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WO2025193173A1 - Capteur, plateforme de détection, système et procédé de détection d'un polynucléotide cible - Google Patents

Capteur, plateforme de détection, système et procédé de détection d'un polynucléotide cible

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Publication number
WO2025193173A1
WO2025193173A1 PCT/SG2025/050184 SG2025050184W WO2025193173A1 WO 2025193173 A1 WO2025193173 A1 WO 2025193173A1 SG 2025050184 W SG2025050184 W SG 2025050184W WO 2025193173 A1 WO2025193173 A1 WO 2025193173A1
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WO
WIPO (PCT)
Prior art keywords
channel portion
sensor
target
sensing
electrode
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/SG2025/050184
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English (en)
Other versions
WO2025193173A9 (fr
Inventor
Zhi Xiang Trifanny YEO
Ali Asgar SALEEM BHAGAT
Chwee Teck Lim
Von Luigi Marcelo VALERIO
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National University of Singapore
Original Assignee
National University of Singapore
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Publication date
Application filed by National University of Singapore filed Critical National University of Singapore
Publication of WO2025193173A1 publication Critical patent/WO2025193173A1/fr
Publication of WO2025193173A9 publication Critical patent/WO2025193173A9/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0663—Whole sensors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces

Definitions

  • the present disclosure relates to a sensor, a sensing platform, a system and a method for detecting a target polynucleotide.
  • Non-small ceil lung cancer comprises nearly 80% of ail lung cancer cases and it poses a significant challenge in global healthcare.
  • NSCLC Non-small ceil lung cancer
  • acquiring genomic information of this cancer serves as the basis for guiding targeted therapy.
  • the use of cancer drugs tailored to specific mutations in the epidermal growth factor receptor (EGFR) has shown enhanced outcomes in patients’ overall survival (OS).
  • OS overall survival
  • current approaches for genetic testing through lung biopsies are invasive and can lead to complications such as infection.
  • certain lung cancer patient populations may also be unsuitable for tissue sampling due to comorbidities or debility, driving the need for alternative diagnostic modalities.
  • ctDNA blood-based circulating tumour DNA
  • ctDNA has been extensively researched in clinical studies across diagnostic, prognostic, and therapeutic monitoring domains. These short nucleotide fragments released from cancer cells contain information on the heterogenous mutations of the primary tumour. For example, in a recent phase II clinical trial involving NSCLC patients, changes in ctDNA levels monitored through next-generation sequencing (NGS) were found to correlate with changes detected through the standard radiographic tumour testing. Additionally, the baseline ctDNA levels were predictive of patient OS.
  • NGS next-generation sequencing
  • PCR real-time polymerase chain reaction
  • digital PCR often rely on amplification-based approaches to enhance sensitivity, which can be resource intensive and time-consuming.
  • microfluidic-based biosensors typically require a red blood cell (REC) depletion step prior to biomarker detection which involve passive sorting utilising a high dilution factor or a sheath flow for efficient RBC depletion.
  • REC red blood cell
  • aspects of the present application relates to a sensor, a sensing platform, a system and a method for detecting a target polynucleotide.
  • a sensor for detecting a target polynucleotide comprising: at least one oligonucleotide probe, wherein the at least one oligonucleotide probe comprises: a target-specific oligonucleotide complementary to a portion of the target polynucleotide: a shorter oligonucleotide anchored to an electrode, one end portion of the target-specific oligonucleotide is complementary to, and forms a duplex with, the shorter oligonucleotide; and a redox molecule attached to the target-specific oligonucleotide, wherein the target-specific oligonucleotide destabilises upon binding to the portion of the target polynucleotide and is released from the shorter oligonucleotide anchored to the electrode, modulating a distance between the redox molecule and a surface of the electrode to cause a change in electrochemical potential detected by
  • a distance between the redox molecule and a surface of the electrode can be modulated to cause a change in electrochemical potential detected by the electrode for detecting the target polynucleotide.
  • this redox-modified target-specific oligonucleotide can flow away from the electrode and out of a sensing platform comprising the sensor, thereby eliminating any potential background signal that could arise if it remains in a proximity of the electrode.
  • This displacement of the redox molecule via the release of the target-specific oligonucleotide of the oligonucleotide probe elicits changes in electron transfer kinetics of the sensor, thereby allowing rapid detection of the target polynucleotide by the electrode.
  • the sensor may comprise the electrode, wherein the surface of the electrode may be modified by dendritic structures formed on the surface of the electrode.
  • the electrode maybe formed using gold.
  • the redox molecule may include a methylene blue (MB) reporter.
  • the redox molecule includes one of: ferrocene, ruthenium complexes, nile blue (NB) and toluidine blue (TB).
  • the target polynucleotide or the at least one oligonucleotide probe may be at least one nucleic acid selected from the group comprising DNA, RNA, PNA and other nucleic acid analogues.
  • the target polynucleotide may be at least one nucleic acid associated with a nonhuman or human disease, genetic variants, forensic, strain identification, environmental and/or food contamination.
  • the target polynucleotide may be a cancer-related polynucleotide.
  • the target polynucleotide may include a base pair mutation, a binding site of the targetspecific oligonucleotide specific to the base pair mutation may be arranged to be included in the one end portion of the target-specific oligonucleotide.
  • the target-specific DNA oligonucleotide may have at least 80% sequence identity to the target DNA sequence or at least 80% sequence identity to the polynucleotide sequence set forth in one of SEQ ID NO: 31 , SEQ ID NO: 32 or SEQ ID NO: 33.
  • the shorter oligonucleotide may have at least 80% sequence identity to the polynucleotide sequence set forth in one of CATGCAG /ThioMC3-D/, GCGGGC /ThioMC3-D/ or TCTCCG /ThioMC3-D/, wherein the polynucleotide sequences CATGCAG /ThioMC3-D/, GCGGGC /ThioMC3-D/ and TCTCCG /ThioMC3- D/ are complementary to the SEQ ID NO: 31 , the SEQ ID NO: 32 and the SEQ ID NO: 33, respectively.
  • the shorter oligonucleotide may have a length of 4 to 10 nucleotide bases.
  • a sensing platform for detecting a target polynucleotide
  • the sensing platform comprising: a microfluidic device adapted to receive a sample comprising a cellular fraction and a substantially cell-free faction including the target polynucleotide, and to extract the substantially cell-free fraction from the sample; and a preceding sensor, the sensor adapted to receive the substantially cell-free fraction from the microfluidic device for detecting the target polynucleotide.
  • the microfluidic device may comprise: a sample inlet for receiving the sample; a contraction channel portion configured to focus the cellular fraction into a first stream in a central region of the contraction channel portion and the substantially cell-free faction into a second stream in a peripheral region of the contraction channel portion; an expansion channel portion comprising a widened channel portion and at least one bifurcation channel portion fluidly connected to a peripheral region of the widened channel portion, the widened channel portion having a channel width larger than the contraction channel portion and is arranged to receive the first stream having the cellular fraction, and the at least one bifurcation channel portion arranged to receive a portion of the second stream having the substantially cell-free faction; a waste outlet arranged to couple with the widened channel portion to receive the first stream at a downstream of the expansion channel; a sensing channel portion arranged to couple with the at least one bifurcation channel portion to receive the portion of the second stream having the substantially cell-free faction and to provide the portion of the second stream for sensing using the sensor; and a
  • the contraction channel portion and the expansion channel portion may form a contraction-expansion channel set, and the sensing platform may include a single set of contraction-expansion channel set.
  • the sensing channel portion may comprise an array of micropillars formed on an inner wall of the sensing channel portion, the array of micropillars may be adapted to suspend from the sensing channel portion without being in contact with the sensor.
  • the array of micropillars may include an array of micropillars having a diameter in a range of 10 pm to 100 pm each, and where each of the micropillars may be placed at a distance of 100 pm to 200 pm from one another.
  • the array of micropillars includes an array of micropillars having a diameter of 15 pm each, and wherein each of the micropillars are placed at a distance of 150 pm from one another.
  • the microfluidic device may be integrated with the sensor and the sensing channel portion has an area arranged to overlap with the sensor.
  • An insulation layer may be sandwiched between the microfluidic device and the sensor, the insulation layer may have openings adapted to allow the substantially cell-free fraction extracted from the microfluidic device to be provided to the sensor.
  • the sensing channel portion may comprise support micropillars adapted to support the sensing channel portion on the insulation layer.
  • the contraction channel portion, the expansion channel portion and the sensing channel portion may be coated with a surfactant.
  • a sensing platform for detecting a target polynucleotide
  • the sensing platform comprising: a microfluidic device comprising: a sample inlet adapted to receive a sample comprising a cellular fraction and a substantially cell-free faction including the target polynucleotide; a contraction channel portion configured to focus the cellular fraction into a first stream in a central region of the contraction channel portion and the substantially cell-free faction into a second stream in a peripheral region of the contraction channel portion; an expansion channel portion comprising a widened channel portion and at least one bifurcation channel portion fluidly connected to a peripheral region of the widened channel portion, the widened channel portion having a channel width larger than the contraction channel portion and is arranged to receive the first stream having the cellular fraction, and the at least one bifurcation channel portion arranged to receive a portion of the second stream having the substantially cell-free faction; a waste outlet arranged to couple with the widened channel portion to receive the
  • a system for detecting a target polynucleotide comprising: a preceding sensing platform; a potentiostat configured to receive measurement signals associated with the change in electrochemical potential detected by the electrode for detecting the target polynucleotide and to generate measurement data: and a computer comprising a processor and a data storage device storing computer program instructions operable to cause the processor to: (i) receive the measurement data from the potentiostat; and (ii) generate an analysis report based on the measurement data for detecting the target polynucleotide.
  • the system may comprise a syringe pump operationally connected to the sensing platform, the syringe pump arranged to provide the sample continuously at a sample flow rate
  • the sample flow rate may range from 100 pl/min to 300 pl/min.
  • a method of detecting a target polynucleotide comprising the steps of: (a) providing a sample comprising a cellular fraction and a substantially cell-free faction including the target polynucleotide to a microfluidic device; (b) extracting the substantially cell-free fraction from the sample using the microfluidic device; (c) providing the substantially cell-free fraction to a sensor, the sensor comprising at least one oligonucleotide probe, wherein the at least one oligonucleotide probe comprises: a target-specific oligonucleotide complementary to a portion of the target polynucleotide; a shorter oligonucleotide anchored to an electrode, one end portion of the target-specific oligonucleotide is complementary to, and forms a duplex with, the shorter oligonucleotide; and a redox molecule attached to the target-specific oligonucleot
  • the microfluidic device may comprise: a sample inlet for receiving the sample; a contraction channel portion configured to focus the cellular fraction into a first stream in a central region of the contraction channel portion and the substantially cell-free faction into a second stream in a peripheral region of the contraction channel portion; an expansion channel portion comprising a widened channel portion and at least one bifurcation channel portion fluidly connected to a peripheral region of the widened channel portion, the widened channel portion having a channel width larger than the contraction channel portion and is arranged to receive the first stream having the cellular fraction, and the at least one bifurcation channel portion arranged to receive a portion of the second stream having the substantially cell-free faction; a waste outlet arranged to couple with the widened channel portion to receive the first stream at a downstream of the expansion channel; a sensing channel portion arranged to couple with the at least one bifurcation channel portion to receive the portion of the second stream having the substantially cell-free faction and to provide the portion of the second stream for sensing using the sensor, and a
  • the sample may include whole blood or a 1-fold diluted blood sample.
  • the step of (a) providing the sample to the microfluidic device may comprise providing the sample continuously to allow the target-specific oligonucleotide bound to the portion of the target polynucleotide and released from the shorter oligonucleotide to flow away from the electrode.
  • Embodiments of a sensor, a sensing platform, a system and a method for detecting a target polynucleotide comprises a redox molecule attached to a targetspecific oligonucleotide of a oligonucleotide probe, By having the redox molecule attached to the target-specific oligonucleotide, and the target-specific oligonucleotide adapted to be destabilised upon binding to the portion of the target polynucleotide and is released from the shorter oligonucleotide anchored to the electrode, a distance between the redox molecule and a surface of the electrode can be modulated to cause a change in electrochemical potential detected by the electrode for detecting the target polynucleotide.
  • Figure 2 shows a schematic of a top planar view of a sensing platform of the system of Figure 1 in accordance with an embodiment
  • Figure 3 shows a schematic of a perspective view of various components of the sensing platform of Figure 2 in accordance with an embodiment
  • Figure 5 shows an image illustrating the use of liquid biopsy for detecting tumour DNA in accordance with an embodiment
  • Figure 6 shows a schematic of a top planar view of a sensing platform comprising a microfluidic device and a sensor for detecting a target polynucleotide in accordance with an embodiment
  • Figure 7 shows a series of schematics to illustrate a displacement mechanism of oligonucleotide probes used for detecting a target polynucleotide in accordance with an embodiment
  • Figure 8 shows a flowchart illustrating steps of a method for detecting a target polynucleotide in accordance with an embodiment
  • Figure 9 shows a series of schematics to illustrate measurements performed using a portable potentiostat and measurement data being provided to a computer for analysis in a clinical setting in accordance with an embodiment
  • Figure 10 shows a schematic of a microfluidic device including a blown-up schematic showing micropillars formed on an inner surface of a sensing channel portion of the microfluidic device to provide capillary effect in accordance with an embodiment
  • Figure 11 shows a schematic of a simplified layout of the microfluidic device having various channel portions in accordance with an embodiment
  • Figure 12 shows a schematic of an equivalent circuit of the microfluidic device of Figure 10 in accordance with an embodiment
  • Figures 13A and 13B show photographs of a sensing channel portion of a microfluidic device to illustrate flow profiles without and with micropiliars formed on an inner surface of the sensing channel portion at 2.5 minutes after a sample is provided to the microfluidic device in accordance with embodiments, where Figure 13A shows a photograph of the sensing channel portion without micropiliars formed on the inner surface of the sensing channel portion and Figure 13B shows a photograph of the sensing channel portion without micropillars formed on the inner surface of the sensing channel portion;
  • Figure 14 shows a series of photographs of a sensing channel portion of a microfluidic device with micropillars formed on an inner surface of the sensing channel portion at different times after a sample is provided to the microfluidic device in accordance with an embodiment
  • Figure 15 shows a block diagram of an expansion channel portion of a microfluidic device in accordance with an embodiment system for detecting a target polynucleotide with a micrograph to illustrate a flow profile of 6pm polystyrene beads in the expansion channel portion at a sample flow rate of 100pl/min;
  • Figure 16 shows a schematic to illustrate percent separation efficiency at a plasma outlet (or bifurcation channel portions) and a waste outlet of a microfluidic device using 6pm polystyrene beads in accordance with an embodiment
  • Figure 19 shows a bar chart showing simulation and experimental results relating to volume of a plasma obtained from the plasma outlet as a percentage of a total sample volume in accordance with an embodiment
  • Figures 20A, 20B and 20C show images of an expansion channel portion of a microfiuidic device with different sample flow rates in accordance with embodiments, where Figure 20A shows an image of the expansion channel portion with a sample flow rate of 100 pl/min, Figure 20B shows an image of the expansion channel portion with a sample flow rate of 150 pl/min and Figure 20C shows an image of the expansion channel portion with a sample flow rate of 200 pl/min;
  • Figure 21 shows a bar chart of lengths of a cell-free (CF) zone in the expansion channel portion against sample flow rates for a whole blood sample and for a 1 :1 diluted blood sample in accordance with embodiments;
  • CF cell-free
  • Figure 22 shows a graph of percentages of red-blood cell (RBC) depletion against sample flow rates for a whole blood sample and for a 1 :1 diluted blood sample in accordance with embodiments;
  • RBC red-blood cell
  • Figure 23 shows an amplification plot of fluorescence (RFU) versus cycle threshold (Ct) for various diluted plasma samples in accordance with an embodiment
  • Figure 24 shows an amplification plot of fluorescence (RFU) versus cycle threshold (Ct) for various plasma concentrations for a first set of plasma dilutions in accordance with an embod-ment;
  • Figure 25 shows an amplification plot of fluorescence (RFU) versus cycle threshold (Ct) for various plasma concentrations for a second set of plasma dilutions in accordance with an embodiment
  • Figure 26 shows an amplification plot of fluorescence (RFU) versus cycle threshold (Ct) for various DNA recovery percentages and for a sensing platform of the present disclosure in accordance with an embodiment
  • Figure 27 shows a plot of cycle threshold (Ct) values versus percentage recovery of DNA for a sensing platform of the present disclosure in accordance with an embodiment and other references;
  • Figures 28A, 28B and 28C show schematics of oligonucleotide probes and their nucleotide sequences complementary to target polynucleotides including T790M, L858R and Exon19del in accordance with an embodiment, where Figure 28A shows a schematic of a oligonucleotide probe and its nucleotide sequence for T790M, Figure 28B shows a schematic of a oligonucleotide probe and its nucleotide sequence for L858R, and Figure 28C shows a schematic of a oligonucleotide probe and its nucleotide sequence for Exon19del;
  • Figures 29A and 29B show an illustration of a redox molecule attached to a semihybridised oligonucleotide probe adapted to bind to a target polynucleotide and be released from an electrode, together with a current plot showing a decrease in a measured current upon displacement of the redox molecule, in accordance with an embodiment
  • Figure 31 shows a scanning electron microscope (SEM) image of dendritic structures formed on a surface of an electrode of the sensing platform of Figure 2 in accordance with an embodiment
  • Figures 32A and 32B show plots of electrochemical current versus applied potential based on the square wave voltammetry technique for various molar concentrations of oligonucleotide probes using a sensor having an electrode with a bare surface and a sensor having an electrode with dendritic structures formed on a surface of the electrode in accordance with an embodiment, where Figure 32A shows a plot of electrochemical current versus applied potential for a sensor having an electrode with a bare surface and Figure 32B shows a plot of electrochemical current versus applied potential for a sensor having an electrode with dendritic structures formed on a surface of the electrode;
  • Figures 34A, 34B and 34C show bar graphs of charge transfer resistance for targeted mutations T790M, L.858R and Exon19del measured using sensors with different electrode surfaces in accordance with embodiments, where Figure 34A shows a bar graph of charge transfer resistance for the targeted mutation T790M, Figure 34B shows a bar graph of charge transfer resistance for the targeted mutation L858R and Figure 34C shows a bar graph of charge transfer resistance for the targeted mutation Exon19del;
  • Figures 35A, 35B and 35C show electrochemical impedance spectroscopy (EIS) curves for targeted mutations T790M, L858R and Exon19del measured using sensors with different electrode surfaces in accordance with embodiments, where Figure 35A shows an EIS curve for the targeted mutation T790M, Figure 35B shows an EIS curve for the targeted mutation L858R and Figure 35C shows an EIS curve for the targeted mutation Exon19del;
  • EIS electrochemical impedance spectroscopy
  • Figures 38A, 38B and 38C show plots of measured current versus potential applied based on the square wave voltammetry technique using the sensing platform of Figure 2 for single-strand and double-strand target polynucleotides including mutations T790M,
  • Figures 39A, 39B and 39C show plots of percentage peak height change of a measured current versus concentration of target polynucleotides including mutations T790M, L858R and Exon19del in accordance with an embodiment, where Figure 39A shows a plot of percentage peak height change of a measured current versus concentration of the target polynucleotide including the mutation T790M, Figure 39B shows a plot of percentage peak height change of a measured current versus concentration of the target polynucleotide including the mutation L858R, and Figure 39C shows a plot of percentage peak height change of a measured current versus concentration of the target polynucleotide including the mutation Exon19del;
  • Figures 40A, 40B and 40C show plots of percentage peak height change of a measured current versus allele frequency (AF) of target polynucleotides including mutations T790M, L858R and Exon19del in accordance with an embodiment, where Figure 40A shows a plot of percentage peak height change of a measured current versus AF of target polynucleotides including the mutation T790M, Figure 40B shows a plot of percentage peak height change of a measured current versus AF of target polynucleotides including the mutation L858R, and Figure 40C shows a plot of percentage peak height change of a measured current versus AF of target polynucleotides including the mutation Exon19del;
  • AF allele frequency
  • Figure 41 shows a plot of percentage peak height change of a measured current using the biosensing platform of Figure 2 versus allele frequency (AF) of the corresponding target mutations measured using conventional next-generation sequencing (NGS) technique in accordance with an embodiment
  • Figure 43 shows a schematic for comparing the required processing time of different sample-to-answer processing methods including using the biosensing platform of Figure 2 in accordance with an embodiment
  • Figure 44 shows a plot of measured current versus time measured using the sensing platform of Figure 2 for target polynucleotides including mutations T790M, L858R and Exon19del in accordance with an embodiment
  • Figures 45A, 45B and 45C show bar graphs of measured current versus time measured using the sensing platform of Figure 2 for target polynucleotides including mutations T790M, L858R and Exon19del in accordance with an embodiment, where Figure 45A shows a bar graph of measured current versus time for target polynucleotides including the mutation T790M, Figure 45B shows a bar graph of measured current versus time for target polynucleotides including the mutation L858R and Figure 45C show's a bar graph of measured current versus time for target polynucleotides including the mutation Exon19del;
  • Figure 46 shows a schematic of a design of a microfluidic device including two contraction-expansion sets in accordance with an embodiment
  • Figure 47 shows a schematic of an equivalent circuit of the microfluidic device of Figure 46 in accordance with an embodiment.
  • Exemplary embodiments relate to a sensor, a sensing platform, a system and a method for detecting a target polynucleotide.
  • substantially cell-free fraction refers to a fraction being 90% to 100% cell-free, or preferably about 95% to 100% cell-free.
  • nucleic acid or “nucleic acid sequence,” as used herein, refer to an oligonucleotide, nucleotide, polynucleotide, or any fragment thereof, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • PNA peptide nucleic acid
  • oligonucleotide refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PGR amplification or in a hybridization assay or microarray.
  • oligonucleotide is substantially equivalent to the terms “amplimers,” “primers,” “oligomers,” and “probes,” as these terms are commonly defined in the art.
  • a biological sample suspected of containing lung cancer genome sequences may comprise a bodily fluid: an extract from a cell, chromosome, organelle, or membrane isolated from a celi; a cell; genomic DNA, RNA, or cDNA (in solution or bound to a solid support); a tissue; a tissue print; and the like.
  • NSCLC non-small cell lung cancer
  • oligonucleotides used in the present disclosure may be structurally and/or chemically modified to, for example, prolong their activity in samples potentially containing nucleases, during performance of methods of the disclosure, or to improve shelf-life in a kit.
  • the aptamer and/or inverter and/or signalling nanostructure or any oligonucleotide primers or probes used according to the present disclosure may be chemically modified.
  • said structural and/or chemical modifications include the addition of tags, such as fluorescent tags, radioactive tags, biotin, a 5' tail, the addition of phosphorothioate (PS) bonds, 2'-O- Methyl modifications and/or phosphoramidite C3 Spacers during synthesis.
  • tags such as fluorescent tags, radioactive tags, biotin, a 5' tail, the addition of phosphorothioate (PS) bonds, 2'-O- Methyl modifications and/or phosphoramidite C3 Spacers during synthesis.
  • PS phosphoroth
  • the signalling oligonucleotide was modified for attachment chemistry with a 5’ amino group.
  • Other attachment modifications can be made on the 5’ end such as thiol, acryldite, azide, etc.
  • the term “comprising” or “including” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof.
  • the term “comprising” or “including” also includes “consisting of”.
  • the variations of the word “comprising”, such as “comprise” and “comprises”, and “including”, such as “include” and “includes”, have correspondingly varied meanings.
  • FIG. 1 shows a block diagram of a system 100 for detecting a target polynucleotide in accordance with an embodiment.
  • the system 100 comprises a sensing platform 102 including a microfluidic device 104, and a sensor 106.
  • the microfluidic device 104 is adapted to receive a sample 105 comprising a cellular fraction and a substantially cell- free faction including a target polynucleotide, and to extract the substantially cell-free fraction from the sample.
  • the sensor 106 is adapted to receive the substantially cell- free fraction from the microfluidic device 104 for detecting the target polynucleotide.
  • the microfluidic device 104 is formed in a microfluidic layer 108 and the sensor 106 is formed on a substrate layer 110
  • the sensing platform in the present embodiment also comprises an insulation layer 112 sandwiched between the microfluidic device 104 and the sensor 106.
  • the insulation layer 112 has openings adapted to allow substantially cell-free fraction of a sample extracted from the microfluidic device 104 to be provided to the sensor 106.
  • the system 100 further comprises a potentiostat 114 and a computer 116, The potentiostat 114 is configured to receive measurement signals associated with a change in electrochemical potential detected by the sensor 106 for detecting the target polynucleotide and to generate measurement data for feeding to the computer 116.
  • the microfluidic device 104 includes: (i) a sample inlet (not shown) for receiving the sample 105, (ii) a contraction channel portion 120 configured to focus the cellular fraction into a first stream in a central region of the contraction channel portion 120 and the substantially cell-free faction into a second stream in a peripheral region of the contraction channel portion 120, (iii) an expansion channel portion 122 comprising a widened channel portion and at least one bifurcation channel portion fluidly connected to a peripheral region of the widened channel portion, the widened channel portion having a channel width larger than the contraction channel portion and is arranged to receive the first stream having the cellular fraction, and the at least one bifurcation channel portion arranged to receive a portion of the second stream having the substantially cell-free faction, (iv) a waste outlet (not shown) arranged to couple with the widened channel portion to receive the first stream at a downstream of the expansion channel, and (v) a sensing channel portion 124 arranged to couple with the at
  • Figure 2 shows a schematic of a top planar view of a sensing platform 200 of the system 100 of Figure 1 in accordance with an embodiment
  • the sensing platform 200 comprises the microfluidic device 104 and the sensor 106, and a configuration of the microfluidic device 104 and the sensor 106 interface is shown in relation to Figure 2.
  • the sample for detection includes whole blood or a 1-fold diluted blood sample.
  • an expansion channel portion comprising a widened channel portion and bifurcation channel portions 204 fluidly connected to a peripheral region of the widened channel portion is used in the present embodiment.
  • the widened channel portion having a channel width larger than the contraction channel portion 202 received the RBCs of the sample by leveraging the Zweifach-Fung effect, wherein RBCs are directed into a waste outlet 206 with a higher flow rate
  • the bifurcation channel portions 204 are arranged to receive the substantially cell-free portion of the sample and provide the substantially cell-free portion of the sample to the sensing channel portion 208.
  • each of the two bifurcation channel portions 204 adopting a wavy structure.
  • the wavy structure allows customisation of a fluid resistance of the bifurcation channel portion 204 by providing a handle to adjust an effective channel length of the bifurcation channel portion 204, thereby optimising the fluid resistance and enhancing purity of the substantially cell-free portion obtained.
  • optimised channel lengths of the bifurcation channel portions 204 plasma enrichment exceeding 99% at a flow rate of 100pl/min was obtained.
  • the sensing channel portion 208 has an area arranged to overlap the sensor 210. More particularly, the sensing channel portion 208 overlaps with an electrode (or a working electrode) of the sensor 210 on which at least one oligonucleotide probe is anchored.
  • the sensor 210 includes three working electrodes, a reference electrode and a counter electrode and is adapted to be connected to a potentiostat 114 for detecting target polynucleotides.
  • each of the three working electrodes include an oligonucleotide probe specific to a target mutation of a target polynucleotide.
  • FIG 3 shows a schematic 300 of a perspective view of various components of the sensing platform 200 of Figure 2 in accordance with an embodiment.
  • a microfluidic device 301 is formed on a microfluidic layer 302 which can then be formed on a patterned insulation layer 304 having openings 305.
  • a sensor 306 formed on a substrate layer 308.
  • the microfluidic layer 302, the patterned insulation layer 304 and the substrate layer 308 having the sensor 306 formed thereon can be integrated to form the sensing platform 200.
  • the microfluidic device 301 was fabricated from polydimethylsiloxane (PDMS) and was covalently bonded onto an SU-8 insulation layer patterned on glass.
  • the sensor 306 comprises three gold (Au) working electrodes, a silver/silver chloride (Ag/AgCI) reference electrode and a gold counter electrode, and was fabricated on a glass substrate 308.
  • This sensing platform 200 of the present embodiment serves as a stable and durable platform for continuous blood processing.
  • Figure 4 shows an image 400 of the sensing platform 200 of Figure 2 to illustrate a sample inlet and outlets of the sensing platform in accordance with an embodiment
  • the microfluidic device 104 includes a sample inlet 402 for providing a sample to the microfluidic device 104, a waste outlet 404 for receiving RBCs separated out from the sample by the microfluidic device 104, and a plasma outlet 406 for receiving piasma separated out from the sample by the microfluidic device 104 and for which has passed through the sensor 406.
  • the following descriptions of the sensor, sensing platform and system are directed to providing a POC microfluidic-based biosensing platform for detecting several EGFR mutation biomarkers (i.e., Exon19del, L858R, and T790M) predictive of response to existing NSCLC treatments in an exemplary embodiment, although it should be appreciated that the microfluidic device and the sensor can be adapted to detect other target polynucleotides having other base pair mutations or mutation biomarkers.
  • channel widths and/or lengths of the microfluidic device and/or one or more oligonucleotide probes of the sensor can be designed or adapted for detecting other target polynucleotides in other embodiments.
  • FIG. 5 shows an image 500 illustrating the use of liquid biopsy for detecting tumour DNA (ctDNA) from whole blood sample in accordance with an embodiment.
  • Liquid biopsy offers a minimally invasive approach for ctDNA detection.
  • Figure 6 shows a schematic of a top planar view 600 of a sensing platform comprising a microfluidic device 602 and a sensor 604 for detecting a target polynucleotide in accordance with an embodiment.
  • a syringe pump is used to introduce sample at a controlled rate via a single sample inlet 606 of the microfluidic device 602 into a contraction channel portion (or focusing channel) 608 where plasma is separated from whole blood based on the Fahrseus-Lindqvist principle.
  • a contraction channel portion or focusing channel
  • an expansion channel portion comprising a widened channel portion 610 and bifurcation channel portions 612 fluidly connected to a peripheral region of the widened channel portion 610 is used in the present embodiment.
  • the widened channel portion has a channel width larger than the contraction channel portion 608 to receive RBCs of the sample by leveraging the Zweifach-Fung effect, while the bifurcation channel portions 612 having wavy structures are arranged to receive substantially cell-free plasma comprising ctDNA carrying T790M, L858R and Exon19del mutations of the blood sample, and to provide the substantially cell-free plasma to a sensing channel portion 614.
  • an area of the sensing channel portion 614 overlaps with the sensor 604 and encompass at least sensing portions of three working electrodes 616, 618, 620.
  • each of the three working electrodes 616, 618, 620 includes specific oligonucleotide probes for detecting actionable DNA biomarkers in the EGFR, namely T790M, L858R and Exon19del, respectively, based on a redox-molecule displacement mechanism.
  • the sensor 604 includes a reference electrode 622 and a counter electrode 624. The combination of the three working electrodes 616, 618, 620, the reference electrode 622 and the counter electrode 624 allowing measurements to be obtained by a potentiostat.
  • Figure 7 shows a series of schematics to illustrate a displacement mechanism of oligonucleotide probes used for detecting a target polynucleotide in accordance with an embodiment.
  • a first schematic 702 double-stranded oligonucleotide probes comprising a shorter oligonucleotide (i.e. an anchor strand) for anchoring to an electrode of the sensor and target-specific oligonucleotide complementary to a portion of the target polynucleotide are anchored to a dendritic gold electrode maintaining stable electron transfer
  • a redox molecule is attached to the target-specific oligonucleotide (i.e. a reporter strand).
  • plasma having ctDNA and healthy DNA reaches the sensor in a sample loading process.
  • a portion of a target polynucleotide binds or hybridizes with the target-specific oligonucleotide in a competitive binding process, thereby destabilises the target-specific oligonucleotide from the shorter oligonucleotide.
  • the target-specific oligonucleotide having the attached redox molecule binds with the portion of the target polynucleotide and is displaced and carried along with the flow to be removed from the sensing channel portion of the sensing platform
  • a redox molecule e.g. a redox-active methylene blue reporter
  • the target-specific oligonucleotide e.g. at an end portion or a termini.
  • Binding events between the target polynucleotide and the target-specific oligonucleotide of the oligonucleotide probe induce structural changes of the oligonucleotide probe, thereby modulating a distance between the redox molecule and a surface of the electrode.
  • binding events elicit changes in electron transfer kinetics, causing a change in electrochemical potential detected by the electrode, thereby allowing rapid detection of the target polynucleotide.
  • the oligonucleotide probe is also designed to allow the target polynucleotide to outcompete the shorter oligonucleotide (i.e. an anchor strand) for binding with the target-specific oligonucleotide, thereby destabilising the target- specific oligonucleotide and releasing it from the anchor strand of the oligonucleotide probe. This results in the displacement of the redox molecule within a plasma flow for exiting the sensing platform.
  • the target polynucleotide i.e. an anchor strand
  • Figure 8 shows a flowchart illustrating steps of a method 800 for detecting a target polynucleotide in accordance with an embodiment.
  • a sample comprising a cellular fraction and a substantially cell-free faction including the target polynucleotide is provided to a microfluidic device.
  • the sample may include a whole blood sample or a 1-fold diluted blood sample.
  • the substantially cell-free fraction is extracted from the sample using the microfluidic device.
  • Working of the microfluidic device has been explained in relation to Figures 2 and 6.
  • the substantially cell-free fraction is provided to a sensor.
  • An example of the sensor is shown in Figure 6.
  • the sensor comprises at least one oligonucleotide probe.
  • the at least one oligonucleotide probe comprises: a target-specific oligonucleotide complementary to a portion of the target polynucleotide, a shorter oligonucleotide anchored to an electrode, one end portion of the target-specific oligonucleotide is complementary to, and forms a duplex with, the shorter oligonucleotide, and a redox molecule attached to the target-specific oligonucleotide.
  • the target-specific oligonucleotide of the probe is adapted to destabilise upon binding to the portion of the target polynucleotide and is released from the shorter oligonucleotide anchored to the electrode, modulating a distance between the redox molecule and a surface of the electrode to cause a change in electrochemical potential detected by the electrode. Details of the displacement mechanism of the redox molecule of the probe have been provided in relation to Figure 7.
  • a step 808 a presence of the target polynucleotide is detected by the change in electrochemical potential using the electrode of the sensor. Measurements of the sensor can be provided to a potentiostat for analysis.
  • Figure 9 shows a series of schematics to illustrate measurements performed using a portable potentiostat and measurement data being provided to a computer for analysis in a clinical setting in accordance with an embodiment.
  • the system 100 for detecting a target polynucleotide in the present embodiment is designed to streamline a current ctDNA sample-to-answer workflow to two steps: 1) introduction of a sample (e.g a whole blood sample) to a sample inlet of a microfluidic device of a sensing platform and 2) measurements provided directly to a suitable computer with appropriate software for result analysis.
  • a sample e.g a whole blood sample
  • a microfluidic device of a sensing platform enables a larger scale processing of blood to realize a liquid biopsy through the system, eliminating a limitation of sample volume as compared to existing approaches.
  • a sensing platform comprising a microfluidic device 904 for separating substantially cell-free faction (i.e.
  • a plasma including a target polynucleotide (e.g. ctDNA) from a sample and a sensor 906 for detecting the target polynucleotide, is inserted or operationally coupled to a portable potentiostat 908. Measurements performed using square wave voltammetry can be obtained with using the portable potentiostat 908. Data of these measurements are then transmitted to a computer for analysis.
  • a target polynucleotide e.g. ctDNA
  • a sensor 906 for detecting the target polynucleotide inserted or operationally coupled to a portable potentiostat 908. Measurements performed using square wave voltammetry can be obtained with using the portable potentiostat 908. Data of these measurements are then transmitted to a computer for analysis.
  • measurement data can be transmitted by the portable potentiostat to a computer 912 via Bluetooth. Analysis of the measurement data can be performed using commercially available software on the computer 912. In a proposed clinical setting as shown by a schematic 920, results provided by the analysis can then be used for personalised treatment.
  • the POC mutation assessment using the system of the present embodiment eliminates the need for sample pre-processing and enables direct identification and quantification of nucleic acid targets from a simple blood draw which can facilitate rapid treatment management for patients.
  • Figure 10 shows a schematic of a microfluidic device 1000 including a blown-up schematic showing micropillars formed on an inner surface of a sensing channel portion of the microfluidic device to provide capillary effect in accordance with an embodiment.
  • the capillary effect provided by the micropillars allow uniform distribution of plasma to the entire sensing channel portion of the microfluidic device. This in turns ensure that the plasma flows pass the working electrodes of the sensor of the sensing platform.
  • the microfluidic device 1000 of the sensing platform is designed to separate or extract a substantialiy celi-free fraction (i.e. plasma) from a blood sample based on the Fahrasus-Lindqvist and Zweifach-Fung effects, which are achieved through contraction-expansion geometries using a contraction channel portion and a expansion channel portion of the microfluidic device 1000, Providing the substantially cell-free fraction to the sensor for detection helps to mitigate interference to electrochemical measurements due to the presence of RBCs, thereby improving detection sensitivity of the sensor.
  • the contraction channel portion and the expansion channel portion comprising bifurcation channels and a widened channel portion are shown as 1002 in relation to Figure 10.
  • the substantially cell-free fraction (i.e. plasma) having target polynucleotides is received at a sensing channel portion 1004 of the microfluidic device, where the substantially celi- free fraction (i.e. plasma) is exposed or provided to a sensor of the sensing platform.
  • the plasma 1010 received at the sensing channel portion 1004 flows across an entire area of the sensing channel portion 1004 for sensing by the sensor.
  • micropillars 1012 are formed on an inner wall of the sensing channel portion 1004.
  • the array of micropillars are adapted to suspend from the sensing channel portion without being in contact with the sensor.
  • the capillary effect 1014 provided by the micropillars allow uniform distribution of plasma to the entire sensing channel portion of the microfluidic device. This in turns ensure that the plasma flows pass the working electrodes of the sensor of the sensing platform.
  • the sensing channel portion 1004 formed above a sensing region of the sensor was specifically designed with a radius 200 times larger than a channel width of the bifurcation channel portions to effectively encompass the working electrodes of the sensor.
  • the micropillars includes an array of micropillars having a diameter of 15 pm each, where each of the micropillars are placed at a distance of 150 pm from one another.
  • the sensing channel portion 1004 comprises support micropillars adapted to support the sensing channel portion on the insulation layer.
  • the support micropillars may include four micropillars each having a diameter of 3Q pm.
  • the support micropillars are in contact with the insulation layer and are adapted to prevent the sensing channel portion on top of the sensor (made of flexible PDMS material in this case) from collapsing onto the sensor due to a large diameter of the sensing channel portion.
  • the present microfluidic device provides a higher throughput for plasma extraction. This is shown in Table 1 ,
  • Figure 11 shows a schematic of a simplified layout 1100 of the microfluidic device having various channel portions in accordance with an embodiment.
  • ‘C’ 1102, 1104 corresponds to narrow ‘contraction’ channels
  • ‘E’ 1106 corresponds to an ‘expansion’ channel
  • ‘P’ 1108, 1110 corresponds to ‘plasma’ channels (i.e. bifurcation channel portions) which lead to a sensmg channel portion of the microfluidic device.
  • the first contraction channel C1 1102 focuses RBCs to its mid-section through the Fahraeus-Lindqvist effect, forming a plasma boundary' adjacent to the channel walls.
  • the channel expands into a larger area E 1106 with adjoining bifurcations P 1108, 1110.
  • Plasma is directed through P 1108, 1110 to a sensing channel portion 1112 while RBCs flow towards a waste channel 1104 due to the Zweifach-Fung effect. This ensures minimal presence of RBCs directed to the sensor for reducing interference for detection.
  • a sample inlet 1114 and waste outlets 1116 for the RBCs and plasma (after sensing) are also shown.
  • Figure 12 shows a schematic of an equivalent circuit 1200 of the microfluidic device 1000 of Figure 10 in accordance with an embodiment. Channel portions of the microfluidic device 1000 are labelled using same reference numbers as referred to in Figure 11.
  • Table 2 Summary of parameters in C1 , C2, E and P.
  • An electrical circuit simulator was used to optimize the channel lengths and hydraulic/fluidic resistances of the various channel portions of the microfluidic device 1000 to obtain desired output in microfluidic device.
  • the relationship between hydraulic resistance nd pressure drop, A p R ⁇ Q, where p is the viscosity, L is length, d is the diameter and Q is the volumetric flow rate, allowed us to calculate optimal channel dimensions for the microfluidic device.
  • the parameters for the various channel portions are shown in relation to Table 2.
  • the microfluidic device of the present embodiment is design to direct 14% of the total sample input into a sensing channel portion of the microfluidic device for ctDNA detection.
  • the optimal channel length of the bifurcation channel portions required to achieve a specific plasma output was determined to exceed 37mm in the present embodiment, surpassing the dimension of the sensor.
  • the bifurcation channel portions in the present embodiment are therefore to include a wavy structure to accommodate this length requirement.
  • Figures 13A and 13B show photographs of a sensing channel portion of a microfluidic device to illustrate flow profiles without and with micropillars formed on an inner surface of the sensing channel portion at 2.5 minutes after a sample is provided to the microfluidic device in accordance with embodiments Flow direction of the sample is from the left to right in these photographs.
  • Figure 13A shows a photograph 1302 of a sensing channel portion 1302 without micropillars formed on the inner surface of the sensing channel portion
  • Figure 13B shows a photograph 1310 of a sensing channel portion 1312 without micropillars formed on the inner surface of the sensing channel portion. From these photographs 1302, 1304, it is clear that the presence of micropillars formed in the sensing channel portion helps to ensure uniform fluid distribution within the entire sensing channel portion of the microfluidic device, which in turns promote uniform coverage for the working electrodes of the sensor for detection.
  • Figure 14 shows a series of photographs 1400 of a sensing channel portion of a microfluidic device with micropillars formed on an inner surface of the sensing channel portion at different times after a sample is provided to the microfluidic device in accordance with an embodiment.
  • Flow direction of the sample is from the left to right in these photographs, and is at a controlled sample flow rate of 100 pL/min. Based on the controlled sample flow rate of 100 pL/min, it is shown that the entire sensing channel portion of the microfluidic device is filled in about 160s for the present embodiment.
  • Figure 15 shows a block diagram 1500 of an expansion channel portion 1502 of a microfluidic device in accordance with an embodiment system for detecting a target polynucleotide, with a micrograph 1504 to Illustrate a flow profile of 6 urn polystyrene beads in the expansion channel portion at a sample flow rate of 100 pl/min.
  • the polystyrene beads formed a parabolic flow profile 1506 in the expansion channel portion E1 with a distinct bead-free boundary region.
  • 99.96% of the polystyrene beads went towards a waste channel portion 1508 leading to a waste outlet. Hence, only a low number of beads entered the sensing channel portion.
  • Figure 16 shows a schematic 1600 to illustrate percent separation efficiency at a plasma outlet and a waste outlet of a microfluidic device using 6 urn polystyrene beads in accordance with an embodiment.
  • the plasma outlet relates to used plasma which flows out of the sensing channel portion of the microfluidic device after sensing by the sensor of the sensing platform.
  • 99.96% 1602 of the polystyrene beads went towards the waste channel portion 1508, while 0.04% 1604 of the polystyrene beads passed through the sensing channel portion and exited the plasma outlet.
  • blood samples were collected in an EDTA tube and the microchannels or channel portions of the microfluidic device were coated with a surfactant prior to sample processing.
  • suitable surfactants include Pluronic® F-68 and Pluronic® F-127. These steps help to ensure more accurate plasma separation was achieved.
  • undiluted blood or whole blood
  • 1-fold diluted blood samples were used for microfluidic performance characterisation.
  • 1 ml of blood could be processed in 10 mins.
  • Figure 17 shows an microscopy image 1700 of an expansion channel portion of a microfluidic device visualize under 10x magnification with 1-fold blood dilution in accordance with an embodiment.
  • RBCs in the expansion channel portion of the microfluidic device formed a parabolic flow profile 1702, with cell-free plasma directed towards the bifurcation channel portions 1704, 1706, as shown in Figure 1700.
  • Figures 18A and 18B show information relating to samples collected after microfluidic processing from a plasma outlet and a waste outlet of a microfluidic device in accordance with an embodiment.
  • Figure 18A shows images 1802, 1804 of samples collected after microfluidic processing from the plasma outlet and the waste outlet, respectively.
  • Figure 18B shows a bar chart 1810 of percentage volume and percentage red blood cells (RBCs) of the samples collected after microfluidic processing from the plasma outlet and the waste outlet, where a bar 1812 provides a graphical representation of the percentage volume of the sample collected at the plasma outlet and the waste outlet and a bar 1814 provides a graphical representation of the red blood cells (RBCs) of the sample collected at the plasma outlet and the waste outlet.
  • the bar 1812 indicates that there is about 14% of volume 1816 of the sample being collected at the plasma outlet and 86% of volume 1818 of the sample being collected at the waste outlet.
  • the bar 1814 indicates that there is about 0.3% of RBCs 1820 of the sample being collected at the plasma outlet and 99.7% of RBCs 1822 of the sample being collected at the waste outlet.
  • Figure 19 shows a bar chart 1900 showing simulation results 1902 and experimental results 1904 relating to volume of a plasma obtained from the plasma outlet as a percentage of a total sample volume in accordance with an embodiment
  • Error bars 1906 represent standard deviation across three experimental replicates.
  • Figures 20A, 20B and 20C show images of an expansion channel portion of a microfluidic device with different sample flow rates for 1-fold blood samples (i.e. 1 :1 diluted blood samples) in accordance with embodiments.
  • Figure 21 shows a bar chart 2100 of lengths of a celi-free (CF) zone in the expansion channel portion against sample flow rates for whole blood samples and for 1 :1 diluted blood samples in accordance with embodiments.
  • Experiments for the whole blood samples were performed using sample flow rates of 150 pl/min, 200 pl/min, 250 pl/min and 300 pl/min, while experiments for the whole blood samples were performed using sample flow rates of 100 pl/min, 150 pl/min and 200 pl/min.
  • Data shown in the bar chart 2100 are presented as mean ⁇ standard error of the mean (SEM).
  • Figure 22 shows a graph 2200 of percentages of red-blood cell (RBC) depletion against sample flow rates for whole blood samples and for 1:1 diluted blood samples for the experiments performed in relation to Figure 21 .
  • RBC red-blood cell
  • Quantitative polymerase chain reaction was used to quantify the cycle threshold (Ct) values based on the number of DNA copies recovered in the plasma.
  • Figure 23 shows an amplification plot 2300 of fluorescence (RFU) versus cycle threshold (Ct) values for various diluted plasma samples based on qPCR analysis in accordance with an embodiment.
  • the amplification plot 2302 is associated with a 1 :10 (plasma:water) diluted plasma sample
  • the amplification plot 2304 is associated with a 1 :100 (plasma:water) diluted plasma sample
  • the amplification plot 2306 is associated with a 1:100 (plasma:water) diluted plasma sample.
  • Figure 24 shows an amplification plot 2400 of fluorescence (RFU) versus cycle threshold (Ct) for various plasma concentrations for a first set of plasma dilutions in accordance with an embodiment.
  • the amplification plot 2402 is associated with a 1 :2 (plasma:water) diluted plasma sample
  • the amplification plot 2404 is associated with a 1 :12.5 (plasma: water) diluted plasma sample
  • the amplification plot 2406 is associated with a 1 :25 (plasma:water) diluted plasma sample
  • the amplification plot 2408 is associated with a 1 :50 (plasma:water) diluted plasma sample
  • the amplification plot 2410 is associated with a 1:75 (plasma:water) diluted plasma sample
  • the amplification plot 2412 is associated with a 1 :100 (plasma:water) diluted plasma sample.
  • the blank sample is for control and shows close to zero RFU values.
  • Figure 25 shows an amplification plot of fluorescence (RFU) versus cycle threshold (Ct) for various plasma concentrations for a second set of plasma dilutions in accordance with an embodiment.
  • the amplification plot 2502 is associated with a 1:3 (plasma:water) diluted plasma sample
  • the amplification plot 2504 is associated with a 1 :5 (plasma:water) diluted plasma sample
  • the amplification plot 2506 is associated with a 1 :6 (plasma:water) diluted plasma sample
  • the amplification plot 2508 is associated with a 1 :7.5 (plasma:water) diluted plasma sample
  • the amplification plot 2510 is associated with a 1 :10 (plasma:water) diluted plasma sample
  • the amplification plot 2512 is associated with a 1 :12.5 (plasma:water) diluted plasma sample.
  • labile 3 Summary of plasma dilutions versus cycle threshold values As shown in relation to Figures 24, 25 and Table 3, with qPCR, a noticeable signal inhibition was observed for lower plasma dilutions, resulting in inconsistent Ct values obtained. It is therefore determined that a 1 :10 plasma dilution is typically required for more accurate quantification of the recovery efficiency using qPCR.
  • Figure 26 shows an amplification plot of fluorescence (RFU) versus cycle threshold (Ct) for various DNA recovery percentages and for a sensing platform of the present disclosure in accordance with an embodiment.
  • the amplification plot 2602 is associated with DNA recovery from the plasma outlet of the sensing platform of the present disclosure, while the amplification plots 2604, 2606, 2608, 2610 are associated with reference DNA recovery efficiency curves for 100% DNA recovery, 90% DNA recovery, 50% DNA recovery and 25% DNA recovery, respectively.
  • Figure 27 shows a plot 2700 of cycle threshold (Ct) values versus percentage recovery of DNA for a sensing platform of the present disclosure in accordance with an embodiment and other references.
  • the data point 2702 indicates the cycle threshold value and the corresponding DNA recovery? percentage for the sensing platform of the present disclosure.
  • the sensing platform of the present disclosure is capable of detecting target mutations in target polynucleotides directly from plasma without further dilution. As shown in relation to Figures 26 and 27, by using reference curves generated with Ct values obtained from various recovery efficiencies, it is concluded that processing blood samples containing target polynucleotides (i.e. target DNA) using the microfluidic device of the sensing platform of the present disclosure was able to achieve a Ct value of near 100% of the input DNA at 100 pL/min.
  • target DNA target polynucleotides
  • the oligonucleotide probe comprises: (I) a target-specific oligonucleotide complementary to a portion of the target polynucleotide, (ii) a shorter oligonucleotide anchored to an electrode, where one end portion of the target-specific oligonucleotide is complementary to, and forms a duplex with, the shorter oligonucleotide, and (Hi) a redox molecule attached to the target-specific oligonucleotide.
  • the target-specific oligonucleotide destabilises upon binding to a portion of a target polynucleotide and is released from the shorter oligonucleotide anchored to the electrode, thereby modulating a distance between the redox molecule and a surface of the electrode to cause a change in electrochemical potential detected by the electrode.
  • oligonucleotide probes against T790M, L858R and Exon19del are considered
  • the oligonucleotide probe or a DNA comprises a targetspecific nucleic acid sequence, with a tail end or an end portion of the target-specific nucleic aad sequence being hybridized to a shorter oligonucleotide strand.
  • the shorter oligonucleotide strand anchors the DNA probe to a gold (AU) electrode having an electrode surface modified with dendritic nanostructures.
  • the shorter oligonucleotide strand anchors the DNA probe to the electrode surface via gold-thiol (Au-S) bonds in this case.
  • the target-specific nucleic acid sequence is complementary to a target polynucleotide (in this case, ctDNAs associated with T790M, L858R or Exon19del base pair mutations) and is modified with a methylene blue (MB) reporter redox molecule.
  • a target polynucleotide in this case, ctDNAs associated with T790M, L858R or Exon19del base pair mutations
  • MB methylene blue
  • any mutation target will compete with the anchored shorter oligonucleotide strand, resulting in the removal of the target-specific nucleic acid sequence with the MB reporter redox molecule.
  • the MB reporter redox molecule Once the MB reporter redox molecule is displaced, it will be directed by the flow path towards the plasma outlet of the microfluidic device.
  • a decrease in electrochemical signal represents detection of a target mutation, which is correlated to a concentration of the target ctDNA.
  • Figures 28A, 28B and 28C show schematics of oligonucleotide probes and their nucleotide sequences complementary to target polynucleotides including T790M, L858R and Exon19del mutations in accordance with an embodiment.
  • Figure 28A shows a schematic of a oligonucleotide probe 2800 and its nucleotide sequence 2802 for the T790M mutation
  • Figure 28B shows a schematic of a oligonucleotide probe 2810 and its nucleotide sequence 2812 for the L858R mutation
  • Figure 28C shows a schematic of a oligonucleotide probe 2820 and its nucleotide sequence 2822 for the Exon19del mutation.
  • Figure 29A shows an illustration 2900 of a redox molecule attached to a semihybridised oligonucleotide probe adapted to bind to a target polynucleotide and be released from an electrode in accordance with an embodiment.
  • the semi-hybridised oligonucleotide probe 2902 includes a target-specific nucleic acid sequence 2904, with a tail end or an end portion of the target-specific nucleic acid sequence being hybridized to a shorter oligonucleotide strand 2906.
  • the shorter oligonucleotide strand 2906 is anchored to a surface 2908 of an electrode of a sensor.
  • the redox molecule 2910 (in this case, MB reporter) is attached to the same end portion of the target-specific nucleic acid sequence 2904 as shown.
  • the target-specific nucleic acid sequence 2904 binds with the target polynucleotide 2912 to form a bound target-specific nucleic acid sequence complex 2914 and is destabilized, thereby being released into a plasma flow, leaving the shorter oligonucleotide strand 2906 remaining anchored to the surface of the electrode. This results in a decrease to the current measured by the electrode.
  • Figure 29B shows a current plot 2920 showing a decrease in a measured current upon displacement of the redox molecule 2910, in accordance with an embodiment.
  • the current plot 2920 shows peak current height changes recorded every 2 mins at baseline and after addition of the target, over a 30 min period. From the current plot 2920, it is shown that the electrochemical signal obtained was found to remain stable up to 20 mins following target introduction as the MB reporter are displaced and flow out of the sensing platform.
  • Figure 30A shows an illustration 3000 of a stem-loop oligonucleotide probe 3002 with a redox molecule 3004 where the stem-loop oligonucleotide probe 3002 is adapted to unwind upon binding to a target polynucleotide and be released from an electrode in accordance with an embodiment.
  • one end of the stem-loop oligonucleotide probe 3002 is anchored to an electrode surface 3006 and the other end of the stemloop oligonucleotide probe 3002 has the redox molecule 3004 attached.
  • the stem-loop oligonucleotide probe 3002 Upon binding with a target polynucleotide 3008, the stem-loop oligonucleotide probe 3002 unwinds and causes the bound hairpin structure of the stem-loop oligonucleotide probe 3010 to open. This distances the MB reporter from the electrode surface 3006, thereby altering the Faradaic efficiency and leads to a decrease in charge transfer, theoretically reducing the electrochemical signal obtained. However, the stem-loop oligonucleotide probe 3002 and the redox molecule 3004 in this case are not displaced, released or detached from the electrode surface 3006.
  • Figure 30B shows a current plot 3020 showing a decrease in a measured current when the stem-loop oligonucleotide probe is unwound.
  • the current plot 3020 shows peak current height changes recorded every 2 mins at baseline and after addition of the target.
  • the sample flow rate used in this experiment is 100 pl/min.
  • a gradual increase in the electrochemical signal was observed following target hybridisation for this case. This might be due to the disruption of the anchored probe configuration by fluid movement from the microfluidic device.
  • FIG 31 shows a scanning electron microscope (SEM) image 3100 of dendritic structures formed on a surface of an electrode of the sensing platform 200 of Figure 2 in accordance with an embodiment.
  • the SEM image 3100 was obtained using a SEM at magnifications of 35000x.
  • dendritic gold (Au) nanostructures are electrodeposited on a bare Au surface of the electrode.
  • the SEM image 3100 confirmed the leaf-like dendritic Au structures extruding from the bare Au electrode surface, thereby significantly increasing an effective surface area of the electrode.
  • Figures 32A and 32B show plots of electrochemical current versus applied potential based on the square wave voltammetry technique for various molar concentrations of oligonucleotide probes using a sensor having an electrode with a bare surface and a sensor having an electrode with dendritic structures formed on a surface of the electrode in accordance with an embodiment.
  • Figure 32A shows a plot 3200 of electrochemical current versus applied potential for a sensor having an electrode with a bare gold (Au) surface.
  • the graph 3202 is associated with a molar concentration of 1 pM of the oligonucleotide probe (or DNA probe), the graph 3204 is associated with a molar concentration of 5 pM of the DNA probe and the graph 3206 is associated with a molar concentration of 10 pM of the DNA probe.
  • Figure 32B shows a plot 3210 of electrochemical current versus applied potential for a sensor having an electrode with dendritic gold structures formed on a surface of the electrode.
  • the graph 3212 is associated with a molar concentration of 1 pM of the oligonucleotide probe (or DNA probe)
  • the graph 3214 is associated with a molar concentration of 5 pM of the DNA probe
  • the graph 3216 is associated with a molar concentration of 10 pM of the DNA probe.
  • Peak current heights obtained from the data shown in the plots 3200, 3210 have been summarised in Figure 33 for easy comparison.
  • Figure 33 shows a plot 3300 of electrochemical current versus molar concentrations of oligonucleotide probes measured using a sensor having an electrode with a bare gold surface and a sensor having an electrode with dendritic gold structures formed on a gold surface of the electrode in accordance with an embodiment.
  • Data representing the bare gold surface of the electrode are shown as 3302, while data representing dendritic goid structures formed on the gold surface of the electrode are shown as 3304.
  • Data are presented as mean + standard error of the mean.
  • the dendritic gold structures-modified surface exhibited a 100x increase in current response compared to the bare goid surface electrode when measured with DNA probe having a redox reporter molecule.
  • a 10 pM concentration of the DNA probe formed on bare Au generated similar electrochemical signal intensity to a 1 pM concentration of the DNA probe formed on dendritic gold structures-modified surface.
  • EIS electrochemical impedance spectroscopy
  • Figures 34A, 34B and 34C show bar graphs of charge transfer resistance for targeted mutations T790M, L858R and Exon19del measured using sensors with electrode surfaces at different fabrication stages in accordance with embodiments.
  • Figure 34A shows a bar graph 3400 of charge transfer resistance for the targeted mutation T790M for various electrode surface conditions.
  • the charge transfer resistance 3402 is associated with the fabrication stage of having bare Au electrode surface
  • the charge transfer resistance 3404 is associated with the fabrication stage of having deposited dendritic Au on the Au electrode surface
  • the charge transfer resistance 3406 is associated with the fabrication stage of having DNA probes added to the deposited dendritic Au
  • the charge transfer resistance 3408 is associated with the fabrication stage of having DNA probes added and after MCH treatment
  • the charge resistance 3410 is associated with the stage of having target polynucleotides introduced to the sensor.
  • Figure 34B shows a bar graph 3420 of charge transfer resistance for the targeted mutation L858R for various electrode surface conditions.
  • the charge transfer resistance 3422 is associated with the fabrication stage of having bare Au electrode surface
  • the charge transfer resistance 3424 is associated with the fabrication stage of having deposited dendritic Au on the Au electrode surface
  • the charge transfer resistance 3426 is associated with the fabrication stage of having DNA probes added to the deposited dendritic Au
  • the charge transfer resistance 3428 is associated with the fabrication stage of having DNA probes added and after MCH treatment
  • the charge resistance 3430 is associated with the stage of having target polynucleotides introduced to the sensor.
  • Figure 34C shows a bar graph 3440 of charge transfer resistance for the targeted mutation Exon19del for various electrode surface conditions.
  • the charge transfer resistance 3442 is associated with the fabrication stage of having bare Au electrode surface
  • the charge transfer resistance 3444 is associated with the fabrication stage of having deposited dendritic Au on the Au electrode surface
  • the charge transfer resistance 3446 is associated with the fabrication stage of having DNA probes added to the deposited dendritic Au
  • the charge transfer resistance 3448 is associated with the fabrication stage of having DNA probes added and after MCH treatment
  • the charge resistance 3450 is associated with the stage of having target polynucleotides introduced to the sensor.
  • FIGS 35A, 35B and 35C show electrochemical impedance spectroscopy (EIS) curves for targeted mutations T790M, L858R and Exon19del measured using sensors with different electrode surfaces in accordance with embodiments.
  • EIS electrochemical impedance spectroscopy
  • FIG 35A shows an EIS curve 3500 for the targeted mutation T790M for various electrode surface conditions.
  • the EIS curve 3502 is associated with the fabrication stage of having bare Au electrode surface
  • the EIS curve 3504 is associated with the fabrication stage of having deposited dendritic Au on the Au electrode surface
  • the EIS curve 3506 is associated with the fabrication stage of having DNA probes added to the deposited dendritic Au
  • the EIS curve 3508 is associated with the fabrication stage of having DNA probes added and after MCH treatment
  • the EIS curve 3510 is associated with the stage of having target polynucleotides introduced to the sensor.
  • Figure 35B shows an EIS curve 3520 for the targeted mutation L858R for various electrode surface conditions.
  • the EIS curve 3522 is associated with the fabrication stage of having bare Au electrode surface
  • the EIS curve 3524 is associated with the fabrication stage of having deposited dendritic Au on the Au electrode surface
  • the EIS curve 3526 is associated with the fabrication stage of having DNA probes added to the deposited dendritic Au
  • the EiS curve 3528 is associated with the fabrication stage of having DNA probes added and after MCH treatment
  • the EIS curve 3530 is associated with the stage of having target polynucleotides introduced to the sensor.
  • Figure 35C shows an EIS curve 3540 for the targeted mutation Exon19de for various electrode surface conditions.
  • the EIS curve 3542 is associated with the fabrication stage of having bare Au electrode surface
  • the EIS curve 3544 is associated with the fabrication stage of having deposited dendritic Au on the Au electrode surface
  • the EIS curve 3546 is associated with the fabrication stage of having DNA probes added to the deposited dendritic Au
  • the EiS curve 3548 is associated with the fabrication stage of having DNA probes added and after MCH treatment
  • the EiS curve 3550 is associated with the stage of having target polynucleotides introduced to the sensor.
  • bar charts 3400, 3420, 3440 as shown in Figures 34A to 34C also show a significant decrease in resistance after the target was introduced (i.e. comparing the surface condition of after MCH treatment and after having target polynucleotides introduced).
  • the charge transfer resistance for target mutation T790M was decreased from 1259.8 Q to 559 Q
  • the charge transfer resistance for target mutation L858R was decreased from 517.3 Q to 228.2 Q
  • the charge transfer resistance for target mutation Exon19del was decreased from 1159.5Q to 327.9 £1.
  • nucleic acid having the target mutations T790M and L858R was extracted from H1975 cells, while synthetic single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) having the target mutation Exon19del was used.
  • the H1975 cells were first cultured in a T75 flask at 37°C with 5% CO2 in RPMI-1640 supplemented with 10% FBS and 1% Pen/Strep. Genomic DNA was then extracted at 70% cell confluency using the GeneJET kit, and the region of interest was amplified using a Veriti thermal cycler (Applied Biosystems) with the settings: 95°C for 30 s, 35 cycles of 95°C for 15 s, 55°C for 45 s, 68°C for 30 s, and 72°C for 5 mins.
  • Veriti thermal cycler Applied Biosystems
  • Figure 36 shows results 3600 of the gel electrophoresis of different amplicon sizes for targeted mutations T790M and L858R region extracted from H1975 cells in accordance with an embodiment.
  • the gel electrophoresis was imaged in a ChemiDoc imager (BioRad)
  • Figures 37A and 37B show Sanger sequencing results for targeted mutations L858R and T790M regions extracted from H1975 cells in accordance with an embodiment.
  • Figure 37A shows Sanger sequencing results 3700 for the targeted mutation L858R
  • Figure 37B shows Sanger sequencing results 3710 for the targeted mutation T790M.
  • Figures 38A, 38B and 38C show plots of measured current versus potential applied based on the square wave voltammetry technique using the sensing platform of Figure 2 for single-strand and double-strand target polynucleotides including mutations T790M, L858R and Exon19del, and for healthy DNA, in accordance with an embodiment.
  • Figure 38A shows a plot 3800 of measured current versus potential applied for singlestrand and double-strand target polynucleotides including the mutation T790M and for healthy DNA, where a curve 3802 is associated with the healthy DNA, a curve 3804 is associated with the double-strand target polynucleotides including the mutation T790M and a curve 3806 is associated with the single- strand target polynucleotides including the mutation T790M.
  • Figure 38B shows a plot 3810 of measured current versus potential applied for singlestrand and double-strand target polynucleotides including the mutation L858R, and for healthy DNA, where a curve 3812 is associated with the healthy DNA, a curve 3814 is associated with the double-strand target polynucleotides including the mutation L858R and a curve 3816 is associated with the single-strand target polynucleotides including the mutation L858R.
  • Figure 38C shows a plot 3820 of measured current versus potential applied for singlestrand and double-strand target polynucleotides including the mutation Exon19del and for healthy DNA, where a curve 3822 is associated with the healthy DNA, a curve 3824 is associated with the double-strand target polynucleotides including the mutation Exon19del and a curve 3826 is associated with the single-strand target polynucleotides including the mutation Exon19deL
  • Figures 39A, 39B and 39C show plots of percentage peak height change of a measured current versus concentration of target polynucleotides including mutations T790M, L858R and Exon19del in accordance w.th an embodiment
  • Figure 39A shows a plot 3900 of percentage peak height change of a measured current versus concentration of the target polynucleotide including the mutation T790M
  • Figure 39B shows a plot 3910 of percentage peak height change of a measured current versus concentration of the target polynucleotide including the mutation L858RM
  • Figure 39C shows a plot 3920 of percentage peak height change of a measured current versus concentration of the target polynucleotide including the mutation Exon19del.
  • the senor exhibited a linear response over a clinically relevant concentration range of between 1 pg/mL and 10 ng/mL for target polynucleotides including the mutations T790M, L858R, and Exon19del. Further, high correlation coefficients were achieved for each of these mutations, with a R 2 value for T790M being 0.95, a R 2 value for L858R being 0.96, and R 2 for Exon19del being 0.86.
  • the linear regression equations for the respective EGFR targets were also computed as follows:
  • mutated copies may constitute a small percentage of cell free DNA
  • AF mutant allele frequency
  • Figures 40A, 40B and 40C show plots of percentage peak height change of a measured current versus allele frequency (AF) for target polynucleotides including mutations T790M, L858R and Exon19del in accordance with an embodiment
  • Figure 40A shows a plot 4000 of percentage peak height change of a measured current versus AF for target polynucleotides including the mutation T790M
  • Figure 40B shows a plot 4010 of percentage peak height change of a measured current versus AF for target polynucleotides including the mutation L858R
  • Figure 40C shows a plot 4020 of percentage peak height change of a measured current versus AF for target polynucleotides including the mutation Exon19del.
  • the AF detection limit used was from 0,001% to 10%
  • target DNAs with 0.01% T790M and L858R mutations were successfully discriminated by the sensor of the present embodiment, while for target DNA with the Exon19del mutation, the sensor was able to detect mutant copies in as low as 0.001% AF.
  • the high sensitivity of the present sensors is beneficial for preventing false negative even with a high background from cell lysis.
  • sensing sensitivity of the sensor was assessed using ten plasma samples from metastatic NSCLC patients. Measurements below the threshold defined by the reference curve were classified as negative for mutation, while those exceeding the threshold were marked as positive. Each plasma sample was tested for the target mutations T790M, L858R and Exon19del, with 30 independent tests conducted in total.
  • Figure 41 shows a plot 4100 of percentage peak height change of a measured current using the biosensing platform of Figure 2 versus allele frequency (AF) of the corresponding target mutations measured using conventional next-generation sequencing (NGS) technique in accordance with an embodiment.
  • the performance of the sensor of the present disclosure was also evaluated with and without the microfluidic plasma separation.
  • Figures 42A, 42B and 42C show bar graphs of percentage peak height change of a measured current using the sensor of Figure 6 for non-enriched and enriched plasma samples having target polynucleotides including mutations T790M, L858R and Exon19del in accordance with an embodiment.
  • Figure 42A shows a bar graph 4200 of percentage peak height change of a measured current for non-enriched and enriched plasma samples having target polynucleotides including the mutation T790M
  • Figure 42B shows a bar graph 4210 of percentage peak height change of a measured current for non-enriched and enriched plasma samples having target polynucleotides including the mutation L858R
  • Figure 42C shows a bar graph 4220 of percentage peak height change of a measured current for nonenriched and enriched plasma samples having target polynucleotides including the mutation Exon19del.
  • the clinical samples processed without enrichment showed reduced sensitivity, especially at low AF levels where non-enriched samples could not be discriminated from the control. This underscores the importance of plasma separation as incorporated in the sensing platform of the present disclosure.
  • the samples processed through the microfluidic device maintained h-gh sensitivity towards all three target biomarker mutations T790M, L858R and Exon19del, even at low AF concentrations. This minimised the risk of false negatives for biosensing measurements obtained using the sensing platform of the present disclosure.
  • Figure 43 shows a schematic for comparing the required processing time of different sample-to-answer processing methods including using the sensing platform of Figure 2 in accordance with an embodiment.
  • the clinical process flow for NGS, targeted NGS, qPCR and the method employing the sensing platform of the present disclosure are labelled as 4302, 4304, 4306 and 4308, respectively, in Figure 43.
  • the integration of microfluidic enrichment and biosensor detection of the present sensing platform provides a streamlined workflow, delivering results from 1mL of blood within 10 mins as compared to conventional methods that could require hours due to the separate steps involved.
  • the shelf-life of the sensor of the present embodiment is also evaluated.
  • the sensors were prepared and stored in a dry state over a period of five weeks.
  • Figure 44 shows a plot 4400 of measured current versus time measured using the sensing platform of Figure 2 for target polynucleotides including mutations T790M, L858R and Exon19del in accordance with an embodiment.
  • the data points for the mutations T790M, L858R and Exon19del are labelled as 4402, 4404 and 4406, respectively.
  • Figures 45A, 45B and 45C show bar graphs of measured current versus time measured using the sensing platform of Figure 2 for target polynucleotides including mutations T790M, L858R and Exon19del, at week 1 and week 5, in accordance with an embodiment.
  • Figure 45A shows a bar graph 4500 of measured current versus time for target polynucleotides including the mutation T790M
  • Figure 45B shows a bar graph 4510 of measured current versus time for target polynucleotides including the mutation L858R
  • Figure 45C shows a bar graph 4520 of measured current versus time for target polynucleotides including the mutation Exon19del.
  • electrochemical response obtained using the sensor remained stable during weekly measurements performed over a period of five weeks. It is also noted that there was no significant difference in measurements was observed between the first and fifth week.
  • Figure 46 shows a schematic 4600 of a design of a microfluidic device including two contraction-expansion sets in accordance with an embodiment.
  • an additional contraction-expansion set i.e. C2 and E2
  • C2 and E2 has been included in the design of Figure 46 between the first contraction-expansion set and the waste outlet channel (now C3).
  • a wavy or sinusoidal structure for bifurcation channel portions modelled as P2 + P2a
  • adjoining the second expansion unit E2 is used to optimise a channel length of the bifurcation channel portions (P2 + P2a)
  • Figure 47 shows a schematic of an equivalent circuit 4700 of the microfluidic device of Figure 46 in accordance with an embodiment.
  • the equivalent circuit 4700 shows two contraction units (C-1 , C-2) and two expansion units (E-1 , E-2), forming two contraction-expansion sets (Cl , E1) and (C2, E2).
  • Table 7 provides a list of targets, primers and probes sequences designed for use in the examples discussed above.
  • Table 7 Targets, primers and probes sequences designed for this disclosure.
  • Ethanol (UN 1170) and acetone (UN 1090) were obtained from Best Chemical Co.
  • RPMI-1640, Heat inactivated fetal bovine serum (FBS), Human plasma, Dulbecco's phosphate-buffered saline, and SeaKem® LE agarose was sourced from Lonza.
  • Penicillin-streptomycin (Pen/Strep) solution, SYBR Safe, and GeneJET kit (Cat. No. K0722) was purchased from Thermo Fisher Scientific.
  • Anti-adherence rinsing solution (Cat. No, 07010) was acquired from STEMCELL Technologies.
  • TAE Buffer (Tris/Acetic Acid/EDTA) was purchased from Bio-Rad Laboratories. All oligonucleotides and TE buffer were acquired from Integrated DNA Technologies.
  • the microfluidic device for ctDNA isolation was designed in AutoCAD 2023. Channel lengths were optimized by calculating hydraulic resistance using the lam-nar flow equation. Values were input into LTSpice (Analog Devices, Inc.), where it was treated analogous to electrical resistance. Simulated output for each circuit path was expressed as a percentage of the desired volume, and channel lengths were adjusted accordingly.
  • the silicon wafer mold and PDMS replica were salinized with APTES for 6 hours. PDMS (1 :10 ratio) was poured over the mold and cured overnight at 70°C. Inlets and outlets were bored using a 0.07 mm biopsy puncher. Bonding of the PDMS mold and glass was facilitated by oxygen plasma treatment (15W) followed by 70°C oven heating for 1 hour.
  • Matrices for fluidic and parade profile characterization included 1x PBS, undiluted whole blood, and 1x PBS-diluted whole blood (1:1). Before introducing the sample, the device was primed with 0.1% anti-adherence solution then rinsed with 1x PBS. Blood samples were mixed by inverting the tube six times LTSpice-simulated volumes were compared to actual experimental output by measuring plasma outlet volume. Particle flow profiles were assessed using 6pm polystyrene beads (106 beads/mL) in 1x PBS under an 1X71 Olympus inverted microscope.
  • Microfluidic performance was evaluated with undiluted and diluted blood at flow rates of 100-300pUmin using a syringe pump (Fusion 200, Chemyx Inc,), with 10 min equilibration per increment, Channel measurements were captured via a Fastcam Mini AX50 high-speed camera (Photron) and analysed with Fiji (imaged).
  • RBC depletion efficiency was calculated by comparing RBCs in the plasma outlet to the total RBCs from both waste and plasma outlets using a C-chip Neubauer Improved (Incyto).
  • DNA probes for T790M, L858R, and Exon19 p.E746_A750del were designed based on sequences near the mutations (Table 7), The epidermal growth factor receptor gene (ID: 1956) coding region was analysed using Snapgene Viewer (v7.2.0). A singlestranded DNA probe for T790M, forming a step-loop structure, was selected using UNAFold nucleic acid folding simulations. For electrochemical experiments, the stem was modified with a 3' methylene blue and 5' thiol group.
  • Double-stranded probes for T790M, L858R, and Exon19del were simulated using an online prediction tool, with one strand modified for electrode attachment and the other with a methylene blue conjugate. Lyophilized oligonucleotides (100 pM) were resuspended in TE buffer and stored at 4°C.
  • the multichannel sensor comprising three working electrodes (WEs), a counter electrode (CE), and a reference electrode (RE) was designed using AutoCAD 2022 and then printed on a polyethylene terephthalate (PET) mask (Nanoimprint Tech). Electrode layers were fabricated in a cleanroom using photolithography. Glass slides (75 mm x 25 mm) were cut to 50 mm, cleaned with isopropyl alcohol (IPA), dried with N2, and treated with oxygen plasma (20 seem, 20 RF power, 120 secs). S1818 photoresist was spin-coated (2000 rpm) and baked at 115°C for 1 min. The coated surface was aligned on the PET mask, exposed with a mask aligner (12 s), and developed in MF-319 for 1 min. This process was repeated for WEs, RE, and CE.
  • WEs working electrodes
  • CE counter electrode
  • RE reference electrode
  • the base Au electrode layer (50 nm) was deposited using a NANO 36 thermal evaporator (Kurt J Lesker), with Cr (30 nm) acting as adhesion layer for Au Ag (200 nm) was deposited on the RE using an AJA electron beam evaporator. Every layer underwent sonication in acetone to remove unbound material. An SU8-3050 insulation layer was added cover the electrode perimeter. To facilitate the deposition of Au nanostructures on the WEs, acetone and ethanol was flushed to clean the surface followed by FeCh (0.1 M) treatment of the Ag RE to form Ag/AgCl.
  • Dendritic Au was formed by chronoamperometric deposition (0 V for 5 s, -0.8 V for 2 min) of 6 mM Au solution.
  • the gold solution was prepared from 0.05 M Au, L-cysteine, and NH3. After deposition, sensors were washed with DI water and air-dried. Au nanostructures were characterized with a FEI Verios 460L scanning electron microscope.
  • Methylene blue-modified DNA probes were reduced with 10 mM TCEP overnight at 4“C then diluted to 20 uM with DI water. All preparations were kept on ice to preserve sequence stability. 5 pM DNA probes forT790M, L858R, and Exon19del were added to respective WEs, ensuring full surface contact with the probe solution. Electrodes were incubated at 4°C for at least 18 hours. Sensors were then washed with autoclaved DI water, air-dried, treated with MCH for 1 hour, and washed again before air-drying for use in experiments.
  • H1975 cells were cultured in a T75 flask at 37°C with 5% CO2 in RPMI-1640 supplemented with 10% FBS and 1% Pen/Strep, Genomic DNA was extracted at 70% cell confluency using the GeneJET kit.
  • the region of interest (Table 7) was amplified using a Veriti thermal cycler (Applied Biosystems) with the settings: 95°C for 30 s, 35 cycles of 95°C for 15 s, 55°C for 45 s, 68°C for 30 s, and 72°C for 5 mins. Gel electrophoresis was used to validate amplicon sizes, stained with SYBR Safe and run at 70V for 90 mins. Gel was imaged in a ChemiDoc imager (Bio-Rad), and amplicon purity was assessed with a nanodrop then sequenced for mutation analysis (Axil Scientific) ( Figures 36, 37A, 37B).
  • Electrochemical impedance spectroscopy (EIS) and linear regression for T790M and L858R probes were acquired using nucleic acid extracted from H1975 ceils.
  • EIS Electrochemical impedance spectroscopy
  • ssDNA synthetic single-stranded DNA
  • dsDNA double-stranded DNA
  • Oligonucleotides were purchased lyophilized, resuspended in nuclease-free water, quantified via Nanodrop, and diluted as needed.
  • SWV square wave voltammetry
  • Impedance data was collected using KsFe(CN)6/K4Fe(CN)8 (5 mM) in 0.1 M KCI (0.1 MHz to 0.01 Hz, 5 mV amplitude) and fitted to a Randles circuit. All electrochemical measurements were performed in triplicate.
  • the sensing platform (microfluidic sensor) was fabricated by assembling the microfluidic device with the sensor. To avoid DNA probe de-hybridization at higher temperatures, which could impact electrochemical readings, only the PDMS portion was treated with oxygen plasma (15W) and was aligned on top the glass electrode. The PDMS-to-glass electrode was adhered at room temperature overnight. Characterisation of specificity and detection limit
  • microfluidic sensor was primed with anti-adherence rinsing solution (e.g. surfactants), then flushed with 1x PBS. Healthy human plasma served as the baseline.
  • anti-adherence rinsing solution e.g. surfactants
  • samples were prepared by spiking dsDNA and ssDNA into 1-fold PBS-diluted blood.
  • dsDNA of T790M, L858R, and Exon19del were spiked into 1-fold plasma. After mixing, the samples were transferred to a 10 mL syringe and processed at 100 pL/min through the integrated sensor. SWV was performed at the end using the settings outlined above.
  • T790M mutation (T790M-167, Table 7) was targeted in 1-fold blood.
  • a microfluidic sensor was prepared with L858R and Exon19del probes on the WE surface.
  • An initial pre-amplification step using PCR was performed to generate sufficient amplicon for qPCR detection in plasma, with settings: 95°C for 30 s, 26 cycles of 95°C for 15 s, 55°C for 45 s, 68°C for 30 s, and a final 72°C for 5 min.
  • the amplicon 500 ng/pL
  • varying plasma concentrations were diluted in deionized water and analysed using qPCR with settings: 95“C for 30 s, 35 cycles of 95 n C for 15 s, 55°C for 45 s, 68”C for 30 s, and a final 72°C for 5 min.
  • a reference curve was generated with amplicon dilutions, simulating recovery from 5% to 100%.
  • DNA recovery efficiency was assessed by comparing Ct results from the plasma outlet to reference values from diluted amplicons.
  • plasma from clinical samples (T790M: patient 4, L858R: patient 2, Exon19: patient 10) was spiked into healthy blood.
  • the blood was centrifuged at 2000xg, 4°C for 15 mins, and the plasma was replaced with the clinical sample.
  • the mixture was pipette-mixed and inverted several times. Two experimental parameters were used. (1) non-enriched spiked blood samples were analysed directly on the biosensor using a straight microfluidic channel at 100 pL/min, and (2) the entire sample volume was processed on the integrated biosensor at the same flow rate. Plasma without target DNA served as the control. SWV measured the change in peak height after full sample processing.
  • sensors with dendritic Au were fabricated at week 0 following the protocol above, DNA probes for T790M, L858R, and Exon19del were incubated overnight, washed with autoclaved DI water, dried, and treated with MCH for 1 hour. Sensors were stored at 4°C after drying. SWV measurements (potential: 0.5V to 0V, step: 5 mV, frequency: 25 Hz, amplitude: 20 mV) were taken weekly at room temperature for five weeks using nine electrodes (three per biomarker).
  • Prism 10 (Version 10.2.2) was used to for a graphical representation of the data as well as statistical analysis. For data represented with column graphs, the mean with standard deviation was plotted. All results were performed in triplicates unless otherwise indicated. Discussion
  • Liquid biopsy provides a less invasive method for identifying actionable mutations to personalise treatment and monitor treatment efficacy.
  • any delays in blood processing in labs can hinder timely treatment initiation, potentially worsening patient outcomes.
  • variability in the blood processing workflow due to a lack of standardization complicates cross-study comparisons, potentially resulting in inconsistent findings
  • a POC liquid biopsy system offers a reliable, rapid, and cost- effective alternative, enabling more timely treatment decisions.
  • the microfluidic electrochemical sensing platform of the exemplary embodiment is capable of detecting three actionable mutations commonly found in NSCLC patients, i.e., T790M, L858R, and Exon 19 deletion, from blood samples with results wirelessly transmitted for analysis.
  • Previous studies have focused on either microfluidic or electrochemical systems as separate units, and integrated platforms often suffered from low throughput or have to analyse plasma samples post-centrifugation.
  • the present sensing platform addresses these issues through a high-throughput, continuous ctDNA capture from blood samples, which significantly enhances its clinical utility.
  • the sensing platform functionality is achieved through a microfluidic architecture that enables efficient blood fractionation.
  • the DNA probes exhibited high affinity for both dsDNA and ssDNA, ensuring effective capture of target ctDNA
  • the detection limit of the exemplary sensor was evaluated to minimize false positives. Exon19 deletion targets were detected at 0.001% AF, while T790M and L858R mutations were detected at 0.01% AF. These limits surpass those that can be detected with current instruments, with reduced processing times and reagent requirements, potentially lowering the overall costs.
  • Table 8 Summary of features of the exemplary sensing platform and their advantages
  • the integrated sensing platform of the present disclosure could continuously process blood and quantify NSCLC ctDNA from whoie blood samples within 10 mins.
  • the microfluidic Fahrasus-Lindqvist effect was leveraged for blood separation, allowing for high purity plasma ctDNA to be captured by DNA probes on nanostructure-modified electrodes of the sensor.
  • the microfluidic device of the exemplary sensing platform is capable of achieving 99.7% plasma enrichment with 1-fold blood dilution at a throughput of 100 pl/min. Th-s enables operation with minimally diluted blood, preserving ctDNA copies while maintaining high throughput.
  • the microfluidic device includes microchannels engineered for passive blood separation and a micropillar array to facilitate uniform plasma flow over the electrode of the sensor.
  • the sensor comprises semihybridized DNA probes (see e.g. Figure 29A) with a detachable target-specific oligonucleotide having a redox molecule attached. Upon hybridization with a circulating tumour DNA (ctDNA) target, the target-specific oligonucleotide together with the redox molecule are released and transported to the outlet for generating a measurable change in electrochemical signal as detected by the sensor.
  • ctDNA circulating tumour DNA
  • incorporating parallel sensor capabilities can potentially enhance throughput with shorter processing time.
  • the compact design provided by the exemplary sensing platform eliminates the need for bulky processing equipment, making it an invaluable point-of- care tool in settings lacking adequate screening facilities.
  • the integrated sensing platform comprising the exemplary microfluidic device and sensor therefore holds promise for blood-based testing, offering rapid sample-to-answer responses that could prove beneficial in numerous clinical scenarios, particularly for personalized medicine applications such as genotyping
  • This POC sensing platform which is developed through the integration of microfluidic and electrochemical technology offers a rapid and efficient tool for detecting actionable mutations, which can potentially bring advancement to cancer decision-making and improve clinical outcome for patients.
  • the target is a cancer-related polynucleotide.
  • the cancer is non-small cell lung cancer.
  • the target-specific DNA oligonucleotide is directed to EGFR mutation T790M and has the polynucleotide sequence set forth in SEQ ID NO: 31 (5'- CTGCATGATGAGCTGCAGCGG-3’).
  • the shorter oligonucleotide that is tethered to a substrate has the polynucleotide sequence set forth in (5’-CATGCAG-3’).
  • the target-specific DNA oligonucleotide is directed to EGFR mutation L858R and has the polynucleotide sequence set forth in SEQ ID NO: 32 (5’- GCCCGCCCAAAATCTGTGAT-3’). In some embodiments the shorter oligonucleotide that is tethered to a substrate has the polynucleotide sequence set forth in: (5’- GCGGGC-3’). In some embodiments the target-specific DNA oligonucleotide is directed to EGFR mutation Exon19del and has the polynucleotide sequence set forth in SEQ ID NO: 33 (5’-CGGAGATGTTTTGATAGCGA-3’). In some embodiments the shorter oligonucleotide that is tethered to a substrate has the polynucleotide sequence set forth in (5’-TCTCCG-3’).
  • the method is to detect a disease in a subject:
  • the disease is cancer.
  • the cancer is Non-small cell lung cancer
  • the sensing platform can be adapted to detect other cancers by including additional relevant gene targets and/or designing the oligonucleotide probe to include other relevant target-specific oligonucleotide and/or other shorter oligonucleotide.
  • other suitable redox molecule besides the MB reporter can be used.
  • the redox molecule may be attached at other portions of the target-specific oligonucleotide. In some embodiments, more than one redox molecules can be attached to the target-specific oligonucleotide.
  • channel portions of the microfluidic device can be optimised or customised to suit other samples (other than whole blood or 1 :1 diluted blood sample) with targeted sample fraction extraction for sensing by the sensor, in some embodiments, more than one contraction-expansion channel sets can be used in the microfluidic device.
  • the target-specific DNA oligonucleotide may have at least 70%, 80%, or 90% sequence identity to the target DNA sequence or at least 70%, 80% or 90% sequence identity to the polynucleotide sequence set forth in one of SEQ ID NO: 31 , SEQ ID NO: 32 or SEQ ID NO: 33.
  • the shorter oligonucleotide may have at least 70%, 80% or 90% sequence identity to the polynucleotide sequence set forth in one of CATGCAG ZThioMC3-D/, GCGGGC /ThioMC3-D/ or TCTCCG /ThioMC3-D/, wherein the polynucleotide sequences CATGCAG /ThioMC3-D/, GCGGGC /ThioMC3-D/ and TCTCCG /ThioMC3- D/ are complementary to the SEQ ID NO: 31 , the SEQ ID NO: 32 and the SEQ ID NO: 33, respectively.
  • the microfluidic device can be integrated directly to the sensor without the insulation layer.
  • other two-dimensional and/or three-dimensional nanostructures can be formed on an electrode surface of the sensor (e.g. working electrode surface) to modified an effective surface area of the electrode.
  • the micropillars may be absent from the sensing channel portion. In some embodiments, the micropillars may have other suitable diameters beside 15 pm, for example, 5, 10, 20, 25 or 30 pm.
  • An electrode of the sensor may be formed using silver, platinum or carbon.
  • each of the micropillars are placed at a distance from 100 pm to 200 pm. In some embodiments, a diameter of the micropillars can have a range of 10 pm to 100 pm.
  • a channel width of the various channel portions of the microfluidic device may be 10 pm to 200 pm. In embodiments, a channel height of the various channel portions of the microfluidic device may be 10 pm to 200 pm. In embodiments, a channel length of the various channel portions of the microfluidic device may be 500 pm to 50,000 pm.
  • a channel width, a channel height and a channel length of the various channel portions of the microfluidic device can be adjusted or optimised based on e.g. a fluid resistance and/or pressure drop as required for the microfluidic device, in view of the samples used and the requirements for separation.
  • a target-specific oligonucleotide of an oligonucleotide probe of a sensor has a length of 15 to 30 nucleotide bases, or 20 to 25 nucleotide bases In some embodiments, a shorter oligonucleotide of the oligonucleotide probe has a length of 4 to 15 bases, or 5 to 15 bases or 10 to 15 bases. These lengths may vary in embodiments, depending on the sequence of interest, and/or target mutations have more than one base pairs.
  • microfluidic device e.g. other materials other than PDMS etc.
  • sensor e.g. different methods for metals deposition or different metals/electrical conductors used
  • sensing platform e.g. other form beside the layered structure
  • system e.g. other communication technique beside Bluetooth

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Abstract

L'invention concerne un capteur de détection d'un polynucléotide cible. Dans un mode de réalisation, le capteur comprend : au moins une sonde oligonucléotidique, ladite au moins une sonde oligonucléotidique comprenant un oligonucléotide spécifique à une cible complémentaire d'une partie du polynucléotide cible, un oligonucléotide plus court ancré à une électrode, une partie d'extrémité de l'oligonucléotide spécifique à une cible étant complémentaire de l'oligonucléotide plus court et formant un duplex avec celui-ci et une molécule redox fixée à l'oligonucléotide spécifique à une cible, l'oligonucléotide spécifique à une cible se déstabilise lors de la liaison à la partie du polynucléotide cible, et étant libéré de l'oligonucléotide plus court ancré à l'électrode, modulant une distance entre la molécule redox et une surface de l'électrode pour provoquer un changement de potentiel électrochimique détecté par l'électrode pour détecter le polynucléotide cible. L'invention concerne également des modes de réalisation en relation avec une plateforme de détection, un système et un procédé de détection d'un polynucléotide cible.
PCT/SG2025/050184 2024-03-14 2025-03-14 Capteur, plateforme de détection, système et procédé de détection d'un polynucléotide cible Pending WO2025193173A1 (fr)

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SG10202400712R 2024-03-14
SG10202400712R 2024-03-14

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WO2025193173A1 true WO2025193173A1 (fr) 2025-09-18
WO2025193173A9 WO2025193173A9 (fr) 2025-11-06

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PCT/SG2025/050184 Pending WO2025193173A1 (fr) 2024-03-14 2025-03-14 Capteur, plateforme de détection, système et procédé de détection d'un polynucléotide cible

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