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 .
Receipt of claim amendments and arguments filed on May 8, 2026 is acknowledged. Claims 1-11 are currently pending.
Priority
The Instant application claims priority as follows:
PNG
media_image1.png
34
332
media_image1.png
Greyscale
PNG
media_image2.png
20
370
media_image2.png
Greyscale
Applicant previously elected the
PNG
media_image3.png
298
632
media_image3.png
Greyscale
, in the reply filed on November 21, 2025.
Claims 10-11 remain withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected inventions, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on November 21, 2025.
Rejections and objections not reiterated herein have been withdrawn.
Examination
The claims have been amended and have overcome the 102(a)(1) rejection over Green et al.
The 103(a) rejection over Margret Ryan in view of Green et al. is maintained for the reasons of record and those below. The elected patentably indistinct group of species wherein R1 is the group myo-inositol phosphoryl was previously searched and found unpatentable over the art. The species wherein R1 is a phosphoryl group was also found unpatentable over the art and rejected in the prior Office Action. Therefore, examination has not been expanded from that performed for the Office Action mailed on 02/13/2026.
Subject matter outside of the examined scope remains withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected inventions, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on November 21, 2025.
Claim Rejections - 35 USC § 103
The following is a quotation of pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
The factual inquiries set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under pre-AIA 35 U.S.C. 103(a) 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 1-9 are rejected under pre-AIA 35 U.S.C. 103(a) as been unpatentable over Margret Ryan et al. (Analytical Biochemistry 214, 548-556 (1993)) in view of Green et al. (ACS Cent. Sci. 2017, 3, 349-358) and Rankin et al. (J. Dairy Sci. (2010) 93:5538-5551).
Chemiluminescent adamantylidene dioxetanes, their use in detection and their mechanism were well-known in the art. See the cited references and references contained therein.
Teachings of Green et al.
Green discloses improved chemiluminescent adamantylidene 1,2-dioxetanes of formula
PNG
media_image4.png
274
580
media_image4.png
Greyscale
,
PNG
media_image5.png
98
184
media_image5.png
Greyscale
,
PNG
media_image6.png
114
294
media_image6.png
Greyscale
and
PNG
media_image7.png
82
182
media_image7.png
Greyscale
for the detection of analytes or enzymes of interest, such as β-galactosidase, alkaline phosphatase, hydrogen peroxide and GSH; wherein the improvement lies on the presence of acrylic acid, acrylate and acrylonitrile electron-withdrawing substituents at the ortho position of the phenolic oxygen. Green found a striking enhancement on the chemiluminescence emission/ efficiency with this substitution. See whole document, particularly the conclusion.
Thus, Green teaches a method comprising providing a medium comprising analytes or enzymes of interest, specifically, β-galactosidase, alkaline phosphatase (AP), hydrogen peroxide and GSH; adding the improved targeted-chemiluminescent adamantylidene dioxetane compounds of formula 2 through 9; and detecting the emitted light.
Green also taught that for activation by β-galactosidase, the phenols were masked with the enzyme-responsive substrate (triggering substrate) of formula
PNG
media_image8.png
75
108
media_image8.png
Greyscale
(colored blue in the reference); and that “to avoid steric interference as a result of the ortho substituent at the enzyme cleavage site, a short self-immolative spacer was installed between the phenolic oxygen and the galactose substrate as previously described.” The short self-immolative spacer is of formula:
PNG
media_image9.png
86
58
media_image9.png
Greyscale
. See page 353.
Green further disclosed that the activation of the chemiluminescence probes is based on removal of a protecting group from the phenolic moiety. Therefore, different phenol protecting groups could be incorporated as triggering substrates for various analytes or enzymes. To demonstrate this modular feature, they synthesized three additional probes for detection of the analytes hydrogen peroxide, glutathione (GSH) and the enzyme alkaline phosphatase (AP), in which the enzyme-responsive substrate (triggering substrate) was specific to these targets. Probe 8
PNG
media_image7.png
82
182
media_image7.png
Greyscale
was equipped with a phosphate group as a substrate for alkaline phosphatase (AP). See page 354 and 355 for probes with different dedicated triggering substrates.
Teachings of Ryan et al.
Ryan teaches a method for sensitive detection of bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) in bacterial cultures of B. cereus, Cytophaga sp. and E. coli MM294 (pIC), by adding the bacterial culture to the chemiluminescent probe LUMI-PI (7)
PNG
media_image10.png
190
406
media_image10.png
Greyscale
, and capturing the emitted light. See pages 551-553. The reference teaches that S. aureus, L. monocytogenes and C. novyi are also known to secrete active PI-PLC into the growth medium, and that phosphatidylinositol (myo-inositol phosphoryl)
PNG
media_image11.png
198
182
media_image11.png
Greyscale
is a triggering substrate for bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). See at least page 548.
Ascertainment of the Difference Between Scope the Prior Art and the Claims
(MPEP §2141.012)
The difference between Applicant’s method and the prior art’s method lies in the compounds used for the detection.
The instant claims require providing a medium comprising a target microorganism, adding a dioxetane of formula:
PNG
media_image12.png
160
518
media_image12.png
Greyscale
or
PNG
media_image13.png
182
244
media_image13.png
Greyscale
, or
PNG
media_image14.png
84
184
media_image14.png
Greyscale
, and detecting the emitted light.
Ryan used phosphatidylinositol (myo-inositol phosphoryl) as enzyme-responsive substrate (triggering substrate) in their probe LUMI-PI (7)
PNG
media_image10.png
190
406
media_image10.png
Greyscale
, which does not have the enhancements to the adamantylidene-dioxetane core probes made by Green.
Finding of Prima Facie Obviousness Rationale and Motivation
(MPEP §2142-2143)
The references are in the same field of endeavor, i.e. improved enzymatically cleavable chemiluminescent adamantylidene 1,2-dioxetanes. It would have been prima facie obvious to one of ordinary skill in the art wanting to perform sensitive detection of bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) in bacterial cultures of B. cereus, Cytophaga sp., S. aureus, L. monocytogenes and C. novyi, to use the enhanced adamantylidene-dioxetane core probes used in probes 3 to 9 of Green with the enzyme-responsive substrate (triggering substrate) phosphatidylinositol (myo-inositol phosphoryl)
PNG
media_image11.png
198
182
media_image11.png
Greyscale
of Ryan et al. The artisan would have expected that the detection would work since Ryan et al. taught the use of adamantylidene dioxetanes with phosphatidylinositol (myo-inositol phosphoryl) as the enzyme-responsive substrate (triggering substrate) to detect said bacteria. Further, a skilled artisan would have reasonably predicted that such a substitution would afford a striking enhancement on the chemiluminescence emission/ efficiency because this is what Green teaches that the enhanced adamantylidene-dioxetane core probes achieve.
Finding of prima facie obviousness
The Supreme Court in KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) identified a number of rationales to support a conclusion of obviousness which are consistent with the proper "functional approach" to the determination of obviousness as laid down in Graham. See MPEP 2143.
Examples of rationales that may support a conclusion of obviousness include:
(A) Combining prior art elements according to known methods to yield predictable results;
(B) Simple substitution of one known element for another to obtain predictable results;
(C) Use of known technique to improve similar devices (methods, or products) in the same way;
(D) Applying a known technique to a known device (method, or product) ready for improvement to yield predictable results;
(E) "Obvious to try" – choosing from a finite number of identified, predictable solutions, with a reasonable expectation of success;
(F) Known work in one field of endeavor may prompt variations of it for use in either the same field or a different one based on design incentives or other market forces if the variations are predictable to one of ordinary skill in the art;
(G) Some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention.
Applying KSR example rationales (A), (B) and (G), the claimed process would have been prima facie obvious.
Regarding claim 8, Green teaches a method comprising providing a medium comprising alkaline phosphatase (ALP), adding the improved targeted-chemiluminescent adamantylidene dioxetane compound of formula 8; and detecting the emitted light. Green does not teach providing a dairy product medium comprising a target organism and pasteurizing it.
However, alkaline phosphatase (ALP) is the most widely used marker of the adequacy of milk pasteurization since it is inactivated at temperatures slightly higher than those required for elimination of pathogens. Chemiluminescent assays for ALP analysis are based on the ALP-mediated dephosphorylation of adamantyl 1,2-dioxetan substrates (e.g., adamantyl-1,2-dioxetane phenylphosphate). See at least Rankin et al. at page 5543.
Thus, the ordinary skilled artisan would have been motivated to use the enhanced chemiluminescent adamantylidene dioxetane compound of formula 8 of Green to test the adequacy of milk pasteurization. The desire of scientists to improve upon what is already generally known would have provided the motivation to do this.
Applicant’s arguments have been carefully considered but were found unpersuasive
First, Applicant argues that the present invention addresses fundamentally different “real-life” detection challenges that are different from those in Green. Applicant argues that suitable chemiluminescent probe for detection of microorganisms has four requirements. See page 14 of the arguments. In response, the claims as they stand do not require the particular interpretation as argued by Applicant. For example, no specific “real-life” detecting conditions is found in the claims. In addition, Green’s probes were designed and tested for “real-life” conditions. See Applicant’s remarks at page 14.
With respect to the Ryan reference, the examiner pointed out the failures of Ryan alone, and that Green cures its deficiencies. Applicant further argues that Ryan discloses LUMI-PI
PNG
media_image10.png
190
406
media_image10.png
Greyscale
, which has the critical structural feature of a methoxy substituent on the dioxetane ring itself, and that this is structurally distinct from the present invention. However, it is not correct that this is structurally distinct from the present invention because, all the compounds in instant claim 4 (see claimed compounds above) have the same methoxy substituent on the dioxetane ring itself.
Applicant further argues that since Ryan reports that their probe is successful in detecting bacterial PI-PLC and the detection limit is described as the “most sensitive procedure for detection of PI-PLC activity reported to date”, Ryan’s results would discourage modification of the probe. The examiner must disagree because the artisan has the general objective and desire to improve a product or a process. That is exactly what both references, Ryan and Green, set to do.
Applicant argues about Green. 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).
Applicant further argues that the obviousness rejection requires changing the dioxetane core structure; maintaining the myo-inositol phosphoryl trigger of Ryan; ensuring the self-immolative linker compatibility; and adapting to microbiological rather than cell imaging conditions, wherein each one of these changes introduces variables that could affect enzymatic recognition and cleavage; luminescence kinetics; quantum yield; stability in bacterial media; and cell permeability. The examiner is not persuaded. First, no compound modification is required of any kind for using the improved targeted-chemiluminescent adamantylidene dioxetane compound of formula 8 of Green. Second, maintaining the myo-inositol phosphoryl trigger of Ryan and ensuring the self-immolative linker compatibility do not incur any changes; these are all present when using the enhanced adamantylidene-dioxetane core probes used in probes 3 to 9 of Green with the enzyme-responsive substrate (triggering substrate) phosphatidylinositol (myo-inositol phosphoryl)
PNG
media_image11.png
198
182
media_image11.png
Greyscale
of Ryan et al. Third, “adapting to microbiological rather than cell imaging conditions” is not a limitation of the claims, and in addition, it is unclear what this encompasses. Thus, this is clearly a use of the enhanced chemiluminescent probes of Green in which the triggering substrate is phosphatidylinositol (myo-inositol phosphoryl)
PNG
media_image11.png
198
182
media_image11.png
Greyscale
of Ryan et al. The only modification required to arrive at the claimed invention is that the beta-galactoside triggering substrate in blue in the compounds of Green is replaced with the triggering substrate myo-inositol phosphoryl of Ryan. Note that Green discussed that different triggering substrates could be incorporated at that position to target other analytes or enzymes:
PNG
media_image15.png
60
376
media_image15.png
Greyscale
In the alternative, it can also be said that the modification is a simple substitution of the chemiluminescent dioxetane in the probe of Ryan for the improved chemiluminescent dioxetane probe of Green. Since Green described all the benefits and improvements of their modified chemiluminescent dioxetane probes, this would have clearly motivated the artisan to do this substitution.
Next, Applicant alleges that the claimed compounds have unexpected results over some other detection systems, MUCAP and D-luciferin-6-O-beta-D-galactopyranoside. Applicant directs the examiner’s attention to Examples 5 and 10 as evidence. The examiner is not persuaded because the compared compounds of Examples 5 and 10 do not contain the chemiluminescent dioxetane core of formula
PNG
media_image16.png
100
172
media_image16.png
Greyscale
that is present in both prior art Ryan and Green. The comparison is to compounds that are not relied upon for any rejection, and that would provide no particular expectation regarding the claimed combination or substitution. MPEP 716.02(e) notes that an affidavit or declaration under 37 CFR 1.132 must compare the claimed subject matter with the closest prior art to be effective to rebut a prima facie case of obviousness.
In addition, applicant states that
PNG
media_image17.png
192
644
media_image17.png
Greyscale
The examiner must disagree because it is Green’s disclosure which states at page 353:
PNG
media_image18.png
76
386
media_image18.png
Greyscale
, and at the conclusion:
PNG
media_image19.png
170
378
media_image19.png
Greyscale
.
Applicant also argues that regarding claim 8, Rankin relates to milk pasteurization validation rather than live microorganism detection, and that Rankin detects absence of enzyme activity while the present invention detects presence of enzyme activity. This argument is found unpersuasive. There is nothing in the claim that requires live microorganism detection, detection of absence of enzyme activity or presence of enzyme activity.
New grounds of rejection necessitated by amendments
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 8 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 8 depends of claim 1, which recites
PNG
media_image20.png
102
630
media_image20.png
Greyscale
. Numeral (i) does not require the presence of or detection of a microorganism, as required by the preamble and step a) of claim 1. Numeral (i) does not further limit claim 1 and does not include all the limitations of claim 1.
Numeral (ii) appears to not require the presence of a microorganism in the provided dairy product medium. Numeral (ii) does not further limit claim 1 and does not include all the limitations of claim 1.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Conclusion
Claims 1-9 are rejected. No claim is in condition for allowance.
Note to Applicants: Not every piece of prior art found in the search has been applied against the instant claims. See MPEP 904.03.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to VALERIE RODRIGUEZ-GARCIA whose telephone number is (571)270-5865. The examiner can normally be reached Monday-Friday 9:30am-5:30pm.
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, Clinton Brooks can be reached at 571-270-7682. 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.
/VALERIE RODRIGUEZ-GARCIA/Primary Examiner, Art Unit 1621
Prosecution Insights