DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
2. Applicant's election with traverse of Group II in the reply filed on March 25, 2026 is acknowledged. The traversal is on the grounds the method claims, represented by independent claim 1, uses the molecular beacon recited in claims 8-10, which was specifically designed for
CRISPR/Cas activity detection. Any alternative use (e.g., SNP detection via Cas enzymes) of the
molecular beacon of Group II would still fall within the method of Group I. This argument has been fully considered but is not persuasive. The MB probe of Group II could be used in methods of detection that do not require Cas enzymes. Just because the MB has a substrate for Cas in the loop section does not mean that it can only be used in methods which use Cas. The requirement is still deemed proper and is therefore made FINAL.
3. Claims 1-10 are currently pending.
Claims 1-7 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on March 25, 2026.
Claim Rejections - 35 USC § 103
4. 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.
5. Claims 8 and 10 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (ACS Sens 2020 5, 557-562) in view of Wang (Bioactive Materials 17 1/7/2022 360-368) and Green (ACS Nano 4/27/2021 15, 9101-9110).
Regarding Claims 8 and 10 Zhang discloses a an electrochemical biosensor based on Cas12a-mediated interfacial cleaving of hairpin DNA (hpDNA) reporter (see Scheme 1 shown below). First, crRNA, which contains a repeat sequence and a programmable target specific sequence, can bind into Cas12a for the formation of a Cas12a-crRNA duplex. Then, the Cas12a-crRNA duplex can specifically recognize and cut target DNA based on crRNA sequence and the protospacer adjacent motif (PAM) located in the target. After that, nonspecific nearby ssDNA can be digested by a Cas12a-crRNA-target DNA ternary complex. Here, an hpDNA consisting of a thiol group at the 5′ terminus and a methylene blue (MB) tag at the 3′ terminus is covalently modified to a gold electrode (GE) through S−Au bonding. In the absence of the target, the Cas12a cannot cleave the hpDNA reporter. So, the formation of a stem-loop structure brings the MB tag near the GE surface, resulting in a high redox response being detected. In the presence of the target, the ssDNase activity of Cas12a is activated, cleaving the loop region of hpDNA into short fragments and leading to the dissociation of the stem part of hpDNA. The melting
temperature (Tm) of the duplex stem decreases from about 48 °C to lower than 10 °C, therefore releasing the MB from GE and decreasing the peak current. Therefore, the established electrochemical biosensor could convert per target recognition event into numerous disintegration of the hpDNA reporter on the interface for highly sensitive electrochemical DNA biosensing (pages 558-559).
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Thus Zhang teaches a MB probe attached to a gold electrode surface, wherein the loop portion of the MB probe is a substrate for a Cas12 enzyme and wherein the gold electrode quenches the signal of the methylene blue tag on the 3’ end of the MB. Zhang further teaches a Cas enzyme having a guide RNA complementary to a target nucleic acid.
Zhang does not teach a MB attached to a quantum dot (QD). Zhang does not teach a MB that comprises a quencher that is configured as a FRET partner of the QD. Zhang does not teach that the MB positions the quencher to quench QD fluorescence. Additionally Zhang does not teach the target nucleic acid is a marker of a disease.
However Wang teaches that quantum dot molecular beacon (QD-MB) functionalized MoS2 (QD-MB @ MoS2) fluorescent probes were designed from the dual detection of multiple myeloma (MM) related miRNA-155 and miRNA-150 (abstract). The design principle and preparation process are shown in Fig. 1. The 3′ of MB is labeled with biotin and the 5′ with was modified with 15 cytosines (PolyC15). The biotin can be coupled with the streptavidin on the surface of QDs, and PolyC15 can absorb on MoS2 nanosheets by van der Waals force(Fig. 1(a), Step 1). At this time, QD is moving closer to MoS2, resulting in significant quenching of QD fluorescence. In the presence of target miRNA, the hybridization of miRNA and MB results in the formation of DNA/RNA heteroduplex, leading to the QD moving away from the MoS2 nanosheet and partially restoring the fluorescence of QD (Fig. 1(a), step 2). In the following step with the assistance of the DSN molecule, the MB in the MB/RNA heteroduplex can be cut into fragments by DSN, resulting in the release of QDs and miRNAs from MoS2 nanosheets (Fig. 1, step 3), which enhances the fluorescence of the QD and allows the target miRNA circulated in the cycling signal amplification (Fig. 1(b), step 4) (abstract, page 361, col 2).
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Thus Wang teaches a MB attached to a quantum dot (QD). Wang teaches a MB that comprises a quencher (MoS2) that is configured as a FRET partner of the QD. Wang teaches that the MB positions the quencher to quench QD fluorescence. Wang teaches the target nucleic acids are markers of multiple myeloma.
Accordingly, 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 Zhang by attaching the MB to a QD and modifying the MB so that it comprises a quencher capable of quencher the signal from the QD as suggested by Wang. One of skill in the art would have been motivated to attach the MB to a QD particularly since Wang teaches that QD is a new type of fluorescence materials with distinct advantages of strong intensity, high stability, strong anti-bleaching ability, wide excitation spectrum, narrow emission spectrum, and adjustable emission wavelength [5–9]. The effective bandgap of QDs is increased with the decrease of particle radius, resulting in a blue shift of their absorption and emission. Therefore, the position of their excitation and emission can be adjusted only by changing their size. Moreover, the advantage of a wide excitation spectrum can realize the simultaneous excitation of different QDs under the same excitation. Because the fluorescence spectrum of QDs is relatively narrow, the fluorescence peaks of multiple QDs excited at the same time are not easy to overlap, which can prevent the crosstalk and interruption. Overall, these excellent fluorescence characteristics of QDs make it possible to realize simultaneous detection of biomolecules (page 360).
The combined references do not teach a QD attached to a MB by a peptide-peptide nucleic acid (PNA). The combined references do not teach that a peptide-PNA comprises a PNA sequence and a polyhistidine sequence for binding to the QD. The combined references do not teach that a PNA sequence binds to a nucleic acid sequence of the MB.
However Green discloses conjugation of quantum dots to DNA nanostructures with peptide-PNA. Green teaches that the conjugation of nanoparticle-binding peptides and peptide nucleic acids (PNA) can produce peptide-PNA with distinct NP-binding and DNA-binding domains. Green demonstrates a simple application of this method to conjugate semiconductor quantum dots (QDs) directly to DNA nanostructures by means of a peptide-PNA with a six-histidine peptide motif that binds to the QD surface. Green teaches that with this method, we achieved greater than 90% capture efficiency for multiple QDs on a single DNA nanostructure while preserving both site specificity and precise spatial control of QD placement (abstract).
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Thus Green teaches QD attached to a nucleic acid sequence by a peptide-peptide nucleic acid (PNA). Green teaches that a peptide-PNA comprises a PNA sequence and a polyhistidine sequence for binding to the QD. Green teaches that a PNA sequence binds to a nucleic acid thereby attaching the nucleic acid to the QD.
Accordingly, 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 QD-MB of Zhang in view Wang by using a peptide-PNA to attach the QD to the MB as suggested by Green. In the instant case Wang teaches that a biotin on the 3’ end of MB can be coupled with streptavidin on the surface of the QD (page 361, col 2). Green teaches that to label QD with DNA, the most common strategy employed to date is the use of biotinylated DNA in conjunction with streptavidin immobilized on the QD surface. Green teaches that while this method is efficient it also has several disadvantages (page 9101-9102). Green teaches they have developed a new method which uses peptide-PNAs to conjugate DNA to QD (abstract). One of skill in the art would have been motivated to use peptide-PNAs to conjugate DNA to QD particularly since Green teaches that with this method, we achieved greater than 90% capture efficiency for multiple QDs on a single DNA nanostructure while preserving both site specificity and precise spatial control of QD placement (abstract).
6. Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang (ACS Sens 2020 5, 557-562) in view of Wang (Bioactive Materials 17 1/7/2022 360-368) and Green (ACS Nano 4/27/2021 15, 9101-9110) as applied to claim 8 above and in further view of Mahani (Microchimica Acta (2019) 186:132).
The teachings of Zhang, Wang, and Green are presented above.
The combined references do not teach a MB when the quencher is a dye.
However Mahani discloses that a carbon quantum dot (CQD) labeled molecular beacon was synthesized and applied to the detection of microRNA-21. The molecular beacon (MB) was labeled with the CQDs at the 5′ end, and with Black Hole Quencher 1 (BHQ1) at the 3′ end. The two labels act as the donor and acceptor parts of a FRET system, respectively. Only weak fluorescence is observed in the absence of microRNA-21, and in the presence of scrambled or mismatched sequences. However, in the presence of microRNA-21, fluorescence intensity of the CQDs at 460 nm (excitation at 360 nm) recovers. The hybridization of the hairpin structure of the MB with microRNA-21 opens the loop of MB. Consequently, the distance between the BHQ1 quencher and the CQDs is increased and fluorescence changes. The probe has high sensitivity (with a 0.3 nM limit of detection) and specificity. It can distinguish between microRNA-21 and its single mismatch mutant and hence represents a valuable tool for the early cancer diagnosis (abstract, Fig 1).
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Thus Mahani teaches a MB when the quencher is a dye (Black hole quencher 1).
Accordingly, 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 QD-MB of Zhang, Wang, and Green by using a dye as the quencher as suggested by Mahani. In the instant case Mahani teaches using a dye (black hole quencher 1) to quench the signal of a quantum dot. One of skill in the art would have been motivated to use this dye as a quencher on a QD-MB since it was known in the art that these two labels (quantum dots and BHQ1) act as donor and acceptor parts of a FRET system.
7. Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMANDA HANEY whose telephone number is (571)272-8668. The examiner can normally be reached Monday-Friday, 8:15am-4:45pm EST.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Wu-Cheng Shen can be reached at 571-272-3157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/AMANDA HANEY/Primary Examiner, Art Unit 1682
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