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 .
Election/Restrictions
Applicant’s election without traverse of Group I, claims 141-158 in the reply filed on 3/26/2026 is acknowledged. Accordingly, the claims 158-161 are withdrawn from consideration.
Priority
This application is a 371 of PCT/EP2022/056361 filed 03/11/2022.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 11/8/2023 Is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Specification
5. The disclosure is objected to because of the following informalities:
(a) The use of the terms “Irgacure 651, Irgacure 184, Irgacure 907, Irgacure 2959”, “SYBR Green, SYBR Gold, Syto 9, Syto 80, SYBR 14, Syto 41, Syto 60, Syto 62, Syto 64, DAPI, Hoechst, ToPro3, Draq5, Draq7, RedDotl, RedDot2, Evagreen, FITC, FAM, PE, APC, Cy3, Cy5, Cy7, PerCP, AF488, AF647, AF555, CF488 CF555, CF647, Atto 488, Atto 565, Brilliant violet”, Triton X-100, Tween-20, NP-40 which are a trade names or a marks used in commerce, has been noted throughput in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Rejections - 35 USC § 112
6. 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.
7. Claims 141-158 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.
(a) Claims 141-158 are indefinite and appear to lack proper antecedent basis in the claim 141 in the step c. at the recitation of “whereby one or more reaction products are produced as a result of the nucleic acid amplification reaction“ because no actual step of performing a nucleic acid amplification reaction is recited. Rather the claims merely recited mixing the sample with polymer hydrogel reagent and nucleic acid amplification reagents being capable of generating a reaction product. Being capable of doing something does not necessarily result in performing the activity. Likewise, incubating the sample as recited in the step c. does not necessarily equate to performing a nucleic acid amplification reaction. Accordingly, the claim language does provide proper antecedent basis such that one can draw the conclusion recited therein and accordingly the claim is indefinite.
(b) Claims 148 contains the trademark/trade name “Irgacure 651, Irgacure 184, Irgacure 907, Irgacure 2959”. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe dye and, accordingly, the identification/description is indefinite.
(c) Claim 153 contains the trademark/trade name “SYBR Green, SYBR Gold, Syto 9, Syto 80, SYBR 14, Syto 41, Syto 60, Syto 62, Syto 64, DAPI, Hoechst, ToPro3, Draq5, Draq7, RedDotl, RedDot2, Evagreen, FITC, FAM, PE, APC, Cy3, Cy5, Cy7, PerCP, AF488, AF647, AF555, CF488 CF555, CF647, Atto 488, Atto 565 and Brilliant violet”, . Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe dye and, accordingly, the identification/description is indefinite.
Claim Rejections - 35 USC § 103
8. 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.
9. 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.
10. This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
11. Claim(s) 141-158 is/are rejected under 35 U.S.C. 103 as being unpatentable over Di Carlo et al {Di Carlo, use interchangeably herein} (US 20190381497, December 19, 2019) in view of Pregibon et al {Pregibon, used interchangeably herein} (WO 22011156432, December 2011, cited as CA-2802049 -C on PTO-892).
The claims are directed to a method of detecting and/or analyzing a biological particle in a sample, said method comprising the steps of: a. mixing the sample with polymer hydrogel reagents and nucleic acid amplification reagents, the nucleic acid amplification reagents being capable of generating a reaction product in the vicinity of biological particles having a specific target, wherein the polymer hydrogel reagents comprise a photoinitiator; b. inducing curing of the mixture to a hydrogel using electromagnetic radiation; c. incubating the sample, whereby one or more reaction products are produced as a result of the nucleic acid amplification reaction; d. recording one or more images of the sample.
Regarding claim 141-158, Di Carlo teaches at paragraphs [0156] – [0157]: [0156] In an alternative embodiment, the PEG gels can be functionalized to allow immobilization of low-molecular-weight species by attachment to the gel matrix. For example, acrydite modification on the 5′ end of one of the primers in a polymerase chain reaction can be used to covalently link the amplified DNA to the hydrogel matrix. Alternatively, the primers can be linked to the hydrogel matrix using biotinylated primers that bind specifically to streptavidin immobilized within the hydrogel matrix. The amplified DNA linked to the matrix can then be assayed using intercalating dyes within the solution surrounded by an oil phase or once transferred to an aqueous phase. Other readout approaches that are sequence specific can also be incorporated, such as by hybridizing complementary fluorophore labeled nucleic acid probes to the immobilized and amplified nucleic acids. In other nucleic acid amplification reactions, e.g. loop-mediated isothermal amplification (LAMP) or rolling circle amplification, the nucleic acids produced are much longer and can be physically entrapped in the hydrogel matrix without the ability to leave, however, still allow exchange of other reagents and dyes (e.g., intercalator dyes such as EvaGreen® or SYBR® Green, or molecular beacons or other fluorophore labeled complementary sequences) upon transferring back to an aqueous external phase. Protein targets can be covalently linked to the gel matrix with the addition of a crosslinking agent and upon exposure to a crosslinking reaction. For example, N-(3-((4-benzoylphenyl) formamido)propyl) methacrylamide can be used along with photo-activation to covalently link proteins to the hydrogel matrix. A UV light source capable of providing 350-360 nm ˜1.8 J/cm.sup.2 of light can be used to link proteins on particle-drops 20. This process can be performed while particle-drops 20 are in an oil suspension to covalently link protein targets to the interior region 14 of the drop-carrier particle 12. The drop-carrier particles 12 can then be transferred back to an aqueous solution for further immune-labeling of protein biomarkers and analysis in aqueous solution.
[0157] An alternative embodiment to entrap molecular or cellular targets within a particle-drop 20 includes polymerizing a pre-polymer solution that acts as the internal aqueous phase 18 of the particle drop 20 following capture of cells, molecules, or other products of amplification reactions within the particle-drop 20. The internal polymerization reaction also covalently links the target molecules or amplification products into the hydrogel or physically entraps cells or larger molecules within the hydrogel. Polymerization and covalent linkage or entrapment can be initiated with exposure to light (UV, white light) with the appropriate photoinitiator (e.g., Irgacure, Eosin Y), exposure to heat, or exposure to a pH change. For example, the LAMP reaction can proceed within 4% 4-arm PEG vinylsulfone, PEG dithiol precursor. Following this polymerization process, the drop-carrier particles 12 can be exchanged into an aqueous solution for downstream reactions, labeling, and flow cytometric or other readout processes.
At paragraphs [0116] – [0117], Di Carlo teaches in another embodiment, the hydrophilic precursor may include PEGDA while the hydrophobic precursor may include 1,6-Hexanediol diacrylate (HDA). PEGDA and HDA are used with an ultraviolet crosslinked transparent thiolene-based optical adhesive, NOA89 available from Norland Products, Inc. which is also used as the photoinitiator.
[0117] In another embodiment, the hydrophilic precursor may include PEGDA while the hydrophobic precursor may include a mixture of HDA and lauryl acrylate with lauryl acrylate ranging from between 0 to 60% of the mixture on a volume basis. The photoinitiator (PI) used in this embodiment is 2-hydroxy-2-methylpropiophenone. (see also Figure 13).
With regards to the step of curing the mixtures to the hydrogel and time frame for curing as recited in the step c., Di Carlo teaches to simultaneously photo-crosslink the two polymer regions, the curing time for PPGDA and PEGDA is optimized to be within one (1) second by adding 1.32 and 2.60 percentage of PI respectively. The flow rates of PPGDA, PPGDA+photoinitiator, PEGDA+photoinitiator, and PEGDA are 1.6, 1.6, 0.4, and 0.4 mL/min respectively. In addition to the design of the polymer precursor cross-section, there are an infinite degree of freedom to design the second pattern exposed on top of the flow stream to determine the final 3D shape of the drop-carrier particles 12. The shape of the optical mask 64 for one demonstration is designed to be a rectangular slit with dimensions of 140 micrometers parallel to the flow direction and 600 micrometers perpendicular to the flow direction respectively. There are more than a hundred transparent apertures 65 (e.g., slits) designed to be in an array on a chrome mask 64. To accelerate the speed of photopolymerization of PPGDA and PEGDA, the power of UV light source 62, which is collimated by an adaptor, is designed to be ˜4 W/cm.sup.2 on the optical mask 64 (paragraph [0119]).
Di Carlo also teaches that magnetic particles may be contained in one or more of the polymer or prepolymer components that is flowed through the microfluidic device during the drop carrier particle formation process. The magnetic particles enable the drop-carrier particles to be manipulated by an externally applied magnetic field which could be a permanent magnet or an electromagnet (para. [0098]). This teaching support that the curing of the mixture may be induced by electromagnetic radiation.
With regards to the limitation of recording one or more images of the sample as recited in the step d., Di Carlo teaches reacted particle-drops can be pooled in a new aqueous solution, or read out using standard microscopy, cost-effective wide-field lensless imaging, or flow cytometry; leading to low-cost complete solutions that can democratize digital molecular and single-cell assays in all research labs, and galvanize the development of point-of-care digital diagnostics that will ultimately improve health. ([0006], [0092]). Di Carlo teaches that imaging of the particle-drops may include imaging the particle-drops with a fluorescence and/or bright-field microscope ([0108]. Di Carlo further teaches that the particle-drops can be passed through a microfluidic channel and analyzed and/or sorted using flow cytometry optical setups and sorting approaches known in the art. Particle-drops size can be exchanged back into an aqueous solution to pass through standard flow cytometer systems. [0132]). Particle-drops 20 may also be collected in wells of a microtiter plate. Fluorescent signal for nucleic acid amplification within particle-drops 20 can be accomplished using intercalator dyes or specific molecular affinity probes with quencher/fluorophore pairs. For protein recognition, an antibody-conjugated with an enzyme that turns over a fluorogenic or chromogenic substrate can be used. Other optical readout approaches known in the art for digital assays can be used in a similar manner for Particle-drops 20. In the event that drop-carrier particles 12 coalesce, small amounts of surfactant (e.g., 0.1-1% Pluronic) can be added following formation of the particle-drop emulsion 20 to further stabilize the separate aqueous compartments [0132].
At paragraph [0134], Di Carlo teaches that the sample may include cells, proteins, nucleic acids, and the like. Following rapid fractionation and exposure to the sample solution, solutions containing particle-drops 20 can be heated to perform amplification (e.g., nucleic acid amplification) and generate an optical signal (e.g., intercalator fluorescence). These signals can be read-out using low-cost Smartphone-based fluorescence imagers, traditional flow cytometers, or other microscope-based devices. At paragraph [0147], Di Carlo teaches Signal that accumulates in the particle drops can be read for fluorescent intensity using microscopy of other fluorescence imaging technique to count the number of particles-drops with intensity above a threshold that were considered positive which yields the target’s concentration in the sample . Di Carlo teaches that for example to capture the amplified signal on the drop-carrier particle 12 tyramide signal amplification or catalyzed reporter deposition (CARD) techniques can be used. For example, tyramide biotin (or tyramide Alexa Fluor® 488) can be covalently linked to neighboring tyrosine residues within peptides or proteins attached to the interior hydrophilic hydrogel matrix of the drop-carrier particle 12. This covalent linkage is catalyzed by the presence of horse radish peroxidase attached to the secondary antibody. Following reaction and high efficiency linkage to the drop-carrier particle 12 while emulsified to localize signal, these drop-carrier particles 12 can be transferred to an aqueous phase for readout by flow cytometry for example, or reacted with streptavidin-conjugated to fluorophore and then run through a flow cytometer. The amount of particles with signal above threshold can be counted by gating on the flow cytometer to determine the concentration in the sample. Multiplexing can be conducted of multiple biomolecules by simultaneously mixing particles with different barcoding schemes as discussed herein that include separate capture antibodies targeting the set of biomolecules of interest. This multiplexed assay can use fluorescent colors of the barcoded drop-carrier particles 12 or scatter signatures to distinguish different assays [0147]. Di Carlo teaches that the densities of the PEGDA is 1.12 g/cm3 , PPGDA is 1.01 g/cm3 [0161].
Di Carlo also provides advantages of the method. The reference teaches [0138] There are a number of applications for particle-drops 20 with unique benefits because a set of solid-phases is associated with fluid drops 18 of substantially uniform volume. Many of these applications are enabled by isolating single entities (e.g., single-molecules or single-cells). Single-cell secretion analysis is an area that significantly benefits from an associated solid-phase to capture secreted molecules from cells within the aqueous phase internal to the particle-drop 20 but prevent cross-talk between neighboring cells in neighboring particle-drops 20. For example, a secretion capture moiety may be present on the drop-carrier particle 12 with affinity which depends on the application. A secondary reporter molecule (e.g., antibody, aptamer, or enzyme) may also be present that binds to the secreted molecule at a different site. The secondary reporter molecule may have an attached fluorophore such that the accumulation of the secreted molecule and accumulation of the secondary reporter molecule leads to a local increase in fluorescence signal intensity which can be observed and quantified.
While Di Carlo teaches various aspects of the instant invention, Di Carlo does not expressly teach recording one or more images as after analysis. However, such techniques in combination with the teachings of Di Carlo could be inherent based on the devices use in the method as taught by Di Carlo, e.g., mobile-phone based fluorescence imaging device, cytometer or microscope-based devices [0134].
In similar embodiment to Di Carlo, Pregibon et al teach an improved method and system for detection and quantification of target analytes by scanning multifunctional particles using in expensive or portable detection systems such as standard flow cytometers ([0003]). Pregibon teaches that multiple events are recorded non-contemporaneously or contemporaneously. The reference teaches that in some embodiments the events are recorded using a flow cytometer ([0006]). Pregibon teaches that the recording step comprises recording temporal signals from the one or more interrogation regions. In some embodiments the temporal signal comprises fluorescent and or scatter signals (see paragraph [0016]. Pregibon et al teaches the use of polymer particles [0093], wherein the polymer particles may be formed by steps of chain polymerization. The amount and kind of radical initiator may comprise of phot-active imitator (e.g., UV or infrared), thermally-active initiator, or chemical initiator, or the amount of heat or light employed, my be used to control polymerization rate or modify molecular weights of resulting polymerase [0101] –[0102]. Pregibon et al teach that the particles may contain detectable moieties that generate fluorescent, luminescent and/or scatter signal [0105]. Pregibon also teaches that particles in some embodiments are or comprise of magnetic particles that is magnetically responsive particles [0109]. Pregibon et al teach that methods and composition taught there can be used to detect and/or quantify any target analytes which may comprise a protein, peptide, hormone, hapten, antigen, receptor, enzyme, nucleic acid, cells, tissues, viruses, bacteria, fungi, algae, and combinations recited therein [0160].
With regarding to recording events, Pregibon et al teach that wherein particles travel through a cytometer, it is excited with an illumination spot while detectors are used to monitor several parameters of illumination and emitted light. By setting a threshold on one of these parameters in a triggering channel, a user can define the instances that he cytometer software will record events. Events are typically reported with the height and area observed in each channel, along with the event width and a time-stamp of when the event occurred ([0175]).
It would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date to have modified the method of Di Carlo to encompass a recording event as taught by Pregibon. Such modification is within the ordinary artisan’s capabilities since both the methods of Di Carlo and Predgibon et al are of similar scope, using similar modes of operation such that such modifications would not negatively alter or modify the results of detecting and/or analyzing a biological particle in a sample as taught by both Di Carlo and Predgibon. Further the ordinary artisan would have been motivated to include a recording event as taught by Predgibon and implied by Di Carlo for the obvious advantage of data analysis and record-keeping. The combination of the cited prior art is prima facie obvious in the absence of secondary consideration.
Conclusion
12. No claims are allowed. Any inquiry concerning this communication or earlier communications from the examiner should be directed to CYNTHIA B WILDER whose telephone number is (571)272-0791. The examiner can normally be reached Flexible.
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/CYNTHIA B WILDER/Primary Examiner, Art Unit 1681
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