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
Claims 1-16 are pending.
Response to Amendment
Applicant amended claims 1-10 and added new claims 11-16.
Applicant narrowed the scope of claim 4 by limiting the speed of the centrifugation steps.
The objection to the specification is withdrawn in view of the amendment.
The objection to claims 2, 5, and 7 is withdrawn in view of the amendment.
The rejection of claim 4 under 35 U.S.C. 103 is withdrawn in view of the amendment.
Claim Objections
Claims 4 and 6 are objected to because of the following informalities:
Regarding claim 4, Applicant may consider amending the claim to recite “the method of claim 1 further comprises”.
In claim 6, the acronym “DAPI” should be replaced by the full name 4′,6-diamidino-2-phenylindole the first time it appears.
Appropriate correction is required.
Maintained Rejection
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 1-10 remain rejected and claims 11-16 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 is drawn to a method for identifying a bacterium in a biological sample. The claim recites the steps of staining and fixing a single living bacterium isolated from a biological sample, extracting the metabolites from the single living bacterium, and performing a mass spectrometry detection of the metabolites. The claim is indefinite because it is not clear which one of the recited steps identifies the single living bacterium in a biological sample as required by the method. There is no connection between the metabolites extracted, the mass spectrometry performed, and the identification of the single living bacterium in a biological sample. The objective and goal of the method of identifying a single living bacterium in a biological sample is not accomplished.
Claims 2-16 which depend from claim 1 do not cure the indefiniteness and are also rejected.
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.
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.
Claims 1-2 and 5-10 remain rejected and claims 11-14 and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Powless (Diagnostics 9, no. 3 (2019): 122, of record in Office Correspondence mailed on 03/09/2026) in view of Benn (Analyst 144.23 (2019): 6944-6952, of record in Office Correspondence mailed on 03/09/2026), Tian (Analytical chemistry 89.9 (2017): 5050-5057, of record in Office Correspondence mailed on 03/09/2026) and Pan (Analytical chemistry 86.19 (2014): 9376-9380, of record in Office Correspondence mailed on 03/09/2026).
Regarding claims 1-2, 5-7, 10-14, and 16, Powless teaches obtaining a urine sample containing bacteria, centrifuging the sample, decanting the supernatant, resuspend the cells in acridine orange (AO) staining agent, and transferring the stained sample onto a glass slide. Powless teaches that Escherichia coli is the predominant bacteria in urinary tract infections (page 1 first para.). Powless teaches that AO is a fluorescent vital dye that stains live bacteria (page 2 first line of last para.) and teaches a single AO-stained bacterium (Figure 2) (i.e. stained single living bacterium). Powless does not teach the bacterium is fixed.
However, Benn teaches immobilizing live cells of E. coli on a slide treated with poly-L-lysine or gelatine for microscopic analysis in solution and teaches single-cell techniques relies on cells remaining immobilized to a substrate (Abstract, Fig 1, page 6944 right column second para., page 6945 right column second para.). Benn teaches bacteria were added to the slides and incubated for 5, 15, or 30 minutes (page 6945 right column “Bacteria immobilization”).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method taught by Powless by transferring the cells onto a glass slide treated with poly-L-lysine, as suggested by Benn. One of ordinary skill in the art would be motivated to do so in order to trap and better visualize and image the cells. Since Powless and Benn teach a desire to stain and visualize single bacterial cells, there is a reasonable expectation of success.
Powless and Benn do not teach performing mass spectrometry on the single bacterial cell.
However, Tian teaches isolating a single cell of E. coli, incubating it with an antibiotic, and performing a mass spectrometry on the single cell (title, Abstract, page 5051 “Sample Preparation of Aggregated and Isolated E. coli Cells on Si”).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to further modify the method taught by Powless by performing mass spectrometry and the single isolated bacterial cell as suggested by Tian. One of ordinary skill in the art would be motivated to do so in order to identify, analyze, and chemically map small molecules in the single cell bacterium including antibiotics. Since Powless teaches a desire to study single cell bacterium and since Tian teaches the feasibility of performing mass spectrometry on a single bacterium cell, there is reasonable expectation of success.
Powless, Benn, and Tian do not teach injecting an extraction solution into a capillary, positioning the capillary under a microscope to contact the single living bacterium
However, Pan teaches a living single-cell mass spectrometry method and teaches the living single-cell is placed on a microscope slide under a microscope, teaches a probe with a solvent (i.e., capillary with extraction solution) contacting the single living cell under the microscope, and teaches extracting in-situ the metabolites from the single living cell and performing mass spectrometry detection of the metabolites (Title, Abstract, Figure 1). Pan teaches the probe also contains nano-ESI emitter (i.e., electrode) (Figure 1). Pan teaches that to ionize the sampled species for MS analysis, an ionization voltage is transmitted throughout the solution inside the probe (page 9377 right column last para.). Pan teaches the tip size is < 10 μm and also teaches prior art using tip size of 1-5 μm (Abstract, page 9377 left column first para.). Pan teaches the solvent can be methanol, water, acetic acid, or acetonitrile (page 9377 right column last para.).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to further modify the method taught by Powless by contacting the single living bacterium with a capillary holding a solvent, extracting in-situ the metabolites and performing mass spectrometry detection, as suggested by Pan. One of ordinary skill in the art would be motivated to do so in order to accurately identify the bacterium and quantify various molecules of the cell, as taught by Pan. Since Powless teaches a desire to identify a single cell and since Pan teaches single mass spectrometry method to identify and analyze a single cell, there is a reasonable expectation of success.
Regarding claims 8-9, Tian teaches performing mass spectrometry on 2 different E. coli strains, strain with vector control and strain with TetA (i.e., establishing a bacterium comparison database). Tian teaches converting the mass spectrometry data to 2D plots and tables (i.e., dimensionality reduction and visual analysis) to compare the strains’ metabolite profiles obtained from the mass spectrometry (Abstract figure, page 5055 left column last para., Table 2)
Claim 3 remains rejected under 35 U.S.C. 103 as being unpatentable over Powless, Benn, Tian and Pan as applied to claim 1, and further in view of Burillo (PloS one 9.1 (2014): e86915, of record in Office Correspondence mailed on 03/09/2026)
Regarding claim 3, Powless, Benn, Tian, and Pan do not teach performing a step of precipitating a large particle interfering substance from the biological sample via a low-speed centrifugation.
However, Burillo teaches centrifuging a urine sample comprising bacteria at 1,500 revolutions per minute (rpm) for 10 min to remove leukocytes, collecting the supernatant, and then centrifuging the supernatant at 13,000 rpm for 2 min (page 2 “MALDI-TOF Mass Spectrometry” a)).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to further modify the method taught by Powless by performing a step of precipitating leukocytes via a low-speed centrifugation prior to staining the bacteria and by optimizing the speed and duration of the centrifugation. One of ordinary skill in the art would be motivated to do so in order to obtain a supernatant free of leukocytes and other contaminants and enriched for the target bacteria.
New Rejection Necessitated by the Amendment
Claims 4 and 15 are rejected under 35 U.S.C. 103 as being unpatentable over Powless, Benn, Tian and Pan as applied to claim 1, and further in view of Burillo (PloS one 9.1 (2014): e86915, of record in Office Correspondence mailed on 03/09/2026) and Fleischman (The Fleischman lab, CFSE staining of E. coli, published 2014, of record in Office Correspondence mailed on 03/09/2026).
Powless, Benn, Tian, and Pan do not teach precipitating a large particle interfering substance from the biological sample via a low-speed centrifugation of 2000rpm- 4000rpm and do not teach performing a high-speed centrifugation of 10000rpm-14000rpm for 5min-10min to wash away the staining agent.
However, Burillo teaches centrifuging a urine sample comprising bacteria at 1,500 revolutions per minute (rpm) for 10 min to remove leukocytes and collecting the supernatant. Burillo also teaches using centrifugation at 13,000 rpm for 2 min to precipitate the bacteria and separate them from supernatant comprising detergent. (page 2 “MALDI-TOF Mass Spectrometry” a)).
Fleischman teaches staining E. coli using a fluorescent dye and teaches incubating the cells with the stain and centrifuging the cells at 4000g for 5 minutes to remove the excess stain (Protocol step 4).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to further modify the method taught by Powless by optimizing the low-speed centrifugation and its duration in order to precipitate the leukocytes and thus separate them from the bacteria as suggested by Burillo and by optimizing the high-speed centrifugation and its duration in order to precipitate the bacteria and separate them from the excess staining agent and other compounds in the supernatant, as suggested by Burillo and Fleischman. One of ordinary skill in the art would be motivated to do so in order to first collect whole bacterial cells while minimizing contamination from leukocytes and then to remove unbound dye or other compounds, and thus prevent background staining and ensure clear visualization of the bacteria. Since Burillo teaches a low-speed centrifugation is required to precipitate leukocytes but not bacteria and a high-speed centrifugation is required to separate bacteria from a supernatant, and since Fleischman teaches a desire to remove excess stain from the bacteria, there is a reasonable expectation of success.
Regarding claim 15, Fleischman teaches staining E. coli in the dark using a fluorescent dye at a concentration of 10 µM (Materials, protocol step 4). One of ordinary skill in the art would be motivated to stain the cells in the dark using the florescent dye at a concentration of 10 µM in order to effectively stain the cells without damaging its structure and to prevent a photobleaching of the dyes.
Response to Arguments
Applicant's arguments filed 06/09/2026 have been fully considered but they are not persuasive.
Applicant argues the amendment overcomes the indefiniteness rejection of the claims.
Prior to the amendment, claim 1 recited staining and fixing a bacterium, extracting metabolites of the fixed single living bacterium and then performing mass spectrometry detection. The limitation was understood as performing the mass spectrometry on the bacterial cell. With the amendment, there seems to be a missing step linking the extraction of the metabolites/performing a mass spectrometry detection of the metabolites to the identification of the single living bacterium as required by the claim. The goal of the method is not accomplished as it is not clear at which point the single living bacterium was identified in a biological sample.
Applicant argues a person having ordinary skill in the art would not be motivated to combine the disclosures of Powless, Benn, Tian, and Pan to arrive at the present invention with a reasonable expectation of success. Applicant argues Powless relates to point-of-care urine microscopy analysis, Benn relates to nanoscale atomic force microscopy (AFM) imaging of live bacteria, Tian relates to the field of antibacterial drug development, and Pan relates to a novel single-probe MS single-cell mass spectrometry technique, and there is no motivation to combine these methods. Applicant argues that the disclosures of Burillo and Fleischman do not overcome the deficiencies the inherent contradictions of Powless, Benn, Tian, and Pan.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, Powless teaches obtaining E. coli from a urine sample, staining the bacteria using acridine orange, and transferring the stained bacteria onto a glass slide. Benn is relied upon for teaching that single-cell techniques relies on cells remaining immobilized to a substrate and for teaching immobilizing live cells of E. coli on a slide treated with poly-L-lysine. Tian is relied upon for teaching isolating a single cell of E. coli, incubating it with an antibiotic, and performing a mass spectrometry on the single cell. Pan is relied upon for teaching a living single-cell mass spectrometry method. Since Powless teaches a desire to stain and place a single living E. coli on microscope slide, and since Ben teaches immobilizing the cell on a slide, and since Tian and Pan teach performing mass spectrometry on a single-cell, there is a reasonable expectation of success.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to MARY A CRUM whose telephone number is (571)272-1661. The examiner can normally be reached M-F 8:00-5:00 CT with alternate Fridays off.
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/MARY A CRUM/Examiner, Art Unit 1657
/THANE UNDERDAHL/Primary Examiner, Art Unit 1699
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