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 1-6 and 8-18 are pending and examined herein.
Priority
The present application, filed 12/13/2024, is a 371 of PCT/SG2022/050590, filed 08/19/2022, which claims foreign priority of SG10202109134Q, filed 08/20/2021.
Information Disclosure Statement
The Information Disclosure Statement(s) filed 02/13/2024 and 06/25/2024 are acknowledged and have
been considered.
Specification
The disclosure is objected to because of the following informalities: The description of FIG. 1A in paragraph [0012] is inconsistent with the drawing. Paragraph [0012] states that FIG. 1A is a general schematic illustration showing cellulose fibers (CF) SERS substrate fabrication and treatment of the cellulose fibers SERS substrate for biomarker proteins sensing. However, FIG. 1A depicts protein immobilization, blocking, and antibody immobilization on an already formed silver nanoparticle-decorated cellulose-fiber membrane, whereas fabrication of the substrate is depicted in FIG. 1B. Paragraphs [00107] and [00118] further reflect this inconsistency. Appropriate correction is required.
Claim Objections
Claim 1 is objected to because of the following informalities: The abbreviation “SERS” is used without first setting forth the corresponding terminology. Appropriate correction is required to recite “surface enhanced Raman spectroscopy (SERS)” upon first occurrence in the claim.
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.
Claims 15-18 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 15 recites “a method of determining a state of wound healing in a diabetic individual.” However, the body of the claim merely recites contacting a wound sample with a thiolating agent, immobilizing one or more modified proteins on the SERS-active nanoparticles, contacting the immobilized proteins with antibody-associated Raman probes, and subjecting the membrane to SERS spectroscopy to generate one or more SERS signals corresponding to one or more biomarkers. The claim does not recite any step, comparison, threshold, reference value, or other criterion by which the generated SERS signals are used to determine the state of wound healing. Accordingly, it is unclear whether the recited “determining a state of wound healing” is satisfied merely by generating biomarker-specific SERS signals, whether the signals must be quantified to determine corresponding biomarker concentrations, or whether the signals or concentrations must be compared with a reference, control, healing range, or non-healing range to classify the wound-healing state. Therefore, the metes and bounds of claim 15 cannot be determined with reasonable certainty. Appropriate correction is required.
Claims 16-18 depend from claim 15 and therefore incorporate all of the limitations of claim 15, including the indefinite limitation directed to “determining a state of wound healing in a diabetic individual.” Therefore, since claims 16-18 depend from and incorporate the indefinite limitations of claim 15 without curing the ambiguity, the metes and bounds of claims 16-18 cannot be determined with reasonable certainty. Appropriate correction is required.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1, 4, 5, 8, 11, 13 and 14 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Park et al. (Plasmonic Schirmer Strip for Human Tear-Based Gouty Arthritis Diagnosis Using Surface-Enhanced Raman Scattering. ACS Nano. Vol. 11, No. 1, January 2017 – IDS dated 02/13/2024).
For purposes of examination under the broadest reasonable interpretation, Park’s flexible and porous cellulose-paper strip, which is configured for direct collection of human tears from the lower eyelid, constitutes the recited biocompatible flexible and porous polymer membrane because claim 1 does not recite specific characteristics required to be biocompatible. Furthermore, Park’s discrete nanoscale Au nanoislands formed directly on the cellulose fibers constitute the recited SERS-active nanoparticles because claim 1 does not further limit the morphology, shape, or method of formation of the nanoparticles.
Regarding claim 1, Park teaches a biocompatible SERS-active membrane configured to detect biomarkers by disclosing a plasmonic Schirmer strip for on-demand, rapid, and simple identification of biomarkers in human tears, and that this functional paper strip enables noninvasive diagnosis of disease-related biomarkers and healthcare monitoring using human tears (Abstract, p. 438). Park further discloses that the device is suitable for biological contact and sample collection by disclosing an efficient collection of human tears by inserting a paper strip into the lower eyelid of the human eye (p. 438; Fig. 1b). Also, Park discloses that the Au nanoislands were positioned 10 mm away from the folding crease for eye safety and that predetermined patterns were used to avoid direct contact between Au nanoislands and the human eye for safety (Results and Discussion, p. 440; Fig. 3a). Park further discloses that all procedures were carried out under the approval of the KAIST Institutional Review Board (Experimental Methods, p. 441). Accordingly, Park’s strip is biocompatible for its disclosed biological-contact and tear-collection use.
Additionally, Park teaches the recited flexible and porous polymer membrane by disclosing that paper contains cellulose nanofiber and microfiber matrices whose hygroscopic micro/nanopores induce strong capillary driven lateral flow (p. 438). Park further discloses that the strip was fabricated on Whatman chromatography paper (Results and Discussion, p. 439), and that the configuration maintains the hygroscopic micro/nanopores for an efficient collection of human tears (p. 438; Figs 1a-1c). Park further discloses that Figure 1 identifies cellulose micro/nanofibers and demonstrates the paper strip and its open cellulose-fiber network, while the inset clearly shows hygroscopic micro/nanopores for capillary driven lateral flow (p. 439; Fig. 1b-1c). Also, Park discloses that the strip width of 5 mm helps an efficient collection of tears without the edge effect, and the folding crease clearly indicates the folding line for the conventional Schirmer test (p. 439, Fig. 1a; p. 440; Figs. 3a). The disclosed paper strip necessarily possess sufficient flexibility to be folded and positioned at the lower eyelid. Hence, Park’s paper strip is therefore a flexible and porous polymer membrane comprising cellulose.
Moreover, Park teaches SERS-active nanoparticles formed on the flexible and porous polymer membrane by disclosing that the diagnostic strip features gold nanoislands directly and evenly formed on the top surface of cellulose fibers (Abstract, p. 438), and that an Au thin film in Volmer−Weber mode directly forms the nanoislands on the top surface of cellulose fibers (Results and Discussion, p. 439). Park further discloses SEM images clearly showing nanogap-rich Au nanoislands on the top surface of cellulose fibers (Results and Discussion, p. 439; Figs. 1-2). These discrete nanoscale gold structures are SERS-active because Park teaches that the nanoislands provide plasmonic enhancement in SERS signals for identification of tear molecules (Abstract, p. 438), and the nanogaps between Au nanoislands on a single cellulose fiber or neighboring fibers create volumetric electromagnetic hot spots and thus result in strong plasmonic enhancement of SERS signals (Results and Discussion, p. 439).
Lastly, Park expressly demonstrates biomarker detection by disclosing that the uric acid in human tears was quantitatively detected at physiological levels (25−150 μM) by using SERS (Abstract, p. 438). Park further reports that the strip provides highly sensitive SERS detection of biomarkers in tears, and a diagnostic guideline for monitoring diverse biomarkers in human tears such as glucose, cortisol, and ascorbic acid (Conclusions, p. 441).
Regarding claim 4, as discussed above, Park expressly teaches gold by disclosing gold nanoislands directly and evenly formed on the top surface of cellulose fibers (Abstract, p. 438).
Regarding claim 5, Park expressly discloses that Au film thicknesses from 4 to 10 nm were fabricated under a constant deposition rate of 0.5 Å/s (Results and Discussion, p. 439). Park teaches that the deposited gold forms nanoislands, rather than a continuous film, over the pertinent thicknesses by teaching the formation of Au became nanoislands from 4 to 8 nm with decreasing nanogaps (Results and Discussion, p. 439; Fig. 2a). Figure 2a identifies plasmonic Schirmer strips having a film thickness of 4, 6, 8, and 10 nm at 0.5 Å/s and further states that Precisely controlled deposition of a Au thin film forms the Au nanoislands in Volmer−Weber mode (Results and Discussion, p. 439; Fig. 2a). Park further discloses that Au nanoislands of 8 nm in film thickness and 0.5 Å/s in deposition rate were finally selected for the plasmonic Schirmer strip (Results and Discussion, p. 440). Park likewise teaches that Au nanoislands in Volmer−Weber mode were directly formed on cellulose micro/nanofiber matrices by controlling the film thickness and the deposition rate during thermal evaporation (Conclusions, p. 441).
Regarding claim 8, as discussed above, Park teaches a method of forming the biocompatible SERS-active polymer membrane of claim 1. Park teaches providing a flexible and porous polymer membrane comprising cellulose by disclosing paper contains cellulose nanofiber and microfiber matrices whose hygroscopic micro/nanopores induce strong capillary-driven lateral flow (p. 438). Figure 1 shows the cellulose micro/nanofiber matrix and the hygroscopic micro/nanopores of the paper strip (p. 439, Fig. 1b-1c). Furthermore, Park teaches depositing SERS-active nanoparticles on the flexible and porous polymer membrane by disclosing that a Au thin film in Volmer−Weber mode directly forms the nanoislands on the top surface of cellulose fibers (Results and Discussion, p. 439), and delicate control of film thickness and deposition rate enables formation of Au nanoislands in Volmer−Weber mode (Experimental Methods, p. 441).
Regarding claim 11, Park teaches that the plasmonic Schirmer strip was simply fabricated on Whatman chromatography paper by using thermal evaporation of an Au thin film (Results and Discussion, p. 439), and that fabrication includes thermal evaporation of an Au thin film on a paper substrate (Experimental Methods, p. 441). Park further discloses that delicate depending on the film thickness and the deposition rate, an Au thin film in Volmer−Weber mode directly forms the nanoislands on the top surface of cellulose fibers (Results and Discussion, p. 439).
Regarding claim 13, Park expressly discloses that Au deposition rates from 0.2 to 2.0 Å/s were used under a constant Au film thickness of 8 nm (Results and Discussion, p. 439). Figure 2a likewise identifies an 8 nm film deposited at rates including 2.0 Å/s at 8 nm and explains that the resulting gold structures are nanoislands (Results and Discussion, p. 439; Fig. 2a). A deposition rate of 2.0 Å/s is 0.2 nm/s. Depositing the expressly disclosed 8 nm thickness at 0.2 nm/s necessarily requires 40 seconds. Thus, Park’s expressly disclosed combination of an 8 nm deposited thickness and a 2.0 Å/s deposition rate necessarily results in a deposition duration of 40 seconds, which is within the claimed duration of up to 60 seconds.
Regarding claim 14, refer to the discussion above. The disclosed range of 0.2–2.0 Å/s corresponds to 0.02–0.2 nm/s. The entire disclosed range is therefore within the claimed rate of up to 1 nm/s. Also, Park selected Au nanoislands of 8 nm in film thickness and 0.5 Å/s in deposition rate for the operative plasmonic Schirmer strip (Results and Discussion, p. 440), wherein 0.5 Å/s corresponds to 0.05 nm/s and is likewise within the claimed range.
Accordingly, Park discloses, expressly or inherently, each and every limitation of claims 1, 4, 5, 8, 11, 13 and 14. Accordingly, claims 1, 4, 5, 8, 11, 13 and 14 are anticipated under 35 U.S.C. 102.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over Park et al. in view of Sun et al. (Dual Functional PDMS Sponge SERS Substrate for the On-Site Detection of Pesticides Both on Fruit Surfaces and in Juice. Analyst. Vol. 143, No. 11, May 2018).
With respect to the teachings of Park, see the discussion above, which applies equally here. However, Park differs from the instant claim in failing to expressly teach or specify that the elastomeric polymer comprises a silicon-based elastomer or the elastomeric polymer comprises polydimethylsiloxane.
Sun teaches the additionally claimed polydimethylsiloxane limitation. In particular, Sun teaches a versatile dual-functional polydimethylsiloxane (PDMS) sponge Surface Enhanced Raman Scattering (SERS) substrate fabricated for detecting analytes on surfaces and in solution (Abstract, p. 2689).. Sun further teaches that the disclosed SERS substrate is based on PDMS sponge modified with Ag NPs, and expressly characterizes the universal SERS substrate as a flexible porous PDMS sponge (Introduction, p. 2690). Sun explains that the porous PDMS sponge can be used not only for surface sampling but also for absorption of analytes from liquid based on its flexibility and porous structures, and that the three-dimensional PDMS sponge contains plenty of pores, thereby permitting absorption of a large quantity of Ag nanoparticles and formation of additional SERS hot spots (Introduction, p. 2690).
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 Park’s flexible and porous SERS-active polymer membrane by selecting Sun’s porous polydimethylsiloxane sponge as the elastomeric polymer membrane material. Sun expressly provides the reason for making this modification by teaching that a flexible porous PDMS sponge is suitable for SERS analysis, supports deposited Ag nanoparticles, conforms to surfaces, and absorbs analytes from liquid through its porous structure. The proposed modification would preserve Park’s liquid-sampling and analyte-collection function while providing the expressly claimed silicon-based elastomeric material. One of ordinary skill in the art would have had a reasonable expectation of success because Sun actually fabricates a flexible, porous PDMS elastomer using a pore-forming template, deposits SERS-active Ag nanoparticles thereon, and demonstrates both surface sampling and liquid absorption followed by SERS detection. Accordingly, substituting Sun’s known porous PDMS SERS membrane for Park’s porous polymer membrane would have been a technically compatible and predictable modification that would have produced the claimed SERS-active membrane comprising polydimethylsiloxane without rendering Park’s membrane inoperative for its intended sampling purpose.
Claims 3 and 9 are rejected under 35 U.S.C. 103 as being unpatentable over Park et al. in view of Qu et al. (CN 104792765 A, refer to English translation provided).
With respect to the teachings of Park, see the discussion above, which applies equally here. However, Park differs from the instant claim in failing to expressly teach or specify that the flexible and porous polymer membrane is functionalized with an anchoring agent to render an amine functional group or a mercapto functional group for binding to a SERS-active nanoparticle, wherein the anchoring agent comprises the recited group (claim 3); nor, teach contacting the flexible and porous polymer membrane with an anchoring agent to provide an amine or mercapto functional group functionalized thereon for binding to a SERS-active nanoparticle (claim 9).
However, Qu teaches the additionally claimed membrane functionalization and anchoring-agent treatment. In particular, Qu teaches a SERS active substrate prepared using silver nanoparticles ([0010], p. 10). Qu further teaches that the pretreated functionalized adsorbent carrier is added to the high-concentration silver nanoparticle solution and that the resulting product is the silver nanoparticle functionalized adsorbent carrier ([0018], p. 14). Qu further teaches that pretreatment of the functionalized adsorbent carrier includes adding the dried adsorbent carrier to a 10% (v/v) 3-mercaptopropyltrimethoxysilane ethanol solution, incubating at room temperature for 24 h, removing the 3-mercaptopropyltrimethoxysilane ethanol solution, washing the adsorbent carrier three times with ethanol, and then drying it by blowing air at 30-40°C to obtain the functionalized adsorbent carrier ([0021], p. 16). The expressly disclosed 3-mercaptopropyltrimethoxysilane is the same mercapto-containing anchoring agent recited in claims 3 and 9.
Furthermore, Qu teaches first treating a paper support with 3-mercaptopropyltrimethoxysilane and thereafter associating silver nanoparticles with the treated paper. In particular, Qu discloses weighing 0.01g of fluffy paper scraps, put them into a centrifuge tube, add 12mL of 10% 3-mercaptopropyltrimethoxysilane ethanol solution, followed by incubation, washing, and drying (Example 5, [0050], p. 33). Qu thereafter teaches adding the functionalized paper scraps obtained in step (2) to the concentrated silver nanoparticle solution and obtain the paper scraps functionalized with silver nanoparticles (Example 5, [0051], pp. 33-34). The resulting silver nanoparticle functionalized paper scrap substrate is then used to make a test strip, and the resulting cotton swab-type SERS active substrate is sealed and stored. (Example 5, [0052], p. 34). Additionally, Qu confirms the technical result of the functionalization treatment. Specifically, Qu discloses that the silver nanoparticles are uniformly and densely assembled on the surface of the cotton fiber, forming a large number of SERS "hot spots", which can produce a good SERS enhancement effect, thereby promoting better SERS performance of the functionalized active substrate (Example 4, [0047], p. 31; Fig. 1).
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 Park’s cellulose-paper SERS membrane and method of forming the membrane by applying Qu’s expressly taught paper-functionalization procedure—contacting Park’s paper support with 3-mercaptopropyltrimethoxysilane before associating the SERS-active metal nanoparticles with the support. Qu provides an express teaching, suggestion, and motivation for this modification because it is directed to the same technical field of paper-based SERS substrates employing noble-metal nanoparticles and expressly teaches treating fluffy paper scraps with 3-mercaptopropyltrimethoxysilane before loading the paper with silver nanoparticles and forming the resulting material into a SERS test strip. One of ordinary skill in the art would have been motivated to apply Qu’s expressly taught paper-functionalization procedure to Park’s paper-based SERS platform because both references are directed to paper-supported SERS substrates employing noble-metal nanoparticles for Raman detection, and Qu expressly teaches that 3-mercaptopropyltrimethoxysilane ethanol solution pretreatment of the paper support enables subsequent nanoparticle attachment and formation of an operative SERS substrate. One of ordinary skill in the art would have had a reasonable expectation of success because Qu expressly performs the 3-mercaptopropyltrimethoxysilane ethanol solution treatment on paper scraps, subsequently loads the functionalized paper with silver nanoparticles, and forms the resulting material into a paper-based SERS test strip. Qu further demonstrates on a fibrous cotton carrier that the same treatment produces silver nanoparticles uniformly and densely assembled on the fiber surface, numerous SERS hot spots, a good SERS enhancement effect, and better SERS performance. Moreover, Park and Qu both concern paper-based or fibrous SERS supports bearing noble-metal nanostructures for Raman detection. Thus, the proposed modification would have applied Qu’s expressly demonstrated paper-functionalization and nanoparticle-loading procedure to Park’s analogous cellulose-paper SERS platform for the same known purpose of supporting SERS-active metal nanostructures, with a reasonable expectation that the functionalized paper would predictably support the nanoparticles and remain operative for SERS detection.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Park et al. in view of Perumal et al. (Development of a Novel SERS Biosensing Platform with Flexible Substrates for Monitoring Wound Healing. Abstract of Oral Presentation at WUWHS 2020 Global Health Changing Lives, Abu Dhabi, UAE, March 8-12, 2020 – IDS dated 02/13/2024; https://doi.org/10.12968/jowc.2020.29.Sup7b.1).
With respect to the teachings of Park, see the discussion above, which applies equally here. However, Park differs from the instant claim in failing to expressly teach or specify that the biomarkers comprise a matrix metalloproteinase, tumor necrosis factor alpha, and/or an interleukin.
However, Perumal teaches the additionally claimed biomarkers in the same flexible SERS biosensing context. In particular, Perumal teaches that that growth factors and pro-inflammatory cytokines, such as interleukins, matrix metalloproteinase (MMP), and tumour necrosis factor alpha, play an important role in chronic wound healing (Background and Aims, p. 169). Perumal further teaches potential of developing a Surface Enhanced Raman Spectroscopy (SERS) based flexible biosensor as a viable point-of-care platform to monitor the changes of these biomarkers to track the trajectory of wound healing (Background and Aims, p. 169). Perumal further discloses developing a sensitive SERS immunoassay using flexible and transparent PDMS, as well as a paper substrate, and that these functionalized flexible SERS substrates can be used for the collection of wound exudates for the sensitive detection of various wound specific biomarkers based on their fingerprint Raman spectra (Method, p. 169). However, Perumal does not expressly teach or specify that the flexible PDMS or paper substrate is porous, as recited in claim 1.
Furthermore, Perumal reports actual detection of biomarkers falling within each alternative recited in claim 6. Specifically, Perumal teaches detecting for example, TNF-α (100 pg to 0.1 µg ml-1), MMP-9 (0.1 to 5 µg ml-1) and IL-1α (1 ng to 1 µg ml-1), in a serum solution mimicking wound exudates (Results, p. 169). Lastly, Perumal teaches that flexible SERS substrates have promise as diagnostic tools, as well as potential to be incorporated into wound dressings, and that this could provide an affordable and reliable sensing platform for the point-of-care monitoring of biomarkers to assess the healing trajectory of chronic wounds (Conclusion, p. 169).
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 Park’s flexible paper-based SERS biomarker-detection membrane to detect one or more wound-healing biomarkers comprising a matrix metalloproteinase, tumor necrosis factor alpha, and/or an interleukin, as expressly taught by Perumal. Perumal provides an express teaching, suggestion, and motivation for the modification because it identifies interleukins, matrix metalloproteinases, and TNF-α as biologically important chronic-wound biomarkers and expressly teaches detecting TNF-α, MMP-9, and IL-1α using a flexible SERS immunoassay that includes a paper substrate for wound-exudate collection and wound-healing monitoring. One of ordinary skill in the art therefore would have been motivated to configure Park’s operative cellulose-paper SERS membrane to detect the biomarkers identified by Perumal so that Park’s sensitive SERS platform could be used to monitor biomolecular changes associated with wound-healing trajectory. One of ordinary skill in the art would have had a reasonable expectation of success because Perumal reports actual SERS detection of TNF-α, MMP-9, and IL-1α at clinically relevant concentrations in a serum solution mimicking wound exudates using flexible SERS substrates, including a paper substrate, while Park already demonstrates an operative flexible cellulose-paper SERS platform for detecting biomarkers in biological samples. Accordingly, the proposed modification would have applied Perumal’s expressly demonstrated selection and SERS detection of wound-healing biomarkers to Park’s analogous paper-based SERS platform for the same known biomarker-detection function, with a reasonable expectation that the resulting membrane would predictably detect at least one of the biomarker classes recited in claim 6.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Park et al. in view of Sun et al. and Kumar et al. (A Facile Method for Fabrication of Buckled PDMS Silver Nanorod Arrays as Active 3D SERS Cages for Bacterial Sensing. Chemical Communications. Vol. 51, No. 62, August 2015).
With respect to the teachings of Park, see the discussion above, which applies equally here. However, Park differs from the instant claim in failing to expressly teach or specify that the flexible and porous polymer membrane comprises an elastomeric polymer and that the membrane is stretched up to 60% before depositing the SERS-active nanoparticles.
With respect to the teachings of Sun, see the discussion above, which applies equally here. Sun teaches a flexible and porous polymer membrane comprising an elastomeric polymer. However, Sun does not expressly teach stretching the porous elastomeric membrane before depositing the SERS-active nanoparticles.
Kumar teaches this additional fabrication limitation. In particular, Kumar teaches a method for fabricating a highly sensitive flexible Ag nanorod (AgNR) array based SERS substrates on buckled poly(dimethylsiloxane) (PDMS), in which the AgNRs were deposited on 30% pre-stretched PDMS using oblique angle deposition (p. 12411). Kumar further explains that the fabrication route includes mounting the PDMS film on a strain stage, stretching the PDMS to a designated initial strain value, and thereafter performing growth of Ag nanorods over the pre-stretched PDMS film (Scheme 1, p. 12412). Kumar further teaches that, for AgNRs deposited over pre-stretched elastomeric PDMS films subjected to initial mechanical strain, release of the strain forms periodic wrinkles (p. 12412). Kumar explains that this AgNR-PDMS buckled system increases the number of hotspots and also provides better entrapment of the analyte onto the AgNRs giving rise to enhancement in the Raman signal (p. 12411).
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 Park’s SERS-active porous polymer membrane and fabrication method by first employing Sun’s flexible porous PDMS sponge as the elastomeric polymer membrane and then applying Kumar’s known pre-deposition stretching technique by stretching that elastomeric membrane within the claimed range before depositing the SERS-active silver nanostructures. Sun expressly provides the reason for the first modification by teaching that a porous PDMS elastomer can retain Ag nanoparticles, absorb liquid analytes, perform surface sampling, and function as a sensitive SERS substrate, thereby preserving rather than defeating Park’s analyte-collection purpose. Kumar expressly provides the reason for the second modification by teaching that depositing Ag nanorods on prestretched elastomeric PDMS and subsequently releasing the strain produces periodic surface buckling, increases SERS hotspot density, increases analyte–metal contact area, improves analyte entrapment, and enhances Raman intensity. One of ordinary skill in the art therefore would have been motivated to apply Kumar’s prestretch-before-deposition technique to Sun’s porous PDMS SERS membrane to improve the morphology, hotspot density, and SERS response of the membrane while retaining the porous liquid-sampling architecture supplied by Sun. The modification would have required only mechanically tensioning Sun’s already-elastomeric flexible, porous PDMS substrate during silver deposition, rather than replacing the porous substrate or altering its liquid absorbing architecture. One of ordinary skill in the art would have had a reasonable expectation of success because Sun demonstrates that porous PDMS is a functional elastomeric SERS support capable of receiving and retaining deposited Ag nanoparticles, while Kumar demonstrates that elastomeric PDMS can be mechanically prestretched to 30%, coated with SERS-active silver nanorods while under strain, and released to produce enhanced SERS performance. The proposed modification therefore applies Kumar’s established PDMS fabrication technique to Sun’s closely related porous PDMS SERS substrate for the same recognized purpose of supporting silver SERS structures and increasing Raman enhancement, and would have predictably produced a flexible and porous elastomeric membrane stretched within the claimed range before deposition of the SERS-active nanoparticles.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Park et al. in view of Salomon et al. (WO 2017/033185 A1).
With respect to the teachings of Park, see the discussion above, which applies equally here. However, Park differs from the instant claim in failing to expressly teach or specify that depositing the SERS-active nanoparticles is carried out at a deposition current of 10 mA to 20 mA.
However, Salomon teaches current as an operating parameter during evaporative physical vapor deposition for forming nanoporous metal films. Salomon teaches physical vapor deposition (PVD) process, wherein the deposition is performed at a power of less than about 90W or at current ranging from about 0.5 mA to about 100 mA (Abstract, p. 1). Salomon further discloses that the PVD process is an evaporative deposition and that evaporative deposition is a common method of thin-film deposition (p. 29). Salomon further teaches that the source material is evaporated in a vacuum. The vacuum allows vapor particles to travel directly to the substrate, where they condense back to a solid state (p. 29). Salomon further teaches that, in evaporative deposition, the deposition source comprises a metal or a metal oxide source and an energy source that evaporates the metal or metal oxide (p. 29). Salomon expressly teaches that the energy source operates at a current ranging from about 1 mA to about 10 mA, from about 10 mA to about 20 mA, from about 20 mA to about 30 mA, from about 30 mA to about 40 mA, from about 40 mA to about 50 mA, from about 50 mA to about 60 mA, from about 60 mA to about 70 mA, from about 70 mA to about 80 mA, from about 80 mA to about 90 mA, or from about 90 mA to about 100 mA (p. 29). Thus, Salomon expressly discloses use of a 10-20 mA operating current in the evaporative-deposition process, which is the same numerical current range recited for carrying out deposition in claim 12.
Also, Salomon provides an operative evaporative-deposition embodiment. Example 4 teaches evaporative deposition of a nanoporous metal film using a BESTEC machine, and Table 5 identifies the evaporation-process parameters as current 1–100 mA, stage rotation 5 rpm, and pellets distance from stage 45 cm (Example 4, Table 5, p. 39). This working disclosure confirms that electrical current is an operative process parameter of Solomon’s evaporative metal-deposition apparatus and that the expressly disclosed 10-20 mA interval lies within the 1-100 mA range employed by the evaporative-deposition embodiment. Additionally, Salomon connects the deposited nanoporous metal films to the same SERS field as Park. Salomon teaches that the nanoporous metal-based film is configured for use in optical sensing, including chemical or biological sensors based on surface enhanced Raman spectroscopy (SERS) enhanced signals or other plasmonic-based sensors (p. 35). Lastly, Salomon teaches that the deposited metal may comprise noble metals including Au and Ag (pp. 6, 27–28, and 31–32).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to carry out Park’s thermal-evaporation deposition of Au onto the cellulose-paper substrate using an evaporative-deposition operating current of about 10 mA to about 20 mA, as expressly identified by Salomon. Salomon provides an express reason for the modification because it teaches evaporative deposition as a thin-film-deposition process in which a metal source is evaporated in vacuum and deposited onto a substrate, expressly identifies 10–20 mA as an operating-current range for the energy source performing that evaporation, demonstrates an operative evaporative-deposition embodiment in which current is a controlled process parameter, and teaches that the resulting nanoporous noble-metal films are suitable for chemical or biological sensors based on SERS-enhanced signals. One of ordinary skill in the art therefore would have been motivated to employ Salomon’s expressly disclosed 10–20 mA current interval when conducting Park’s closely analogous thermal evaporation of Au because both processes use electrically operated evaporation equipment to deposit noble-metal nanostructures for plasmonic sensing. One of ordinary skill would have had a reasonable expectation of success because Salomon demonstrates evaporative metal deposition over an operative current range encompassing 10–20 mA, while Park independently demonstrates successful thermal evaporation of Au to form SERS-active nanoislands on cellulose paper. Thus, the proposed modification would have applied Salomon’s known evaporative-deposition operating parameter to Park’s analogous noble-metal thermal-evaporation process according to its established function of forming plasmonic metal nanostructures suitable for SERS detection.
Claims 15-18 are rejected under 35 U.S.C. 103 as being unpatentable over Park et al. in view of Perumal et al., Haddada et al. (Gold Nanoparticle-Based Localized Surface Plasmon Immunosensor for Staphylococcal Enterotoxin A (SEA) Detection. Analytical and Bioanalytical Chemistry. Vol. 409, No. 26, October 2017) and Vo-Dinh et al. (US 7,267,948 B2).
With respect to the teachings of Park, see the discussion above, which applies equally here. However, Park differs from the instant claim in failing to expressly teach or specify the following: a method of determining a state of wound healing in a diabetic individual that includes contacting a sample extracted from a wound with a thiolating agent to form modified biomarker proteins, immobilizing the modified proteins on the SERS-active nanoparticles, providing Raman probes attached to corresponding antibodies, contacting the probes with the immobilized modified proteins, and generating corresponding biomarker SERS signals (claim 15); contacting the biocompatible SERS-active polymer membrane with a serum albumin to have the serum albumin immobilized on surfaces of the flexible and porous polymer membrane which are not occupied by the one or more modified proteins (claim 16); the thiolating agent comprises a Traut’s reagent (claim 17); the one or more antibodies comprise an antibody of matrix metalloproteinase, an antibody of tumor necrosis factor alpha, and/or antibody of interleukin (claim 18).
Regarding claim 15, as discussed above, Perumal teaches the additionally claimed diabetic-wound context, wound sample, wound-healing biomarkers, and flexible SERS immunoassay. Perumal teaches that one of the prevalent comorbidities is the formation of foot ulcers and that monitoring and tracking the response of diabetic foot ulcers (DFU) to treatment is difficult for clinicians (Background and Aims, p. 169). Perumal further teaches that growth factors and pro-inflammatory cytokines, such as interleukins, matrix metalloproteinase (MMP), and tumour necrosis factor alpha, play an important role in chronic wound healing (Background and Aims, p. 169), and describes a Surface Enhanced Raman Spectroscopy (SERS) based flexible biosensor as a viable point-of-care platform to monitor the changes of these biomarkers to track the trajectory of wound healing (Background and Aims, p. 169). Also, as discussed above, Perumal teaches a sensitive SERS immunoassay using flexible and transparent PDMS, as well as a paper substrate, and that these functionalized flexible SERS substrates can be used for the collection of wound exudates for the sensitive detection of various wound specific biomarkers based on their fingerprint Raman spectra (Method, p. 169). However, Perumal does not expressly teach or specify that the flexible PDMS or paper substrate is porous, as recited in claim 1. Lastly, Perumal reports detection of TNF-α, MMP-9, and IL-1α in a serum solution mimicking wound exudates and in the process of monitoring their presence in wound fluid to track changes in these biomarkers as wounds heal (Results, p. 169).
Haddada teaches the additionally claimed treatment of a protein biomarker with a thiolating agent to form a modified protein and immobilization of the modified protein on gold nanoparticles. Specifically, Haddada teaches preparing AuNP bioconjugates by covalently attaching anti-Staphylococcal enterotoxin A (SEA) antibody (Ab) or SEA to AuNPs, and that this was achieved by reacting Traut’s reagent with lysine residues of both proteins to generate thiol groups that bind to gold atoms on AuNp surface (Abstract, p. 6227). Haddada further teaches that SEA and anti-SEA antibody were treated by Traut’s reagent to convert some of their primary amines to thiol groups followed by conjugation with the AuNP (Introduction, p. 6228). Thus, Haddada expressly treats the protein antigen itself—not merely the antibody—with a thiolating agent to form a thiol-modified protein for attachment to gold. Additionally, Haddada’s experimental procedure teaches that Traut’s reagent (85 μM) was reacted with anti-SEA (0.32 mg/mL, 0.55 μM) in 50Mm phosphate buffer pH 8.0 at room temperature to generate sulfhydryl groups, and that the same procedure was applied to prepare AuNP-SEA bioconjugate using a solution of thiolated SEA at 24 μg/mL (Experimental Section, p. 6228). Haddada further explains that this coupling method ensures a covalent immobilization of the protein molecules on the surface of the gold nanoparticles by formation of Au-S bonds (Results and Discussion, p. 6229). Moreover, Haddada teaches that Figure 1A depicts SEA treatment with 2-Iminothiolane (Traut’s reagent), formation of an SH-containing SEA protein, and subsequent attachment of the modified SEA to AuNPs (Fig. 1A, p. 6229).
Vo-Dinh teaches the additionally claimed antibody-bearing Raman probes, specific antibody-protein recognition, contacting the probes with the immobilized proteins, and generation of SERS signals. Vo-Dinh teaches a Raman integrated sensor system for the detection of targets including biotargets, wherein the sampling platform is preferably a Raman active surface-enhanced scattering (SERS) platform, and the receptors can include at least one protein receptor (Abstract, p. 1). Vo-Dinh further teaches that antibody probes may also be used with the invention, preferably with other receptor probe types, and such antibodies may then be used as specific probes to identify an analyte of interest (col. 12, p. 28). Furthermore, Vo-Dinh teaches that SERS based immunoassay measurement strategies can be divided into three categories: competitive, direct, and sandwich assays. The assays can be performed in a SERS active medium, such as in solution using sol-gel or on a SERS active solid or gel substrate. Labels can be fluorescent labels, Raman or SERS active molecules. Figure 5 expressly depicts competitive, direct, and sandwich antibody assays employing a employing a SERS/RAMAN label, SERS/RAMAN antigen, and SECOND SERS/RAMAN labeled antibody (col. 12, p. 28; Fig. 5), thereby teaching Raman probes associated with antibodies that bind antigen targets in immunoassay formats. Lastly, Vo-Dinh discloses Figure 13 which further teaches contacting a metal SERS surface with a Protein with -SH moiety in aqueous buffer, resulting in attachment of the SH-containing protein to the Ag surface (col. 20, p. 32; Fig. 13). These disclosures teach the claimed use of immobilized thiol-containing proteins together with antibody-based Raman probes for target-specific SERS detection.
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 Park’s flexible, porous, gold-nanoisland SERS membrane for determining the state of wound healing in a diabetic individual by collecting wound exudate from a diabetic foot ulcer and detecting wound-healing protein biomarkers, as suggested by Perumal’s express identification of diabetic foot ulcers as wounds requiring biomarker monitoring, its teaching that flexible SERS substrates can be used to collect wound exudates, and its objective of monitoring those biomarkers in wound fluid as wounds heal; further modifying Park’s membrane by treating the wound-healing protein biomarkers with Traut’s reagent to generate sulfhydryl-containing modified proteins and immobilizing those modified proteins on Park’s gold nanoislands through Au-S attachment, as expressly taught by Haddada; and thereafter contacting the immobilized proteins with corresponding antibody-bearing Raman probes and performing SERS detection according to the immunoassay formats taught by Vo-Dinh. Perumal provides an express reason to adapt Park’s flexible paper-based SERS substrate for the clinical monitoring of diabetic wound-healing biomarkers, Haddada provides an express reason to employ Traut’s-reagent thiolation because it converts protein amines into gold-binding thiol groups that enable stable covalent immobilization while preserving antigen-antibody recognition, and Vo-Dinh provides an express reason to employ antibody-based SERS/Raman probes because they specifically recognize protein analytes and generate target-responsive Raman signals in established SERS immunoassays. One of ordinary skill in the art would have had a reasonable expectation of success because Perumal demonstrates sensitive SERS detection of wound-specific protein biomarkers in a serum solution mimicking wound exudates and expressly teaches using flexible SERS substrates for collecting wound exudates and monitoring those biomarkers in wound fluid, Haddada experimentally demonstrates successful protein thiolation, Au-S immobilization, and retention of antibody affinity following conjugation, and Vo-Dinh teaches operative SERS immunoassays using Raman-labeled antibodies and SH-containing proteins immobilized on SERS-active metal surfaces. The proposed modifications therefore would have combined expressly taught and technically complementary wound-sample collection, protein surface chemistry, antibody-recognition, and SERS detection techniques according to their established functions to obtain a predictable wound-healing assay.
Regarding claim 16, Haddada teaches the additionally claimed serum-albumin blocking limitation. In particular, after forming protein-functionalized gold nanoparticles, Haddada teaches that bovine serum albumin (BSA) was added to the bioconjugate solution at a final concentration of 0.25% w/v to block non-specific binding sites (Experimental section, p. 6228). Haddada further teaches that the same procedure was applied to prepare AuNP-SEA bioconjugate using a solution of thiolated SEA at 24 μg/mL (Experimental section, p. 6229), thereby applying the BSA-blocking procedure after immobilization of the thiolated protein antigen on the gold nanoparticles. Figure 1A likewise depicts thiolated SEA attached to AuNPs followed by addition of BSA, centrifugation, and redispersion (Fig. 1A, p. 6229). Also, Haddada describes subsequent BSA backfilling of the SEA-functionalized nanoparticles and reports that upon covalent attachment of anti-SEA Ab to AuNP, the LSPR band underwent a red-shift of 8 nm (λmax = 528 nm) and an additional shift of 1 nm (λmax=529nm) was seen upon further blocking by BSA (Fig. 2). The same trend was observed upon SEA grafting to the nanoparticles (Results and discussion, p. 6230; Fig. 2). These disclosures teach contacting a gold-nanoparticle surface already occupied in part by immobilized modified proteins with serum albumin so that the serum albumin blocks the remaining unoccupied surface sites.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further modify the method of claim 15 by contacting Park’s gold-nanoisland-bearing flexible membrane, after immobilization of the thiolated wound-biomarker proteins, with serum albumin according to Haddada’s expressly taught BSA-backfilling procedure. Haddada provides an express teaching, suggestion, and motivation for the modification because it adds BSA after protein immobilization specifically to block nonspecific binding sites on the remaining gold surface. One of ordinary skill in the art would have recognized that applying the same blocking treatment to the unoccupied regions of Park’s gold-bearing membrane would reduce nonspecific adsorption during the subsequent antibody-probe assay. A reasonable expectation of success would have existed because Haddada actually performs the sequence of protein thiolation, immobilization on gold, and subsequent BSA blocking, and confirms successful BSA backfilling of both antibody- and SEA-functionalized AuNPs. Thus, the proposed modification would have applied Haddada’s demonstrated serum-albumin passivation step to the analogous unoccupied regions of the protein-functionalized SERS-active gold surface for its expressly taught purpose.
Regarding claim 17, refer to the discussion above and here. Haddada expressly teaches this limitation. Haddada identifies 2-iminothiolane hydrochloride (Traut’s reagent) as the thiolating reagent and teaches that SEA and anti-SEA antibody were treated by Traut’s reagent to convert some of their primary amines to thiol groups followed by conjugation with the AuNP (Introduction, p. 6228). Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to perform the thiolation step of the method of claim 15 using Traut’s reagent, as expressly taught by Haddada. Haddada provides a direct teaching, suggestion, and motivation for selecting Traut’s reagent because it converts primary amines of a protein antigen into thiol groups that bind to gold nanoparticles, thereby enabling covalent immobilization through Au–S bonds. One of ordinary skill in the art would have had a reasonable expectation of success because Haddada actually uses Traut’s reagent to thiolate the protein antigen SEA, prepares a thiolated SEA–AuNP bioconjugate using that same procedure, and demonstrates that the resulting modified antigen remains suitable for antibody recognition and biosensing. Thus, selecting Traut’s reagent for thiolating the wound-biomarker proteins in the method of claim 15 would have been a predictable use of Haddada’s expressly demonstrated protein-thiolation reagent for the same known purpose of forming gold-binding sulfhydryl groups.
Regarding claim 18, refer to the discussion above and here. Perumal teaches the additionally claimed wound-healing biomarker targets. While, Vo-Dinh teaches using antibodies as analyte-specific probes in SERS immunoassays. As discussed above, Vo-Dinh teaches antibody probes may also be used with the invention, preferably with other receptor probe types, and such antibodies may then be used as specific probes to identify an analyte of interest (col. 12, p. 28). Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to perform the antibody-based Raman-probe detection method of claim 15 using an antibody specific for MMP-9, TNF-α, or IL-1α, as suggested by Perumal and Vo-Dinh. Perumal provides an express reason to select these antibodies because it identifies MMP-9, TNF-α, and IL-1α as wound-specific biomarkers relevant to monitoring chronic wound healing and expressly detects those biomarkers using a SERS immunoassay, while Vo-Dinh teaches using analyte-specific antibodies, including SERS/Raman-labeled antibodies, to recognize corresponding protein antigens. One of ordinary skill in the art would have had a reasonable expectation of success because Perumal demonstrates SERS detection of the recited biomarker classes in a wound-exudate model, and Vo-Dinh teaches established direct and sandwich immunoassay formats in which antibodies specifically bind their corresponding protein targets and provide Raman/SERS detection. Thus, selecting antibodies directed to Perumal’s expressly identified MMP, TNF-α, or interleukin biomarkers for use in Vo-Dinh’s antibody-based Raman-probe format would have been a predictable application of the disclosed immunoassay technique to the expressly identified wound-healing analytes.
Ultimately, claims 2, 3, 6, 9, 10, 12, and 15-18 are rejected under 35 U.S.C. 103 as being unpatentable over the cited prior art because the references collectively teach or suggest all of the claimed limitations, and it would have been obvious to one of ordinary skill in the art to combine the teachings as set forth above with a reasonable expectation of success.
Conclusion
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 July 30, 2026
Prosecution Insights