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 .
Applicant’s arguments and amendments have been thoroughly reviewed and considered. Claim 2 has been canceled. Claims 13-15 and 19-20 remain withdrawn. Claims 21-22 have been added. Claims 1, 3-8, 10-12, 16-18, and 21-22 are pending and are examined on the merits herein.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 5/28/2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Response to Applicant’s Amendments
Nucleotide and/or Amino Acid Sequence Disclosure
Applicant’s previous Sequence Listing was found to be defective. Applicant has submitted a new Sequence Listing dated 5/28/2026, but this listing has also been marked as defective. See below.
Claim Objections
Claims 1 and 17 were objected to due to informalities. In light of Applicant’s amendments to the claims submitted 5/28/2026, these objections have been withdrawn. However, see new grounds of objection below.
35 USC 112(b) Rejections
Claims 1-8, 10-12, and 16-18 were objected to due to various indefiniteness issues. In light of Applicant’s amendments to the claims submitted 5/28/2026, these rejections have been withdrawn for all currently pending claims, but see new grounds of rejection below. Claim 2 has been canceled, and so this rejection has been rendered moot.
35 USC 103 Rejections
Claims 1, 3-8, 10-12, and 16-18 were rejected under 35 U.S.C. 103 as being unpatentable over Gines et al. (WO 2017/141068 A1) in view of Chen et al. (Chem. Commun., 2018).
Applicant’s arguments and amendments have been thoroughly reviewed and considered. In light of Applicant’s amendments to the claims submitted 5/28/2026, these rejections have been withdrawn, but see new grounds of rejection and “Response to Applicant’s Arguments” below.
Claim 2 was rejected under 35 U.S.C. 103 as being unpatentable over Gines et al. (WO 2017/141068 A1), in view of Chen et al. (Chem. Commun., 2018), and further in view of Rondelez et al. (WO 2017/140815 A1).
This claim has been canceled, and so this rejection has been rendered moot.
Response to Applicant’s Arguments
Regarding the 35 USC 103 Rejections presented in the Non-Final Rejection mailed 1/28/2026, Applicant argues that the invention of Gines, the primary reference, lacks the sensitivity required for single-molecule detection, and uses Figure 2F of Gines et al. (Nature Nanotechnology, 2017; cited in Applicant’s IDS), hereby Gines 2, to support this point. “Poor sensitivity” at low concentrations is allegedly “fundamentally incompatible with the digital readout of Applicant’s claimed invention,” (Remarks, page 18).
It is noted that as claimed, the instant claims do require absolute quantification of target biomolecules, and so single-molecule level detection is also required. In the Non-Final Rejection mailed 1/28/2026, this is not taught by Gines (the primary reference) alone. Para. 30 of the Non-Final Rejection states, “Additionally, Gines does not discuss target hybridization following a Poissonian distribution, and though the reference teaches detecting target biomolecules (e.g. Figures 8, 10, and 12), it is unclear if absolute quantification of target particles occurs. Gines does discuss measuring concentrations of targets (paras. 26 and 81), and teaches the use of multiple target concentrations (para. 72), including small concentrations (para. 86).” The teachings of Chen are then used, which does note digital amplification, absolute quantification of target sequences, a Poissonian distribution, as well as high sensitivity at low cDNA concentrations (where a LOD of 0.3 x 10-17 M was determined; Figure 1, page 292, column 2, para. 2 and page 293, column 1, para. 1). In combining Gines in view of Chen, the methods of Chen used would impart these benefits to Gines. Thus, this combination of references arrives at a highly sensitive, absolute quantification method.
Applicant points to Gines 2, and particularly Figure 2f, to show that “particles functionalized with both the amplification oligonucleotide and the leak-absorption oligonucleotide fail to respond even at trigger concentrations as high as 2.5nM.” Figure 2f is focused on PαB particles, which are bistable particles that contain two oligonucleotides (Figure 2e). Applicant notes that the two oligonucleotides on this particle are analogous to the amplification and leak-absorption oligonucleotides, and so this particle does not read on the claimed particles, which require a conversion oligonucleotide. The PαB particles also do not read on the particles described by Gines as used in the rejection, as these particles contain all four nucleotides described in step (a) of instant claim 1. Gines 2 also does not subject particles to the same conditions as Gines in view of Chen, as the former utilizes a two-dimensional microchamber with fluorescence imaging (Figure 1), while the latter performs amplification in reaction tubes and utilizes flow methods for detection (Scheme 1 of Chen). It is not clear that the results shown in Gines 2 in terms of a lack of detection at lower target concentrations with PαB would be applicable to Gines in view of Chen. Furthermore, the instant claims do not require a particular target concentration be used, so long as the Poissonian distribution described in step (c) is met. Therefore, it is not clear that low concentrations as described in Gines 2 would be necessary or required in the claimed invention.
Additionally, Applicant states that the particles of Gines rely on distinct particle types with distinct bistable switches and amplification signals, whereas the instant invention uses a universal signal that is the same for all targets (Remarks, page 19). When distinct signals are used for each target, Applicant argues that crosstalk is generated in a multiplex reaction, as is shown in Rondelez et al. (ACS Sensors, 2020; cited in Applicant’s IDS), hereby Rondelez 2 (Remarks, pages 20-21).
In the current claims, the amplification oligonucleotide is not given a particular structure in terms of universality or target specificity. In the instant specification, an “amplification oligonucleotide” is defined as “an oligonucleotide which is able to exponentially amplify the trigger (signal sequence),” (page 12, para. 3). There is no requirement that a universal or generic sequence be used, and so such a feature is not required in the instant claims. In Rondelez 2, cross-talk is shown when isothermal amplification is performed on two targets simultaneously (Figure 1). In developing this amplification system, Rondelez 2 uses four oligonucleotides that overlap with those of the instant claims, though these do not appear to be associated with a particle, and so it is not clear that the specific cross-talk results shown by Rondelez 2 would be repeated in Gines in view of Chen (page 2431, columns 1-2 joining para.). Applicant states that, “To mitigate this effect, the inventors [Rondelez 2] had to introduce additional components, called cross-inhibitory templates, which are designed to counterbalance the crosstalk.” The use of such cross-inhibitory sequences is not prohibited by the instant claims, as the claims comprise the listed steps. Furthermore, in Gines, the use of multiplex reactions is recited. Particularly, Example 4 on pages 36-37 (and associated Figure 16) shows the use of two particles with two different reporters, where both can be detected. Para. 62 states, “This result demonstrates that simultaneous measurements of various targets can be performed using differently programmed microspheres in the same solution. This highlights the potential for massive multiplexing capabilities of CompuSpheres because it shows that different microspheres can perform different tasks while being immersed in the same solution.” Therefore, it is not clear that the issues described by Rondelez 2 would be applicable to the functionalized particles of Gines and Gines in view of Chen, particularly when Gines generally teaches the multiplexing capabilities of their invention (e.g. paras. 19, 24, 67, 74, and 76 and Figure 1).
On pages 21-22 of their Remarks, Applicant discusses para. 37 of the Non-Final Rejection. This paragraph discusses previous claim 2, which has been canceled in the instant claims, but the cited reference, Rondelez, is additionally discussed as the context of the reference is relevant to the instant invention and the Gines and Chen references. Page 22 specifically highlights the alleged differences between Applicant’s instant invention and the three cited references.
It is noted that Rondelez is only combined with Gines in view of Chen to arrive at the invention of now canceled claim 2, specifically in the process of adding the third and fourth claimed oligonucleotides later in the method of claim 1 during step (d), rather than including them in the particles of step (a). This limitation is now incorporated into newly amended claim 1. In considering Gines, in view of Chen, and in view of Rondelez, Applicant does not appear to specifically provide arguments against the rationale for the combination presented in para. 37 of the Non-Final Rejection. Instead, Applicant compares each individually cited reference to the alleged benefits of the instant invention. In response to Applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Additionally, of the benefits described by the table on page 22 of Applicant’s Remarks, the only one allegedly not provided by any of the three cited reference is a universal amplifier, which as pointed out above, is not currently required by Applicant’s claimed invention. Applicant also states that while Gines is a multiplex invention, it is not “multiplexable,” – this distinction is not entirely clear, though is likely stems from Applicant’s discussion of Rondelez 2 above. Applicant states that in the instant invention, specificity is encoded entirely in the particle barcode. While the claimed barcodes allow “the discrimination of the multiple target biomolecules,” such a function is generally possible for barcodes in the art, see for example paras. 26-27 and 60 of Gines. Applicant also states that the leak-absorption templates are also universal/non-specific, but this function is also not claimed, and is not required in the definition of such sequences as described on page 12, para. 7 of the instant specification. Thus, generally, it is not clear that the allegedly critical aspects of Applicant’s claimed invention are actually required, and as written, the instant claims do not appear to clearly distinguish themselves from the combination of Gines, in view of Chen, and in view of Rondelez.
On pages 24-25, Applicant discusses secondary considerations as they relate to the instant invention – in particular, long-felt but unresolved need, unexpected results, praise, and commercial success.
MPEP 716.04 discusses secondary considerations of long-felt need, and states that it requires the establishment “that an art recognized problem existed in the art for a long period of time without solution. The relevance of long-felt need and the failure of others to the issue of obviousness depends on several factors. First, the need must have been a persistent one that was recognized by those of ordinary skill in the art. In re Gershon, 372 F.2d 535, 539, 152 USPQ 602, 605 (CCPA 1967) ("Since the alleged problem in this case was first recognized by appellants, and others apparently have not yet become aware of its existence, it goes without saying that there could not possibly be any evidence of either a long felt need in the . . . art for a solution to a problem of dubious existence or failure of others skilled in the art who unsuccessfully attempted to solve a problem of which they were not aware."); Orthopedic Equipment Co., Inc. v. All Orthopedic Appliances, Inc., 707 F.2d 1376, 217 USPQ 1281 (Fed. Cir. 1983) (Although the claimed invention achieved the desirable result of reducing inventories, there was no evidence of any prior unsuccessful attempts to do so.).” Applicant cites the problem of enzymes adsorbing onto particles, which can lead to false-positive results. Applicant cites art that was published after the effective filing date of the claimed invention (Jet et al., Journal of American Chemical Society, 2025) to support that this issue was a known problem in the art. This issue of enzyme adsorption is described in Figure 5 and pages 37 and 43-44 of the instant specification. However, Applicant does not provide evidence of a long-felt need regarding this problem, as they do not provide examples of art discussing this problem (and a lack of solution) before the effective filing date of the claimed invention.
Applicant briefly mentions unexpected results, but does not provide any details about said results. Applicant is directed to MPEP 716.02 for a discussion of the requirements regarding this secondary consideration.
Applicant also mentions praise regarding the claimed invention, particularly the CNRS Bronze Medal. This can be considered industry praise as is relevant to the secondary considerations described in MPEP 716, though it is not clear that this praise is specific to the claimed invention, as it appears to be related to the research generally conducted by instant inventor Gines.
Finally, Applicant discusses licensing. While licensing can be associated with commercial success secondary considerations, MPEP 716.03(b) discusses commercial success, and notes “licensing is a secondary consideration which must be carefully appraised as to its evidentiary value because licensing programs may succeed for reasons unrelated to the unobviousness of the product or process, e.g., license is mutually beneficial or less expensive than defending infringement suits.” The commercial success must clearly be derived from the claimed invention and its functions and advantages. Applicant has not currently provided evidence of such, as the cited CNRS article generally discusses the research of the instant inventors, and does not appear to discuss the specifically claimed instant invention.
Thus, overall, Applicant’s arguments are not considered persuasive to obviate the use of Gines, Chen, and Rondelez. Due to Applicant’s amendments to the claims, new grounds of rejection are provided below, but the relevant portions of these references to the newly amended portions of the claims are reiterated.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency - This application fails to comply with the requirements of 37 CFR 1.821 - 1.825. This application contains a “Sequence Listing” as a PDF file (37 CFR 1.821(c)(2)) or as physical sheets of paper (37 CFR 1.821(c)(3)). A copy of the "Sequence Listing" in computer readable form (CRF) has been submitted; however, the content of the CRF does not comply with one or more of the requirements of 37 CFR 1.822 through 1.824, as indicated in the "Error Report" that indicates the "Sequence Listing" could not be accepted. Refer to attachment or document "Computer Readable Form (CRF) for Sequence Listing – Defective" dated 5/28/2026.
Required response – Applicant must provide:
A replacement "Sequence Listing" part of the disclosure, as described above in item 1); together with
An amendment specifically directing its entry into the application in accordance with 37 CFR 1.825(b)(2);
A statement that the "Sequence Listing" includes no new matter as required by 37 CFR 1.825(b)(5); and
A statement that indicates support for the amendment in the application, as filed, as required by 37 CFR 1.825(b)(4).
If the replacement "Sequence Listing" part of the disclosure is submitted according to item 1) a) or b) above, Applicant must also provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required incorporation-by-reference paragraph, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter and
An amendment to the specification to remove the “Sequence Listing previously submitted as a PDF file (37 CFR 1.821(c)(2)) or as physical sheets of paper (37 CFR 1.821(c)(3))
If the replacement "Sequence Listing" part of the disclosure is submitted according to item 1) c) or d) above, Applicant must also provide:
A CRF in accordance with 1.821(e)(1) or 1.821(e)(2) as required by 37 CFR 1.825(b)(6)(ii); and
Statement according to item 2) a) or b) above.
Claim Objections
Claim 21 is objected to because of the following informality: in line 2, “multiple biomolecules” should read “multiple target biomolecules.” Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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 1, 3-8, 10-12, 16-18, and 21-22 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.
Claim 1 recites in step a) that the particles must be functionalized with a conversion oligonucleotide (the first oligonucleotide) and one or more of a reporting oligonucleotide (the second nucleotide), an amplification oligonucleotide (the third oligonucleotide), and a leak absorption oligonucleotide (the fourth nucleotide). However, in the newly amended wherein clause, it is stated that the third and fourth oligonucleotide must be added in step d), and thus would not be functionalized on the particles. Thus, step a) would comprise only functionalizing the particles with the first and second oligonucleotide (as one or more of the second, third, and fourth oligonucleotides must be added). However, this wherein clause also states that the second oligonucleotide is specifically added to the particle when present, which appears to contradict the requirements of the claim as a whole, where the second oligonucleotide must be present on the particles. Additionally, step d) states that optionally, oligonucleotides are added, but based on the newly added wherein clause, this optionality no longer appears to be accurate. In step f) the claim also states “the amplification oligonucleotide on the particle carrying the target biomolecule,” but these oligonucleotides are added in step d) and are not specifically stated to be functionalized onto the particle. Similar language is used in step g), which states “the amplification oligonucleotide of each particle.” These factors in sum render the scope of the claim indefinite.
If it is Applicant’s intention, it is recommended that step a) explicitly state that the particles are functionalized with the conversion and reporting oligonucleotides, and that step d) explicitly cite the addition of the amplification and leak absorption oligonucleotides. The subsequent steps should then be amended to clarify that the amplification oligonucleotide itself is not functionalized with the particle.
Claims 3-8, 10-12, 16-18, and 21-22 are rejected based on their dependence on rejection claim 1.
Claim 21 is also rejected because the limitation “allowing the sensitivity of the detection of multiple biomolecules to increase” is an unclear functional limitation. It is generally unclear what this increase in sensitivity is relative to, and Applicant does not point to an additional functional limitation other than what is already presented in claim 1 that would lead to an increase in sensitivity. Thus, the scope of the claim is indefinite, and prior art will be considered to read on the claim if it teaches the method of claim 1 and also generally discusses sensitivity associated with the method.
Claim 22 is also rejected because “the amplified” sequence, first appearing in line 4, lacks antecedent basis, and “an amplified sequence” does not appear earlier in the claim or in claim 1, from which this claim depends. Additionally, the term “amplified sequence” is unclear in the context of the claim as a whole. Claim 22 discusses concentrations of the third and fourth oligonucleotide, where the third oligonucleotide is the amplification oligonucleotide and the fourth oligonucleotide is the leak absorption oligonucleotide. The amplification oligonucleotide is presumably needed to create amplified sequences, and so would be reacting with the target biomolecules to produce amplified sequences. Thus, in the first scenario of claim 22, “the amplified sequence” will be interpreted to mean “the target biomolecules.” In the second scenario of claim 22, amplification is eliminated, and so no amplified sequences would be produced, and so “the amplified sequence” will also be interpreted to mean “the target biomolecules.”
Claim Interpretation
In claim 1, an amplification signal is generated by an amplification oligonucleotide, and this signal is then detected and/or measured. The term “signal” in this context is taken to encompass the production of a particular sequence, and is not necessarily detectable on its own (see pages 11-12 of the instant specification, which note that amplification oligonucleotides produce a signal sequence that the reporter oligonucleotide may then translate to a detectable signal). Claim 1 also does not specifically recite that direct detection/measurement of the amplification signal must occur. Thus, the instant claims do not require the amplification signal to be detected/measured directly, and so detection/measurement may occur indirectly by measuring the signal of a reporter oligonucleotide.
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.
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.
Claims 1, 3-8, 10-12, 16-18, and 21-22 are rejected under 35 U.S.C. 103 as being unpatentable over Gines et al. (WO 2017/141068 A1) in view of Chen et al. (Chem. Commun., 2018), and in view of Rondelez et al. (WO 2017/140815 A1).
Gines teaches methods of molecular computing involving microspheres with particular DNA modules attached (Abstract). Mesoporous particles are functionalized in solution with a mixture of DNA modules and a fluorescent barcode element that allows for multiplexing (paras. 24 and 26-27 and Figure 1; instant claims 3 and 18). These DNA modules can be conversion, reporting, amplification, and leak-absorbing oligonucleotides (para. 26 and (f) of para. 27). The particles are then exposed to one or more biomolecular targets (such as DNA or RNA; instant claims 10 and 11), an enzyme mixture is added, and the resulting composition is incubated at a constant temperature, allowing amplification to occur (para. 26). Detection of the amplification of specific targets in the sample can then be done by detecting the fluorescent barcodes and signals from the reporter oligonucleotide (which generates a fluorescent signal based on amplification products, see para. 18). Different barcodes may be used in the same solution (para. 39), particularly to detect several different targets (paras. 26 and 60). Gines teaches that the particles in the solution can perform identical yet independent functions (para. 89), and can thus act independently. The enzyme mixture can contain one or more of polymerase, nickase, and exonuclease (claim 17; instant claim 5). dNTPs and buffers are also included in the solution (Figure 1 and para. 26, and see mixtures in Figures 5, 7, and 11, for example). The particles specifically contemplated by Gines are porous microspheres and have a mean diameter of 34 μm (para. 40; instant claims 8 and 16).
Gines generally teaches the multiplexing capabilities of their invention, and thus teaches that multiple different target biomolecules can be used (e.g. paras. 19, 24, 67, 74, and 76 and Figure 1). Additionally, Example 4 on pages 36-37 (and associated Figure 16) shows the use of two particles with two different reporters, where both can be detected. Para. 62 states, “This result demonstrates that simultaneous measurements of various targets can be performed using differently programmed microspheres in the same solution. This highlights the potential for massive multiplexing capabilities of CompuSpheres because it shows that different microspheres can perform different tasks while being immersed in the same solution.” This provides evidence that multiple sequences can be successfully detected with the oligonucleotides/particles of Gines.
The constant temperature contemplated by Gines is 45°C (para. 48 and Figures 5, 7, and 11 for example; instant claim 7).
Gines teaches that their invention can be applied to miRNA targets for diagnostic purposes to detect diseases such as cancer (paras. 79-81; instant claim 12).
However, though Gines mentions droplet methods, the reference seems to mainly contemplate these in the context of PCR and continuous flow/microfluidics methods (para. 75), which are not recited in the method of their invention. Additionally, Gines does not discuss target hybridization following a Poissonian distribution, and though the reference teaches detecting target biomolecules (e.g. Figures 8, 10, and 12), it is unclear if absolute quantification of target particles occurs. Gines does discuss measuring concentrations of targets (paras. 26 and 81), and teaches the use of multiple target concentrations (para. 72), including small concentrations (para. 86).
Chen teaches a BEAMing LAMP reaction. The basic methodology is shown in Scheme 1. Molecules are captured on beads in solution. The beads are then mixed with LAMP reagents and water-in-oil droplet emulsions are formed (instant claim 6). LAMP then occurs, the beads are washed, and then flow cytometry is used to detect targets (page 291, final para through page 292, para. 1; instant claim 4). Chen also teaches taking microscopy images of the beads by simply shaking them in solution (Figure 2 and page 293, columns 1-2 joining para.), where the detectable beads can be easily identified. The droplets had diameters of 5–13 µm, equating to a volume of 0.523-9.202 pL (page 293, column 1, para. 2; instant claim 17). Chen teaches that this method is highly sensitive, accurate, can be useful for diagnostics, and improves detection rates of targets (page 294, columns 1-2 joining para.). These methods allow for absolute quantification of target sequences (Abstract, page 293, column 1, para. 1, page 294, column 1, paras. 1-2, and Figures 1 and 3). The reference mentions that when little target DNA is present, most incubated beads will contain zero or one hybridized target (page 292, column 1, para. 1), and that the pattern for droplet analysis in detecting beads that do and do not have targets follows a Poisson distribution (page 293, column 1, para. 1).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Chen to add droplet methodology to the overall method of Gines. Specifically, this would involve using the water-in-oil droplet methodology of Chen with the particles of Gines before performing subsequent isothermal amplification and detection. Gines teaches that droplets in flow systems are not useful for the suspensions and solutions of their invention as it is difficult to fabricate and program the microparticles and requires the use of multiple distinct probes for multiplexing (para. 75). However, Gines already solves this second problem via the use of multiple barcodes. Chen teaches a mechanism by which droplets can be created in solution (i.e. not in a flow) for isolation of particles during amplification, solving the first problem presented by Gines, and shows that fluorescent reporters can be successfully detected later with the use of flow methods in order to absolutely quantify target sequences. By utilizing droplets, beads can be further separated from one another – Chen teaches that most droplets created in their method contained only single beads (page 293, column 1, para. 2), so this would be particularly useful for Gines, which teaches the use of multiple targets in a single solution, as distinct targets would be further separated from one another. Chen teaches sample concentrations for use in their method (e.g. page 292, column 2, paras. 2-3, page 293, column 1, para. 1, and page 294, column 1, para. 1), and teaches that the empty beads present are due to sample concentrations, and so the ordinary artisan would be capable of providing samples at low enough concentrations (such as those taught by Chen) that a Poissonian distribution of target-particle hybridization would be possible for each desired target. Utilizing this type of distribution would also allow for copy number calculations, as shown in Chen, and so this could provide a check on the absolute quantification values derived from droplet fluorescence calculations (page 293, column 1, para. 1). Chen also details several benefits regarding the utility of this method as described in the above paragraph that would motivate the ordinary artisan. There would be a reasonable expectation of success with this combination as the only change to the pre-analysis method steps of Gines would be the addition of the water-in-oil emulsions to create the droplets and a subsequent washing step – these are techniques that would well-known to the ordinary artisan, as evidenced by Chen and Gines (see Gines paras. 26, 46, 56, and 71 for examples of washing the recited particles).
Gines also teaches that DNA modules can be added after the initial functionalization of the particles. Para. 66 details particles being created with the amplification and leak-absorbing oligonucleotides, and then the conversion oligonucleotide being added later (Figure 19). However, this addition is not done with the addition of the amplification mixture, as is required in instant claim 1. It is noted that the amplification and leak-absorbing oligonucleotides are considered a bistable system when paired together (para. 64), and that the purpose of the leak-absorbing oligonucleotide is to avoid unspecific amplification when amplification is initiated (para. 16).
Rondelez teaches methods of isothermal amplification while eliminating background noise (Abstract). This is done by preparing a mixture including buffer and enzymes, and adding amplification, leak-absorption, and conversion oligonucleotides (para. 8). A reporting probe can also be added (para. 19). This reference teaches that reaction mixtures with nucleic acid templates and enzymes can be prone to leaking reactions (para. 34), and that the use of these oligonucleotides may absorb such leaks (para. 35). The leak-absorption and amplification oligonucleotides are also complementary to one another, and their hybridization can decrease background amplification (para. 35). These oligonucleotides can be included in the final mixtures used for amplification (e.g. paras. 61, 66, 67, 79, and 82).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to combine the teachings of Gines in view of Chen and Rondelez to arrive at the invention of instant claim 1. Though Gines teaches four functional oligonucleotides, these do not all need to be present on every particle at the outset of the method, as shown in Example 5. Gines also details the importance of the leak-absorbing oligonucleotide in preventing unspecific amplification, providing reason to pair this oligonucleotide with the amplification oligonucleotide specifically. Rondelez teaches that oligonucleotides with identical functions to those of Gines can be added with amplification mixtures during isothermal amplification to prevent background noise. In Rondelez, the leak-absorbing and amplification oligonucleotides are also clearly functionally linked. Thus, it would be prima facie obvious to the ordinary artisan that the leak-absorbing and amplification oligonucleotides could be added with the amplification mixture in Gines in view of Chen while still allowing for the reduction of nonspecific amplification. By not including these oligonucleotides on the particles of Gines in view of Chen, more conversion and reporter oligonucleotides can be included on the particles, which would allow for the capture and detection of additional target sequences (Gines paras. 17-18), allowing for more accurate results. Additionally, by adding the amplification and leak-absorbing oligonucleotides in the amplification mixture (i.e. later in the method of Gines in view of Chen), this may prevent any premature hybridization of the two oligonucleotides before amplification can occur. There would be a reasonable expectation of success with this combination because Rondelez shows that these oligonucleotides can be added with an amplification mixture and still result in effective target amplification with background noise reduction. Also, the basic principles of the method of Gines in view of Chen would not be changing, as all four oligonucleotides would still be present together in solution with amplification reagents and target sequences in order to perform amplification (and later detection) reactions.
Thus, claims 1, 3-12, and 16-18 are prima facie obvious over Gines, in view of Chen, and in view of Rondelez.
Regarding claims 21 and 22, Rondelez teaches an embodiment in para. 8 where the amplification oligonucleotide is referred to as a first oligonucleotide and a leak-absorption oligonucleotide is referred to as a second oligonucleotide. In para. 16, the reference states, “concentrations of the first and second oligonucleotides are selected so that a reaction of the first oligonucleotide is faster than a reaction of the second oligonucleotide at high concentration of the amplified sequence but the reaction on the second oligonucleotide is faster than the reaction of the first oligonucleotide at low concentration of the amplified sequence, thereby effectively eliminating amplification unless the stimulus threshold is crossed.” This is the same language found in instant claim 22. Para. 31 of Rondelez explains that this setup is able to eliminate background non-triggered amplification, and allows for ultra-sensitive and ultra-specific detection. Gines describes a similar scenario in para. 16, “In yet another method, the modules include a first and second template, the first template is an amplification template, the second template absorbs leak reaction and avoids unspecific spontaneous amplification when the microsphere contacts with the mixture of enzymes, so that DNA is amplified exponentially only when the first template receives stimulation above a predetermined concentration threshold for a specific target specie.”
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to provide the amplification and leak-absorption oligonucleotides at concentrations that allow them to function as described above in Rondelez and Gines. Both references note that this would further decrease nonspecific amplification, and would allow amplification of a target to proceed only when a threshold value of said target is reached. This would decrease false positive results, and therefore would increase the accuracy of the assay, which would motivate the ordinary artisan, particularly when the assay is being used for disease diagnostic purposes, as described in the teachings of Gines above. There would be a reasonable expectation of success as both Gines and Rondelez teach this relationship regarding the concentration of the amplification and the leak-absorption oligonucleotide, and this change would not alter the structure or function of either oligonucleotide – it would simply change the concentration(s) at which they are provided.
Gines notes that adjusting the amplification threshold can adjust sensitivity (para. 81). Chen states on page 292, column 2, paras. 1-2 notes that their BEAMing LAMP is a highly sensitive method, and on page 294, Chen states that their method has desirable sensitivity. Rondelez also teaches that their methods are sensitive (e.g. paras. 6, 17, and 32). These discussions of sensitivity read on instant claim 21 as described above in the 35 USC 112(b) Rejections, particularly as all three references teach sensitive methods, and thus the combination of teachings described above, which contain the same oligonucleotides as Gines and Rondelez and the same droplet detection methods of Chen, would also be drawn to an overall sensitive assay.
Thus, claims 21-22 are prima facie obvious over Gines, in view of Chen, and in view of Rondelez.
Conclusion
No claims are currently allowable.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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/F.F.G./Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
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