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 .
Status of the Claims
The Amendment filed December 11, 2025 has been entered. Claims 24, 31-32, 38 and 39 have been amended; and claims 1-23 have been withdrawn. Claims 24-39 are currently examined herein.
Status of the Rejection
Applicant’s amendments to the Claims have overcome each objection previously set forth in the Non-Final Office Action mailed October 2, 2025.
New grounds of claim objection are necessitated by the amendment as outlined below.
All calim interpretations for claims 31 and 38 under 35 U.S.C. 112(f) are essentially maintained and modified in response to the amendment.
All 35 U.S.C. § 103 rejections from the previous office action are withdrawn in view of the Applicant’s amendment.
New grounds of rejection under 35 U.S.C. § 103 are necessitated by the amendments as outlined below.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 11/12/2025 has been considered by the examiner.
Claim Objection
Claims 23, 31-32 and 38 are objected to because of the following informalities:
Claim 23: please amend “one or more biomarkers of interest” in lines 10-11 to – the one or more biomarkers of interest --.
Claim 31: please amend “extracellular vesicles” in line 5 to -- the extracellular vesicles--.
Claim 32: please amend “extracellular vesicles comprising one or more biomarkers of interest” in lines 6-7 to – the extracellular vesicles comprising the one or more biomarkers of interest--.
Claim 38: please amend “extracellular vesicles” in line 7 to – the extracellular vesicles--; “the one or more EV-derived biomarkers of interest” to -- the one or more --.
Appropriate correction is required.
Claim Interpretation
The following is a quotation of 35 U.S.C. 112(f):
(f) Element in Claim for a Combination. – An element in a claim for a combination may be expressed as a means or step for performing a specified function without the recital of structure, material, or acts in support thereof, and such claim shall be construed to cover the corresponding structure, material, or acts described in the specification and equivalents thereof.
The following is a quotation of pre-AIA 35 U.S.C. 112, sixth paragraph:
An element in a claim for a combination may be expressed as a means or step for performing a specified function without the recital of structure, material, or acts in support thereof, and such claim shall be construed to cover the corresponding structure, material, or acts described in the specification and equivalents thereof.
The claims in this application are given their broadest reasonable interpretation using the plain meaning of the claim language in light of the specification as it would be understood by one of ordinary skill in the art. The broadest reasonable interpretation of a claim element (also commonly referred to as a claim limitation) is limited by the description in the specification when 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, is invoked.
As explained in MPEP § 2181, subsection I, claim limitations that meet the following three-prong test will be interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph:
(A) the claim limitation uses the term “means” or “step” or a term used as a substitute for “means” that is a generic placeholder (also called a nonce term or a non-structural term having no specific structural meaning) for performing the claimed function;
(B) the term “means” or “step” or the generic placeholder is modified by functional language, typically, but not always linked by the transition word “for” (e.g., “means for”) or another linking word or phrase, such as “configured to” or “so that”; and
(C) the term “means” or “step” or the generic placeholder is not modified by sufficient structure, material, or acts for performing the claimed function.
Use of the word “means” (or “step”) in a claim with functional language creates a rebuttable presumption that the claim limitation is to be treated in accordance with 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph. The presumption that the claim limitation is interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, is rebutted when the claim limitation recites sufficient structure, material, or acts to entirely perform the recited function.
Absence of the word “means” (or “step”) in a claim creates a rebuttable presumption that the claim limitation is not to be treated in accordance with 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph. The presumption that the claim limitation is not interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, is rebutted when the claim limitation recites function without reciting sufficient structure, material or acts to entirely perform the recited function.
Claim limitations in this application that use the word “means” (or “step”) are being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, except as otherwise indicated in an Office action. Conversely, claim limitations in this application that do not use the word “means” (or “step”) are not being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, except as otherwise indicated in an Office action.
Claims 31 and 38, “means for receiving a first biological sample … into the solid-state device”, is being interpreted under 35 U.S.C. 112(f) . Prong 1: mans for (uses “means for”), prong 2: receiving a first biological sample … into the solid-state device (functional language), prong 3: sufficient structure for performing the function not recited. Therefore, claims 31 and 38 invoke 112(f). The corresponding structure for performing the functions is described in the specification. For example, flowchart 200 may begin at step 202 where the biological sample is placed within the system and passed to the sample preparation chamber 204 [para. 0083, Fig.2].
Claims 31 and 38, “means for transitioning the first biological sample comprising the extracellular vesicles to a dielectrophoresis (DEP) chamber” (for both claims 31 and 38) and “applying an AC to the first biological sample through one or more electrodes of the DEP chamber” (for claim 38 only), is being interpreted under 35 U.S.C. 112(f) . Prong 1: mans for (uses “means for”), prong 2: transitioning the first biological sample comprising the extracellular vesicles to a dielectrophoresis (DEP) chamber and applying an AC to the first biological sample through one or more electrodes of the DEP chamber (functional language), prong 3: sufficient structure for performing the function not recited. Therefore, claims 31 and 38 invoke 112(f). The corresponding structure for performing the functions is described in the specification. For example, one or more syringes 510 of the detection apparatus may be actuated by one or more motors 520, wherein motor 520 may contain one or more gears [para. 0092, Fig.5A]. RF AC waveform generator may apply one or more of a particular waveform across one or more electrodes (e.g., reference electrode 920, counter electrode 930, working electrode 940) [para. 0114].
Claims 31 and 38, “means for passing/transitioning the first biological sample through/via a microfluidics (MF) channel to a tagging chamber”, is being interpreted under 35 U.S.C. 112(f) . Prong 1: mans for (uses “means for”), prong 2: passing/transitioning the first biological sample through/via a microfluidics (MF) channel to a tagging chamber (functional language), prong 3: sufficient structure for performing the function not recited. Therefore, claims 31 and 38 invokes 112(f). The corresponding structure for performing the functions is described in the specification. For example, one or more syringes 510 of the detection apparatus may be actuated by one or more motors 520, wherein motor 520 may contain one or more gears [para. 0092, Fig.5A].
Claim 31, “means for processing the first biological sample in the tagging chamber” is being interpreted under 35 U.S.C. 112(f) . Prong 1: mans for (uses “means for”), prong 2: processing the first biological sample in the tagging chamber (functional language), prong 3: sufficient structure for performing the function not recited. Therefore, claim 31 invokes 112(f). The corresponding structure for performing the functions is described in the specification. For example, in one or more tagging chambers (e.g., tagging chambers 640, 642, 644), EV and/or biomarker specific labels may be cleaved from EV 102 and subsequently chemically bonded to one or more sensors (e.g., sensor 570), wherein the labels may be digitally quantified or quantified through suitable means [para. 0100].
Claims 31 and 38, “means for passing/transitioning the one or more biomarkers of interest of the extracellular vesicles with the respective tags//the first biological sample to a sensor chamber/one or more detector chambers”, is being interpreted under 35 U.S.C. 112(f) . Prong 1: mans for (uses “means for”), prong 2: passing/transitioning the one or more biomarkers of interest with respective tags//the first biological sample to a sensor chamber/one or more detector chambers (functional language), prong 3: sufficient structure for performing the function not recited. Therefore, claims 31 and 38 invoke 112(f). The corresponding structure for performing the functions is described in the specification. For example, one or more syringes 510 of the detection apparatus may be actuated by one or more motors 520, wherein motor 520 may contain one or more gears [para. 0092, Fig.5A].
Claim 31, “means for digitally determining a quantity of the one or more biomarkers of interest of the extracellular vesicles with the respective tags though one or more processes performed in the sensor chamber”, is being interpreted under 35 U.S.C. 112(f) . Prong 1: mans for (uses “means for”), prong 2: digitally determining a quantity of the one or more biomarkers of interest of the extracellular vesicles with the respective tags though one or more processes performed in the sensor chamber (functional language), prong 3: sufficient structure for performing the function not recited. Therefore, claim 31 invokes 112(f). The corresponding structure for performing the functions is described in the specification. For example, one or more detector arrays 710, 720, 730 [para. 0102, Fig.7].
Claim 38, “means for assigning a tag to each of the one or more biomarkers of interest of the first biological sample”, is being interpreted under 35 U.S.C. 112(f) . Prong 1: mans for (uses “means for”), prong 2: assigning a tag to each of the one or more biomarkers of interest of the first biological sample (functional language), prong 3: sufficient structure for performing the function not recited. Therefore, claim 38 invokes 112(f). The corresponding structure for performing the functions is described in the specification. For example, the one or more biological samples may be processed and assigned one or more tags in a plurality of chambers, such as tagging chamber “1” 640, and/or tagging chamber “N” 644 [para. 0102, Fig.7].
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 24-26, 28-29 and 31 are rejected under 35 U.S.C. 103 as being unpatentable over Lo et al. (US20150368635A1), and in view of Tian et al. (Microfluidic separation, detection, and engineering of extracellular vesicles for cancer diagnostics and drug discovery, Accounts of materials research, 2022, 3, 498-510), Moral-Zamora et al. (Combined dielectrophoretic and impedance system for on-chip controlled bacteria concentration: application to Escherichia Coli, Electrophoresis, 2015, 36, 1130-1141), and Hadady et al. (AC electrokinetic isolation and detection of extracellular vesicles from dental pulp stem cells: Theoretical simulation incorporating fluid mechanics, Electrophoresis, 2021, 42, 2018-2026).
Regarding claim 24, Lo teaches a method for isolating, on a solid-state device, one or more biomarkers of interest in a first biological sample (method for capturing, concentrating and isolating molecules in a biofluid such as blood plasma or whole blood on a microfluidic device [title, abstract and Figs. 1A-1C]; Because miRNAs are linked to over 100 diseases, including many types of cancers, they can be used as biomarkers for disease diagnosis [para. 0022] ; Fig.1A shows DNA/RNA capture, release & recapture, and detection; and the target biomolecules [e.g., nucleic acids such as DNA and/or RNA, including miRNA] [para. 0062]; miRNAs are deemed as the one or more biomarkers of interest), the method comprising:
receiving the first biological sample into the solid-state device (Figs. 1A-1C show receiving blood plasma or whole blood from a sample vial or container 158 into the device 110 via the inlet 116 [para. 0044]),
transitioning the first biological sample to a dielectrophoresis (DEP) chamber, wherein one or more processes are performed on the first biological sample in the DEP chamber (lab-on-a-chip device structured to include three sections. The device includes a first section 101 that captures molecules [e.g., including nucleic acids such as double- or single-stranded DNA or RNA including miRNA] from a fluidic sample introduced and flowed through the molecular capture section 101 at high flow rate [para. 0032]; The device 110 includes electric field assisted capture and release functionalities, for example, the device 110 can be used to capture nucleic acids [e.g., DNA or RNA, such as miRNA] contained in the blood that are at a concentration in a femtomolar range or less. [para. 0034]; the device 110 can use electrophoretic and/or dielectrophoretic forces to enable selective capturing of biomolecules in the capture region based on the characteristic frequency response of the dielectric permittivity of the biomolecule versus that of the medium [para. 0041]; Fig.1B shows an array of electrodes 113 formed on a surface within the microfluidic channel 112 along a parallel direction of the microfluidic channel, e.g., constituting a capture region of the device 110. The array of electrodes 113 are operable to produce an electric field across the microfluidic channel 112 that can create an electrostatic attractive force on the molecules [e.g., nucleic acids] to immobilize them within the capture region of the device 110 [para. 0035]; the capture section is deemed as the DEP “chamber”, which will be further modified by Moral-Zamora and Hadady as outlined below);
passing the first biological sample through a microfluidics (MF) channel to a tagging chamber (The device 110 includes a microchamber 114 formed on the substrate 111 at the end of the microfluidic channel 112 to receive the captured molecules after they are released from the capture region [para.0036; Fig.1B]; the microchamber 114 is deemed as the tagging chamber and the microfluidic channel 112 is deemed as the MF channel);
processing the first biological sample in the tagging chamber, wherein the one or more biomarkers of interest of the first biological sample are tagged based on one or more characteristics (the microchamber 114 can include molecular probes capable of binding the molecules in the chamber 114 for detection, e.g., via hybridization for examples when the target molecules are nucleic acids [para. 0036]; In some implementations, for example, molecular probes can be functionalized to the surface of the substrate 111 in the recapture section 114 for assistance in optical detection and characterization of the recaptured miRNAs. For example, the molecular probes can be configured to have complimentary base pairs specific to the target miRNA, and thereby hybridize only with the target miRNAs recaptured in the recapture section 114 [para. 0046]; the target miRNAs are deemed as the one or more biomarkers of interest of the first biological sample, which are tagged by molecular probes having complimentary base pairs specific to the target miRNA);
passing the one or more biomarkers of interest with the respective tags to a sensor chamber (the microchamber 114 can serve as an interface section that introduces any desired amount of miRNAs to the detection region 115 [para. 0047]; the device 110 can further include one or more detection regions or chambers 115 formed on the substrate 111 to provide an optical or electrical interrogation section to characterize [e.g., quantify] the presence, concentration, and/or properties of the captured molecules [para. 0037]; each detection region/chamber 115 is deemed as the sensor chamber); and
digitally determining a quantity of the one or more biomarkers of interest with the respective tags through one or more processes performed in the sensor chamber (the device 110 can further include one or more detection regions or chambers 115 formed on the substrate 111 to provide an optical or electrical interrogation section to characterize [e.g., quantify] the presence, concentration, and/or properties of the captured molecules [para. 0037]).
In the above, the capture section of the microfluidic channel is deemed as the DEP chamber. However, Lo is silent to the caption section is a “chamber”. Thus, Lo is silent to the following limitations: (1) the one or more biomarkers of interest in the first biological sample is extracellular vesicle-derived (EV-derived) biomarkers of interest in the first biological sample containing extracellular vesicles, wherein the extracellular vesicles comprise the one or more biomarker of interest; and (2) wherein the DEP caption section is a DEP chamber.
Tian teaches extracellular vesicles (EVs) are cell-derived submicron bioparticles composed of lipid bilayer membrane and molecular cargos, acting as important mediators of physiopathological cellular processes. The analysis and engineering of EVs hold significant therapeutic potential in noninvasive cancer diagnostics and innovative drug delivery systems. Tian further reviews the state-of-the-art advances in the development of microfluidic platforms for EV separation, detection and engineering. These EV-associated biomarkers reveal great potential for the diagnosis, monitoring, and prognosis of cancer (abstract; TOC graph; Fig.1). Fig.1 shows a solid-state device (microfluidic platform) for efficient separation, detection, and engineering EV-derived biomarkers of interest in a biological sample containing EVs, wherein the EVs comprise at least one biomarker of interest (the 3rd paragraph in Col. 1 on page 500; detection of EV miRNA [section 3.1.3]). Tain further teaches DEP-based isolation of EVs from undiluted plasma. By exploration of the difference between the dielectric properties of EVs and plasma, EVs could be accumulated at the DEP high-field regions around, while small plasma proteins remailed unaffected by the DEP field. After washing step, the purified EVs are eluted from the microfluidic device using a reverse electric field. Moreover, ACE chip could be integrated with in situ immunofluorescence detection for analysis EV proteins in plasma samples. The levels of EV-associated proteins (glycan-1 and CD63) in plasma from pancreatic cancer patients were found to be much higher than those from health controls (Fig.3a and section 2.2). Section 3.1.3 details thermophoretic fluorescence detection of EV RNAs and Fig.6 shows thermophoretic fluorescence sensing EV mRNAs. FDT contained two recognition sequences complementary to the target mRNA, and the ends of two sequences of FDT were labelled with Cy3 and Cy5, respectively. In the presence of target mRNA inside EVs, an increased signal was observed arising from the close proximity of Cy3 and Cy5, due to the hybridization of recognition sequences and mRNA (section 3.1.3).
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 method for isolating and detecting the one or more biomarkers (miRNA) in blood plasma in Lo to isolate and detect one or more biomarkers of interest such as miRNA in the first biological sample (blood plasma) containing extracellular vesicles, wherein the extracellular vesicles comprise the one or more biomarkers of interest (such as miRNA), as taught by Tian, since it would allow for efficient separation and detection EV-associated biomarkers, which reveal great potential for the diagnosis, monitoring, and prognosis of cancer (the 3rd paragraph in Col. 1 on page 500; the first paragraph in section 2.2; and abstract in Tian).
With the above modification, the one or more biomarkers of interest (miRNAs) in Lo is accordingly modified to the one or more biomarkers of interest of the extracellular vesicles (EV miRNAs) in the first biological sample containing EVs.
Modified Lo is silent to the following limitations: (2) wherein the DEP caption section is a DEP chamber;
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Lo further teaches an exemplary microfluidic device 500 comprising an inlet, micro-chamber 1, micro-chamber 2 and outlet which are sequentially connected by a microfluidic channel [para. 0068].
Moral-Zamora teaches a bacterial autonomous controlled concentrator prototype consisting of a DEP chamber connecting a microfluidic channel on either side to preconcentrate the sample (abstract, Figs. 1-2, and annotated Fig.2 in Mora-Zamora). Figs.1-2 show the DEP chamber comprises two IDEs in the PDMS microfluidic chamber for concentrating the target sample (section 3.1). Although Mora-Zamora uses the DEP chamber comprising IDEs to preconcentrate/capture bacteria instead of EVs, Hadady does teach a DEP chamber comprising two IDEs for isolating EVs based on DEP (section 2.2 and abstract). Thus a DEP chamber comprising IDEs is capable of isolating EVs.
Given the teachings of Lo regarding the capture section configured to receive the sample, capture/concentrate the target molecules by DEP, and release the captured/concentrated target molecules to the second section at the downstream [para. 0032; Figs. 1A-1C], and micro-chambers connected by a microfluidic channel (Fig.5); the teachings of Mora-Zamora regarding a DEP chamber connecting a microfluidic channel at the upstream and downstream of the DEP chamber, wherein the DEP chamber is configured to receive a sample from inlet port of the microfluidic channel, capture/concentrate the target molecules by DEP within the DEP chamber; and the teachings of Hadady regarding a DEP chamber comprising two IDEs for isolating EVs based on DEP, 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 capture section of the microfluidic channel in Lo to a DEP chamber connecting the microfluidic channel on the upstream and downstream of the DEP chamber, and further modify the array of electrodes in Lo to IDEs disposed on the bottom wall of the DEP chamber, as taught by combined Mora-Zamora and Hadady, since the microfluidic chamber with IDEs (DEP chamber) would remove the need of huge and expensive devices and would make the system smaller with better functionalities (the first paragraph in Col. 2 on page 1139 in Mora-Zamora), and would allow for isolation of EVs on IDEs by DEP (concluding remarks in Hadady).
Regarding claim 25, modified Lo teaches the method of claim 24, and Lo teaches wherein the first biological sample is received via the microfluidics channel (Figs.1B-1C show the sample is received via inlet 116 of the microfluidic channel 112 [para. 0044]).
Regarding claim 26, modified Lo teaches the method of claim 24, wherein the one or more biomarkers of interest of the first biological sample is tagged with a particular label of a plurality of labels (Lo teaches the molecular probes can be configured to have complimentary base pairs specific to the target miRNA, and thereby hybridize only with the target miRNAs recaptured in the recapture section 114 [para. 0046]. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have the EV miRNAs been tagged in the same way with a particular label of a plurality of labels, as taught by Lo, since it would allow to detect specific miRNAs [para. 0046 in Lo]).
Regarding claim 28, modified Lo teaches the method of claim 24, wherein the DEP chamber comprises one or more electrode arrays (As outlined in the rejection of claim 24 above, the capture section of the microfluidic channel in Lo is modified to the DEP chamber by Mora-Zamora and Hadady, wherein the DEP chamber comprises two IDEs [see Figs. 1-2 in Mora-Zamora; section 2.2 in Hadady]).
Regarding claim 29, modified Lo teaches the method of claim 28, wherein each of the one or more electrode arrays are arranged in an interdigitated configuration (IDEs as shown in Figs. 1-2 in Mora-Zamora; interdigitated electrode array in section 2.2 in Hadady).
Regarding claim 31, Lo teaches a solid-state device for isolating at least one biomarker of interest (the device 110 as shown in Figs. 1A-1C includes an array of electrodes 113 formed on a surface within the microfluidic channel 112 along a parallel direction of the microfluidic channel, e.g., constituting a capture region of the device 110. The array of electrodes 113 are operable to produce an electric field across the microfluidic channel 112 that can create an electrostatic attractive force on the molecules [e.g., nucleic acids] to immobilize them within the capture region of the device 110 [para. 0035; abstract; title]; Because miRNAs are linked to over 100 diseases, including many types of cancers, they can be used as biomarkers for disease diagnosis [para. 0022] ; Fig.1A shows DNA/RNA capture, release & recapture, and detection; and the target biomolecules [e.g., nucleic acids such as DNA and/or RNA, including miRNA] [para. 0062]; miRNAs are deemed as the at least one biomarker of interest), the device comprising:
means for receiving a first biological sample into the solid-state device (Fig.1C shows blood plasma or whole blood received by the device 110 [e.g., via the inlet 116] from a sample vial or container 158 [para. 0044]),
means for transitioning the first biological sample to a dielectrophoresis (DEP) chamber, wherein one or more processes are performed on the first biological sample in the DEP chamber (lab-on-a-chip device structured to include three sections. The device includes a first section 101 that captures molecules [e.g., including nucleic acids such as double- or single-stranded DNA or RNA including miRNA] from a fluidic sample introduced and flowed through the molecular capture section 101 at high flow rate [para. 0032]; The device 110 includes electric field assisted capture and release functionalities, for example, the device 110 can be used to capture nucleic acids [e.g., DNA or RNA, such as miRNA] contained in the blood that are at a concentration in a femtomolar range or less. [para. 0034]; the device 110 can use electrophoretic and/or dielectrophoretic forces to enable selective capturing of biomolecules in the capture region based on the characteristic frequency response of the dielectric permittivity of the biomolecule versus that of the medium [para. 0041]; Fig.1B shows an array of electrodes 113 formed on a surface within the microfluidic channel 112 along a parallel direction of the microfluidic channel, e.g., constituting a capture region of the device 110. The array of electrodes 113 are operable to produce an electric field across the microfluidic channel 112 that can create an electrostatic attractive force on the molecules [e.g., nucleic acids] to immobilize them within the capture region of the device 110 [para. 0035]; the serum sample is introduced to the microfluidic device 100 and enters the molecular capture section 101 at a high flow rate [e.g., greater than 30 μL/min] [para. 0033]; the capture section of the microfluidic channel is deemed as the DEP chamber; due to the presence of flow, it must have a means for transitioning the first biological sample to the caption section wherein the first biological sample is captured/concentrated [see DNA/RNA capture in 101 in Fig.1A; miRNA capture section and capture circulating miRNA from blood plasma in Fig.1C; para. 0033]);
means for passing the first biological sample through a microfluidics (MF) channel to a tagging chamber (The device 110 includes a microchamber 114 formed on the substrate 111 at the end of the microfluidic channel 112 to receive the captured molecules after they are released from the capture region [para.0036; Fig.1B]; the immobilized molecules [e.g., nucleic acids] can be released from the capture region by implementing at least one of the following exemplary processes. In one exemplary process, the device 110 can be operated to remove or reduce the applied electric field [and thereby release the immobilized molecules from the capture region] while flowing another fluid to fluidically transfer the released molecules to the recapture region 114 of the device 110 [para. 0038]; the microchamber 114 is deemed as the tagging chamber and the microfluidic channel 112 is deemed as the MF channel);
means for processing the first biological sample in the tagging chamber, wherein one or more biomarkers of interest of the first biological sample are tagged based on one or more characteristics (the microchamber 114 can include molecular probes capable of binding the molecules in the chamber 114 for detection, e.g., via hybridization for examples when the target molecules are nucleic acids [para. 0036]; In some implementations, for example, molecular probes can be functionalized to the surface of the substrate 111 in the recapture section 114 for assistance in optical detection and characterization of the recaptured miRNAs. For example, the molecular probes can be configured to have complimentary base pairs specific to the target miRNA, and thereby hybridize only with the target miRNAs recaptured in the recapture section 114 [para. 0046]; the target miRNAs are deemed as the one or more biomarkers of interest of the first biological sample, which are tagged by molecular probes having complimentary base pairs specific to the target miRNA);
means for passing the one or more biomarkers of interest with the respective tags to a sensor chamber (the microchamber 114 can serve as an interface section that introduces any desired amount of miRNAs to the detection region 115 [para. 0047]; the device 110 can further include one or more detection regions or chambers 115 formed on the substrate 111 to provide an optical or electrical interrogation section to characterize [e.g., quantify] the presence, concentration, and/or properties of the captured molecules [para. 0037]; each detection region/chamber 115 is deemed as the sensor chamber); and
means for digitally determining a quantity of the one or more biomarkers of interest with the respective tags though one or more processes performed in the sensor chamber (the device 110 can further include one or more detection regions or chambers 115 formed on the substrate 111 to provide an optical or electrical interrogation section to characterize [e.g., quantify] the presence, concentration, and/or properties of the captured molecules [para. 0037]; a process 153 can be performed to use optical or electrical characterization schemes to detect specific binding events of target miRNAs [para. 0046]).
In the above, the capture section of the microfluidic channel is deemed as the DEP chamber. However, Lo is silent to the caption section is a “chamber”. Lo teaches wherein the first biological sample (blood plasma or whole blood) containing the one or more biomarkers of interest (target miRNAs), but does not explicitly teach wherein the first biological sample containing extracellular vesicles, and the one or more biomarkers of interest is EV-derived biomarkers.
Tian teaches extracellular vesicles (EVs) are cell-derived submicron bioparticles composed of lipid bilayer membrane and molecular cargos, acting as important mediators of physiopathological cellular processes. The analysis and engineering of EVs hold significant therapeutic potential in noninvasive cancer diagnostics and innovative drug delivery systems. Tian further reviews the state-of-the-art advances in the development of microfluidic platforms for EV separation, detection and engineering. These EV-associated biomarkers reveal great potential for the diagnosis, monitoring, and prognosis of cancer (abstract; TOC graph; Fig.1). Fig.1 shows a solid-state device (microfluidic platform) for efficient separation, detection, and engineering EV-derived biomarkers of interest in a biological sample containing EVs, wherein the EVs comprise at least one biomarker of interest (the 3rd paragraph in Col. 1 on page 500; detection of EV miRNA [section 3.1.3]). Tain further teaches DEP-based isolation of EVs from undiluted plasma. By exploration of the difference between the dielectric properties of EVs and plasma, EVs could be accumulated at the DEP high-field regions around, while small plasma proteins remailed unaffected by the DEP field. After washing step, the purified EVs are eluted from the microfluidic device using a reverse electric field. Moreover, ACE chip could be integrated with in situ immunofluorescence detection for analysis EV proteins in plasma samples. The levels of EV-associated proteins (glycan-1 and CD63) in plasma from pancreatic cancer patients were found to be much higher than those from health controls (Fig.3a and section 2.2). Section 3.1.3 details thermophoretic fluorescence detection of EV RNAs and Fig.6 shows thermophoretic fluorescence sensing EV mRNAs. FDT contained two recognition sequences complementary to the target mRNA, and the ends of two sequences of FDT were labelled with Cy3 and Cy5, respectively. In the presence of target mRNA inside EVs, an increased signal was observed arising from the close proximity of Cy3 and Cy5, due to the hybridization of recognition sequences and mRNA (section 3.1.3).
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 device for isolating and detecting the one or more biomarkers (miRNAs) in blood plasma in Lo to isolate and detect one or more biomarkers of interest such as miRNAs in the first biological sample (blood plasma) containing extracellular vesicles, wherein the extracellular vesicles comprise the one or more biomarkers of interest (such as EV miRNAs), as taught by Tian, since it would allow for efficient separation and detection EV-associated biomarkers, which reveal great potential for the diagnosis, monitoring, and prognosis of cancer (the 3rd paragraph in Col. 1 on page 500; the first paragraph in section 2.2; and abstract in Tian).
With the above modification, the first biological sample of blood plasma in Lo is modified to the first biological sample of blood plasma containing EVs, and the one or more biomarkers of interest (miRNAs) in Lo is accordingly modified to the one or more biomarkers of interest of the EVs (EV miRNAs).
Modified Lo is silent to the following limitations: wherein the DEP caption section is a DEP chamber.
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Lo further teaches an exemplary microfluidic device 500 comprising an inlet, micro-chamber 1, micro-chamber 2 and outlet which are sequentially connected by a microfluidic channel [para. 0068].
Moral-Zamora teaches a bacterial autonomous controlled concentrator prototype consisting of a DEP chamber connecting a microfluidic channel on either side to preconcentrate the sample (abstract, Figs. 1-2, and annotated Fig.2 in Mora-Zamora). Figs.1-2 show the DEP chamber comprises two IDEs in the PDMS microfluidic chamber for concentrating the target sample (section 3.1). Although Mora-Zamora uses the DEP chamber comprising IDEs to preconcentrate/capture bacteria instead of EVs, Hadady does teach a DEP chamber comprising two IDEs for isolating EVs based on DEP (section 2.2 and abstract). Thus a DEP chamber comprising IDEs is capable of isolating EVs.
Given the teachings of Lo regarding the capture section configured to receive the sample, capture/concentrate the target molecules by DEP, and release the captured/concentrated target molecules to the second section at the downstream [para. 0032; Figs. 1A-1C], and micro-chambers connected by a microfluidic channel (Fig.5); the teachings of Mora-Zamora regarding a DEP chamber connecting a microfluidic channel at the upstream and downstream of the DEP chamber, wherein the DEP chamber is configured to receive a sample from inlet port of the microfluidic channel, capture/concentrate the target molecules by DEP within the DEP chamber; and the teachings of Hadady regarding a DEP chamber comprising two IDEs for isolating EVs based on DEP, 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 capture section of the microfluidic channel in Lo to a DEP chamber connecting the microfluidic channel on the upstream and downstream of the DEP chamber, and further modify the array of electrodes in Lo to IDEs disposed on the bottom wall of the DEP chamber, as taught by combined Mora-Zamora and Hadady, since the microfluidic chamber with IDEs (DEP chamber) would remove the need of huge and expensive devices and would make the system smaller with better functionalities (the first paragraph in Col. 2 on page 1139 in Mora-Zamora), and would allow for isolation of EVs on IDEs by DEP (concluding remarks in Hadady).
Claim 27 is rejected under 35 U.S.C. 103 as being unpatentable over Lo, Tian, Mora-Zamora, and Hadady, as applied to claim 26 above, and in view of Zhu et al. (Avenues toward microRNA detection in vitro: a review of technical advances and challenges, Computational and structural biotechnology Journal, 2019, 17, 904-916).
Regarding claim 27, modified Lo teaches the method of claim 26, and Lo further teaches wherein the plurality of labels (molecular probes) can be configured to have complimentary base pairs specific to the target miRNA, and thereby hybridize only with the target miRNAs recaptured in the recapture section 114 [para. 0046]. But Lo does not explicitly teach wherein the molecular probes are ssDNA.
Lo is silent to wherein the plurality of labels include at least an antibody label, metal nanoparticle (MNP) label, or single-stranded DNA (ssDNA) label.
Zhu teaches single-stranded DNA or RNA probes are frequently used in the detectors for miRNA detection as the biological recognition element. Fig.1a shows ssDNA probe hybridizing with the target miRNA to form the rigid DNA-RNA hetero-duplex structure, resulting in the signal change of corresponding biosensor (section 2.1.1 on page 905).
Given the teachings of Lo regarding the molecular probes having complimentary base pairs specific to the target miRNA, and thereby hybridize only with the target miRNAs recaptured in the recapture section, and the teaching of Zhu regarding ssDNA probes are frequently used in the detectors for miRNA detection as the biological recognition element, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use ssDNA as the molecular probes, as taught by Zhu, since ssDNA probes have been frequently used in the detectors for miRNA detection as the biological recognition element (section 2.1.1 and Fig.1a in Zhu).
Claim 30 is rejected under 35 U.S.C. 103 as being unpatentable over Lo, Tian, Mora-Zamora, and Hadady, as applied to claim 29 above, and in view of Reyes-Hernandez et al. (US20170097319A1).
Regarding claim 30, modified Lo teaches the method of claim 29, and Lo is silent to wherein each of the one or more electrode arrays comprises at least a working electrode, reference electrode, and counter electrode.
Mora-Zamora further teaches wherein the DEP chamber comprises two IDEs and two lateral electrodes (see Figs. 1-2), wherein the four electrodes are used to perform the impedance measurement to quantity the amount of concentrated bacterial concentrated by DEP in the DEP chamber (section 3.2.2; Fig.6).
Reyes-Hernandez teaches dielectrophoresis device comprising receiving a first biological sample containing the bioparticles of interest onto a DEP electrode array (abstract; [para. 0095-0097]), wherein the DEP electrode array comprises at least a working electrode, a reference electrode, and a counter electrode allowing for monitoring impedance of the bioparticles (first/second/third/fourth electrodes 112/114/116/118, and reference electrodes 190/194 in Figs. 63-64 [para. 0159-0162]; abstract).
Since Lo teaches a device for isolating and detecting biomolecules using a DEP electrode array; Lo as modified by Mora-Zamora and Hadady teaches wherein the DEP electrode array is arranged in an interdigitated configuration (as outlined in the rejection of claim 29 above); Reyes-Hernandez discloses where bioparticles may be manipulated using a DEP electrode array comprising working, reference, and counter electrodes; and Mora-Zamora teaches impedance measurement using four-electrode method comprising the DEP electrode array and two additional electrodes to quantify the concentrated bacteria concentration concentrated by DEP, it would have been obvious to one of ordinary skill in the art to modify the electrode array of the DEP chamber in modified Lo by providing at least a working electrode, a reference electrode, and a counter electrode, as taught by Reyes-Hernandez, since this would simply allow incorporating electrodes to detect impedance at the array position, thus indicating the amount of the captured bioparticles (corresponding to the EVs) by the DEP forces ([para. 0159-0162 in Reyes-Hernandez]; Fig.6 and abstract in Mora-Zamora).
Claims 32-33, 35-36 and 38 are rejected under 35 U.S.C. 103 as being unpatentable over Lo, and in view of Tian, Mora-Zamora, Hadady, and Huff et al. (US20180275088A1).
Regarding claim 32, Lo teaches a method for isolating, on a solid-state device,
one or more biomarkers of interest in a first biological sample (method for capturing, concentrating and isolating molecules in a biofluid such as blood plasma or whole blood on a microfluidic device [title, abstract and Figs. 1A-1C]; Because miRNAs are linked to over 100 diseases, including many types of cancers, they can be used as biomarkers for disease diagnosis [para. 0022] ; Fig.1A shows DNA/RNA capture, release & recapture, and detection; and the target biomolecules [e.g., nucleic acids such as DNA and/or RNA, including miRNA] [para. 0062]; miRNAs are deemed as the one or more biomarkers of interest), the method comprising:
receiving the first biological sample into the solid-state device (Figs. 1A-1C show receiving blood plasma or whole blood from a sample vial or container 158 into the device 110 via the inlet 116 [para. 0044]), wherein the first biological sample comprises the one or more biomarkers of interest (molecules [e.g., including nucleic acids such as double- or single-stranded DNA or RNA including miRNA] from a fluidic sample introduced [para. 0032]; miRNA in Fig.1C) and is input via a microfluidics channel (MF) (Figs.1B-1C show the sample is input via inlet 116 of the microfluidic channel 112);
transitioning, via the MF channel, the first biological sample to a dielectrophoresis (DEP) chamber and applying an alternating current (AC) to the first biological sample through one or more electrodes of the DEP chamber (lab-on-a-chip device structured to include three sections. The device includes a first section 101 that captures molecules [e.g., including nucleic acids such as double- or single-stranded DNA or RNA including miRNA] from a fluidic sample introduced and flowed through the molecular capture section 101 at high flow rate [para. 0032]; The device 110 includes electric field assisted capture and release functionalities, for example, the device 110 can be used to capture nucleic acids [e.g., DNA or RNA, such as miRNA] contained in the blood that are at a concentration in a femtomolar range or less. [para. 0034]; the device 110 can use electrophoretic and/or dielectrophoretic forces to enable selective capturing of biomolecules in the capture region based on the characteristic frequency response of the dielectric permittivity of the biomolecule versus that of the medium [para. 0041]; Fig.1B shows an array of electrodes 113 formed on a surface within the microfluidic channel 112 along a parallel direction of the microfluidic channel, e.g., constituting a capture region of the device 110. The array of electrodes 113 are operable to produce an electric field across the microfluidic channel 112 that can create an electrostatic attractive force on the molecules [e.g., nucleic acids] to immobilize them within the capture region of the device 110 [para. 0035]; an AC electrical potential can be applied across the peripheral electrodes 113 a parallel along the microfluidic channel 112 to produce an AC electric field. For example, the applied AC electric field may cause a negative dielectrophoretic (NDEP) effect in high electrolytic solutions to provide the molecular capture force of the exemplary nucleic acids in the capture region [para. 0042]; the capture section is deemed as the DEP chamber);
transitioning, via the MF channel, the first biological sample from the DEP chamber to a tagging chamber (the device 110 includes a microchamber 114 formed on the substrate 111 at the end of the microfluidic channel 112 to receive the captured molecules after they are released from the capture region [para.0036; Fig.1B]; the immobilized molecules [e.g., nucleic acids] can be released from the capture region by implementing at least one of the following exemplary processes. In one exemplary process, the device 110 can be operated to remove or reduce the applied electric field [and thereby release the immobilized molecules from the capture region] while flowing another fluid to fluidically transfer the released molecules to the recapture region 114 of the device 110 [para. 0038]; the microchamber 114 is deemed as the tagging chamber);
assigning a tag to each of the one or more biomarkers of interest (the microchamber 114 can include molecular probes capable of binding the molecules in the chamber 114 for detection, e.g., via hybridization for examples when the target molecules are nucleic acids [para. 0036]; In some implementations, for example, molecular probes can be functionalized to the surface of the substrate 111 in the recapture section 114 for assistance in optical detection and characterization of the recaptured miRNAs. For example, the molecular probes can be configured to have complimentary base pairs specific to the target miRNA, and thereby hybridize only with the target miRNAs recaptured in the recapture section 114 [para. 0046]; the target miRNAs are deemed as the one or more biomarkers of interest of the first biological sample, which are tagged by molecular probes having complimentary base pairs specific to the target miRNA);
transitioning the first biological sample to one or more detector chambers (the microchamber 114 can serve as an interface section that introduces any desired amount of miRNAs to the detection region 115 [para. 0047]; the device 110 can further include one or more detection regions or chambers 115 formed on the substrate 111 to provide an optical or electrical interrogation section to characterize (e.g., quantify) the presence, concentration, and/or properties of the captured molecules [para. 0037]; each detection region/chamber 115 is deemed as the detector chamber); and
digitally determining, by one or more detectors within the one or more detector chambers, a quantity of the one or more biomarkers of interest based on the assigned tags (the device 110 can further include one or more detection regions or chambers 115 formed on the substrate 111 to provide an optical or electrical interrogation section to characterize [e.g., quantify] the presence, concentration, and/or properties of the captured molecules [para. 0037]; a process 153 can be performed to use optical or electrical characterization schemes to detect specific binding events of target miRNAs [para. 0046]).
In the above, the capture section of the microfluidic channel is deemed as the DEP chamber. Thus, Lo is silent to the following limitations: (1) the one or more biomarkers of interest in the first biological sample (target miRNAs in blood plasma) is extracellular vesicle-derived (EV-derived) biomarkers of interest in the first biological sample containing extracellular vesicles, wherein the extracellular vesicles comprise the one or more biomarker of interest; (2) the caption section is a “chamber”; (3) the first biological sample is transitioned to the one or more detector chambers via the MF channel; (4) wherein each detector chamber is programmed for a particular biomarker of interest.
Tian teaches extracellular vesicles (EVs) are cell-derived submicron bioparticles composed of lipid bilayer membrane and molecular cargos, acting as important mediators of physiopathological cellular processes. The analysis and engineering of EVs hold significant therapeutic potential in noninvasive cancer diagnostics and innovative drug delivery systems. Tian further reviews the state-of-the-art advances in the development of microfluidic platforms for EV separation, detection and engineering. These EV-associated biomarkers reveal great potential for the diagnosis, monitoring, and prognosis of cancer (abstract; TOC graph; Fig.1). Fig.1 shows a solid-state device (microfluidic platform) for efficient separation, detection, and engineering EV-derived biomarkers of interest in a biological sample containing EVs, wherein the EVs comprise at least one biomarker of interest (the 3rd paragraph in Col. 1 on page 500; detection of EV miRNA [section 3.1.3]). Tain further teaches DEP-based isolation of EVs from undiluted plasma. By exploration of the difference between the dielectric properties of EVs and plasma, EVs could be accumulated at the DEP high-field regions around, while small plasma proteins remailed unaffected by the DEP field. After washing step, the purified EVs are eluted from the microfluidic device using a reverse electric field. Moreover, ACE chip could be integrated with in situ immunofluorescence detection for analysis EV proteins in plasma samples. The levels of EV-associated proteins (glycan-1 and CD63) in plasma from pancreatic cancer patients were found to be much higher than those from health controls (Fig.3a and section 2.2). Section 3.1.3 details thermophoretic fluorescence detection of EV RNAs and Fig.6 shows thermophoretic fluorescence sensing EV mRNAs. FDT contained two recognition sequences complementary to the target mRNA, and the ends of two sequences of FDT were labelled with Cy3 and Cy5, respectively. In the presence of target mRNA inside EVs, an increased signal was observed arising from the close proximity of Cy3 and Cy5, due to the hybridization of recognition sequences and mRNA (section 3.1.3).
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 method for isolating and detecting the one or more biomarkers (miRNAs) in blood plasma in Lo to isolate and detect one or more biomarkers of interest such as miRNAs in the first biological sample (blood plasma) containing extracellular vesicles, wherein the extracellular vesicles comprise the one or more biomarkers of interest (such as EV miRNAs), as taught by Tian, since it would allow for efficient separation and detection EV-associated biomarkers, which reveal great potential for the diagnosis, monitoring, and prognosis of cancer (the 3rd paragraph in Col. 1 on page 500; the first paragraph in section 2.2; and abstract in Tian).
With the above modification, the one or more biomarkers of interest (miRNAs) in Lo is accordingly modified to the one or more biomarkers of interest of the extracellular vesicles (EV miRNAs) in the first biological sample containing EVs.
Modified Lo is silent to the following limitations: (2) the caption section is a “chamber”; (3) the first biological sample is transitioned to the one or more detector chambers via the MF channel; (4) wherein each detector chamber is programmed for a particular biomarker of interest.
Lo further teaches an exemplary microfluidic device 500 comprising an inlet, micro-chamber 1, micro-chamber 2 and outlet which are sequentially connected by a microfluidic channel [para. 0068].
Moral-Zamora teaches a bacterial autonomous controlled concentrator prototype consisting of a DEP chamber connecting a microfluidic channel on either side to preconcentrate the sample (abstract, Figs. 1-2, and annotated Fig.2 in Mora-Zamora). Figs.1-2 show the DEP chamber comprises two IDEs in the PDMS microfluidic chamber for concentrating the target sample (section 3.1). Although Mora-Zamora uses the DEP chamber comprising IDEs to preconcentrate/capture bacteria instead of EVs, Hadady does teach a DEP chamber comprising two IDEs for isolating EVs based on DEP (section 2.2 and abstract). Thus a DEP chamber comprising IDEs is capable of isolating EVs.
Given the teachings of Lo regarding the capture section configured to receive the sample, capture/concentrate the target molecules by DEP, and release the captured/concentrated target molecules to the second section at the downstream [para. 0032; Figs. 1A-1C], and micro-chambers connected by a microfluidic channel (Fig.5); the teachings of Mora-Zamora regarding a DEP chamber connecting a microfluidic channel at the upstream and downstream of the DEP chamber, wherein the DEP chamber is configured to receive a sample from inlet port of the microfluidic channel, capture/concentrate the target molecules by DEP within the DEP chamber; and the teachings of Hadady regarding a DEP chamber comprising two IDEs for isolating EVs based on DEP, 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 capture section of the microfluidic channel in Lo to a DEP chamber connecting the microfluidic channel on the upstream and downstream of the DEP chamber, and further modify the array of electrodes in Lo to IDEs disposed on the bottom wall of the DEP chamber, as taught by combined Mora-Zamora and Hadady, since the microfluidic chamber with IDEs (DEP chamber) would remove the need of huge and expensive devices and would make the system smaller with better functionalities (the first paragraph in Col. 2 on page 1139 in Mora-Zamora), and would allow for isolation of EVs on IDEs by DEP (concluding remarks in Hadady).
With the above modification, the DEP chamber and the tagging chamber (chamber 114) are connected via the microfluidic channel. Since the microfluidic channel connects the DEP chamber and the tagging chamber, and Fig.5 in Lo shows the device comprising inlet, micro-chamber 1, micro-chamber 2 and outlet which are sequentially connected by a microfluidic channel [para. 0068 in Lo], 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 device by using the microfluidic channel to connect the tagging chamber (chamber 114 in Fig.1B of Lo) and the one or more detector chambers (chambers 115 in Fig.1B of Lo), since it would allow to transport the sample containing the target miRNAs into the detector chambers for detection [para. 0045 in Lo]. Furthermore, one skilled in the art could have used the microfluidic channel to connect the tagging chamber and the one or more detector chambers in the same way to the microfluidic channel connecting the DEP chamber and the tagging chamber, yielding predictable results (MPEP 2143(I)(D)). With this modification, modified Lo teaches the first biological sample is transitioned to the one or more detector chambers via the MF channel.
Modified Lo is silent to: (4) wherein each detector chamber is programmed for a particular biomarker of interest.
Lo does disclose multiple detector chambers (Fig.1B shows multiple detection regions or chambers 115), and further teaches exemplary implementations using the device 500 to capture, enrich, and detect miRNAs were performed. For example, among the over 2,000 types of mature miRNAs known, some 88 miRNAs have been reported to show different expression levels in non-small cell lung cancer (NSCLC) patients. Moreover, for example, the combination of three circulating miRNAs (e.g., miR-155, miR-197, and miR-182) are quite unique to NSCLC. The disclosed lab-on-a-chip technology platform can be implemented for miRNA detection suitable for clinical applications, including capture, enrichment, detection, and characterization of miR-155, miR-182 and miR-197 for NSCLC clinical applications [para. 0075]. In other implementations, for example, the detection region 115 can be modified to include the molecular probes on the surface of the substrate 111 in that region, where the extracted miRNAs can be transferred to the detection region 115 such that they bind (e.g., hybridize) to the molecular probes having complimentary base pairs specific to the target miRNA. A process 153 can be performed to use optical or electrical characterization schemes to detect specific binding events of target miRNAs. [para. 0046].
Huff teaches a method for biomarker analysis comprising a device for manipulating a sample (abstract; para [0437]) using DEP forces
[para.0150], where a biomarker may be tagged with a label [para. 0240-0242], and the device may be programmed to perform electrochemical sensing of an analyte (Devices and systems that are programmed to carry out the disclosed methods are also
provided. Also provided herein are instruments that are programmed to operate a cartridge that includes an array of electrodes for actuating a droplet and further includes an electrochemical species sensing region [abstract]; device may be programmed to perform analyte analysis as disclosed herein, including any optional mixing, incubating, and washing steps ... may include a data acquisition module for processing electrical signals from the nanopore device or module [para. 0358]; In certain embodiments, the device in FIG. 58A may include a housing that includes processor 413 which is operably connected to a memory that contains programming for using the
detection chips [para. 0504]).
Given the teachings of Lo regarding the device comprising multiple detection regions/chambers for detecting multiple biomarkers (multiple target miRNAs [para. 0045; Fig.1B]) and the detection region includes the molecular probes having complimentary base pairs specific to the target miRNA for detecting specific binding events of the target miRNA [para. 0046], and the teachings of Huff regarding a DEP device programmed to manipulate a biological sample and operate detection regions and biomarkers may be tagged with a label for detection, 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 method and device of modified Lo to program each detector chamber for a particular biomarker of interest, since it would provide a lab-on-a-chip platform comprising distinct detection chambers with particular molecular probes having complimentary base pairs specific to the target miRNAs for detecting the specific target miRNAs, and accordingly allow for multiplexed detection of a large number of biomarkers such as miR-155, miR-182 and miR-197 for NSCLC clinical applications [para. 0046, 0075 in Lo], and would automate the analyte analysis [para. 0026 in Huff].
Regarding claim 33, modified Lo teaches the method of claim 32, wherein the tag defines a particular label of a plurality of labels (Lo teaches the molecular probes can be configured to have complimentary base pairs specific to the target miRNA, and thereby hybridize only with the target miRNAs recaptured in the recapture section 114 [para. 0046]. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have the EV miRNAs been tagged in the same way with a tag defining a particular label of a plurality of labels, as taught by Lo, since it would allow to detect specific miRNAs [para. 0046 in Lo]).
Regarding claim 35, modified Lo teaches the method of claim 32, wherein a first biomarker of interest of the first biological sample is assigned a first tag based on a particular characteristic (Lo teaches the molecular probes can be configured to have complimentary base pairs specific to the target miRNA, and thereby hybridize only with the target miRNAs recaptured in the recapture section 114 [para. 0046]. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to detect the EV miRNAs in the same way as detecting the miRNA in Lo by providing a first biomarker of interest of the first biological sample been assigned a first tag based on a particular characteristic, since it would allow for detecting specific target miRNA [para. 0046 in Lo]).
Regarding claim 36, modified Lo teaches the method of claim 32, and Lo is silent to wherein a second biomarker of interest of the first biological sample is assigned a second tag based on a particular characteristic.
Lo further teaches the disclosed microfluidic techniques, systems, and devices can detect the presence and abundance level of specific nucleic acids from blood and biofluids (e.g., saliva, sputum, urine, etc.) from patients. Many kinds of nucleic acids are present in the blood and biofluids and they are promising candidates as disease markers for diagnosis and prognosis of heart, lung, liver, central nerve system, chronic and infectious diseases, as well as cancer [para. 0027]. Exemplary implementations using the device 500 to capture, enrich, and detect miRNAs were performed. For example, among the over 2,000 types of mature miRNAs known, some 88 miRNAs have been reported to show different expression levels in non-small cell lung cancer (NSCLC) patients. Moreover, for example, the combination of three circulating miRNAs (e.g., miR-155, miR-197, and miR-182) are quite unique to NSCLC. The disclosed lab-on-a-chip technology platform can be implemented for miRNA detection suitable for clinical applications, including capture, enrichment, detection, and characterization of miR-155, miR-182 and miR-197 for NSCLC clinical applications [para. 0075].
Since Lo discloses a method for isolating and detecting biomarkers of interest using biomarker-specific tags [para. 0046], and multiplexed detection of multiple biomarkers is desired [para. 0027, 0075], it would have been obvious to one of ordinary skill in the art to modify the method in modified Lo to include a second biomarker of interest of the first biological sample been assigned a second tag based on a particular characteristic, such as a second miRNA assigned a second molecular probe which hybridizes to said miRNA, since this would simply allow multiplexed detection of biomarkers in the sample by using multiple labels specific for different biomarkers potentially found in the biological sample, thereby increasing the versatility, applicability, and therefore value of the method of modified Lo for multiplexed detection of multiple target miRNAs.
Regarding claim 38, Lo teaches a solid-state device for isolating at least one biomarker of interest (the device 110 as shown in Figs. 1A-1C includes an array of electrodes 113 formed on a surface within the microfluidic channel 112 along a parallel direction of the microfluidic channel, e.g., constituting a capture region of the device 110. The array of electrodes 113 are operable to produce an electric field across the microfluidic channel 112 that can create an electrostatic attractive force on the molecules [e.g., nucleic acids] to immobilize them within the capture region of the device 110 [para. 0035; abstract; title]; Because miRNAs are linked to over 100 diseases, including many types of cancers, they can be used as biomarkers for disease diagnosis [para. 0022] ; The microfluidic device 100 can be implemented for miRNA capture and detection using blood or other biofluids [para.0032]; miRNAs are deemed as the at least one biomarker of interest), the solid-state device comprising:
means for receiving a first biological sample into the solid-state device (Fig.1C shows blood plasma or whole blood received by the device 110 [e.g., via the inlet 116] from a sample vial or container 158 [para. 0044]), wherein the first biological sample comprises one or more biomarkers of interest (molecules including nucleic acids such as double- or single-stranded DNA or RNA including miRNA from a fluidic sample introduced [para. 0032]) and is input via a microfluidics (MF) channel (Figs. 1B-1C shows the blood plasma is input via the inlet 116 of a MF channel 112);
means for transitioning, via the MF channel, the first biological sample to a dielectrophoresis (DEP) chamber and applying an alternating current (AC) to the first biological sample through one or more electrodes of the DEP chamber (lab-on-a-chip device structured to include three sections. The device includes a first section 101 that captures molecules [e.g., including nucleic acids such as double- or single-stranded DNA or RNA including miRNA] from a fluidic sample introduced and flowed through the molecular capture section 101 at high flow rate [para. 0032]; The device 110 includes electric field assisted capture and release functionalities, for example, the device 110 can be used to capture nucleic acids [e.g., DNA or RNA, such as miRNA] contained in the blood that are at a concentration in a femtomolar range or less. [para. 0034]; the device 110 can use electrophoretic and/or dielectrophoretic forces to enable selective capturing of biomolecules in the capture region based on the characteristic frequency response of the dielectric permittivity of the biomolecule versus that of the medium [para. 0041]; Fig.1B shows an array of electrodes 113 formed on a surface within the microfluidic channel 112 along a parallel direction of the microfluidic channel, e.g., constituting a capture region of the device 110. The array of electrodes 113 are operable to produce an electric field across the microfluidic channel 112 that can create an electrostatic attractive force on the molecules [e.g., nucleic acids] to immobilize them within the capture region of the device 110 [para. 0035]; an AC electrical potential can be applied across the peripheral electrodes 113 a parallel along the microfluidic channel 112 to produce an AC electric field. For example, the applied AC electric field may cause a negative dielectrophoretic (NDEP) effect in high electrolytic solutions to provide the molecular capture force of the exemplary nucleic acids in the capture region [para. 0042]; the serum sample is introduced to the microfluidic device 100 and enters the molecular capture section 101 at a high flow rate, e.g., greater than 30 μL/min [para. 0033]; the capture section of the microfluidic channel is deemed as the DEP chamber; due to the presence of flow, it must have a means for transitioning the first biological sample to the caption section wherein the first biological sample is captured/concentrated [see DNA/RNA capture in 101 in Fig.1A; miRNA capture section and capture circulating miRNA from blood plasma in Fig.1C; para. 0033] by the DEP force generated by applying an AC field to the electrodes 113);
means for transitioning, via the MF channel, the first biological sample from the DEP chamber to a tagging chamber (The device 110 includes a microchamber 114 formed on the substrate 111 at the end of the microfluidic channel 112 to receive the captured molecules after they are released from the capture region [para.0036; Fig.1B]; the immobilized molecules (e.g., nucleic acids) can be released from the capture region by implementing at least one of the following exemplary processes. In one exemplary process, the device 110 can be operated to remove or reduce the applied electric field [and thereby release the immobilized molecules from the capture region] while flowing another fluid to fluidically transfer the released molecules to the recapture region 114 of the device 110 [para. 0038]; the microchamber 114 is deemed as the tagging chamber);
means for assigning a tag to each of the one or more biomarkers of interest of the first biological sample (the microchamber 114 can include molecular probes capable of binding the molecules in the chamber 114 for detection, e.g., via hybridization for examples when the target molecules are nucleic acids [para. 0036]; In some implementations, for example, molecular probes can be functionalized to the surface of the substrate 111 in the recapture section 114 for assistance in optical detection and characterization of the recaptured miRNAs. For example, the molecular probes can be configured to have complimentary base pairs specific to the target miRNA, and thereby hybridize only with the target miRNAs recaptured in the recapture section 114 [para. 0046]);
means for transitioning the first biological sample to one or more detector chambers (the microchamber 114 can serve as an interface section that introduces any desired amount of miRNAs to the detection region 115 [para. 0047]; the device 110 can further include one or more detection regions or chambers 115 formed on the substrate 111 to provide an optical or electrical interrogation section to characterize (e.g., quantify) the presence, concentration, and/or properties of the captured molecules [para. 0037]; each detection region/chamber 115 is deemed as the claimed detector chamber); and
means for digitally determining, by one or more detectors within the one or more detector chambers, a quantity of the one or more biomarkers of interest based on the assigned tags (the device 110 can further include one or more detection regions or chambers 115 formed on the substrate 111 to provide an optical or electrical interrogation section to characterize [e.g., quantify] the presence, concentration, and/or properties of the captured molecules [para. 0037]; a process 153 can be performed to use optical or electrical characterization schemes to detect specific binding events of target miRNAs [para. 0046]).
In the above, the capture section of the microfluidic channel is deemed as the DEP chamber. Thus, Lo is silent to the following limitations: (1) the one or more biomarkers of interest in the first biological sample (target miRNAs in blood plasma) is extracellular vesicle-derived (EV-derived) biomarkers of interest in the first biological sample containing extracellular vesicles, wherein the extracellular vesicles comprise the one or more biomarker of interest; (2) the caption section is a “chamber”; (3) the first biological sample is transitioned to the one or more detector chambers via the MF; (4) wherein each detector chamber is programmed for a particular biomarker of interest.
Tian teaches extracellular vesicles (EVs) are cell-derived submicron bioparticles composed of lipid bilayer membrane and molecular cargos, acting as important mediators of physiopathological cellular processes. The analysis and engineering of EVs hold significant therapeutic potential in noninvasive cancer diagnostics and innovative drug delivery systems. Tian further reviews the state-of-the-art advances in the development of microfluidic platforms for EV separation, detection and engineering. These EV-associated biomarkers reveal great potential for the diagnosis, monitoring, and prognosis of cancer (abstract; TOC graph; Fig.1). Fig.1 shows a solid-state device (microfluidic platform) for efficient separation, detection, and engineering EV-derived biomarkers of interest in a biological sample containing EVs, wherein the EVs comprise at least one biomarker of interest (the 3rd paragraph in Col. 1 on page 500; detection of EV miRNA [section 3.1.3]). Tain further teaches DEP-based isolation of EVs from undiluted plasma. By exploration of the difference between the dielectric properties of EVs and plasma, EVs could be accumulated at the DEP high-field regions around, while small plasma proteins remailed unaffected by the DEP field. After washing step, the purified EVs are eluted from the microfluidic device using a reverse electric field. Moreover, ACE chip could be integrated with in situ immunofluorescence detection for analysis EV proteins in plasma samples. The levels of EV-associated proteins (glycan-1 and CD63) in plasma from pancreatic cancer patients were found to be much higher than those from health controls (Fig.3a and section 2.2). Section 3.1.3 details thermophoretic fluorescence detection of EV RNAs and Fig.6 shows thermophoretic fluorescence sensing EV mRNAs. FDT contained two recognition sequences complementary to the target mRNA, and the ends of two sequences of FDT were labelled with Cy3 and Cy5, respectively. In the presence of target mRNA inside EVs, an increased signal was observed arising from the close proximity of Cy3 and Cy5, due to the hybridization of recognition sequences and mRNA (section 3.1.3).
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 device for isolating and detecting the one or more biomarkers (miRNAs) in blood plasma in Lo to isolate and detect one or more biomarkers of interest such as miRNAs in the first biological sample (blood plasma) containing extracellular vesicles, wherein the extracellular vesicles comprise the one or more biomarkers of interest (such as EV miRNAs), as taught by Tian, since it would allow for efficient separation and detection EV-associated biomarkers, which reveal great potential for the diagnosis, monitoring, and prognosis of cancer (the 3rd paragraph in Col. 1 on page 500; the first paragraph in section 2.2; and abstract in Tian).
With the above modification, the one or more biomarkers of interest (miRNAs) in Lo is accordingly modified to the one or more biomarkers of interest of the extracellular vesicles (EV miRNAs) in the first biological sample containing EVs.
Modified Lo is silent to the following limitations: (2) the caption section is a “chamber”; (3) the first biological sample is transitioned to the one or more detector chambers via the MF channel; (4) wherein each detector chamber is programmed for a particular biomarker of interest.
Lo further teaches an exemplary microfluidic device 500 comprising an inlet, micro-chamber 1, micro-chamber 2 and outlet which are sequentially connected by a microfluidic channel [para. 0068].
Moral-Zamora teaches a bacterial autonomous controlled concentrator prototype consisting of a DEP chamber connecting a microfluidic channel on either side to preconcentrate the sample (abstract, Figs. 1-2, and annotated Fig.2 in Mora-Zamora). Figs.1-2 show the DEP chamber comprises two IDEs in the PDMS microfluidic chamber for concentrating the target sample (section 3.1). Although Mora-Zamora uses the DEP chamber comprising IDEs to preconcentrate/capture bacteria instead of EVs, Hadady does teach a DEP chamber comprising two IDEs for isolating EVs based on DEP (section 2.2 and abstract). Thus a DEP chamber comprising IDEs is capable of isolating EVs.
Given the teachings of Lo regarding the capture section configured to receive the sample, capture/concentrate the target molecules by DEP, and release the captured/concentrated target molecules to the second section at the downstream [para. 0032; Figs. 1A-1C], and micro-chambers connected by a microfluidic channel (Fig.5); the teachings of Mora-Zamora regarding a DEP chamber connecting a microfluidic channel at the upstream and downstream of the DEP chamber, wherein the DEP chamber is configured to receive a sample from inlet port of the microfluidic channel, capture/concentrate the target molecules by DEP within the DEP chamber; and the teachings of Hadady regarding a DEP chamber comprising two IDEs for isolating EVs based on DEP, 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 capture section of the microfluidic channel in Lo to a DEP chamber connecting the microfluidic channel on the upstream and downstream of the DEP chamber, and further modify the array of electrodes in Lo to IDEs disposed on the bottom wall of the DEP chamber, as taught by combined Mora-Zamora and Hadady, since the microfluidic chamber with IDEs (DEP chamber) would remove the need of huge and expensive devices and would make the system smaller with better functionalities (the first paragraph in Col. 2 on page 1139 in Mora-Zamora), and would allow for isolation of EVs on IDEs by DEP (concluding remarks in Hadady).
With the above modification, the DEP chamber and the tagging chamber (chamber 114) are connected via the microfluidic channel. Since the microfluidic channel connects the DEP chamber and the tagging chamber, and Fig.5 in Lo shows the device comprising inlet, micro-chamber 1, micro-chamber 2 and outlet which are sequentially connected by a microfluidic channel [para. 0068 in Lo], 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 device by using the microfluidic channel to connect the tagging chamber (chamber 114 in Fig.1B of Lo) and the one or more detector chambers (chambers 115 in Fig.1B of Lo), since it would allow to transport the sample containing the target miRNAs into the detector chambers for detection [para. 0045 in Lo]. Furthermore, one skilled in the art could have used the microfluidic channel to connect the tagging chamber and the one or more detector chambers in the same way to the microfluidic channel connecting the DEP chamber and the tagging chamber, yielding predictable results (MPEP 2143(I)(D)). With this modification, modified Lo teaches the first biological sample is transitioned to the one or more detector chambers via the MF.
Modified Lo is silent to: (4) wherein each detector chamber is programmed for a particular biomarker of interest.
Lo does disclose multiple detector chambers (Fig.1B shows multiple detection regions or chambers 115), and further teaches exemplary implementations using the device 500 to capture, enrich, and detect miRNAs were performed. For example, among the over 2,000 types of mature miRNAs known, some 88 miRNAs have been reported to show different expression levels in non-small cell lung cancer (NSCLC) patients. Moreover, for example, the combination of three circulating miRNAs (e.g., miR-155, miR-197, and miR-182) are quite unique to NSCLC. The disclosed lab-on-a-chip technology platform can be implemented for miRNA detection suitable for clinical applications, including capture, enrichment, detection, and characterization of miR-155, miR-182 and miR-197 for NSCLC clinical applications [para. 0075]. In other implementations, for example, the detection region 115 can be modified to include the molecular probes on the surface of the substrate 111 in that region, where the extracted miRNAs can be transferred to the detection region 115 such that they bind (e.g., hybridize) to the molecular probes having complimentary base pairs specific to the target miRNA. A process 153 can be performed to use optical or electrical characterization schemes to detect specific binding events of target miRNAs [para. 0046].
Huff teaches a method for biomarker analysis comprising a device for manipulating a sample (abstract; para [0437]) using DEP forces
[para.0150], where a biomarker may be tagged with a label [para. 0240-0242], and the device may be programmed to perform electrochemical sensing of an analyte (Devices and systems that are programmed to carry out the disclosed methods are also
provided. Also provided herein are instruments that are programmed to operate a cartridge that includes an array of electrodes for actuating a droplet and further includes an electrochemical species sensing region [abstract]; device may be programmed to perform analyte analysis as disclosed herein, including any optional mixing, incubating, and washing steps ... may include a data acquisition module for processing electrical signals from the nanopore device or module [para. 0358]; In certain embodiments, the device in FIG. 58A may include a housing that includes processor 413 which is operably connected to a memory that contains programming for using the
detection chips [para. 0504]).
Given the teachings of Lo regarding the device comprising multiple detection regions/chambers for detecting multiple biomarkers (multiple target miRNAs [para. 0045; Fig.1B]) and the detection region includes the molecular probes having complimentary base pairs specific to the target miRNA for detecting specific binding events of the target miRNA [para. 0046], and the teachings of Huff regarding a DEP device programmed to manipulate a biological sample and operate detection regions and biomarkers may be tagged with a label for detection, 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 method and device of modified Lo to program each detector chamber for a particular biomarker of interest, since it would provide a lab-on-a-chip platform comprising distinct detection chambers with particular molecular probes having complimentary base pairs specific to the target miRNAs for detecting the specific target miRNAs, and accordingly allow for multiplexed detection of a large number of biomarkers such as miR-155, miR-182 and miR-197 for NSCLC clinical applications [para. 0046, 0075 in Lo], and would automate the analyte analysis [para. 0026 in Huff].
Claim 34 is rejected under 35 U.S.C. 103 as being unpatentable over Lo, Tian, Mora-Zamora, Hadady, and Huff, as applied to claim 33 above, and Zhu et al. (Avenues toward microRNA detection in vitro: a review of technical advances and challenges, Computational and structural biotechnology Journal, 2019, 17, 904-916).
Regarding claim 34, modified Lo teaches the method of claim 33, and Lo further teaches wherein the plurality of labels (molecular probes) can be configured to have complimentary base pairs specific to the target miRNA, and thereby hybridize only with the target miRNAs recaptured in the recapture section 114 [para. 0046]. But Lo does not explicitly teach wherein the molecular probes are ssDNA.
Lo is silent to wherein the plurality of labels includes at least an antibody label, metal nanoparticle (MNP) label, or single-stranded DNA (ssDNA) label.
Zhu teaches single-stranded DNA or RNA probes are frequently used in the detectors for miRNA detection as the biological recognition element. Fig.1a shows ssDNA probe hybridizing with the target miRNA to form the rigid DNA-RNA hetero-duplex structure, resulting in the signal change of corresponding biosensor (section 2.1.1 on page 905).
Given the teachings of Lo regarding the molecular probes having complimentary base pairs specific to the target miRNA, and thereby hybridize only with the target miRNAs recaptured in the recapture section, and the teaching of Zhu regarding ssDNA probes are frequently used in the detectors for miRNA detection as the biological recognition element, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use ssDNA as the molecular probes, as taught by Zhu, since ssDNA probes have been frequently used in the detectors for miRNA detection as the biological recognition element (section 2.1.1 and Fig.1a in Zhu).
Claim 37 is rejected under 35 U.S.C. 103 as being unpatentable over Lo, Tian, Mora-Zamora, Hadady, and Huff, as applied to claim 32 above, and in view of Reyes-Hernandez et al. (US20170097319A1).
Regarding claim 37, modified Lo teaches the method of claim 32, and Lo is silent to wherein each of the one or more electrode arrays comprises at least a working electrode, reference electrode, and counter electrode.
Mora-Zamora further teaches wherein the DEP chamber comprises two IDEs and two lateral electrodes (see Figs. 1-2), wherein the four electrodes are used to perform impedance measurement to quantity the amount of concentrated bacterial concentrated by DEP in the DEP chamber (section 3.2.2; Fig.6).
Reyes-Hernandez teaches dielectrophoresis device comprising receiving a first biological sample containing the bioparticles of interest onto a DEP electrode array (abstract; [para. 0095-0097]), wherein the DEP electrode array comprises at least a working electrode, a reference electrode, and a counter electrode allowing for monitoring impedance of the trapped bioparticle of interest (first/second/third/fourth electrodes 112/114/116/118, and reference electrodes 190/194 in Figs. 63-64 [para. 0159-0162]; abstract).
Since Lo teaches a device for isolating and detecting biomolecules using a DEP electrode array; Reyes-Hernandez discloses where bioparticles may be manipulated using a DEP electrode array comprising working, reference, and counter electrodes; and Mora-Zamora teaches impedance measurement using four-electrode method to quantify the concentrated bacteria concentration concentrated by DEP, it would have been obvious to one of ordinary skill in the art to modify the one or more electrodes of the DEP chamber in modified Lo to include at least a working electrode, a reference electrode, and a counter electrode, as taught by Reyes-Hernandez, since this would simply allow incorporating electrodes to detect impedance at the array position, thus indicating the amount of the captured biomarkers by the DEP forces ([para. 0159-0162 in Reyes-Hernandez]; Fig.6 and abstract in Mora-Zamora).
Claim 39 is rejected under 35 U.S.C. 103 as being unpatentable over Lo, and in view of Tian, Mora-Zamora, Hadady, Bhargava et al. (WO2019204510A1), and Reyes-Hernandez.
Regarding claim 39, Lo teaches a solid-state device (the device 110 as shown in Figs. 1A-1C) comprising:
a microfluidics channel (microfluidic channel 112 in Fig.1B), wherein the microfluidics channel receives a biological sample (Figs.1B-1C show blood plasma or whole blood received by the device 110 via the inlet 116 of the microfluidic channel from a sample vial or container 158 [para. 0044]);
one or more actuated valves (on-chip valves [para. 0007, 0067-0068 ]; Fig.5), wherein each of the one or more actuated valves connects a plurality of the microfluidics channels (see Fig.5; the device 500 includes a first valve 515 positioned in the microfluidic channel between the first and second microchambers 510 and 520 to control the flow of the fluid through the device 500 between these chambers. The device 500 includes a second valve 525 positioned in the microfluidic channel between the second microchamber 520 and the outlet 521 to control the flow of the fluid out of the device 500 from the second microchamber 520 [para. 0007, 0068]);
one or more dielectrophoresis (DEP) chamber(s) (a first capture section 101 that captures molecules [e.g., including nucleic acids such as double- or single-stranded DNA or RNA including miRNA] from a fluidic sample introduced and flowed through the molecular capture section 101 at high flow rate [para. 0032]; the capture section of the microfluidic channel is deemed as the DEP chamber);
one or more tagging chambers (microchamber 114 in Fig.1B), wherein the biological sample is assigned one or more tags (the microchamber 114 can include molecular probes capable of binding the molecules in the chamber 114 for detection, e.g., via hybridization for examples when the target molecules are nucleic acids [para. 0036]; In some implementations, for example, molecular probes can be functionalized to the surface of the substrate 111 in the recapture section 114 for assistance in optical detection and characterization of the recaptured miRNAs. For example, the molecular probes can be configured to have complimentary base pairs specific to the target miRNA, and thereby hybridize only with the target miRNAs recaptured in the recapture section 114 [para. 0046]);
one or more detector chambers (one or more detection regions or chambers 115 in Fig.1B [para. 0037]), wherein the biological sample and associated one or more tags are processed (the device 110 can further include one or more detection regions or chambers 115 formed on the substrate 111 to provide an optical or electrical interrogation section to characterize [e.g., quantify] the presence, concentration, and/or properties of the captured molecules [para. 0037]; a process 153 can be performed to use optical or electrical characterization schemes to detect specific binding events of target miRNAs [para. 0046]); and
one or more detectors, wherein the one or more detectors are operable to quantify the one or more tags (the device 110 can further include one or more detection regions or chambers 115 formed on the substrate 111 to provide an optical or electrical interrogation section to characterize [e.g., quantify] the presence, concentration, and/or properties of the captured molecules [para. 0037]; a process 153 can be performed to use optical or electrical characterization schemes to detect specific binding events of target miRNAs [para. 0046]; therefore, it must have one or more detectors operable to quantify the one or more tags in order to quantify concentration of the captured molecules).
Lo further teaches the disclosed microfluidic devices, systems, and techniques can extract target molecules as the fluid flows through a microfluidic channel [para. 0030, 0032]. In some implementations, for example, after flowing through the desired amount of serum sample, the channel can be washed by RNAse-free buffer solution [para. 0055]. After the desired amount of serum (e.g., 0.5 to 1 mL) flows through the device 400 (e.g., removed from the outlet 431), in which the targeted nucleic acids (e.g., DNAs and/or RNAs, such as miRNA) are captured at the filter region 425 via the charged particles 401, the device 400 can be washed (e.g., with isopropanol) and a controlled amount of elusion buffer can be introduced from inlet 421 connected to the first microchamber 420 to release the captured DNAs and RNAs [para. 0066].
Lo is silent to the following limitations: (1) the biological sample of blood plasma comprising extracellular vesicles; wherein the extracellular vesicles of the biological sample are assigned one or more tags; (2) the caption region is a “chamber”; (3) a syringe; (4) one or more motors, wherein each motor contains one or more gears; (5) one or more reservoirs, wherein the one or more reservoirs connect to the microfluidics channel; and (6) wherein each DEP chamber contains at least one or more working electrodes, one or more reference electrodes, and one or more counter electrodes.
Tian teaches extracellular vesicles (EVs) are cell-derived submicron bioparticles composed of lipid bilayer membrane and molecular cargos, acting as important mediators of physiopathological cellular processes. The analysis and engineering of EVs hold significant therapeutic potential in noninvasive cancer diagnostics and innovative drug delivery systems. Tian further reviews the state-of-the-art advances in the development of microfluidic platforms for EV separation, detection and engineering. These EV-associated biomarkers reveal great potential for the diagnosis, monitoring, and prognosis of cancer (abstract; TOC graph; Fig.1). Fig.1 shows a solid-state device (microfluidic platform) for efficient separation, detection, and engineering EV-derived biomarkers of interest in a biological sample containing EVs, wherein the EVs comprise at least one biomarker of interest (the 3rd paragraph in Col. 1 on page 500; detection of EV miRNA [section 3.1.3]). Tain further teaches DEP-based isolation of EVs from undiluted plasma. By exploration of the difference between the dielectric properties of EVs and plasma, EVs could be accumulated at the DEP high-field regions around, while small plasma proteins remailed unaffected by the DEP field. After washing step, the purified EVs are eluted from the microfluidic device using a reverse electric field. Moreover, ACE chip could be integrated with in situ immunofluorescence detection for analysis EV proteins in plasma samples. The levels of EV-associated proteins (glycan-1 and CD63) in plasma from pancreatic cancer patients were found to be much higher than those from health controls (Fig.3a and section 2.2). Section 3.1.3 details thermophoretic fluorescence detection of EV RNAs and Fig.6 shows thermophoretic fluorescence sensing EV mRNAs. FDT contained two recognition sequences complementary to the target mRNA, and the ends of two sequences of FDT were labelled with Cy3 and Cy5, respectively. In the presence of target mRNA inside EVs, an increased signal was observed arising from the close proximity of Cy3 and Cy5, due to the hybridization of recognition sequences and mRNA (section 3.1.3).
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 device for isolating and detecting target miRNAs in blood plasma in Lo to isolate and detect miRNAs of extracellular vesicles in the biological sample (blood plasma) containing the extracellular vesicles, wherein the extracellular vesicles are assigned one or more tags for the detection of the EV miRNAs (In the presence of target mRNA inside EVs, an increased signal was observed arising from the close proximity of Cy3 and Cy5, due to the hybridization of recognition sequences and mRNA [section 3.1.3 in Tian]), as taught by Tian, since it would allow for efficient separation and detection EV-associated biomarkers, which reveal great potential for the diagnosis, monitoring, and prognosis of cancer (the 3rd paragraph in Col. 1 on page 500; the first paragraph in section 2.2; and abstract in Tian).
With the above modification, the biological sample of blood plasma in Lo is modified to the biological sample containing EVs (blood plasma containing EVs).
Modified Lo is silent to the following limitations: (2) the caption region is a “chamber”; (3) a syringe; (4) one or more motors, wherein each motor contains one or more gears; (5) one or more reservoirs, wherein the one or more reservoirs connect to the microfluidics channel; and (6) wherein each DEP chamber contains at least one or more working electrodes, one or more reference electrodes, and one or more counter electrodes.
Lo further teaches an exemplary microfluidic device 500 comprising an inlet, micro-chamber 1, micro-chamber 2 and outlet which are sequentially connected by a microfluidic channel [para. 0068].
Moral-Zamora teaches a bacterial autonomous controlled concentrator prototype consisting of a DEP chamber connecting a microfluidic channel on either side to preconcentrate the sample (abstract, Figs. 1-2, and annotated Fig.2 in Mora-Zamora). Figs.1-2 show the DEP chamber comprises two IDEs in the PDMS microfluidic chamber for concentrating the target sample (section 3.1). Although Mora-Zamora uses the DEP chamber comprising IDEs to preconcentrate/capture bacteria instead of EVs, Hadady does teach a DEP chamber comprising two IDEs for isolating EVs based on DEP (section 2.2 and abstract). Thus a DEP chamber comprising IDEs is capable of isolating EVs.
Given the teachings of Lo regarding the capture section configured to receive the sample, capture/concentrate the target molecules by DEP, and release the captured/concentrated target molecules to the second section at the downstream [para. 0032; Figs. 1A-1C], and micro-chambers connected by a microfluidic channel (Fig.5); the teachings of Mora-Zamora regarding a DEP chamber connecting a microfluidic channel at the upstream and downstream of the DEP chamber, wherein the DEP chamber is configured to receive a sample from inlet port of the microfluidic channel, capture/concentrate the target molecules by DEP within the DEP chamber; and the teachings of Hadady regarding a DEP chamber comprising two IDEs for isolating EVs based on DEP, 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 capture section of the microfluidic channel in Lo to a DEP chamber connecting the microfluidic channel on the upstream and downstream of the DEP chamber, and further modify the array of electrodes in Lo to IDEs disposed on the bottom wall of the DEP chamber, as taught by combined Mora-Zamora and Hadady, since the microfluidic chamber with IDEs (DEP chamber) would remove the need of huge and expensive devices and would make the system smaller with better functionalities (the first paragraph in Col. 2 on page 1139 in Mora-Zamora), and would allow for isolation of EVs on IDEs by DEP (concluding remarks in Hadady).
Moral-Zamora further teaches a syringe placed on an infusion micropump so as to obtain a continuous flow rate (section 3.5).
Given the teachings of Lo regarding fluid flows through a microfluidic channel [para. 0033], and the teachings of Mora-Zamora regarding the use of a syringe placed on an infusion micropump to provide a continuous flow rate, 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 device by providing a syringe placed on a micropump, as taught by Mora-Zamora, since it would provide a continuous flow rate (section 3.5 in Mora-Zamora).
Modified Lo is silent to the following limitations: (4) one or more motors, wherein each motor contains one or more gears; (5) one or more reservoirs, wherein the one or more reservoirs connect to the microfluidics channel; and (6) wherein each DEP chamber contains at least one or more working electrodes, one or more reference electrodes, and one or more counter electrodes.
Bhargava teaches a solid-state microfluidic device using DEP forces (In some instances, the force is a dielectrophoretic force. In some instances, methods comprise switching the dielectrophoretic force polarity, thereby repelling particles when they are initially attracted to an electrode, and vice versa. This process may be automated [para. 0061]; Disclosed herein, in some aspects, are microfluidic systems for performing an assay for a target analyte in a sample [para. 078]), comprising one or more motors, wherein each motor contains one or more gears (The fluid logic may comprise a
motorized gantry, a motorized mover, an array of valves, a rotary shear valve, or a combination thereof [para. 0139]; the pump is a peristaltic pump [para. 0114]); one or more reservoirs, wherein the one or more reservoirs connect to microfluidics channels (the pump may be used to draw a fluid containing a reagent, solution, sample, or combination thereof, from a fluid reservoir of a system disclosed herein. The pump may draw the fluid through the fluidic logic [para. 0108]).
Given the teachings of Lo regarding the use of different buffers to wash the device [para. 0055, 0066, 0069-0070], 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 device of modified Lo to include one or more motors, wherein each motor contains one or more gears; and one or more reservoirs, wherein the one or more reservoirs connect to the microfluidics channel, as taught by Bhargava, since this would simply allow incorporating peristaltic pumps to delivery different buffers, reagents, and/or solutions from different reservoirs into the microfluidic device to wash the device and release the captured DNA/RNA (which corresponds to the EVs in modified Lo) [para. 0069 in Lo; 0157-0158 in Bhargava ].
Modified Lo is silent to the following limitations: (6) wherein each DEP chamber contains at least one or more working electrodes, one or more reference electrodes, and one or more counter electrodes.
Reyes-Hernandez teaches dielectrophoresis device comprising receiving a first biological sample containing the bioparticles of interest onto a DEP electrode array (abstract; [para. 0095-0097]), wherein the DEP electrode array comprises at least a working electrode, reference electrode, and counter electrode to monitor impedance of the trapped bioparticles (first/second/third/fourth electrodes 112/114/116/118, and reference electrodes 190/194 in Figs. 63-64 [para. 0159-0162]).
Mora-Zamora also teaches wherein the DEP chamber comprises two IDEs and two lateral electrodes (see Figs. 1-2), wherein the four electrodes are used to perform impedance measurement to quantify the amount of concentrated bacteria concentrated by DEP (section 3.2.2; Fig.6).
Since Lo teaches a device for isolating and detecting biomolecules using a DEP electrode array; Reyes-Hernandez discloses where bioparticles may be manipulated using a DEP electrode array comprising working, reference, and
counter electrodes; and Mora-Zamora teaches impedance measurement to quantify the captured/enriched/concentrated bacteria concentration concentrated by DEP, it would have been obvious to one of ordinary skill in the art to modify the electrode array of the DEP chamber in modified Lo to include at least a working electrode, a reference electrode, and a counter electrode within the DEP chamber, as taught by Reyes-Hernandez, since this would simply allow incorporating electrodes to detect impedance at the array position, thus indicating the amount of the captured bioparticles (corresponding to EVs in modified Lo) by the DEP forces ([para. 0159-0162 in Reyes-Hernandez]; Fig.6 and abstract in Mora-Zamora).
Response to Arguments
Applicant's arguments, see Remarks Pgs. 11-21, filed 12/11/2025, with respect to the 35 U.S.C. § 103 rejections have been fully considered, and all 103 rejections from the previous office action are withdrawn.
Applicant’s Argument #1:
Regarding claims 24-26, 28-29 and 31, Applicant argues at pages 12-14 that the combined prior art fails to teach EV-derived biomarkers of interest and the first biological sample containing EVs. Lo merely discloses capturing, concentrating, and isolating molecules that are free within a fluid and teaches away from isolating extracellular vesicle-derived (EV-derived) biomarkers on a solid-state device. Moral-Zamora does not make up for the deficiencies of Lo, as it teaches a prototype for concentrating bacteria.
Examiner’s Response #1:
Applicant’s arguments have been fully considered, but are moot in view of the new grounds of rejection above. Note that Lo teaches isolating and detecting miRNA in blood plasma, and does not explicitly teach wherein the miRNAs are from EVs of the blood plasma. The new prior art of Tian teaches isolating and detecting biomarkers such as miRNA from EVs of blood plasma on a microfluidic device (solid-state device), as outlined in the new grounds of rejection above. Lo does not teach away isolating extracellular vesicle-derived (EV-derived) biomarkers on a solid-state device since Lo teaches one alternative is to collect miRNAs encapsulated in exosomes, but the steps required to collect exosomes [para. 0023]. Thus, the disclosed device can be used to collect miRNAs from EVs if the EVs are collected.
Applicant’s Argument #4:
Regarding claims 27 and 30, Applicant argues at pages 14-15 that dependent claims 27 and 30 are allowable for the same reasons of the independent claim 24 above.
Examiner’s Response #2:
As outlined in the new grounds of rejection for claim 24 above, claim 24 is still unpatentable over the prior art of the record.
Applicant’s Argument #3:
Regarding claims 32-33, 35-36 and 38, applicant argues at pages 15-17 that
the combined prior art fails to teach EV-derived biomarkers of interest and the first biological sample comprises EVs comprising one or more biomarkers of interest.
Examiner’s Response #3:
Applicant’s arguments have been fully considered, but are moot in view of the new grounds of rejection above. Note that Lo teaches isolating and detecting miRNA in blood plasma, and does not explicitly teach wherein the miRNAs are from EVs of the blood plasma. The new prior art of Tian teaches isolating and detecting biomarkers such as miRNA from EVs of blood plasma on a microfluidic device (solid-state device), as outlined in the new grounds of rejection above.
Applicant’s Argument #4:
Regarding claims 34 and 37, applicant argues at pages 17-18 that the dependent claims 34 and 37 are allowable for their dependence on the independent claim 32.
Examiner’s Response #4:
As outlined in the new grounds of rejection for claim 32 above, claim 32 is still unpatentable over the prior art of the record.
Applicant’s Argument #5:
Regarding claim 39, applicant argues at pages 18-20 that the combined prior art fails to teach a biological sample comprising EVs, wherein the EVs of the biological sample are assigned one or more tags, recited in the amended claim 39.
Examiner’s Response #5:
Applicant’s arguments have been fully considered, but are moot in view of the new grounds of rejection above. Note that Lo teaches isolating and detecting miRNAs in blood plasma, and the target miRNAs are assigned one or more tags for detection, and does not explicitly teach wherein the miRNAs are from EVs of the blood plasma. The new prior art of Tian teaches isolating and detecting biomarkers such as miRNA from EVs of blood plasma on a microfluidic device (solid-state device), as outlined in the new grounds of rejection above.
Conclusion
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).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SHIZHI QIAN whose telephone number is (571)272-3487. The examiner can normally be reached Monday-Thursday 8:00 am-5:00 pm.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Luan V Van can be reached on 571-272-8521. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/SHIZHI QIAN/Examiner, Art Unit 1795