DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on September 22, 2025 has been entered.
Status of the Claims
Claims 1-26 were pending.
Claims 1, 19, 20, and 26 are amended.
Claims 1-26 are examined herein.
Withdrawn Rejections
The rejection of claim 26 under 35 U.S.C. 102 is withdrawn in view of claim 26 amendments. The amendments necessitated a new prior art search and new grounds of rejection have been found in view of newly found prior art reference of Cretich et al. (Expert Rev Mol Diagn. 2013 Nov;13(8):863-73). Claim 26 is rejected under 35 U.S.C. 103 as unpatentable over Hasenbank in view of Heurich, Mittermayar, and Cretich.
The rejection of claims 1-25 under 35 U.S.C. 103 is withdrawn in view of claims 1, 19, and 20 amendments. The amendments necessitated a new prior art search and new grounds of rejection have been found in view of newly found prior art reference of Cretich et al.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-13, 15, 18, and 24-25 are rejected under 35 U.S.C. 103 as being unpatentable over Hasenbank et al. (PGPub 2007/0202559) in view of Heurich et al. (Sensors and Actuators B: Chemical, Volume 156, Issue 1, 2011, Pages 162-168), Mittermayar (Austrian Patent AT505850), and Cretich et al. (Expert Rev Mol Diagn. 2013 Nov;13(8):863-73).
Regarding claims 1, 8-12, 15, 18, and 24-25, Hasenbank teaches “Chemical sensor enhanced by direct coupling of redox enzyme to conductive surface” (Title) and “a method, apparatus and system for detecting electrochemical oxidoreduction activity mediated by a redox enzyme” (Abstract). Hasenbank teaches method “comprises exposing the immobilized redox enzyme to conditions that effect oxidation or reduction of the enzyme, and detecting oxidation or reduction of the substrate” ([0007]).
Hasenbank also teaches a method comprising introducing simultaneously a reporter enzyme substrate, an electroactive mediator, and a precipitating agent to an electrode, thereby forming an electroactive mediator precipitating composition that reacts with the reporter enzyme conjugate to form an electroactive precipitate locally adsorbed at the surface of the electrode.
Specifically, Hasenbank teaches “enzyme precipitation system involving the enzyme horseradish peroxidase (HRP), and its calorimetric substrate, 3,3',5,5'-tetramethylbenzidine (TMB)” and “HRP is first oxidized by hydrogen peroxide; the oxidized HRP then, in turn, oxidizes a molecule of TMB into an insoluble blue form that, upon reaction with a precipitating agent, results in precipitate formation” ([0046]). HRP is the reporter enzyme, 3,3',5,5'-tetramethylbenzidine (TMB) is the electroactive mediator and hydrogen peroxide is the reporter enzyme substrate of the instant invention. Dextran sulfate is the precipitating agent and TMB is a part of the precipitate ([0007]).
Additionally, Hasenbank teaches analyte immobilized on gold surface ((Fig. 11.1).
Hasenbank fails to teach an electrochemical sensor comprising an electrode comprising a capture probe immobilized on a surface of the electrode, a target analyte bound to the capture probe, and a reporter enzyme conjugate bound to the target analyte; applying a voltage to the electrode, wherein the voltage corresponds to the standard redox potential of the electroactive precipitate; and measuring a current generated from the electrode of the electrochemical sensor to quantify an unknown concentration of the target analyte from the sample.
Heurich teaches “An electrochemical sensor based on carboxymethylated dextran modified gold surface for ochratoxin A analysis” (Title). Heurich also teaches providing an electrochemical sensor comprising an electrode comprising a capture probe immobilized on a surface of the electrode and a reporter enzyme; applying a voltage to the electrode, wherein the voltage corresponds to the standard redox potential of the electroactive precipitate; and measuring a current generated from the electrode of the electrochemical sensor to detect the target analyte.
Specifically, Heurich teaches “An indirect competitive enzyme-linked immunosorbent assay (ELISA) format was constructed by immobilizing ochratoxin A conjugate using passive adsorption or covalent immobilization via amine coupling to a carboxymethylated dextran (CMD) hydrogel on the gold working electrode” (Abstract). Therefore, Heurich teaches an electrochemical sensor comprising an electrode and a reporter enzyme conjugate (Fig. 1).
Additionally, Heurich teaches “low background current is optimal for enzyme activity determination when a small amount of catalysis product (TMBox) needs to be measured in the presence of high concentrations of substrate. A working potential of -150mV was chosen for this sensor for chronoamperometric measurements” (pg. 165, Col.2, 2nd paragraph, and Fig. 2). Working potential of -150 mV approximately corresponds to -0.2 V recited in [00186] of the instant specification. The fact that Heurich observed current during the analysis (Fig. 1) indicates that the applied voltage of −150 mV corresponded to the standard redox potential of the TMB precipitate. Fig. 1 teaches exposing the reporter enzyme conjugate to a reporter enzyme substrate and an electroactive mediator to at least partially oxidize or reduce (see Fig. 1 for converting TMB(ox) into TMBRed form) the electroactive mediator.
Heurich also teaches the electrode is a planar electrode. Specifically, Heurich teaches “Stainless steel screens with a screen mesh size of 200 counts per inch were used to print the electrodes, which meets the limitation of claim 15 for a planar electrode. The electrodes were printed onto 250 µm thick Melinex polyester sheet. Typical film thickness of screen-printed sensors ranged from 10 to 50 µm” (pg. 163, Col. 2, last paragraph).
Heurich also teaches the generated current corresponds to a current derived from reduction of the fully or partially oxidized electroactive precipitate (claim 18) or a current
derived from oxidation of the fully or partially reduced electroactive precipitate (claim 25). Specifically, Heurich teaches “Fig. 2 shows the cyclic voltammograms at increasing scan rates displaying the characteristic TMB double shoulder on the positive scan, which are the result from the two 1-electron oxidation steps of TMB. The negative scan is illustrating a 2-electron reduction step” (pg. 165, Col. 1, last paragraph and Col. 2, 1st paragraph; Fig. 1). One having ordinary skill in the art practicing methods taught by Hasenbank and Heurich would necessarily expect the current corresponding to a reduction or oxidation current derived from reduction of the fully or partially oxidized or reduced electroactive precipitate to be present during the assay. Hasenbank teaches tetramethylbenzidine (TMB) is the precipitate ([0007]).
Hasenbank and Heurich do not specifically teach the detection method of claims 1 and 26 wherein the target analyte is captured in a sandwich configuration with the reporter enzyme conjugate bound to the target analyte. Heurich teaches a competitive ELISA method with BSA immobilized on the gold electrode. Both sandwich and competitive assay formats are widely known in the field of assays - Cretich teaches both assay formats in Fig. 2A and Fig. 2B respectively. Specifically, Cretich teaches two assay formats utilizing different capture reagents immobilized on a solid surface, but using the same method of detection of the formed capture complex – a secondary antibody labeled with a generic label.
Fig. 2A illustrates the sandwich assay configuration of instant invention using antibody planar microarray as an example, with antibodies immobilized on a solid surface for capture of biomarkers from a sample. The bound analytes are visualized using fluorescently tagged detection antibodies (pg. 865, col. 1, par. 5). The planar microarray of Cretich is equivalent to the electrode of instant invention comprising a capture probe immobilized on a surface of the electrode.
Fig. 2B illustrates the competitive ELISA method of Heurich, using antigen planar microarrays as an example, with target analytes (proteins, peptides, or small molecules) immobilized on the support and detected using a labeled secondary antibody (pg. 865, col. 1, par. 6). Both approaches share the same kind of the label – a labeled secondary antibody. The electrochemical sensor and detection method taught by Hasenbank and Heurich function as an electrochemical label and therefore, can be used with both sandwich and competitive assay formats, since a wide variety of different labels based on different detection principles have been successfully applied to both competitive and sandwich assays.
Additionally, Heurich teaches quantifying ochratoxin A using current measurement “the amount of bound ochratoxin A was then determined using chronoamperometry at a set potential of −150 mV” (pg. 164, Col. 2, section 2.2.4 “Amperometric immunosensor for ochratoxin A”). Although, Heurich teaches using current measurement in a competitive assay format, this approach is equally applicable to a sandwich assay format as well, because it is the label enzyme (HRP) and its substrate (TMB) that provide the target analyte detection using current measurement. In the sandwich format the amount of the reporter conjugate bound to the electrode via the target analyte and the capture probe will be proportional to the amount of the target analyte. Therefore, Heurich in view of Cretich teach measuring a current generated from the electrode of the electrochemical sensor to quantify an unknown concentration of the target analyte from the sample.
Finally, Heurich teaches the reporter enzyme conjugate comprises an enzyme conjugated with an antibody (Claim 10) and the reporter enzyme conjugate comprises horseradish peroxidase (HRP) (Claims 11 and 12) - an anti-ochratoxin A antibody, which is later detected using anti-rabbit–IgG-HRP conjugate (pg. 164, Col. 2, 1st paragraph). The anti-rabbit–IgG-HRP conjugate is an anti-rabbit IgG antibody conjugated to HRP, which meets the limitations of claims 10-12. Although, Heurich teaches using the reporter enzyme conjugate conjugated with an antibody configured for use in the competitive assay of ochratoxin A, this approach is equally applicable to a sandwich assay format as well, wherein the antibody-HRP conjugate will be bound to the target analyte bound to the capture probe on the electrode. Therefore, Heurich in view of Cretich teach the reporter enzyme conjugate comprises an enzyme conjugated with an antibody and the reporter enzyme conjugate comprises horseradish peroxidase (HRP).
Hasenbank, Heurich, and Cretich fail to teach introducing simultaneously a reporter enzyme substrate, an electroactive mediator, and a precipitating agent to the electrode.
Mittermayar teaches “a method, a microfluidic device, and a kit for the detection and / or determination of bacteria associated with periodontitis from a biological sample.” (pg. 1, Description). The reference teaches “the detection is carried out colorimetrically, wherein tetramethylbenzidine, preferably dissolved in polyvinylpyrrolidone, is added to commercial tetramethylbenzidine reaction mixtures, the color reaction being added, whereby the visual signal is amplified by the formation of complementary colors” (pg. 4, 9th paragraph). Polyvinylpyrrolidone is another name for a pyrrolidinone polymer of claim 9.
Additionally, Mittermayar teaches premixing the electroactive mediator and the precipitating agent “[f]or the preparation of the color reaction solution TMB and TMB/PVP solution and Proclin 200 are mixed or can also be a commercially available precipitate-forming TMB solutions are used” (pg. 5, 3rd sentence from the end), where PVP is polyvinylpyrrolidone. Therefore, Mittermayar teaches simultaneous introduction of an electroactive mediator and a precipitating agent as a TMB/PVP solution.
Mittermayar does not explicitly teach simultaneous addition of the reporter enzyme substrate together with the electroactive mediator and the precipitating agent. However, the reporter enzyme substrate and the electroactive mediator are both substrates of the reporter enzyme and their simultaneous presence is required for proper functioning of the reporter enzyme. Therefore, Mittermayar inherently teaches simultaneous introduction of the reporter enzyme substrate, the electroactive mediator, and the precipitating agent. All three components are present in the same composition, meeting the limitation of claim 24 reciting the reporter enzyme substrate, the electroactive mediator and the precipitating agent are comprised in a same composition.
Finally, Mittermayar teaches using PVP as a precipitate-forming reagent for TMB (pg. 5, 3rd sentence from the end), therefore, PVP can be used as a TMB precipitating agent instead of dextran sulfate, meeting the limitations of claims 8-9, reciting the precipitating agent is a pyrrolidinone polymer.
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to modify the method of visual detection of the reaction results of Hasenbank by employing measuring the generated current to detect a target analyte as taught by Heurich, in order to provide electrochemical detection method using inexpensive, mass-produced, disposable sensors (Heurich, pg. 163, Col. 1, 2nd paragraph). One having ordinary skill in the art would have been motivated to make such a change because electrochemical detection allows a wide linear dynamic detection range of 0.01–100 µg/L (Heurich, Abstract). The use of such combination would have been desirable to those of ordinary skill in the art because small, portable diagnostic devices with disposable sensors are useful in the field of clinical diagnosis, environmental and food analysis (Heurich, pg. 163, Col. 1, 2nd paragraph) and these portable devices can directly detect analytes from wide range of samples without time-consuming sample dilution or concentrating steps.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because methods of Hasenbank and Heurich are similarly drawn to detection methods utilizing TMB as an electroactive mediator.
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method for electrochemical detection of Hasenbank and Heurich by employing it in a sandwich format assay as taught by Cretich, in order to provide benefits of electrochemical detection with its wide linear dynamic detection range to sandwich assays, as an obvious matter of using of known technique (electrochemical detection of Hasenbank and Heurich) to improve similar methods (sandwich assays) in the same way.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references of Hasenbank, Heurich and Cretich because Hasenbank and Heurich teach electrochemical detection as a label for immunoassay and Cretich teaches a generic approach of detecting captured analyte molecules in both competitive and sandwich formats on a solid support using a generic labeled secondary antibody.
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified a method for electrochemical detection of Hasenbank, Heurich, and Cretich by combining the reporter enzyme substrate and the electroactive mediator with the precipitating agent (polyvinylpyrrolidone) as taught by Mittermayar, in order to form a single solution containing all three reaction components. One having ordinary skill in the art would have been motivated to make such a change because of obvious convenience of adding only one solution containing all three reaction components.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references of Hasenbank, Heurich, and Cretich with Mittermayar because Hasenbank teaches that the reporter enzyme substrate and the electroactive mediator can be successively combined in one reaction ([0046]) and the precipitating agent (polyvinylpyrrolidone) of Mittermayar reacts with a product of the reporter enzyme reaction.
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified a method for electrochemical detection of Hasenbank, Heurich, and Cretich by employing polyvinylpyrrolidone (a pyrrolidinone polymer) as a precipitating agent as taught by Mittermayar, in order to provide an electrochemical detection method, as an obvious matter of simple substitution of one known element (polyvinylpyrrolidone of Mittermayar, pg. 4, 9th paragraph) for another (dextran sulfate of Hasenbank [0007]).
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references with Mittermayar because Mittermayar teaches that polyvinylpyrrolidone is compatible with electrochemical detection.
Regarding claims 2 and 3, Hasenbank teaches the electrochemical sensor comprises a plurality of electrodes and a plurality of target analytes. Specifically, Hasenbank teaches “the invention further provides a method, apparatus and system for detecting a plurality of analytes” ([0011]), and “Design for a multi-analyte assay. The 3x5 array of rectangular regions (and the two large rectangular regions on either side of the array) were coated with gold. Each region of the array, since it is electrically isolated from the other regions, may be treated as an independent measurement” ([0019] and Fig. 7).
Regions of the array, electrically isolated from the other regions, correspond to the plurality of electrodes of claim 2 and one of the electrodes is the first electrode of the instant invention. The 3x5 electrode array meets the limitation of claim 3 for at least two electrodes in the plurality of electrodes.
Regarding claims 4 and 5, Hasenbank teaches the electroactive mediator is 3,3',5,5'-tetramethylbenzidine “a substrate capable of producing a detectable signal upon oxidation or reduction include a colorimetric or fluorogenic enzyme substrate, such as tetramethylbenzidine (TMB)” ([0007]), meeting the limitations of claims 4 and 5.
Regarding claims 6 and 7, Hasenbank teaches the reporter enzyme substrate is hydrogen peroxide “the specific enzymatic catalytic cycle is as follows: HRP is first oxidized
by hydrogen peroxide” ([0046]), meeting the limitations of claims 6 and 7. Hasenbank does not explicitly call hydrogen peroxide a reporter enzyme substrate, but since hydrogen peroxide interacts with the reporter enzyme and gets consumed in the course of this reaction, it is one of the substrates of this reaction.
Regarding claim 13, Hasenbank teaches the electrochemical sensor comprises one or more microfluidic flow cells “For most experiments, a microfluidic flow cell was then assembled from laminate sheets of Mylar and adhesive, and the pre-patterned gold-coated microscope slide was used as the bottom layer of the flow cell” ([0048]).
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Hasenbank in view of Heurich, Cretich, and Mittermayar, as applied to claim 1 above, and further in view of Henkens et al. (IDS; USP 6,391,558).
The teachings of Hasenbank, Heurich, Cretich, and Mittermayar have been set forth above.
Hasenbank, Heurich, Cretich, and Mittermayar fail to teach a method using an electrochemical sensor comprising one or more open wells.
Henkens teaches “Electrochemical detection of nucleic acid sequences” (Title) and “The system utilizes biological probes such as nucleic acid or peptide nucleic acid probes which are complementary to and specifically hybridize with selected nucleic acid segments in order to generate a measurable current when an amperometric potential is applied. The electrochemical signal can be quantified” (Abstract). Henkens also teaches the electrochemical sensor comprises one or more open wells.
Specifically, Henkens teaches “The biosensor is preferably small in size for easy handling, and the working electrode may have a surface area from about 0.001 mm2 to about 100 mm2. The array may comprise several sample wells including 4, 96, 384, 1536-well configurations or larger” (Col. 5, lines 44-49) and “The three electrodes are contained within a bean-shaped depression which serves as a sample well” (Col. 20, lines 53-55).
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method for electrochemical detection method of Hasenbank, Heurich, Cretich, and Mittermayar by employing open wells as taught by Henkens, in order to provide an electrochemical detection method using sensors in an open well format, as an obvious matter to try, namely choosing from a finite list of suitable, art recognized/known alternatives for a cell design: open or enclosed (e.g., microfluidic format).
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because using electrochemical cell with an open well is a traditional approach for prototyping electrochemical sensors prior to mass production.
Claims 16 and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Hasenbank in view of Heurich, Cretich, and Mittermayar, as applied to claim 1 above, and further in view of Gordon et al. (WO 2010/003212).
The teachings of Hasenbank, Heurich, Cretich, and Mittermayar have been set forth above.
Hasenbank, Heurich, Cretich, and Mittermayar fail to teach a counter electrode and a reference electrode.
Gordon teaches “A biosensing device for detecting a presence of target biomolecules is provided, including at least one working electrode having a systematic array of nano-electrode wires projecting vertically from an electrode pad” (Abstract). Gordon also teaches electrochemical sensor further comprises (a) a counter electrode and a reference electrode (claim 16) and (b) a positive control electrode and/or a negative control electrode (claim 17).
Specifically, Gordon teaches “expression "electrochemical sensor" refers to an electrochemical system that determines the presence and concentration of a chemical material through measurements of electrical signal in a solution between a working electrode and counter electrode such as induced by a redox reaction or electrical potential from the release or absorption of ions” (pg. 9, lines 24-28), and “The potential of the working electrode is measured against a reference electrode which is typically a stable, well-behaved electrochemical half-cell such as silver/silver chloride” (pg. 10, lines 2-4).
Additionally, Gordon teaches “The biosensing device further includes a top assembly extending over the bottom assembly and including a reference electrode and at least one counter electrode” (pg. 5 lines 1-2), and “assembly may have any appropriate number of working and control electrodes as required for a given application of the biosensing device. In its simplest form, the bottom assembly may have 2 electrodes, a working electrode for detecting the target biomolecules and a negative control electrode for measuring the background noise used to determine the detection threshold limit. In another embodiment, a biosensing device containing 16 electrodes may be considered where 14 of them are working electrodes having biosensor probes that interact with different target biomolecules, one electrode being the negative control electrode used to measure the background noise, and one electrode being the positive control electrode used to measure a positive signal from a known biomolecule intentionally added to the analyte solution to ensure that the biosensing device is functioning” (pg. 16, lines 17-24).
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method for electrochemical detection of Hasenbank, Heurich, Cretich, and Mittermayar by employing additional electrodes as taught by Gordon, in order to provide an electrochemical detection method using sensors, comprising of working, control and reference electrodes, as well as, positive and negative control electrodes. One having ordinary skill in the art would have been motivated to make such a change because a pair of working and control electrodes provides measurements related to the amount of analyte and is a minimal number of electrodes in a sensor; the reference electrode provides a stable, well-behaved electrochemical half-cell such as silver/silver chloride, for measuring the potential of the working electrode (Gordon, pg. 10, lines 2-4); and the positive and negative control electrodes provide integrated into the sensor equivalents of the positive and negative assay reactions (Gordon, pg. 16, lines 17-24). The use of such combination would have been desirable to those of ordinary skill in the art because integrating all electrodes into one sensor simplifies detection of analytes in remote locations and point-of-care settings.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because electrodes taught by Gordon are already integral parts of many electrochemical sensors, built on similar principles as the sensors of Hasenbank and Heurich.
Claims 19-20 are rejected under 35 U.S.C. 103 as being unpatentable over Hasenbank in view of Heurich, Cretich, and Mittermayar, and further in view of Henkens.
Regarding claims 19-20, Hasenbank teaches “Chemical sensor enhanced by direct coupling of redox enzyme to conductive surface” (Title) and “invention provides a method, apparatus and system for detecting electrochemical oxidoreduction activity mediated by a redox enzyme” (Abstract). Hasenbank teaches method “comprises exposing the immobilized redox enzyme to conditions that effect oxidation or reduction of the enzyme, and detecting oxidation or reduction of the substrate” ([0007]).
Hasenbank also teaches a kit for electrochemical detection of a target analyte in a sample comprising:
a fluid-contact surface and an electrode immobilized on at least a portion of the fluid-contact surface, wherein the electrode is functionalized with a capture probe bound to a target analyte;
a reporter enzyme conjugate configured to be immobilized on the electrode via the target analyte bound to the capture probe; and
an electroactive mediator precipitating composition comprising a reporter enzyme substrate, an electroactive mediator, and a precipitating agent, wherein the electroactive mediator precipitating composition is configured to react with the reporter enzyme conjugate to at least partially oxidize or reduce the electroactive mediator which forms an electroactive precipitate locally adsorbed at a surface of the electrode.
Specifically, Hasenbank teaches “enzyme precipitation system involving the enzyme horseradish peroxidase (HRP), and its calorimetric substrate, 3,3',5,5'-tetramethylbenzidine (TMB)” and “HRP is first oxidized by hydrogen peroxide; the oxidized HRP then, in turn, oxidizes a molecule of TMB into an insoluble blue form that, upon reaction with a precipitating agent, results in precipitate formation” ([0046]). HRP is the reporter enzyme, 3,3',5,5'-tetramethylbenzidine (TMB) is the electroactive mediator and hydrogen peroxide is the reporter enzyme substrate of the instant invention. Dextran sulfate is the precipitating agent and TMB is a part of the precipitate ([0007]). Additionally, Hasenbank teaches analyte immobilized on gold surface (Fig. 11.1) and TMB forming blue precipitate locally on the gold surface (Fig. 11.7).
Hasenbank fails to teach an electrochemical sensor comprising an electrode functionalized with a capture probe bound to a target analyte.
Heurich teaches “An electrochemical sensor based on carboxymethylated dextran modified gold surface for ochratoxin A analysis” (Title). Heurich also teaches the electrochemical sensor comprising an electrode functionalized with a capture probe bound to a target analyte.
Specifically, Heurich teaches “An indirect competitive enzyme-linked immunosorbent assay (ELISA) format was constructed by immobilizing ochratoxin A conjugate using passive adsorption or covalent immobilization via amine coupling to a carboxymethylated dextran (CMD) hydrogel on the gold working electrode” (Abstract). Therefore, Heurich teaches an electrochemical sensor comprising an electrode. BSA molecule immobilized on the gold electrode is a capture probe, ochratoxin A is a target analyte bound to the capture probe, and secondary antibody-HRP conjugate is a reporter enzyme conjugate bound to the target analyte (Fig. 1). As such, the HRP is configured via its secondary antibody and a primary antibody to be immobilized via the target analyte bound to the capture probe. Fig. 1 teaches exposing the reporter enzyme conjugate to a reporter enzyme substrate and an electroactive mediator to at least partially oxidize or reduce (see Fig. 1 for converting TMB(ox) into TMBRed form) the electroactive mediator.
Heurich teaches indirect ELISA assay (pg. 164, col. 1, section “2.2.3. Indirect competitive immunoassay for ochratoxin A”) and the capture probe-analyte conjugate binds analyte-specific antibody “polyclonal antibody raised against ochratoxin A” (pg. 164, col. 2, par. 1).
Hasenbank and Heurich do not specifically teach electrochemical detection and/or quantification of an unknown concentration of target analyte and a sensor, wherein the capture probe is directly immobilized on at least a portion of the electrode.
Heurich teaches a competitive ELISA method with BSA immobilized on the gold electrode. Electrochemical sensor and detection method taught by Heurich can be used with both sandwich and competitive assay formats. While Heurich teaches a competitive ELISA method using electrochemical detection by HRP enzyme and TMB substrate, Cretich teaches both sandwich and competitive assay methods utilizing different capture reagents immobilized on a solid surface, but using the same method of detection of the formed capture complex – a secondary antibody labeled with a generic label (Fig. 2A and Fig. 2B respectively).
Specifically, Cretich teaches two assay formats utilizing different capture reagents immobilized on a solid surface, but using the same method of detection of the formed capture complex – a secondary antibody labeled with a generic label.
Fig. 2A illustrates the sandwich assay configuration of instant invention using antibody planar microarray as an example, with antibodies immobilized on a solid surface for capture of biomarkers from a sample. The bound analytes are visualized using fluorescently tagged detection antibodies (pg. 865, col. 1, par. 5). The planar microarray of Cretich is equivalent to the electrode of instant invention comprising a capture probe immobilized on a surface of the electrode.
Fig. 2B illustrates the competitive ELISA method of Heurich, using antigen planar microarrays as an example, with target analytes (proteins, peptides, or small molecules) immobilized on the support and detected using a labeled secondary antibody (pg. 865, col. 1, par. 6). Both approaches share the same kind of the label – a labeled secondary antibody. The electrochemical sensor and detection method taught by Heurich function as an electrochemical label and therefore, can be used with both sandwich and competitive assay formats, since prior art recognizes wide variety of different labels based on different detection principles successfully applied to both competitive and sandwich assay.
Additionally, Cretich teaches sandwich format assay, wherein the capture probe is immobilized on a solid support (Fig. 2A). Therefore, Heurich in view of Cretich teaches the capture probe is directly immobilized on at least a portion of the electrode.
Regarding the kit format, Henkens teaches “Such kits contain monitors, reagents and procedures that can be utilized in a clinical or research setting or adapted for either the field laboratory or on-site use. In particular, kits comprising the disclosed biosensor or biosensor array, or an apparatus comprising the biosensor in an integrated chip form, or a system that includes any of a number of means for detecting the captured target molecule and measuring the electrochemical signal produced subsequent to target capture, along with appropriate instructions, are contemplated. Kits comprising electrodes with one or more capture or reporter probes attached to the electrode surface” (Col. 9, lines 10-18).
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to modify the sensor and reagents for visual detection of the reaction results of Hasenbank by employing an electrode-based sensor as taught by Heurich, in order to provide a kit for electrochemical detection using inexpensive, mass-produced, disposable sensors (Heurich, pg. 163, Col. 1, 2nd paragraph). One having ordinary skill in the art would have been motivated to make such a change because electrochemical detection allows a wide linear dynamic detection range of 0.01–100 µg/L (Heurich, Abstract). The use of such combination would have been desirable to those of ordinary skill in the art because small, portable diagnostic devices with disposable sensors are useful in the field of clinical diagnosis, environmental and food analysis (Heurich, pg. 163, Col. 1, 2nd paragraph). The use of such combination in the form of a kit would have been desirable to those of ordinary skill in the art because kits provide a convenience of different reagents preassembled for specific assays and the kit format saves money and resources, and reduces waste. Using immobilized capture probes is inherent to assays involving target capture on a solid surface.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because methods of Hasenbank and Heurich are similarly drawn to detection methods utilizing TMB as an electroactive mediator.
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method for electrochemical detection of Hasenbank and Heurich by employing it in a sandwich format assay as taught by Cretich, in order to provide benefits of electrochemical detection with its wide linear dynamic detection range to sandwich assays, as an obvious matter of using of known technique (electrochemical detection of Hasenbank and Heurich) to improve similar methods (sandwich assays) in the same way.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references of Hasenbank, Heurich and Cretich because Hasenbank and Heurich teach electrochemical detection as a label for immunoassay and Cretich teaches a generic approach of detecting captured analyte molecules in both competitive and sandwich formats on a solid support using a generic labeled secondary antibody.
Claims 21-23 are rejected under 35 U.S.C. 103 as being unpatentable over Hasenbank in view of Heurich, Cretich, and Mittermayar, as applied to claim 1 above, and further in view of Akter et al. (Anal Chem. 2012 Aug 7;84(15):6407-15).
The teachings of Hasenbank, Heurich, Cretich, and Mittermayar have been set forth above.
Hasenbank, Heurich, Cretich, and Mittermayar do not specifically teach the reporter enzyme conjugate comprises a plurality of enzymes, the target analyte is bound to a plurality of reporter enzyme conjugates, and the target analyte is bound to a nanoparticle functionalized with a second capture probe for binding with the target analyte.
Claim 23 recites the target analyte is bound to a plurality of reporter enzyme conjugates. This limitation is interpreted as a single molecule of a target analyte bound to a conjugate comprising a plurality of reporter enzymes.
Regarding claim 22, Akter teaches an electrochemical immunosensor based on multiwall carbon nanotubes (MWCNTs)/gold nanoparticles (AuNPs) (Abstract). Akter also teaches nanoparticles bound to an electrode and functionalized with second capture probe.
Specifically, Akter teaches an electrochemical immunosensor based on gold nanoparticles (AuNPs) attached to a gold electrode and functionalized with monoclonal anti-PSA antibody (Abstract), where PSA is a target analyte. AuNPs are attached to a gold electrode and functionalized with monoclonal anti-PSA antibody (PSA is prostate specific antigen). A polyclonal anti-PSA antibody – the second capture probe of the instant claim (Ab2, pg. 6409, Col. 2, section “Fabrication of the Immunosensor Probe and the Sensing Procedure”) is conjugated with carbon nanotubes-HRP complexes (id.). The HRP reporter enzymes are adapted with the aid of anti-PSA polyclonal antibody for immobilizing on an electrode via PSA target analyte bound to the capture probe (monoclonal anti-PSA antibody).
Regarding claims 21 and 23, Akter teaches amplified detection of PSA “using a higher number of horseradish peroxidase (HRP) molecules attached on MWCNTs” (Abstract). Multiple HRPs attached to the carbon nanotubes meet the limitations of claim 21 reciting the reporter enzyme conjugate comprises a plurality of enzymes and claim 23 reciting the target analyte is bound to a plurality of reporter enzyme conjugates.
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Hasenbank, Heurich, Cretich, and Mittermayar by employing a nanoparticle bound to the target electrode and functionalized with a second capture probe for binding with the target analyte as taught by Akter, in order to provide a better sensitivity of the assay by using a higher number of HRP molecules attached to MWCNTs (Akter, Abstract). One having ordinary skill in the art would have been motivated to make such a change because increasing assay sensitivity is an ultimate quest in the art. The use of such combination would have been desirable to those of ordinary skill in the art because it is widely known in the art that better assay sensitivity is highly desirable for variety of reasons.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because methods of Hasenbank, Heurich, and Akter are similarly drawn to HRP as a reporter enzyme. Cretich is generic with respect to specific label mechanism.
Claim 26 is rejected under 35 U.S.C. 103 as being unpatentable over Heurich in view of Cretich.
Regarding claim 26, Heurich teaches a method of using a disposable electrochemical immunosensor for ochratoxin A analysis (Abstract), the method comprising:
specifically binding the target analyte to a capture probe immobilized on the surface of an electrode – BSA molecule immobilized on the gold electrode is conjugated to a capture probe - ochratoxin A, which specifically binds the primary antibody (which is equivalent to the target analyte in a competitive assay of Heurich) (Fig. 1);
target analyte (the primary antibody) specifically bound to the capture probe (ochratoxin A), and a reporter enzyme conjugate bound to the target analyte - the secondary antibody-HRP conjugate binds to the primary antibody (Fig. 1);
exposing the reporter enzyme conjugate to a reporter enzyme substrate and an electroactive mediator to at least partially oxidize or reduce (see Fig. 1 for converting TMB(ox) into TMBRed form) the electroactive mediator which forms an electroactive precipitate on the surface of the electrode from the electroactive mediator – specifically, hydrogen peroxide (substrate) and TMB (electroactive mediator) are added to the immunosensor; and
measuring a current from oxidizing or reducing the electroactive precipitate to detect the target analyte (Fig. 2).
Heurich does not specifically teach the detection method of claim 26 comprising introducing a sample comprising an unknown concentration of the target analyte to an electrode.
Heurich teaches a competitive ELISA method with BSA immobilized on the gold electrode. Electrochemical sensor and detection method taught by Heurich can be used with both sandwich and competitive assay formats. While Heurich teaches a competitive ELISA method using electrochemical detection by HRP enzyme and TMB substrate, Cretich teaches both sandwich and competitive assay methods utilizing different capture reagents immobilized on a solid surface, but using the same method of detection of the formed capture complex – a secondary antibody labeled with a generic label (Fig. 2A and Fig. 2B respectively).
Specifically, Cretich teaches two assay formats utilizing different capture reagents immobilized on a solid surface, but using the same method of detection of the formed capture complex – a secondary antibody labeled with a generic label.
Fig. 2A illustrates the sandwich assay configuration of instant invention using antibody planar microarray as an example, with antibodies immobilized on a solid surface for capture of biomarkers from a sample. The bound analytes are visualized using fluorescently tagged detection antibodies (pg. 865, col. 1, par. 5). The planar microarray of Cretich is equivalent to the electrode of instant invention comprising a capture probe immobilized on a surface of the electrode.
Fig. 2B illustrates the competitive ELISA method of Heurich, using antigen planar microarrays as an example, with target analytes (proteins, peptides, or small molecules) immobilized on the support and detected using a labeled secondary antibody (pg. 865, col. 1, par. 6). Both approaches share the same kind of the label – a labeled secondary antibody. The electrochemical sensor and detection method taught by Heurich function as an electrochemical label and therefore, can be used with both sandwich and competitive assay formats, since prior art recognizes wide variety of different labels based on different detection principles successfully applied to both competitive and sandwich assay.
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method for electrochemical detection of Heurich by employing it in a sandwich format assay as taught by Cretich, in order to provide benefits of electrochemical detection with its wide linear dynamic detection range to sandwich assays, as an obvious matter of using of known technique (electrochemical detection of Heurich) to improve similar methods (sandwich assays) in the same way.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references of Heurich and Cretich because Heurich teaches electrochemical detection as a label for immunoassay and Cretich teaches a generic approach of detecting captured analyte molecules in both competitive and sandwich formats on a solid support using a generic labeled secondary antibody.
Response to Arguments
Applicant’s arguments filed September 22, 2025 have been fully considered.
Applicant argues that “independent claim 26 has been amended to further recite that the step of introducing a sample comprising an unknown concentration of the target analyte to an electrode” and “[t]hus, the interpretation of the primary antibody of Heurich as the target analyte by the Patent Office is improper in view of amended independent claim 26. The argument is persuasive; therefore, the rejection of claim 26 under 102 is withdrawn.
However, claim 26 amendments necessitated a new prior art search and new grounds of rejection have been found in view of Cretich et al.
Briefly, Cretich teaches generic sandwich and competitive assay formats with generic labels (Fig. 2A and 2B). Both formats are widely known in the art and have been successfully implemented with a wide variety of labels and detection methods. In the sandwich assay format, typically, a sample comprises an unknown concentration of the target analyte as recited in claim 26. It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method for electrochemical detection of Hasenbank and Heurich by employing it in a sandwich format assay as taught by Cretich, in order to provide benefits of electrochemical detection with its wide linear dynamic detection range to sandwich assays, as an obvious matter of using of known technique (electrochemical detection of Hasenbank and Heurich) to improve similar methods (sandwich assays) in the same way. Therefore, claim 26 is rejected under 35 U.S.C. 103 as unpatentable over Heurich in view of Cretich.
Applicant argues that “independent claim 1 has been amended to clarify that the target analyte is from a sample and that measuring a current generated from the electrode of the electrochemical sensor quantifies an unknown concentration of the target analyte from the sample” and the prior art does not teach the amended limitation (pg. 8, par. 2). The argument is persuasive; therefore, the rejection of claims 1-25 under 35 U.S.C. 103 is withdrawn. The amendments necessitated a new prior art search and new grounds of rejection have been found in view of newly found prior art reference of Cretich et al. is withdrawn. Please, see brief explanation of Cretich teachings in response #1 above.
Applicant argues with the Patent Office motivation statement for combination of Hasenbank and Heurich. Specifically, Applicant argues that” one of ordinary skill in the art would not have looked to make the combination as asserted by the Patent Office, as the invention of Hasenbank is related to amplifying chemical sensor measurements. See, e.g., Hasenbank technical field and Col 4, lines 46-52. The invention of Hasenbank is already related to improving sensor sensitivity (i.e., via signal amplification), and thus one of ordinary skill in the art would not have looked to change the mode of operation to electrochemical detection of Heurich as asserted by the Patent Office, as doing so would have eliminated the signal amplification from the chemical sensor measurements described in Hasenbank” (pg. 8, par. 3).
The argument is not persuasive, because the Patent Office motivation for electrochemical detection is based on a wide linear dynamic detection range, while Applicant argues “improving sensor sensitivity” – these are two entirely different assay parameters.
Applicant arguments on pg. 8, last par. - pg. 12 are moot because the rejection of claims 1-25 under 35 U.S.C. 103 has been withdrawn and the claims are being rejected in view of newly found prior art reference of Cretich (see §103 rejections above).
Double patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP §§ 706.02(l)(1) - 706.02(l)(3) for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/process/file/efs/guidance/eTD-info-I.jsp.
Claims 1-4 and 13-18 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 6, 7, 9-13, 15, and 18 of U.S. Patent No. 10,753,940.
Claim 1 of ‘940 recites a method for detecting a target analyte in a sample, comprising:
(a) introducing a sample comprising a target analyte into an electrochemical sensor comprising a fluid-contact surface and an analyte-specific electrode immobilized on at least a portion of the fluid-contact surface, wherein the analyte-specific electrode is functionalized with a first capture probe for specific binding with the target analyte;
(b) allowing the target analyte to bind with the capture probe on the analyte-specific electrode, thereby forming a complex comprising the target analyte and the capture probe on a surface of the analyte-specific electrode;
(c) labeling the complex with a label probe, wherein the label probe binds specifically with the target analyte and the label probe is conjugated with at least one reporter enzyme;
(d) introducing simultaneously a reporter enzyme substrate, an electroactive mediator and a precipitating agent, forming an electroactive mediator precipitating composition, into the electrochemical sensor, wherein a reaction of the electroactive mediator precipitating composition with the at least one reporter enzyme conjugated with the label probe forms an electroactive precipitate locally adsorbed at the surface of the analyte-specific electrode;
(e) applying a voltage to the electrochemical sensor, wherein the voltage corresponds to the standard redox potential of the electroactive precipitate; and
(f) measuring a current generated from the analyte-specific electrode of the electrochemical sensor to detect the target analyte;
wherein the target analyte is not a nucleic acid.
Instant claim 1 recites the same method for detecting a target analyte in a sample. Target analyte which is not a nucleic acid anticipates a generic target analyte of instant claim 1.
Claim 6 of ‘940 recites plurality of electrodes. Instant claim 2 recites the electrode is a first electrode of a plurality of electrodes.
Claim 7 of ‘940 recites the analyte-specific electrodes are adapted to detect different target analytes. Instant claim 3 recites the plurality of electrodes are adapted to detect different target analytes.
Claim 9 of ‘940 recites the electrochemical sensor comprises one or more microfluidic flow cells. Instant claim 13 recites the electrochemical sensor comprises one or more microfluidic flow cells.
Claim 10 of ‘940 recites the electrochemical sensor comprises one or more open wells. Instant claim 14 recites the electrochemical sensor comprises one or more open wells.
Claim 11 of ‘940 recites the analyte-specific electrode is a planar or 3-dimensional electrode. Instant claim 15 recites the electrode is a planar or 3-dimensional electrode.
Claim 12 of ‘940 recites the fluid-contact surface further comprises a counter electrode and a reference electrode immobilized thereon. Instant claim 16 recites the electrochemical sensor further comprises a counter electrode and a reference electrode.
Claim 13 of ‘940 recites the fluid-contact surface further comprises a positive control electrode and/or a negative control electrode immobilized thereon. Instant claim 17 recites the electrochemical sensor further comprises a positive control electrode and/or a negative control electrode.
Claim 15 of ‘940 recites the generated current corresponds to a reduction or oxidation current derived from reduction of the fully or partially oxidized electroactive mediator. Instant claim 18 recites the generated current corresponds to a current derived from reduction of the fully or partially oxidized electroactive precipitate.
Claim 18 of ‘940 recites the electroactive mediator is selected from the group consisting of 3,3′,5,5′-tetramethylbenzidine (TMB), o-phenylenediamine dihydrochloride (OPD), 2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid] (ABTS), p-Nitrophenyl Phosphate (PNPP), 3,3′-diaminobenzidine (DAB), 4-chloro-1-naphthol (4-CN), 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), nitro blue tetrazolium (NBT), methylene blue, hydroquinone, ferrocene derivatives, and any combination thereof. Instant claim 4 recites the electroactive mediator is selected from the group consisting of 3 ,3' ,5 ,5'-tetramethylbenzidine (TMB), o-phenylenediamine dihydrochloride (OPD), 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid] (ABTS), p-Nitrophenyl Phosphate (PNPP), 3,3'-diaminobenzidine (DAB), 4-chloro-1-naphthol (4-CN), 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), nitro blue tetrazolium (NBT), methylene blue, hydroquinone, ferrocene derivatives, and any combination thereof.
Conclusion
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/ALEXANDER ALEXANDROVIC VOLKOV/
Examiner, Art Unit 1677
/REBECCA M GIERE/Primary Examiner, Art Unit 1677