EP4665869A1 - Methods and devices for using enzymatic amplification and fragmentation to detect biomarkers with nanopores - Google Patents

Abstract

Methods and devices for nanopore-based biomarker detection that utilize immunocapture, isothermal rolling circle amplification, and sequence-specific fragmentation of the product to release multiple DNA reporter molecules for nanopore detection. These DNA fragment reporters produce sets of nanopore signals that form distinctive fingerprints. This fingerprint signature therefore allows the identification and quantification of biomarker analytes.

Description

Methods and Devices for Using Enzymatic Amplification and Fragmentation to Detect Biomarkers with Nanopores

RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63/485,528, filed on February 16, 2023. The entire teachings of the above application(s) are incorporated herein by reference.

INCORPORATION BY REFERENCE OF MATERIAL IN XML

[0002] This application incorporates by reference the Sequence Listing contained in the following extensible Markup Language (XML) file being submitted concurrently herewith: a) File name: 5200_2362_001_SL.xml; created February 16, 2024, 30,845 Bytes in size.

GOVERNMENT SUPPORT

[0003] This invention was made with government support under Grant No. R01- HG011087 and Grant No. F32-EB029777 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0004] Progress toward applying nanopores as highly sensitive, quantitative diagnostic tools has been impeded by several challenges. One major limitation is the insufficient sensitivity of nanopores in detecting disease biomarkers, which are typically present at pM or lower concentrations in biological fluids, while a second limitation is the general absence of unique nanopore signals for different analytes.

SUMMARY

[0005] An embodiment according to the invention provides methods and devices for nanopore-based biomarker detection that utilizes immunocapture, isothermal rolling circle amplification, and sequence-specific fragmentation of the product to release multiple DNA reporter molecules for nanopore detection. These DNA fragment reporters produce sets of nanopore signals that form distinctive fingerprints, or clusters. This fingerprint signature therefore allows the identification and quantification of biomarker analytes. [0006] One embodiment is a method for detecting presence of a biomarker in a sample using a nanopore. The method comprises capturing the biomarker using an immobilized antibody specific to the biomarker, and performing an isothermal nucleic acid amplification to produce an amplified product of the biomarker, the amplified product comprising a nucleic acid sequence. The amplified product is fragmented using a restriction enzyme specific to the nucleic acid sequence of the amplified product to thereby release multiple sequence fragment molecules to be detected by a nanopore. A voltage is applied to a solution containing the multiple sequence fragment molecules to thereby induce the multiple sequence fragment molecules to pass through the nanopore. A nanopore blockade fingerprint signal is generated for the multiple sequence fragment molecules passing through the nanopore, the generating being automated using a computer processor based on electrical signals generated from the multiple sequence fragment molecules passing through the nanopore; and the presence of the biomarker in the sample is detected based on determining, in an automated fashion using a computer processor, a unique correspondence of the nanopore blockade fingerprint signal with the presence of the biomarker.

[0007] The method may further comprise performing the isothermal amplification to produce the amplified product of the biomarker, by performing a rolling circle amplification (RCA). Capturing the biomarker using the immobilized antibody specific to the biomarker may comprise forming a sandwich immunocomplex for the biomarker. The capturing the biomarker using the immobilized antibody specific to the biomarker may comprise performing an enzyme-linked immunosorbent assay (ELISA) for the biomarker. The capturing the biomarker using the immobilized antibody specific to the biomarker further may comprise hybridizing circular DNA templates with primers that are conjugated with secondary antibodies in an enzyme-linked immunosorbent assay (ELISA) structure for the biomarker. The method may comprise performing in situ cleavage and amplification (INCA) of the circular DNA templates, wherein the circular DNA templates comprise a site that is not amenable to restriction digestion, thereby enabling simultaneous rolling circle amplification (RCA) and digestion of the amplified product. The site that is not amenable to restriction digestion may comprise a methylated site. The circular DNA templates may comprise a DNA hairpin structure, the DNA hairpin structure comprising a restriction enzyme recognition site that allows isothermal enzymatic digestion to permit fragmenting the amplified product using the restriction enzyme. The method may comprise introducing a highly processive polymerase to initiate rolling circle amplification (RCA) of the circular DNA templates. Further, the method may comprise initiating rolling circle amplification (RCA) of the circular DNA templates to produce single stranded Deoxyribonucleic Acid (ssDNA) comprising repetitive complimentary circular DNA template sequences, the ssDNA being cleaved by the restriction enzyme to produce the multiple sequence fragment molecules.

[0008] In other related embodiments, generating the nanopore blockade fingerprint signal may be based on at least one of a nanopore dwell time, a nanopore fractional current blockade rate, and a nanopore blockade interevent time for the multiple sequence fragment molecules passing through the nanopore. Generating the nanopore blockade fingerprint signal may comprise determining a cluster signal pattern in the nanopore dwell time and the nanopore fractional current blockade rate for the multiple sequence fragment molecules passing through the nanopore. The method may further comprise quantifying, in an automated fashion using a computer processor, the abundance of the biomarker in the sample. The quantifying may be based on a nanopore blockade interevent time for the multiple sequence fragment molecules passing through the nanopore.

[0009] In further related embodiments, the applying the voltage to the solution containing the multiple sequence fragment molecules to thereby induce the multiple sequence fragment molecules to pass through the nanopore may comprise inducing the multiple sequence fragment molecules to pass through a biological nanopore. The biological nanopore may comprise an a-hemolysin nanopore. The biological nanopore may be inserted into a lipid bilayer; and may be inserted into an aperture in a silicon-based substrate. The nanopore may comprise an aperture diameter of between about 10 pm and about 100 pm. The biomarker may comprise at least one of an amino acid, a protein, a messenger Ribonucleic Acid (mRNA), a Deoxyribonucleic Acid (DNA), and a single nucleotide polymorphism (SNP). The method may further comprise compromising the base-pairing stability of the multiple sequence fragment molecules being passed through the nanopore, such as by passing the multiple sequence fragment molecules through a solution of guanidinium chloride.

[0010] Another embodiment comprises a device for detecting presence of a biomarker in a sample using a nanopore. The device comprises an amplification and restriction module configured to (i) capture the biomarker using an immobilized antibody specific to the biomarker; (ii) perform an isothermal nucleic acid amplification to produce an amplified product of the biomarker, the amplified product comprising a nucleic acid sequence; (iii) fragment the amplified product using a restriction enzyme specific to the nucleic acid sequence of the amplified product to thereby release multiple sequence fragment molecules to be detected by a nanopore. Further, the device comprises the nanopore, and a voltage source configured to apply a voltage to a solution containing the multiple sequence fragment molecules to thereby induce the multiple sequence fragment molecules to pass through the nanopore. A computer processor module is configured to, in an automated fashion, (i) generate a nanopore blockade fingerprint signal for the multiple sequence fragment molecules passing through the nanopore, the generating being based on electrical signals generated from the multiple sequence fragment molecules passing through the nanopore; and (ii) detect the presence of the biomarker in the sample based on determining a unique correspondence of the nanopore blockade fingerprint signal with the presence of the biomarker.

[0011] In further related embodiments, the amplification and restriction module may be configured to perform the isothermal amplification to produce the amplified product of the biomarker by performing a rolling circle amplification (RCA). The amplification and restriction module may be configured to form a sandwich immunocomplex for the biomarker, such as by performing an enzyme-linked immunosorbent assay (ELISA) for the biomarker. Circular DNA templates may be hybridized with primers that are conjugated with secondary antibodies in an enzyme-linked immunosorbent assay (ELISA) structure for the biomarker. The amplification and restriction module may be configured to perform in situ cleavage and amplification (INCA) of the circular DNA templates, wherein the circular DNA templates comprise a site that is not amenable to restriction digestion, such as a methylated site, thereby enabling simultaneous rolling circle amplification (RCA) and digestion of the amplified product. The circular DNA templates may comprise a DNA hairpin structure, which comprises a restriction enzyme recognition site that allows isothermal enzymatic digestion to permit fragmenting the amplified product using the restriction enzyme. The amplification and restriction module may be configured to introduce a highly processive polymerase to initiate rolling circle amplification (RCA) of the circular DNA templates. Further, the amplification and restriction module may be configured to initiate rolling circle amplification (RCA) of the circular DNA templates to produce single stranded Deoxyribonucleic Acid (ssDNA) comprising repetitive complimentary circular DNA template sequences, the ssDNA being cleaved by the restriction enzyme to produce the multiple sequence fragment molecules.

[0012] In other, related embodiments, the computer processor module may be configured to generate the nanopore blockade fingerprint signal based on at least one of a nanopore dwell time, a nanopore fractional current blockade rate, and a nanopore blockade interevent time for the multiple sequence fragment molecules passing through the nanopore. The nanopore blockade fingerprint signal may be generated by determining a cluster signal pattern in the nanopore dwell time and the nanopore fractional current blockade rate for the multiple sequence fragment molecules passing through the nanopore. The computer processor module may be further configured to quantify the abundance of the biomarker in the sample, such as based on a nanopore blockade interevent time for the multiple sequence fragment molecules passing through the nanopore. The nanopore may comprise a biological nanopore, such as an a-hemolysin nanopore. The device may further comprise a lipid bilayer, the biological nanopore being inserted into the lipid bilayer; and may further comprise a silicon-based substrate, the biological nanopore being inserted into an aperture in the silicon- based substrate. The nanopore may comprise an aperture diameter of between about 10 pm and about 100 pm. The biomarker may comprise at least one of an amino acid, a protein, a messenger Ribonucleic Acid (mRNA), a Deoxyribonucleic Acid (DNA), and a single nucleotide polymorphism (SNP). The amplification and restriction module may further comprise a solution, such as guanidinium chloride, configured to compromise the basepairing stability of the multiple sequence fragment molecules being passed through the nanopore.

[0013] Another embodiment comprises an immunoassay for detecting presence of a biomarker in a sample using a nanopore. The immunoassay comprises an immobilized antibody specific to the biomarker, the immobilized antibody configured to capture the biomarker; a polymerase configured to perform an isothermal nucleic acid amplification to produce an amplified product of the biomarker, the amplified product comprising a nucleic acid sequence; and a restriction enzyme, specific to the nucleic acid sequence of the amplified product, configured to fragment the amplified product to thereby release multiple sequence fragment molecules to be detected by a nanopore.

[0014] In further related embodiments, the polymerase may be configured to perform a rolling circle amplification (RCA). The immobilized antibody specific to the biomarker may comprise at least part of a sandwich immunocomplex for the biomarker, such as an enzyme- linked immunosorbent assay (ELISA) for the biomarker. The immunoassay may further comprise circular DNA templates configured to be hybridized with primers that are conjugated with secondary antibodies in an enzyme-linked immunosorbent assay (ELISA) structure for the biomarker. The circular DNA templates may comprise a site that is not amenable to restriction digestion, such as a methylated site, thereby enabling simultaneous rolling circle amplification (RCA) and digestion of the amplified product. The circular DNA templates may comprise a DNA hairpin structure, the DNA hairpin structure comprising a restriction enzyme recognition site that allows isothermal enzymatic digestion to permit fragmenting the amplified product using the restriction enzyme. A highly processive polymerase may be used to initiate rolling circle amplification (RCA) of the circular DNA templates. The highly processive polymerase may be configured to initiate the rolling circle amplification (RCA) of the circular DNA templates to produce single stranded Deoxyribonucleic Acid (ssDNA) comprising repetitive complimentary circular DNA template sequences, the ssDNA being cleaved by the restriction enzyme to produce the multiple sequence fragment molecules. The biomarker may comprise at least one of an amino acid, a protein, a messenger Ribonucleic Acid (mRNA), a Deoxyribonucleic Acid (DNA), and a single nucleotide polymorphism (SNP). The immunoassay may further comprise a solution configured to compromise the base-pairing stability of the multiple sequence fragment molecules being passed through the nanopore, such as a solution of guanidinium chloride.

[0015] Another embodiment is a method of identifying a species from a sample. The method comprises capturing a species-loaded complex, the species-loaded complex comprising: (i) the species, isolated from the sample, (ii) a carrier particle, (iii) a first speciesspecific binder, binding the species to the carrier particle (iv) a second species-specific binder bound to the species, (v) a DNA primer, bound to the second species-specific binder, and (vi) a circular DNA template comprising a restriction enzyme recognition site, bound to the DNA primer. The circular DNA template is amplified while the species-loaded complex is captured to generate a long DNA strand. The generated DNA strand is fragmented to form a mixture of smaller DNA molecules. A voltage is applied to the mixture, the voltage being sufficient to translocate the DNA molecules through a nanopore; and an electrical current change produced as a result of the translocation of the DNA molecules through the nanopore is detected, thereby permitting identification of the species.

[0016] In further related embodiments of the method, detecting the electrical current change may comprise detecting a temporary reduction in ionic current through the nanopore produced by transient occlusion of the nanopore by the DNA molecules during the translocation of the DNA molecules through the nanopore produced by the applying the voltage. The carrier particle may comprise a magnetic bead or a plate. The first speciesspecific binder and the second species-specific binder may comprise a pair of antibodies, each of the antibodies binding to different epitopes of the same species. The species may comprise at least one of a nucleic acid molecule, a protein, and a polypeptide. The species may comprise at least a portion of at least one of a biomarker, a circulating tumor cell nucleic acid, and a circulating tumor protein. The circular DNA template may comprise at least one 5-methylcytosine site. The sample may be a blood sample. The first species-specific binder may be chemically immobilized on the carrier particle. The circular DNA template may comprise a restriction enzyme recognition site, and the generated DNA strand may be fragmented by restriction enzymes, which may comprise Alul, and may comprise Rsal. Amplifying the circular DNA template may be done by rolling circle amplification method. The diameter of the nanopore may be about 50 pm. The DNA molecules in the DNA mixture may be between about 4bp and about 50 bp in length, such as between about 4bp and about 10 bp in length. The sample may be a biological sample. The sample may contain the species at pM levels.

[0017] Another embodiment is a species-loaded complex to permit identification and quantification of a species in a sample. The species-loaded complex comprises: (i) the species, isolated from the sample, (ii) a carrier particle, (iii) a first species-specific binder, binding the species to the carrier particle, (iv) a second species-specific binder bound to the species, (v) a DNA primer, bound to the second species-specific binder, and (vi) a DNA strand comprising at least one copy of a circular DNA template comprising a restriction enzyme recognition site, bound to the DNA primer.

[0018] In further related embodiments, the circular DNA template may comprise a restriction enzyme recognition site. The circular DNA template may comprise at least one 5- methylcytosine site. The first species-specific binder and the second species-specific binder may comprise a pair of antibodies, each of the antibodies binding to different epitopes of the same species. The species may comprise at least one of a nucleic acid molecule, a protein, and a polypeptide. The species may comprise at least a portion of at least one of: a biomarker, a circulating tumor cell nucleic acid, and a circulating tumor protein. The sample may be a blood sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee. [0020] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0021] FIG. 1 is a schematic diagram illustrating isothermal rolling circle amplification (RCA) for nanopore-based biomarker quantification in accordance with an embodiment of the invention. Panel (A) shows a schematic of RCA-based assay: Capture antibody is conjugated to magnetic beads, and detector antibody is conjugated with DNA primer and template. After the formation of the sandwich immunocomplex, RCA is performed using phi29 polymerase. The circular DNA template contains a DNA hairpin structure that carries a restriction enzyme recognition site. Restriction enzyme-based cleavage produces DNA fragments used as reporter molecules for nanopore-based detection. Panel (B) shows a schematic of reporter molecule detection using nanopore system. DNA reporter molecules of various sizes generated from panel A were added to the cis chamber of the flow cell. A 50 /im wedge-on-pillar aperture as well as a PBD11-PEO8 bilayer, which has an «-hemolysin pore inserted, was used for detection of reporter molecules in a guanidinium chloride denaturing environment at high voltage. The inset shows an SEM image of a 50 //m wedge-pillar aperture.

[0022] FIG. 2 is a schematic diagram illustrating how amplification and sequencespecific cleavage produce nanopore signal fingerprints in accordance with an embodiment of the invention. Panel (A) is a schematic workflow of DNA reporter molecule generation on streptavidin beads. Possible sequences and structures of DNA reporter molecules produced from Alul D6 template (SEQ ID NO: 2), a (SEQ ID NO: 10), b (SEQ ID NO: 10-11), c (SEQ ID NO: 12) and d (SEQ ID NO: 12) (Panel B); and Alul D7 template (SEQ ID NO: 4), a (SEQ ID NO: 10), b (SEQ ID NO: 10-11), c (SEQ ID NO: 13) and d (SEQ ID NO: 13) (Panel C); and the corresponding 20% native PAGE gel stained by GelRed (Panel D). Lane 1 : O’RangeRuler 5 bp DNA Ladder. Lane 2: 6 bp DNA hairpins. Lanes 3 and 4: Reporter molecules generated from circular template Alul D6; lane 3 is Alul cleavage for 30 min, and lane 4 is 60 min. Lane 5: Reporter molecules generated from circular template Alul D7. [0023] FIG. 3 shows possible sequence and structure of DNA reporter molecules produced from Rsal DI template (SEQ ID No: 6), a (SEQ ID NO: 14), b (SEQ ID NO: 14), c (SEQ ID NO: 15) and d (SEQ ID NO: 16, 17) (Panel E), a (SEQ ID NO: 14), b (SEQ ID NO: 14), c (SEQ ID NO: 15) and d (SEQ ID NO: 12, 12); and Rsal D2 template (SEQ ID No: 8), a (SEQ ID NO: 18), b (SEQ ID NO: 18, 19), c (SEQ ID NO: 15) and d (SEQ ID NO: 19) (Panel F) and the corresponding 20% native PAGE gel, in accordance with an embodiment of the invention. All restriction digestion was performed at 37 °C for 1 h. Both ladders are O’RangeRuler 5 bp DNA Ladder. In addition, FIG. 3 shows scatter plot of fractional blockade versus dwell time of reporter molecules produced from Alul D6 (Panel G), Alul D7 (Panel H), Rsal DI (Panel I), and Rsal D2 (Panel J). Bayesian Gaussian mixture model with full covariance type, random state 1, 1 x 10-9 convergence threshold, 10,000 initiation number, and random initiation parameters were used for clustering populations in scatter plots. Each cluster is labeled with a number, and the current trace of representative events from each cluster is shown on the right. The concentration of the initial circular DNA template was 10 nM except for Alul D6, which is 2 nM. Experiments were performed in 1 M GdmCl, 1 M KC1, 50 mM Tris, pH 7.6, at 250 mV applied bias. The current signal was lowpass-filtered at 10 kHz.

[0024] FIG. 4 shows an investigation of DNA reporter capture rate versus initial circular template concentration, in accordance with an embodiment of the invention. Current traces recorded from no circular template control (Panel A), 16 pM (Panel B), 80 pM (Panel C), 400 pM (Panel D), and 2 nM (Panel E) initial circular DNA template concentration. (Panel F) Log-log plot of capture rate (s— 1) or RCA product mass concentration as a function of initial circular DNA template concentration (pM). The lower limit of detection (LOD) was measured by three standard deviations above the blank. Experiments were performed in 1 M GdmCl, 1 M KC1, 50 mM Tris, pH 7.6, at 250 mV applied bias and lowpass filtered at 10 kHz.

[0025] FIG. 5 shows antibody quantification using methylated circular DNA template in accordance with an embodiment of the invention. Scatter plot of DNA reporter molecules generated from unmethylated templates (Panel A) and methylated templates (Panel B) immobilized on streptavidin beads. Scatter plots of methylated template immobilized with antibody-conjugated beads and reaction performed for 1 h (Panel C) and 2 h (Panel D). Beads/Stv stands for streptavidin beads and Beads/ Ab stands for HE4 antigen conjugated beads. All scatter plots were clustered by Bayesian Gaussian mixture model with full covariance type, random state 1, 1 x 10 ⁹ convergence threshold, 10000 initiation number, and random initiation. (Panel E) Capture rate as a function of HE4 concentration. The dashed lines denote assay LODs which were calculated by three standard deviations above the blank. Data were collected from three replicates. Representative current traces were recorded from blank control (Panel F), 40 pM (Panel G), 200 pM (Panel H), and 1 nM HE4 (Panel I). Nanopore measurements were performed in 1 M GdmCl, 1 M KC1, 50 mM Tris, pH 7.6, at 250 mV applied bias. The current signal was lowpass filtered at 10 kHz.

[0026] FIG. 6 shows HE4 concentration measurements in human plasma using INCA in accordance with an embodiment of the invention. Representative current traces recorded from blank control (Panel A) and 500 pM HE4 in 4-fold diluted plasma samples (Panel B) and corresponding scatter plots (Panels C and D). The INCA reaction was performed for 2 h. Bayesian Gaussian mixture model with full covariance type, random state 1, 1 x 10 ⁹ convergence threshold, 10000 initiation number, and random initiation was used for clustering. (Panel E) Capture rate measurement of corresponding HE4 sample plotted on the calibration curve. LOD was measured by three standard deviations above the blank. Experiments were performed in 1 M GdmCl, 1 M KC1, 50 mM Tris, Ph 7.6, at 250 mV applied bias. The current signal was lowpass filtered at 10 kHz.

[0027] FIG. 7 shows native PAGE characterization of template ligation in accordance with an embodiment of the invention. (Panel A) Alul cleavable template ligation. Lane 1-3: linear, circularized Alul D5 template and the Exonuclease I and Exonuclease III treatment of circularized Alul D5 templates. Lane 4: O'RangeRuler 5 bp DNA Ladder. Lane 5-7: linear, circularized Alul D6 template and the Exonuclease I and Exonuclease III treatment of circularized Alul D6 templates. Lane 8-10: linear, circularized Alul D7 template and the Exonuclease I and Exonuclease III treatment of circularized Alul D7 templates. (Panel B) Rsal cleavable template ligation. Lane 1-3: linear, circularized Rsal DI template and the Exonuclease I and Exonuclease III treatment of circularized Rsal DI templates. Lane 4: O'RangeRuler 5 bp DNA Ladder. Lane 5-7: linear, circularized Rsal D2 template and the Exonuclease I and Exonuclease III treatment of circularized Rsal D2 templates. Exonuclease I and Exonuclease III treatment is to confirm successful ligation. Because Exonuclease I and Exonuclease III act upon linear ssDNA and dsDNA respectively, while leaving circularized DNA intact. The gel was run with 20% polyacrylamide and stained with Gelred. The image was visualized with a Biorad system. All circularized DNA samples show a single band which indicates a high ligation efficiency.

[0028] FIG. 8 shows optimization of the rolling circle amplification reaction in accordance with an embodiment of the invention. RCA product concentration (ng/pL) as a function of (Panel A) dNTP concentration, (Panel B) phi29 activity, (Panel C) different surfaces for immobilizing primer-template hybrid, (Panel D) salt type and salt concentration, (Panel E) magnetic beads concentration, (Panel F) reaction time. (Panel G) Three DNA templates, Alul D5 (SEQ ID NO: 20), Alul D6 (SEQ ID NO: 2), and Alul D7 (SEQ ID NO: 4), were designed to produce RCA products that could be cleaved by restriction enzyme Alul (Panel G) and two DNA templates, Rsal DI (SEQ ID NO: 6), and Rsal D2 (SEQ ID NO: 8) were designed to produce RCA products that could be cleaved by restriction enzyme Rsal (Panel H). The three Alul cleavable templates share the same primer sequence (labeled with an underline). The difference in the sequence is labeled in red and the same sequence is labeled in black. Same color coding is also used in Rsal cleavable template. Inset shows the 0.3% agarose gel electrophoresis result of formamide denatured RCA product and 20 kbp Nolimit DNA as a reference. All primer-template hybrids were immobilized on streptavidin- coated magnetic beads except for (Panel A) and (Panel B) in which the primer-template hybrid was not immobilized on any surface, and (Panel C) in which primer-template hybrid was immobilized on either streptavidincoated magnetic beads or streptavidin-coated plates. The reaction time is 1 hour except for 2 hours for (Panel D). All reaction conditions were 10 mM (NH₄)₂SO4, 10 mM MgCh, 50 mM Tris-HCl pH 7.5, 4 mM DTT, 0.05% Tween-20, 1 mM dNTP, 2 U/pL phi29 polymerase, 1 pg/pL 2.8 pm magnetic beads, 37 °C and 10 nM initial template concentration unless stated otherwise.

[0029] FIG. 9 shows sequence (SEQ ID NO: 21), structure, current trace of 8bp DNA hairpin in accordance with an embodiment of the invention. Multiple steps were observed in a single event with first level blockade around 58% blockade. And the largest blockade level in a single event could up to 96 %, Some events have more than 100 s dwell time and only leave alpha-hemolysin upon reversing the voltage polarity. All measurements were performed with IM KC1, 20 mM Tris, pH 7.6, at 300 mV applied bias, and the current signal was lowpass filtered at 10 kHz.

[0030] FIG. 10 shows sequence, structure, dwell time versus blockade scatter plot and the current trace of DNA hairpins in accordance with an embodiment of the invention. (Panel A) 4 bp (SEQ ID NO: 22), 5 bp (SEQ ID NO: 23), 6 bp (SEQ ID NO: 10), and 7 bp (SEQ ID NO: 24) DNA hairpin sequences and the predicted secondary structure. (Panel B) Scatter plot of dwell time versus fractional blockade for the four-hairpin mixture. (Panel C) Current trace of the four-hairpin mixture translocation through alpha-hemolysin pore. All measurements were performed with IM KC1, 20 Mm Tris, pH 7.6, at 250 mV applied bias, and each hairpin concentration was 250 nM. The current signal was lowpass filtered at 10 kHz. [0031] FIG. 11 shows sequence, structure, current trance, and dwell time versus blockade scatter plot of DNA hairpin in accordance with an embodiment of the invention. Sequences, predicted structures, dwell time versus fractional blockade scatter plot, and a current trace of 6 bp (SEQ ID NO: 10), 7 bp 1 mismatch (SEQ ID NO: 25), and 8 bp 2 mismatch (SEQ ID NO: 26) hairpin. Mismatches in the hairpin reduce the unfolding energy resulting in reduced dwell time. All measurements were performed with IM KC1, 20 mM Tris, pH 7.6, at 300 mV applied bias, and the current signal was lowpass filtered at 10 kHz.

[0032] FIG. 12 shows continuous current traces and the scatter plots of reporter molecules in accordance with an embodiment of the invention. Ionic current trace obtained from reporter molecules of Alul D6 (Panel A), Alul D7 (Panel B), Rsal DI (Panel C), and Rsal D2 (Panel D). A corresponding scatters plot is shown on the left. All measurements were performed with IM GdmCl, IM KC1, 20 mM Tris, pH 7.6, at 250 mV applied bias, and the current signal was lowpass filtered at 10 kHz.

[0033] FIG. 13 shows an ionic current trace and scatter plot of Alul D6 reporter molecules heat incubated in IM GdmCl, in accordance with an embodiment of the invention. Alul D6 reporter molecules with an initial circular template concentration of 10 nM were incubated at 37 °C with 1 M GdmCl, 1 M KC1, 50mM Tris, pH 7.6 for 1 h. Then nanopore measurement was performed with the same buffer, at 250 mV applied bias, lowpass filtered at 10 kHz. The current trace (Panel A), and scatter plot (Panel B) were obtained.

[0034] FIG. 14 shows distinctive scatter plots and reproducibility between different pores, in accordance with an embodiment of the invention. Overlayed scatter plots of Alul D7 (Panel A), Rsal DI (Panel B), and Rsal D2 (Panel C) over Alul D6. The Alul D6 is shown as blue and Alul D7, Rsal DI, and Rsal D2 are shown in green, purple, and brown, respectively. Scatter plot obtained from multiple independent experiment of Alul D6 reporter molecules (Panels D-F), Alul D7 reporter molecules (Panels G-H), Rsal D2 reporter molecules (Panel I). All measurements were performed with IM GdmCl, IM KC1, 20 mM Tris, pH 7.6, at 250 mV applied bias, and the current signal was lowpass filtered at 10 kHz.

[0035] FIG. 15 shows capture rate comparison of different templates in accordance with an embodiment of the invention. (Panel A) Comparison of different circular DNA templates at initial concentration of 10 nM. (Panel B) Capture rate of Rsal D2 at different initial concentrations. Nanopore experiments were performed in 1 M GdmCl, 1 M KC1, 50 mM Tris, pH 7.6, at 250 mV applied bias. The current signal was lowpass-filtered at 10 kHz. [0036] FIG. 16 shows IFN gamma detection using Rsal D2 in accordance with an embodiment of the invention. Magnetic beads and primer-Rsal D2 template hybrid were conjugated with interferon-gamma capture antibody and detector antibody. After the formation of the sandwich ELISA structure, an RCA reaction was performed, and reporter molecules were released by the Rsal enzyme. Nanopore measurements were conducted in 1 M GdmCl, 1 M KC1, 50 mM Tris, pH 7.6 buffer. The current trace (Panel A) and scatter plot of dwell time versus fractional blockade (Panel B) were obtained.

[0037] FIG. 17 shows characterization of methylated Alul D6 template in accordance with an embodiment of the invention. (Panel A) Circularization of the linear template. The linear and circularized template shows a different migration speed on 20% native PAGE gel. Treatment of linear template with Exol and ExoIII shows complete digestion while the circularized template is resistant to Exol and ExoIII. (Panel B) Alul resistant assay of methylated Alul D6. After circularization, the templates were titrated with Alul. 20 % native PAGE and Gelred was used for visualization. The distorted band is due to the high concentration of Alul protein in the sample. (Panel C) RCA reaction with methyl-AluI D6. The reaction condition was 10 mM (NH4)2SO4, 10 mM MgC12, 50 mM Tris-HCl pH 7.5, 4 mM DTT, 1 mM dNTP, 2 U/pL phi29 polymerase, 1 pg/pL 2.8 pm magnetic beads, 37 °C for Ih. After the reaction, the sample and the 20 kbp Nolimit DNA were incubated in 95% formamide, lOmM EDTA, pH 8.2, at 90 °C for 15 min. And 0.3 % agarose gel was used to compare the relative length.

[0038] FIG. 18 shows influence of reaction parameters for INCA in accordance with an embodiment of the invention. To optimize the yield of as well as understand the possible structure and size reporter molecules produced via INCA. We changed the dNTP concentration, phi29 unit, Alul unit and compared the results. No significant difference was observed in the range of parament we choose. The reaction temperature and time were 37 °C, and 1 h. the primer-template hybrid was immobilized on streptavidin beads.

[0039] FIG. 19 shows a comparison of unmethylated and methylated Alul D6, in accordance with an embodiment of the invention. Current trace of methylated (Panel A) and unmethylated Alul D6 (Panel B). Both initial template concentrations were 400 pM and immobilized on 1 pg/pl 2.8 pm magnetic beads. Unmethylated Alul D6 was RCA amplified first and then cleavage by Alul while methylated Alul D6 was done via INCA.

[0040] FIG. 20 shows overlayed scatter plots of methylated Alul D6 on streptavidin coated beads (Panel C), methylated Alul D6 on antibody coated beads (Panel D) (Panel E) over unmethylated Alul D6, in accordance with an embodiment of the invention. The unmethylated Alul D6 was shown as blue. After reporter molecule generation, nanopore measurements were performed in 1 M GdmCl, 1 M KC1, 50 mM Tris, pH 7.6, 250 mV applied bias, lowpass filtered at 10 kHz.

[0041] FIG. 21 shows HE4 quantification using methylated circular DNA template and 1 h reaction time, in accordance with an embodiment of the invention. Representative current traces recorded from blank control (A), 40 pM (B), 200 pM (C), and 1 nM HE4 (D). Experiments were performed in IM GdmCl, IM KC1, 50 mM Tris, pH 7.6, at 250 mV applied bias. The current signal was lowpass filtered at 10 kHz.

[0042] FIG. 22 shows a schematic of LAMP assay for nanopore amplification of reporter molecules, in accordance with an embodiment of the invention. After the formation of sandwich ELISA, a heat release of aptamer molecules, loop-mediated isothermal amplification, and 80 °C heat inactivation of reaction, we will have long dsDNA products. And by using restriction enzymes to digest long dsDNA products into DNA fragments, which are reporter molecules in this schematic, an alphahemolysin pore can be used for biomarker quantification.

[0043] FIG. 23 is a schematic block diagram illustrating components of a device for detecting presence of a biomarker in a sample using a nanopore, in accordance with devices, methods and other features of embodiments taught herein.

DETAILED DESCRIPTION

[0044] A description of example embodiments follows.

[0045] Proteins play important roles in biological processes and thus can serve as valuable biomarkers for various diseases. Detection of many protein-based biomarkers for clinical diagnostics relies on centralized laboratory tests that use enzyme-linked immunosorbent assay (ELISA) (1,2). These assays have advantages of high sensitivity, high accuracy, and high-throughput sample processing (2). As healthcare trends toward patientcentered models, the concept of point-of-care diagnostic devices has become more commonplace. A point-of-care diagnostic device is portable, easy to operate, easily accessible for people living in remote areas, and convenient for monitoring chronic diseases (1,2). While there exist hand-held devices that can detect a single analyte, multifunctional immunoassay platforms have generally remained on benchtop formats (2), as these technologies are usually based on sandwich ELISA immunocomplex formation which is quantified using fluorescence measurements and therefore require bulky equipment that integrates sample fluidics with cameras, mechanical stages, light sources, and optics. Hence, alternative strategies need to be explored in order to develop point-of-care immunoassay devices.

[0046] Over the past three decades, biological nanopores have been developed and evolved into reliable biosensors capable of probing biophysical properties at the singlemolecule level, identifying various biomolecules (3-12), studying enzyme kinetics (13-15), and sequencing DNA and RNA (16,17), and currently nanopores are among a handful of candidate tools for single-molecule protein sequencing (18-20). The portability of nanopore sensors and the fast measurement times nanopores can deliver position nanopores as ideal choices for point-of-care diagnostic applications. In nanopore sensing, typically a biological porin such as a protein toxin (21-23) or a synthetic DNA origami pore (24-26) spontaneously inserts into a thin organic membrane that separates two chambers filled with electrolyte solutions (27,28). Applying a voltage across the membrane induces a highly localized electric field across the pore, leading to a steady-state ionic current signal. This electric field draws charged molecules to the nanopore, and as a result, the ionic current is partially occluded. The occlusion time, amplitude of the current blockade, and signal fluctuations can be used to extract the size, charge, and conformation of the molecules.

[0047] Furthermore, the frequency of each molecular species being captured by nanopores, known as the capture rate, is a function of the molecular concentration. Nevertheless, two prerequisites must be met for general biosensing applications: (i) a signal amplification method must be adopted to enhance the capture rates of rare clinically relevant biomarkers, and (ii) reporter molecules compatible with the nanopore of choice must be developed, as nanopores are very sensitive to size and charge of analytes, which imposes a major limitation on the use of nanopores for sensing proteins with a wide range of molecular weights and charges. The reporter molecules for a given biomarker can be a group of molecules comprising distinct sizes and structures that produce distinct signal patterns (or fingerprints) in the dwell time versus fractional current blockade parameter space.

[0048] Surrogate reporters for biomarkers such as star-like DNA probes (11) and ssDNA barcodes (29) have been previously demonstrated using nanopore sensors. While these studies demonstrate the quantification ability of nanopores, sensing low concentrations remains as a major bottleneck for adopting nanopores as tools for biomarker detection. To fill this gap, here we present an amplification scheme that generates a large number of short DNA molecules as reporter molecules for biomarker quantification which enhances the capture rate of specific molecules and produces events with distinctive fingerprints. In this scheme, we combine isothermal nucleic acid amplification and a sandwich ELISA immunocomplex (30,31), with controlled sequence-specific cleavage to enhance the sensitivity of nanopores. Rolling circle amplification (RCA) with the highly processive enzyme phi29 polymerase generates ultralong ssDNA (~100 knt), an ideal product for signal amplification (32-34). We employ this isothermal amplification method that is rapid, cost- effective, easy-to-use, and more tolerant to inhibitory components from crude samples than polymerase chain reaction (PCR), another enzyme-based amplification method (35). Restriction enzyme-based digestion of the RCA product generates many DNA fragments of different sizes and structures, which are detected using an a-hemolysin nanopore (Figure 1 A). Our measurements reveal an identifiable nanopore fingerprint for a biomarker linked to a circular DNA template. We quantify pM concentrations of a representative protein biomarker, human epididymis protein 4 (HE4), with only 5 min of recording time. We also developed an in situ cleavage and amplification (INCA) assay that reduces reaction times and wash steps by using a circular template containing a 5-methylcytosine site that is not amenable to restriction digestion, thereby enabling simultaneous RCA and digestion of the product. Using this approach, we demonstrate the detection of low pM levels of HE4. Our scheme employs reliable and commercially available reagents and circumvents the need for complex DNA nanoparticle synthesis and expensive DNA modifications.

[0049] In one embodiment according to the present invention, the disclosure relates to a method for electrically detecting biomarkers in serum or any sample. The method is based on nanopore-based biomarker detection that utilizes immunocapture, isothermal rolling circle amplification, and sequence-specific fragmentation of the product to release multiple DNA reporter molecules for nanopore detection. These DNA fragment reporters produce sets of nanopore signals that form distinctive fingerprints, or clusters. This fingerprint signature therefore allows the identification and quantification of biomarker analytes.

[0050] Briefly, an example embodiment of the method comprises the following steps: [0051] 1) A primary antibody for the biomarker of interest is chemically immobilized on microbeads or microplates in a manner that allows the epitope of the antibody to have steric access to a biomarker of interest.

[0052] 2) A solution that contains the biomarker, say a protein in serum, is introduced to the immobilized antibody (i.e., bead suspension). [0053] 3) After incubation which allows the biomarkers to bind to the immobilized antibodies, the surface is washed several times so that only specific biomarkers bind to the microbeads or microplates via primary antibody.

[0054] 4) DNA primer-conjugated secondary antibodies are introduced for the formation of sandwich ELISA structure (primary antibody -biomarker-secondary antibody) on microbeads or microplates.

[0055] 5) The surface is washed several times, then a solution containing the circular

DNA templates is introduced for hybridization with the primers that are conjugated with secondary antibodies. The circular DNA templates are designed to contain a DNA hairpin structure, which is used for restriction enzyme cleavage.

[0056] 6) A solution containing high processive polymerase, dNTP, DTT, etc. is introduced to initiate rolling circle amplification (RCA) of the circular DNA

[0057] 7) After incubation for an appropriate amount of time and temperature, the primers are extended into ultra-long highly repetitive concatemer ssDNAs by the polymerase via RCA. This results in ultralong (~70 knt) ssDNA that contains repetitive complimentary circular DNA template sequences. The long concatemer ssDNA is also immobilized on the surface via primary antibody-biomarker-secondary antibody complex.

[0058] 8) The surface is washed several times to remove all biomolecules not bound to the surface, and then a restriction enzyme is introduced. Since the circular DNA template has a DNA hairpin sequence, the long concatemer ssDNA also has a DNA hairpin sequence. The restriction enzyme recognizes the encoded DNA hairpin sequence and cleaves at that specific site. The long ssDNA concatemer is now cleaved into DNA fragments that have different sizes and shapes.

[0059] 9) Due to the precise shapes and lengths of the fragments, these fragments are used as reporter molecules for biomarker quantification using a nanopore. The solution containing reporter molecules is added to the nanopore chamber.

[0060] 10) When the reporter molecules are passing through the pore, a distinctive nanopore ionic current signal is produced based on the size and shape of the reporter molecule. The amount of ionic current being blocked by a certain reporter molecule is called blockade and the amount of time for the reporter molecule to pass through to pore is called the dwell time. The time between two molecules passing through the pore is called interevent time. [0061] 11) Since the reporter molecule is comprised of different sizes and shapes of

DNA fragments, a distinctive blockade-dwell time spectrum is generated which link to the sequence design of a circular DNA template.

[0062] 12) The interevent time is linked to the concentration of reporter molecules, which is directly associated with the original concentration of the biomarker.

[0063] 13) By extracting the interevent time of reporter molecules, the original concentration of the biomarker could be recovered. RCA amplification provides manifold enhancement in sensitivity (-100).

[0064] The above-mentioned method is using two steps for generating reporter molecules (RCA and restriction digestion). Also described herein is a steps method for faster and more sensitive biomarker quantification: The circular DNA template is methylated at the site of restriction in order to protect the circular template from action by a restriction enzyme. This allows RCA and restriction simultaneously, which avoids the formation of very large ssDNA concatemers which creates a gel-like precipitate that inhibits RCA and post-RCA cleavage. [0065] Further embodiments are described herein, and are discussed below in connection with experiments carried out in accordance with embodiments of the invention.

[0066] Reporter Molecule Generation Using RCA

[0067] Generally, biosensing with biological nanopore platforms is conducted under low applied bias and at relatively high (nM-//M) analyte concentrations (21, 36). At 120 mV, DNA hairpin detection was usually conducted at 1 z/M concentrations (21). While increasing the applied voltage typically increases the molecular capture rate, this advantage is limited by the maximum voltage that can be applied while maintaining a stable membrane. To extend this voltage range, we have previously developed a voltage-stable lipid bilayer platform that allows regularly applied voltages up to 300 mV. With a high applied bias, capture rates increase by as much as 10-fold, thereby reducing detection limits (28). However, improving the detection limit and expanding the dynamic range of nanopore sensing to pM levels would vastly increase the utility of nanopore-based sensing of biomarkers. To bridge this gap, we combine immunocapture and RCA of a short circular DNA template to generate a DNA mass that, upon cleavage using restriction enzymes, releases a combination of DNA fragments for nanopore-based detection. Here, a high processivity enzyme, phi29 polymerase, was used to amplify the circular DNA template into a highly repetitive concatemer product. Restriction enzymes, which are known to recognize and cleave DNA at specific sequences, were used to cleave the RCA concatemer product into fragments. We chose the restriction enzymes Alul and Rsal because these two enzymes are active even when cleaving near the end of a DNA duplex. A feature in our design is a DNA hairpin secondary structure within the circular DNA template, which contains a restriction enzyme recognition site that allows isothermal enzymatic digestion (FIG. 1, Panel A). Upon digestion of the RCA concatemer product, a distinctive collection of DNA fragments is obtained, which produces fingerprint signals during nanopore detection.

[0068] We combine here a few of our recent developments in our nanopore sensing platform. First, use of a 50 pm diameter wedge-on-pillar (WOP) (28) aperture allows high voltage recordings (FIG. 1, Panel B), which further pushes down the detection limit. Second, use of guanidinium chloride (GdmCl) facilitates passage of the DNA reporter molecules through the pore by compromising the Watson-Crick base-pairing stability. To use GdmCl for detection, we employed the chemically resilient polymer bilayer membrane poly(l,2- butadiene)n-b-poly-(ethylene oxide)s (PBD11-PEO8) in our platform (12). The primer and templates used in this study are listed in the Supporting Information (Table 1). Successful ligation was confirmed by Exonuclease I and Exonuclease III treatment, since Exonuclease I and Exonuclease digest linear ssDNA and dsDNA while leaving circularized DNA intact (FIG. 7). We performed an extensive optimization of the RCA reaction performance as a function of dNTP concentration, phi29 activity, surface (magnetic beads vs ELISA plates), salt type and concentration, magnetic bead concentration, and RCA reaction time (FIG. 8). Following optimization, we first characterized the DNA reporter molecules generated from four different primer-template designs on a simple streptavidin-biotin model system (FIG. 2, Panel A). Since the long ssDNA formed by RCA reaction can hybridize to itself in various ways, there are multiple restriction products that one can obtain. We outline here four different possible DNA fragments, labeled as “a”, “b”, “c”, and “d” (FIG. 2, Panel A), and whose structures are estimated based on PAGE electrophoresis migration speeds. For both Alul D6 (FIG. 2, Panel B) and Alul D7 (FIG. 2, Panel C), the estimated migration speeds for DNA fragments a, b, c, and d correspond to 8, 16, 23, and 46 bp, respectively (gel shown in FIG. 2, Panel D). The minor appearance of other bands around 50 bp dsDNA is possibly due to the formation of higher-order structures. Similarly, the rough migration speeds of DNA fragments produced from Rsal DI and Rsal D2 correspond to 7, 14, 24, 48 bp, and 10, 20, 21, 42 bp, respectively (FIG. 3, Panels E,F). Larger molecular weight bands here are possibly due to incomplete restriction enzyme cleavage, as indicated by a digestion time-course study (FIG. 2, Panel D, lanes 3 and 4). [0069] Generally, a blunt dsDNA hairpin that is longer than 8 bp is difficult to translocate through an a-hemolysin pore without a denaturing agent (FIG. 9) (21, 28), because the free energy of duplex melting is too high. To facilitate this process, we conducted our measurements in a denaturing buffer consisting of 1 M GdmCl, 1 M KC1, and 50 mM Tris, pH 7.6, and used a 250 mV applied bias. Since the product digest consists of several fragments of varying degrees of stability, several types of signals should be obtained. Indeed, we observe multiple populations in the scatter plots of dwell time versus fractional blockade (FIG. 3, Panels G-J). Employing a Bayesian Gaussian mixture model for clustering these populations, we obtain three clusters of events for Alul D6, and four clusters for Alul D7. We reason that this occurs because Alul cleavage produces two identical 6 bp hairpins; hence, two populations merge into one (FIG. 2, Panels B, C). Comparing the clustered scatter plots, both Alul D6 and Alul D7 have a cluster “1” centered at ~55% fractional blockade. We have previously shown that the 6 bp hairpin produces characteristic “shoulder-spike” events with a lower-level blockade around 55% and a sharp deep blockade at the end (FIGS. 10 and 11) (21). When examining the single translocation events, this population also has a characteristic two-level blockade pattern (FIG. 3, Panels G and H, cluster 1). We hypothesize that cluster l’s in Alul D6 and Alul D7 are translocation events from 6 bp hairpin molecules. The reduction in dwell time is attributed to the action of 1 M GdmCl (12) which does not alter the pore structure yet expedites DNA duplex denaturation to facilitate a diffusion-limited turnover of capture events.

[0070] Other than the 6 bp hairpin, which is DNA fragment “a”, Alul D6 and Alul D7 can produce fragment “b” that also has 6 bp at both ends, and therefore, it is likely that fragments b and a are in the same cluster. During translocation of molecules a and b, the poly-T4 loop is docked on the a-hemolysin pore mouth, since it will adopt a conformation that is not ready to enter the vestibule, and the double-strand stem enters the vestibule. As the voltage exceeds the energy barrier, the double-strand stem will unzip, and the DNA will translocate the pore as a ssDNA strand forms (21). DNA fragments c and d have similar structures but subtle differences in their sequences; that is, Alul D6 has a lower GC content, thus likely corresponding to the broader distribution of clusters 2 and 3 in Alul D6 compared to Alul D7.

[0071] Likewise, Rsal DI and Rsal D2 produce 4 bp and 8 bp hairpins (FIG. 3, Panels E, F), which also have signature “shoulder- spike” events as shown in our nanopore measurement (FIG. 3, Panels I, J, cluster 1). The blockade level matches with previous measurements (FIG. 11). For reference, we present continuous current traces recorded from reporter molecules generated from four circular templates in FIG. 12. Another interesting observation is that when incubating the Alul D6 sample with 1 M GdmCl, 1 M KC1, and 20 mM Tris buffer at 30 °C for 1 h, the nanopore events occur faster, which may be due to the majority of reporter molecules translocating in ssDNA form due to denaturation by GdmCl (FIG. 13).

[0072] The scatter plots demonstrate a distinctive pattern based on reporter molecules. As an example, we overlaid scatter plots of Alul D7, Rsal DI, and Rsal D2 over Alul D6 (FIG. 21 Panels A-C). The reproducibility across multiple independent experiments is shown in FIG. 14 Panels D-I. Thus, by designing the sequence and structure of different RCA templates, we can create characteristic “fingerprints” that have the potential for identifying multiple distinct biomarkers.

[0073] Nanopore Quantification

[0074] Next, we investigated the limit of detection for reporter molecules amplified from Alul D6 using streptavidin-coated beads and a biotinylated primer as a model system. FIG. 4 Panels A-E shows representative 20 s current traces recorded at different initial circular template concentrations. At the lowest concentration, a 5 min current recording provided 142 events. Capture rates were obtained by fitting exponential curves (37) to the distribution of inter-event time. As shown in the log-log plot in FIG. 4 Panel F, the concentration versus capture rate curve follows a power law with an exponent of 0.63. This exponent is slightly different from the RCA product versus template concentration power exponent of 0.72 (see FIG. 8 Panel C), the slight difference being possible composition of short versus long DNA fragments in the overall DNA mass, which impacts capture by the nanopore. We also note that for the lowest RCA product concentrations, Qubit quantification results had a large variance, while our nanopore capture rates showed a low variance across multiple experiments. Measured from three blank replicates (no antigen present in sample), the mean capture rate of the blank control was 0.148 s ' . Based on the mean value (p) and standard deviation (c) of the blank control, an LOD of 14.4 pM is calculated by LOD = 3o + p. Importantly, these results show that combining RCA amplification with high voltages enables detection of DNA reporter molecules above 16 pM with a single a-hemolysin pore in a few minutes of detection time. We also compared capture rates between different templates as shown in FIG. 15 Panel A. The difference in capture rate was similar to the difference in overall RCA product mass, as shown in FIG. 8 Panels G, H. To demonstrate whether there is a difference in capture rate when using non-AluI D6 primer and template, we tested Rsal D2 at initial template concentrations of 10 and 2 nM, the results of which are shown in FIG. 15 Panel B.

[0075] INCA Assay and HE4 Quantification

[0076] Having successfully demonstrated the rapid detection of low pM concentrations of circular templates using model streptavidin beads, we next applied our platform toward protein biomarker detection. Our first trial in detecting a representative cytokine, interferon-y, utilized the Rsal D2 template, showing an 8 bp hairpin cluster in the dwell time versus fractional blockade scatter plot. However, as fewer target molecules are expected to be captured by the antibodies compared to the high-affinity streptavidin-biotin system, the capture rate of reporter molecules was significantly lower than that of the streptavidin bead system, at only 0.5 s ' even at 10 nM (FIG. 16). We hypothesized that in situ cleavage of the RCA product during the amplification reaction can increase reporter molecule yield. To achieve our goal, we exploited the methylation-sensitive property of the Alul restriction enzyme. We created an Alul-resistant circular template methyl-AluI D6 by the addition of two methylated cytosines in the Alul recognition site. Because the incorporated nucleotides are not methylated, the RCA product can be cleaved as phi29 polymerase is acting on the template. The circularization of linear methyl-AluI D6, resistance to Alul digestion, and the successful RCA reaction on the methylated template were confirmed by gel electrophoresis (FIG. 17). Phi29 polymerase has a high processivity that could incorporate dNTP ats 2280 nt/min at 30 °C (38). In contrast, restriction enzyme cleavage speeds are generally slow. To make sure cleavage is suitable for detection, we varied a few different reaction parameters such as dNTP concentration, phi29 polymerase concentration, and Alul enzyme concentration, and the results were not very different within the range of concentration tested (FIG. 18). The scatter plots of DNA reporter molecules generated from unmethylated and methylated Alul D6, both captured on streptavidin beads at an initial template concentration of 400 pM, are shown in FIG. 5, Panel A, B. Clustering with a Bayesian Gaussian mixture model shows three populations, with the 6 bp hairpin denoted as population 1. The corresponding trace is shown in FIG. 19 Panels A, B. Consistent with our hypothesis that in situ cleavage of the RCA product would increase reporter molecule yield, the methylated template improved the nanopore capture rate by nearly 4-fold over the unmethylated template (13.07 versus 3.43 s '). Next, we applied the INCA platform to the detection of the protein biomarker HE4, using capture antibody-conjugated magnetic beads and a detector antibody conjugated to the annealed primer and methylated template pair. Consistent with our results, nanopore data with methylated template and antibody conjugated beads show similar population clusters as with streptavidin beads (FIG. 5 Panels C, D). The overlaid scatter plots are shown in FIG. 20 Panels C- E. Here we note that population 3 from the reporter molecules generated from the methylated template demonstrates larger blockades and long dwell time events, possibly due to a relatively large amount of larger reporter DNA fragment present in the mixture.

[0077] We measured the concentration dependence on capture rates for both 1 and 2 h reaction times and found that 2 h RCA reaction times show an approximate 1.7-fold higher capture rates than 1 h reaction times (FIG. 5 Panel E). The LODs for a 1 h and a 2 h reaction are 28.8 pM and 7.4 pM, respectively. The current traces for 2 and 1 h reaction times at various HE4 concentrations are shown in FIGS. 5 Panels F-I and FIG. 21, respectively. The INCA assay not only increases the yield of reporter molecules but also reduces the need for washing steps between RCA and restriction enzyme treatment.

[0078] Finally, we explored the INCA performance in biological fluids. As a proof of concept, we measured HE4 in 4-fold diluted human serum. The representative current traces and corresponding scatterplots of the blank control and 500 pM HE4 are shown in FIG. 6 Panels A-D. Clustering with a Bayesian Gaussian mixture model shows consistent results as discussed above. The calculated capture rate at 500 pM HE4 in serum was 8.58 s ', which correspond to 199 nM. The recovery is 37.6%. The low recoveries could be attributed to matrix effects from interfering components in biological fluids. This issue could be addressed by multiple strategies, such as further dilutions, addition of blocking agents/detergents, etc. [0079] Experimental Conclusions

[0080] We have developed a robust biological nanopore platform for biomarker detection based on isothermal amplification of DNA reporters followed by enzymatic DNA fragmentation. Our signal amplification assay bridges the gap between biological nanopores and point-of-care biomarker quantification by allowing rapid biomarker detection at pM levels. As a proof of concept, we demonstrate its capability of quantifying a representative protein biomarker, HE4, with 7.4 pM LOD.

[0081] One of the advantages of RCA as a signal amplification method is its high adaptability toward a wide range of targets, including proteins, mRNA, and single nucleotide polymorphisms (SNPs) (35,39). For example, with the addition of T4 ligase and padlock probes, the assay can be modified for SNP detection. In addition, compared to other isothermal amplification methods, RCA can be conducted at relatively low temperatures and does not require fine thermal control, thus facilitating integration into a portable and simple device (35).

[0082] However, to create a distinct spectrum, we can also employ loop-mediated isothermal amplification, which can potentially provide higher sensitivities and a wider dynamic range. As shown in FIG. 22, after the formation of a sandwich immunocomplex, heat-mediated release of aptamer molecules, and loop-mediated isothermal amplification, long dsDNA products are generated that can be digested by restriction enzymes into DNA fragments similar to the reporter molecules employed in this article. Thus, our main idea of a nanopore spectrum is highly versatile and can be applied to other nucleic acid amplification methods.

[0083] Further improvements can be made to further expand this method and improve detection limits. Enhancing the capture rates of DNA fragments by shifting from wild-type a- hemolysin to an electroosmotically enhanced a-hemolysin mutant (40), applying salt gradients across the pore (41), scaling up to an array of pores to integrate the detection events in similar sample volumes, and further reducing the sample volume via an automated microfluidic system can potentially improve analytical sensitivity to femtomolar or potentially attomolar levels. Further work will focus on achieving even lower LODs, faster reaction times, and multiplexing, which are desirable for many clinical applications, especially in biological fluids such as saliva that can be noninvasively collected but contain much lower biomarker concentrations.

[0084] Methods: SU-8 Wedge-Pillar Aperture Fabrication

[0085] The SU-8 aperture was fabricated on a 500 pm thick (100) Si wafer with 200 pm silicon dioxide and a 50 nm thick silicon nitride layer coated on both sides. The 200 pm silicon dioxide buried underneath the silicon nitride serves to reduce the capacitive noise of the chips. The wafer was the first pattern with an array of 1 mm squares using the standard photolithography method. Then the wafer was etched by 150 W 1 min of SF6 reactive ion etching, 50 min of buffered oxide etch (BOE). Next, on the backside, the wafer was spun coated with 25 pm thick SU-8 3025, soft baked at 95 °C, constant power 275 W for 12.5 s, post exposure baked at 95 °C for 4 min 30 s, and developed for 6 min. Greyscale photolithography was used to create wedge-pillars as we discussed in previous work. Finally, the 500 pm Si layer, silicon dioxide layer, and silicon nitride layer were removed using standard KOH, BOE, SF6 reactive ion etching while a single side etcher was used to protect the SU-8 wedge-on-pillar aperture on the backside.

[0086] Polymer Bilayer Painting and Nanopore Measurement

[0087] First, the wedge-pillars aperture was pretreated with 1 pL of hexane dissolved PBD11-PEO8 (Catalog #P41807C-BdEO; PolymerSource, Montreal, Quebec, Canada) (5 mg/mL) on each side. After the hexane evaporated, the chip was mounted on our customized flow cell. The trans chamber was filled with 1 M GdmCl, 1 M KC1, 50 mM Tris, pH 7.6 electrolyte, and the cis chamber was filled 50 pL of reporter molecules sample plus 150 pL of 1.33 M GdmCl, 1.33 M KC1, 66.5 mM Tris pH 7.6 so that the final electrolyte concentration is the same as the trans. A Ag/AgCl pair was inserted into the electrolyte and connected to the patch amplifier (Axopatch 200B; Molecular Devices, San Jose, CA). Decane-dissolved PBD11-PEO8 (20 mg/mL) was painted across the aperture using a pipet. After confirmation of bilayer formation by checking the capacitance, 0.5 pL of 25 pg/mL a-hemolysin was added to the cis chamber until single-channel insertion was observed. Current signals were collected at 250 kHz sampling rates, lowpass filtered to 10 kHz, and analyzed using Pyth-Ion. For this analysis, the threshold value for the current to detect events was 30% of the open pore current value.

[0088] Rolling Circle Amplification Assay on Streptavidin Beads Phi29 polymerase was purchased from Enzymatics (P7020-HC-L); restriction enzymes Alul (R0137S), Rsal (R0167S), Exol (M0293S), ExoIII (M0206S), and dNTPs (N0447L) were purchased from New England Biolabs (NEB); CircLigase I was purchased from Lucigen (CL411 IK); and all oligonucleotides were purchased from Integrated DNA Technologies (IDT). The linear template ligation conditions were 500 nM single-stranded linear DNA, 50 mM MOPS, 10 mM KC1, 5 mM MgC12, 2.5 mM MnCh, 50 pM ATP, 1 mM DTT, 5 U/pL CircLigase I, pH 7.5, 60 °C for 10 h ,and 80 °C for 10 min for inactivation. Later, the circular templates were mixed with primer at a 1 : 1 ratio and incubated at 37 °C for 15 min.

[0089] A suspension of streptavidin magnetic beads (Dynabeads M-270 Streptavidin, Invitrogen) was transferred to an autoclaved PCR tube and washed with 2* Binding& Washing buffer (2 M NaCl, 1 mM EDTA,10 mM Tris-HCl, pH 7.5) 3 times. The biotinylated primer-template hybrid was bound at the condition of 50 pL of the final concentration of 1 pg/pL streptavidin magnetic beads, 1 M NaCl, 0.5 mM EDTA,5 mM Tris- HCl, pH 7.5, 25 °C for 30 min with gently shaking with tube rotator (Roto-Therm). After DNA binding, beads were washed 3 times with 50 pL of 1 x Binding& Washing buffer, moved to a new autoclaved PCR tube, and washed 2 times with 50 pL of 1 x phi29 reaction buffer (100 mM (NH^SC , 100 mM MgCh, 500 mM Tris-HCl, 40 mM DTT, pH 7.5). The reaction volume is 50 pL. Generally, the reaction was carried out in 1 mM dNTPs, 2 U/pL phi29 polymerase, 1 pg/pL 2.8 pm magnetic beads, 10 mM (NHThSCh, 10 mM MgC12, 50 mM Tris-HCl, 4 mM DTT, 0.05% Tween-20, pH 7.5 with gentle mixing at 37 °C for 1 h unless indicated otherwise. The reaction was stopped by the addition of EDTA to a final concentration of 50 mM.

[0090] For characterizing RCA reaction yield, the samples were washed 5 times with Binding& Washing buffer and incubated at 90 °C for 30 min in 50 uL of 95% formamide, 10 mM EDTA, pH 8.2. After the sample was cooled down, Qubit ssDNA dye (Invitrogen Q10212) was used and later quantified by Qubit Fluorometer. 0.3% agarose gel was run at 50 V at 4 °C for 1 h and later stained by Gelred.

[0091] For producing reporter molecules, the samples were washed 5 times with 1 x NEB4 buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT, pH 7.9). The restriction enzyme cleavage experiments were performed in 50 pL reaction volume, 1 ^x NEB4 buffer, 20 U restriction enzyme, with gentle mixing at 37 °C for 1 h. In situ cleave and amplification (INCA) was performed in 1 mM dNTPs, 2 U/pL phi29 polymerase, 0.4 U/pL restriction enzyme, 1 pg/pL 2.8 pm magnetic beads, 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM magnesium 490 acetate, 4 mM DTT, 0.05% Tween-20, pH 7.9, and 50 pL reaction volume. After enzyme digestion, magnetic beads were separated, and the 50 pL of supernatants were mixed with 150 pL of 1.33 M GdmCl 1.33 M KC1, 66.5 mM Tris pH 7.6 so that final GdmCl and KC1 concentrations are 1 M. The reporter molecules were characterized by 20% native PAGE which are run at 160 V for 2 h, stained with Gelred, and visualized with a Biorad PharosFX imaging system.

[0092] For the streptavidin plates experiment, the same RCA reaction conditions were used except the reaction volume is 100 pL and the biotinylated primer binding is 1 h instead of 30 min. The plate was purchased from Thermofisher (Nunc immobilizer streptavidin).

[0093] Capture Antibody Conjugation to Beads for HE4 Assay

[0094] For conjugation, 4.2 x 10⁸ paramagnetic carboxylated beads (Homebrew Singleplex beads, Quanterix) were washed three times with 300 pL of Bead Wash Buffer (Quanterix) and two times with 300 pL of cold Bead Conjugation Buffer (Quanterix) before resuspending in 291 pL of cold Bead Conjugation Buffer. The carboxyl groups on the beads were activated by adding 9 pL of freshly dissolved l-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC) and shaken at 4 °C for 30 min. The beads were then washed once with 300 pL of cold Bead Conjugation Buffer and resuspended in 300 pL of 0.167 mg/mL capture antibody (MAB62741, R&D Systems) in cold Bead Conjugation Buffer. Antibody conjugation was carried out by shaking the beads at 4 °C for 2 h. The beads were then washed twice with 300 pL of Bead Wash Buffer before resuspending in 300 pL of Bead Blocking Buffer (Quanterix) and shaking at room temperature for 30 min. After blocking, the beads were washed with 300 pL of Bead Wash Buffer and 300 pL of Bead Diluent (Quanterix) and resuspended in Bead Diluent for storage at 4 °C. The beads were counted with a Beckman Coulter Z1 Particle Counter.

[0095] Detector Antibody Conjugation

[0096] The primer-template hybrid for conjugation to detector antibody was prepared by first annealing a 5' azide-modified primer (36.1 pM) and a linear methylated Alul D6 template (37.9 pM) in NEBNext Quick Ligation Buffer (New England Biolabs). The mixture was heated at 95 °C for 2 min and allowed to slowly cool to room temperature over 1.5 h. The template was then ligated by adding T4 DNA ligase and incubating at room temperature for 3 h. After ligation, a 7K MWCO Zeba spin desalting column (Thermo Fisher Scientific) was used to buffer exchange the primer-template pair into phosphate buffered saline (PBS) with 1 mM EDTA. The detector antibody (AF6274, R&D Systems) was reconstituted to 1 mg/mL in PBS and incubated with a 20-fold molar excess of dibenzocyclooctyne-PEG4-N- hydroxysuccinimidyl ester (DBCO-PEG4-NHS, MilliporeSigma) at room temperature for 30 min before purification with a 10K Amicon Ultra-0.5 mL centrifugal filter in PBS with 1 mM EDTA. The DBCO-modified detector antibody was then mixed with a 2-fold molar excess of the ligated primer-template and incubated at 4 °C overnight. The antibody-DNA conjugate was aliquoted and stored at -80 °C in PBS with 5 mM EDTA, 0.1% BSA, and 0.02% sodium azide.

[0097] HE4 INCA Assay

[0098] Immunoassays were performed in 96-well plates (Greiner Bio-One, 655096). For each sample to be measured, 1 x 106 beads diluted in 10 pL of Homebrew Sample Diluent (Quanterix) were added to 100 pL of sample diluted in Homebrew Sample Diluent. The plate was sealed and shaken at room temperature for 1 h before washing three times with System Wash Buffer 1 (Quanterix) using a BioTek 405 TS Microplate Washer. The beads were then resuspended in 100 pL of detector antibody-DNA conjugate (0.6 pg/mL diluted in Homebrew Sample Diluent) and shaken at room temperature for 15 min. After washing 10 times with System Wash Buffer 1, the beads were transferred to a new 96-well plate and resuspended in 60 pL of RCA reaction mix consisting of 1 U/pL phi29, 0.2 U/pL Alul, 1 mM dNTP, 0.05% Tween-20, and l x NEBuffer 4. The plate was shaken at 37 °C for 1.5 h, and the RCA reaction was quenched by adding 6 pL of 500 mM EDTA before nanopore measurements of the supernatant.

[0099] Template Ligation, optimization of rolling circle amplification, and PAGE characterization of reporter molecules

[00100] Table 1 Sequences of primers and templates used in this study [00101] Optimization nanopore characterization of reporter molecules

[00102] We optimized the RCA reaction performance as a function of dNTP concentration, phi29 activity, surface (magnetic beads versus ELISA plates), salt type and concentration, magnetic bead concentration, and RCA reaction time. Circular template Alul design 6 (Alul D6) was used in all optimization experiments. After the reaction was completed, we used formamide to disrupt streptavidin-biotin binding to release immobilized nucleic acids from the surface as well as denature the secondary structure of the RCA product. The total mass concentration of the RCA product was quantified using Qubit single strand staining dye, and the RCA product length was visualized using 0.3% agarose gel electrophoresis.

[00103] As shown in FIG. 8 Panel A, the RCA product mass concentration increased and then decreased with dNTP concentration. At low concentration of dNTP, both RCA product mass and RCA product length, as shown in the agarose gel in the inset, are lower. This could be due to the long waiting time for the next dNTP to diffuse to the phi29 polymerase amplification site. On the other hand, at high dNTP concentration, dNTP will chelate magnesium (Mg²⁺) which then influences polymerase activity and results in a low RCA product mass. When increasing phi29 polymerase activity, we observed an increase and then decrease in RCA product mass (FIG. 8 Panel B) (43). The increase in product mass at low phi29 polymerase activity could be attributed to increasing amounts of enzyme involved in the reaction, while the decrease at high phi29 polymerase activity might be due to increased glycerol concentration (co-solvent in the phi29 stock solution).

[00104] To quantify the RCA product mass and length on surfaces, the primer and template conjugate were immobilized on streptavidin-coated surfaces, and 95% formamide, lOmM EDTA, pH 8.2, 90 °C 30 min incubation was performed to break the streptavidinbiotin bound after the reaction was finished. Generally, plates have a lower surface-to-volume ratio and phi29 polymerases need to diffuse and bind to primer-template hybrids which are located at the bottom of the ELISA well. Both factors lead to a lower binding of phi29 polymerases to primer-template hybrids. Thus, comparing the two different surfaces, magnetic beads showed higher RCA product mass when the initial circular template concentration was the same (FIG. 8 Panel C), however, the average RCA product length is shorter than in the bead reaction (FIG. 8 Panel C inset). [00105] Salt is of importance for bioassays. We examined eight different salts, and our result shows that 10 mM NH3SO4 provides the highest RCA rate and LiCl is the lowest (FIG. 8 Panel D). When comparing LiCl, NaCl, and KC1, which are salts with different cations and the same anion, we observe that K⁺ gives the highest enzyme performance. Compared to KC1, K2SO4, and KCH3CO2, SO₄ ²' and CFbCCh’ all perform better , in accordance with the Hofmeister series of ion-specific effects on proteins and their biological functions (44, 45). Based on the Hofmeister series, the order of cations being hydrated in the buffer is K⁺ > Na⁺ > Lit Strongly hydrated ions tend to have higher surface tension, lower hydrocarbon solubility, and stabilize native protein structure (44). Compared with SCh²' and acetate', Cl' is poorly hydrated and tends to facilitate protein unfolding and denaturation (45). An interesting observation is that the difference in salt type and concentration does not affect RCA product length (FIG. 8 Panel D inset). The RCA product mass shows an increase and then decrease as a function of beads concentration (FIG. 8 Panel E), which might be caused by the absorption of phi29 polymerase and DNA oligos to the surface of the beads. And finally, the RCA product mass and length show an increase as a function of time as expected (FIG. 8 Panel F) (42).

[00106] The circular templates include a DNA hairpin structure that requires unfolding by phi29 polymerase during RCA. Thus, the sequence and the length of DNA hairpin structure can potentially influence the speed of phi29 polymerase. To examine this hypothesis, and to optimize our final DNA reporter molecule yield, we design several circular templates with the same primer sequence. The first set of circular templates is cleaved by the Alul restriction enzyme (FIG. 8 Panel G). The primer binding sequence is underlines, and the difference in sequences is marked in red. Considering the steric hindrance, we inserted several poly adenosines (poly-A) between the primer binding sequence and the DNA hairpin secondary structure. While the remaining lengths of DNA hairpins are the same 13 bps, Alul design 5 (Alul D5) has poly-A2, Alul design 6 (Alul D6) and Alul design 7 (Alul D7) have poly-A6 between the primer binding sequence and the DNA hairpins. The first base pair that phi29 polymerase needs to unfold in DNA hairpin structures is the AT pair for Alul D6, and the GC pair for Alul D5 and Alul D7. The results show that Alul D6 has both the highest RCA product mass and length followed by Alul D7 and Alul D5. Thus, we reason that the overall steric hindrance, the kinetics of phi29 unfolding first base pair, and the overall GC content will influence the efficiency of amplification. The second set of circular templates os cleaved by restriction enzyme Rsal (FIG. 8 Panel H). For these two Rsal cleavable templates, the length of DNA hairpin secondary structures is 12 bp for Rsal DI and 13 bp for Rsal D2. Poly-A6 has been put in between the primer binding sequence and the DNA hairpin secondary structure, and the GC content of the DNA hairpin stem is almost the same, with Rsal D2 slightly lower. We did not observe a strong RCA product mass and length difference between these two templates. Overall, this result implies that by fine-tuning the steric hindrance, hairpin stem length, and GC content, phi29 performance can be optimized.

[00107] Further Example Method and Device Embodiments

[00108] FIG. 23 is a schematic block diagram illustrating components of a device 2300 for detecting presence of a biomarker in a sample 2301 using a nanopore 2310, in accordance with devices, methods and other features of embodiments taught herein.

[00109] The device 2300 includes an amplification and restriction module 2305 configured to (i) capture the biomarker using an immobilized antibody specific to the biomarker; (ii) perform an isothermal nucleic acid amplification to produce an amplified product of the biomarker, the amplified product comprising a nucleic acid sequence; and (iii) fragment the amplified product using a restriction enzyme specific to the nucleic acid sequence of the amplified product to thereby release multiple sequence fragment molecules to be detected by a nanopore. For example, the amplification and restriction module 2305 can include components taught herein such as immobilized antibodies specific to the biomarker (for example, a sandwich immunocomplex for the biomarker that includes an ELISA assay for the biomarker), polymerases configured to perform an isothermal nucleic acid amplification (such as an isothermal rolling circle amplification), restriction enzymes, and circular DNA templates taught herein, such as circular DNA templates that include a DNA hairpin structure including one or more restriction enzyme recognition sites that allows isothermal enzymatic digestion to permit fragmenting the amplified product using the restriction enzyme.

[00110] In other examples, the amplification and restriction module 2305 can be configured to perform in situ cleavage and amplification (INCA) of the circular DNA templates, wherein the circular DNA templates comprise a site that is not amenable to restriction digestion, such as a methylated site, thereby enabling simultaneous rolling circle amplification (RCA) and digestion of the amplified product. The amplification and restriction module 2305 can be configured to introduce a highly processive polymerase (for example, phi29 polymerase) to initiate rolling circle amplification (RCA) of the circular DNA templates. The amplification and restriction module 2305 can be configured to initiate rolling circle amplification (RCA) of the circular DNA templates to produce single stranded Deoxyribonucleic Acid (ssDNA) comprising repetitive complimentary circular DNA template sequences, the ssDNA being cleaved by the restriction enzyme to produce the multiple sequence fragment molecules, in accordance with techniques taught herein. The biomarker can include at least one of an amino acid, a protein, a messenger Ribonucleic Acid (mRNA), a Deoxyribonucleic Acid (DNA), and a single nucleotide polymorphism (SNP). The amplification and restriction module 2305 can further include a solution, such as guanidinium chloride, configured to compromise the base-pairing stability of the multiple sequence fragment molecules being passed through the nanopore 2310.

[00111] Further, the device 2300 includes the nanopore 2310, and a voltage source 2315 configured to apply a voltage to a solution containing the multiple sequence fragment molecules to thereby induce the multiple sequence fragment molecules to pass through the nanopore 2310. For example, the nanopore 2310 can be between about 10 nm and about 100 nm in diameter, such as about 50 nm in diameter, and can, for example, be fabricated by techniques taught herein. The nanopore 2310 can comprise a biological nanopore, such as an a-hemolysin nanopore. The device can include a lipid bilayer, with the biological nanopore being inserted into the lipid bilayer; and may further comprise a silicon-based substrate, with the biological nanopore being inserted into an aperture in the silicon-based substrate. The voltage source 2315 can, for example, apply a voltage such as between about 100 mV and about 350 mV, more particularly between about 250 mV and about 300 mV, to a solution containing the multiple sequence fragment molecules (such as fragment molecules generated by restriction enzyme cleavage taught herein) to thereby induce those fragments to pass through the nanopore as taught herein.

[00112] The device 2300 also includes a computer processor module 2320 that is configured to, in an automated fashion, (i) generate a nanopore blockade fingerprint signal for the multiple sequence fragment molecules passing through the nanopore, the generating being based on electrical signals generated from the multiple sequence fragment molecules passing through the nanopore; and (ii) detect the presence of the biomarker in the sample based on determining a unique correspondence of the nanopore blockade fingerprint signal with the presence of the biomarker. For example, the computer processor module 2320 can be configured to generate the nanopore blockade fingerprint signal based on at least one of a nanopore dwell time, a nanopore fractional current blockade rate, and a nanopore blockade interevent time for the multiple sequence fragment molecules passing through the nanopore. As another example, the nanopore blockade fingerprint signal can be generated by determining a cluster signal pattern in the nanopore dwell time and the nanopore fractional current blockade rate for the multiple sequence fragment molecules passing through the nanopore. With reference to FIG. 3, for example, the “nanopore blockade fingerprint signal” can include data generated by a computer processor within module 2320 that indicates that module 2320 has identified, in an automated fashion, a “cluster” or other data pattern in the electrical signals collected as the multiple fragment molecules pass through the nanopore 2310. For example, in FIG. 3, Panel G, clusters 1, 2, and 3 are identified as clusters in the data for nanopore dwell time and fractional current blockade rate, shown here as a two- dimension graph of those two parameters. Such clusters can be recognized automatically by processor 2320 using scatter plots of those parameters that are clustered by a Bayesian Gaussian mixture model as taught herein, or by any other suitable pattern recognition technique. The computer processor module 2320 can be further configured to quantify the abundance of the biomarker in the sample, such as based on a nanopore blockade interevent time for the multiple sequence fragment molecules passing through the nanopore. For example, the interevent time can be directly linked to the concentration of reporter molecules, which is directly associated with the original concentration of the biomarker, using pre- established standards for those concentrations as being correlated with the interevent time. For example, as shown in connection with FIG. 4, capture rates can be obtained by fitting exponentional curves to the distribution of interevent times, and then a log-log plot can be used to determine a curve of concentration versus capture rate, all being performed in a fashion that can be automated to be performed by a computer processor module 2320 based on recorded interevent times from measured electrical signals. It will be appreciated that suitable electrical measurement components 2325 can be included in device 2300 in connection with nanopore 2310 for purposes of supplying electrical signals to be used by computer processor 2320, in accordance with embodiments taught herein. The computer processor module 2320 can output an appropriate signal (such as data displayed to a user) indicated detection of the biomarker, and can also additionally output such data for quantification of the biomarker, such as an indication of a concentration of the biomarker in the sample.

[00113] Example Features of Embodiments

[00114] Among other features discussed herein, some examples of features of embodiments include: [00115] - Fast enzymatic steps (~1 hour)

[00116] - Compatible with multiplexing to detect multiple biomarkers

[00117] - Compatible with existing instrumentation for deployment

[00118] Example Advantages of Embodiments

[00119] Among other advantages, some examples of advantages of embodiments include: [00120] - Purely electrical detection (most other methods employ light, which is more cumbersome)

[00121] - pM level detection is not easy using nanopores (sensitive)

[00122] - Ability to detect multiple agents simultaneously is useful for monitoring disease

[00123] - Electrical counters using nanopores are less expensive than optical sensors.

[00124] - Embodiments of the technology are sensitive and rapid.

[00125] Example Uses of Embodiments

[00126] Among other uses, some examples of uses of embodiments include:

[00127] - Cancer biomarker detection

[00128] - Disease biomarker detection

[00129] - PCR-free virus detection

[00130] - Rapid biomarker quantification (clinic, field).

[00131] - Virus detection (for example, using ELISA-type assays)

[00132] Definitions

[00133] The terms “a” or “an” as used herein in the specification may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more.

[00134] The term “comprise,” or variations such as “comprises” or “comprising,” as used herein may be used to imply the inclusion of a stated element or integer or group of elements or integers, but not the exclusion of any other element or integer or group of elements or integers.

[00135] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[00136] Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[00137] The term “amplification” as used herein, encompasses amplification of RNA and DNA molecules. Amplification of RNA and DNA could be achieved by reverse transcription using an RNA or DNA polymerase. “Isothermal amplification” refers to enzyme-based methods that can be performed to amplify nucleic acids including DNA, at a single temperature.

[00138] The term “antibody” is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments as long as they exhibit the desired biological property. The antibody may be derived from any species and may be IgM, IgG (e.g. IgGl, IgG2, IgG3, or IgG4), IgD, IgA, or IgE, for example. The desired biological activity is binding to a biomarker (e.g. HE4 antigen).

[00139] The term “biological sample” encompasses a variety of sample types obtained from an organism and can be used in a diagnostic or monitoring assay. The term encompasses blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The term encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components. The term encompasses a clinical sample, and also includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. The term “biological sample” is meant to distinguish between a sample in a clinical setting from a sample that may be a recombinant sample or derived from a recombinant sample.

[00140] As used herein, the term “biomarker” refers to a molecule that is associated either quantitatively or qualitatively with a biological change. Examples of biomarkers include polypeptides, proteins or fragments of a polypeptide or protein; and polynucleotides, such as a gene product, RNA or RNA fragment; and other body metabolites. In certain embodiments, a “biomarker” means a compound that is differentially present (i.e., increased or decreased) in a biological sample from a subject or a group of subjects having a first phenotype (e.g., having a disease or condition) as compared to a biological sample from a subject or group of subjects having a second phenotype (e.g., not having the disease or condition or having a less severe version of the disease or condition). A biomarker may be differentially present at any level, but is generally present at a level that is increased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100%, by at least 110%, by at least 120%, by at least 130%, by at least 140%, by at least 150%, or more; or is generally present at a level that is decreased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by 100% (i.e., absent). A biomarker is preferably differentially present at a level that is statistically significant (e.g., a p-value less than 0.05 and/or a q-value of less than 0.10 as determined using, for example, either Welch's T-test or Wilcoxon's rank-sum Test).

[00141] As used herein, a “computer processor” refers to a hardware component capable of executing software instructions to perform computational tasks related to the operation and analysis of data from the methods and devices disclosed in the patent application. In some embodiment, a computer processor may include one or more processing units capable of executing software instructions, as well as associated memory, input/output interfaces, and other components necessary for its operation. In some embodiments, a computer processor may be configured to execute algorithms, data analysis routines, signal processing operations, or control commands related to the operation of the nanopore-based sensing system and the analysis of biomarker data obtained from the system. [00142] As used herein, “current blockade” refers to a phenomenon wherein the flow of electric current through a nanopore is obstructed or modulated due to the presence of particles, molecules or biomarker fragments passing through the nanopore.

[00143] As used herein, “dwell time” refers to the duration that a particle, molecule or biomarker fragment spends within the nanopore during a sensing or measurement process. [00144] As used herein, the term “enzyme” is intended to mean a molecule that catalytically modifies another molecule. Enzymes can include proteins, as well as certain other types of molecules such as polynucleotides. Examples of enzymes that also are proteins include polymerases, exonucleases and helicases. As used herein, a “polymerase” is intended to mean an enzyme having an active site that assembles polynucleotides by polymerizing nucleotides into polynucleotides. A polymerase can bind a primed single stranded polynucleotide template, and can sequentially add nucleotides to the growing primer to form a polynucleotide having a sequence that is complementary to that of the template. In some embodiments, a polymerase is Phi29 polymerase.

[00145] As used herein, a “fragment” encompasses a portion of a larger molecule that has been enzymatically cleaved or fragmented into smaller molecular units which can be detected and quantified using techniques described herein.

[00146] As used herein, an “immunocomplex” refers to a molecular complex formed by the specific binding of an antibody to its target antigen. In the context of present disclosure, immunocomplex formation occurs when an antibody, which is typically immobilized on a solid support such as a magnetic bead or microplate well, binds specifically to its target antigen present in a biological sample. This binding event results in the formation of the immunocomplex, where the antibody and antigen are linked together. In subsequent steps of the assay, additional reagents may be introduced to the system to facilitate enzymatic amplification and fragmentation, leading to enhanced sensitivity and specificity in biomarker detection.

[00147] As used herein, “immobilized” refers to the state of being securely attached or fixed to a solid surface or support material. Specifically, in methods involving enzyme-linked immunosorbent assay (ELISA) techniques, biomolecules such as antibodies or antigens may be immobilized onto a solid support, such as a microplate well or a nanopore surface. This immobilization allows for the selective capture and retention of target molecules, enhancing the sensitivity and specificity of the assay. In some embodiments, antibodies or antigens can be covalently attached to a solid support through chemical reactions, such as amine coupling or thiol chemistry. In some embodiments, antibodies or antigens can be immobilized onto a solid support through physical adsorption, where non-covalent interactions, such as electrostatic forces, hydrophobic interactions, or van der Waals forces, hold the antibody molecules onto the surface of the support material. In some embodiments, antibodies or antigens can be immobilized onto a solid support using streptavidin/biotin interactions. For instance, if the antibody contains a biotin tag, it can be captured by streptavidin immobilized on the solid support. In some embodiments, antibodies or antigens can be immobilized onto a solid support using click chemistry reactions, such as azide-alkyne cycloaddition or thiol-ene reactions. In this approach, functional groups introduced onto the antibody or the solid support react specifically with complementary functional groups, forming stable covalent bonds.

[00148] As used herein, a “lipid bilayer” refers to a two-layered structure composed of lipid molecules arranged with their hydrophobic tails facing inward and their hydrophilic heads facing outward, thereby forming a barrier between two aqueous compartments. In some embodiments, biological nanopores may be inserted into lipid bilayers to create artificial membrane systems.

[00149] As used herein, the term "nucleic acid" refers to the genetic material that undergoes amplification through described amplification techniques, ultimately producing an amplified product comprising a nucleic acid sequence corresponding to the biomarker of interest. In some embodiments, nucleic acid may comprise DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). IN some embodiments, DNA is ssDNA (single stranded deoxyribonucleic acid). In some embodiments, RNA is mRNA (messenger ribonucleic acid). In some embodiments, nucleic acid molecule is circular.

[00150] As used herein, the term “pore” is intended to mean a structure that includes an aperture that permits molecules to cross there through from a first side of the pore to a second side of the pore. That is, the aperture extends through the first and second sides of the pore. Molecules that can cross through an aperture of a pore can include, for example, ions or water-soluble molecules such as nucleic acids, proteins, nucleotides, and amino acids. The pore can be disposed within a barrier. When at least a portion of the aperture of a pore has a width of 100 nm or less, e.g., 10 nm or less, or 2 nm or less, the pore can be, but need not necessarily be, referred to as a “nanopore.”

[00151] The terms “polypeptide”, “peptide”, and “protein”, as used herein, refer to a molecule composed of amino acids linked by a peptide bond. [00152] The term “restriction enzyme” refers to a type of enzyme that recognizes specific DNA sequences and cleaves the DNA at or near these recognition sites. Each restriction enzyme typically recognizes a specific DNA sequence, known as its recognition sequence or restriction site, and cleaves the DNA at or near this site, resulting in fragments of DNA with defined ends. In some embodiments, a restriction enzyme is Alul. In some embodiments, a restriction enzyme is Rsal. “Restriction digestion” refers to digestion of a DNA samples through using restriction enzymes.

[00153] The terms “sample”, “patient sample”, “biological sample” and the like, encompass a variety of sample types obtained from a patient, individual, or subject and can be used in a diagnostic or monitoring assay. The patient sample may be obtained from a healthy subject, a diseased patient or a patient having associated symptoms of brain injury. Moreover, a sample obtained from a patient can be divided and only a portion may be used for diagnosis. Further, the sample, or a portion thereof, can be stored under conditions to maintain sample for later analysis. The definition specifically encompasses blood and other liquid samples of biological origin (including, but not limited to, peripheral blood, serum, plasma, cord blood, amniotic fluid, cerebrospinal fluid, urine, saliva, stool and synovial fluid), solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. In certain embodiment, a sample comprises cerebrospinal fluid. In a specific embodiment, a sample comprises a blood sample. In another embodiment, a sample comprises a plasma sample. In yet another embodiment, a serum sample is used. The definition of “sample” also includes samples that have been manipulated in any way after their procurement, such as by centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washed, or enriched for certain cell populations. The terms further encompass a clinical sample, and also include cells in culture, cell supernatants, tissue samples, organs, and the like. Samples may also comprise fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks, such as blocks prepared from clinical or pathological biopsies, prepared for pathological analysis or study by immunohi stochemi stry .

[00154] The term “specifically binding” refers to binding between two molecules, for example, an antigen and an antibody, characterized by the ability of a molecule (antigen) to associate with another specific molecule (antibody) even in the presence of many other diverse molecules, i.e., to show preferential binding of one molecule for another in a heterogeneous mixture of molecules. The binding which occurs between such paired species may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions. When the interaction of the two species produces a non-covalently bound complex, the binding which occurs is typically electrostatic, hydrogen-bonding, or the result of lipophilic interactions. Accordingly, “specific binding” occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody/antigen or enzyme/ substrate interaction. In particular, the specific binding is characterized by the binding of one member of a pair to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs. Thus, for example, an antibody typically binds to a single epitope and to no other epitope within the family of proteins. In some embodiments, specific binding between an antigen and an antibody will have a binding affinity of at least 10 ⁶ M. In other embodiments, the antigen and antibody will bind with affinities of at least 10 ⁷ M, 10 ⁸ M to 10 ⁹ M, 10 ¹⁰ M, 10 ^{1 1} M, or 10 ¹² M. As used herein, the terms “specific binding” or “specifically binding” when used in reference to the interaction of an antibody and a protein or peptide means that the interaction is dependent upon the presence of a particular structure (i.e., the epitope) on the protein.

[00155] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.

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[00203] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[00204] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

CLAIMS What is claimed is:

1. A method for detecting presence of a biomarker in a sample using a nanopore, the method comprising: capturing the biomarker using an immobilized antibody specific to the biomarker; performing an isothermal nucleic acid amplification to produce an amplified product of the biomarker, the amplified product comprising a nucleic acid sequence; fragmenting the amplified product using a restriction enzyme specific to the nucleic acid sequence of the amplified product to thereby release multiple sequence fragment molecules to be detected by a nanopore; applying a voltage to a solution containing the multiple sequence fragment molecules to thereby induce the multiple sequence fragment molecules to pass through the nanopore; generating a nanopore blockade fingerprint signal for the multiple sequence fragment molecules passing through the nanopore, the generating being automated using a computer processor based on electrical signals generated from the multiple sequence fragment molecules passing through the nanopore; and detecting the presence of the biomarker in the sample based on determining, in an automated fashion using a computer processor, a unique correspondence of the nanopore blockade fingerprint signal with the presence of the biomarker.

2. The method of Claim 1, wherein the performing the isothermal amplification to produce the amplified product of the biomarker comprises performing a rolling circle amplification (RCA).

3. The method of Claim 1, wherein capturing the biomarker using the immobilized antibody specific to the biomarker comprises forming a sandwich immunocomplex for the biomarker.

4. The method of Claim 3, wherein the capturing the biomarker using the immobilized antibody specific to the biomarker comprises performing an enzyme-linked immunosorbent assay (ELISA) for the biomarker.

5. The method of Claim 4, wherein the capturing the biomarker using the immobilized antibody specific to the biomarker further comprises hybridizing circular DNA templates with primers that are conjugated with secondary antibodies in an enzyme- linked immunosorbent assay (ELISA) structure for the biomarker.

6. The method of Claim 5, comprising performing in situ cleavage and amplification (INCA) of the circular DNA templates, wherein the circular DNA templates comprise a site that is not amenable to restriction digestion, thereby enabling simultaneous rolling circle amplification (RCA) and digestion of the amplified product.

7. The method of Claim 6, wherein the site that is not amenable to restriction digestion comprises a methylated site.

8. The method of Claim 5, wherein the circular DNA templates comprise a DNA hairpin structure, the DNA hairpin structure comprising a restriction enzyme recognition site that allows isothermal enzymatic digestion to permit fragmenting the amplified product using the restriction enzyme.

9. The method of Claim 8, comprising introducing a highly processive polymerase to initiate rolling circle amplification (RCA) of the circular DNA templates.

10. The method of Claim 8, comprising initiating rolling circle amplification (RCA) of the circular DNA templates to produce single stranded Deoxyribonucleic Acid (ssDNA) comprising repetitive complimentary circular DNA template sequences, the ssDNA being cleaved by the restriction enzyme to produce the multiple sequence fragment molecules.

11. The method of Claim 1, wherein generating the nanopore blockade fingerprint signal is based on at least one of a nanopore dwell time, a nanopore fractional current blockade rate, and a nanopore blockade interevent time for the multiple sequence fragment molecules passing through the nanopore.

12. The method of Claim 11, wherein generating the nanopore blockade fingerprint signal comprises determining a cluster signal pattern in the nanopore dwell time and the nanopore fractional current blockade rate for the multiple sequence fragment molecules passing through the nanopore.

13. The method of Claim 1, further comprising quantifying, in an automated fashion using a computer processor, the abundance of the biomarker in the sample.

14. The method of Claim 13, wherein the quantifying is based on a nanopore blockade interevent time for the multiple sequence fragment molecules passing through the nanopore.

15. The method of Claim 1, wherein the applying the voltage to the solution containing the multiple sequence fragment molecules to thereby induce the multiple sequence fragment molecules to pass through the nanopore comprises: inducing the multiple sequence fragment molecules to pass through a biological nanopore.

16. The method of Claim 15, wherein the biological nanopore comprises an a-hemolysin nanopore.

17. The method of Claim 15, wherein the biological nanopore is inserted into a lipid bilayer.

18. The method of Claim 15, wherein the biological nanopore is inserted into an aperture in a silicon-based substrate.

19. The method of Claim 15, wherein the nanopore comprises an aperture diameter of between about 10 pm and about 100 pm.

20. The method of Claim 1, wherein the biomarker comprises at least one of an amino acid, a protein, a messenger Ribonucleic Acid (mRNA), a Deoxyribonucleic Acid (DNA), and a single nucleotide polymorphism (SNP).

21. The method of Claim 1, further comprising: compromising the base-pairing stability of the multiple sequence fragment molecules being passed through the nanopore.

22. The method of Claim 21, wherein compromising the base-pairing stability comprises passing the multiple sequence fragment molecules through a solution of guanidinium chloride.

23. A device for detecting presence of a biomarker in a sample using a nanopore, the device comprising: an amplification and restriction module configured to (i) capture the biomarker using an immobilized antibody specific to the biomarker; (ii) perform an isothermal nucleic acid amplification to produce an amplified product of the biomarker, the amplified product comprising a nucleic acid sequence; (iii) fragment the amplified product using a restriction enzyme specific to the nucleic acid sequence of the amplified product to thereby release multiple sequence fragment molecules to be detected by a nanopore; the nanopore; a voltage source configured to apply a voltage to a solution containing the multiple sequence fragment molecules to thereby induce the multiple sequence fragment molecules to pass through the nanopore; and a computer processor module configured to, in an automated fashion, (i) generate a nanopore blockade fingerprint signal for the multiple sequence fragment molecules passing through the nanopore, the generating being based on electrical signals generated from the multiple sequence fragment molecules passing through the nanopore; and (ii) detect the presence of the biomarker in the sample based on determining a unique correspondence of the nanopore blockade fingerprint signal with the presence of the biomarker.

24. The device of Claim 23, wherein the amplification and restriction module is configured to perform the isothermal amplification to produce the amplified product of the biomarker by performing a rolling circle amplification (RCA).

25. The device of Claim 23, wherein the amplification and restriction module is configured to capture the biomarker using the immobilized antibody specific to the biomarker comprises by forming a sandwich immunocomplex for the biomarker.

26. The device of Claim 25, wherein the amplification and restriction module is configured to capture the biomarker using the immobilized antibody specific to the biomarker comprises by performing an enzyme-linked immunosorbent assay (ELISA) for the biomarker.

27. The device of Claim 26, wherein the amplification and restriction module is configured to capture the biomarker using the immobilized antibody specific to the biomarker by hybridizing circular DNA templates with primers that are conjugated with secondary antibodies in an enzyme-linked immunosorbent assay (ELISA) structure for the biomarker.

28. The device of Claim 27, wherein the amplification and restriction module is configured to perform in situ cleavage and amplification (INCA) of the circular DNA templates, wherein the circular DNA templates comprise a site that is not amenable to restriction digestion, thereby enabling simultaneous rolling circle amplification (RCA) and digestion of the amplified product.

29. The device of Claim 28, wherein the site that is not amenable to restriction digestion comprises a methylated site.

30. The device of Claim 27, wherein the circular DNA templates comprise a DNA hairpin structure, the DNA hairpin structure comprising a restriction enzyme recognition site that allows isothermal enzymatic digestion to permit fragmenting the amplified product using the restriction enzyme.

31. The device of Claim 30, wherein the amplification and restriction module is configured to introduce a highly processive polymerase to initiate rolling circle amplification (RCA) of the circular DNA templates.

32. The device of Claim 30, wherein the amplification and restriction module is configured to initiate rolling circle amplification (RCA) of the circular DNA templates to produce single stranded Deoxyribonucleic Acid (ssDNA) comprising repetitive complimentary circular DNA template sequences, the ssDNA being cleaved by the restriction enzyme to produce the multiple sequence fragment molecules.

33. The device of Claim 23, wherein the computer processor module is configured to generate the nanopore blockade fingerprint signal based on at least one of a nanopore dwell time, a nanopore fractional current blockade rate, and a nanopore blockade interevent time for the multiple sequence fragment molecules passing through the nanopore.

34. The device of Claim 33, wherein the computer processor module is configured to generate the nanopore blockade fingerprint signal by determining a cluster signal pattern in the nanopore dwell time and the nanopore fractional current blockade rate for the multiple sequence fragment molecules passing through the nanopore.

35. The device of Claim 23, wherein the computer processor module is further configured to quantify the abundance of the biomarker in the sample.

36. The device of Claim 35, wherein the computer processor module is configured to quantify the abundance of the biomarker in the sample based on a nanopore blockade interevent time for the multiple sequence fragment molecules passing through the nanopore.

37. The device of Claim 23, wherein the nanopore comprises a biological nanopore.

38. The device of Claim 37, wherein the biological nanopore comprises an a-hemolysin nanopore.

39. The device of Claim 37, further comprising a lipid bilayer, the biological nanopore being inserted into the lipid bilayer.

40. The device of Claim 37, further comprising a silicon-based substrate, the biological nanopore being inserted into an aperture in the silicon-based substrate.

41. The device of Claim 23, wherein the nanopore comprises an aperture diameter of between about 10 pm and about 100 pm.

42. The device of Claim 23, wherein the biomarker comprises at least one of an amino acid, a protein, a messenger Ribonucleic Acid (mRNA), a Deoxyribonucleic Acid (DNA), and a single nucleotide polymorphism (SNP).

43. The device of Claim 23, wherein the amplification and restriction module further comprises a solution configured to compromise the base-pairing stability of the multiple sequence fragment molecules being passed through the nanopore.

44. The device of Claim 43, wherein the solution configured to compromise the basepairing stability comprises guanidinium chloride.

45. A immunoassay for detecting presence of a biomarker in a sample using a nanopore, the immunoassay comprising: an immobilized antibody specific to the biomarker, the immobilized antibody configured to capture the biomarker; a polymerase configured to perform an isothermal nucleic acid amplification to produce an amplified product of the biomarker, the amplified product comprising a nucleic acid sequence; and a restriction enzyme, specific to the nucleic acid sequence of the amplified product, configured to fragment the amplified product to thereby release multiple sequence fragment molecules to be detected by a nanopore.

46. The immunoassay of Claim 45, wherein the polymerase is configured to perform a rolling circle amplification (RCA).

47. The immunoassay of Claim 45, wherein the immobilized antibody specific to the biomarker comprises at least part of a sandwich immunocomplex for the biomarker.

48. The immunoassay of Claim 47, wherein the sandwich immunocomplex for the biomarker comprises an enzyme-linked immunosorbent assay (ELISA) for the biomarker.

49. The immunoassay of Claim 48, further comprising circular DNA templates configured to be hybridized with primers that are conjugated with secondary antibodies in an enzyme-linked immunosorbent assay (ELISA) structure for the biomarker.

50. The immunoassay of Claim 49, wherein the circular DNA templates comprise a site that is not amenable to restriction digestion, thereby enabling simultaneous rolling circle amplification (RCA) and digestion of the amplified product.

51. The immunoassay of Claim 50, wherein the site that is not amenable to restriction digestion comprises a methylated site.

52. The immunoassay of Claim 49, wherein the circular DNA templates comprise a DNA hairpin structure, the DNA hairpin structure comprising a restriction enzyme recognition site that allows isothermal enzymatic digestion to permit fragmenting the amplified product using the restriction enzyme.

53. The immunoassay of Claim 52, comprising a highly processive polymerase to initiate rolling circle amplification (RCA) of the circular DNA templates.

54. The immunoassay of Claim 53, wherein the highly processive polymerase is configured to initiate the rolling circle amplification (RCA) of the circular DNA templates to produce single stranded Deoxyribonucleic Acid (ssDNA) comprising repetitive complimentary circular DNA template sequences, the ssDNA being cleaved by the restriction enzyme to produce the multiple sequence fragment molecules.

55. The immunoassay of Claim 45, wherein the biomarker comprises at least one of an amino acid, a protein, a messenger Ribonucleic Acid (mRNA), a Deoxyribonucleic Acid (DNA), and a single nucleotide polymorphism (SNP).

56. The immunoassay of Claim 45, further comprising a solution configured to compromise the base-pairing stability of the multiple sequence fragment molecules being passed through the nanopore.

57. The immunoassay of Claim 56, wherein the solution configured to compromise the base-pairing stability comprises a solution of guanidinium chloride.

58. A method of identifying a species from a sample, the method comprising: capturing a species-loaded complex, the species-loaded complex comprising:

(i) the species, isolated from the sample, (ii) a carrier particle, (iii) a first speciesspecific binder, binding the species to the carrier particle (iv) a second speciesspecific binder bound to the species, (v) a DNA primer, bound to the second speciesspecific binder, and (vi) a circular DNA template comprising a restriction enzyme recognition site, bound to the DNA primer; amplifying the circular DNA template while the species-loaded complex is captured to generate a long DNA strand; fragmenting the generated DNA strand to form a mixture of smaller DNA molecules; applying a voltage to the mixture, the voltage being sufficient to translocate the DNA molecules through a nanopore; and detecting an electrical current change produced as a result of the translocation of the DNA molecules through the nanopore, thereby permitting identification of the species.

59. The method of Claim 58, wherein detecting the electrical current change comprises detecting a temporary reduction in ionic current through the nanopore produced by transient occlusion of the nanopore by the DNA molecules during the translocation of the DNA molecules through the nanopore produced by the applying the voltage.

60. The method of Claim 58, wherein the carrier particle comprises a magnetic bead.

61. The method of Claim 58, wherein the carrier particle comprises a plate.

62. The method of claim 58, wherein the first species-specific binder and the second species-specific binder comprise a pair of antibodies, each of the antibodies binding to different epitopes of the same species.

63. The method of Claim 58, wherein the species comprises at least one of: a nucleic acid molecule, a protein, and a polypeptide.

64. The method of Claim 63, wherein the species comprises at least a portion of at least one of: a biomarker, a circulating tumor cell nucleic acid, and a circulating tumor protein.

65. The method of Claim 58, wherein the circular DNA template comprises at least one 5- methylcytosine site.

66. The method of Claim 58, wherein the sample is a blood sample.

67. The method of Claim 58, wherein the first species-specific binder is chemically immobilized on the carrier particle.

68. The method of claim 58, wherein the circular DNA template comprises a restriction enzyme recognition site.

69. The method of Claim 68, wherein the generated DNA strand is fragmented by restriction enzymes.

70. The method of Claim 69, wherein the restriction enzymes comprise Alul.

71. The method of Claim 69, wherein the restriction enzymes comprise Rsal.

72. The method of claim 58, wherein amplifying the circular DNA template is done by rolling circle amplification method.

73. The method of claim 58, wherein the diameter of the nanopore is about 50 pm.

74. The method of claim 58, wherein the DNA molecules in said DNA mixture are between about 4bp and about 50 bp in length.

75. The method of claim 58, wherein the DNA molecules in said DNA mixture are between about 4bp and about 10 bp in length.

76. The method of Claim 58, wherein the sample is a biological sample.

77. The method of Claim 58, wherein the sample contains said species at pM levels.

78. A species-loaded complex to permit identification and quantification of a species in a sample, the species-loaded complex comprising:

(i) the species, isolated from the sample,

(ii) a carrier particle,

(iii) a first species-specific binder, binding the species to the carrier particle,

(iv) a second species-specific binder bound to the species,

(v) a DNA primer, bound to the second species-specific binder,

(vi) a DNA strand comprising at least one copy of a circular DNA template comprising a restriction enzyme recognition site, bound to the DNA primer.

79. The species-loaded complex of Claim 78, wherein the circular DNA template comprises a restriction enzyme recognition site.

80. The species-loaded complex of Claim 78, wherein the circular DNA template comprises at least one 5-methylcytosine site.

81. The species-loaded complex of Claim 78, wherein the first species-specific binder and the second species-specific binder comprise a pair of antibodies, each of the antibodies binding to different epitopes of the same species.

82. The species-loaded complex of Claim 78, wherein the species comprises at least one of: a nucleic acid molecule, a protein, and a polypeptide.

83. The species-loaded complex of Claim 78, wherein the species comprises at least a portion of at least one of: a biomarker, a circulating tumor cell nucleic acid, and a circulating tumor protein.

84. The species-loaded complex of Claim 78, wherein the sample is a blood sample.