DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of the Claims
Claims 1-18 and 20-23 are pending and examined herein.
Priority
The present application, filed 05/17/2024, is a continuation of PCT/US2022/050631, filed 11/21/2022, which claims benefit of U.S. Provisional Patent Application 63/281,509, filed 11/19/2021. The benefit is acknowledged and the claims examined herein are treated as having an effective filing date of 11/19/2021.
Information Disclosure Statement
The Information Disclosure Statement(s) filed 05/22/2024 are acknowledged and have been considered. However, the reference “EL-FAHAM, AYMAN , et al., "Peptide Coupling Reagents, More than a Letter Soup", Chem. Rev., Vol. 111, No. 11, 2011, 6557-6602” was not considered because, although a copy of the cover page was provided, a complete copy of the publication was not provided in the file wrapper.
Claim Objections
Claim 8 is objected to under 37 CFR 1.75 as being a substantial duplicate of claim 7. Claims 7 and 8 depend from claim 1 and recite substantially the same subject matter, differing only in the use of “a” versus “the” before carboxyaryl porphyrin carboxamide. Accordingly, claims 7 and 8 are so close in content that they cover the same subject matter. Appropriate correction is required. See MPEP § 608.01(m).
The claims are objected to because the numerical sequence of the claims is improper. Specifically, claim 19 is omitted, such that the claims proceed from claim 18 directly to claim 20. Applicant is required to correct the claim numbering to provide a consecutive numerical sequence of claims. 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 2, 8, and 14 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.
Regarding claim 2, the claim recites “the compensation method of claim 1.” However, claim 1 is directed to a compensation bead, and claim 2 does not recite steps defining a compensation method, but instead further recites structural limitations of the compensation bead, including the covalent bonding of the carboxyaryl porphyrin to the amine functionalized bead via an amide bond and the absence of a linker. Accordingly, it is unclear whether claim 2 is intended to further limit the compensation bead of claim 1 or is instead intended to recite a compensation method, thereby rendering the scope of claim 2 indefinite. For purposes of compact prosecution, claim 2 will be interpreted as further limiting the compensation bead of claim 1, rather than reciting a separate method, such that the carboxyaryl porphyrin is covalently bonded to the amine functionalized bead via an amide bond, wherein the carboxy portion is present in the carboxyaryl porphyrin and the amine portion is part of the amine functionalized bead, in the absence of a linker. Appropriate correction is required.
Regarding claim 8, the claim recites “the carboxyaryl porphyrin carboxamide.” However, claim 8 depends directly from claim 1, and claim 1 recites a carboxyaryl porphyrin, but does not previously introduce or otherwise recite a carboxyaryl porphyrin carboxamide. Although claim 1 recites that the carboxyaryl porphyrin is covalently bonded with the amine group of the amine functionalized polymer bead, claim 1 does not specify that the covalent bond forms a carboxamide. Further, while claim 7 introduces a carboxyaryl porphyrin carboxamide, claim 8 does not depend from claim 7, and therefore claim 7 does not provide antecedent basis for the carboxyaryl porphyrin carboxamide recited in claim 8. Accordingly, it is unclear what antecedent element is intended by the carboxyaryl porphyrin carboxamide, thereby rendering the scope of claim 8 indefinite. For purposes of compact prosecution, claim 8 will be interpreted such that the recited carboxyaryl porphyrin carboxamide refers to the carboxyaryl porphyrin of claim 1 covalently bonded with the amine group through a carboxamide bond. Appropriate correction is required.
Regarding claim 14, the claim recites “the EDC.” However, claim 14 depends from claim 9, and claim 9 does not previously introduce or otherwise recite EDC. Although EDC is recited in claim 12, claim 14 does not depend from claim 12. Accordingly, it is unclear what antecedent element is intended by the EDC, thereby rendering the scope of claim 14 indefinite. For purposes of compact prosecution, claim 14 will be interpreted as further limiting the method of claim 9 to require EDC and the carboxyaryl porphyrin to be present at a mol/mol ratio of between about 0.35 to about 25.91, wherein EDC is employed in the reaction between the amine group on the polymer bead surface and the carboxyaryl porphyrin. Appropriate correction is required.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1, 3, 4, and 6 are rejected under 35 U.S.C. 103 as being unpatentable over Pitner et al. (US 2019/0264102 A1) in view of Kabe et al. (Porphyrin Accumulation in Mitochondria Is Mediated by 2-Oxoglutarate Carrier. The Journal of Biological Chemistry. Vol. 281, No. 42, October 2006 – IDS dated 05/22/2024), Castriciano et al. (Spectroscopic Investigation and Molecular Modeling on Porphyrin/PAMAM Supramolecular Adduct. Photochemistry and Photobiology. Vol. 87, No. 2, March 2011), as evidenced by Ohtsu et al. (Selective Ligand Purification Using High-Performance Affinity Beads. Analytical Biochemistry. Vol. 338, No. 2, March 2005 – IDS dated 05/22/2024).
Regarding claim 1, for the limitation a compensation bead for use in a flow cytometer, Pitner teaches fluorescent polymeric microparticles and beads useful as calibration standards for flow cytometry and specifically identifies compensation controls and instrument setup controls as exemplary applications ([0138], p. 14). Pitner further states that the hydroporphyrin beads have potential uses as calibration standards for instrument set-up and for determining compensation values for overlapping fluorophores used in flow cytometry ([0176], p. 19). Regarding a polymer bead having a diameter of between about 3 μm to about 18 μm, Pitner expressly employs 5.43 μm polystyrene (PS) beads and demonstrates their fluorescence under 405-nm excitation ([0170], p. 18; Figs. 15–16).
Regarding a polymer bead bearing a fluorescent porphyrinic species, Pitner teaches that the polymeric matrix comprises polystyrene, which is optionally in the form of a bead, and that a porphyrinic macrocycle may be non-covalently associated with the exterior of the bead, covalently attached to a surface of the bead, or any combination thereof ([0120], p. 12). This disclosure is particularly significant because Pitner itself expressly contemplates both surface-association mechanisms and combinations thereof rather than limiting the fluorescent species to the internal-doping embodiments of its examples. Regarding the requirement that the carboxyaryl porphyrin be bonded to the exterior surface via a non-covalent bond and via a covalent bond, Pitner therefore supplies the overall bead architecture and expressly teaches the availability of both exterior noncovalent association and surface covalent attachment, including any combination thereof ([0120], p. 12).
Regarding the functional requirement that, when suspended in media, the bead-bound porphyrin exhibit fluorescence spectra comparable with the fluorescence spectra of the carboxyaryl porphyrin, Pitner reports that suspensions of hydroporphyrin-stained polystyrene beads in aqueous buffer showed fluorescence properties (e.g., sharp emission bands) that were nearly superimposable over the spectra of the same hydroporphyrins dissolved in THF ([0177], p. 19). Pitner additionally states that its dye-loaded beads maintain the same large Stokes shifts and sharp narrow fluorescence emission spectra as displayed by the hydroporphyrins when characterized in organic solvents ([0175], p. 19).
Pitner also provides an express reason to employ surface-associated porphyrinic macrocycles in a flow-cytometric bead system: it identifies such particles as compensation/calibration standards and teaches that porphyrinic macrocycles provide sharp narrow emission peaks, large effective Stokes shifts, reduced background from other non-dye bead components such as polystyrene, and consequently cleaner and more reliable negative fluorescence control signals and more accurate positive signals ([0175]-[0176], [0179], pp. 19–20). Pitner therefore directs one of ordinary skill toward attachment arrangements that preserve the advantageous optical behavior of the porphyrinic macrocycle.
Pitner, however, does not expressly teach that the bead is amine functionalized, that an amine group is covalently bonded to the exterior surface of the polymer bead, or specifically that the fluorescent species is a carboxyaryl porphyrin covalently bonded with the amine group, while also providing noncovalent association with the amino-functionalized polymer surface.
Kabe teaches these missing chemical features. Kabe employs glycidylmethacrylate-covered glycidylmethacrylate-styrene copolymer core beads and explains that the SG beads possess an extremely large surface area producing a relatively high binding capacity (p. 31730). Kabe specifically uses the carboxyaryl porphyrins PdTCPP and TCPP and teaches that PdTCPP or TCPP was incubated with one equivalent of N-hydroxysuccinimide in N,N-dimethylformamide, after which the succinated porphyrin was incubated with SGNEGDEN beads (Experimental Procedures, pp. 31730–31731). More particularly, Kabe states that the reaction product was 80% single succinated PdTCPP and that succinated PdTCPP was then conjugated to amino-modified SG beads (Fig. 3A, p. 31732). Figure 3A depicts the resulting amide linkage between the porphyrin carboxyl functionality and bead-associated amino functionality. Kabe also identifies PdTCPP as palladium meso-tetra(4-carboxyphenyl)porphyrin and reports excitation at approximately 400 nm (Fig. 1A; p. 31729).
Ohtsu provides evidentiary support for the chemistry and surface functionality of the same SG-bead platform. Ohtsu describes SG beads as GMA–styrene copolymer core beads and states that their extremely large surface area results in a relatively high capacity (p. 246). Ohtsu further expressly refers to unreacted amino groups on the beads and demonstrates NHS/EDC-mediated surface coupling (pp. 246, 248). Thus, Ohtsu corroborates that the amino functionality relied upon in Kabe is bead-associated surface chemistry capable of covalent coupling.
Castriciano supplies the complementary noncovalent interaction specifically between a carboxyaryl porphyrin and amino functionality of a polymer. Castriciano teaches noncovalent adducts (TPPC@PAMAM) between meso-tetrakis(4-carboxyphenyl)porphyrin (TPPC) and polyamidoamine PAMAM dendrimer and identifies electrostatic interactions between the anionic carboxylate groups of TPPC and the protonated amino groups of the PAMAM dendrimer as important in stabilizing the adduct (Abstract, p. 292). Castriciano further teaches a noncovalent synthetic procedure producing stable supramolecular adducts whose structure is mainly stabilized through electrostatic interactions acting between the anionic carboxylate groups of the porphyrin and the charged protonated nitrogen atoms of the dendrimer, with the resulting species exhibiting good solubility in water up to millimolar concentration, where they are stable for months (p. 295). This provides a direct chemical basis for noncovalent association between a carboxyaryl porphyrin and an amino-functionalized polymer surface. Castriciano additionally reports that, at appropriate PAMAM/TPPC ratios, the fluorescence emission spectra exhibit bands at 650 and 712 nm that are very similar to those of the parent porphyrin (p. 295), supporting preservation of the porphyrin’s characteristic fluorescence following association with an amino-containing polymer.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Pitner’s 5.43-μm polystyrene compensation bead, for which Pitner expressly permits a porphyrinic macrocycle to be noncovalently associated with the bead exterior, covalently attached to the bead surface, or present by a combination thereof, by providing accessible surface amino functionality as demonstrated by Kabe/Ohtsu and employing Kabe’s demonstrated carboxyaryl-porphyrin-to-amino-bead conjugation together with the complementary noncovalent carboxylate/amino association taught by Castriciano. The modification would implement the very combined surface-attachment modes contemplated by Pitner using chemically compatible attachment mechanisms known for carboxyaryl porphyrins, while retaining the narrow porphyrin-derived fluorescence that Pitner identifies as advantageous for compensation and calibration. One of ordinary skill would have had a reasonable expectation of success because Kabe actually produced PdTCPP/TCPP-conjugated amino-modified polymer beads, Castriciano actually obtained stable aqueous TPPC/amino-polymer noncovalent adducts whose fluorescence remained very similar to that of the parent porphyrin, and Pitner demonstrates that porphyrin-loaded polystyrene beads preserve porphyrin-like spectra and function in flow cytometry. The proposed modification therefore represents the use of known carboxyaryl-porphyrin/amino-polymer attachment techniques to improve the similar Pitner flow-cytometry bead in the same way, namely to provide the combined surface attachment expressly contemplated by Pitner while preserving the porphyrin fluorescence required for compensation.
Regarding claim 3, for the polymer being selected from the recited group including polystyrene, Pitner expressly teaches that the polymeric matrix comprises polystyrene, which is optionally in the form of a bead ([0120], p. 12).
Regarding claim 4, for a bead diameter of about 5 μm to about 15 μm, Pitner teaches 5.43 μm PS beads ([0170], p. 18; Figs. 15–16), which fall within the claimed range. Pitner also expressly depicts flow-cytometric experiments using 5.4 μm diameter polystyrene beads ([0023], p. 5; Fig. 4) and 5.43 μm diameter beads ([0026]-[0027], p. 5; Figs. 7–8).
Regarding claim 6, regarding excitation at about 400 nm, Pitner teaches that its fluorescent polymeric particle population is capable of excitation by light at 405 nm ([0124], p. 12). Regarding emission at about 600 nm to about 800 nm, Pitner teaches emission wavelength bands between about 590 nm and about 750 nm ([0124], p. 12), which overlap and substantially fall within the claimed range. Pitner experimentally confirms that its beads retain sharp emission bands under a common violet (405 nm) laser excitation over the emission wavelength range from 590 nm to 750 nm ([0178], p. 19).
Claims 2, 7, and 8 are rejected under 35 U.S.C. 103 as being unpatentable over Pitner et al., Kabe et al., Castriciano et al., as evidenced by Ohtsu et al., as applied to claim 1 above, and further in view of Covolan et al. (Preparation of Aminated Polystyrene Latexes by Dispersion Polymerization. Macromolecules. Vol. 33, No. 18, September 2000) and Kazenwadel et al. (Optimization of Enzyme Immobilization on Magnetic Microparticles Using 1-Ethyl-3-(3-Dimethylaminopropyl)Carbodiimide (EDC) as a Crosslinking Agent. Analytical Methods, Vol. 7, No. 24, January 2015).
Regarding claim 2, with respect to the teachings of Pitner et al., Kabe et al., Castriciano et al., see the discussion above, which applies equally here. These references differ from the instant claims in failing to expressly teach or specify the claimed no-linker amide architecture.
Regarding an amine-functionalized polymer architecture suitable for direct coupling, Covolan teaches aminated polystyrene-based latexes produced by copolymerization of Boc-p-aminostyrene and styrene, followed by deprotection to form p-aminostyrene/styrene copolymers (Abstract, p. 6685). Covolan explains that functional groups including amino, exposed on the surfaces of the constituting particles can be used in immobilization by ionic or covalent bindings of biological molecules (p. 6685). Importantly, Covolan’s surface amines arise from aminostyrene units of the polymer itself rather than from a subsequently grafted peptide spacer. XPS analysis showed free amino groups on the particle surfaces, with the detected nitrogen nearly exclusively attributable to the amino groups of the aminate styrene units, and Covolan concludes that the amino groups tend to distribute on the microsphere surface (p. 6691). Covolan further supplies an affirmative reason to employ this architecture: it explains that surface-grafting procedures involve a variety of reactions making them rather complex and time-consuming and are often susceptible of altering the inherent latex morphological properties, whereas its copolymerization procedure provides aminated latexes under one pot reaction conditions and is by far more convenient than more cumbersome and time-consuming systems based on nitration of polystyrene followed by reduction of nitro groups (pp. 6685, 6691).
Kazenwadel confirms that direct carboxyl-to-amine coupling can be performed without introducing an intervening spacer. Kazenwadel explains that zero-length crosslinkers only serve as activators, but do not introduce spacer atoms while connecting molecules (p. 10291), and specifically teaches that 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is one of the most commonly used cross linking molecules, and that EDC only activates carboxy-groups and mediates the linkage with superficial primary amino groups without the introduction of any spacer molecules, it can be classed among the so-called zero-length crosslinking agents (p. 10293).
Kabe, as discussed above, supplies the complementary porphyrin chemistry: succinated PdTCPP was then conjugated to amino-modified SG beads (Fig. 3A, p. 31732).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further modify the compensation bead of claim 1 by employing Covolan’s aminostyrene/styrene polymer architecture, in which reactive amino functionality is provided by aminostyrene units distributed at the microsphere surface, and directly coupling a carboxyl group of the carboxyaryl porphyrin to that surface amino functionality according to Kabe’s demonstrated carboxyaryl-porphyrin/amine coupling and the zero-length coupling principle disclosed by Kazenwadel. A skilled artisan would have had an affirmative reason to select Covolan’s polymer-integrated surface-amine architecture because Covolan expressly identifies graft-based surface-functionalization procedures as complex, time-consuming, and potentially morphology-altering, while its copolymerized aminated latex provides accessible surface amino functionality through a more convenient architecture. One of ordinary skill would have had a reasonable expectation of success because Covolan experimentally confirms free amino groups attributable to aminostyrene units at the microsphere surface, Kabe experimentally conjugates carboxyaryl porphyrin to amino-modified polymer beads, and Kazenwadel establishes that carboxyl/primary-amine coupling can be effected without introducing spacer atoms. The resulting predictable structure places the amide directly between the porphyrin carboxyl functionality and the polymer-associated amino functionality, thereby providing the claimed absence of an intervening linker.
Regarding claims 7 and 8, the combination applied to claim 1 teaches the underlying compensation bead and Kabe teaches covalent conjugation of carboxyaryl porphyrin to amino-modified polymer beads. The combination, however, does not expressly teach or specify no linker covalently connecting a carboxyaryl porphyrin carboxamide and the surface of the polymer bead.
Covolan teaches aminated polystyrene microspheres having free amino groups on the particle surfaces, with the surface nitrogen attributable to amino groups of the aminate styrene units (p. 6691). Thus, the reactive amino functionality is furnished by the aminostyrene units of the polymer architecture itself rather than by the separately grafted SGNEGDEN-type spacer architecture used in Kabe. Kazenwadel teaches the relevant direct-coupling principle: zero-length crosslinkers do not introduce spacer atoms while connecting molecules, and EDC mediates linkage of carboxyl groups with superficial primary amino groups without the introduction of any spacer molecules (pp. 10291, 10293). Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further modify the bead of claim 1 by employing Covolan’s polymer-integrated surface-amino architecture and forming Kabe’s carboxyaryl-porphyrin carboxamide directly with that surface amino functionality using the zero-length coupling approach disclosed by Kazenwadel, thereby avoiding an intervening covalent linker between the porphyrin carboxamide and the aminated polymer surface. The skilled artisan would have had reason to make this modification because Covolan expressly identifies conventional graft-based functionalization as complex, time-consuming, and potentially morphology-altering, whereas its aminostyrene/styrene copolymer provides reactive amino functionality as part of a convenient aminated latex architecture. A reasonable expectation of success existed because Covolan experimentally confirms free polymer-associated amino groups at the microsphere surface, Kabe demonstrates successful carboxyaryl-porphyrin coupling to amino-modified polymer beads, and Kazenwadel establishes that zero-length carboxyl/amine coupling introduces no spacer atoms. The modification therefore predictably provides the claimed carboxamide attachment without an intervening linker.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Pitner et al., Kabe et al., Castriciano et al., as evidenced by Ohtsu et al., as applied to claim 1 above, and further in view of Schmutz et al. (Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry. Journal of Visualized Experiments. No. 123, May 2017) and Gibco (HBSS, no calcium, no magnesium, no phenol red. Catolog number: 14175095. October 2017. HBSS, no calcium, no magnesium, no phenol red - Thermo Fisher Scientific).
With respect to the teachings of Pitner et al., Kabe et al., Castriciano et al., see the discussion above, which applies equally here. These references differ from the instant claims in failing to expressly teach or specify suspending the bead in DPBS, HBSS, or a combination thereof at a pH of about 6.5–7.5.
Regarding the media being selected from DPBS, HBSS, or a combination thereof, Schmutz teaches preparation of single-stained commercial compensation bead microspheres, expressly referred to as beads, for spectral flow cytometry (Protocol §2, p. 3). Schmutz instructs adding 2 mL of Hank's Balanced Salted Solution (HBSS) 1% FCS, centrifuging, discarding the supernatant, and resuspending the pellet in 100 µL of HBSS 1% FCS before the beads are acquired by the spectral flow cytometer (Protocol §§2–4, pp. 3–4). Thus, Schmutz directly teaches HBSS as a suspension medium for compensation beads used for flow-cytometric spectral measurements. Schmutz, however, does not expressly teach or specify that the HBSS used to suspend the compensation beads has a pH of about 6.5-7.5.
Gibco teaches a commercially available HBSS, no calcium, no magnesium, no phenol red, catalog no. 14175095. The archived product page expressly identifies the product as Hanks’ Balanced Salt Solution (HBSS) and states that Gibco offers a variety of HBSS formulations. Gibco further specifies that this HBSS has a pH Range: 6.7 - 7.8 (Specifications, p. 2), which overlaps the claimed range of about 6.5–7.5.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further modify the compensation bead system of claim 1 by suspending the compensation beads in HBSS as taught by Schmutz and, in implementing Schmutz’s expressly taught HBSS suspension medium, to select the commercially available Gibco HBSS formulation having a disclosed pH range of 6.7–7.8 that overlaps the claimed range of about 6.5–7.5. A skilled artisan would have had reason to make this modification because Schmutz specifically teaches HBSS as the medium for washing and resuspending compensation beads immediately before flow-cytometric acquisition, while Gibco provides a commercially available HBSS formulation suitable for implementing that expressly taught medium and having a specified pH range overlapping the claimed range. One of ordinary skill would have had a reasonable expectation of success because Schmutz actually prepares and acquires compensation beads following resuspension in HBSS, and Gibco expressly identifies its product as HBSS and specifies a pH range of 6.7–7.8. Thus, selecting the known Gibco HBSS formulation, whose disclosed pH range overlaps the claimed range, to implement Schmutz’s expressly taught HBSS suspension medium would predictably provide an HBSS suspension medium encompassing pH values within the claimed range of about 6.5–7.5.
Claims 9-12, 14, 17, 18, and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Pitner et al., Kabe et al., Castriciano et al., as evidenced by Ohtsu et al., as applied to claim 1 above, and further in view of Scanone et al. (Photodynamic Properties and Photoinactivation of Microorganisms Mediated by 5,10,15,20-Tetrakis(4-Carboxyphenyl)Porphyrin Covalently Linked to Silica-Coated Magnetite Nanoparticles. Journal of Photochemistry and Photobiology. A, Chemistry. Vol. 346, September 2017)
Regarding claim 9, with respect to the teachings of Pitner et al., Kabe et al., Castriciano et al., see the discussion above, which applies equally here. The combination therefore teaches the underlying compensation bead, an amino-functionalized polymer bead, a carboxyaryl porphyrin, activation of the carboxyl-bearing porphyrin, and subsequent conjugation to the amino-functionalized bead. However, the combination does not as expressly set forth the claimed process sequence of reacting a preactivated carboxyaryl porphyrin with the surface amine and then separating unreacted carboxyaryl porphyrin and collecting the carboxyaryl porphyrin carboxamide functionalized bead for use as the compensation bead.
Scanone directly supplies that process. Scanone teaches TCPP, i.e., 5,10,15,20-Tetrakis(4-carboxyphenyl)porphyrin (TCPP), and states that TCPP was covalently bound to amino-functionalized nanoparticles via carbodiimide activation (Abstract, p. 452). Scanone further states that TCPP was attached through amide bonds (Introduction, p. 453). More particularly, Scanone prepares amino-functionalized particles and then teaches: a solution of N-hydroxysuccinimide (NHS, 20 μmol in 1.5 mL PBS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 20 μmol in 1.5 mL PBS) was added dropwise to the solution of TCPP (5 μmol) in 1 mL N,N-dimethylformamide (DMF). The mixture was stirred for 2 h at 25 °C to activate TCPP. After that, 15 mg MNPSiNH2 was added to the solution of TCPP/EDC/NHS (Materials and Methods §2.2, p. 454). Scanone then teaches the claimed separation and recovery sequence: nanoparticles were washed several times with ethanol and water to eliminate the excess of TCPP followed by MNPSiNH-TCPP were resuspended in 5 mL water obtaining a stock suspension of ~3 mg MNPSiNH-TCPP/mL (Materials and Methods §2.2, p. 454). Thus, Scanone expressly separates unreacted/excess TCPP from the TCPP-functionalized particles and thereafter collects/resuspends the functionalized particles.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method for preparing the compensation bead taught by the Pitner/Kabe/Castriciano combination by employing Scanone’s demonstrated preactivation and post-reaction purification sequence, namely activating the carboxyl groups of TCPP with EDC/NHS, reacting the activated TCPP with the amino-functionalized particulate surface, and thereafter washing away excess unreacted TCPP and recovering the resulting TCPP-functionalized particles. The skilled artisan would have had reason to make this modification because Kabe already relies on activated carboxyaryl porphyrin for conjugation to amino-modified polymer beads, while Scanone provides a complete experimentally demonstrated process for implementing that same carboxyaryl-porphyrin/amine coupling and removing excess porphyrin after conjugation, thereby furnishing a purified porphyrin-functionalized particulate product suitable for the intended optical use. One of ordinary skill would have had a reasonable expectation of success because Scanone actually activates TCPP, reacts it with an amino-functionalized particulate surface, washes away excess TCPP, and recovers the TCPP-functionalized particles, while Kabe independently demonstrates successful TCPP/PdTCPP conjugation to amino-modified polymer beads. The proposed modification therefore applies Scanone’s known TCPP/amine-particle conjugation and purification technique to the analogous carboxyaryl-porphyrin/amino-bead system of the combination to obtain the predictable purified compensation bead.
Regarding claim 10, the combination applied to claim 9 teaches reacting activated TCPP in solution with amino-functionalized particles and subsequently removing excess TCPP. Regarding carboxyaryl porphyrin in a solution of the reacting step, Scanone expressly teaches adding the amino-functionalized particles to the solution of TCPP/EDC/NHS and allowing the mixture to react for 24 hours (Materials and Methods §2.2, p. 454). Following the reaction, Scanone teaches that the particles were washed several times with ethanol and water to eliminate the excess of TCPP (Materials and Methods §2.2, p. 454). The excess TCPP being removed is therefore unreacted TCPP remaining in the reaction mixture. Scanone’s express removal of excess TCPP remaining from the TCPP-containing reaction solution satisfies the first recited alternative. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of claim 9 such that unreacted carboxyaryl porphyrin remaining in the reaction solution is separated from the functionalized beads, as taught by Scanone, because removal of excess unreacted TCPP provides the desired TCPP-functionalized particulate product free from residual unbound porphyrin. One of ordinary skill in the art would have had a reasonable expectation of success because Scanone actually reacts TCPP with amino-functionalized particles and thereafter washed several times with ethanol and water to eliminate the excess of TCPP, while retaining and resuspending the resulting TCPP-functionalized particles
Regarding claim 11, the references applied to claim 9 already teach the alternative limitation. Regarding a covalent bond, Kabe teaches succinated PdTCPP was then conjugated to amino-modified SG beads and depicts the carboxamide linkage in Fig. 3A (p. 31732). Scanone independently teaches that TCPP was covalently bound to amino-functionalized particles and states that the attachment occurs through amide bonds (Abstract, p. 452; Introduction, p. 453). Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of claim 9 such that the amine group of the polymer bead surface is bonded to the carboxyaryl porphyrin by a covalent bond, as taught by Kabe and Scanone, because covalent attachment through the porphyrin carboxyl functionality and surface amino functionality provides stable immobilization of the porphyrin on the particulate surface. One of ordinary skill in the art would have had a reasonable expectation of success because Kabe demonstrates conjugation of activated PdTCPP to amino-modified polymer beads, and Scanone independently demonstrates that TCPP was covalently bound to amino-functionalized particles through amide bonds, thereby establishing that the claimed carboxyaryl-porphyrin/amine coupling predictably produces the recited covalent bond.
Regarding claim 12, the claim 9 combination already includes Scanone, which directly teaches this limitation. As discussed above, Scanone expressly teaches preactivating TCPP with EDC/NHS in a DMF-containing reaction medium before introducing the amino-functionalized particulate substrate (Materials and Methods §2.2, p. 454). Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to employ Scanone’s EDC/NHS preactivation procedure in the method of claim 9 by incubating TCPP with EDC and NHS in a DMF-containing medium before contacting the activated TCPP with the amino-functionalized bead. The skilled artisan would have had reason to employ this sequence because the purpose of the activation step is to render the TCPP carboxyl functionality reactive toward the surface amino functionality before the coupling reaction, thereby implementing the carboxamide attachment required by the underlying method. One of ordinary skill would have had a reasonable expectation of success because Scanone actually performs this sequence with TCPP and an amino-functionalized particulate surface and obtains covalently TCPP-functionalized particles, while Kabe independently demonstrates activated TCPP/PdTCPP conjugation to amino-modified polymer beads. The modification therefore predictably applies an experimentally successful TCPP activation/coupling procedure to the analogous carboxyaryl-porphyrin/amino-bead reaction.
Regarding claim 14, for EDC and carboxyaryl porphyrin present at a mol/mol ratio of between about 0.35 to about 25.91, Scanone uses EDC, 20 μmol with TCPP (5 μmol) (Materials and Methods §2.2, p. 454), thereby providing an EDC:TCPP molar ratio of: 20 μmol EDC : 5 μmol TCPP = 4:1. The disclosed ratio of 4:1 falls squarely within the claimed approximately 0.35–25.91 range. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to employ Scanone’s experimentally demonstrated EDC:TCPP molar ratio of 4:1 in the TCPP activation step of the claim-9 method because Scanone uses that ratio specifically to activate the carboxyl-bearing TCPP for subsequent covalent attachment to an amino-functionalized particulate surface—the same chemical transformation required by the modified claim-9 process. One of ordinary skill would have had a reasonable expectation of success because Scanone actually uses the 4:1 EDC:TCPP ratio, activates TCPP, couples the activated TCPP to amino-functionalized particles, and obtains the covalently functionalized particulate product. Thus, selecting Scanone’s demonstrated ratio within the claimed range would predictably provide effective carboxyl activation for the analogous TCPP/amine-bead coupling.
Regarding claim 17, for the composition of the bead, the combination applied to claim 9 already teaches this limitation. Pitner expressly teaches a polystyrene bead as the polymeric matrix and experimentally employs polystyrene beads in its flow-cytometric porphyrinic-bead embodiments.
Regarding claim 18, for the diameter of the compensation bead, the combination applied to claim 9 already teaches the claimed range. Pitner experimentally uses 5.43 μm polystyrene beads for its porphyrinic flow-cytometric bead compositions. The disclosed 5.43 μm diameter falls squarely within the claimed approximately 5–20 μm range.
Regarding claim 20, the combination applied to claim 9 teaches the additional spectral limitation of claim 20. Regarding excitation of about 400 nm, Pitner teaches flow-cytometric excitation of its porphyrinic beads using approximately 405 nm excitation. Regarding emission of between about 600 nm to about 800 nm, Pitner teaches porphyrinic bead fluorescence within approximately 590–750 nm, which substantially overlaps and includes wavelengths within the claimed 600–800 nm interval.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Pitner et al., Kabe et al., Castriciano et al., Scanone et al., as evidenced by Ohtsu et al., as applied to claims 1, 9, and 12 above, and further in view of Kuznetsova et al. (Water-Soluble Cyanine Dyes for Biological Microchip Technology. Russian Chemical Bulletin. Vo. 56, No. 12, December 2007).
With respect to the teachings of Pitner et al., Kabe et al., Castriciano et al., Scanone et al., see the discussion above, which applies equally here. However, the combination does not expressly teach or specify the specific intermediate workup of combining the activated ester solution with water, filtering it, and using the resulting activated-ester-containing filtrate before the amine-coupling reaction.
Kuznetsova teaches precisely this process topology for an NHS-activated fluorescent carboxyl compound. Kuznetsova teaches that succinimide esters 9a–h were obtained in quantitative yields under the action of N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide in anhydrous DMF and subsequently uses those activated esters to label an amino-containing oligonucleotide (p. 2439). More particularly, Kuznetsova teaches N-Hydroxysuccinimide (69 mg, 0.6 mmol) and a solution of dicyclohexylcarbodiimide (62 mg, 0.3 mmol) in anhydrous DMF (500 µL) were added to a solution of the carboxyl-containing fluorescent dye in anhydrous DMF (11 mL); after activation, the mixture was stirred at ~20 °C for 5 days, diluted with water, and filtered. The filtrate was put on the top of an RP-18 column (Experimental, p. 2442). Kuznetsova thereafter demonstrates that the resulting succinimide ester remains useful for subsequent reaction with an amino-containing substrate: the succinimide ester of dye 9a–h (~0.6 mg) was added to the amino-containing oligonucleotide mixture and allowed to react (Experimental, p. 2442). Thus, the reference demonstrates that an activated fluorescent carboxyl compound can undergo the claimed water-addition/filtration workup and thereafter remain competent for amine conjugation.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further modify Scanone’s preactivated TCPP/DMF procedure by applying Kuznetsova’s demonstrated aqueous filtration workup to the activated fluorescent carboxyl compound before its subsequent reaction with the amino-functionalized bead, namely by adding water to the activated-ester-containing DMF solution, filtering the resulting mixture, and retaining the activated-esters-containing filtrate for the subsequent amine-coupling step. A skilled artisan would have had reason to make this modification because Kuznetsova demonstrates the filtration as an intermediate workup of an NHS-activated fluorescent carboxyl compound prepared by carbodiimide chemistry in DMF while preserving the activated ester for subsequent amine labeling, thereby providing a practical purification/workup step before conjugation rather than altering the underlying coupling chemistry. One of ordinary skill would have had a reasonable expectation of success because Kuznetsova actually subjects a carbodiimide/NHS-activated fluorescent carboxyl compound in DMF to water addition and filtration and thereafter successfully employs the resulting succinimide ester for reaction with an amino-containing substrate, while Scanone independently demonstrates that activated TCPP prepared by EDC/NHS chemistry in DMF reacts successfully with an amino-functionalized particulate surface. The proposed modification therefore predictably applies a demonstrated pre-conjugation workup for an activated fluorescent carboxyl compound to the analogous activated TCPP coupling process.
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Pitner et al., Kabe et al., Castriciano et al., Scanone et al., as evidenced by Ohtsu et al., as applied to claims 1 and 9 above, and further in view of Kazenwadel.
With respect to the teachings of Pitner et al., Kabe et al., Castriciano et al., Scanone et al., see the discussion above, which applies equally here. The claim-9 combination already teaches several of the recited solvent/buffer components. Scanone teaches PBS solution of 10 mM pH = 7.0 (Materials and Methods §2.1, p. 454), uses TCPP (5 μmol) in 1 mL N,N-dimethylformamide (DMF) during activation, adds aqueous EDC/NHS solutions thereto, and subsequently introduces the amino-functionalized particles into that reaction solution (Materials and Methods §2.2, pp. 454). Scanone therefore teaches a DMF/aqueous reaction environment and independently identifies pH 7.0 PBS.
Howver, to the extent the claim is interpreted to require the reacting solution itself to be controlled within approximately pH 6–7 while comprising at least one of the recited solution components.
Kazenwadel provides the additional teaching regarding the pH conditions suitable for EDC-mediated carboxyl/primary-amine coupling. Kazenwadel explains that EDC is one of the most commonly used crosslinking molecules, that it only activates carboxy-groups and mediates the linkage with superficial primary amino groups without the introduction of any spacer molecules, and that a neutral pH value around 7.0 during the immobilization process is required according to the standard protocols (p. 10293). Kazenwadel further experimentally employs 0.01 M MES-buffer at pH 5.3 and 0.01 M sodium phosphate buffer at pH 6.0 and 7.0 in its coupling studies (Materials and Methods, p. 10294). Thus, Kazenwadel directly demonstrates EDC-mediated coupling chemistry using buffered aqueous media and expressly evaluates sodium phosphate-buffered coupling conditions at pH 6.0 and 7.0, both falling within the claimed approximately 6–7 interval. Kazenwadel also explains why the reaction environment matters: the pH-value influences enzyme binding efficiency and reports that adjustment of coupling pH significantly changes binding performance (p. 10295).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to conduct the Scanone-modified carboxyaryl-porphyrin/amine coupling in a reaction medium containing the recited DMF/aqueous components and to maintain the coupling environment at approximately pH 6–7 using the buffer conditions in view of Kazenwadel’s teaching that approximately neutral pH conditions are appropriate for EDC-mediated carboxyl/primary-amine coupling. A skilled artisan would have had a specific reason to make this modification because Kazenwadel teaches that pH is a relevant reaction parameter for EDC-mediated carboxyl/primary-amine coupling and expressly identifies neutral pH around 7.0 in standard protocols, while Scanone performs the same fundamental carboxyl-bearing TCPP/amine-surface conjugation in DMF together with aqueous PBS-containing EDC/NHS solutions. Maintaining the reaction environment in the approximately pH 6–7 region would therefore provide conditions known to support the intended EDC-mediated carboxyl-to-primary-amine coupling rather than being an arbitrary selection of a known buffer. One of ordinary skill would have had a reasonable expectation of success because Scanone actually obtains covalently TCPP-functionalized amino particles using EDC/NHS in the disclosed DMF/aqueous system, and Kazenwadel experimentally demonstrates EDC coupling in MES/phosphate-buffered media including pH 6.0 and 7.0. The modification would therefore predictably preserve the same carboxyl/amine coupling chemistry while employing a known operative reaction environment.
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Pitner et al., Kabe et al., Catriciano et al., Scanone et al., as evidenced by Ohtsu et al., as applied to claims 1 and 9 above, and further in view of Purvis et al. (US 2010/0285594 A1).
With respect to the teachings of Pitner et al., Kabe et al., Catriciano et al., Scanone et al., see the discussion above, which applies equally here. However, the combination does not expressly teach or specify that the compensation beads have a median fluorescence intensity of between about 140 to about 1500.
Purvis teaches fluorescent microbeads for fluorescence-detector calibration and expressly teaches that fluorescence intensity of a population of particles collected as single-particle data may be summarized into a single numerical value by calculating the mean, median, geometric mean, etc., and that median fluorescence intensity (MFI) is a preferred statistic because it is less influenced by extreme outliers than mean fluorescence intensity ([0067], p. 9). Purvis further teaches determining the MFI of fluorescent-labeled polymer microbead populations and using microbead populations within the linear range of the instrument for fluorescence calibration ([0067], p.12). Regarding the claimed fluorescence-intensity range, Purvis expressly measures the raw observed MFI (FL1|FL2|FL3) of each microbead population in the kit and reports, inter alia, an MFI of 1,255 for Population 3, an MFI of 500 for Population 6, and an MFI of 500 for Population 7 and Population 8, each falling within the claimed range of about 140 to about 1500 ([0090]–[0098], p. 11). Purvis further teaches that Population 6 comprises surface-bound FITC microbeads and uses the measured FL1 MFI of 495 after background correction for fluorescence calibration ([0111], p. 11).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the flow-cytometric use of the compensation beads prepared according to the Pitner/Kabe/Castriciano/Scanone combination such that the fluorescent bead population has an MFI within the claimed range, such as an MFI of about 500, as taught by Purvis. A skilled artisan would have had reason to make this modification because Purvis teaches selecting fluorescent microbead populations having median fluorescence intensities within the linear detection range of the fluorescence detector to be calibrated, thereby providing bead fluorescence suitable for reliable fluorescence-detector calibration. One of ordinary skill would have had a reasonable expectation of success because Purvis actually demonstrates flow-cytometric measurement of fluorescent microbead populations, including surface-labeled fluorescent microbeads, having measured MFI values within the claimed range. Accordingly, applying Purvis’s known fluorescence-intensity selection to the flow-cytometric compensation beads of the combination would predictably provide compensation beads having an MFI between about 140 and about 1500.
Claim 21 is rejected under 35 U.S.C. 103 as being unpatentable over Pitner et al., Kabe et al., Catriciano et al., Scanone et al., as evidenced by Ohtsu et al., as applied to claims 1 and 9 above, and further in view of Covolan et al. and Kazenwadel et al.
With respect to the teachings of Pitner et al., Kabe et al., Catriciano et al., Scanone et al., see the discussion above, which applies equally here. However, the combination does not expressly teach or specify that there is no linker on the polymer bead surface.
As discussed above, Covolan provides a polymer-integrated surface-amino architecture particularly suited to the claimed absence of a surface linker. Kazenwadel further confirms that direct carboxyl/amine coupling can be implemented without introducing a spacer. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the amino-functionalized polymer-bead architecture used in the Pitner/Kabe/Castriano/Scanone method by employing Covolan’s p-aminostyrene/styrene polymer bead, in which the surface amino functionality is supplied by aminostyrene units incorporated into the polymer itself, and to employ Kazenwadel’s zero-length EDC coupling principle when coupling the carboxyaryl porphyrin to those surface amino groups. A skilled artisan would have had a specific reason to make this modification because Covolan identifies post-polymerization grafting approaches as complex, time-consuming, and potentially morphology-altering and instead provides polymer microspheres having free surface amino groups attributable to aminostyrene units, while Kazenwadel teaches that EDC mediates carboxyl-to-superficial-primary-amine linkage without introducing spacer molecules. The modification would therefore simplify the surface architecture and avoid an additional surface spacer while retaining the amino functionality required for porphyrin attachment. One of ordinary skill would have had a reasonable expectation of success because Covolan experimentally establishes accessible free amino groups on the polymer-particle surface, Kabe/Scanone demonstrate successful carboxyaryl-porphyrin coupling to surface amino functionality, and Kazenwadel establishes that EDC-mediated carboxyl/primary-amine coupling can proceed as zero-length coupling without introduction of a spacer molecule.
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Pitner et al., Kabe et al., Catriciano et al., Scanone et al., as evidenced by Ohtsu et al., as applied to claims 1 and 9 above, and further in view of Wang et al. (Bead mediated separation of microparticles in droplets. PLoS ONE. Vol. 12, No. 3, March 2017).
With respect to the teachings of Pitner et al., Kabe et al., Catriciano et al., Scanone et al., see the discussion above, which applies equally here. However, the combination does not expressly teach or specify performing that separation through a filter of about 5 μm porosity and thereafter removing the retained functionalized beads from that filter.
Wang supplies the precise physical separation operation. Regarding filtering through a filter of about 5 μm, Wang teaches surface-functionalized polystyrene beads and expressly states: beads are separated from non-bound particles by a 5 μm filter (Materials and Methods, p. 3). Regarding removing the functionalized beads from the filter, Wang immediately teaches: the beads are washed off the filter and resuspended in PBS (Materials and Methods, p. 3). Wang additionally employs 14–17.9 μm diameter polystyrene beads functionalized with streptavidin in the bead experiments (p. 2), demonstrating application of the separation approach to surface-functionalized polymer beads whose size is greater than the filter dimension. Wang reports that beads larger than 10 μm were captured with 100% efficiency in its size-based capture experiments, further supporting the predictability of retaining larger functionalized beads while smaller material passes through.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Scanone’s separation of excess TCPP from the functionalized particulate product, as incorporated into the claim-9 method, by selecting compensation beads within Wang’s disclosed 14–17.9 μm diameter range, which also falls within the approximately 3–18 μm range required by claim 1, and employing Wang’s demonstrated size-based separation technique in which the mixture is passed through a 5 μm filter such that the larger functionalized beads are retained on the filter, followed by washing the retained beads from the filter and recovering them. A skilled artisan would have had reason to make this modification because the claim-9 process requires separation of unreacted soluble/excess porphyrin from the functionalized compensation beads, and Wang demonstrates that a 5 μm filter provides a direct physical means for retaining substantially larger surface-functionalized polymer beads while allowing smaller non-bound material to be separated, followed by straightforward recovery of the retained beads from the filter. Applying that demonstrated bead-retention and recovery operation to Scanone’s purification process would provide a physical means for retaining the functionalized compensation beads while separating the unreacted/excess TCPP-containing liquid phase. One of ordinary skill would have had a reasonable expectation of success because the underlying Pitner bead system permits bead diameters within the claimed approximately 3–18 μm range, Scanone demonstrates that excess TCPP can be removed after porphyrin functionalization, and Wang actually employs 14–17.9 μm surface-functionalized polystyrene beads, which fall within the claimed approximately 3–18 μm range, and demonstrates separation of surface-functionalized polymer beads from non-bound material using a 5 μm filter followed by washing the retained beads from that filter. Wang further reports 100% capture for beads larger than 10 μm, supporting predictable retention of the selected larger functionalized beads by the 5 μm filter. The proposed modification therefore substitutes Wang’s demonstrated size-based bead-retention and operation for Scanone’s washing-based separation to accomplish the same purification objective while providing the claimed physical separation and bead-recovery steps.
Claim 23 is rejected under 35 U.S.C. 103 as being unpatentable over Pitner et al., Kabe et al., Catriciano et al., as evidenced by Ohtsu et al., as applied to claim 1 above, and further in view of Scanone et al. and Kazenwadel et al.
With respect to the teachings of Pitner et al., Kabe et al., Catriciano et al., see the discussion above, which applies equally here. However, the combination does not expressly teach or specify the particular one-step EDC-containing preparation recited in claim 23.
Regarding reacting an amine present on a polymer bead surface that forms an amine functionalized bead with a carboxyaryl porphyrin, as discussed above, Kabe teaches amino-modified polymer beads and TCPP/PdTCPP conjugation through carboxyl/amine chemistry. Scanone independently teaches amino-functionalized particles and TCPP covalently attached through amide formation. Regarding with a carboxyaryl porphyrin and EDC, Scanone expressly uses 5,10,15,20-Tetrakis(4-carboxyphenyl)porphyrin (TCPP) and adds 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 20 μmol) to TCPP (5 μmol) before introducing the amino-functionalized particles (Materials and Methods §2.2, p. 454). Regarding a solution containing DMF, Scanone expressly teaches TCPP (5 μmol) in 1 mL N,N-dimethylformamide (DMF) (Materials and Methods §2.2, p. 454). Regarding water, Scanone’s EDC/NHS solutions are aqueous and its TCPP-functionalized particles are subsequently washed with water and resuspended in water. Thus, the process employs both DMF and aqueous components. Regarding the alternatively recited buffered conditions, Kazenwadel teaches EDC-mediated carboxyl/primary-amine coupling and explains that a neutral pH value around 7.0 during the immobilization process is required according to the standard protocols (p. 10293). Kazenwadel experimentally evaluates 0.01 M MES-buffer pH 5.3 and 0.01 M sodium phosphate buffer, pH 6.0 and 7.0 in the coupling process. The reference therefore corroborates the operability of approximately pH 6–7 aqueous conditions for EDC-mediated carboxyl/primary-amine coupling.
Regarding producing a carboxyaryl porphyrin carboxamide functionalized bead, Scanone teaches that TCPP is covalently bound to amino-functionalized particles through amide bonds and experimentally determines amino groups bonded to TCPP. Regarding separating an unreacted carboxyaryl porphyrin from the amine functionalized bead bonded with carboxyaryl porphyrin, Scanone teaches that the particles were washed several times with ethanol and water to eliminate the excess of TCPP (Materials and Methods §2.2, p. 454). Regarding collecting the carboxyaryl porphyrin carboxamide functionalized bead for use as the compensation bead, Scanone thereafter teaches that the TCPP-functionalized particles were resuspended in 5 mL water obtaining a stock suspension (Materials and Methods §2.2, p. 454), thereby recovering/collecting the purified TCPP-functionalized particulate product.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of preparing the Pitner/Kabe/Castriano compensation bead by employing Scanone’s experimentally demonstrated EDC-mediated TCPP coupling procedure—contacting carboxyl-bearing TCPP in a DMF/aqueous reaction system with an amino-functionalized particulate surface, forming the covalent carboxamide-functionalized product, removing excess TCPP, and recovering the functionalized particles—and, where an approximately neutral aqueous coupling environment is employed, to use the approximately pH 6–7 coupling conditions taught by Kazenwadel. A skilled artisan would have had reason to make these modifications because the underlying combination requires attachment of a carboxyaryl porphyrin to surface amino functionality while retaining a purified particulate product, and Scanone supplies a complete experimentally successful process directed to that same TCPP/amine-surface chemical transformation, while Kazenwadel identifies the reaction environment relevant to efficient EDC-mediated carboxyl/primary-amine coupling. One of ordinary skill would have had a reasonable expectation of success because Kabe independently demonstrates carboxyaryl-porphyrin conjugation to amino-modified polymer beads, Scanone actually performs EDC-mediated TCPP coupling to amino-functionalized particles followed by removal of excess TCPP and recovery of the product, and Kazenwadel demonstrates EDC-mediated carboxyl/primary-amine coupling under approximately neutral aqueous conditions. Applying those demonstrated process conditions to the claim-1 compensation bead would therefore predictably produce the claimed purified carboxyaryl-porphyrin carboxamide-functionalized compensation bead.
Conclusion
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/E.O./Examiner, Art Unit 1677
/BAO-THUY L NGUYEN/Supervisory Patent Examiner, Art Unit 1677 September 23, 2026
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