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 4/16/2025, 4/16/2025, 4/16/2025 has been considered by the examiner.
Election/Restrictions
Applicant's election of Group I, Claims 1-13 and 19-20, without traverse in the reply filed on 01/29/2026 is acknowledged.
Specification
Applicant is reminded of the proper language and format for an abstract of the disclosure.
The abstract should be in narrative form and generally limited to a single paragraph on a separate sheet preferably within the range of 50 to 150 words in length. The abstract should describe the disclosure sufficiently to assist readers in deciding whether there is a need for consulting the full patent text for details.
The language should be clear and concise and should not repeat information given in the title. It should avoid using phrases which can be implied, such as, "The disclosure concerns," "The disclosure defined by this invention," "The disclosure describes," etc. In addition, the form and legal phraseology often used in patent claims, such as "means" and "said," should be avoided.
The abstract of the disclosure is objected to because “circuitry configured to conduct SWV to apply the electric current to the conductive substrate; and circuitry configured to conduct KDM to determine concentration of the analyte based changes in the electric current caused by conformational changes in the aptamer upon an interaction between the aptamer and the analyte” appear on the sheet of abstract. Correction is required. See MPEP § 608.01(b).
35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, requires the specification to be written in “full, clear, concise, and exact terms.” The specification is replete with terms which are not clear, concise and exact. The specification should be revised carefully in order to comply with 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112. Examples of some unclear, inexact or verbose terms used in the specification are: In Fig.9, the 3rd equation for calculating the target concentration contradicts to the 2nd equation since the equation for calculating [target] cannot be derived from the 2nd equation. Furthermore, this instant application is similar to applicant’s own journal publication: Nguyen et al. (Label-free, real-time monitoring of cytochrome C drug response in microdissected tumor biopsies with a multi-well aptasensor platform, Science Advances, 2024, 10, eadn5875), wherein the analyte concentration is calculated by equation (2) on page 14, which was derived from equation 1 on page 14. Note that the equation (1) on page 14 of the above journal publication is essentially the same as the 2nd equation in Fig.9 of this instant application. Thus, the equation for calculating the analyte concentration in [para. 0018, 0052, 0138] in PG-pub and the 3rd equation in Fig.9 of the instant specification are not correct, and also contradict to the equation 2 for calculating the analyte concentration in [para. 0124] in Pg-Pub.
Claim Objection
Claims 1-2, 7, 12-13, and 19-20 are objected to because of the following informalities:
Claim 1: please amend “concentration of the analyte” in Ln 7 to – the concentration of the analyte--.
Claim 2: please amend “an electrical current” in Ln 2 to – [[an]] the electrical current--; “circuitry configured to conduct SWV” to – the circuitry configured to conduct SWV--; “circuitry configured to conduct KDM” to – the circuitry configured to conduct KDM--; “concentration of the analyte” to --the concentration of the analyte--.
Claim 7: please amend “the 3’ terminus” to – [[the]] a 3’ terminus--.
Claim 12: please amend “concentration of the analyte” (two places) to – the concentration of the analyte--.
Claim 13: please amend “according to SWV” to -- according to the SWV--; “according to KDM” to -- according to the KDM--.
Claim 19: please amend “a change in an electric current” to -- a change in [[an]] the electric current--.
Claim 20: please amend “the aptamer operably connected to the conductive substrate;” to -- the aptamer operably connected to the conductive substrate[[;]].--.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 2, 4-10 and 12-13 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as failing to set forth the subject matter which the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the applicant regards as the invention.
Regarding claim 2, claim 2 recites wherein the aptamer comprises: a redox reporter and a nucleic acid aptamer. The ordinary meaning of an aptamer is a short, synthetic, single-stranded DNA or RNA molecule. Where applicant acts as his or her own lexicographer to specifically define a term of a claim contrary to its ordinary meaning, the written description must clearly redefine the claim term and set forth the uncommon definition so as to put one reasonably skilled in the art on notice that the applicant intended to so redefine that claim term. Process Control Corp. v. HydReclaim Corp., 190 F.3d 1350, 1357, 52 USPQ2d 1029, 1033 (Fed. Cir. 1999). The term “aptamer” in claim 2 is used by the claim to mean a complex comprising a redox reporter and a nucleic acid aptamer, while the accepted meaning is a single-stranded DNA or RNA molecule without a redox reporter. The term is indefinite because the specification does not clearly redefine the term. Therefore, the scope of claim 2 is indefinite. Claims 4-10 are further rejected by virtue of their dependence upon and because they fail to cure the deficiencies of indefinite claim 2.
Regarding claim 12, claim 12 recites an equation to calculate concentration of the analyte. As explained in the specification objection, the equation contradicts to the 2nd equation in Fig.9 and equation 2 in [para. 0124] in PG-Pub of the instant specification. A claim, although clear on its face, may also be indefinite when a conflict or inconsistency between the claimed subject matter and the specification disclosure renders the scope of the claim uncertain as inconsistency with the specification disclosure or prior art teachings may make an otherwise definite claim take on an unreasonable degree of uncertainty. In re Moore, 439 F.2d 1232, 1235-36, 169 USPQ 236, 239 (CCPA 1971); In re Cohn, 438 F.2d 989, 169 USPQ 95 (CCPA 1971); In reHammack, 427 F.2d 1378, 166 USPQ 204 (CCPA 1970) [see MPEP 2173.03]. Thus, the scope of claim 12 is indefinite.
Regarding claim 13, claim 13 recites “when executed by the processor”, wherein “the processor” lacks antecedent basis. Thus, the scope of claim 13 is indefinite.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 20 rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. The limitations of claim 20 are already recited in claim 19. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 102
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 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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-2, 4-5, 11, 13, and 19-20 are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Plaxco et al. (US20210196161A1). Mirceski et al. (Square-wave voltammetry, ChemTexts, 2018, 4, 17) is an evidence for claim 11.
Regarding claim 1, Plaxco teaches an integrated electrochemical aptamer-based (E-AB) sensor (an E-AB sensing platform as shown in Fig.1A [para. 0009]) configured to measure a concentration of an analyte (measuring the concentration of a target species in a sample by the use of an electrochemical sensor [para. 0091; claim 1]; thus the E-AB sensor is configured to perform the intended use of measuring a concentration of an analyte), the integrated E-AB sensor comprising:
an aptamer operably connected to a conductive substrate (an aptamer 102 bound to an electrode substrate 101 in Fig.1A [para.0009]);
circuitry configured to conduct square wave voltammetry (SWV) to apply an electric current to the conductive substrate (Fig.1B depicts a representative square wave voltammetry trace for this type of E-AB sensor wherein peak current is higher in the presence than in the absence of target [para. 0009]; All SWV measurements were performed using a three-electrode setup and with a CH Instruments electrochemical workstation [para. 0121]; the CHI electrochemical workstation is deemed as the circuitry configured to conduct SWV to apply an electric current to the conductive substrate); and
circuitry configured to conduct kinetic differential measurement (KDM) (the scope of the invention encompasses non-transitory computer-readable recording media having stored thereon data and/or an encoding program that causes a computer to execute series of operations. The computer may comprise any general purpose computer, processor, embedded processor, mobile device, or other computing device [para.0087]; Kinetic differential measurements correction techniques applied to the measurements provide a corrected signal [para. 0014]; the computer is deemed as the circuitry configured to conduct KDM) to determine concentration of the analyte based on changes in the electric current caused by conformational changes in the aptamer upon an interaction between the aptamer and the analyte (calculating the concentration of the target species by application of a mathematical relationship between selected measure of current decay and the concentration of the target species in the sample [claim 1]; the scope of the invention encompasses computer programs, software, and operations which enable the acquisition and interpretation of electrochemical sensor outputs to measure target concentration [para. 0008]; aptamer undergoes a conformational change upon target binding [para. 0097, 0105; Fig.1A]; signals via a binding-induced conformational change that alters electron transfer from the reporter, leading in turn to an easily measurable electrochemical output [para. 0109; Fig.1B]).
Regarding claim 2, Plaxco teaches the integrated E-AB sensor of claim 1, wherein the aptamer comprises:
a redox reporter (methylene blue redox reporter 103 in Fig.1A [para. 0009]) configured to modify an electric current to and/or from the conductive substrate of the integrated E-AB sensor (When target 105 binds to the aptamer, the resulting conformation change alters the proximity of the reporter 103 to the substrate 101, increasing the rate of electron transfer 104, and Fig.1B depicts a representative SWV for this type of E-AB sensor wherein peak current is higher in the presence than in the absence of target [para. 0009; Figs.1A-1B ]; thus the MB redox reporter is configured to perform the claimed function above); and
a nucleic acid aptamer (aptamer 102 in Fig.1A [para.0009]; the aptamer may comprise a DNA aptamer, RNA aptamer, or an aptamer comprising non-natural nucleic acids [para. 0022]) comprising a polynucleotide sequence (nucleic acid aptamer inherently comprises a polynucleotide sequence) configured to interact with the analyte (target 105 binds to the aptamer [para. 0009]), wherein an interaction between the polynucleotide sequence and the analyte causes a conformational change in the nucleic acid aptamer, a positional change in the redox reporter, and a change in the electric current of the integrated E-AB sensor (When target 105 binds to the aptamer, the resulting conformation change alters the proximity of the reporter 103 to the substrate 101, increasing the rate of electron transfer 104 [para. 0009; Figs. 1A-1B]; current flows are altered by target binding and are assessed by voltammetric methods including SWV [para. 0016]);
wherein the electric current is applied with the circuitry configured to conduct SWV (All SWV measurements were performed using a three-electrode setup and with a CH Instruments electrochemical workstation [para. 0121]; thus the CHI electrochemical workstation is configured to apply the electric current) and the change in the electric current is processed with the circuitry configured to conduct KDM to determine concentration of the analyte (non-transitory computer-readable recording media having stored thereon data and/or an encoding program that causes a computer to execute series of operations [para. 0087]; calculating the concentration of the target species by application of a mathematical relationship between selected measure of current decay and the concentration of the target species in the sample [claim 1]; the scope of the invention encompasses computer programs, software, and operations which enable the acquisition and interpretation of electrochemical sensor outputs to measure target concentration [para. 0008]).
Regarding claim 4, Plaxco teaches the integrated E-AB sensor of claim 2, wherein the nucleic acid aptamer comprises deoxyribonucleic acid (DNA) (DNA aptamer [para. 0022]) and the polynucleotide sequence comprises a DNA sequence (DNA aptamer inherently comprises a DNA sequence).
Regarding claim 5, Plaxco teaches the integrated E-AB sensor of claim 2, wherein the redox reporter comprises methylene blue (MB) (methylene blue redox reporter 103 in Fig.1A [para. 0009]).
Regarding claim 11, Plaxco teaches the integrated E-AB sensor of claim 1, wherein the circuitry configured to conduct SWV uses a combined square wave applied to the conductive substrate of the integrated E-AB sensor (For the SWV measurements, the sensors were interrogated from 0.0 V to −0.5 V versus Ag/AgCl, using an amplitude of 50 mV, potential step sizes of 1-5 mV, and varying frequencies from 10 Hz to 500 Hz. All SWV measurements were performed using a three-electrode setup and with a CH Instruments electrochemical workstation [para. 0121]. As evidenced by Mirceski, SWV does use a combined square wave as shown in Fig.5b. Thus, the CHI electrochemical workstation is configured to use a combined square wave applied to the conductive substrate of the integrated E-AB sensor).
Regarding claim 13, Plaxco teaches the integrated E-AB sensor of claim 1, wherein the circuitry configured to conduct SWV and the circuitry configured to conduct KDM are implemented as a non-transitory computer-readable storage medium having instructions stored thereon which, when executed by a processor (the scope of the invention encompasses non-transitory computer-readable recording media having stored thereon data and/or an encoding program that causes a computer to execute series of operations. wherein the data and/or series of operations causes the operation of an electrochemical sensing system to effect the methods of the invention. the non-transitory computer-readable recording media may effect operations such as: controlling a potentiostat or equivalent device to deliver a series of stepped voltage pulses to a deployed electrochemical sensor; controlling a data collection device to record sensor current outputs following the delivery of voltage pulses; controlling a processor to perform calculations that derive one or more selected measures of current decay from sensor output data; storing a calibration curve that relates a selected measure of current decay to target concentration; controlling a processor to calculate target concentration based on sensor outputs and a stored calibration curve; storing instructions for the performance of the methods of the invention; and other operations of the invention [para.0087-0088]), configure the processor to:
apply the electric current according to SWV (controlling a potentiostat or equivalent device to deliver a series of stepped voltage pulses to a deployed electrochemical sensor [para. 0088]);
detect the change in the electric current (controlling a data collection device to record sensor current outputs following the delivery of voltage pulses [para. 0088]); and
process the change in the electric current according to KDM (controlling a processor to perform calculations that derive one or more selected measures of current decay from sensor output data; storing a calibration curve that relates a selected measure of current decay to target concentration; controlling a processor to calculate target concentration based on sensor outputs and a stored calibration curve [para. 0088]; Kinetic differential measurements correction techniques applied to the measurements provide a corrected signal [para. 0014]).
Regarding claim 19, Plaxco teaches a kit (kits and systems [para. 0090]) for measurement of an analyte (for calculating target concentration [para. 0090-0091]; an electrochemical sensor is any sensor that is capable of measuring the concentration of a target species in a sample [para. 0021]), the kit comprising:
an integrated E-AB sensor (E-AB sensor in Fig.1A [para. 0009]), comprising:
an aptamer operably connected to a conductive substrate of the E-AB sensor (an aptamer 102 bound to an electrode substrate 101 in Fig.1A [para.0009]);
circuitry configured to conduct SWV to apply an electric current to the conductive substrate (Fig.1B depicts a representative square wave voltammetry trace for this type of E-AB sensor wherein peak current is higher in the presence than in the absence of target [para. 0009]; All SWV measurements were performed using a three-electrode setup and with a CH Instruments electrochemical workstation [para. 0121]; the CHI electrochemical workstation is deemed as the circuitry configured to conduct SWV to apply an electric current to the conductive substrate); and
circuitry configured to conduct KDM (the scope of the invention encompasses non-transitory computer-readable recording media having stored thereon data and/or an encoding program that causes a computer to execute series of operations. The computer may comprise any general purpose computer, processor, embedded processor, mobile device, or other computing device [para.0087]; Kinetic differential measurements correction techniques applied to the measurements provide a corrected signal [para. 0014]; the computer is deemed as the circuitry configured to conduct KDM) to determine concentration of the analyte based on a change in an electric current caused by a conformational change in the aptamer upon an interaction between the aptamer and the analyte (calculating the concentration of the target species by application of a mathematical relationship between selected measure of current decay and the concentration of the target species in the sample [claim 1]; the scope of the invention encompasses computer programs, software, and operations which enable the acquisition and interpretation of electrochemical sensor outputs to measure target concentration [para. 0008]; aptamer undergoes a conformational change upon target binding [para. 0097, 0105; Fig.1A]; signals via a binding-induced conformational change that alters electron transfer from the reporter, leading in turn to an easily measurable electrochemical output [para. 0109; Fig.1B]); and
instructions for a use of the kit in a method for measuring the analyte with the kit (instructions for performing the methods of the invention [para. 0090]; [para. 0048] details a method of measuring the concentration of a target species in a sample by the use of an E-AB sensor).
Regarding claim 20, Plaxco teaches the kit of claim 19, wherein the integrated E-AB sensor comprises:
the aptamer operably connected to the conductive substrate (an aptamer 102 bound to an electrode substrate 101 in Fig.1A [para.0009]);
the circuitry configured to conduct SWV to apply the electric current to the conductive substrate (as outlined in the rejection of claim 19 above, the CHI electrochemical workstation is configured to conduct SWV to apply the electric current to the conductive substrate); and
the circuitry configured to conduct KDM to determine the concentration of the analyte based on changes in the electric current caused by conformational changes in the aptamer upon the interaction between the aptamer and the analyte (as outlined in the rejection of claim 19 above, the computer is configured to conduct KDM to determine the concentration of the analyte based on the change in the electric current caused by the conformational change in the aptamer upon the interaction between the aptamer and the analyte).
Note: for compact prosecution claim 20 is examined as if it included the portion “circuitry configured to conduct SWV to apply the electric current to the conductive substrate; and circuitry configured to conduct KDM to determine concentration of the analyte based changes in the electric current caused by conformational changes in the aptamer upon an interaction between the aptamer and the analyte” above the abstract. Note that claims should be completely separated from the abstract and any other documents.
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 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.
Claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Plaxco, as applied to claim 1 above, and in view of Pluznick et al. (US20190064161A1).
Regarding claim 3, Plaxco teaches the integrated E-AB sensor of claim 1, and is silent to wherein a form factor of the integrated E-AB sensor comprises a dip-stick form factor.
Pluznick teaches an E-AB sensor comprises a dip-stick form factor (One preferred ligand binding part of Olfr90 is an aptamer representing the binding site of Olfr90 and the aptamer is labeled with a tag, a fluorophore, for example. An Olfr90, or the ligand binding part thereof, such as an aptamer is preferably attached to a surface such as a strip or a dip stick [para. 0008; claim 17]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the E-AB sensor to comprise a dip-stick form factor, as taught by Pluznick, since Pluznick teaches an alternative suitable configuration of the E-AB sensor having the aptamer attached to a surface such as a dip stick [para. 0008; claim 17].
Claims 6-7 are rejected under 35 U.S.C. 103 as being unpatentable over Plaxco, as applied to claim 2 above, and in view of Plaxco et al. (WO2020252401A1; hereinafter Plaxco’401).
Regarding claims 6-7, Plaxco teaches the integrated E-AB sensor of claim 2, and Plaxco teaches wherein the redox reporter is linked to the nucleic acid aptamer (an aptamer 102 is modified with a methylene blue redox reporter 103 [para. 0009]; Fig.1A shows the MB redox reporter is linked to the distal end of the aptamer 102).
Plaxco does not explicitly teaches wherein the redox reporter is linked to the nucleic aptamer “at a terminus of the nucleic acid aptamer” (of claim 6); and wherein the terminus is the 3′ terminus of the nucleic acid aptamer (of claim 7).
Plaxco’401 teaches an E-AB sensor (claims 1 and 4; E-AB sensor [para. 0100]), wherein the aptamer is functionalized with -SH at the 5’ end for attachment to the SAM functionalized gold electrode surface and functionalized at the 3’ end with methylene blue [para. 0026]. Thus, Plaxco’401 teaches wherein the redox reporter of methylene blue is linked to the nucleic acid aptamer at the 3’ terminus of the nucleic acid aptamer.
Given the teachings of Plaxco regarding methylene blue (MB) redox reporter is linked to the nucleic acid aptamer at the distal end (see Fig.1A), and the teachings of Plaxco’401 regarding methylene blue is linked to the nucleic acid aptamer at the 3’ terminus of the nucleic acid aptamer, It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the E-AB sensor of Plaxco to have the MB redox reporter linked to the nucleic acid aptamer at the 3’ terminus of the nucleic acid aptamer, as taught by Plaxco’401, since Plaxco’401 teaches it would be suitable to functionalize the aptamer at the 3’ end for attaching the MB redox reporter [para. 0026].
Claims 8-9 are rejected under 35 U.S.C. 103 as being unpatentable over Plaxco, as applied to claim 2 above, and in view of Poturnayova et al. (Optimization of cytochrome c detection by acoustic and electrochemical methods based on aptamer sensors, Sensors and Actuators B: Chemical, 2017, 238, 817-827).
Regarding claims 8-9, Plaxco teaches the integrated E-AB sensor of claim 2, and is silent to: (1) wherein the analyte comprises Cytochrome C (Cyt-C) and the polynucleotide sequence comprises a DNA sequence specific for interaction with Cyt-C (of claim 8); and (2) wherein the analyte comprises Cyt-C and the polynucleotide sequence comprises a DNA sequence specific for interaction with Cyt-C, wherein the DNA sequence specific for interaction with Cyt-C comprises a DNA sequence that is at least 80% identical to SEQ ID NO:1 (of claim 9).
Poturnayova teaches an E-AB sensor for the detection of Cyt-C (title and abstract), wherein the aptamer 40 specific for interaction with Cyt-C comprises a DNA sequence:
5′- CC GTG TCT GGG GCC GAC CGG CGC ATT GGG TAC GTT GTT GC-3′ (see apt 40 in Fig.1). Note that the SEQ ID NO:1 of this instant application is: 5′-Thio-C6-Disulfide/CC GTG TCT GGG GCC GAC CGG CGC ATT GGG TAC GTT GTT GC/—NH2-3′ [para. 0016 ] in PG-Pub of this instant specification. Thus, the disclosed DNA sequence of the aptamer 40 is essentially the same as the claimed SEQ ID NO:1 except the modifiers at the 5’ terminus and the 3’ terminus.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the nucleic acid aptamer in Plaxco with the aptamer 40 having the DNA sequence of 5′- CC GTG TCT GGG GCC GAC CGG CGC ATT GGG TAC GTT GTT GC-3′ and take the modified E-AB sensor for detecting analyte of Cyt-C, as taught by Poturnayova, since it would allow to detect the analyte of Cyt-C, which takes part in essential biological functions as electron carrier and metal-ion liaison (Introduction in Poturnayova).
With the substituted aptamer 40 as the aptamer specific for detecting Cyt-C as the analyte, modified Plaxco teaches: (1) wherein the analyte comprises Cytochrome C (Cyt-C) and the polynucleotide sequence comprises a DNA sequence specific for interaction with Cyt-C (claim 8); and (2) wherein the analyte comprises Cyt-C and the polynucleotide sequence comprises a DNA sequence specific for interaction with Cyt-C, wherein the DNA sequence specific for interaction with Cyt-C comprises a DNA sequence that is at least 80% identical to SEQ ID NO:1 (claim 9).
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Plaxco, as applied to claim 2 above, and in view of Poturnayova et al. (Optimization of cytochrome c detection by acoustic and electrochemical methods based on aptamer sensors, Sensors and Actuators B: Chemical, 2017, 238, 817-827), Catanante et al. (Sensitive analytical performance of folding based biosensor using methylene blue tagged aptamers, Talanta, 2016, 153, 138-144), Kelley et al. ( US20230107004A1), and McHenry et al. (Voltammetry peak tracking for longer-lasting and reference-electrode-free electrochemical biosensors, Biosensors, 2022, 12, 782).
Regarding claim 10, Plaxco teaches the integrated E-AB sensor of claim 2, and is silent to wherein the analyte comprises Cyt-C and the polynucleotide sequence comprises a DNA sequence specific for interaction with Cyt-C, wherein the DNA sequence specific for interaction with Cyt-C is SEQ ID NO:1.
Poturnayova teaches an E-AB sensor for the detection of Cyt-C (title and abstract), wherein the aptamer 40 specific for interaction with Cyt-C comprises a DNA sequence: 5′- CC GTG TCT GGG GCC GAC CGG CGC ATT GGG TAC GTT GTT GC-3′ (see apt 40 in Fig.1). Note that the SEQ ID NO:1 of this instant application is: 5′-Thio-C6-Disulfide/CC GTG TCT GGG GCC GAC CGG CGC ATT GGG TAC GTT GTT GC/—NH2-3′ [para. 0016 ] in PG-Pub of this instant specification. Thus, the disclosed DNA sequence of the aptamer 40 is essentially the same as the claimed SEQ ID NO:1 except the modifiers at the 5’ terminus and the 3’ terminus.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the nucleic acid aptamer in Plaxco with the aptamer 40 having the DNA sequence of 5′- CC GTG TCT GGG GCC GAC CGG CGC ATT GGG TAC GTT GTT GC-3′ and take the modified E-AB sensor for detecting analyte of Cyt-C, as taught by Poturnayova, since it would allow to detect the analyte of Cyt-C, which takes part in essential biological functions as electron carrier and metal-ion liaison (Introduction in Poturnayova).
With the substituted aptamer 40 as the aptamer specific for detecting Cyt-C as the analyte, modified Plaxco teaches: wherein the analyte comprises Cyt-C and the polynucleotide sequence comprises a DNA sequence specific for interaction with Cyt-C, wherein the DNA sequence specific for interaction with Cyt-C is essentially the same as SEQ ID NO:1 EXCEPT the modifiers at the 5’ terminus and the 3’ terminus.
Modified Plaxco is silent to wherein the modifier at the 5’ terminus is Thio-C6-Disulfide, and the modifier at the 3’ terminus is NH2.
Plaxco further teaches methylene blue (MB) redox reporter 103 is linked to the aptamer 102 at its distal end, and the other end of the aptamer 102 is linked to the electrode substrate 101 ([para. 0009] and Fig.1A).
Catanante teaches E-AB sensor comprising methylene blue as the redox reporter (title and abstract). Typically, the aptamer is modified at the 5’-end for electrode attachment (e.g., thoilated) and at the distal, 3’-end, with a redox probe molecule such as methylene blue (MB) (the first paragraph in Introduction on page 138).
Kelley teaches an E-AB sensor comprising a receptor (which is an aptamer) and a redox reporter (abstract; claims 1, 26-27 and [para. 0035]), and one of the probe sequences is 5′-SH-MC6-TAC CAG CTA TTG TAT CTA ATA AGA-NH2-3′ [para. 0157, 0171]. The redox report is methylene blue ( claim 16; a methylene blue reporter molecule was conjugated to a ssDNA probe [para. 0216]). Thus, Kelley teaches an E-AB sensor wherein the redox reporter is MB, and the modifier of the aptamer at the 3’ terminus is NH2, and the 5’ end of the aptamer is thoilated containing SH group.
Given the teachings of Plaxco regarding MB redox reporter is linked to the aptamer at its distal end; the teachings of Catanante regarding aptamer being typically modified at the 3’-end for MB redox probe and the 5’ end of the aptamer being modified with a modifier containing -SH group for electrode attachment; and the teachings of Kelley regarding the MB redox reporter and the aptamer modified at the 3’-end with an amino modifier (i.e., NH2) and modified at the 5’ end with a modifier containing a thiol group -SH, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the 3’ terminus of the aptamer 40 in modified Plaxco with an amino modifier to provide NH2 at the 3’ terminus, as taught by combined Plaxco, Catanante, and Kelley, since it would allow to attach the MB redox reporter at the 3’ terminus of the aptamer. Note that the 5’ end containing a thiol group -SH would be used for electrode attachment since Catanante teaches the aptamer is modified at the 5’-end for electrode attachment (e.g., thoilated).
Modified Plaxco is silent to wherein the modifier at the 5’ terminus is Thio-C6-Disulfide.
McHenry teaches electrochemical aptamer-based sensor (abstract and Fig.1) comprising aptamer, [/5ThioMC6-D/CGAGG GTACC GCAAT AGTAC TTATT GTTCG CCTAT TGTGG GTCGG/3MeBlN/] with conjugated methylene blue and thiol ends (section 2.1). Fig.1 shows one end of the aptamer is attached to the electrode and the distal end of the aptamer is linked to the redox reporter. Since the 3’-end is conjugated with methylene blue redox reporter, the 5’-end of the aptamer is then attached to the electrode. Thus, McHenry teaches wherein the aptamer is modified at the 5’-end with a modifier of ThioMC6-D for attaching the aptamer to the electrode.
Given the teachings of Catanante regarding the aptamer is typically modified at the 5’-end for electrode attachment (e.g., thoilated); and the teachings of McHenry regarding the aptamer is modified at the 5’-terminus with a modifier of ThioMC6-D for attaching the aptamer to the electrode , it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention modify the 5’-terminus of the aptamer 40 in modified Plaxco with a modifier of ThioMC6-D, as taught by combined Catanante and McHenry, since it would allow to attach the aptamer to the electrode at the 5’-terminus.
With the above modifications of the modifiers at the 3’ terminus and the 5’-terminus, the modified aptamer has the DNA sequence of SEQ ID: No:1.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Plaxco, as applied to claim 1 above, and in view of Downs et al. (Improved calibration of electrochemical aptamer-based sensors, Scientific reports, 2022, 12, 5535).
Regarding claim 12, Plaxco teaches the integrated E-AB sensor of claim 1, and is silent to wherein the circuitry configured to conduct KDM to determine the concentration of the analyte is configured to calculate the concentration of the analyte according to:
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109
450
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wherein: [Analyte] is the concentration of the analyte, optionally in ng/mL; KDMmin is KDM observed in absence of the analyte; KDMmax is KDM expected at saturation of the analyte; and nH is Hill coefficient.
Downs teaches an E-AB sensor (title, abstract and Fig.1), and also teach KDM to determine analyte concentration (see Fig.1 and equations 1-2) using the equation (2):
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94
656
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Note that the above equation (2) for calculating the target analyte concentration is derived from the equation (1):
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74
618
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The above equation (1) is the same as the 2nd equation in Fig.9 of this instant application. Thus, Downs teaches the equation (2), which is derived from the 2nd equation in Fig.9 of this instant application, to calculate the analyte concentration.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the computer program(s)/software in the computer of Plaxco to calculate the concentration of the analyte according to the equation (2) of Downs, since it would allow to calculate the target concentration based on the E-AB sensor output (see Figs. 1D to 1E; the first paragraph on -page 2 in Downs).
Note: As outlined in the specification objection, the claimed equation is inconsistent with the 2nd equation in Fig.9. Examiner interprets the analyte concentration is calculated based on the 2nd equation in Fig.9 by rearranging the 2nd equation (which is the same as equation 1 in Downs) to derive the equation (which is equation 2 in Downs) for target concentration.
Conclusion
The prior arts made of record and not relied upon are considered pertinent to applicant's disclosure: Heeger et al. (US20050112605A1) teaches an E-AB sensor as shown in Fig.1. Xiao et al. (US20210396706A1) teaches an E-AB sensor for detection of fentanyl opioids. Heikenfeld et al. (US20250116626A1) teaches E-AB sensors with signal amplification via multiple redox reporters. Choi et al. (Magnetic response of mitochondria-targeted cancer cells with bacterial magnetic nanoparticles, ChemComm, 2012, 48, 7474-7476) teaches Cyt-C aptamer using the following oligonucleotide: 5’-CCG TGT CTG GGG CCG ACC GGC GCA TTG GGT ACG TTG TTG C-3’, and the DNA sequence is essentially the same the SEQ ID NO:1 except the modifiers at the 3’ and 5’ ends.
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/SHIZHI QIAN/Examiner, Art Unit 1795
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