CTFR 18/044,371 CTFR 90066 DETAILED ACTION Notice of Pre-AIA or AIA Status 07-03-aia AIA 15-10-aia The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA. 07-06 AIA 15-10-15 In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status. Priority Acknowledgment is made of the present application as a proper National Stage (371) entry of PCT Application No. PCT/US2021/049493, filed 09/08/2021, which claims benefit under 35 U.S.C. 119(e) to provisional application No. 63/076,259, filed 09/09/2020. Status of the Claims Claims 1-10, 12-15 and 17-19 are pending; claims 1, 12-14, 17 and 18 are amended; no claims are withdrawn; claims 11 and 16 are canceled. Claims 1-10, 12-15 and 17-19 are examined below. Claim Rejections - 35 USC § 103 07-20-aia AIA 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. 07-21-aia AIA Claim (s) 1-4, 9-10, 12-15 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Appleyard et al., Bar-coded hydrogel microparticles for protein detection: synthesis, assay and scanning, 6(11), (2011), p.1761-1774 in view of JP2005505762A (English Machine Translation obtained via PE2E), ThermoFisher Scientific. “Tyramide signal amplification (TSA).” Sept. 19, 2015. Internet Archive Wayback Machine: https://web.archive.org/web/20150919000142/https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/immunofluorescence/tyramide-signal-amplification-tsa.html. Accessed 12/10/2025, Liu et al., Methods for Generating Hydrogel Particles for Protein Delivery, 44(6), (2016), p. 1946-1958 and Horati et al., Particle-Templated Emulsification for Microfluidics-Free Digital Biology, Anal. Chem., 90, (2018), p.9813-9820 . Appleyard et al. teach a method comprising the use of a particle based assay system, the method/system comprising probe incorporated hydrogel microparticles (antibody probe covalently incorporated throughout a probe region of the particle), a reporter capturing agent that forms an affinity complex with captured analyte of interest on or within the hydrogel microparticles (capture and detection within particles using an antibody sandwich technique), see for example, abstract, page 1761, col. 2, paragraph 2, hydrogel particles allowing bulk immobilization of capture probes within the hydrated 3D environment, and providing enhanced binding capacity over surface-functionalization techniques; see also page 1764, col. 2, end of column and Figure 2). Appleyard report their method (at the examples) using a fluorescent reporter, however, see in the introduction, Appleyard acknowledge that in common sandwich applications, paired antibodies are used where the second reporter antibody reports the capture event through fluorescence, chemiluminescent, colorimetric or radiation (see reference to ELISA, enzyme based detection). Appleyard is teaching a system substantially as claimed, however fails to teach the system comprising a catalytic reporter (see as noted Appleyard using a fluorescent reporter), and substrate molecules that react with the catalytic reporter to generate one or more signaling molecules. Additionally, although the mixture of particles in solution of Appleyard appear to read on the limitation “contained in an emulsion”, the reference does not specifically describe the solution as an emulsion and as such, Appleyard et al. also fails to teach the plurality of hydrogel microparticles are contained in particle-templated emulsions formed by mixing a suspension containing the plurality of hydrogel microparticles and the substrate molecules with an oil phase. Similar to Appleyard, is JP2005505762A (hereinafter referred to as ‘762), which also teaches a particle-based assay system and method for detecting an analyte of interest, the method using a system of ‘762, the system comprising a plurality of microparticles having analyte capturing agents disposed thereon (see for example, translation page 2, abstract, and paras [0002], [0007], [0015], see also paras [0027] and [0028], at para [0028] the microparticles are preferably swellable, microparticles that expand or become more porous when incorporated in a suitable medium). ‘762 further teach a catalytic reporter that forms an affinity complex with captured analyte of interest on the hydrogel microparticles and substrate molecules that react with the catalytic reporter to generate signaling molecule, for example tyramide signal amplification (see para [0034], ‘762 teach tyrosine residues linked to the microparticles, HPR- linked target probes and fluorescent tyramide derivatives). See as cited above, ‘762 teaches swellable microparticles, microparticles that expand and become more porous, para [0028]. ThermoFisher also teach tyramide signal amplification techniques visualize low abundance targets that are not detectable by conventional means (see page 1, describing it as a technique that provides unprecedented sensitivity without compromising resolution) See however, Liu et al., Liu reviews methods for generating hydrogel particles (see abstract, Liu et al. acknowledges how hydrogels have long been investigated for their potential in carrying and delivering proteins). Liu reviews common methods of making such materials. At page 4, paragraph 2, Liu teach emulsion polymerization as an art recognized technique for forming hydrogel microparticles (loading protein during or post synthesis, see paragraphs 2-3). Liu teach at page 4, paragraph 2, forming an emulsion using either oil or aqueous (inverse emulsion) phase containing monomer, performing a mixing/homogenizing step to generate droplets of monomer in aqueous phase, surrounded by the oil phase. See further at this citation, Liu teach the polymerized particles can be washed and optionally lyophilized (freeze-dried) for storage (paragraph 2). Hatori et al. teach the technique particle-templated emulsification, a method to encapsulate samples in monodispersed emulsions without microfluidics (abstract). Hatori teach their approach (particle template emulsification) as a way for generating compartmentalized reactions in monodispersed droplets with vortexing, that the method uses the particles to “template” the formation of droplets of similar size under agitation with oil, this technique offers a way to introduce additional components to the droplets (see page 9813, col. 2, para 2). The method involves mixing hydrogel particles with oil (see scheme presented at Figure 1, described at the caption). Hatori et al. teach particle-templated emulsification results in emulsion having droplets of similar size to the original particles, and thus, monodispersed (page 9816, col. 1, para 1). Hatori et al. also teach advantages (in addition to creating droplets of uniform size) are that the method is simple, scalable and compatible/gentle on biological samples (page 9819, col. 1, para 3 and col. 2, conclusions). It would have been prima facie obvious to one having ordinary skill in the art prior to the effective filing date of the claimed invention to have modified the system of Appleyard in order to provide tyramide signal amplification as the reporter of the system, specifically to have modified the system of Appleyard et al. such to provide as reporter HRP labeled antibody, provide substrate to produce signaling molecule, that binds tyrosine (tyrosine labeled hydrogel microparticle), as in ‘762. In particular, the modification would be a simple substitution of one art recognized labeling technique for another, where TSA provides the additional benefit of improved sensitivity, thereby further accommodating low abundance target detection (ThermoFisher). In particular, both were art recognized labeling techniques applicable to sandwich type assays (for example, see Appleyard, and also ‘762), TSA having the additional benefit of improved sensitivity. One having ordinary skill in the art would have a reasonable expectation of success using TSA, given that ‘762 demonstrate its feasibility, binding HRP to target, introducing tyramide, causing signal molecule to be captured at tyrosine linked to the microparticles, Appleyard further already demonstrating the ability to covalently link capture substances to the polymeric matrix of their hydrogel microparticles. It would have been further prima facie obvious to one having ordinary skill in the art to have modified Appleyard et al., in order to have generated the hydrogel particles using emulsion technique (Liu and Hatori), namely by way of particle-template emulsification (PTE) (i.e., by mixing, via vortex, the suspension containing the plurality of microparticles and the substrate molecules with an oil phase as Hatori), as a simple substitution of one known way of making/providing the hydrogel particles (Appleyard) for another (Hatori), one specifically motivated as a result of the advantages of PTE as taught by Hatori (specifically, generates compartmentalized components, droplets having similar size related to the particle size as a result of the particle used as a template, the method a simple, scalable method, recognized as gentle/compatible with biological samples). In particular, the prior art contained the base system and method of using said system, as taught by Appleyard et al., further PTE is an art recognized technique for producing hydrogel microparticles consistent with those in Appleyard et al., the technique known and available to those of ordinary skill for producing hydrogel microparticles suspended in an emulsion (Horati). One having ordinary skill would have found it obvious to use such a technique and the result would have been predictably generation of protein loaded/bound hydrogel microparticles of uniform size (still would have achieved hydrogel microparticles). Regarding claim 2, see as cited above, Appleyard is teaching a system comprising porous hydrogel microparticles, analyte capturing agent located within the hydrogel (within the pores of the microparticles, see citations above). Regarding claims 3-4 and 9-10, the combination of the cited art addresses catalytic reporter that is the species HRP (see as cited above, referring to the combination of Appleyard in view of ‘762 and ThermoFisher) and signal capturing moiety disposed within the hydrogel microparticles (tyrosine). Regarding claim 12, see as discussed in detail above, the combination of the cited art is teaching droplets uniform in volume. Regarding claim 13, the combination of the cited art (Appleyard in view of ‘762), teaches a method as claimed. See for example, Appleyard teach methods comprising steps of incubating the plurality of hydrogel microparticles in sample solution containing analyte of interest, incubating with reporter (e.g., shown at Figure 2), Appleyard as modified by ‘762, and further ThermoFisher, further results in a method further comprising exposing microparticles to substrate that reacts to cause signal molecule to be produced and captured at the signal capturing agent at the hydrogel matrix (tyrosine, see ‘762). Regarding claim 14, see at Figure 3, demonstrating detection by fluorescence sorter (a flow through system for detecting and sorting the detected signals). Regarding claim 15, Appleyard teach wash steps between each of the binding/assay steps (see for example page 1769, regarding protein assay, washing after incubation of sample with microparticles). Regarding claim 19, see also as cited above, Liu recognized that hydrogel microparticles my further be lyophilized (freeze dried) in order to store the microparticles, as such it would have been further obvious to have provided the hydrogel particles either as an emulsion or in a dried state (such as freeze dried, for example to be rehydrated with addition of sample) in order to provide the hydrogel microparticle reagent for later used (i.e., so that they can be stored for use at a later time). One having ordinary skill in the art would have had a reasonable expectation of success further providing the hydrogel microparticles in a dry state (as in Liu) since the art specifically discloses the ability to do so for storage purposes . 07-22-aia AIA Claim (s) claims 5 and 6 are rejected under 35 U.S.C. 103 as being unpatentable over Appleyard et al. in view of JP2005505762A, ThermoFisher, Liu et al. and Horati , as applied to claim 1 above, and further in view of Krevolin et al., EP 0514173A2 . Appleyard et al. and the cited prior art teach a system substantially as claimed, see as cited in detail previously above detecting signal produced between enzymatic label and substrate (HRP and tyramide). However, the combination of the cited art fails to teach an enzyme substrate combination that is β-galactosidase and fluorescein-β-galactopyranoside (claim 5) or resorufin-β-D galactopyranoside (claim 6). However, see for example, β-galactosidase and fluorescein-β-galactopyranoside or resorufin-β-D galactopyranoside, these are other art recognized examples of enzyme-substrate pair known to produce a detectable signal (see for example, Krevolin et al., page 10, lines 48-55, Krevolin teaching these combinations suitable for spectrophotometric of fluorometric analysis). It would have been further prima facie obvious to have modified the enzyme/substrate pair as taught by the combination of Appleyard et al. and the cited prior art, in order to provide for example β-galactosidase and fluorescein-β-galactopyranoside (thereby producing signal molecule that is fluorescein) or resorufin-β-D galactopyranoside (thereby producing signal molecule that is resorufin), as a simple substitution of one known enzyme/catalytic labeling substance used for producing a detectable signal for another. The art recognized these reporter/substrate combinations as available and known to those of ordinary skill in the prior art (Krevolin). It would have been obvious to have provided these in place of that as taught by ‘762 and the results would have been predictable (one would have a reasonable expectation of success given one would have predictably produced a detectable signal, see Krevolin teaching suitable for spectrophotometric of fluorometric analysis) . 07-22-aia AIA Claim (s) claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Appleyard et al. in view of JP2005505762A, ThermoFisher, Liu et al. and Horati , as applied to claim 1 above, and further in view of Gildor et al., US PG Pub No. 2019/0094214A1 . Appleyard et al. and the cited prior art teach a system substantially as claimed, see as cited in detail previously above detecting signal produced between enzymatic label and substrate (HRP and tyramide). However, the combination of the cited art fails to teach an enzyme substrate combination that HRP and ADHP to produce fluoresce resorufin (claim 7). However, see further Gildor et al., Gildor et al. teach regarding enzyme labels, that HRP used with ADHP (10-acetyl -3, 7-dihydroxyphenoxazine) in the presence of hydrogen peroxide yields detectable resorufin (para [0086]). Consistent with reasoning previously above, presently it would have been similarly prima facie obvious to have modified the enzyme/substrate pair as taught by the combination of Appleyard et al. and the cited prior art, in order to provide as the substrate for HRP, ADHP as the signal producing system as a simple substitution of one known enzyme/catalytic labeling substance used for producing a detectable signal for another. The art recognized these reporter/substrate combinations as available and known to those of ordinary skill in the prior art (Gildor). It would have been obvious to have provided ADHP as the substrate in place of that as taught by ‘762 and the results would have been predictable (one would have a reasonable expectation of success given one would have predictably produced a detectable signal, see Gildor et al.) . 07-22-aia AIA Claim (s) claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Appleyard et al. in view of JP2005505762A, ThermoFisher, Liu et al. and Horati et al ., as applied to claim 1 above, and further in view of Tanaka et al., EP0683396A1 . Appleyard et al. and the cited prior art teach a system substantially as claimed, see as cited in detail previously above detecting signal produced between enzymatic label and substrate (HRP and tyramide). However, the combination of the cited art fails to teach an enzyme substrate combination alkaline phosphatase and 4-methylumbelliferonyl phosphate (claim 8). However, see for example Tanaka, Tanaka teach another art recognized detectable signal producing enzyme substrate pair is alkaline phosphatase and 4-methylumbelliferonyl phosphate (see col. 7, lines 19-36). Consistent with reasoning previously above, presently it would have been similarly prima facie obvious to have modified the enzyme/substrate pair as taught by the combination of Appleyard et al. and the cited prior art, in order to provide alkaline phosphatase and 4-methylumbelliferonyl phosphate as the signal producing system as a simple substitution of one known enzyme/catalytic labeling substance used for producing a detectable signal for another. The art recognized these reporter/substrate combinations as available and known to those of ordinary skill in the prior art (Tanaka et al.). It would have been obvious to have provided alkaline phosphatase and 4-methylumbelliferonyl phosphate in place of that as taught by ‘762 and the results would have been predictable (one would have a reasonable expectation of success given one would have predictably produced a detectable signal, see Tanaka et al.) . 07-21-aia AIA Claim (s) 17 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Appleyard et al. in view of ‘762, ThermoFisher, Liu et al. and Hatori, as applied to claim 13, and further in view of Le Goff et al., Hydrogel microparticles for biosensing, Eur. Polym. J., 72, (2015), p.386-412 and Rakszewska et al., One drop at a time: toward droplet microfluidics as a versatile tool for single-cell analysis, Nature Publishing Group, 6, (2014), (11 pages) . Appleyard et al. and the cited art teach a method substantially as claimed (see as cited above, further forming an emulsion). However, the combined cited prior art is silent to whether analysis is performed on the microparticle following a break of the emulsion (claim 17), or the microparticles in the emulsion (claim 18). The prior art supports that for flow through analyses of single particles, analysis can be conducted by breaking an emulsion or on an emulsion. For example, Le Goff is a review of the use of hydrogel microparticles for biosensing (see abstract), Le Goff supports that flow through analysis can be conducted either as an emulsion (see for example page 24, section ii., referring to hydrodynamic focusing to generate an emulsion of dispersed phase into a sheath stream of the continuous oil phase). See further, Rakszewska et al., which is an example in the art which forms demonstrates an emulsion containing single components is broken and subsequently sent through a flow cytometry for detection (page 7, col. 1, para 2). It appears based on the prior art, that for methods involving flow through detection (for example flow cytometry) of fluorescently labeled components, whether sample is provided in the form of an emulsion, or that emulsion is broken, is insignificant to detection (see as examples, Le Goff and Rakszewska). Given that presence in an emulsion or as a broken emulsion appears ancillary to flow through detection methods, it would have been prima facie obvious to one having ordinary skill to have either performed detection by breaking the emulsion and performing analysis, or by subjecting the microparticles in the emulsion to analysis. Further, since the art demonstrates the ability to detect targets present as either an emulsion or a broken emulsion, one having ordinary skill would have a reasonable expectation of success . Response to Arguments 07-37 AIA Applicant's arguments filed 03/03/2026 have been fully considered but they are not persuasive for the following reasons . At remarks pages 7 Applicant refers to the amendments to the claims, to recite particle-templated emulsion. Regarding the previous rejection of claims under 35 U.S.C. 103, Applicant argues (remarks pages 8-12) that the previously cited art (Liu) use emulsions that trap reaction products, not particle-templated emulsion (remarks pages 10-12). See the amended grounds of rejection as set forth in detail above. Further, at remarks page 12, regarding the rejection of claim 17 and the citation of Le Goff, Applicant argues the reference is only related to how emulsions can be generated using a flow-focusing device with T-junctions, that there is no disclosure of subjecting emulsions with hydrogel microparticles to visualization or fluorescence assay. However, this argument is not persuasive. The prior art (e.g., Le Goff) is considered to support that for flow through analyses of single particles, analysis can be conducted on an emulsion, Le Goff is an example in the art performing a flow through analysis on a sample considered to be an emulsion. The reference is not cited independently as anticipating the claimed invention, rather it is the combination which addresses the claim. Conversely to Le Goff, Rakszewska et al., is an example in the art which forms demonstrates an emulsion containing single components is broken and subsequently sent through a flow cytometry for detection (page 7, col. 1, para 2). It appears, based on prior art, that either performing analysis as in emulsion or following breaking of an emulsion is ancillary to flow through or fluorescence analyses. The prior art suggest either type of sample state is suitable and there is nothing of record suggesting it would be unexpected that either would be successful. For these reasons, Applicant’s arguments are not persuasive, and the claims are rejected as indicated in detail above (the rejections amended in response to Applicant’s amendments to the claims). Conclusion 07-40 AIA 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. Correspondence Any inquiry concerning this communication or earlier communications from the examiner should be directed to ELLEN J MARCSISIN whose telephone number is (571)272-6001. The examiner can normally be reached M-F 8:00am-4:30pm. 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, Bao-Thuy Nguyen can be reached at 571-272-0824. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000. /ELLEN J MARCSISIN/Primary Examiner, Art Unit 1677 Application/Control Number: 18/044,371 Page 2 Art Unit: 1677 Application/Control Number: 18/044,371 Page 3 Art Unit: 1677 Application/Control Number: 18/044,371 Page 4 Art Unit: 1677 Application/Control Number: 18/044,371 Page 5 Art Unit: 1677 Application/Control Number: 18/044,371 Page 6 Art Unit: 1677 Application/Control Number: 18/044,371 Page 7 Art Unit: 1677 Application/Control Number: 18/044,371 Page 8 Art Unit: 1677 Application/Control Number: 18/044,371 Page 9 Art Unit: 1677 Application/Control Number: 18/044,371 Page 13 Art Unit: 1677 Application/Control Number: 18/044,371 Page 14 Art Unit: 1677 Application/Control Number: 18/044,371 Page 15 Art Unit: 1677
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