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 .
DETAILED ACTION
Applicant’s amendment filed on 02 September 2025 is entered. Claims 1-4, 7, 11-12, and 17 are amended, and claims 5-6, 8-10, 13-16, and 18-20 are canceled. Claims 1-4, 7, 11-12, and 17 are pending and under examination.
Priority
Receipt is acknowledged of the English language translation of the certified copies of papers required by 37 CFR 1.55. Applicant has perfected their claim to the foreign priority filing date of CN202111583690.4 filed on 22 December 2021. Accordingly, the effective filing date of the invention is now 22 December 2021.
Drawings
The drawings are objected to because:
Figure 4 labels the y-axis as “Intensity ratio (600 cm-1/700 cm-1)”, but experimentally the intensity ratio is the signal intensity at 600 cm--1 and 677 cm--1 (instant specification [0047]). It is suggested to amend the y-axis label to “Intensity ratio (600 cm-1/677 cm-1)” to obviate the objection.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Objections
Claims 1 and 3 are objected to because of the following informalities:
In claims 1 and 3, the phrase “incubating resulting mixtures” in line 6 of claim 1 and line 8 of claim 3 should add a definite article “the” before the term “resulting”; and the phrase “an SERS” incorrectly uses the indefinite article “an” intended to precede vowel sounds before a consonant sound “S”, and should be corrected to “a SERS”.
In claim 3, the term “gold nanoparticle colloid” needs the indefinite article “a” before it.
Appropriate correction is required.
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 1-4, 7, 11-12, and 17 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.
(Maintained) Claim 2 recites the phrase “…adding DMSO and a colloid of gold nanoparticles…” on line 4. It is unclear where the DMSO and colloid of gold nanoparticles are added. Claim 3 recites the same issue in step 4.
(Maintained) Claim 2 recites the phrase “substituting the ratio of the SERS signal of the reaction product to the SERS signal of DMSO…into a quantification model…to obtain a β-GAL activity of the seawater sample”. It is unclear what the verb “substituting” means in the context of the quantification model. It is unclear what value is substituted with ratio of the SERS signal of the reaction product to the SERS signal of DMSO in the quantification model.
(Maintained) Claim 2 recites the phrase “directly detecting SERS signals of a reaction product and a SERS signal of DMSO” on lines 5-6. It is unclear if the SERS signals of the reaction product and DMSO are determined individually in their own separate solutions, or if both SERS signals are determined from one solution comprising both the reaction product and DMSO. Claim 3 recites the same issue in step 4.
(Maintained) Claim 3 step 1 recites the phrase “taking 180µL of each of a BCIG solution, a β-GAL solution, DMSO, and a solution obtained after a reaction of BCIG and β-GAL, adding 180µL of DMSO, adding 200µL of a gold nanoparticle colloid, and subjecting each of resulting mixtures to SERS analysis on a machine”. It is unclear if the 180µL DMSO and 200µL gold nanoparticle colloid are added to a combination of “a BCIG solution, a β-GAL solution, DMSO, and a solution obtained after a reaction of BCIG and β-GAL”, or if the 180µL DMSO and 200µL gold nanoparticle colloid are added to each “a BCIG solution, a β-GAL solution, DMSO, and a solution obtained after a reaction of BCIG and β-GAL” individually to create four different solutions. The limitations of 180µL, or 200 µL of a solution without setting forth the concentration of each solution renders the claim indefinite as to the amount of each of BCIG, β-GAL, DMSO and gold nanoparticle colloid that is required.
(New necessitated by amendment) Claim 1 lines 21-23 recite “an average value of β-GAL activity data of the standard solutions with different β-GAL activities refers to the peak intensity at the Raman shift of 600 cm-1 divided by the peak intensity at the Raman shift of 677 cm-1.” It is unclear if the phrase “β-GAL activity data of the standard solutions…refers to the peak intensity at the Raman shift of 600 cm-1 divided by the peak intensity at the Raman shift of 677 cm-1” is intended to mean that the β-GAL activities of the standard solutions are the Raman shift peak intensities of 600 cm-1 divided by 677 cm-1, or if the Raman shift peak intensities of 600 cm-1 divided by 677 cm-1 are used to determine the β-GAL activities of the standard solutions. Claim 3 recites the same issue.
(New necessitated by amendment) Claim 2 line 5 recites the term “a reaction product”. It is unclear if the “reaction product” is the chemical product of the reaction of seawater and BCIG, the chemical product of the reaction of seawater, BCIG, DMSO, and the colloid of gold nanoparticles, or some chemical reaction product not adequately described.
(New necessitated by amendment) Claim 3 lines 32-33 recite “the SERS intensity ratio refers to a ratio of the SERS signal to the SERS signal of the DMSO.” It is unclear if the first term “the SERS signal” in the ratio is referring to the SERS signal of the standard solutions, the SERS signal of a given sample, or the SERS signal of a different solution not adequately described.
Claims 4 and 7 depend on claim 1, claim 11 depends on claim 2, and claims 12 and 17 depend on claim 3, so these dependent claims are indefinite for the same reasons as their parent claims.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
(New necessitated by amendment) Claims 1 and 7 are rejected under 35 U.S.C. 103 as being unpatentable over Stevenson et al. (Analysis of intracellular enzyme activity by surface enhanced Raman scattering, Analyst, 2013, 138, 6331) in view of Lin et al. (A universal strategy for the incorporation of internal standards into SERS substrates to improve the reproducibility of Raman signals, Analyst, 2021, 146, 7168), Goodacre et al. (Recent developments in quantitative SERS: Moving towards absolute quantification, Trends in Analytical Chemistry 102 (2018) 359-368), Cowcher et al. (Portable, Quantitative Detection of Bacillus Bacterial Spores Using Surface-Enhanced Raman Scattering, Anal. Chem. 2013, 85, 3297−3302), Kiernan et al. (Indigogenic substrates for detection and localization of enzymes, Biotechnic & Histochemistry, (2007) 82:2, 73-103), Apte et al. (Rapid detection of sewage contamination in marine waters using a fluorimetric assay of β-d-galactosidase activity. Science of The Total Environment. Volume 141, Issues 1–3, 25 January 1994, Pages 175-180), Burkowska et al. (SERS of dimethyl sulphoxide on silver electrodes and on silver chloride sol, J. Electroanal. Chem., 251 (1988) 339-348), and Kode et al. (Raman Labeled Nanoparticles: Characterization of Variability and Improved Method for Unmixing, J. Raman Spectrosc. 2012 July 1; 43(7): 895–905), and as evidenced by In Vivo Chem (Vat Blue 2, https://www.invivochem.com/product/V67365, accessed 21 May 2025).
Regarding claim 1, Stevenson teaches the use of surface enhanced Raman scattering (SERS) to detect β-galactosidase (β-GAL) activity by mixing and incubating β-GAL with 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (BCIG or X-gal) and dimethylformamide (DMF) to generate a solution comprising DMF and a blue indicator 5,5'-dibromo-4,4'-dichloro-indigo that can be measured by SERS to generate a SERS signal intensity peak at 598 cm-1 (Stevenson p. 6332, left column, Abstract and Fig. 1). Although Stevenson teaches the SERS peak intensity is at 598 cm-1, not 600 cm-1 as claimed, MPEP 2144.05(I) states that “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)”. In the instant case, a prima facie case of obviousness exists because the Raman shift value of 598 cm-1 of Stevenson is very close to the claimed Raman shift value of 600 cm-1.
Stevenson teaches the use of colloidal gold nanoparticles with an average diameter of 20nm to adsorb the 5,5'-dibromo-4,4'-dichloro-indigo and enhance its measured SERS signal (Stevenson Pg. 6331 paras.1-2 and Pg. 6332 paras. 4 and 8).
Stevenson does not teach the preparation of a plurality of β-GAL standard solutions or dimethyl sulfoxide (DMSO).
Lin teaches preparation of solutions with different known concentrations of an analyte “thiram” together with 0.5 µM of internal standard 4-MBA to generate standard solutions, and measurement of said standard solutions by SERS to generate different SERS signals for each standard solution (Lin Pg. 7169 sec. 2.4, Pg. 7174 sec 3.5, and Fig. 6). The thiram concentrations of the standard solutions were measured quantitatively by referencing a SERS peak intensity mean ratio of the SERS peak intensity of analyte thiram (1382 cm-1) to the SERS peak intensity of internal standard 4-MBA (1079 cm-1), which generated a standard curve with a linear regression equation (Lin Pg. 7174 sec 3.5 and Fig. 6).
However, Stevenson and Lin do not teach a logarithm value of an activity/concentration of the standard solutions plotted against a SERS signal ratio of the SERS signals of the standard solutions to the SERS signal of DMSO, nor the use of DMSO.
Goodacre teaches that the use of internal standards in SERS results in unequal competition between the target analyte and the internal standard on the metal nanoparticle surface, meaning that SERS signals may vary as a function of overall concentration and the ratios of analyte to internal standards are not linear (Goodacre Pg. 364 sec 5 para. 3).
However, Stevenson, Lin, and Goodacre do not teach that the ratios of analyte to internal standards can result in a standard curve having a logarithmic relationship requiring logarithmic transformation of the plotted standard curve, nor the use of DMSO.
Cowcher teaches the logarithmic transformation of a calibration (standard) curve when the untransformed calibration curve is nonlinear (Cowcher Fig. 3).
However, Stevenson, Lin, Goodacre, and Cowcher do not teach the use of DMSO.
Kiernan teaches that many indoxyl substrates are insoluble in water, but soluble in DMSO, and DMSO is preferred to more toxic DMF in methods for detecting β-GAL” (Kiernan Pg. 77 Sec. Synthetic indoxyl substrates for hydrolytic enzymes). The product of the reaction of β-GAL and BCIG is 5,5'-dibromo-4,4'-dichloro-indigo (also known as Vat Blue 2) is soluble in DMSO, as evidenced by In Vivo Chem (pg. 3).
Stevenson, Lin, Goodacre, Cowcher, and Kiernan do not teach quantitative detection of β-GAL in seawater.
Apte teaches that rapidly detecting β-GAL activities of seawater could be used as an early warning indicator of sewage contamination of coastal waters because β-GAL is an enzyme marker of fecal coliform bacteria (Apte abstract).
Stevenson, Lin, Goodacre, Cowcher, Kiernan, and Apte do not teach the limitation “…wherein the standard curve is fitted using the following standard equation: y = 0.784 * x + 0.004, with a correlation coefficient R2= 0.936, wherein x represents a logarithm value of an activity of a β-GAL-active standard solution and y represents a ratio of an SERS signal of the β-GAL-active standard solution to the SERS signal of the DMSO”. However, this limitation is interpreted as a property of the resultant standard curve quantification model achieved by performing the active steps of the instant method, but does not recite any required active method steps or structural limitations for the claimed method for constructing a quantification model for quantitative detection of β-galactosidase in seawater. Thus, where the art teaches each and every method step of constructing a quantification model for quantitative detection of β-galactosidase in seawater, this property of the resultant standard curve quantification model is considered necessarily present in resultant standard curve quantification model.
Stevenson, Lin, Goodacre, Cowcher, Kiernan, and Apte do not teach that the SERS signal peak intensity of DMSO is at a Raman shift of 677 cm-1.
Burkowska teaches that DMSO produces a SERS spectrum of 678, 710, and 2925 cm-1 (Burkowska Fig. 1 and Results para. 1). MPEP 2144.05(I) states that “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)”. In the instant case, a prima facie case of obviousness exists because the Raman shift value of 678 cm-1 of Burkowska is very close to the claimed Raman shift value of 677 cm-1.
Since Stevenson taught that the Raman shift value the β-GAL and BCIG (X-gal) indicator byproduct 5,5'-dibromo-4,4'-dichloro-indigo is 598 cm-1, and Burkowska taught the Raman shift value of DMSO is 678 cm-1, and Lin taught that plotting a ratio of the SERS signal of standard solutions of known analyte concentrations to the SERS signal of an internal standard versus the known concentrations of the standard solutions would produce a standard curve useful for easily calculating an given sample’s unknown analyte concentration based on a SERS signal intensity of said sample, one of ordinary skill in the art would have found it obvious that the ratio of the peak intensity at a Raman shift of 600 cm-1 (corresponding to the peak for analyte 5,5'-dibromo-4,4'-dichloro-indigo) to the peak intensity at a Raman shift of 677 cm-1 (corresponding to the peak for internal standard and solvent DMSO) is the data determined by SERS measurement of the known β-GAL activities of the standard solutions.
Stevenson in view of Lin, Goodacre, Cowcher, Kiernan, Apte, and Burkowska do not teach the use of an ordinary least squares method to linearly fit the β-GAL activity and a SERS intensity ratio in the standard curve to obtain a standard equation.
Kode teaches that the ordinary least squares method is commonly used to determine linear regressions when all component spectra are known, and that the advantage of this approach is its simplicity of implementation. Kode also teaches that the resulting linear fit can expressed as linear functions of respective concentrations (Kode Pg. 3 para. 1).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the present invention to create a standard curve quantification model for quantifying β-GAL in a sample by preparing a set of β-GAL standard solutions with different known concentrations, mixing and incubating these standard solutions with BCIG to produce blue indicator 5,5'-dibromo-4,4'-dichloro-indigo in the standard solutions, adding DMSO and colloidal gold nanoparticles to the standard solutions, conducting SERS on said standard solutions, and plotting a standard curve of the logarithm of the different β-GAL activities of each standard solution against the ratio of the SERS signal of the β-GAL solution to the SERS signal of the DMSO.
One of ordinary skill in the art would have been motivated to do so in order to rapidly and easily detect sewage contamination of marine waters with high sensitivity by measuring the SERS signal intensity of the seawater sample and comparing that SERS signal intensity with a standard curve to calculate the β-GAL activity of the sample.
One of ordinary skill in the art would have had reasonable expectations of success because Apte taught that β-GAL is an enzyme marker of fecal coliform bacteria, and that rapidly detecting and quantifying β-GAL activities of seawater could be used as an early warning indicator of sewage contamination of coastal waters (Apte abstract). Stevenson taught that β-GAL can be quantified by using the reaction of β-GAL and BCIG to produce a blue indicator 5,5'-dibromo-4,4'-dichloro-indigo and measuring the SERS signal of that mixture, using colloidal gold nanoparticles as the SERS enhancing substrate. This quantification method of Stevenson could then be used to plot a standard curve by quantifying the β-GAL activities of standard solutions with several different known activities of β-GAL against the ratio of their respective SERS signals to the SERS signal of an internal standard, which is expected to be successful because Lin taught the preparation of standard curves by plotting a ratio of the SERS signal of standard solutions of known analyte concentrations to the SERS signal of an internal standard versus the known concentrations of the standard solutions, thereby generating a non-linear standard curve. It would then be reasonably expected, in view of Goodacre and Cowcher, that logarithmically transforming the non-linear standard curve by taking the logarithm of the known β-GAL activity values would produce a linear standard curve with a linear regression equation useful for easily calculating an seawater sample’s unknown β-GAL activity based on a SERS signal intensity of the seawater sample.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the present invention to perform an ordinary least squares method to linearly fit the β-GAL activities and the SERS signal intensity ratio in the standard curve produced by constructing a quantification model for quantitative detection of β-galactosidase in seawater taught by Stevenson in view of Lin, Goodacre, Cowcher, Kiernan, Apte, and Burkowska. One of ordinary skill in the art would have been motivated to do so in order to produce a linear fit standard curve with a linear fir equation that can be easily used to calculate the β-GAL activity of a given sample that has been measured by SERS. One of ordinary skill in the art would have had reasonable expectations of success because the SERS spectra of both β-GAL and DMSO were determined in the obvious method taught by Stevenson in view of Lin, Goodacre, Cowcher, Kiernan, Apte, and Burkowska, and Lin taught that plotting a ratio of the SERS signal of standard solutions of known analyte concentrations to the SERS signal of an internal standard versus the known concentrations of the standard solutions would produce a standard curve useful for easily calculating an given sample’s unknown analyte concentration based on a SERS signal intensity of said sample. Since Kode teaches that the ordinary least squares method is commonly used to determine a linear regression when all component spectra are known, One of ordinary skill in the art would have reasonably expected that an ordinary least squares method would be a simple means of generating a standard curve with a linear equation that can be used to calculate unknown β-GAL activities of a given SERS measured sample.
Regarding claim 7, the limitation “…wherein β-GAL activity data of the standard solutions with different β-GAL activities have an average relative standard deviation (RSD) of less than 15%” is interpreted as an intended result for the average relative standard deviation of the β-GAL activity data of the standard solutions which is achieved by performing the active steps of the instant method, but does not recite any required active method steps or structural limitations for the claimed method for constructing a quantification model for quantitative detection of β-galactosidase in seawater. Thus, where the art teaches each and every method step of constructing a quantification model for quantitative detection of β-galactosidase in seawater, these results of the average relative standard deviation of the β-GAL activity data of the standard solutions is considered necessarily present in the β-GAL activity data of the standard solutions.
(Maintained) Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over Stevenson et al. (Analysis of intracellular enzyme activity by surface enhanced Raman scattering, Analyst, 2013, 138, 6331) in view of Apte (Rapid detection of sewage contamination in marine waters using a fluorimetric assay of β-d-galactosidase activity. Science of The Total Environment. Volume 141, Issues 1–3, 25 January 1994, Pages 175-180), Lin (A universal strategy for the incorporation of internal standards into SERS substrates to improve the reproducibility of Raman signals, Analyst, 2021, 146, 7168), Kiernan (Indigogenic substrates for detection and localization of enzymes, Biotechnic & Histochemistry, (2007) 82:2, 73-103), and O’Haver (Worksheets for Analytical Calibration Curves, 2008, https://terpconnect.umd.edu/~toh/models/CalibrationCurve.html , accessed 22 May 2025) and as evidenced by In Vivo Chem.
Stevenson teaches the use of surface enhanced Raman scattering (SERS) to detect β-galactosidase (β-GAL) activity by mixing and incubating β-GAL with 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (BCIG or X-gal) to generate a solution comprising a blue indicator 5,5'-dibromo-4,4'-dichloro-indigo that can be measured by SERS to generate a SERS signal intensity peak at ~600cm-1 (Stevenson Abstract and Fig. 1). Stevenson teaches the use of colloid containing gold nanoparticles with an average diameter of 20nm to adsorb the 5,5'-dibromo-4,4'-dichloro-indigo and enhance its measured SERS signal (Stevenson Pg. 6331 paras.1-2 and Pg. 6332 paras. 4 and 8).
Stevenson does not teach quantitative detection of β-GAL in seawater, the use of DMSO, the calculation of a SERS signal ratio of a sample and DMSO, or the comparison of the sample’s SERS signal ratio with a quantification standard curve to obtain a β-GAL activity of a seawater sample.
Apte teaches a simple and rapid method for the detection of fecal coliform bacteria in seawater by detecting β-GAL, and states that rapidly detecting β-GAL activities of seawater could be used as an early warning indicator of sewage contamination of coastal waters because β-GAL is an enzyme marker of fecal coliform bacteria (Apte abstract).
Stevenson and Apte do not teach the use of DMSO, the calculation of a SERS signal ratio of a sample and DMSO, or the comparison of the sample’s SERS signal ratio with a quantification standard curve to obtain a β-GAL activity of a seawater sample.
Kiernan teaches that many indoxyl substrates are insoluble in water, but soluble in DMSO, and DMSO is preferred to more toxic DMF in methods for detecting β-GAL” (Kiernan Pg. 77 Sec. Synthetic indoxyl substrates for hydrolytic enzymes). The product of the reaction of β-GAL and BCIG is 5,5'-dibromo-4,4'-dichloro-indigo (also known as Vat Blue 2) is soluble in DMSO, as evidenced by In Vivo Chem (pg. 3).
Stevenson, Kiernan, and Apte do not teach the calculation of a SERS signal ratio of a sample and DMSO, or the comparison of the sample’s SERS signal ratio with a quantification standard curve to obtain a β-GAL activity of a seawater sample.
Lin teaches preparation of solutions with different known concentrations of an analyte “thiram” together with 0.5 µM of internal standard 4-MBA to generate standard solutions, and measurement of said standard solutions by SERS to generate different SERS signals for each standard solution (Lin Pg. 7169 sec. 2.4, Pg. 7174 sec 3.5, and Fig. 6). The thiram concentrations of the standard solutions were measured quantitatively by referencing a SERS peak intensity mean ratio of the SERS peak intensity of analyte thiram (1382 cm-1) to the SERS peak intensity of internal standard 4-MBA (1079 cm-1), which generated a standard curve with a linear regression equation (Lin Pg. 7174 sec 3.5 and Fig. 6).
Stevenson, Kiernan, Apte, and Lin do not teach the comparison of the sample’s SERS signal ratio with the quantification standard curve to obtain a β-GAL activity of the seawater sample.
O’Haver teaches that quantitative measurement of the composition of samples usually requires the preparation of a standard or calibration curve by preparing and measuring a series of solutions with known concentrations and fitting that data to a line or curve which produces an equation that is used to convert sensor readings from unknown samples into concentrations (O’haver Pgs. 1-2 bridging para.).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the present invention to measure the β-GAL activities of seawater samples by mixing the seawater sample with BCIG, DMSO, and gold nanoparticle colloid and then subjecting the mixture to SERS measurement and calculating a SERS signal ratio of the mixture with the SERS signal of the DMSO, using those SERS signal results to calculate a ratio of SERS signals of the mixture and the DMSO, then ultimately calculating the concentration of the β-GAL in the seawater sample.
One of ordinary skill in the art would have been motivated to do so in order to rapidly, easily, and sensitively detect sewage contamination of marine waters with high sensitivity by measuring the SERS signal intensity of the seawater sample and comparing that SERS signal intensity with a standard curve to calculate the β-GAL activity of the sample.
One of ordinary skill in the art would have a reasonable expectation of success because Apte taught that β-GAL is an enzyme marker of fecal coliform bacteria, and that rapidly detecting and quantifying β-GAL activities of seawater could be used as an early warning indicator of sewage contamination of coastal waters (Apte abstract). Stevenson taught that β-GAL can be quantified by using the reaction of β-GAL and BCIG to produce a blue indicator 5,5'-dibromo-4,4'-dichloro-indigo and measuring the SERS signal of that mixture, using gold nanoparticle colloid as the SERS enhancing substrate. This quantification method of Stevenson could then be used to plot a standard curve by quantifying the β-GAL activities of standard solutions with several different known activities of β-GAL against the ratio of their respective SERS signals to the SERS signal of an internal standard, which is expected to be successful because Lin taught the preparation of standard curves by plotting a ratio of the SERS signal of standard solutions of known analyte concentrations to the SERS signal of an internal standard versus the known concentrations of the standard solutions, thereby generating a standard curve. That standard curve would then be used to calculate the activity of β-GAL in the seawater sample in view of O’Haver.
(New necessitated by amendment) Claims 3 and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Stevenson in view of Apte, Lin, Goodacre, Cowcher, Kiernan, O’Haver, Decho (US 8546071 B2, published 01 October 2013), Burkowska, and Kode, and as evidenced by In Vivo Chem.
Regarding claim 3, Stevenson teaches the use of surface enhanced Raman scattering (SERS) to detect β-galactosidase (β-GAL) activity by mixing and incubating β-GAL with 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (BCIG or X-gal) to generate a solution comprising a blue indicator 5,5'-dibromo-4,4'-dichloro-indigo that can be measured by SERS to generate a SERS signal intensity peak at ~600cm-1 (Stevenson Abstract and Fig. 1). Although Stevenson teaches the SERS peak intensity is at 598 cm-1, not 600 cm-1 as claimed, MPEP 2144.05(I) states that “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)”. In the instant case, a prima facie case of obviousness exists because the Raman shift value of 598 cm-1 of Stevenson is very close to the claimed Raman shift value of 600 cm-1.
Stevenson teaches the use of a gold nanoparticle colloid with an average diameter of 20nm to adsorb the 5,5'-dibromo-4,4'-dichloro-indigo and enhance its measured SERS signal (Stevenson Pg. 6331 paras.1-2 and Pg. 6332 paras. 4 and 8).
However, Stevenson does not teach the preparation of a plurality of β-GAL standard solutions to produce a quantification model standard curve, a logarithm value of an activity/concentration of the standard solutions plotted against a SERS signal ratio of the SERS signals of the standard solutions to the SERS signal of DMSO, the quantitative detection of β-GAL in seawater, the use of DMSO, the calculation of a SERS signal ratio of a sample and DMSO, or the comparison of the sample’s SERS signal ratio with a quantification standard curve to obtain a β-GAL activity of a seawater sample, nor the preparation of a BCIG solution, a β-GAL solution, DMSO, and a solution obtained after a reaction of BCIG and β-GAL, adding DMSO and colloidal gold nanoparticles to said solutions, and subjecting each of those solutions to SERS analysis.
Apte teaches a simple and rapid method for the detection of fecal coliform bacteria in seawater by detecting β-GAL, and states that rapidly detecting β-GAL activities of seawater could be used as an early warning indicator of sewage contamination of coastal waters because β-GAL is an enzyme marker of fecal coliform bacteria (Apte abstract).
However, Stevenson and Apte do not teach the preparation of a plurality of β-GAL standard solutions to produce a quantification model standard curve, a logarithm value of an activity/concentration of the standard solutions plotted against a SERS signal ratio of the SERS signals of the standard solutions to the SERS signal of DMSO, the use of DMSO, the calculation of a SERS signal ratio of a sample and DMSO, or the comparison of the sample’s SERS signal ratio with a quantification standard curve to obtain a β-GAL activity of a seawater sample, nor the preparation of a BCIG solution, a β-GAL solution, DMSO, and a solution obtained after a reaction of BCIG and β-GAL, adding DMSO and colloidal gold nanoparticles to said solutions, and subjecting each of those solutions to SERS analysis.
Lin teaches preparation of solutions with different known concentrations of an analyte “thiram” together with 0.5 µM of internal standard 4-MBA to generate standard solutions, and measurement of said standard solutions by SERS to generate different SERS signals for each standard solution (Lin Pg. 7169 sec. 2.4, Pg. 7174 sec 3.5, and Fig. 6). The thiram concentrations of the standard solutions were measured quantitatively by referencing a SERS peak intensity mean ratio of the SERS peak intensity of analyte thiram (1382 cm-1) to the SERS peak intensity of internal standard 4-MBA (1079 cm-1), which generated a standard curve with a linear regression equation (Lin Pg. 7174 sec 3.5 and Fig. 6).
However, Stevenson, Apte, and Lin do not teach a logarithm value of an activity/concentration of the standard solutions plotted against a SERS signal ratio of the SERS signals of the standard solutions to the SERS signal of DMSO, the use of DMSO, or the comparison of the sample’s SERS signal ratio with a quantification standard curve to obtain a β-GAL activity of a seawater sample, nor the preparation of a BCIG solution, a β-GAL solution, DMSO, and a solution obtained after a reaction of BCIG and β-GAL, adding DMSO and colloidal gold nanoparticles to said solutions, and subjecting each of those solutions to SERS analysis.
Goodacre teaches that the use of internal standards in SERS results in unequal competition between the target analyte and the internal standard on the metal nanoparticle surface, meaning that SERS signals may vary as a function of overall concentration and the ratios of analyte to internal standards are not linear (Goodacre Pg. 364 sec 5 para. 3).
However, Stevenson, Apte, Lin, and Goodacre do not teach that the ratios of analyte to internal standards can result in a standard curve having a logarithmic relationship requiring logarithmic transformation of the plotted standard curve, the use of DMSO, or the comparison of the sample’s SERS signal ratio with a quantification standard curve to obtain a β-GAL activity of a seawater sample, nor the preparation of a BCIG solution, a β-GAL solution, DMSO, and a solution obtained after a reaction of BCIG and β-GAL, adding DMSO and colloidal gold nanoparticles to said solutions, and subjecting each of those solutions to SERS analysis.
Cowcher teaches the logarithmic transformation of a calibration (standard) curve when the untransformed calibration curve is nonlinear (Cowcher Fig. 3).
However, Stevenson, Apte, Lin, Goodacre, and Cowcher do not teach the use of DMSO, or the comparison of the sample’s SERS signal ratio with the quantification standard curve to obtain a β-GAL activity of the seawater sample, nor the preparation of a BCIG solution, a β-GAL solution, DMSO, and a solution obtained after a reaction of BCIG and β-GAL, adding DMSO and colloidal gold nanoparticles to said solutions, and subjecting each of those solutions to SERS analysis.
Kiernan teaches that many indoxyl substrates are insoluble in water, but soluble in DMSO, and DMSO is preferred to more toxic DMF in methods for detecting β-GAL” (Kiernan Pg. 77 Sec. Synthetic indoxyl substrates for hydrolytic enzymes). The product of the reaction of β-GAL and BCIG is 5,5'-dibromo-4,4'-dichloro-indigo (also known as Vat Blue 2) is soluble in DMSO, as evidenced by In Vivo Chem (pg. 3).
Stevenson, Apte, Lin, Goodacre, Cowcher, and Kiernan do not teach the comparison of the sample’s SERS signal ratio with the quantification standard curve to obtain a β-GAL activity of the seawater sample, nor the preparation of a BCIG solution, a β-GAL solution, DMSO, and a solution obtained after a reaction of BCIG and β-GAL, adding DMSO and colloidal gold nanoparticles to said solutions, and subjecting each of those solutions to SERS analysis.
O’Haver teaches that quantitative measurement of the composition of samples usually requires the preparation of a standard or calibration curve by preparing and measuring a series of solutions with known concentrations and fitting that data to a line or curve which produces an equation that is used to convert sensor readings from unknown samples into concentrations (O’Haver Pgs. 1-2 bridging para.).
However, Stevenson, Apte, Lin, Goodacre, Cowcher, Kiernan, and O’Haver do not teach the preparation of a BCIG solution, a β-GAL solution, DMSO, and a solution obtained after a reaction of BCIG and β-GAL, adding DMSO and colloidal gold nanoparticles to said solutions, and subjecting each of those solutions to SERS analysis.
Claim 3 step a recites the preparation and SERS measurement of blank control solutions that contain BCIG, β-GAL, DMSO, and a solution obtained after a reaction of BCIG and β-GAL, but none of the solutions contain the seawater sample.
Decho teaches an assay that involves the measurement of indicator 5,5'-dibromo-4,4'-dichloro-indigo derived from the reaction of X-gal (BCIG, 5-bromo-4-chloro-3-indolyl-β-D-galactopyransoside) with β-GAL coupled with quorum sensing signal molecules acyl-homoserine lactones (AHL) to detect quorum sensing signals in a cell-lysate extract (Decho abstract and col. 5-6 bridging para.). Decho teaches the preparation and measurement of control/blank solutions comprising the constitutive components of the assay in order to determine if there is measurable production of β-galactosidase in each of those components without the addition of the β-GAL inducing molecule AHL (negative controls), and also with the addition of all of the components together (positive control) (Decho Fig. 5A, col. 8 lns. 5-26, and col. 10 lns. 5-12). In Fig. 5A, the “X-gal only” testing bar is analogous to the BCIG solution of instant claim 3 step A, the CFL and AHL testing bar is analogous to the β-GAL solution in instant claim 3 step a because AHL induces β-GAL expression in the CFL (Decho cols. 7-8 bridging para.), and the CFL, AHL, and X-gal test bar (positive control) is analogous to the BCIG and β-GAL solution of instant claim 3 step a because the AHL induces expression of β-GAL and the X-gal (BCIG) reacts with the β-GAL to produce the blue indicator molecule 5,5'-dibromo-4,4'-dichloro-indigo (Decho Fig. 5A).
Decho also teaches the measurement of the β-GAL expression in the background solution in the form of the CFL and AHL as well as CFL and X-gal test bars (Decho Fig. 5A). While the background solution is not directly measured, it is shown to not have any significant production of the 5,5'-dibromo-4,4'-dichloro-indigo indicator of β-GAL presence on its own because the AHL only and X-Gal only bars show zero expression, and the CFL and AHL as well as CFL and X-gal test bars show zero and significantly minimal levels of 5,5'-dibromo-4,4'-dichloro-indigo indicator of β-GAL concentration, respectively (Decho Fig. 5A). With all the data of Fig. 5A taken together, one of ordinary skill in the art would conclude that the background CFL solution does not produce significant quantities of 5,5'-dibromo-4,4'-dichloro-indigo indicator of β-GAL concentration on its own without the combination of AHL and X-gal together. The indirect measurement of the background CFL solution control as taught by Decho is analogous to the DMSO control solution of instant claim 3 step a because the DMSO of claim 3 is a background solvent of the measurement method of claim 3.
Decho does not teach the specific amounts of 180 µL of each of a BCIG solution, a β-GAL solution, DMSO, and a solution obtained after a reaction of BCIG and β-GAL, 180 µL of DMSO, or 200 µL of a colloidal gold nanoparticle. MPEP §2144.05(II)(A) states “[g]enerally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955)”. In the instant case, because Stevenson and Kiernan teach the SERS measurement of BCIG and β-GAL solutions using DMSO and colloidal gold nanoparticles, one of ordinary skill in the art would be able to determine an appropriate quantity and concentration of the constitutive components of the assay to acquire a satisfactory SERS measurement result.
Stevenson in view of Apte, Lin, Goodacre, Cowcher, Kiernan, O’Haver, and Decho do not teach that the SERS signal peak intensity of DMSO is at a Raman shift of 677 cm-1.
Burkowska teaches that DMSO produces a SERS spectrum of 678, 710, and 2925 cm-1 (Burkowska Fig. 1 and Results para. 1). MPEP 2144.05(I) states that “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)”. In the instant case, a prima facie case of obviousness exists because the Raman shift value of 678 cm-1 of Burkowska is very close to the claimed Raman shift value of 677 cm-1.
Since Stevenson taught that the Raman shift value the β-GAL and BCIG (X-gal) indicator byproduct 5,5'-dibromo-4,4'-dichloro-indigo is 598 cm-1, and Burkowska taught the Raman shift value of DMSO is 678 cm-1, and Lin taught that plotting a ratio of the SERS signal of standard solutions of known analyte concentrations to the SERS signal of an internal standard versus the known concentrations of the standard solutions would produce a standard curve useful for easily calculating an given sample’s unknown analyte concentration based on a SERS signal intensity of said sample, one of ordinary skill in the art would have found it obvious that the ratio of the peak intensity at a Raman shift of 600 cm-1 (corresponding to the peak for analyte 5,5'-dibromo-4,4'-dichloro-indigo) to the peak intensity at a Raman shift of 677 cm-1 (corresponding to the peak for internal standard and solvent DMSO) is the data determined by SERS measurement of the known β-GAL activities of the standard solutions.
Stevenson in view of Apte, Lin, Goodacre, Cowcher, Kiernan, O’Haver, Decho, and Burkowska do not teach the use of an ordinary least squares method to linearly fit the β-GAL activity and a SERS intensity ratio in the standard curve to obtain a standard equation.
Kode teaches that the ordinary least squares method is commonly used to determine linear regressions when all component spectra are known, and that the advantage of this approach is its simplicity of implementation. Kode also teaches that the resulting linear fit can expressed as linear functions of respective concentrations (Kode Pg. 3 para. 1).
Stevenson in view of Apte, Lin, Goodacre, Cowcher, Kiernan, O’Haver, Decho, Burkowska, and Kode do not teach the limitation “…wherein the standard curve is fitted using the following standard equation: y = 0.784 * x + 0.004, with a correlation coefficient R2= 0.936, wherein x represents a logarithm value of an activity of a β-GAL-active standard solution and y represents a ratio of an SERS signal of the β-GAL-active standard solution to the SERS signal of the DMSO”. However, this limitation is interpreted as a property of the resultant standard curve quantification model achieved by performing the active steps of the instant method, but does not recite any required active method steps or structural limitations for the claimed method for constructing a quantification model for quantitative detection of β-galactosidase in seawater. Thus, where the art teaches each and every method step of constructing a quantification model for quantitative detection of β-galactosidase in seawater, this property of the resultant standard curve quantification model is considered necessarily present in resultant standard curve quantification model.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the present invention to: 1) measure the β-GAL activities of seawater samples by mixing the seawater sample with BCIG, DMSO, and colloidal gold nanoparticles and then subjecting the mixture to SERS measurement and calculating a SERS signal ratio of the mixture with the SERS signal of the DMSO, using those SERS signal results to calculate a ratio of SERS signals of the mixture and the DMSO, then ultimately calculating the concentration of the β-GAL in the seawater sample using a standard curve; 2) produce and measure blank positive and negative control solutions comprising the constitutive components of the SERS measuring solution; and 3) create a standard curve quantification model for quantifying β-GAL in the seawater sample by preparing a set of β-GAL standard solutions with different known concentrations, mixing and incubating these standard solutions with BCIG to produce blue indicator 5,5'-dibromo-4,4'-dichloro-indigo in the standard solutions, adding DMSO and colloidal gold nanoparticles to the standard solutions, conducting SERS on said standard solutions, and plotting a standard curve of the logarithm of the different β-GAL activities of each standard solution against the ratio of the SERS signal of the β-GAL solution to the SERS signal of the DMSO.
One of ordinary skill in the art would have been motivated to do so in order to rapidly, easily, and sensitively detect sewage contamination of marine waters with high sensitivity by measuring the SERS signal intensity of the seawater sample and comparing that SERS signal intensity with a standard curve to calculate the β-GAL activity of the sample.
One of ordinary skill in the art would have had reasonable expectations of success in measuring the β-GAL activities of seawater samples because Apte taught that β-GAL is an enzyme marker of fecal coliform bacteria, and that rapidly detecting and quantifying β-GAL activities of seawater could be used as an early warning indicator of sewage contamination of coastal waters (Apte abstract). Stevenson taught that β-GAL can be quantified by using the reaction of β-GAL and BCIG to produce a blue indicator 5,5'-dibromo-4,4'-dichloro-indigo and measuring the SERS signal of that mixture, using colloidal gold nanoparticles as the SERS enhancing substrate. This quantification method of Stevenson could then be used to plot a standard curve by quantifying the β-GAL activities of standard solutions with several different known activities of β-GAL against the ratio of their respective SERS signals to the SERS signal of an internal standard, which is expected to be successful because Lin taught the preparation of standard curves by plotting a ratio of the SERS signal of standard solutions of known analyte concentrations to the SERS signal of an internal standard versus the known concentrations of the standard solutions, thereby generating a non-linear standard curve. It would then be reasonably expected, in view of Goodacre and Cowcher, that logarithmically transforming the non-linear standard curve by taking the logarithm of the known β-GAL activity values would produce a linear standard curve with a linear regression equation that would then be used to calculate the activity of β-GAL in the seawater sample in view of O’Haver. One of ordinary skill in the art would have had reasonable expectations of success in preparing and measuring blank control solutions comprising the constitutive components of the SERS measuring solution because Decho taught the measurement of such constitutive control solution, and the importance of performing such measurements: to determine that the 5,5'-dibromo-4,4'-dichloro-indigo is not produced by any constitutive product except for the combination of the BCIG and β-GAL in one solution.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the present invention to perform an ordinary least squares method to linearly fit the β-GAL activities and the SERS signal intensity ratio in the standard curve produced by the obvious method of constructing a quantification model for quantitative detection of β-galactosidase in seawater taught by Stevenson in view of Apte, Lin, Goodacre, Cowcher, Kiernan, O’Haver, Decho, Burkowska, and Kode. One of ordinary skill in the art would have been motivated to do so in order to produce a linear fit standard curve with a linear fir equation that can be easily used to calculate the β-GAL activity of a given sample that has been measured by SERS. One of ordinary skill in the art would have a reasonable expectation of success because the SERS spectra of both β-GAL and DMSO were determined in the obvious method taught by Stevenson in view of Apte, Lin, Goodacre, Cowcher, Kiernan, O’Haver, Decho, Burkowska, and Kode, and Lin taught that plotting a ratio of the SERS signal of standard solutions of known analyte concentrations to the SERS signal of an internal standard versus the known concentrations of the standard solutions would produce a standard curve useful for easily calculating an given sample’s unknown analyte concentration based on a SERS signal intensity of said sample. Since Kode teaches that the ordinary least squares method is commonly used to determine a linear regression when all component spectra are known, One of ordinary skill in the art would have reasonably expected that an ordinary least squares method would be a simple means of generating a standard curve with a linear equation that can be used to calculate unknown β-GAL activities of a given SERS measured sample
Regarding claim 17, the limitation “…wherein β-GAL activity data of the standard solutions with different β-GAL activities have an average relative standard deviation (RSD) of less than 15%” is interpreted as an intended result for the average relative standard deviation of the β-GAL activity data of the standard solutions which is achieved by performing the active steps of the instant method, but does not recite any required active method steps or structural limitations for the claimed method for constructing a quantification model for quantitative detection of β-galactosidase in seawater. Thus, where the art teaches each and every method step of constructing a quantification model for quantitative detection of β-galactosidase in seawater, these results of the average relative standard deviation of the β-GAL activity data of the standard solutions is considered necessarily present in the β-GAL activity data of the standard solutions.
(New necessitated by amendment) Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Stevenson, Lin, Goodacre, Cowcher, Kiernan, Apte, Burkowska, and Kode as applied to claims 1 and 7 above, and further in view of Benz et al. (SERS of Individual Nanoparticles on a Mirror: Size Does Matter, but so Does Shape, J. Phys. Chem. Lett. 2016, 7, 2264−2269).
Stevenson teaches that in order to improve assay sensitivity, SERS needs adsorption of the analyte of interest (such as 5,5'-dibromo-4,4'-dichloro-indigo measured in Stevenson Fig. 1) onto a suitable enhancing surface, such as gold nanoparticles, to enhance sensitivity. Stevenson continues to teach that the sensitivity of SERS can be enhanced when the plasmonic resonance frequencies/wavelengths of the nanoparticles can be coupled with the excitation light frequencies/wavelengths used in SERS, and also that sensitivity can be further enhanced by using an analyte which has an electronic transition (such as an absorption maxima wavelength) coincident with the Raman excitation light frequencies/wavelengths (Stevenson pg. 6331 para. 2). Stevenson teaches that the 5,5'-dibromo-4,4'-dichloro-indigo has an absorption maxima at ~635 nm (Stevenson Pg. 6332 last para. and Fig 1), which matches the surface plasmon resonance of the gold nanoparticles (~630nm aggregated) and the used excitation wavelength of 632.8nm, thereby resulting in a large enhancement of Raman scattering when detecting 5,5'-dibromo-4,4'-dichloro-indigo (Stevenson Fig. 1).
However, Stevenson, Lin, Goodacre, Cowcher, Kiernan, Apte, Burkowska, and Kode do not teach a colloidal gold nanoparticle with a particle size of 70 nm.
Benz teaches that gold nanostructures have plasmonic resonances that can be tuned to an excitation light wavelength to greatly enhance SERS signal intensity, and that scaling the size of the nanoparticles can be an easy method to tune plasmonic resonance and also the strength of the resonant enhancement (Benz Pg. 2264 para. 2). Benz teaches that different sizes of gold nanoparticles have different plasmonic resonant frequencies, such as dipolar coupled and quadrupolar coupled resonance which resonate at different frequencies/wavelengths. Benz teaches that the 70nm sized gold nanoparticle had resonance positions at ~600nm and ~700nm wavelengths (Benz Fig. 3). Benz also teaches that increasing the size of the nanoparticles always produced larger SERS emission intensities irrespective of nanoparticle tuning to excitation wavelength (Benz abstract and Fig. 3d).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the present invention to use 70nm sized gold nanoparticles in the obvious method of constructing a quantification model for quantitative detection of β-galactosidase in seawater taught by Stevenson in view of Lin, Goodacre, Cowcher, Kiernan, Apte, Burkowska, and Kode.
One of ordinary skill in the art would have been motivated to do so in order to enhance the Raman signal intensity and sensitivity of detection of the β-GAL and BCIG (X-gal) indicator byproduct 5,5'-dibromo-4,4'-dichloro-indigo in the SERS assay of β-GAL activities.
One of ordinary skill in the art would have a reasonable expectation of success because Benz teaches that 70nm sized gold nanoparticles have resonance positions at ~600nm (Benz Fig. 3), and Stevenson teaches that the β-GAL and BCIG (X-gal) indicator byproduct 5,5'-dibromo-4,4'-dichloro-indigo has an absorption maxima at ~635 nm (Stevenson Pg. 6332 last para. and Fig 1). Therefore, the 5,5'-dibromo-4,4'-dichloro-indigo detection sensitivity of the SERS assay would be greatly enhanced because the ~600nm plasmonic resonance wavelength of the 70nm gold nanoparticles is coupled with the 635nm absorption maxima wavelength of the 5,5'-dibromo-4,4'-dichloro-indigo analyte (Stevenson pg. 6331 para. 2). Furthermore, it is known that modulating the size of the gold nanoparticles used in a SERS assay is an easy way to tune the plasmonic resonant wavelengths of the gold nanoparticles to the resonances of the excitation light wavelength and the analyte’s electron transition (light absorption) wavelength, which results in an enhanced Raman signal intensity and analyte detection sensitivity (Benz Pg. 2264 para. 2).
(Maintained) Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Stevenson, Apte, Lin, Kiernan, and O’Haver as applied to claim 2 above, and further in view of Benz.
Stevenson teaches that in order to improve assay sensitivity, SERS needs adsorption of the analyte of interest (such as 5,5'-dibromo-4,4'-dichloro-indigo measured in Stevenson Fig. 1) onto a suitable enhancing surface, such as gold nanoparticles, to enhance sensitivity. Stevenson continues to teach that the sensitivity of SERS can be enhanced when the plasmonic resonance frequencies/wavelengths of the nanoparticles can be coupled with the excitation light frequencies/wavelengths used in SERS, and also that sensitivity can be further enhanced by using an analyte which has an electronic transition (such as an absorption maxima wavelength) coincident with the Raman excitation light frequencies/wavelengths (Stevenson pg. 6331 para. 2). Stevenson teaches that the 5,5'-dibromo-4,4'-dichloro-indigo has an absorption maxima at ~635 nm (Stevenson Pg. 6332 last para. and Fig 1), which matches the surface plasmon resonance of the gold nanoparticles (~630nm aggregated) and the used excitation wavelength of 632.8nm, thereby resulting in a large enhancement of Raman scattering when detecting 5,5'-dibromo-4,4'-dichloro-indigo (Stevenson Fig. 1).
However, Stevenson, Apte, Lin, Kiernan, and O’Haver do not teach a colloidal gold nanoparticle with a particle size of 70 nm.
Benz teaches that gold nanostructures have plasmonic resonances that can be tuned to an excitation light wavelength to greatly enhance SERS signal intensity, and that scaling the size of the nanoparticles can be an easy method to tune plasmonic resonance and also the strength of the resonant enhancement (Benz Pg. 2264 para. 2). Benz teaches that different sizes of gold nanoparticles have different plasmonic resonant frequencies, such as dipolar coupled and quadrupolar coupled resonance which resonate at different frequencies/wavelengths. Benz teaches that the 70nm sized gold nanoparticle had resonance positions at ~600nm and ~700nm wavelengths (Benz Fig. 3). Benz also teaches that increasing the size of the nanoparticles always produced larger SERS emission intensities irrespective of nanoparticle tuning to excitation wavelength (Benz abstract and Fig. 3d).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the present invention to use 70nm sized gold nanoparticles in the obvious method of quantitatively detecting β-GAL in seawater based on SERS as taught by Stevenson, Apte, Lin, Kiernan, and O’Haver.
One of ordinary skill in the art would have been motivated to do so in order to enhance the Raman signal intensity and sensitivity of detection of the β-GAL and BCIG (X-gal) indicator byproduct 5,5'-dibromo-4,4'-dichloro-indigo in the SERS assay of β-GAL activities in seawater samples.
One of ordinary skill in the art would have a reasonable expectation of success because Benz teaches that 70nm sized gold nanoparticles have resonance positions at ~600nm (Benz Fig. 3), and Stevenson teaches that the β-GAL and BCIG (X-gal) indicator byproduct 5,5'-dibromo-4,4'-dichloro-indigo has an absorption maxima at ~635 nm (Stevenson Pg. 6332 last para. and Fig 1). Therefore, the 5,5'-dibromo-4,4'-dichloro-indigo detection sensitivity of the SERS assay would be greatly enhanced because the ~600nm plasmonic resonance wavelength of the 70nm gold nanoparticles is coupled with the 635nm absorption maxima wavelength of the 5,5'-dibromo-4,4'-dichloro-indigo analyte (Stevenson pg. 6331 para. 2). Furthermore, it is known that modulating the size of the gold nanoparticles used in a SERS assay is an easy way to tune the plasmonic resonant wavelengths of the gold nanoparticles to the resonances of the excitation light wavelength and the analyte’s electron transition (light absorption) wavelength, which results in an enhanced Raman signal intensity and analyte detection sensitivity (Benz Pg. 2264 para. 2).
(New necessitated by amendment) Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Stevenson in view of Apte, Lin, Goodacre, Cowcher, Kiernan, O’Haver, Decho, Burkowska, and Kode as applied to claims 3 and 17 above, and further in view of Benz.
Stevenson teaches that in order to improve assay sensitivity, SERS needs adsorption of the analyte of interest (such as 5,5'-dibromo-4,4'-dichloro-indigo measured in Stevenson Fig. 1) onto a suitable enhancing surface, such as gold nanoparticles, to enhance sensitivity. Stevenson continues to teach that the sensitivity of SERS can be enhanced when the plasmonic resonance frequencies/wavelengths of the nanoparticles can be coupled with the excitation light frequencies/wavelengths used in SERS, and also that sensitivity can be further enhanced by using an analyte which has an electronic transition (such as an absorption maxima wavelength) coincident with the Raman excitation light frequencies/wavelengths (Stevenson pg. 6331 para. 2). Stevenson teaches that the 5,5'-dibromo-4,4'-dichloro-indigo has an absorption maxima at ~635 nm (Stevenson Pg. 6332 last para. and Fig 1), which matches the surface plasmon resonance of the gold nanoparticles (~630nm aggregated) and the used excitation wavelength of 632.8nm, thereby resulting in a large enhancement of Raman scattering when detecting 5,5'-dibromo-4,4'-dichloro-indigo (Stevenson Fig. 1).
However, Stevenson in view of Apte, Lin, Goodacre, Cowcher, Kiernan, O’Haver, Decho, Burkowska, and Kode do not teach a colloid with gold nanoparticles with a particle size of 70 nm.
Benz teaches that gold nanostructures have plasmonic resonances that can be tuned to an excitation light wavelength to greatly enhance SERS signal intensity, and that scaling the size of the nanoparticles can be an easy method to tune plasmonic resonance and also the strength of the resonant enhancement (Benz Pg. 2264 para. 2). Benz teaches that different sizes of gold nanoparticles have different plasmonic resonant frequencies, such as dipolar coupled and quadrupolar coupled resonance which resonate at different frequencies/wavelengths. Benz teaches that the 70nm sized gold nanoparticle had resonance positions at ~600nm and ~700nm wavelengths (Benz Fig. 3). Benz also teaches that increasing the size of the nanoparticles always produced larger SERS emission intensities irrespective of nanoparticle tuning to excitation wavelength (Benz abstract and Fig. 3d).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the present invention to use 70nm sized colloidal gold nanoparticles in the obvious method of quantitatively detecting β-GAL in seawater based on SERS as taught by Stevenson in view of Apte, Lin, Goodacre, Cowcher, Kiernan, O’Haver, Decho, Burkowska, and Kode.
One of ordinary skill in the art would have been motivated to do so in order to enhance the Raman signal intensity and sensitivity of detection of the β-GAL and BCIG (X-gal) indicator byproduct 5,5'-dibromo-4,4'-dichloro-indigo in the SERS assay of β-GAL activities in seawater samples.
One of ordinary skill in the art would have had reasonable expectations of success because Benz teaches that 70nm sized gold nanoparticles have resonance positions at ~600nm (Benz Fig. 3), and Stevenson teaches that the β-GAL and BCIG (X-gal) indicator byproduct 5,5'-dibromo-4,4'-dichloro-indigo has an absorption maxima at ~635 nm (Stevenson Pg. 6332 last para. and Fig 1). Therefore, the 5,5'-dibromo-4,4'-dichloro-indigo detection sensitivity of the SERS assay would be greatly enhanced because the ~600nm plasmonic resonance wavelength of the 70nm gold nanoparticles is coupled with the 635nm absorption maxima wavelength of the 5,5'-dibromo-4,4'-dichloro-indigo analyte (Stevenson pg. 6331 para. 2). Furthermore, it is known that modulating the size of the gold nanoparticles used in a SERS assay is an easy way to tune the plasmonic resonant wavelengths of the gold nanoparticles to the resonances of the excitation light wavelength and the analyte’s electron transition (light absorption) wavelength, which results in an enhanced Raman signal intensity and analyte detection sensitivity (Benz Pg. 2264 para. 2).
Response to Arguments
Applicant's arguments filed 02 September 2025 have been fully considered but they are not persuasive.
Regarding Applicant’s argument that the amendment to Figure 4 updated the y-axis as recommended by the Office Action thereby obviating the objection (Remarks pg. 9 sec. III), amended Figure 4 still has the y-axis labeled as “Intensity ratio (600 cm-1/700 cm-1)”, but experimentally the intensity ratio is the signal intensity at 600 cm--1 and 677 cm--1 (instant specification [0047]). It is suggested to amend the y-axis label to “Intensity ratio (600 cm-1/677 cm-1)” to obviate the objection.
Regarding Applicant’s argument that the claims were amended as recommended by the Office Action thereby obviating the objection (Remarks pg. 10 sec. B), some of the objected issues remain. The phrase “incubating resulting mixtures” in line 6 of claim 1 and line 8 of claim 3 should add a definite article “the” before the term “resulting”. In claims 1 and 3, the phrase “an SERS” incorrectly uses the indefinite article “an” intended to precede vowel sounds before a consonant sound “S”, and should be corrected to “a SERS”.
Regarding Applicant’s arguments that claims 1-3, 8, and 9 have been amended to obviate the rejection (Remarks pg. 10 sec. IV), some of the rejected issues remain. For the reasons outlined in the maintained 112b rejections above, claims 2-3 still recite indefinite limitations that must be corrected.
Regarding Applicant’s arguments that none of the cited references teach or suggest the use of the peak intensity of DMSO at a Raman shift of 677 cm-1 and the peak intensity of β-GAL at a Raman shift of 600 cm-1, or the standard equation obtained by fitting the standard curve using the standard equation y = 0.784 * x + 0.004, with a correlation coefficient R2= 0.936, wherein the SERS intensity ratio refers to a ratio of the SERS signal to the SERS signal of the DMSO (Remarks pg. 12 last para. through pg.17 para. 2), one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Stevenson teaches the detection of β-GAL activity using the reaction of β-GAL reacting with BCIG to take a SERS measurement in order to determine the peak intensity of β-GAL at a Raman shift of 600 cm-1.
Kiernan teaches that DMSO is a preferred solvent in methods for detecting β-GAL, and In Vivo Chem provides evidence that the product of β-GAL reacting with BCIG (5,5'-dibromo-4,4'-dichloro-indigo) is soluble in DMSO. Burkowska teaches that DMSO produces a SERS spectrum of 678, 710, and 2925 cm-1, so the claimed DMSO peak of 677 cm-1 is obvious over Burkowska due to the values being very close to each other.
Lin taught that plotting a ratio of the SERS signal of standard solutions of known analyte concentrations to the SERS signal of an internal standard versus the known concentrations of the standard solutions would produce a standard curve useful for easily calculating a given sample’s unknown analyte concentration based on a SERS signal intensity of said sample.
Thus, one of ordinary skill in the art would have found it obvious that the ratio of the peak intensity at a Raman shift of 600 cm-1 (corresponding to the peak for analyte 5,5'-dibromo-4,4'-dichloro-indigo) to the peak intensity at a Raman shift of 677 cm-1 (corresponding to the peak for internal standard and solvent DMSO) can be used to construct a standard curve for easily calculating a seawater sample’s unknown β-GAL activity based on the SERS signal intensity ratio.
Although Lin does not teach the claimed standard equation y = 0.784 * x + 0.004, with a correlation coefficient R2= 0.936, Lin does teach the process of plotting a standard curve based on a ratio of the SERS signal of standard solutions of known analyte concentrations to the SERS signal of an internal standard versus the known concentrations of the standard solutions. Thus, the combination of references Stevenson, Lin, Goodacre, Cowcher, Kiernan, Apte, and Burkowska teach or suggest the fitting of a standard curve using the same standard curve preparation process as the instant invention. Thus, one of ordinary skill in the art would have found the claimed standard equation y = 0.784 * x + 0.004, with a correlation coefficient R2= 0.936 obvious in view of the combination of Stevenson, Lin, Goodacre, Cowcher, Kiernan, Apte, and Burkowska.
Regarding Applicant’s arguments that none of the cited references teach or suggest mathematically dividing the peak intensity of β-GAL at a Raman shift of 600 cm-1 by the peak intensity of DMSO at a Raman shift of 677 cm-1 to construct a quantification model for quantitative detection of β-GAL in seawater (Remarks pg. 17 para. 3), Lin taught that plotting a ratio of the SERS signal of standard solutions of known analyte concentrations to the SERS signal of an internal standard versus the known concentrations of the standard solutions would produce a standard curve useful for easily calculating a given sample’s unknown analyte concentration based on a SERS signal intensity of said sample.
Thus, one of ordinary skill in the art would have found it obvious to construct a quantification model by calculating the ratio of the peak intensity at a Raman shift of 600 cm-1 (corresponding to the peak for analyte 5,5'-dibromo-4,4'-dichloro-indigo) to the peak intensity at a Raman shift of 677 cm-1 (corresponding to the peak for internal standard and solvent DMSO), and plotting that ratio versus known concentrations of standard solutions to construct a standard curve for easily calculating a seawater sample’s unknown β-GAL activity based on the SERS signal intensity ratio.
Ratios are calculated by dividing the first value by the other value, so in the instant case the ratio is calculated by dividing the peak intensity of analyte 5,5'-dibromo-4,4'-dichloro-indigo at a Raman shift of 600 cm-1 by the peak intensity of DMSO at a Raman shift of 677 cm-1.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/Alexander M Duryee/Examiner, Art Unit 1657
/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657