DETAILED ACTION
Response to Amendment
This is a final office action in response to a communication filed on August 26, 2025. Claims 1-8 and 11-16 are pending in the application.
Status of Objections and Rejections
All rejections under 35 U.S.C. §112 from the previous office action are withdrawn in view of Applicant’s amendment.
All rejections under 35 U.S.C. §103 from the previous office action are maintained.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim(s) 1-7 is/are rejected under 35 U.S.C. 103 as being unpatentable over Jin (CN 104833713B) in view of Fan (L. Fan, Label-free and highly selective electrochemical aptasensor for detection of PCBs based on nickel hexacyanoferrate nanoparticles/reduced graphene oxides hybrid, Biosensors and Bioelectronics, 2019 Vol. 145, 111728, pp. 1-9), and further in view of Noritomi (US 2016/0319232), and further in view of Murphy (US 2017/0246690), and further in view of Huang (CN 106353387A).
Regarding claim 1, Jin teaches a method for preparing an biosensing chip (Title: preparation method for simple enzyme biosensor; p. 3, para. 2: good for the production of chips), wherein the method comprises the following preparation steps:
step 1: preparation of synthetic solution A and synthetic solution B and synthesis of hexacyanoferrate (p. 2: step (1)), wherein
synthetic solution A is an anionic acid solution (p. 2, step (1): e.g., K3Fe(CN)6), synthetic solution B is a cationic acid solution (p. 2, step (1): e.g., FeCl3 aqueous solution), and the two synthetic solutions have the same ion concentration (p. 2, step (1): an equimolar amount of FeCl3 aqueous solution); synthetic solutions A and B are dropwise added at the same rate, and are stirred after the addition (p. 2, step (1): the obtained mixed solution); and the mixed solution of solutions A and B is centrifuged and cleaned several times and then transferred to deionized water to obtain a hexacyanoferrate suspension (p. 2, step (1): centrifuged and filtered to obtain a Prussian blue mud);
step 2: preparation of hexacyanoferrate/carbon mixed ink (p. 2, step (2)); and
step 3: preparation of the biosensing chip (p. 2, step (3)-(4)), wherein
the hexacyanoferrate/carbon mixed ink obtained in step 2 (p. 2, step (2)) is fixed on a support by the screen-printing technique to form a working electrode (p. 2, step (3)); glutaraldehyde is added to an enzyme solution to obtain a mixed enzyme solution (p. 2, step (4)); the mixed enzyme solution is evenly applied on the working electrode (p. 2, step (4): the enzyme solution is uniformly applied to the working electrode), and the working electrode is dried at low temperature to obtain the biosensing chip (p. 2, step (4): dried at a low temperature to be enzyme biosensor).
Jin does not disclose the hexacyanoferrate Prussia blue mud is a nickel hexacyanoferrate suspension or synthesis of gold nanoparticles/nickel hexacyanoferrate/carbon mixed ink, or centrifugal cleaning and drying are conducted after the addition to obtain gold nanoparticles/nickel hexacyanoferrate mixed powder; and carbon ink is added to the powder and mixed evenly to obtain gold nanoparticles/nickel hexacyanoferrate/carbon mixed ink.
However, Fan teaches synthesis of nickel hexacyanoferrate nanoparticles (NiHCF NPs)/reduced graphene oxides (rGO) hybrids ([Abstract]). In the hybrids, rGO with large area and good conductivity supplies more space for loading NiHCF NPs to improve the conductivity of the hybrids, and NiHCF NPs as a signal probe exhibits well-defined peaks with highly reversible redox ability and good stability ([Abstract]). To prepare NiHCF NPs/rGO hybrids (p. 2, col. 2, section 2.2), K3[Fe(CN)6] was slowly added to rGO solution for Fe[(CN)6]3- to be adsorbed on rGO sheets, and then the same volume of Ni(NO3)2 with K3[Fe(CN)6] was injected into the mixture to form NiHCF NPs/rGO hybrids (p. 2, col. 2, para. 4). The NiHCF NPs/rGO hybrids were prepared through centrifugation and rinsed by using deionized water (p. 2, col. 2, para. 4). Further, Noritomi teaches electrochemical sensors used to detect a gas or liquid sample (¶3). The enzyme body 3 may be obtained by immobilizing the enzyme 5 to a molecular aggregate entrapped in the water pool 4 (Fig. 1; ¶114). To increase the efficiency of the enzyme reaction or the efficiency of detection of the enzyme reaction product, nanoparticles (NPs) may be dispersed in the interior, on the surface, or in the periphery of the molecular aggregate, e.g., gold nanoparticles (AuNPs) (¶126).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Jin by replacing Fe ions in Prussian blue and the carbon ink with Ni ions as taught by Fan and incorporating gold nanoparticles as taught by Noritomi to obtain the final product of gold nanoparticle/nickel hexacyanoferrate/carbon mixed ink because nickel hexacyanoferrate would exhibit well-defined peaks with highly reversible redox ability and good stability (Fan, [Abstract]) and gold nanoparticles would help immobilization of the enzyme and increase the efficiency of the enzyme reaction or the efficiency of detection of the enzyme reaction product (Noritomi, ¶126).
The designations “the two synthetic solutions have the same pH value; and are dropwise added at the same rate, and are stirred after the addition, and then synthetic solution B is dropwise added at the same rate, and the solution is centrifuged and cleaned several times and then transferred to deionized water” and “centrifugal cleaning and drying are conducted after the addition” to obtain gold nanoparticles/nickel hexacyanoferrate mixed powder; and carbon ink is “added to the powder and mixed evenly” to obtain gold nanoparticles/nickel hexacyanoferrate/carbon mixed ink are routine experimental operations for reaction of two solutions to obtain and precipitate the final product. Here, Fan teaches the pH of the mixed solution of the two solutions would be adjusted to 3.0 (p. 2, col. 2, para. 4). During the fabrication of NiHCF NPs/rGO hybrids, the solution was slowly added under stirring to prevent agglomeration (p. 2, col. 2, para. 4). The NiHCF NPs/rGO hybrids were prepared through centrifugation and rinsed by using deionized water (p. 2, col. 2, para. 4). It would be obvious to mix two solutions having the same pH value in replacement of adjusting the mixed solution to a final pH value because mixing of two solutions having the same pH value would yield nothing more than predictable results of obtaining a mixed solution at the same pH value. MPEP 2143(I)(A). Further, it would be obvious to slowly add the solution, e.g., dropwise adding the solution, and precipitate solid product by centrifugation and wash by deionized water, to one of ordinary skill in the art and because these steps would not yield nothing more than predictable results.
Jin, Fan, and Noritomi do not disclose wherein a chloroauric acid solution is dropwise added to the nickel hexacyanoferrate suspension, a reducing solution is dropwise added after the addition.
However, Murphy teaches synthesis of metal nanoparticles (title). The method comprises: (1) preparing a metal precursor mixture in a first aqueous liquid medium; (2) preparing a reducing agent mixture in a second aqueous liquid medium; (3) combining the metal precursor mixture with the reducing agent mixture (¶¶56-61). The metal precursor compound may be tetrachloroautic acid (i.e., chloroauric acid) (¶76), and the reducing agent is ascorbic acid (¶99).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Jin, Fan, and Noritomi by incorporating the recited steps for synthesis of gold nanoparticles during the preparation of gold nanoparticles/nickel hexacyanoferrate/carbon mixed ink as taught by Murphy because chloroauric acid solution is a suitable gold precursor and the ascorbic acid is suitable agent for gold nanoparticle synthesis. Here, the claimed limitations are obvious because all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination yielded nothing more than predictable results. MPEP 2143(I)(A). Further, the designation “dropwise added” is a routine experimental operation and would yield nothing more than predictable results for mixing two solutions for reaction.
Jin, Fan, and Noritomi do not disclose the biosensing chip is ethanol biosensing chip and the enzyme solution is ethanol dehydrogenase solution.
However, Huang teaches an electrochemical testing strip coated with modification enzyme membrane for testing a specific target analysis substance ([Abstract]). The enzyme for detecting ethanol is ethanol dehydrogenase (p. 3).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Jin, Fan, and Noritomi by using ethanol dehydrogenase for ethanol biosensing as taught by Huang because ethanol dehydrogenase is a suitable enzyme for electrochemical testing of ethanol. Here, the claimed limitations are obvious because all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination yielded nothing more than predictable results. MPEP 2143(I)(A).
Regarding claim 2, Jin, Fan, Noritomi, Murphy, and Huang disclose all limitations of claim 1. Jin teaches the concentration of the K3Fe(CN)6 and FeCl3 aqueous solutions in the step (1) during preparation of Prussian blue slurry ranges from 1 to 100 mM/L (p. 2), which is 0.01 to 1 M. Fan teaches the pH value of the mixed solution was adjusted to 3.0, and the same volume of Ni(NO3)2 with K3Fe(CN)6 (10.0 mL) was injected into the mixture and stirred for 4h (p. 2, section 2.2), which is at a rate of 42 µL/min. Murphy teaches the reaction temperature is typically from about 3 C to about 35 C (¶104).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Jin, Fan, Noritomi, Murphy, and Huang by adjusting the reaction conditions as claimed because these conditions are routine experimental parameters and can be optimized through routine experimentation. MPEP 2144.05 (II)(B). Further, in the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). MPEP 2144.05(I). Similarly, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985). MPEP 2144.05(I).
Regarding claim 3, Jin, Fan, Noritomi, Murphy, and Huang disclose all limitations of claim 1. Jin further discloses wherein in step 1, the anion donor is K3[Fe(CN)6], and the cation donor is FeCl3 (p. 2, step (1)). Fan further discloses preparation of NiHCF NPs/rGO hybrids, using the anion donor K3[Fe(CN)6], and cation donor Ni(NO3)2 (p. 2, col. 2, section 2.2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Jin, Fan, Noritomi, Murphy, and Huang by substituting FeCl3 with NiCl2 as suggested by Jin and Fan because metal chloride is a suitable cationic reagent for preparation of Prussian blue slurry (Jin, p. 2, step (1)) and substitution of Prussia blue with nickel hexacyanoferrate (NiHCF) would display well-defined, reversible and reproducible responses acting as an electron transfer mediator (p. 2, col. 1, para. 3). The combination of Jin and Fan would render the cation donor NiCl2 obvious using a transition metal chloride reagent to substitute ferric with nickel, i.e., using NiCl2 instead of FeCl3, to prepare NiHCF.
Regarding claim 4, Jin, Fan, Noritomi, Murphy, and Huang disclose all limitations of claim 1. Jin teaches the centrifugation rate of step (1) is from 3,000 r/min to 10,000 r/min, and the centrifugation time is from 3 min to 30 min (p. 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Jin, Fan, Noritomi, Murphy, and Huang by adjusting the centrifugation rate and time as claimed because these conditions are routine experimental parameters and can be optimized through routine experimentation. MPEP 2144.05 (II)(B). In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). MPEP 2144.05(I). Similarly, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985). MPEP 2144.05(I). Further, it would be obvious to adjust the centrifugal times and volume of deionized water as claimed because there are routine experimental parameters and can be optimized through routine experimentation. MPEP 2144.05 (II)(B).
Regarding claim 5, Jin, Fan, Noritomi, Murphy, and Huang disclose all limitations of claim 1, including wherein in step 2, the reducing solution is ascorbic acid (Murphy, ¶99).
Regarding claim 6, Jin, Fan, Noritomi, Murphy, and Huang disclose all limitations of claim 1, but fails to teach wherein in step 2, the molar ratio of chloroauric acid to the reducing substance in the reducing solution is 1:3-1:9.
However, Murphy teaches the molar ratio of reducing agent-to-metal precursor compound is from typically from about 0.5:1 to about 16:1 (¶103), which is from 1:2 to 1:16 for the molar ratio of chloroauric acid to the reducing substance in the reducing solution.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Jin, Fan, Noritomi, Murphy, and Huang by adjusting the molar ratio of chloroauric acid and the reducing substance as claimed because it represents a suitable molar ratio of the reactants for synthesis of gold nanoparticles and can be optimized through routine experimentation. MPEP 2144.05 (II)(B). In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). MPEP 2144.05(I). Similarly, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985). MPEP 2144.05(I).
Regarding claim 7, Jin, Fan, Noritomi, Murphy, and Huang disclose all limitations of claim 1. Jin further discloses the mass ratio of Prussian blue and the carbon ink is 1: (1 to 99) and the support is PVC (p. 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Jin, Fan, Noritomi, Murphy, and Huang by adjusting the molar ratio of metal cyanoferrate and carbon ink as claimed because it represents a suitable molar ratio for preparation of electrode can be optimized through routine experimentation. MPEP 2144.05 (II)(B). In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). MPEP 2144.05(I). Similarly, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985). MPEP 2144.05(I).
Claim(s) 8 is/are rejected under 35 U.S.C. 103 as being unpatentable over Jin, Fan, Noritomi, Murphy, Huang, and further in view of Hu (CN 108267494), Yang (CN 106950270), and Wang (CN 110596218).
Regarding claim 8, Jin, Fan, Noritomi, Murphy, and Huang disclose all limitations of claim 1. Jin further teaches the low drying temperature is preferably 0 ⁰C to 10 ⁰C (p. 3), but fails to teach wherein in step 3, the concentration of ethanol dehydrogenase solution is 0.1-1U/µL, the volume percent of glutaraldehyde in the mixed enzyme solution being 0.5%-2%, the volume of the mixed enzyme solution applied on the working electrode being 1-5µl.
However, Hu teaches an electrochemical sensor using PPase enzyme which has a final concentration ranges from 0.0 ~ 2.0 U/L (p. 6). Yang teaches an electrode in detecting the concentration of CEA, using glutaraldehyde as a crosslinking agent, which has a volume percentage of 0.25% to 1% (p. 2). Wang teaches preparation of an enzyme biosensor by dripping 2 µL of the mixed enzyme solution on the electrode (p. 3).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Jin, Fan, Noritomi, Murphy, and Huang by adjusting the concentration of the enzyme, the volume percentage of the crosslinker, the volume of the enzyme solution to be applied on the electrode, and the temperature for drying the biosensor as claimed as suggested by Hu, Yang, and Wang. In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). MPEP 2144.05(I). Similarly, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985). MPEP 2144.05(I).
Response to Arguments
Applicant’s arguments have been considered but are unpersuasive.
Applicant agrees that Fan explored the substitution of Fe with Ni to form NiHCF, but asserts that there is no evidence suggesting that such substitution would consistently yield well-defined cubic nanostructures (p. 2, last para.). This argument is unpersuasive because Jin teaches a method for preparing an biosensing chip by synthesizing a hexacyanoferrate/carbon mixed ink (Jin, p. 2), and Fan teaches nickel hexacyanoferrate would provide highly reversible redox ability and good stability for generating well-defined peaks (Fan, p. 2, col. 2, section 2.2). Thus, it would have been obvious to one ordinary skill in the art to substitute Fe with Ni to form NiHCF as taught by Fan to generate well-defined peaks of the biosensing chip, regardless of the recognition of well-defined cubic nanostructures, because Fan teaches, suggests, and motivates one ordinary skill in the art to use Ni for a desirable result. Applicant argues the substitution would need further experimental optimization (p. 3, para. 1). However, Applicant fails to provide evidence to show the required experimental optimization is “undue”. Instead, with the guidance of Fan’s teaching, the experimental optimization would be routine optimization and yield nothing more than predictable results.
Applicant argues the incorporation of gold nanoparticles is not for enzyme immobilization as in Noritomi, but to improve conductivity of the NiHCF matrix and thus enhance electrochemical properties pertinent for NADH detection (p. 3, para. 2). In another word, Applicant argues the motivation and technical rationale of the combination of Jin, Fan, and Noritomi is distinct from the instant invention (p. 3, para. 3). This argument is unpersuasive. The fact that applicant has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious. See Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985).
Applicant argues Murphy merely describes the general chemical reduction of metal precursors using reductants, but does not address the specific integration or synergy between gold nanoparticles and NiHCF in a biosensing context or their effect on electron transfer relevant to ethanol detection (p. 4, para. 1). Applicant seems to argue the combination with Murphy is based on hindsight reasoning. In response, 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 the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Applicant argues Huang fails to appreciate the tailored configuration of enzyme, nanostructure, and electrode interface in the instant invention, i.e., selective ethanol and NADH detection under optimized conditions as claimed in claims 2-8 ( p. 4, last para.). This argument is unpersuasive. For electrochemically sensing an analyte, a specific enzyme is required; for example, ethanol dehydrogenase is used for sensing ethanol as taught by Huang. By pointing out that both alcohol oxidase and ethanol dehydrogenase are commonly used enzymes (p. 5, para. 1), Applicant fails to demonstrate that use of ethanol dehydrogenase is non-obvious to one of ordinary skill in the art.
Applicant argues the instant invention establishes a distinct and optimized set of experimental parameters, tailored to the sensitive and selective detection of ethanol (p. 5, para. 4), and these parameters are not simply results of routine optimization (p. 5, last para.) because the prior art references have no consideration for ethanol-specific challenges (p. 5, para. 3). This argument is unpersuasive. Examiner notes that 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, Jin teaches a method of using hexacyanoferrate/carbon mixed ink to prepare a biosensing chip. Fan teaches the metal Ni is better than Fe to achieve redox ability and good stability, and Noritomi teaches that gold nanoparticles would increase the efficiency of the enzyme reaction or the efficiency of detection. Thus, it would have been obvious to one of ordinary skill in the art to combine Jin, Fan, and Noritomi to arrive at the claimed invention of preparing a biosensing chip due to the desired results by substituting Fe with Ni and using gold nanoparticles. Further, ethanol dehydrogenase is a specific enzyme to detect ethanol, as taught by Huang, and one of ordinary skill in the art would be motivated to tailor the biosensing chip to ethanol biosensor using its specific enzyme, i.e., ethanol dehydrogenase. The dependent claims further limit the synthesis parameters, e.g., pH values, temperature, injection rate, centrifugal rate, centrifugal time, the volume of deionized water, etc., which are taught in the cited prior art references and would be able to be optimized through routine experimentation. These optimizations do not amount to “undue” experimentation because Applicant fails to point out the unpredictability of these experimental parameter or any of these experimental parameters yield unpredictable or surprising results.
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Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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/C. SUN/Primary Examiner, Art Unit 1795
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