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 .
Information Disclosure Statement
The information disclosure statement (IDS) submitted on October 23rd, 2023 and February 12th, 2026 are 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.
Election/Restrictions
Election was made without traverse in the reply filed on February 12th, 2026.
Claim Status
Claims 8-14, 16-17, 20, 22-23, 27-31, 37-42, and 44-50 have been canceled. Claims 1-7, 15, 18-19, 21, 24-26, 32-36, and 43 are pending and discussed in this Office Action.
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 1-7, 15, 18-19, 21, 32-33, 36, and 43 are rejected under 35 U.S.C. 103 as being unpatentable over Vu et al. (“Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis” Nat Commun 13, 169 (2022). https://doi.org/10.1038/s41467-021-27798-0), in view of Katzlinger (US 10379046 B2; published August 13th, 2019).
Regarding instant claim 1, Vu teaches a method comprising contacting the biological sample with a plurality of detectably labeled probes for detecting multiple analytes, wherein each detectably labeled probe is configured to directly or indirectly bind to a different analyte or product thereof and comprises a detectable label (p. 1, right column, para 3; Fig. 1a-c).
Vu further teaches that the detectable labels comprise a plurality of fluorophores exhibiting distinct fluorescence emission lifetimes, wherein one detectable label has a first signal emission lifetime and a second detectable label has a second signal emission lifetime different from the first. (p. 3, left column, para 1; p. 1, right column, para 3; Fig. 3). The second signal emission lifetime is longer than the first signal emission lifetime (p. 4, left column, para 3).
Vu further teaches detecting signals associated with the detectable labels at one or more locations in the biological sample (Fig. 1; p.2, right column, para 2).
Vu further teaches generating a combinatorial fluorescent signature code assigned to each analyte target using a codebook, wherein each code corresponds to an analyte of the multiple analytes, thereby identifying the analyte at one or more locations in the biological sample (p. 4, right column, para 2; Fig, 1e-f).
Vu does not teach detecting signals associated with the detectable labels, or absence thereof, during a detection time interval t1 and a detection time interval t2 at one or more locations in the biological sample, wherein the onset of t2 is later than the onset of t1,wherein signals associated with the first detectable label are detectable during t1 and not during t2, and wherein signals associated with the second detectable label are detectable during t2, such that the presence or absence of signal at each shared fixed time window that generates a signal code sequence. Vu instead uses Fluorescence Lifetime Imaging and Microscopy (FLIM). FLIM is an imaging technique that leverages the intrinsic fluorescence lifetime properties of fluorescent dyes. It creates microscopic images by mapping based on the length of fluorescence lifetimes. Fluorescence lifetime refers to the time it takes for molecules (such as fluorescent dyes) to emit photons and return to their ground state after being excited by light. In the simplest terms, it is defined as the time it takes for fluorescence intensity to decrease to 1/e of its initial value after excitation.
Katzlinger, in the same field of endeavor of detecting multiple analytes in a biological sample using fluorescent labels with different emission lifetimes, teaches that a first fluorescent label, a transition metal chelates of ruthenium (Ru(II)) has a first fluorescence emission lifetime in a range of about 0.1 μs to 10 μs, and a second fluorescence emission lifetime in a range of about 100 μs to fluorescence about 1 ms, wherein the second fluorescence emission lifetime is at least 5 times longer than the first fluorescence emission lifetime (claim 1; claim 4-5; column 6, line 32-53).
Katzlinger further teaches that the larger lifetime difference enables temporal separation of the two label signals, specifically that Ru, whose half-life is ~1 μsec is detected with a shorter time integration window of 2 μsec during which its signal is present and then absent in subsequent longer windows; Eu, whose half-life is ~ 800 μsec is detected with longer integration window of 1000 μsec during which its signal persists and remains detectable (column 19, line 45-50). Katzlinger further teaches using a combination of spectral and temporal differences to separate signals from multiple labels with cross talk reduced to below 0.01% (column 19, line 42-45).
Katzlinger further teaches that TFF detection with a 50 μs delay significantly reduces background from autofluorescence (column 17, line 31-36). Katzlinger further teaches contacting the sample with a primary antibody that specifically binds the first analyte and a primary antibody that specifically binds the second analyte, followed by a first fluorescent antibody conjugate that specifically binds the first antibody and comprises the first fluorescent label, and a second fluorescent antibody conjugate that specifically binds the second antibody and comprises the second fluorescent label, thereby indirectly binding the detectable labels to their respective analytes (claim 11).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of Vu to replace its nanosecond lifetime FLIM labels with the microsecond to millisecond lifetime labels of Katzlinger and to detect labels in shared fixed detection time windows where the presence or absence of each label signal at each time window encodes analyte identity.
Regarding instant claim 2, Vu, in view of Katzlinger teaches method of claim 1. Katzlinger further teaches that the sample may comprise at least one additional fluorescent label bound to an additional analyte, wherein the additional fluorescent label has a label-specific fluorescence emission lifetime that is at least an order of magnitude different from both the first and second fluorescence emission lifetimes (claim 13; column 3, line 42-52).
Regarding instant claim 3, Vu, in view of Katzlinger teaches method of claim 2. Katzlinger further teaches that when three labels are used, the first fluorescence emission lifetime, the second fluorescence emission lifetime, and the label-specific fluorescence emission lifetime are each at least an order of magnitude different from one another (claim 13). Given that Katzlinger’s first label ((Ru(II) has a lifetime of ~ 1 μsec and second label (Eu) has a lifetime of ~ 800 μsec (column 19, line 45-50). A third label having a lifetime at least an order of magnitude different from Eu’s ~ 800 μsec lifetime would have a lifetime if at least ~8 ms in the longer direction. One of ordinary skill in the art would recognize that at a third detection time interval t3 with an onset positioned after the ~ 800 μs decay period of Eu, signals from both the first (~ 1 μs) and second (~ 800 μs) labels would predictably be absent given their substantially shorter lifetimes, while a third label with a lifetime of ~ 8 ms or longer would remain detectable.
Regarding instant claim 4, Vu, in view of Katzlinger teaches method of claim 3. As discussed above, it is established that the third detection time interval t3 during which a third long lifetime label is detectable while the first and second labels are not. One of ordinary skill in the art would recognize that incorporating the presence or absence of the third label’s signal during t3 into the combinatorial signal code sequence in the same manner that Vu teaches, incorporating the presence or absence of signals in multiple spectral/lifetime components into a unique combinatorial code for each analyte (p. 4, right column, para2).
Regarding instant claim 5, Vu, in view of Katzlinger teaches method of claim 1. Vu teaches multiplexed mRNA detection in fixed cells and tissues using a single round of staining and imaging (p. 2, left column, para 3). Although Vu teaches detection in a single cycle, Vu acknowledges that multi-cycle sequential probe hybridization methods exist in the same field, “[o]ther emerging spatial transcriptomics technologies such as seqFISH can offer greater multiplexing capabilities but requires many rounds of sample re-labeling, imaging, indexing, and error-prone image registration” (p. 9, right column, para 2). Katzlinger similarly teaches its multiplexed TRF detection method has application to, “immunoassays, protein arrays, and other multiplexed biological assays” beyond the Western blot context (column 19, line 31-33).
Regarding instant claim 6, Vu, in view of Katzlinger teaches method of claim 1. Vu teaches generating a unique combinatorial fluorescent signature code that is functioning as a signal code sequence for each of multiple analytes simultaneously at the same spatial locations, wherein each target gene transcript is assigned a distinct code and all codes are decoded simultaneously (Fig. 3l-m; p. 6).
Regarding instant claim 7, Vu, in view of Katzlinger teaches method of claim 5. As discussed above, applying the spectral and temporal differences to separate signals from multiple sequential cycles is an obvious extension to the single cycle method. One of ordinary skill in the art would recognize that using the same shared detection time intervals t1/t2 and t1s/t2s across all cycles rather than defining different time windows for each cycle would be more practical implementation, since the time windows are determined by the lifetimes of the labels.
Regarding instant claim 15 Vu, in view of Katzlinger teaches method of claim 1. Vu teaches that the detectable signals are fluorescence emissions from fluorophores (p. 2, left column, para 3; p. 2, right column, para 3; p. 3, right column para 2). Katzlinger further teaches that the detectable signals are fluorescence or phosphorescence from long-lifetime labels, specifically transition metal chelates of ruthenium chelates of europium which also emit by a phosphorescent mechanism (column 6, line 32-59).
Regarding instant claim 18 Vu, in view of Katzlinger teaches method of claim 1. Katzlinger teaches the definition of emission lifetime as the operational basis for its TRF detection. Conventional TRF detection involves exciting a fluorescent label with a short pulse of light, then typically waiting a certain time after excitation before measuring the remaining long-lived fluorescent signal (column 5, line 63-66). Katzlinger further teaches FIG. 4B plots intensity of the lamp excitation pulse and fluorescence decay as a function of time, with time=0 corresponding to the initiation of the excitation pulse. FIG. 4B also shows the period of time during which measurement may be taken relative to the preceding excitation pulse (column 17, line 46-52). FIG. 4B shows the decay of fluorescence signal as a function of time following cessation of the excitation pulse, illustrating that the emission lifetime is the interval between the offset of the excitation stimulus and the point at which the label fluorescence signal decays to an undectable level.
Regarding instant claim 19, Vu, in view of Katzlinger teaches method of claim 1. Katzlinger further teaches that the first fluorescence emission lifetime is in a range of 0.1 μs to 10 μs (claim 5) and the second fluorescence emission lifetime is in a range of 100 μs to 1 ms (claim 4).
Regarding instant claim 21, Vu, in view of Katzlinger teaches method of claim 1. Vu teaches interrogating spectral and lifetime characteristics of labeled moieties using multiple fluorescent detection channels (Fig. 1d; p. 3, left column, para 1 p. 7, left column, para 2). Katzlinger similarly teaches detection using separate emission filters and detectors each configured for a different emission wavelength range (claim 18-19; column 19, line 48-50).
Regarding instant claim 32 Vu, in view of Katzlinger teaches method of claim 1. Vu teaches that each analyte is a nucleic acid analyte, specifically mRNA transcripts (Fig. 1a; p. 2, right column para 3-4; p. 5, right column para 3 – p. 6). Katzlinger further teaches that analytes include nucleic acids (column 9, line 21-28).
Regarding instant claim 33 Vu, in view of Katzlinger teaches method of claim 1. Vu further teaches an indirect probe binding wherein each detectably labeled probe binds to a primary probe that directly binds to the mRNA analyte (Fig. 1b-c; p. 2, right column, para 3). Katzlinger similarly teaches an indirect probe binding using a primary antibody that specifically binds the analyte and a secondary fluorescent antibody conjugate that specifically binds the primary antibody and comprises the fluorescent label (claim 11; column 17, line 41-43).
Regarding instant claim 36 Vu, in view of Katzlinger teaches method of claim 1. Vu teaches application of the method on human melanoma skin biopsy FFPE tissue sections (p. 7, right column, para 3).
Regarding instant claim 43 Vu, in view of Katzlinger teaches method of claim 1. Vu further teaches that the method is performed in situ in the biological sample, with labeled targets detected at their native spatial locations in intact fixed cells and FFPE tissue samples (Fig. 1a; p. 7, right column, para 3; p. 9, left column, para 1; p. 11, right column, para 2).
Claims 24-26 are rejected under 35 U.S.C. 103 as being unpatentable over Vu et al. (“Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis” Nat Commun 13, 169 (2022). https://doi.org/10.1038/s41467-021-27798-0), in view of Katzlinger (US 10379046 B2; published August 13th, 2019), as applied to claims 1-7, 15, 18-19, 21, 32-33, 36, and 43 above, and further in view of Swager et al. (US 20210120193 A1; published April 22nd, 2021).
Regarding instant claim 24, Vu, in view of Katzlinger teaches method of claim 1. Neither Vu nor Katzlinger teaches that one or more of the detectable labels comprise a thermally activated delayed fluorescence (TADF) emitter or a phosphorescent emitter.
Swager, in the same field of endeavor of detecting chemical and biological species using emissive labels with time-resolved emission properties teaches both TADF emitters and phosphorescent emitters as detectable labels for biological diagnostic assays. Swager teaches TADF emitters having delayed fluorescence with lifetimes longer than 10 ns and less than 50 microseconds, wherein the TADF mechanism involves molecular systems with single and triplet excited states that are sufficiently close in energy to allow thermal equilibration [208; 204]. Swager further teaches phosphorescent emitters based on europium (III) lanthanide chelates with delayed-phosphorescence lifetimes, including specific Eu (III) complexes incorporating carbazole containing ligands and 1,10-phenanthroline ligand [409-412]. Swager further teaches that TADF materials and phosphorescent emitters may be attached to biological species and used to produce signals in assays [390].
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of Vu, in view of Katzlinger, to substitute one or more of the detectable labels with a TADF emitter or phosphorescent emitter as taught by Swager. Since Vu, Katzlinger, and Swager are all in the same field of endeavor of detecting biological analytes using labeled probes that exploit time-resolved emission properties, one of ordinary skill in the art would combine the three teachings with a reasonable expectation of success (see MPEP 2141 (III)).
Regarding instant claim 25, Vu, in view of Katzlinger teaches method of claim 24. Swager teaches carbazole as a structural moiety optionally substituted with C1-C6 alkyl as a structural component of phosphorescent emitter compositions, disclosing unsubstituted carbazole [409] and 9-Ethylcarbazole wherein the carbazole is substituted with a C2 alkyl group [0607-0608].
Regarding instant claim 26, Vu, in view of Katzlinger and Swager teaches method of claim 24. Swager teaches encapsulation of phosphorescent emissive species in a polymer matrix, specifically demonstrating europium based phosphorescent emitter compositions formulated with PMMA [0079-0080; 0631-0632].
Claims 34-35 are rejected under 35 U.S.C. 103 as being unpatentable over Vu et al. (“Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis” Nat Commun 13, 169 (2022). https://doi.org/10.1038/s41467-021-27798-0), in view of Katzlinger (US 10379046 B2; published August 13th, 2019), as applied to claims 1-7, 15, 18-19, 21, 32-33, 36, and 43 above, and further in view of Nagendran et al. (“Automated cell-type classification in intact tissues by single-cell molecular profiling” eLife, 7:e30510 (2018). https://doi.org/10.7554/eLife.30510).
Regarding instant claim 34, Vu, in view of Katzlinger and teaches method of claim 33. Neither Vu nor Katzlinger teaches a circularizable primary probe or a 3’ overhang comprising intermediate probe for in situ nucleic acid analyte detection.
Nagendran, in the same field of endeavor of detecting nucleic acid analytes in fixed cells and tissue samples by in situ hybridization of labeled oligonucleotide probes, teaches both of the elected limitations. Nagendran teaches primary probe comprising H probe pairs, wherein the right H probe pairs, wherein the right H probe includes a single-stranded 3’overhang (p. 3, para 2; Fig. 1A).
Nagendran further teaches a circularizable probe, specifically the linear ‘circle’ oligonucleotide that hybridizes to the overhang regions of the H probes via a bridge oligonucleotide and is subsequently ligated to form a covalently closed circle (p. 3, para 2). The linear ‘circle’ oligonucleotide prior to ligation is a circularizable probe, as it is probe that is capable of being circularized upon ligation.
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of Vu, in view of Katzlinger, to use the circularizable probe and 3’ overhang primary probe of Nagendran. Since Vu, Katzlinger, and Nagendran are all un the same field of endeavor of multiplexed in situ nucleic acid analyte detection in fixed cells and tissue samples, one of ordinary skill in the art would combine the three teachings with a reasonable expectation of success. One of ordinary skill in the would have been motivated to make this motivation because Nagendran teaches that its circularizable probe and 3’ overhang generates RCA amplicons with signal-to-noise exceeding cellular and tissue fluorescence background by more than 30-fold (p. 3, para 3), which directly addresses the autofluorescence challenge that Vu identifies as a key limitation. Furthermore, Nagendran’s probe is compatible with FFPE tissue samples (p. 6, para 3), which is the sample type demonstrated by Vu. In addition, it would have been obvious to one of ordinary skill in the art that the known methods of the cited references could have been combined with predictable results, as all three references use in situ hybridization of oligonucleotide probes to detect mRNA targets in fixed cells and tissue samples (see MPEP 2141 (III)).
Regarding instant claim 35, Vu, in view of Katzlinger and Nagendran teaches method of claim 1. Nagendran teaches generating RCA products in situ in fixed tissue samples, wherein rolling-circle replication generates a long single-stranded amplicon of tandem repeats directly at the site of each labeled transcript in the intact tissue (p. 3, para 2).
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
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Claims 1, 24, 25, 32, 33, 35, and 43 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 13, 15, 33, 35, 39, 49, and 51 of Application Number: 18/360,137 in view of Vu et al. (“Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis” Nat Commun 13, 169 (2022). https://doi.org/10.1038/s41467-021-27798-0) and Katzlinger (US 10379046 B2; published August 13th, 2019), as applied to claim 1 above.
Although the claims at issue are not identical, they are not patentably distinct from each other because both the ‘137 application and the instant application claim methods of detecting an analyte in a biological sample using a thermally activated delayed fluorescence (TADF) emitter conjugated to a probe that directly or indirectly binds to the analyte or a product thereof in the biological sample.
Instant claim 1 recites a method comprising contacting a biological sample with a plurality of detectably labeled probes, detecting signals associated with the detectable labels during detection time intervals t1 and t2, and generating a signal code sequence identifying an analyte at one or more locations in the biological sample. Claim 1 of the ‘137 application which recites a method of detecting an analyte in a biological sample comprising contacting the biological sample with a nucleic acid probe conjugated to a TADF emitter that directly or indirectly binds to the analyte or a product or complex thereof in the biological sample, exciting the biological sample with a pulsed light source, and detecting the fluorescence emitted by the TADF emitter at a location in the biological sample after a time period post excitation. Both instant claim 1 and claim 1 of the ‘137 application are directed to detecting a nucleic acid analyte at one or more locations in a biological sample using a labeled nucleic acid probe that directly or indirectly binds to the analyte, with detection occurring after a time delay that exploits the emission lifetime of the label.
Claim 1 of the ‘137 application does not teach detecting signals associated with the detectable labels, or absence thereof, during a detection time interval t1 and a detection time interval t2 at one or more locations in the biological sample, wherein the onset of t2 is later than the onset of t1,wherein signals associated with the first detectable label are detectable during t1 and not during t2, and wherein signals associated with the second detectable label are detectable during t2; and generating a signal code sequence comprising signal codes corresponding to the signals or absence thereof detected during t1 and t2, respectively, at the one or more locations, wherein the signal code sequence corresponds to an analyte of the multiple analytes. However, these additional limitations of instant claim 1 do not render it patentably distinct from claim 1 of the ‘137 application because they would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention. As established in the 35 USC § 103 rejection of claim 1 above over Vu teaches a method comprising contacting the biological sample with a plurality of detectably labeled probes for detecting multiple analytes, wherein each detectably labeled probe is configured to directly or indirectly bind to a different analyte or product thereof and comprises a detectable label (p. 1, right column, para 3; Fig. 1a-c). Vu further teaches that the detectable labels comprise a plurality of fluorophores exhibiting distinct fluorescence emission lifetimes, wherein one detectable label has a first signal emission lifetime and a second detectable label has a second signal emission lifetime different from the first. (p. 3, left column, para 1; p. 1, right column, para 3; Fig. 3). The second signal emission lifetime is longer than the first signal emission lifetime (p. 4, left column, para 3). Vu further teaches detecting signals associated with the detectable labels at one or more locations in the biological sample (Fig. 1; p.2, right column, para 2). Vu further teaches generating a combinatorial fluorescent signature code assigned to each analyte target using a codebook, wherein each code corresponds to an analyte of the multiple analytes, thereby identifying the analyte at one or more locations in the biological sample (p. 4, right column, para 2; Fig, 1e-f). Vu does not teach detecting signals associated with the detectable labels, or absence thereof, during a detection time interval t1 and a detection time interval t2 at one or more locations in the biological sample. Katzlinger teaches that a first fluorescent label, a transition metal chelates of ruthenium (Ru(II)) has a first fluorescence emission lifetime in a range of about 0.1 μs to 10 μs, and a second fluorescence emission lifetime in a range of about 100 μs to fluorescence about 1 ms, wherein the second fluorescence emission lifetime is at least 5 times longer than the first fluorescence emission lifetime (claim 1; claim 4-5; column 6, line 32-53). Katzlinger further teaches that the larger lifetime difference enables temporal separation of the two label signals, specifically that Ru, whose half-life is ~1 μsec is detected with a shorter time integration window of 2 μsec during which its signal is present and then absent in subsequent longer windows; Eu, whose half-life is ~ 800 μsec is detected with longer integration window of 1000 μsec during which its signal persists and remains detectable (column 19, line 45-50). It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of Vu to replace its nanosecond lifetime FLIM labels with the microsecond to millisecond lifetime labels of Katzlinger and to detect labels in shared fixed detection time windows where the presence or absence of each label signal at each time window encodes analyte identity, thereby arriving at the additional limitations of instant claim 1.
Instant claim 24 recites that one or more of the detectable labels comprise a TADF emitter or a phosphorescent emitter. Instant claim 24 is not patentably distinct from claim 1 of the ‘137 application, which recites “[a] method of detecting an analyte in a biological sample, the method comprising: contacting the biological sample with a nucleic acid probe conjugated to a thermally activated delayed fluorescence (TADF) emitter, wherein the nucleic acid probe directly or indirectly binds to the analyte or a product or complex thereof in the biological sample.” Both instant claim 24 and claim 1 of the ‘137 application are therefore directed to detecting a nucleic acid analyte in a biological sample using a TADF emitter conjugated to a nucleic acid probe, and differ only in the specific detection scheme.
Instant claims 25 recites a list of TADF emitter moieties including organoboron moiety, cyanobenzene moiety, dicyanobenzene moiety, diphenyltriazine moiety, diphenylsulfone moiety, naphthalimide moiety, dicyanopyrazino moiety, phenanthrene moiety, carbazole moiety, phenoxazine moiety, triphenylamine moiety, diphenylamide moiety, acridine moiety, and dimethylacridine moiety. These moieties overlap with the electron donor moieties of claim 13 of the ‘137 application (carbazole, phenoxazine, diphenylamine, triphenylamine, acridine, or dimethylacridine) and the electron acceptor moieties of claim 15 of the ‘137 application (cyanobenzene, dicyanobenzene, diphenyltriazine, diphenylsulfone, naphthalimide, dicyanoimidazole, phenylbenzimidazole).
Instant claim 32 recites that each analyte is independently a nucleic acid analyte. Instant claim 32 is not patentably distinct from claim 33 of the ‘137 application, which recites that the analyte comprises an mRNA, and claim 71 of ‘137 application recites that the biological sample is a cell or tissue sample. Both instant claim 32 and claims 33 and 71 of the ‘137 application are directed to detecting a nucleic acid analyte in a biological sample. The difference between instant claim 32, which broadly recites nucleic acid analytes, and claim 33 of the ‘137 application, which specifically recites mRNA, does not render the claims patentably distinct because mRNA is a nucleic acid and extending the method of the ‘137 application to detect nucleic acid analytes other than mRNA would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
Instant claim 33 recites that each detectably labeled probe binds to a primary probe that directly binds to its corresponding analyte, or binds to an intermediate probe that binds directly or indirectly to a primary probe that directly binds to its corresponding analyte. Instant claim 33 is not patentably distinct from claim 35 and 39 of the ‘137 application, which recites that the nucleic acid probe hybridizes to the mRNA analyte (claim 35) and the nucleic acid probe hybridizes to an intermediate probe that hybridizes to an intermediate probe that hybridizes to the mRNA (claim 39). Both the instant claim 33, and claims 35 and 39 of the ‘137 application are directed to the same indirect probe binding wherein a detectably labeled probe binds indirectly to an analyte through one or more intermediate probes.
Instant claim 35 recites that the product of each analyte is a rolling circle amplification (RCA) product generated in situ in the biological sample. Instant claim 35 is not patentably distinct from claim 49 of the ‘137 application, which recites that the product or complex of the intermediate probe is an RCA product, and claim 51 of the ‘137 application, which recites that the product of the nucleic acid probe is generated or detected in situ in the biological sample. Both instant claim 35, and claims 49 and 51 of the ‘137 application are directed to generating an RCA product in situ in the biological sample as the detectable product of analyte-bound probes.
Instant claim 43 teaches that the method is performed in situ of the biological sample. Instant claim 43 is not patentably distinct from claim 51 of the ‘137 application, which recites that the product of the nucleic acid probe is generated or detected in situ in the biological sample. Both instant claim 43 and claim 51 of the ‘137 application are directed to performing detection in situ detection of biological sample.
Both applications further share the common context of detecting a nucleic acid analyte in a biological sample using a TADF labeled probe that directly or indirectly binds to the analyte or a product thereof, in situ in the biological sample. The ‘137 application claims 30, 33, 35, 37, and 39 recite nucleic acid probe hybridization to an analyte or primary probe or intermediate probe, and claim 51 recites in situ detection in the biological sample. Both elements also present in the instant application’s claims 32, 33, 35, and 43.
This is a provisional nonsatutory double patenting rejection because the patentably indistinct claims have not been in fact been patented.
Conclusion
All claims are rejected.
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/NURA M. CHOUDHURY/ Examiner, Art Unit 1683
/ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683
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