fluids / solutions are provided in the following Table. Each replicate (denoted by Rl, R2, R3) was obtained from an independent sample.Table

[0185] Based on the evidence from testing, the inventor observed a measurable difference in the voltage / potential across the chromatography chamber when PVP is bound to the silver nanoparticles (Test Sample) compared to when no PVP present (Positive Control). As expected, the negative control (only buffer) produced the lowest measured voltage. This measurable signal difference between the Test Sample and the Positive Control may be attributed to the capping layer of citrates on the silver nanospheres being replaced by PVP molecules. PVP can displace citrate due to its stronger binding affinity to the surface of the silver nanospheres. This is not a redox chemistry, generating electrons which would lead to a potentiometric difference. Instead, it generates a different colloidal stability, which causes the altered electrochemical properties of the solution in the affinity chromatography column, leading to the measurable voltage difference shown in the Table.346B33215.DOCX

Claims

Attorney Reference No. 10697-2506324THE INVENTION CLAIMED IS:

1. A detection method for quantitative or semi-quantitative analysis of one or more target analytes, comprising:introducing a sample specimen to an affinity chromatography column through a port of the affinity chromatography column, the affinity chromatography column comprising detection antibodies and nanoparticles in an interior of the affinity chromatography column that bind to the one or more target analytes;separating the one or more target analytes that are bound to the detection antibodies and nanoparticles from unbound materials;washing the affinity chromatography column in order to remove the unbound materials from the affinity chromatography column; anddetecting electric current signal(s) representative of a quantity of the one or more target analytes bound to the detection antibodies and nanoparticles within the affinity chromatography column with at least one electrochemical sensor.

2. The method of claim 1, further comprising analyzing the detected electric current signal(s) to determine a concentration or level of the one or more target analytes bound to the detection antibodies and nanoparticles within the affinity chromatography column to make diagnostic conclusions from the sample specimen.

3. The method of claim 1, wherein the detection antibodies and nanoparticles in the affinity chromatography column comprise antibody-conjugated-nanoparticles.

4. The method of claim 1, wherein the one or more target analytes comprises at least one of a clinically relevant biomarker, antigen, pathogen, or protein (e.g., CA125, HE4, corona virus, albumin).

5. The method of claim 1, wherein the one or more target analytes comprises one or more of the following clinically relevant biomarkers suitable for cancer detection: CA125, HE4, VEGF, AP0-A1, Prostatin, Transthyretin, or Transferrin.

6. The method of claim 1, wherein the affinity chromatography column comprises a plurality of beads disposed within the affinity chromatography column comprising 356B33215.DOCXAttorney Reference No. 10697-2506324the detection antibodies and nanoparticles, andwherein the plurality of beads comprises at least one of magnetic beads, silica beads, or beads comprising gold nanoparticles.

7. The method of claim 1, wherein washing the affinity chromatography column comprises introducing a buffer solution through the affinity chromatography column, andwherein the buffer solution comprises at least one of the following types of buffer solutions: a neutral buffer (e.g., PBS or TBS), a mild detergent (e.g., Tween-20 or Triton X-100), a regeneration buffer (e.g., glycine-HCl), or an electrochemical mediator.

8. The method of claim 1, wherein the affinity chromatography column comprises:a column container comprising a first end, a second end, and a sidewall extending therebetween;at least one first connector over the first end of the column container configured for introducing a buffer solution through the at least one first connector into an interior of the column container;at least one second connector over the second end of the column container configured for removing the buffer solution from the interior of the column container through the at least one second connector; andat least one conductive layer comprising at least one intrinsically conducting polymer (ICP) over at least portions of an inner surface of the sidewall providing contact regions for the one or more target analytes and detection antibodies that are bound to the target analytes.

9. The method of claim 8, wherein detecting the electric current signal(s) is performed using electrodes that are electrically connected to the at least one conductive layer of the affinity chromatography column.

10. The method of claim 8, wherein the affinity chromatography column further comprises a degassing chamber and vent membrane positioned between the degassing chamber and the interior of the column container,the method further comprising allowing gasses to escape from the interior of the 366B33215.DOCXAttorney Reference No. 10697-2506324column container to the degassing chamber while washing the affinity chromatography chamber.

11. The method of claim 8, wherein the at least one ICP at least partially covers a bottom side of the inner surface of the sidewall of the column container when the column is in a horizontal orientation.

12. The method of claim 8, wherein the at least one ICP comprises at least one of polyallanine, polypyrrole or Poly(3,4-ethylenedioxythiophene).

13. The method of claim 8, wherein the contact regions on the inner surface of the sidewall comprise grooves for increasing surface area of the contact regions.

14. The method of claim 8, further comprising oscillating the one or more target analytes and detection antibodies that are bound to the target analytes through the interior of the column container by applying electric current to at least two conductive members repeatedly and one after another to introduce laminar flow and oscillating movement of the one or more analytes and bound antibodies between the at least two conductive members.

15. The method of claim 14, wherein the at least two conductive members of the affinity chromatography column comprise:a first conductive member extending axially over a first portion of an exterior surface of the sidewall; anda second conductive member extending axially over a second portion of the exterior surface of the sidewall.

16. The method of claim 8, wherein the port of the affinity chromatography column comprises a sample injection port extending through the sidewall of the column container, the sample injection port comprising a one-way valve for permitting fluid flow into an interior of the column container and restricting fluid flow out of the column container, and wherein introducing the sample specimen to the affinity chromatography column comprises introducing the sample specimen through the one-way valve.376B33215.DOCXAttorney Reference No. 10697-250632417. The method of claim 16, wherein the one-way valve comprises a duckbill valve.

18. The method of claim 8, wherein washing the affinity chromatography column in order to remove the unbound materials from the affinity chromatography column is performed by a buffer solution circulation system comprising a pump fluidly connected between the at least one first connector and the at least one second connector, which introduces the buffer solution into the interior of the column container through the at least one first connector and removes the buffer solution from the column container through the at least one second connector.

19. An affinity chromatography column for quantitative or semi-quantitative analysis of one or more target analytes, the affinity chromatography column comprising:a column container comprising a first end, a second end, and a sidewall extending therebetween;at least one first connector over the first end of the column container configured for introducing fluid through the at least one first connector into an interior of the column container;at least one second connector over the second end of the column container configured for removing fluid from the interior of the column container through the at least one second connector; andat least one conductive layer comprising at least one intrinsically conducting polymer (ICP) over at least portions of an interior surface of the sidewall of the column container providing contact regions for the one or more target analytes and metal nanoparticles that are bound to the target analytes.

20. A detection method for quantitative or semi-quantitative analysis of one or more target analytes, the method comprising:introducing a sample specimen to the interior of the affinity chromatography column of claim 19, wherein the affinity chromatography column comprises detection antibodies that bind to the one or more target analytes through the sample injection port of the affinity chromatography column; anddetecting electric current signal(s) representative of a quantity of the one or 386B33215.DOCXAttorney Reference No. 10697-2506324more target analytes bound to the detection antibodies within the affinity chromatography column with at least one electrochemical sensor.396B33215.DOCX

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