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 .
Status of the Claims
Claims 1, 2, 4, 5, 10, 12, 13, 16, 19, 22, 24-28, and 31 are pending and examined herein. No claims are amended.
Priority
The present application, filed 01/30/2026 is an RCE of U.S. Patent Application 17/413,368 filed, 06/11/2021, which is a 371 of PCT/GB2019/053549, filed 12/13/2019, which claims foreign priority of GB1820413.1, filed 12/14/2018. The priority is acknowledged and the claims examined herein are treated as having an effective filing date of 12/14/2018.
Maintained Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 25 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
First, the term “medium to high binding affinity” in claim 25 is a relative term which renders the claim indefinite. The term “medium to high binding affinity” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The term “medium to high binding affinity” is a relative term that lacks objective boundaries. The claim does not specify any quantitative measure of binding affinity (e.g. dissociation constant), a numerical range, a reference standard, or test conditions under which binding affinity is to be evaluated. Since “medium” and “high” are subjective descriptors and may vary depending on the particular tag/anti-tag pair, assay conditions, and measurement technique, a PHOSITA cannot determine with reasonable certainty what binding interactions fall within the scope of the claim. The specification does not provide a definition or objective criteria that would render the scope of this term reasonably certain. For purposes of compact prosecution, the term “medium to high binding affinity” in claim 25 will be interpreted to mean that the complementary tag/anti-tag pairs possess a binding strength sufficient to immobilize the tagged immune complex at the detection zone within a short incubation time under the operating conditions of the device, such that the tagged immune complex is retained at the electrode while unbound reagents are carried away by fluid flow. Under this interpretation, the term is interpreted broadly to include tag/anti-tag interactions exhibiting binding affinities commonly used in immunoassays and affinity capture systems that enable rapid capture and stable immobilization during electrochemical measurement, without regard to any specific numerical affinity value. Appropriate correction is required.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 2 and 12 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
In particular, claim 2 depends from claim 1 and recites that “the electrochemical detection comprises faradaic or non-faradaic impedance spectroscopy, or differential pulse voltammetry (DPV).” However, claim 2 merely recites intended use limitations relating to the type of signal or detection methodology employed during operation of the device. The recited detection techniques do not require any additional structural features, physical configuration, or integration of components beyond those already recited in claim 1. The device of claim 1 is structurally complete irrespective of whether it is used to perform impedance spectroscopy, chronoamperometry, DPV, or other electrochemical measurements. Accordingly, claim 2 fails to further limit the subject matter of claim 1 and is therefore of improper independent form.
Likewise, claim 12 depends from claim 1 and recites that the immunoassay comprises one or more of the following formats, including sandwich, competitive, inverted competitive, direct, and indirect immunoassays. These formats inherently differ in reagents used, binding sequences, signal generation, and order of assay steps, rather than in the physical structure or configuration of the device itself. Claim 12 does not require any distinct structural modification, physical integration, or configuration of the device of claim 1 to support the recited assay formats. As such, the limitations of claim 12 define intended use and operational context rather than imposing additional structural limitations on the apparatus. Accordingly, claim 12 fails to further limit the subject matter of claim 1 and is therefore of improper independent form.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Maintained Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under pre-AIA 35 U.S.C. 103(a) are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
Claims 1, 4, 13, 27, and 28 are rejected under 35 U.S.C. 103 as being unpatentable over Kim et al. (US 2010/0261286) in view of Fitzgerald et al. (US 20130310277) and Chikkaveeraiah et al. (Microfluidic electrochemical immunoarray for ultrasensitive detection of two cancer biomarker proteins in serum. Biosensors and Bioelectronics. Vol. 26, No. 11, July 2011).
Kim et al. discloses a microfluidic device architecture having physically distinct chambers for reaction and detection, stating that “ in one embodiment, the photoresist layer includes at least three microfluidic regions: a sample inlet chamber or region, a reagent or reaction chamber or region, and at least one detection chamber or region.” (page 15, paragraph [0022]). Kim et al. further states “in a basic use, when a sample inlet chamber receives a liquid sample containing an analyte to be analyzed, the liquid sample is drawn into the sample inlet chamber by capillary action and flows to the reaction chamber where the samples mixes with binding reagents such as labeled antibodies. The labels may comprise fluorescence labels, or electrochemical labels.” (page 15, paragraph [0023]). Next, “as the sample flows out of the reagent chamber, it flows into the detection chamber. A mixing channel may optionally be placed between the reaction chamber and the first detection chamber. Thorough mixing of sample and reagents in a mixing channel insures the reaction of sample analyte and reagents” (page 15, paragraph [0023]). Then, “typically, an immunocomplex is formed between an analyte and a labeled antibody” (page 15, paragraph [0023]). Additionally, Kim et al. teaches that “in the detection chamber(s), an analyte-antibody complex binds to a second antibody which is in turn directly bound to the detection chamber. The analyte-antibody complex is thus captured and immobilized in the detection chamber” (page 15, paragraph [0023]). Also, Kim et al. further teaches electrochemical detection and electrodes, stating that “detection methods include electrochemical detection” (Abstract 1, page 1) and “electrochemical detection of enzyme labeled antigen or antibody or other binding complexes is well established. A silver/silver chloride reference electrode may be used as well as gold electrodes or carbon electrodes” (page 16, paragraph [0034]). Kim et al. further discloses that “the amount of the captured antigen-antibody complex on the electrode surface is related to the capacitance or voltage change of the working electrode” (page 20, paragraph [0099]). Lastly, Kim et al. teaches kits, stating that “it is an object of the present invention to provide new and improved microfluidic devices and assay kits including the same” (page 15, paragraph [0014]); “Fig.4 is a perspective view of the rapid assay kit including the microfluidic device” (page 16, paragraph [0043]).
Although Kim et al. teaches the following: an incubation/reaction chamber comprising reagents that form immune complexes in liquid phase, one or more detection chambers separate from the reaction chamber, immobilization occurring in the detection chamber, and electrochemical detection using electrodes – Kim et al. does not expressly teach: a tag/anti-tag multiplex architecture, an array of different anti-tags immobilized on different working electrodes, multiple electrochemical electrode arrays, and tag reagents supplied in a kit for multiplex tag/anti-tag immunoassays.
On the other hand, Fitzgerald et al. teaches a multiplex tag/anti-tag immunoassay architecture, in which analyte-specific immune reagents carry tags and are captured by corresponding tag-specific antibodies (anti-tags) immobilized at defined locations. Specifically, Fitzgerald et al. states that “the invention describes accurate and flexible methods and kits for conducting multi-analyte microarrays through the use of Tag-specific antibodies and analyte-specific Tag-anchored antibodies (Abstract, page 1). Fitzgerald et al. further states that “the invention is underpinned by Tag specific antibodies attached to a solid support that bind analyte-specific Tag-anchored antibodies. The invention is applicable to traditional competitive and sandwich assay formats, as well as a mixture of the two formats” (page 6, paragraph [0001]). Furthermore, Fitzgerald et al. teaches spatially resolved multiplex capture, stating that “a kit for detecting or determining two or more analytes in a sample comprising two or more Tag-specific antibodies at spatially-defined locations on a solid support and two or more Tag-conjugated analyte-specific antibodies” (page 7, paragraph 0015). Also, Fitzgerald et al. teaches kits comprising tag reagents and optional signal reagents, stating that “another aspect of the invention is a kit for detecting or determining two or more analytes in a sample comprising one or more Tag-specific antibodies and one or more analyte specific antibodies occupying spatially-defined locations on a solid support, and one or more analyte-specific Tag-anchored antibodies. The kits of the invention optionally contain one or more labeled conjugates” (page 7, paragraph 0016).
Chikkaveeraiah et al. teaches “a microfluidic electrochemical immunoassay system for multiplexed detection of protein cancer biomarkers was fabricated using a molded polydimethylsiloxane channel and routine machined parts interfaced with a pump and sample injector” (Abstract, page 4477). Chikkaveeraiah et al. further states that “we extend our earlier microfluidic design to include an 8-electrode AuNP immunoarray featuring capture antibodies attached to the sensors. The system involves a pump and a sample injector interfaced to a 1.5 mm wide, 63 μL volume, polydimethylsiloxane (PDMS) channel cured on a mold and press fitted into a plastic housing with inlets and outlets” (Introduction, paragraph 7, page 4478). Furthermore, Chikkaveeraiah et al. further states that “this assembly houses a disposable 8-electrode microarray in the channel along with symmetrically placed long wire Ag/AgCl reference and Pt counter electrodes” (Introduction, paragraph 7, page 4478). Chikkaveeraiah et al. further teaches immobilization of capture antibodies on electrodes, stating that “electrodes in the array are coated with a dense layer of 5 nm glutathione-decorated gold nanoparticles, which are then derivatized with capture antibodies for the protein analytes” (Figure 1, page 4479). Additionally, Chikkaveeraiah et al. teaches liquid-phase operation and incubation, stating that “we attached capture antibodies (Ab1) for PSA and IL-6 onto individual carboxylated AuNPs electrodes in the array by EDC-NHS amidization. Before each assay, a solution of 2% BSA in PBS containing 0.05% Tween-20 (PBS-T20) was passed into the microfluidic channel with reference and counter electrodes in place to block non-specific binding. Flow was stopped and incubation was allowed for 10 min, followed by washing with PBS-T20. MP-Ab2-HRP with captured PSA and IL-6 were injected to fill the 100 μL sample loop, and injected into the microfluidic channel at 100 μL min−1. When the sample plug was in the clear microfluidic channel, as evidenced by the red-brown color of the particles, flow was stopped for at least 15 min to capture the particles” (Experimental Section 2.5, page 4480). Lastly, Chikkaveeraiah et al. discloses electrochemical signal measurement, stating that “amperometric detection was done at an optimized potential of −0.2 V vs. Ag/AgCl by filling the washed sample loop with a mixture of 1 mM hydroquinone mediator and 100 μM hydrogen peroxide, then injecting at 100 μL min−1” (Experimental Section 2.5, page 4480).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to modify the microfluidic device architecture disclosed by Kim et al. to incorporate the multiplex tag/anti-tag immunoassay architecture taught by Fitzgerald et al. and to implement detection using the electrochemical electrode array system disclosed by Chikkaveeraiah et al. because the references address complementary and technically compatible aspects of multiplex immunoassay design. Kim et al. teaches a microfluidic device having physically distinct chambers, including a reaction chamber in which binding reagents mix with the sample and a separate downstream detection chamber in which immune complexes are captured and immobilized. This architecture establishes a clear separation between liquid-phase immune complex formation and downstream solid-phase capture. However, Kim et al. does not limit the nature of the binding reagents used in the reaction chamber or the capture chemistry used in the detection chamber. The reference is therefore structurally enabling but chemically non-limiting. While, Fitzgerald et al. teaches a tag/anti-tag multiplex immunoassay strategy in which analyte-specific reagents are provided with tags and are captured by tag-specific antibodies immobilized on a solid support. Fitzgerald et al. further teaches that this architecture is applicable to competitive and sandwich assay formats and that it supports multi-analyte detection in a spatially defined format. A person having ordinary skill in the art (PHOSITA) seeking to implement multiplex detection within Kim et al.’s separated microfluidic architecture would have recognized that Fitzgerald et al.’s tag-based capture system provides a modular and scalable approach to multiplexing that is directly compatible with Kim et al.’s downstream detection chamber.
Additionally, Chikkaveeraiah et al. further teaches a microfluidic electrochemical immunoassay device employing a multi-electrode array with capture antibodies immobilized on individual electrodes to allow multiplex electrochemical detection. Since Kim et al. already teaches the use of electrochemical labels and electrodes and Fitzgerald et al. teaches immobilization of tag-specific antibodies on solid supports, a skilled artisan would have recognized that Chikkaveeraiah et al.’s electrode array provides a predictable and technically established platform for implementing electrochemical readout of Fitzgerald et al.’s tag/anti-tag capture system within Kim et al.’s microfluidic structure. The proposed combination therefore does not require altering the fundamental operation of any reference. Rather, it integrates: Kim et al.’s structurally separated microfluidic flow path. Fitzgerald et al.’s multiplex tag-based capture chemistry, and Chikkaveeraiah et al.’s electrochemical electrode array detection system. Each reference performs its known function within the combined system. The combination represents the application of known multiplex chemistry to a known microfluidic platform using a known electrochemical detection array.
Lastly, a PHOSITA would have had a reasonable expectation of success in making this modification because the biochemical and electrochemical principles underlying each system are well-established, compatible, and routinely used in immunoassay engineering. Kim et al. demonstrates that immune complexes formed in a reaction chamber can be transported via microfluidic channels to a separate detection chamber and immobilized on a solid phase. This establishes that separated liquid-phase incubation and downstream capture operate reliably in microfluidic devices. Fitzgerald et al. demonstrates that tag/anti-tag systems provide highly specific and robust immobilization of analyte-specific immune reagents on solid supports. Tag/anti-tag chemistries are well-known in the art for their high specificity and modularity. Chikkaveeraiah et al. demonstrates that capture antibodies immobilized on individual electrodes within a microfluidic channel can generate reliable electrochemical signals corresponding to analyte binding events.
Importantly, none of the references introduce incompatible chemistries or device architectures. Fitzgerald et al.’s immobilized tag-specific antibodies function on generic solid supports, and Chikkaveeraiah et al. explicitly demonstrates antibody immobilization on electrode surfaces. Kim et al. teaches electrochemical labels and electrode use in detection chambers. The physical and chemical interfaces required for integration are therefore already demonstrated in the art. Since the combination involves substituting one known chemistry (tag/anti-tag immobilization) for another within a microfluidic detection chamber that already supports immune complex immobilization, and because electrochemical electrode arrays were known to support multiplex immunodetection, a skilled artisan would have reasonably expected the integrated system to function as intended without undue experimentation. The result would predictably yield a microfluidic device in which tagged immune complexes are formed in a liquid-phase reaction chamber, transported to a separate detection chamber, captured by immobilized anti-tags on individual working electrodes, and detected electrochemically
Claims 2 and 31 are rejected under 35 U.S.C. 103 as being unpatentable over Kim et al., Fitzgerald et al., and Chikkaveeraiah et al. as applied to Claims 1 and 28 above, and further in view of Bard et al (Electrochemical Methods: Fundaments and Application. John Wiley & Sons, 2ND Edition. 2001).
With respect to the teachings of Kim et al., Fitzgerald et al., and Chikkaveeraiah et al., see the discussion above, which applies equally here. These references differ from the instant claims in failing to teach or specify electrochemical detection comprising: faradaic impedance spectroscopy, non-faradaic impedance spectroscopy, chronoamperometry, or differential pulse voltammetry (DPV), and wherein the device comprises multiple working electrodes and there is different anti-tag on each working electrode.
However, Bard et al. teaches electrochemical impedance spectroscopy and distinguishes faradaic and non-faradaic components. First, Bard et al. discloses that “two types of processes occur at electrodes. One kind comprises reactions like those just discussed, in which charges (e.g., electrons) are transferred across the metal-solution interface. Electron transfer causes oxidation or reduction to occur. Since such reactions are governed by Faraday's law (i.e., the amount of chemical reaction caused by the flow of current is proportional to the amount of electricity passed), they are called faradaic processes” (page 9) and “processes such as adsorption and desorption can occur, and the structure of the electrode-solution interface can change with changing potential or solution composition. These processes are called nonfaradaic processes” (page 9). Furthermore, Bard et al. states that “the technique where the cell or electrode impedance is plotted vs. frequency is called electrochemical impedance spectroscopy (EIS)” (page 369), and the “faradaic impedance measurement, in which the cell contains a solution with both forms of a redox couple, so that the potential of the working electrode is fixed” (page 368). Bard et al. further discloses that “we concentrated on the components of the faradaic impedance – series resistance and pseudocapacity. We assumed that they can be extracted readily from direct measurements of the total impedance, which also includes the solution resistance, and the double-layer capacitance” (page 383) – “One approach to obtaining the faradaic impedance from these values is to measure the cell impedance in a separate experiment under identical conditions, but in the absence of the electroactive couple” (page 383). Additionally, Bard et al. discloses that this “this method circumvents the need for separate measurements without the electroactive species, and it eliminates the need to assume that the electroactive species has no effect on the nonfaradaic impedance” (page 384).
Moreover, Bard et al. teaches chronoamperometry as an electrochemical detection method, stating that “current flows subsequently to maintain the fully reduced condition at the electrode surface. The initial reduction has created a concentration gradient that in turn produces a continuing flux of anthracene to the electrode surface. Since this arriving material cannot coexist with the electrode at E2, it must be eliminated by reduction. The flux of anthracene, hence the current as well, is proportional to the concentration gradient at the electrode surface. Note, however, that the continued anthracene flux causes the zone of anthracene depletion to thicken; thus the slope of the concentration profile at the surface declines with time, and so does the current. This kind of experiment is called chronoamperometry, because current is recorded as a function of time” (page 158).
Lastly, Bard et al. teaches differential pulse voltammetry stating that “sensitivities even better than those of normal pulse voltammetry can be obtained with the small-amplitude pulse scheme shown in Figures 7.3.9 and 7.3.10, which show the basis for differential pulse voltammetry. The figures focus on the special case of differential pulse polarography (DPP), but the waveform and measurement strategy are general for the method in its broader sense” (page 286). Bard et al. further states that “DPP resembles normal pulse polarography, but several major differences are evident: (a) The base potential applied during most of a drop's lifetime is not constant from drop to drop, but instead is changed steadily in small increments, (b) The pulse height is only 10 to 100 mV and is maintained at a constant level with respect to the base potential, (c) Two current samples are taken during each drop's lifetime. (d) The record of the experiment is a plot of the current difference, versus the base potential. Obviously the name of the method is derived from its reliance on this differential current measurement” (page 287).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to modify the microfluidic device architecture disclosed by Kim et al., as implemented with the multiplex tag/anti-tag immunoassay architecture taught by Fitzgerald et al. and the electrochemical electrode array of Chikkaveeraiah et al. – by employing the electrochemical detection techniques taught Bard et al. Kim et al. teaches a microfluidic device comprising a reaction chamber, mixing channels, and a detection chamber, and further teaches that labels may comprise “electrochemical labels.” Chikkaveeraiah et al. demonstrates that multiplex electrochemical detection can be performed in a microfluidic channel containing multiple electrodes. However, neither reference restricts the electrochemical measurement to any single technique.
Bard et al. provides the canonical disclosure of electrochemical measurement methods applicable to working electrodes immersed in electrolyte solution, including impedance spectroscopy (faradaic and non-faradaic), chronoamperometry, and differential pulse voltammetry. These methods are not alternative architectures but rather different modes of interrogating the same electrode interface. A PHOSITA designing an electrochemical immunoassay device according to Kim et al. would have been motivated to select from among well-established electrochemical measurement modes in order to optimize: sensitivity, signal-to-noise ratio, detection limits, and/or compatibility with multiplex electrode arrays. Selecting impedance spectroscopy, chronoamperometry, or DPV would have represented the routine application of known electrochemical techniques to a known electrode platform for the predictable purpose of measuring binding events at the electrode surface. The modification does not alter the physical structure of Kim et al.’s device; it merely specifies the mode by which electrical signals from the working electrodes are measured.
Lastly, a PHOSITA would have had a reasonable expectation of success in applying the electrochemical techniques taught by Bard et al. to the microfluidic electrode system disclosed by Kim et al. and Chikkaveeraiah et al. Kim et al. teaches working electrodes in a detection chamber and electrochemical labels. Chikkaeveeraiah et al. demonstrates that multiplex immunoassays that can be measured electrochemically at electrodes within a PDMS microfluidic channel. Bard et al. teaches that impedance spectroscopy, chronoamperometry, and DPV are mature, well-characterized electrochemical methods applicable to working electrodes in electrolyte solutions. There is no technical incompatibility between Kim et al.’s microfluidic detection chamber, Chikkaveeraiah et al.’s electrode array, Fitzgerald et al.’s tag/anti-tag capture strategy, and Bard et al.’s electrochemical measurement techniques. Since these electrochemical techniques are standard tools used to interrogate electrode-solution interfaces and surface-bound species, and because the primary combination already employs working electrodes in a liquid environment for immunoassay detection, a skilled artisan would reasonably expect that applying faradaic or non-faradaic impedance spectroscopy, chronoamperometry, or DPV would successfully detect the immobilized tagged immune complexes. The modification therefore represents the predictable application of established electrochemical measurement techniques to an existing microfluidic electrode-based immunoassay system.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Kim et al., Fitzgerald et al., and Chikkaveeraiah et al. as applied to Claim 1 above, and further in view of Frenz et al. (Reliable Microfluidic On-Chip Incubation of Droplets in Delay-Lines. Lab on a Chip. Vol. 9, No. 10, May 2009).
With respect to the teachings of Kim et al., Fitzgerald et al., and Chikkaveeraiah et al., see the discussion above, which applies equally here. These references differ from the instant claims in failing to teach or specify a microfluidic delay channel between an incubation chamber and one or more detection zones specifically configured such that the delay channel increases the liquid-phase incubation time of the sample and reagents before immune complexes reach downstream capture sites for signal measurement.
Frenz et al. teaches that delay lines (delay channels) in microfluidic systems are used to allow incubation of reactions for precise time periods. Specifically, Frenz et al. states “together with droplet creation, fusion and sorting, the incubation of droplets is one of the most important and essential operations for droplet-based microfluidic assays. This manuscript concerns the development of delay-lines, which are necessary to allow incubation of reactions for precise time periods” (Abstract, page 1344). Additionally, Frenz et al. teaches that incubation occurs prior to downstream measurement, stating that “droplets therefore moved back and forth between the deep channels for incubation and the narrow channels for measurements”(Results and Discussion, page 1348). Frenz et al. further explains that microfluidic channels (delay lines) are used to obtain incubation delays, stating that “for very short reaction times (<1 min) short and narrow microfluidic channels (delay-lines) have been used in which the droplets remain in single-file” (Introduction, paragraph 2, page 1344). Lastly, Frenz et al. further teaches that these delay-line structures enable a broad range of incubation/reaction times in integrated microfluidic systems, stating that “these improvements allow the creation of integrated droplet based microfluidic systems for a wide range of (bio)chemical reactions, containing multiple modules, including delay-lines which allow reaction times of 1 min to >1 hour” (Conclusions, page 1348). These disclosures teach using delay-lines (e.g., microfluidic delay channels) to increase incubation time before subsequent steps/measurements in microfluidic assay systems.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to modify the microfluidic device architecture disclosed by Kim et al., as implemented with the multiplex tag/anti-tag immunoassay architecture taught by Fitzgerald et al. and the electrochemical electrode array of Chikkaveeraiah et al. - by incorporating a microfluidic delay channel as taught by Frenz et al. Kim et al. already teaches a microfluidic system in which a sample flows from a reaction chamber into a detection chamber. However, Kim et al. does not provide a structural solution for precisely controlling or extending incubation time before detection. But, Frenz et al. identifies this exact engineering problem in microfluidic systems and provides a structural solution: a dedicated delay-line positioned between reaction and detection regions to allow controlled incubation for defined time periods. A PHOSITA designing Kim et al.’s device for immunoassay applications would have recognized that reaction kinetics and immune complex formation efficiency depend on sufficient incubation time in the liquid phase. The incorporation of Frenz et al.’s delay line into Kim et al.’s flow path would have been a predictable design improvement to: improve assay sensitivity, allow detection of lower analyte concentrations, enable kinetic measurements, and allow control of reaction time independent of flow rate. This modification does not alter Kim et al.’s fundamental device architecture; it simply lengthens or structurally configures the channel between reaction and detection to achieve controlled incubation -precisely as Frenz et al. teaches. This is a classic application of known microfluidic modules to improve reaction control in a known microfluidic device.
Lastly, a PHOSITA would have had a reasonable expectation of success in making this modification because the references are technically compatible and address the same engineering problem within the same technological field. Kim et al. already teaches a microfluidic device in which a liquid sample flows from a reaction chamber into a detection chamber through microfluidic channels. The operation of Kim et al.’s device inherently depends on sufficient interaction between analyte and binding reagents within the liquid phase prior to detection. Frenz et al., in turn, identifies the need in integrated microfluidic systems for “delay-lines, which are necessary to allow incubation of reactions for precise time periods,” and provides experimentally validated structural configurations that increase incubation time by extending and engineering the channel between reaction and measurement regions. The incorporation of Frenz et al.’s delay-line into Kim et al.’s architecture would not require any change to the fundamental principle of operation of Kim et al.’s device. The modification would merely involve lengthening or structurally configuring the channel between the reaction chamber and detection chamber to provide controlled residence time of the reacting species before they reach the detection zone. Both references employ microfluidic channels fabricated with conventional materials and operate under comparable capillary or pressure-driven flow regimes. Frenz et al. demonstrates that extended incubation times can be achieved reliably without introducing instability or back-pressure problems, thereby confirming that such channel-based delay structures function predictably within microfluidic systems.
Since the modification involves the routine application of a known microfluidic module (a delay-line) to a known microfluidic device (Kim et al.’s reaction-to-detection flow path), and because the biochemical reactions involved are well-understood liquid-phase binding events whose kinetics improve with increased incubation time, a skilled artisan would reasonably expect that integrating Frenz et al.’s delay channel into Kim et al.’s device would successfully increase liquid-phase incubation time prior to detection without comprising device functionality. The modification represents the predictable use of prior art elements according to their established functions, and therefore a reasonable expectation of success would have existed at the time of the invention.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Kim et al., Fitzgerald et al., and Chikkaveeraiah et al. as applied to Claim 1 above, and further in view of Clark et al. (US 2013/0115619 - IDS dated 07/13/2021) and Terpe et al. (Overview of Tag Protein Fusions: From Molecular and Biochemical Fundamentals to Commercial Systems. Applied Microbiology and Biotechnology. Vol. 60, No. 5, January 2002)
With respect to the teachings of Kim et al., Fitzgerald et al., and Chikkaveeraiah et al., see the discussion above, which applies equally here. These references differ from the instant claims in failing to teach that the device comprises an array of six different anti-tags, nor do they disclose that the six different anti-tags are antibodies against fluorescein isothiocyanate (FITC) or biotin, FLAG, His-6, c-Myc, V5, and glutathione S-transferase (GST).
On the other hand, Clark et al. discloses epitope peptide/protein tags and immobilization binding pairs applicable to electrode surface. In particular, Clark et al. states that “preferably the immobilisation tag is selected from the group consisting of a polyarginine tag (Arg-tag), polyhistidine tag (His-tag), FLAG-tag, Strep-tag, c-myc-tag, S-tag, calmodulin-binding peptide, cellulose-binding domain, SBP tag, chitin-binding domain, glutathione S-transferase-tag (GST)” (page 69, paragraph [0155]). Clark et al. further teaches biotin-based immobilization system, stating that “immobilization may utilize one or more binding-pairs to bind or otherwise attach the immobilisation tag to the binding member, including, but not limited to, an antigen-antibody binding pair, hapten/anti-hapten systems, an avidin-biotin binding pair, a streptavidin-biotin binding pair” (page 68, paragraph [0150]). Furthermore, Clark et al. teaches electrode-based immobilization, stating that “preferably the solid surface is an electrode. Preferably, the electrode comprises a binding member which is specific for one of the immobilisation tags” (page 61, paragraph [0026]). Lastly, Clark et al. further teaches microfluidic implementation, disclosing that “if step (b) of the methods of the invention involves the transfer of the sample to a second reaction container then this transfer can be achieved by any method known in the art including, but not limited to, pipetting, pouring or transfer along microchannels between wells in a microfluidic chip” (page 62, paragraph [0044]). These disclosures teach electrode-based immobilization of tagged components, tag-specific binding members, biotin-based binding systems, and microfluidic workflows.
Terpe et al. teaches the use of V5 as an epitope tag suitable for detection, stating that “the bacteriophage T7 and V5 epitopes are interesting tags for sensitive detection” (page 529, paragraph 1). This disclosure teaches V5 as a detection tag, corresponding directly to the V5 tag recited in claim 10.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to modify the microfluidic device architecture disclosed by Kim et al., as implemented with the multiplex tag/anti-tag immunoassay architecture taught by Fitzgerald et al. and the electrochemical electrode array of Chikkaveeraiah et al. - by incorporating the specific epitope tag systems taught by Clark et al. and Terpe et al. Kim et al. teaches a microfluidic device with: a reaction chamber where labeled antibodies form immunocomplexes, a detection chamber where complexes are immobilized, and electrochemical detection at electrodes. While, Fitzgerald et al. teaches that multiplex detection can be achieved through the use of tag-specific antibodies immobilized at spatially defined locations. Clark et al. teaches that multiple epitope peptide/protein tags (His, FLAG, c-Myc, GST) and biotin systems are known immobilization tags compatible with electrode surfaces. Terpe et al. teaches V5 as a known epitope tag suitable for detection.
A skilled artisan implementing a multiplex immunoassay within Kim et al.’s microfluidic architecture would have needed to select specific tag systems to enable parallel detection at different electrodes. Clark et al. provides a well-recognized catalog of commonly used epitope peptide/protein tags and binding-pair systems used in immunoassays and electrode immobilization. Terpe et al. confirms that V5 is a known sensitive detection epitope tag. Hence, the selection of these well-known epitope tags represents a predictable design choice among a finite number of known alternatives for implementing multiplex tag-based detection. No structural modification of Kim et al.’s device is required; only substitution of known tag chemistries. Accordingly, the modification represents the predictable use of known tag systems within a known microfluidic electrochemical immunoassay architecture.
Lastly, a PHOSITA would have had a reasonable expectation of success in making this modification because Kim et al. demonstrates that antibody-analyte complexes form in a reaction chamber and are captured in a detection chamber; Fitzgerald et al. demonstrates that tagged antibodies can be captured by tag-specific antibodies in multiplex format; Clark et al. demonstrates that His, FLAG, c-Myc, GST, and biotin systems are routinely used immobilization tags and explicitly teaches electrode-based immobilization; Terpe confirms V5 as a known epitope peptide/protein tag used for sensitive detection. All references operate within the same technical field of immunoassay tagging and detection. The biochemical principles underlying epitope peptide/protein tags and antibody binding are well understood and routine. The substitution of one known epitope peptide/protein tag for another does not alter the principle of operation of the assay; it merely defines which specific tag/anti-tag pair is used at each electrode. Since each tag system was already known to function reliably in immunoassay detection and because Kim et al.’s architecture already supports antibody capture and electrode-based detection, a skilled artisan would reasonably expect that incorporating the specific epitope tags disclosed by Clark et al. and Terpe et al. into Kim et al.’s device would successfully yield the multiplex array recited in claim 10. The modification would therefore have been technically straightforward and predictable.
Claims 12, 16, 19, 22, 25, and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Kim et al., Fitzgerald et al., and Chikkaveeraiah. as applied to Claim 1 above, and further in view of Wild (The Immunoassay Handbook : Theory and Applications of Ligand Binding, ELISA and Related Techniques. Elsevier Science, 4th edition, 2013).
With respect to the teachings of Kim et al., Fitzgerald et al., and Chikkaveeraiah et al., see the discussion above, which applies equally here. As set forth above, Kim et al. teaches a microfluidic device comprising a reaction chamber and a separate detection chamber in which immunocomplexes are immobilized and detected. Fitzgerald et al. teaches a multiplex tag/anti-tag architecture and states that “the invention is underpinned by Tag specific antibodies attached to a solid support that bind analyte-specific Tag-anchored antibodies. The invention is applicable to traditional competitive and sandwich assay formats, as well as a mixture of the two formats” (page 6, paragraph [0001]). Chikkaveeraiah et al. teaches electrochemical immunoassay detection using electrode arrays. However, these references differ from the instant claims in failing to collectively teach, with sufficient specificity, the full set of immunoassay formats, method implementations, and binding-affinity considerations recited in claims 12, 16, 19, 22, 25, and 26.
On the other hand, Wild teaches immunometric (sandwich) assays, stating that “the simplest type of immunoassay to understand is the immunometric design. An antibody immobilized onto a plastic surface captures the test analyte from the sample, and a different antibody, specific for another part of the analyte molecule, is used as the basis of the signal generation system. This antibody is “labeled,” e.g., with a radioactive isotope. After an incubation to allow the antibodies to bind with the analyte, unbound labeled antibody is washed away. The final stage of the assay involves measurement of the level of signal, which is radioactivity in this example. The signal level in this type of assay is proportional to the analyte concentration in the sample” (Chapter 1.2, page 7). Wild further states that “because the antibodies form a sandwich around the analyte, immunometric assays are also known as sandwich assays” (Chapter 1.2, page 7).
Next, Wild teaches competitive immunoassays, stating that “in this method, the serum is incubated simultaneously with the antibody (this is usually immobilized on a solid surface) and a labeled derivative of the analyte (the labeled analog tracer). During a single incubation period (at 37 °C), the analog tracer competes with the free analyte for the limiting number of antibody binding sites. The amount of tracer binding to the antibody is inversely proportional to the concentration of the analyte” (Chapter 2.6, page 133).
Wild further discloses a competitive immunoassay configuration in which a labeled antibody probe competes with sample antibodies bound to immobilized antigen, producing an inverse signal relationship. Specifically, Wild states that “commercially available tests for total anti-HAV are based on the competitive assay format. Samples containing a mixture of IgG and IgM antibodies are incubated with a complex of monoclonal HAV antibody and the antigen, immobilized on the solid surface using microtiter plates or microsphere beads. The available antibodies from patient sample bind to antigen immobilized on the solid surface (Fig. 2). After a wash step, the complex is incubated with chromophore or fluorophore-conjugated anti-HAV immunoglobulin probe. Anti-HAV antibodies from sample bound to the solid surface in the first step competitively inhibit the binding of conjugated HAV immunoglobulin probe. The labeled immunoglobulin is subsequently measured by an appropriate detection system. The signal intensity is inversely proportional to anti-HAV in the sample” (Chapter 9.17, page 902). This disclosure teaches a competitive assay in which the labeled species is an antibody, rather than the analyte, and in which competition produces an inverse signal relationship, corresponding directly to the competitive inverted immunoassay recited in claims 12c.
Additionally, Wild teaches direct immunoassays for antibody detection using antigen capture and a labeled signal antibody, stating that “the immunoassay has been designed to detect antibodies in a blood sample using the appropriate antigen as the “bait.” Proteins that occur on the surface of a virus can be immobilized onto plastic. They capture specific antibodies for that virus from the sample. As a tracer, a labeled antibody raised in animals against the constant region of human antibody can be used. This is sometimes referred to as the second antibody” (Chapter 1.2, pages 7-8). Wild et al. also teaches indirect immunoassays, stating that “indirect assays employ antigen coupled to the microparticles. Specific antibody in the sample binds to the antigen on the microparticles. After washing, an anti-immunoglobulin alkaline phosphatase-conjugated antibody binds to the complex. After a second washing, the amount of bound alkaline phosphatase is directly proportional to the amount of specific antibody in the sample” (Chapter 7.15, page 593). Lastly, Wild et al. teaches binding affinity as a design consideration, stating that “the selected capture reagent should display high affinity and specific binding (as measured by low background and lack of matrix interference) in the desired matrix (e.g., human blood)” (Chapter 2.8, page 150).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to modify the microfluidic device architecture disclosed by Kim et al., as implemented with the multiplex tag/anti-tag immunoassay architecture taught by Fitzgerald et al. and the electrochemical electrode array of Chikkaveeraiah et al. - by employing the established immunoassay formats and operational principles taught by Wild, in order to perform multiples testing using different assay formats (Claim 12) and to carry out the corresponding method workflows (Claims 16, 19, 22, and 26) with appropriate binding performance (Claim 25). Wild is a canonical immunoassay reference that systemically explains how sandwich, competitive, inverted competitive, direct, and indirect immunoassays function, including signal proportionality relationships and separation logic. A skilled artisan implementing multiplex assays in Kim’s et al.’s device would necessarily need to select a particular immunoassay format depending on analyte type (e.g., large antigen vs small molecule vs. antibody detection). Wild provides the established doctrinal guidance for making those selections. The modification does not alter Kim et al.’s device architecture; it merely applies well-known immunoassay formats to a known microfluidic detection platform. The selection of sandwich versus competitive versus direct versus indirect format represents routine assay design choice among known alternatives. Accordingly, combining Kim et al.’s device with Wild’s immunoassay teachings represents the predictable use of prior art elements according to their established functions.
A PHOSITA would have had a reasonable expectation of success because: Kim et al. demonstrates operable immunocomplex formation in a reaction chamber and immobilization in a detection chamber; Fitzgerald et al. teaches that tag/anti-tag systems are compatible with competitive and sandwich formats; Wild et al. teaches that sandwich, competitive, inverted, direct, and indirect immunoassays are well-established, routine, and function according to predictable biochemical principles. Wild’s disclosure does not require structural modification to Kim et al.’s microfluidic device. The immunochemical interactions (antigen-antibody binding, tracer competition, labeled antibody detection) are all well-understood and compatible with liquid-phase incubation followed by immobilization – exactly the sequence disclosed by Kim et al. The combination therefore would not require undue experimentation and would predictably yield the claimed immunoassay formats.
Claim 24 is rejected under 35 U.S.C. 103 as being unpatentable over Kim et al., Fitzgerald et al., Chikkaveeraiah et al., and Wild as applied to Claims 1 and 16 above, and further in view of Frenz et al.
With respect to the teachings of Kim et al., Fitzgerald et al., and Chikkaveeraiah et al., and Wild, see the discussion above, which applies equally here. These references differ from the instant claims in failing to teach a device in which the microfluidic architecture comprises a microfluidic delay channel between the incubation chamber and the detection zone configured such that the delay channel increases liquid-phase incubation time before immune complexes reach the detection region.
On the other hand, as discussed above, Frenz et al. teaches that delay lines (delay channels) in microfluidic systems are used to allow incubation of reactions for precise time periods (Abstract, page 1344). Also, Frenz et al. explains that incubation occurs prior to downstream measurement (Results and Discussion, page 1348). Frenz et al. further teaches that these delay-line structures provide extended incubation/reaction times in integrated microfluidic systems (Conclusions, page 1348).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to modify the microfluidic device architecture disclosed by Kim et al., as implemented with the multiplex tag/anti-tag immunoassay architecture taught by Fitzgerald et al. and the electrochemical electrode array of Chikkaveeraiah et al. - by incorporating a microfluidic delay channel as taught by Frenz et al, to increase and precisely control liquid-phase incubation time before electrochemical detection. Kim et al. already teaches a device in which as the sample flows out of the reagent chamber, it flows into the detection chamber. However, Kim et al. does not provide structural mechanisms for substantially extending or precisely tuning incubation time between reaction and detection. Frenz et al. directly addresses this technical problem, identifying that delay-lines are “necessary to allow incubation of reactions for precise time periods.” A PHOSITA designing a multiplex immunoassay microfluidic device would recognize that control of incubation time directly affects assay sensitivity, binding completeness, and signal stability. Frenz et al. teaches that incorporation of a delay-line between reaction and detection chambers: “allows longer droplet incubation times” and enables reaction times “of 1 minute to 1 hour.” Since both Kim et al. and Frenz et al. operate in the same technical field of droplet-based microfluidic reaction systems, and because Frenz et al. explicitly describes integration of delay-lines into multi-module microfluidic devices, it would have been an obvious design modification to incorporate Frenz et al.’s delay line architecture into Kim et al.’s device - to improve reaction completeness and temporal control prior to detection. The modification merely adds a known microfluidic structural element (a delay-line channel) to perform its known function (increasing incubation time). This represents a predictable application of a known design element to achieve an expected improvement in assay performance.
Lastly, a PHOSITA would have had a reasonable expectation of success in making this modification because Frenz et al. demonstrates fully functional delay-lines integrated within droplet-based microfluidic systems and confirms that such delay-lines allow reaction times of “of 1 minute to 1 hour.” Frenz et al. further demonstrates compatibility with biochemical assays, stating that “ we validated our delay-line system by measuring the reaction kinetics of the enzyme b-lactamase on-chip: the reactions kinetics were identical to a conventional cuvette-based assay” (Conclusions, page 1348). This disclosure confirms that the delay-line structure is biochemically compatible and does not interfere with reaction integrity. Hence, incorporating a lengthened microfluidic channel configured as a delay-line between the reaction chamber and detection zone would not require altering the fundamental chemistry or detection principles. Rather, it merely extends the fluidic path to increase residence time – a straightforward hydraulic modification. Since Kim et al. provides separated reaction and detection regions; Frenz et al. teaches delay-lines integrated within similar microfluidic systems and demonstrates reliable incubation control and biochemical compatibility – a skilled artisan would reasonably expect that adding a delay-line between Kim et al.’s reaction chamber and detection zone would successfully increase liquid-phase incubation time before immobilization at the anti-tag detection region.
Ultimately, for the reasons set forth above, claims 1, 2, 4, 5, 10, 12, 13, 16, 19, 22, 24-28, and 31 are rejected under 35 U.S.C. 103 as being unpatentable over the cited prior art combinations. The applied prior art, taken in combination, teaches or suggests all of the claimed structural features, assay formats, and method steps, including multiplex tag/anti-tag immunoassay architectures, microfluidic incubation and detection workflows, electrode-based signal measurement, multiple immunoassay formats, controlled incubation timing, and kit configurations. Where individual references do not expressly disclose certain limitations, those limitations are supplied by additional references that address the same technical problems using known and compatible solutions. A PHOSITA would have been motivated to combine these teachings prior to the effective filing date of the claimed invention and would have had a reasonable expectation of success in doing so. Accordingly, the claims, when viewed as a whole, would have been obvious to PHOSITA at the time of the invention.
Response to Arguments
Applicant’s arguments filed 05/18/2026 have been fully considered but are not persuasive.
Claim Rejections Under 35 U.S.C. § 112(b)
With respect to the rejection of claim 25 under 35 U.S.C. § 112(b), Applicant argues that the phrase “medium to high binding affinity,” as used in the context of complementary tag/anti-tag pairs, would be readily understood by a person of ordinary skill in the art because the specification describes binding affinity in qualitative terms such as weak, medium, or strong. Applicant further argues that absolute numerical precision is not required and that the functional language “to allow for relatively short incubation times and rapid immobilization” provides sufficient boundaries. Applicant’s arguments are not persuasive. The fact that the specification states that affinities “can be described as weak, medium or strong” does not provide an objective standard for determining where “medium” binding affinity begins, where “high” binding affinity begins, or which binding interactions fall within the claimed range. The cited disclosure merely repeats qualitative categories without defining them. Although the specification discusses affinity qualitatively and provides example tag/anti-tag systems, the specification does not appear to provide a dissociation constant, association constant, kinetic on-rate or off-rate, binding-strength range, numerical threshold, defined reference tag/anti-tag pair, or standardized assay conditions that objectively distinguish “medium” binding affinity from “high” binding affinity.
Applicant’s reliance on the surrounding language “to allow for relatively short incubation times and rapid immobilization” also does not cure the indefiniteness. The terms “relatively short” and “rapid” are themselves relative terms that do not identify a clear benchmark, reference time, comparison standard, or operating condition. Binding affinity, immobilization rate, and incubation time may vary depending on the tag/anti-tag pair, analyte, antibody or antigen, concentration, flow rate, electrode surface chemistry, temperature, buffer, viscosity, and detection format. Without objective criteria, a person of ordinary skill in the art would be required to speculate as to whether a given binding pair falls within or outside the scope of “medium to high binding affinity.” Accordingly, Applicant’s arguments do not overcome the rejection. Claim 25 remains indefinite because the claim fails to provide objective boundaries for the relative phrase “medium to high binding affinity,” and the specification does not provide a standard for ascertaining the requisite degree with reasonable certainty. The rejection of claim 25 under 35 U.S.C. § 112(b) is maintained.
Claim Rejections Under 35 U.S.C. § 112(d)
With respect to the rejection of claims 2 and 12 under 35 U.S.C. § 112(d), Applicant argues that claim 2 further limits claim 1 because the recited electrochemical detection techniques allegedly require configurations suitable for faradaic or non-faradaic impedance spectroscopy, chronoamperometry, or differential pulse voltammetry. Applicant further argues that claim 12 further limits claim 1 by narrowing the identity, tagging state, and binding relationships of the reagents present in the incubation chamber. Applicant’s arguments are not persuasive. Regarding claim 2, claim 2 depends from claim 1 and recites that “the electrochemical detection comprises faradaic or non-faradaic impedance spectroscopy, chronoamperometry, or differential pulsed voltammetry (DPV).” Claim 2 does not recite any additional device structure beyond the device already recited in claim 1. Claim 2 does not recite, for example, a potentiostat, waveform generator, sinusoidal signal-generation circuitry, differential pulse circuitry, particular electrode dimensions, electrode spacing, reference-electrode arrangement, electroactive label chemistry, redox mediator, frequency range, voltage range, sampling protocol, or other structural component or configuration required for any particular electrochemical measurement mode. Instead, claim 2 merely identifies possible electrochemical detection methodologies by which a signal may be measured during use of the device. Applicant’s argument that different electrochemical techniques may impose constraints on electrode configuration, electronics, surface chemistry, label chemistry, and signal transduction mechanisms is not commensurate with the claim language. Those alleged constraints are not recited in claim 2. Although the claims are interpreted in light of the specification, limitations from the specification or from Applicant’s characterization of how a device might be implemented are not read into the claims. Claim 2 does not recite that the device is “configured to,” “adapted to,” or “capable of” performing any particular electrochemical technique, nor does claim 2 recite the structural features that Applicant now relies upon. Accordingly, Applicant’s argument improperly relies on unclaimed features. Applicant’s reliance on MPEP §§ 2173.05(g) and 2114 is also not persuasive. Those sections recognize that apparatus claims may, in appropriate circumstances, include functional language tied to claimed structure. Claim 2, however, does not recite functional apparatus language that defines a structural component by what it is configured or adapted to do. Rather, claim 2 recites electrochemical detection techniques without reciting additional device structure. Therefore, Applicant’s reliance on functional-claiming principles does not establish that claim 2 further limits the structural subject matter of claim 1.
Regarding claim 12, claim 12 depends from claim 1 and recites that “the immunoassay comprises one or more” of several immunoassay formats, including sandwich, competitive, inverted competitive, direct, and indirect formats. Claim 12 does not recite additional device structure beyond the incubation chamber, reagents, detection zones, electrodes, and anti-tags already recited in claim 1. Rather, the claim identifies assay formats or workflows that may be performed using the device. The recited formats differ in assay protocol, reagent use, binding sequence, and signal relationship, but claim 12 does not expressly recite additional physical structure, reagent placement, chamber configuration, electrode architecture, or structural relationship that further limits the device of claim 1. Applicant argues that claim 12 narrows the “identity, tagging state, and binding relationships” of the reagents. However, the claim language does not clearly recite the specific structural or compositional limitations that Applicant relies upon. To the extent Applicant contends that each assay format inherently requires distinct combinations of tagged species, signal species, and binding interactions, such details are not recited as structural limitations of the device. Claim 12 does not require any additional chamber, additional electrode, particular immobilized reagent arrangement, particular label, particular concentration, or particular spatial organization beyond the device of claim 1. Applicant’s arguments again rely on features not actually recited in the claim. Accordingly, claims 2 and 12 fail to further limit the subject matter of claim 1 and are therefore improper dependent claims under 35 U.S.C. § 112(d). The rejection of claims 2 and 12 under 35 U.S.C. § 112(d) is maintained.
Claim Rejections Under 35 U.S.C. § 103
With respect to the rejections under 35 U.S.C. § 103, Applicant argues that the cited references, whether considered individually or in combination, fail to render the claims obvious. Applicant further argues that the rejection relies on impermissible hindsight, combines references directed to materially different assay paradigms, and would not have resulted in the claimed universal, reagent-programmable electrochemical microfluidic device. Applicant’s arguments have been fully considered but are not persuasive. In response to Applicant’s arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
In response to Applicant’s argument that the Examiner’s conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from Applicant’s disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). In response to Applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, the motivation is found in the combined teachings of the applied references, which address complementary and compatible aspects of multiplex immunoassay device design, including microfluidic reaction/detection architecture, tag/anti-tag multiplex capture chemistry, and electrochemical electrode-array detection.
In response to Applicant’s argument that the rejection improperly bodily incorporates features of secondary references into the primary reference, the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference, nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981).
Kim
Applicant argues that Kim is directed to a conventional microfluidic immunoassay device in which analyte specificity is physically coupled to detection site location, and that Kim does not teach or suggest a tag/anti-tag abstraction layer or decoupling analyte specificity from detection geometry. Applicant also argues that Kim does not disclose a multiplex electrochemical electrode array architecture of the claimed type. Applicant’s arguments are not persuasive because the rejection does not rely on Kim alone to teach the tag/anti-tag multiplex architecture or the complete multiplex electrochemical electrode array. Rather, Kim is relied upon for the microfluidic device architecture and flow-based immunoassay operation. As set forth in the Office Action, Kim teaches a microfluidic device having distinct regions including an inlet chamber, reaction chamber, and detection chamber, with the sample mixing with binding reagents such as labeled antibodies in the reaction chamber, flowing downstream into the detection chamber, and forming/capturing immune complexes in the detection chamber. Kim also teaches electrochemical detection and electrode-based measurement. Thus, Kim provides the separated microfluidic reaction/detection framework in which liquid-phase immune complex formation occurs upstream of downstream capture and detection.
The fact that Kim uses analyte-specific capture antibodies in the detection chamber does not teach away from using tag-specific capture antibodies in the same downstream detection region. Kim demonstrates that a microfluidic device can receive a liquid sample, permit immunoreactive complex formation, transport the complex through a microfluidic flow path, capture the complex at a downstream detection chamber, and measure the captured complex electrochemically. Fitzgerald is relied upon to supply the tag/anti-tag capture chemistry that would have been predictably implemented in Kim’s downstream detection region in place of analyte-specific immobilized capture. Chikkaveeraiah is relied upon to supply the multiplex electrode-array electrochemical implementation. Therefore, Applicant’s argument that Kim alone does not disclose the entire claimed system is not persuasive because the rejection is based on a combination of references. Applicant’s assertion that Kim discourages abstraction of analyte identity away from fixed detection locations is also not persuasive. A reference does not teach away merely because it discloses a different preferred embodiment or does not itself disclose the claimed modification. Kim does not criticize, discredit, or otherwise discourage tag/anti-tag capture chemistry. Kim teaches a structurally enabling microfluidic architecture and recognizes electrochemical detection of immunoreactive complexes. A person of ordinary skill in the art seeking to improve flexibility and multiplexing within Kim’s device would have had reason to look to Fitzgerald’s tag/anti-tag multiplex format, which expressly addresses flexible multi-analyte detection using tag-specific antibodies and analyte-specific tag-anchored antibodies.
Fitzgerald
Applicant argues that Fitzgerald is directed to static microarray assays rather than microfluidic immunoassays, that Fitzgerald involves bulk incubation and optical or chemiluminescent detection, and that Fitzgerald provides no suggestion that its tag/anti-tag chemistry would function in a flowing microfluidic environment or with downstream electrochemical detection. Applicant’s arguments are not persuasive. Fitzgerald is not relied upon for the microfluidic device structure or for the electrochemical electrode-array detection system. Fitzgerald is relied upon for the tag/anti-tag multiplex immunoassay architecture. Fitzgerald teaches using tag-specific antibodies on a solid support to bind analyte-specific tag-anchored antibodies, thereby enabling flexible multi-analyte detection. This teaching is directly relevant to the missing tag/anti-tag multiplex capture chemistry in Kim. The fact that Fitzgerald discloses a biochip or microarray format does not render its tag/anti-tag chemistry incompatible with Kim’s microfluidic architecture. Fitzgerald teaches an immunochemical binding strategy, not a chemistry that is limited to static optical systems. The tag-specific antibody/tag-anchored antibody interaction is a binding interaction capable of occurring when the corresponding species contact one another. Kim already teaches transporting immunoreactive complexes through a liquid microfluidic path to a downstream detection region having immobilized capture molecules. Thus, adapting Fitzgerald’s tag/anti-tag capture chemistry to Kim’s downstream detection region would have involved substituting one known immunoassay capture chemistry for another in a device already designed for liquid-phase immunocomplex formation and downstream immobilization.
Applicant’s argument that Fitzgerald uses 30-minute incubations does not establish incompatibility or teaching away. The claims do not require any particular incubation time for claim 1. Moreover, Kim recognizes the need for mixing and reaction of sample and reagents before downstream detection, including that thorough mixing of sample and reagents insures reaction and that immunocomplexes are formed before capture in the detection chamber. Kim also recognizes that flow speed should be slow enough to allow binding substances to react. A prior-art disclosure of a 30-minute incubation in one embodiment does not criticize, discredit, or preclude use of the same tag/anti-tag binding chemistry in a flow-based device. At most, Applicant identifies differences in implementation, not an incompatibility that would defeat obviousness. Applicant’s argument that Fitzgerald is limited to optical detection is also not persuasive. The rejection does not require bodily importing Fitzgerald’s optical detection into Kim. Fitzgerald is relied upon for tag/anti-tag multiplex capture chemistry, while Kim and Chikkaveeraiah provide the electrochemical detection environment. The combined teachings would have suggested using Fitzgerald’s tag/anti-tag multiplex capture approach in the known electrochemical microfluidic architecture of Kim as further implemented using Chikkaveeraiah’s electrode-array teachings. The claimed electrochemical detection is therefore supplied by the electrochemical references, not by Fitzgerald alone.
Chikkaveeraiah
Applicant argues that Chikkaveeraiah discloses a fundamentally different multiplexing strategy because it patterns different capture antibodies directly on different electrodes, thereby coupling analyte identity to electrode position. Applicant further argues that Chikkaveeraiah relies on off-line analyte capture using magnetic particle-antibody conjugates rather than in situ liquid-phase immune complex formation within a microfluidic incubation chamber. Applicant’s arguments are not persuasive. Chikkaveeraiah is relied upon for its teaching that multiplexed electrochemical immunoassay detection can be implemented in a microfluidic channel using a multi-electrode array with capture antibodies attached to individual electrodes. Chikkaveeraiah demonstrates that an 8-electrode electrochemical immunoarray can be housed in a microfluidic channel with reference and counter electrodes and can generate analyte-dependent electrochemical signals from immunocomplex capture at electrode surfaces. This directly supports the use of a multiplex electrochemical electrode array for immunoassay detection.
The rejection does not require bodily incorporating Chikkaveeraiah’s exact capture antibody patterning strategy or off-line magnetic particle workflow into Kim. Rather, Chikkaveeraiah evidences that microfluidic electrochemical immunoarrays with multiple working electrodes were known and operable for multiplex protein detection. Fitzgerald supplies the tag/anti-tag capture chemistry, and Kim supplies the upstream microfluidic reaction chamber and downstream detection region. The modification would have predictably involved using the known multi-electrode electrochemical detection arrangement of Chikkaveeraiah as the downstream detection zone in Kim, while using Fitzgerald’s tag-specific capture antibodies as the immobilized capture chemistry at the electrode locations. Applicant’s assertion that replacing Chikkaveeraiah’s analyte-specific capture antibodies with tag-specific antibodies would require fundamental re-engineering is not persuasive. Both analyte-specific capture antibodies and tag-specific antibodies are antibody-based capture reagents immobilized on solid supports or electrode-associated surfaces. Chikkaveeraiah teaches immobilizing capture antibodies on individual electrodes, and Fitzgerald teaches tag-specific antibodies immobilized at spatially defined locations on a solid support. Using the known tag-specific antibodies of Fitzgerald as the immobilized capture chemistry at electrode-associated locations of the type taught by Chikkaveeraiah would have been a predictable implementation of one known antibody capture chemistry for another for the purpose of achieving flexible multiplex capture. The immunochemical function remains the same: a surface-bound capture antibody binds its corresponding target-bearing complex at a defined location. The substitution does not require changing the fundamental electrochemical detection principle of the electrode array.
Applicant’s argument that Chikkaveeraiah uses off-line capture also does not defeat the rejection. Kim supplies the in-device liquid-phase reaction chamber and downstream detection flow path. Chikkaveeraiah is not relied upon for the upstream immune-complex formation step. Instead, Chikkaveeraiah is relied upon to show that captured immune complexes at individual electrodes in a microfluidic channel can be detected electrochemically in a multiplex format. Therefore, Applicant’s argument attacks Chikkaveeraiah individually rather than the combination as a whole.
Hindsight and Alleged Incompatibility
Applicant argues that the rejection selectively extracts features from disparate references and recombines them using Applicant’s disclosure as a roadmap. Applicant further argues that the cited references address different technical fields, incompatible assay architectures, and different approaches to multiplexing and detection. Applicant’s arguments are not persuasive. The references are analogous and technically related. Kim is directed to microfluidic immunoassay devices and electrochemical detection. Fitzgerald is directed to multi-analyte immunoassays using tag-specific antibodies and analyte-specific tag-anchored antibodies. Chikkaveeraiah is directed to microfluidic electrochemical immunoarrays for multiplexed protein biomarker detection. The references are within the field of immunoassay detection and are reasonably pertinent to the problem of multiplex detection of analytes using microfluidic/electrode-based platforms and spatially resolved capture chemistries. The rejection does not rely on hindsight. It relies on the predictable use of known prior-art elements according to their established functions. Kim teaches a microfluidic platform in which immune complexes form in a liquid phase and are transported to a downstream detection chamber for immobilization and detection. Fitzgerald teaches that tag-specific antibodies and tag-anchored analyte-specific antibodies provide a flexible multiplex assay architecture. Chikkaveeraiah teaches that multiplexed electrochemical immunoassay detection can be performed in a microfluidic channel using multiple working electrodes. A person of ordinary skill in the art seeking to implement flexible multiplex detection in Kim’s microfluidic immunoassay device would have had reason to use Fitzgerald’s tag/anti-tag capture strategy and Chikkaveeraiah’s multi-electrode electrochemical array because those teachings address complementary aspects of the same design problem.
Applicant’s characterization of the invention as a “universal, reagent-programmable electrochemical microfluidic device” is not sufficient to establish nonobviousness. To the extent Applicant relies on flexible analyte selection, reagent programmability, or decoupling analyte specificity from a manufacturer-fixed capture layout, those concepts are taught or suggested by Fitzgerald’s flexible MAMA approach using Tag-specific antibodies and analyte-specific Tag-anchored antibodies, or are not recited in the claims with the specificity now argued. In response to Applicant’s argument that the references fail to show certain features of the invention, it is noted that the features upon which Applicant relies, including a “universal” device, “reagent-programmable” operation, and complete decoupling of analyte identity from electrode identity as a purported principle of the invention, are not recited in the rejected claims in the manner argued. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Dependent Claims and Additional References
Applicant argues that the additional references Bard, Frenz, Clark, Terpe, and Wild do not cure the alleged deficiencies of Kim, Fitzgerald, and Chikkaveeraiah and merely describe background techniques or alternative implementations. Applicant’s arguments are not persuasive because the primary combination of Kim, Fitzgerald, and Chikkaveeraiah establishes the core microfluidic tag/anti-tag electrochemical multiplex architecture for the reasons discussed above. The additional references are properly relied upon to supply the additional limitations recited in dependent claims.
Bard is relied upon for well-known electrochemical measurement techniques, including impedance spectroscopy, chronoamperometry, and differential pulse voltammetry. The use of these established techniques to interrogate electrode-solution interfaces and surface-bound species would have been a routine and predictable selection among known electrochemical measurement modes for an electrode-based immunoassay device.
Frenz is relied upon for microfluidic delay lines used to increase or control incubation time before downstream measurement. Kim already provides a flow path between reaction and detection regions, and adding a known delay line or extending/configuring the flow path to provide controlled residence time would have been a predictable use of a known microfluidic module for its established function.
Clark and Terpe are relied upon for known tag systems, including epitope and protein tags such as His, FLAG, c-Myc, GST, biotin-related systems, and V5. Their teachings support the obviousness of selecting known tag/anti-tag pairs for multiplex immunoassay implementation. Selecting known tag systems for use in Fitzgerald’s tag/anti-tag multiplex format within Kim’s microfluidic device would have represented a predictable design choice among known alternatives.
Wild is relied upon for known immunoassay formats, including sandwich, competitive, inverted competitive, direct, and indirect immunoassay principles, as well as binding-affinity considerations. Applying these established immunoassay formats to the known microfluidic and tag-based multiplex platform would have been a routine implementation of known assay designs according to their established functions.
Accordingly, Applicant’s arguments do not overcome the rejections of claims 1, 2, 4, 5, 10, 12, 13, 16, 19, 22, 24-28, and 31 under 35 U.S.C. § 103. The applied prior art, taken in combination, teaches or suggests the claimed subject matter, and a person of ordinary skill in the art would have had a reason to combine the teachings with a reasonable expectation of success. The § 103 rejections are maintained.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ELIZABETH OGUNTADE whose telephone number is (571)272-6802. The examiner can normally be reached Monday-Friday 6:00 AM - 3 PM.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Bao-Thuy Nguyen can be reached at 571-272-0824. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/E.O./Examiner, Art Unit 1677
/BAO-THUY L NGUYEN/Supervisory Patent Examiner, Art Unit 1677 June 1, 2026
Prosecution Insights