DETAILED ACTION
Claims 1-9 are pending.
Election/Restrictions
Applicant’s election without traverse of group I, claims 1-9 in the reply filed on 12/12/2025 is acknowledged.
Upon further review and the applicant’s response, in the reply filed on 12/12/2025, the election of species has been withdrawn.
Applicant cancelled claims 10-20 in the claim amendments filed on 12/12/2025.
Claims 1-9 are under examination.
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.
1.Claims 1-9 are 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.
Claim 1 (and thus dependent claims 2-9) recite “a method of making a single atom nanozyme linked immunoassay”. However, there are no active method steps recited that would lead to the use of an immunoassay. The specification does not provide any indication as to what the active method of making a single atom nanozyme linked immunoassay is. The specification simply recites that the single atom nanozyme can be chemically linked to suitable antibodies for detecting other proteins, bacteria, viruses, or other suitable detection targets (see [0012] – [0013], [0045], and [0053]). As the claim does not recite any active method steps for making a single atom nanozyme linked immunoassay, the metes and bounds of the claimed invention cannot be ascertained. Therefore, the claims are indefinite for failing to particularly point out and distinctly claim the subject matter. For the purpose of compact prosecution, the limitation of “a method of making a single atom nanozyme linked immunoassay” is interpreted to encompass a method of making a single atom nanozyme.
Please note that claims 2-9 are also indefinite due to the dependency on indefinite claim 1.
It is noted any interpretation of the claims set forth above to not relieve Applicant of the responsibility of responding to this rejection. If the actual interpretation of the claim is different than posited by the Examiner, additional rejections and art may be readily applied in a subsequent final Office action.
Examiner suggests amending the claim preamble to reflect the active steps that are followed by the preamble. For example, “A method of making a single atom nanozyme structure”, or “A method of making a nanostructure template for use in single atom nanozyme linked immunoassay”.
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 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 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.
2.Claims 1-3 are rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al. “Single-Atom Nanozyme Based on Nanoengineered Fe-N-C Catalyst with Superior Peroxidase-Like Activity for Ultrasensitive Bioassays.” Small (Weinheim an der Bergstrasse, Germany) vol. 15,48 (2019): e1901485. doi:10.1002/smll.201901485.
Regarding claim 1, Cheng teaches a method of making a single atom nanozyme linked immunoassay, the method comprising:
forming a soft template having nanoscale structures in an aqueous solution (see page 1 “In order to synthesize CNT/FeNC, oxidized CNTs were dispersed in pyrrole solution.”);
adding a solution of a monomer and a solution of a metal containing salt into the aqueous solution such that the metal containing salt causes polymerization of the monomer to form nanostructures according to the nanoscale structures of the previously formed soft template in the aqueous solution (see page 1 “In order to synthesize CNT/FeNC, oxidized CNTs were dispersed in pyrrole solution. The pyrrole molecules would be adsorbed on CNTs due to the π–π interaction between carbon plane of CNTs and pyrrole (Scheme 1a). Then ammonium peroxydisulfate acting as an oxidant was added to induce pyrrole polymerization to obtain polypyrrole coated on CNT (CNT/PPy) (Scheme 1b). Subsequently, the hybrid was dispersed in the mixed Fe(NO3)3 and NaCl solution to adsorb metal cations (Scheme 1c). Finally, the CNT/PPy with adsorbate suffered from a high-temperature pyrolysis at N2 and then NH3 to achieve the CNT/FeNC (Scheme 1d), which was used as nanozyme in this work.”);
upon forming the nanostructures, coating the individual nanostructures with a confinement layer in the aqueous solution (see scheme 1, see page 1 “Then ammonium peroxydisulfate acting as an oxidant was added to induce pyrrole polymerization to obtain polypyrrole coated on CNT (CNT/ PPy) (Scheme 1b).” The instant specification teaches that the confinement layer can include a solid molecular structure that covers at least a portion or the entire surface of the individual nanotubes (see [0009]). Polypyrrole is a known solid molecular structure); and
after coating the individual nanostructures with the confinement layer, pyrolyzing the nanostructures coated with the confinement layer to derive the single atom nanozyme (see pages 1-2 “Subsequently, the hybrid was dispersed in the mixed Fe(NO3)3 and NaCl solution to adsorb metal cations (Scheme 1c). Finally, the CNT/PPy with adsorbate suffered from a high-temperature pyrolysis at N2 and then NH3 to achieve the CNT/FeNC (Scheme 1d), which was used as nanozyme in this work.”),
wherein during the pyrolyzing of the nanostructures, the confinement layer at least restricts or completely prevents migration of atoms on the external surface of the individual nanostructures (see scheme 1c and 1d showing the atoms remaining before and after pyrolysis).
The difference between Cheng and the invention as instantly claimed is that Cheng does not teach multiple nanostructures.
While Cheng does not explicitly teach the use of multiple nanostructures, Cheng does teach all of the invention of instant claim 1 as detailed above. Thus, in view of Cheng, one of ordinary skill in the art would reasonably optimize the use of multiple nanostructures.
In the same manner, duplication of parts is not patentably significant. See MPEP 2144.04(VI)(B): B. Duplication of Parts In re Harza, 274 F.2d 669, 124 USPQ 378 (CCPA 1960) (Claims at issue were directed to a water-tight masonry structure wherein a water seal of flexible material fills the joints which form between adjacent pours of concrete. The claimed water seal has a "web" which lies in the joint, and a plurality of "ribs" projecting outwardly from each side of the web into one of the adjacent concrete slabs. The prior art disclosed a flexible water stop for preventing passage of water between masses of concrete in the shape of a plus sign (+). Although the reference did not disclose a plurality of ribs, the court held that mere duplication of parts has no patentable significance unless a new and unexpected result is produced.).
Regarding claim 2, Cheng teaches the nanostructures individually having multiple active sites for catalyzing an oxidation reaction (see page 1 “The synthesis and catalytic mechanism of atomically dispersed CNT/FeNC SAN has been illustrated in Scheme 1. In order to synthesize CNT/FeNC, oxidized CNTs were dispersed in pyrrole solution. The pyrrole molecules would be adsorbed on CNTs due to the π–π interaction between carbon plane of CNTs and pyrrole (Scheme 1a). Then ammonium peroxydisulfate acting as an oxidant was added to induce pyrrole polymerization to obtain polypyrrole coated on CNT (CNT/PPy) (Scheme 1b).” see scheme 1);
the confinement layer coats at least some of the multiple active sites on the individual nanostructures (see scheme 1B); and
the method further includes removing the confinement layer from the individual nanostructures after pyrolyzing the nanostructures (see scheme1D).
Regarding claim 3, Cheng teaches the nanostructures individually have multiple active sites for catalyzing an oxidation reaction, the individual active sites having a single atom of the metal in the metal containing salt covalently connected to additional atoms of the polymerized monomer (see scheme 1, see pages 1-2 “Subsequently, the hybrid was dispersed in the mixed Fe(NO3)3 and NaCl solution to adsorb metal cations (Scheme 1c). Finally, the CNT/PPy with adsorbate suffered from a high-temperature pyrolysis at N2 and then NH3 to achieve the CNT/FeNC (Scheme 1d), which was used as nanozyme in this work. During the synthesis process, NaCl was used to assist Fe in directly converting to atomic Fe–Nx–C moieties, leading to 100% Fe-Nx-C moieties in the final CNT/FeNC SAN (Scheme 1e).);
the confinement layer coats at least some of the multiple active sites having the single atom of the metal in the metal containing salt (see scheme 1c-e); and
the method further includes removing the confinement layer from the individual nanostructures after pyrolyzing the nanostructures (see scheme 1c-d showing the confinement layer being removed after pyrolyzing the nanostructures).
It would have been obvious to one of ordinary skill in the art at the time of the instant application to perform the methods as taught by Cheng, where multiple nanostructures are used, to arrive at the instantly claimed invention. As Cheng shows, a nanostructure can be made with the methods as claimed above, one of ordinary skill would have reasonably created multiple nanostructures in the same manner with a reasonable expectation of success. It would have been prima facie obvious to one of skill in the art to reasonably optimize the use of multiple nanostructures. Thus, claims 1-3 are obvious to one of ordinary skill at the time of filing, especially in the absence of evidence to the contrary.
3.Claims 4-5 are rejected under 35 U.S.C. 103 as being unpatentable over Cheng as applied to claims 1-3 above and in view of Yang et al., “Rod-shape inorganic biomimetic mutual-reinforcing MnO2-Au nanozymes for catalysis-enhanced hypoxic tumor therapy”. Nano Res. 13, 2246–2258 (2020). https://doi.org/10.1007/s12274-020-2844-3.
Regarding claim 4-5, Yang teaches adding potassium permanganate (KMnO4) to the aqueous solution upon forming the nanostructures (see page 2247 “Briefly, dried MSNRs were evenly blended in 4 mL KMnO4 (0.02 M) by sonicating for 15 min.”); and
reducing the KMnO4 to form a magnesium oxide (MnO2) coating on the external surface of the nanostructures (see page 2250 “And oxygen generation catalyst (MnO2) was grown on the surface of MSNR by in situ reduction of KMnO4. As revealed in the TEM images (Fig. 1(b)), a core–shell structure with a shell thickness of about 10 nm was observed, indicating the successful coating of MnO2.”).
It would have been obvious to one of ordinary skill in the art at the time of the application to consider combining the methods of creating single-atom nanozymes taught by Cheng, with the addition of KMnO4 and MnO2 taught by Yang. Yang teaches that oxygen generated catalyst (MnO2) was grown on the surface of the mesoporous silica nanorods (MSNR) by in situ reduction of KMnO4 (see page 2250). Yang provides motivation by teaching that MnO2 provides the benefits of increased oxygen content, thermal sensitivity, and the ability to rapidly catalyze the composition of hydrogen peroxide into oxygen (see page 2257). The artisan would have reasonable expectation of success based on the cumulative disclosure of these prior art references at the time the instant application was filed.
4.Claims 6-8 are rejected under 35 U.S.C. 103 as being unpatentable over Cheng as applied to claims 1-3 above, in view of Yang et al., “Rod-shape inorganic biomimetic mutual-reinforcing MnO2-Au nanozymes for catalysis-enhanced hypoxic tumor therapy”. Nano Res. 13, 2246–2258 (2020). https://doi.org/10.1007/s12274-020-2844-3, and in view of Junseop et al., “Fabrication of Multidimensional Metal/Conducting Polymer Hybrid Nanoparticles and Their Chem/Bio Sensor Applications” Thesis from Graduate School of Seoul National University (2015).
Regarding claim 7, Cheng teaches wherein the individual polypyrrole nanotubes having multiple active sites each having a single iron (Fe) atom covalently connected to additional nitrogen (N) atoms which in turn are covalently connected to additional carbon (C) atoms of the polypyrrole (see scheme 1d-e showing Fe being bonded to nitrogen atoms, and being bonded to carbon atoms, see page 2 “During the synthesis process, NaCl was used to assist Fe in directly converting to atomic Fe–Nx–C moieties, leading to 100% Fe-Nx-C moieties in the final CNT/FeNC SAN (Scheme 1e). The atomically dispersed CNT/FeNC SAN was proposed to possess superior peroxidase-like activity, which means that it can catalyze H2O2 to generate hydroxyl radicals (·OH) though surface Fenton reaction between H2O2 and Fe ion.[12,33]”).
Cheng does not tech the use of iron chloride, potassium permanganate, or magnesium oxide.
Yang teaches adding potassium permanganate (KMnO4) to the aqueous solution upon forming the nanostructures (see page 2247 “Briefly, dried MSNRs were evenly blended in 4 mL KMnO4 (0.02 M) by sonicating for 15 min.”); and
reducing the KMnO4 to form a magnesium oxide (MnO2) coating on the external surface of the nanostructures (see page 2250 “And oxygen generation catalyst (MnO2) was grown on the surface of MSNR by in situ reduction of KMnO4. As revealed in the TEM images (Fig. 1(b)), a core–shell structure with a shell thickness of about 10 nm was observed, indicating the successful coating of MnO2.”).
Junseop teaches nanostructures that contain multiple nanotubes (see figure 6);
adding the solution of the monomer and the solution of the metal containing salt includes adding a solution of pyrrole monomer and a solution of iron chloride (FeCl3) to the aqueous solution such that the iron chloride (FeCl3) causes polymerization of the pyrrole monomer to form the multiple nanotubes of polypyrrole according to the nanoscale structures of the previously formed soft template in the aqueous solution (see page 35 “Uniformly sized polypyrrole (PPy) nanoparticles were previously prepared with PVA, FeCl3, and pyrrole monomer in distilled water, as following (Figure 11).”, see figure 11 showing polymerization, see page 37 “The materials obtained from the electrospray were dispersed in various concentrations of the FeCl3 aqueous solution and stirred for 4 h at 70°C.”).
Regarding claim 8, Cheng teaches wherein the individual polypyrrole nanotubes having multiple active sites each having a single iron (Fe) atom covalently connected to additional nitrogen (N) atoms which in turn are covalently connected to additional carbon (C) atoms of the polypyrrole (see scheme 1d-e showing Fe being bonded to nitrogen atoms, and being bonded to carbon atoms, see page 2 “During the synthesis process, NaCl was used to assist Fe in directly converting to atomic Fe–Nx–C moieties, leading to 100% Fe-Nx-C moieties in the final CNT/FeNC SAN (Scheme 1e). The atomically dispersed CNT/FeNC SAN was proposed to possess superior peroxidase-like activity, which means that it can catalyze H2O2 to generate hydroxyl radicals (·OH) though surface Fenton reaction between H2O2 and Fe ion.[12,33]”).
Cheng further teaches during the pyrolyzing of the formed nanotube, at least restricting or completely preventing the migration of iron (Fe) atoms on the external surface of the nanotube, thereby reducing aggregation of the iron (Fe) atoms during pyrolysis (see scheme 1). While Cheng does not explicitly teach that MnO2 coating at least restricts or prevents migration of iron atoms, one of ordinary skill in the art would have considered swapping out the polypyrrole confinement layer taught by Cheng, with Yang’s MnO2 confinement layer as MnO2 provides the benefit of increasing oxygen content and thermal sensitivity (see page 2257) and MnO2 can rapidly catalyze the composition of endogenous hydrogen peroxide (H2O2) into oxygen (see page 2257).
Cheng does not tech the use of iron chloride, potassium permanganate, or magnesium oxide.
Yang teaches adding potassium permanganate (KMnO4) to the aqueous solution upon forming the nanostructures (see page 2247 “Briefly, dried MSNRs were evenly blended in 4 mL KMnO4 (0.02 M) by sonicating for 15 min.”); and
reducing the KMnO4 to form a magnesium oxide (MnO2) coating on the external surface of the nanostructures (see page 2250 “And oxygen generation catalyst (MnO2) was grown on the surface of MSNR by in situ reduction of KMnO4. As revealed in the TEM images (Fig. 1(b)), a core–shell structure with a shell thickness of about 10 nm was observed, indicating the successful coating of MnO2.”).
Junseop teaches nanostructures that contain multiple nanotubes (see figure 6);
adding the solution of the monomer and the solution of the metal containing salt includes adding a solution of pyrrole monomer and a solution of iron chloride (FeCl3) to the aqueous solution such that the iron chloride (FeCl3) causes polymerization of the pyrrole monomer to form the multiple nanotubes of polypyrrole according to the nanoscale structures of the previously formed soft template in the aqueous solution (see page 35 “Uniformly sized polypyrrole (PPy) nanoparticles were previously prepared with PVA, FeCl3, and pyrrole monomer in distilled water, as following (Figure 11).”, see figure 11 showing polymerization, see page 37 “The materials obtained from the electrospray were dispersed in various concentrations of the FeCl3 aqueous solution and stirred for 4 h at 70°C.”).
Regarding claim 6, Yang teaches coating the individual formed nanotubes includes: adding potassium permanganate (KMnO4) to the aqueous solution upon forming the polypyrrole nanotubes (see page 2247 “Briefly, dried MSNRs were evenly blended in 4 mL KMnO4 (0.02 M) by sonicating for 15 min.”); and
reducing the added potassium permanganate (KMnO4) to form a magnesium oxide (MnO2) coating on the external surface of the individual formed polypyrrole nanotubes (see page 2250 “And oxygen generation catalyst (MnO2) was grown on the surface of MSNR by in situ reduction of KMnO4. As revealed in the TEM images (Fig. 1(b)), a core–shell structure with a shell thickness of about 10 nm was observed, indicating the successful coating of MnO2.”).
Yang does not teach adding iron chloride (FeCl3) to an aqueous solution.
Junseop teaches nanostructures that contain multiple nanotubes (see figure 6);
adding the solution of the monomer and the solution of the metal containing salt includes adding a solution of pyrrole monomer and a solution of iron chloride (FeCl3) to the aqueous solution such that the iron chloride (FeCl3) causes polymerization of the pyrrole monomer to form the multiple nanotubes of polypyrrole according to the nanoscale structures of the previously formed soft template in the aqueous solution (see page 35 “Uniformly sized polypyrrole (PPy) nanoparticles were previously prepared with PVA, FeCl3, and pyrrole monomer in distilled water, as following (Figure 11).”, see figure 11 showing polymerization, see page 37 “The materials obtained from the electrospray were dispersed in various concentrations of the FeCl3 aqueous solution and stirred for 4 h at 70°C.”).
It would have been obvious to one of ordinary skill in the art at the time of the application to consider combining the methods of creating single-atom nanozymes taught by Cheng, with the addition of KMnO4 and MnO2 taught by Yang, and with the teaching of adding a solution of pyrrole monomer and a solution of iron chloride to form multiple nanotubes taught by Junseop. Yang provides motivation by teaching that MnO2 provides the benefits of increased oxygen content, thermal sensitivity, and the ability to rapidly catalyze the composition of hydrogen peroxide into oxygen (see page 2257). Junseop provides motivation by teaching that polypyrrole is a promising conducting polymer because of their easy synthesis, tunable conductivity, reversible redox property, and their environmental stability (see page 14). Junseop provides motivation by teaching that the amount of pyrrole monomer used during fabrication can control the diameter of the nanoparticles (see page 82). Junseop provides further motivation by teaching that the morphology and density are controlled by the concentration of FeCl3 in the aqueous solution (see pages 52-53). The artisan would have reasonable expectation of success based on the cumulative disclosure of these prior art references at the time the instant application was filed.
5.Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al., as applied to claims 1-3 above, and in view of Tao et al. “Conjugation of antibodies and aptamers on nanozymes for developing biosensors.” Biosensors & bioelectronics vol. 168 (2020): 112537. doi:10.1016/j.bios.2020.112537
Regarding claim 8, Cheng teaches the use of single-atom nanozymes for ultrasensitive bioassays (see pages 1).
Cheng does not teach covalently linking the formed nanozyme to an antibody.
Tao teaches covalently linking the nanozyme to an antibody (see page 1 “To use nanozymes, a critical step is to conjugate them to affinity molecules such as antibodies and aptamer via bioconjugation (e.g. a covalent linkage or high affinity biotin/avidin interactions), or via physisorption.”).
It would have been obvious to one of ordinary skill in the art at the time of the application to consider combining the methods of creating single-atom nanozymes taught by Cheng, with the teachings of covalently linking antibodies with nanozymes for use with biosensors taught by Tao. Tao provides motivation by teaching that in order to use nanozymes, a critical step is to conjugate them to affinity molecules such as antibodies via bioconjugation (e.g. a covalent linkage) (see page 1). Tao provides motivation by teaching the use of a nanozyme with a lateral flow strip design (see figure 5e, see page 7 “Developing more cost-effective and time-saving assays with high sensitivity and selectivity is key in biosensor development. Lateral flow immunochromatography has attracted extensive attention for its simplicity, where the best-known example is probably the pregnant test strips (Zhao et al., 2018). This technology has also been used in the nanozyme field. Duan et al. (2015) developed MNP-based immunochromatographic strips using Fe3O4 nanoparticles as a nanozyme probe. This was the first report of introducing the Fe3O4 nanozyme into an immunochromatographic strip and the sensitivity was about 100 times greater than that of a colloidal gold strip, attributable to the catalytic signal amplification (Fig. 5e).”). Tao provides further motivation by teaching that the use of nanozymes in an immunochromatographic strip increased the sensitivity about 100 times greater than that of a colloidal gold strip, attributable to the catalytic signal amplification (see page 7). Lastly, Tao provides motivation by teaching that the nanozyme-strip method can be used for diagnosis of infectious diseases without the use of advanced analytical instruments, making it more accessible and affordable (see page 7). The artisan would have reasonable expectation of success based on the cumulative disclosure of these prior art references at the time the instant application was filed.
Conclusion
No claim is allowed.
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/MCKENZIE A DUNN/ Examiner, Art Unit 1678
/GREGORY S EMCH/ Supervisory Patent Examiner, Art Unit 1678