Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6816—Hybridisation assays characterised by the detection means
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/487—Physical analysis of biological material of liquid biological material
  • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
  • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores

Definitions

• Detection of nano-scale and micro-scale particles has immense clinical utility.
• Currently available methods include immunohistochemistry and nucleic acid-based detection, and cell proliferation is typically required before a sensitive detection can be carried out.
• Molecular detection and quantitation are also important, and can be carried out with various methods depending on the type of the molecule. For instance, a nucleotide sequence can be detected by virtue of its sequence complementarity to a probe or primer, through hybridization and/or amplification, or in fewer occasions, with a protein that recognizes the sequence. A protein, on the other hand, is commonly detected with an antibody that specifically recognizes and binds the protein.
• An enzyme-linked immuno sorbent assay (ELISA) is highly repetitive DNA sequence to be detected by virtue of its sequence complementarity to a probe or primer, through hybridization and/or amplification, or in fewer occasions, with a protein that recognizes the sequence.
• a protein on the other hand, is commonly detected with an antibody that specifically recognizes and binds the protein.
• An enzyme-linked immuno sorbent assay (ELISA) is highly
• Methods and systems for highly sensitive detection of molecules as well as particles, such as tumor cells and pathogenic organisms have broad applications, in particular, clinically, for pathogen detection and disease diagnosis, for instance. Additionally, such detection may: allow for the personalization of medical treatments and health programs; facilitate the search for effective pharmaceutical drug compounds and biotherapeutics; and enable clinicians to identify abnormal hormones, ions, proteins, or other molecules produced by a patient's body and/or identify the presence of poisons, illegal drugs, or other harmful chemicals ingested or injected into a patient.
• the present disclosure provides a method for assaying whether a target molecule or particle is present in a sample, the method comprising: (a) contacting the sample with (i) a fusion molecule comprising a ligand capable of binding to the target molecule or particle and a binding domain, and (ii) a polymer scaffold comprising at least one binding motif to which the binding domain of the fusion molecule is capable of binding, under conditions that allow the target molecule or particle to bind to the ligand and the binding domain to bind to the binding motif; (b) loading the polymer into a device comprising a pore that separates an interior space of the device into two volumes, and configuring the device to pass the polymer through the pore from one volume to the other volume, wherein the device comprises a sensor configured to identify objects passing through the pore; and (c) determining, with the sensor, whether the fusion molecule bound to the binding motif is bound to the target molecule or particle, thereby detecting the presence or absence of the target
• the target molecule is selected from the group consisting of a protein, a peptide, a nucleic acid, a chemical compound, a lipid, a receptor, an ion, and an element.
• the target particle is selected from the group consisting of protein complexes and protein aggregates, peptide aggregates, protein/nucleic acid complexes, fragmented or fully assembled viruses, bacteria, cells, and cellular aggregates.
• step (a) of the method for assaying whether a target molecule or particle is present in a sample is performed prior to step (b).
• step (b) is performed prior to step (a).
• the method further comprises applying a condition suspected to alter the binding between the target molecule or particle and the ligand, and carrying out the determination again.
• the condition is selected from the group consisting of removing the target molecule or particle from the sample, adding an agent that competes with the target molecule or particle or the ligand for binding, and changing the pH, salt concentration, or temperature.
• the binding motif comprises a chemical modification for binding to the binding domain.
• the chemical modification is selected from the group consisting of acetylation, methylation, summolation, glycosylation, phosphorylation, and oxidation.
• the polymer comprises a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a peptide nucleic acid (PNA), a DNA/RNA hybrid, or a polypeptide.
• the polymer is a synthetic scaffold.
• the binding domain is selected from the group consisting of a helix-turn-helix, a zinc finger, a leucine zipper, a winged helix, a winged helix turn helix, a helix-loop-helix and an HMG-box.
• the binding domain is selected from the group consisting of locked nucleic acids (LNAs), PNAs, transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPRs), peptides, dendrimers, and aptamers (DNA and/or protein).
• LNAs locked nucleic acids
• PNAs PNAs
• TALENs transcription activator-like effector nucleases
• CRISPRs clustered regularly interspaced short palindromic repeats
• peptides peptides
• dendrimers dendrimers
• aptamers DNA and/or protein
• the ligand is a protein.
• the ligand is selected from the group consisting of an antibody, an antibody fragment, an epitope, a hormone, a neurotransmitter, a cytokine, a growth factor, a cell recognition molecule, a nucleic acid, a peptide, and a receptor.
• the ligand is an aptamer (e.g., DNA, protein, or DNA/protein).
• the ligand is a small molecule compound.
• the binding domain and the ligand are linked via a covalent bond, a hydrogen bond, an ionic bond, a metallic bond, van der Walls force, a hydrophobic interaction, or a planar stacking interaction, or are translated as a continuous polypeptide, to form the fusion molecule.
• the method further comprises contacting the sample with a detectable label capable of binding to the target molecule, target particle or target/ligand complex.
• the polymer comprises at least two units of the binding motif.
• the polymer comprises at least two different binding motifs.
• the sample is in contact with at least two fusion molecules, each of which comprises a different binding domain capable of binding to a different one of the at least two different binding motifs, and a different ligand capable of binding to a different target molecule or particle; and the sensor is configured to identify whether the fusion molecule bound to each binding motif is bound to a target molecule or particle.
• the senor comprises electrodes further configured to apply a voltage across the two volumes.
• the device comprises an upper chamber, a middle chamber and a lower chamber, wherein the upper chamber is in communication with the middle chamber through a first pore, and the middle chamber is in communication with the lower chamber through a second pore.
• the first pore and second pore are about 1 nm to about 100 nm in diameter. Such pores can be suitable for detecting molecules such as proteins and nucleic acids. In one aspect, the first pore and second pore are as large as about 50,000 nm in diameter, which can be suitable for detecting larger particles such as tumor and bacterial cells.
• the pores are about 10 nm to about 1000 nm apart from each other. In some such aspects, the distance between the pores is sized such that the polymer scaffold may simultaneously extend through both the first and second pores. In other aspects, the pores are more than 1000 nm apart from each other.
• each of the chambers comprises an electrode for connecting to a power supply.
• the method further comprises moving the polymer in a reverse direction after the binding motif passes through at least one pore, such as to identify, again, whether the fusion molecule bound to each binding motif is bound to a target molecule or particle.
• kits, packages or mixtures that detect the presence of a target molecule or particle.
• the kit, package or mixture is comprised of (a) a fusion molecule, which itself is a ligand capable of binding to the target molecule or particle and a binding domain, (b) a polymer scaffold, which is comprised of at least one binding motif to which the binding domain is capable of binding, (c) a device, which is comprised of a pore that separates an interior space of the device into two volumes, wherein the device is configured to allow the polymer to pass through the pore from one volume to the other volume, and wherein the device is further comprised of a sensor configured to identify whether the binding motif is (i) bound to the fusion molecule while the ligand is bound to the target molecule or particle, (ii) bound to the fusion molecule while the ligand is not bound to the target molecule or particle, or (iii) not bound to the fusion molecule.
• the device is further comprised
• the kit, package or mixture further comprises a sample suspected of containing the target molecule or particle.
• the sample further comprises a detectable label capable of binding to the target molecule, particle, ligand/target complex, or ligand/particle complex.
• FIG. 1 illustrates the detection of a target molecule or particle with one embodiment of the presently disclosed method.
• FIG. 2 provides the illustration of a more specific example, where a double- stranded DNA is used as the polymer scaffold, and a human immunodeficiency virus (HIV) envelope protein is used as the ligand. The combination is used to detect an anti-HIV antibody.
• HIV human immunodeficiency virus
• FIG. 3 shows representative and idealized current profiles of three example molecules, demonstrating that binding between a target molecule (or particle) and a fusion molecule can be detected when passing through a nanopore, since it has a different current profile, compared to that of the fusion molecule alone or the DNA alone.
• FIG. 3A shows current profiles consistent with higher salt concentrations (>0.4 M KCI, for example at 1 M KCI) in the experimental buffer and a positive applied voltage, generating a positive current flow through the pore.
• FIG. 3B shows current profiles consistent with lower salt concentrations ( ⁇ 0.4 M KCI, for example at 100 mM KCI) in the experimental buffer and again at a positive applied voltage.
• FIG. 3C shows current profiles consistent with lower salt concentrations ( ⁇ 0.4 M KCI, for example at 100 mM KCI) in the experimental buffer and a negative applied voltage.
• FIG. 4 illustrates the multiplexing capability of the present technology by including different binding motifs in the polymer scaffold. Such multiplexing can be accomplished with one nanopore or more than one nanopore.
• FIGs. 5(l)-(lll) illustrate a nanopore device with at least two pores separating multiple chambers.
• FIG. 5(l) is a schematic of a dual-pore chip and a dual-amplifier electronics configuration for independent voltage control (1 ⁇ 4 or V 2 ) and current measurement (/ ? or l 2 ) of each pore.
• Three chambers, A-C, are shown and are volumetrically separated except by common pores.
• FIG. 5(H) is a schematic where electrically, 1 ⁇ 4 and V 2 are principally applied across the resistance of each nanopore by constructing a device that minimizes all access resistances to effectively decouple and / 2 .
• FIG. 5(111) depicts a schematic in which competing voltages are used for control, with arrows showing the direction of each voltage force.
• FIGs. 6a-6c illustrate a nanopore device having one pore connecting two chambers and example results from its use.
• FIG. 6a depicts a schematic diagram of the nanopore device.
• FIG. 6b depicts a representative current trace showing a blockade event resulting from the passage of a double-stranded DNA passing through the pore.
• FIG. 6c depicts a scatter plot showing the change in current amount ( ⁇ /) vs. translocation time (t D ) for all blockade events recorded over 16 minutes.
• FIGs. 7a-7b depict current traces measured within one embodiment of a nanopore device fabricated in accordance with the present invention.
• the provided current traces show that unbound dsDNA causes current enhancement events at KCI concentrations below 0.4 M.
• Current enhancements appeared as downward shifts in the provided experiment, since the voltage and current are both negative (as in FIG. 3C).
• 5.6 kb dsDNA scaffold causes brief current enhancement events that are 50-70 pA in amplitude and 10-200 microseconds in duration.
• 48 kb Lambda DNA causes current enhancement events 50-70 pA in amplitude and 50-2000 microseconds in duration.
• FIG. 8 depicts a schematic diagram of a polymer scaffold. Specifically, FIG. 8 shows a 5,631 bp dsDNA scaffold and the location of 10 total VspR binding sites. Of the 10 VspR binding sites, 5 are of one 14 base-pair sequence, 3 of a different 18 base pair sequence, and 2 are of a 27 base pair sequence. Also shown are the distances (in base pairs) between the binding sites.
• FIGs. 9a and 9b each show schematic representations of embodiments of a nanopore with a scaffold passing therethrough. Each also shows a resultant current profile associated with the scaffold passage as measured by one embodiment of the disclosed nanopore device.
• FIGs. 9(a) and 9(b) compare events with DNA scaffold alone (a) and VspR-bound DNA (b). Specifically, (a) shows a graphic depicting the 5,631 bp dsDNA scaffold passing through the pore, and a
• Part (b) shows a graphic depicting multiple VspR bound to a dsDNA scaffold that is passing through the pore, and a representative current attenuation event (upward 150 pA shift lasting 1.1 milliseconds) when the VspR-bound scaffold passes through the pore.
• the open channel current is negative, so downward events correspond to current enhancement events, and upward events correspond to current attenuation events (as in FIG. 3C). The shift direction is preserved, even though the baseline is zeroed for display purposes.
• FIG. 10 shows ten more representative current attenuation events depicted in a current profile consistent with the VspR-bound scaffold passing through the pore. All shifts are consistent with current attenuations; the baseline is zeroed for display purposes.
• FIG. 11 shows two representative current events depicted in a current profile captured in an experiment with 5.6 kb dsDNA scaffold and RecA protein at 180 mV and 1 M KCI using a 16-18 nm diameter nanopore.
• the first event is consistent with an unbound dsDNA or possibly a free RecA (or multiple associated RecA proteins) passing through the pore, at 280 pA mean current attenuation lasting 70 microseconds.
• the second event is consistent with RecA-bound scaffold passing through the pore, at 1.1 nA mean current attenuation lasting 2.7 milliseconds. RecA- bound events commonly display deeper blockades with longer duration.
• FIG. 12 depicts four more current profiles, each showing a representative current event consistent with RecA-bound scaffold passing through the pore.
• FIG. 13 shows scatter plots and histograms depicting all 1385 events recorded over 10 minutes in one experiment conducted using embodiments of methods described herein.
• the depicted graphs show: (a) maximum conductance in nS (maximum current shift in pA divided by voltage in mV) vs. time duration in seconds, with time duration on a log-scale; (b) a probability histogram of the maximum conductance shift values; (c) mean conductance (mean current shift divided by voltage) vs.
• FIG. 14(a-c) illustrate results from a nanopore device detecting DNA/RecA complexes and RecA-antibody on DNA/RecA complexes, and the results differentiating these complexes from unbound DNA and also from free RecA.
• FIG. 14(a) is a gel shift assay.
• the DNA/RecA/mAb ARM191 Gel Shift Experiments have lanes: 1 ) Ladder, top rung 5000 bp; 2) Scaffold DNA only in RecA labeling buffer; 3) DNA/RecA complex, 1 :1 RecA protein to theoretical RecA binding sites; 4) DNA/RecA/Ab complex, DNA/Rec incubated with a 1 :2000 dilution of monoclonal Ab ARM 191 ; 5) Scaffold DNA only in Ab labeling buffer; and 6) Scaffold DNA mixed with mAb (ARM 191 ).
• FIG. 14(b) shows representative events for DNA (230 pA, 0.1 ms), DNA/RecA (390 pA, 1.1 ms), and probable DNA/RecA/Ab (860 pA, 1.5 ms). RecA-bound DNA event amplitudes are uniformly smaller than in earlier figures (FIGs. 11 -13) since the pore used to measure these events is considerably larger (27-29 nm in diameter).
• FIG. 14(c) depicts a (i) Scatter plot of
• a RecA alone control experiment 0.5 uM RecA (*) was measured at 180 mV in 1 M KCI with a 20 nm diameter pore, generating 767 events over 10 min. Note that only 0.6% of RecA events exceed a criteria of (600 pA, 0.2 ms) under these conditions.
• three reagents were added in sequence in 1 M LiCI.
• 0.1 uM DNA (EE!) was measured at 200 mV with a 20 nm diameter pore, generating 402 events at 0.1 events/sec. After the pore enlarged to 27 nm, 1.25 nM DNA/ RecA (*) was added, generating 3387 events at 1.44 events/sec. Lastly, 1.25 nM DNA/ RecA/Ab (O) was added generating 4953 events at 4.49 events/sec. Events exceeding the (600 pA, 0.2 ms) criteria grew monotonically from 0% with DNA alone, to 5.2% (176) with DNA/RecA added, and up to 9.8% (485) with DNA/RecA/Ab added. While RecA could have increased event durations in LiCI, as shown for DNA, event amplitudes are unlikely to shift significantly toward the (600 pA, 0.2 ms) criteria.
• FIG. 15 illustrates schematic diagrams of polymer scaffolds consistent with embodiments of the present disclosure.
• FIG. 15(a) shows a 5.6 kb dsDNA scaffold used in various experiments, the scaffold having been engineered to bind 12-mer peptide-nucleic-acid (PNA) molecules, with each PNA having 3 biotinylated sites for binding avidin (e.g., neutravidin, and or monovalent streptavidin).
• avidin e.g., neutravidin, and or monovalent streptavidin
• FIG. 15(b) identifying the 25 distinct PNA binding sites on the scaffold that localize up to 75 avidin biomarker binding sites.
• FIGs. 16(a)-(c) illustrate: (a) a schematic of the 5.6 kb dsDNA scaffold passing through a nanopore; (b) a schematic of a free neutravidin passing through a nanopore; (c) a schematic of the dsDNA labeled with a PNA passing through a nanopore, the PNA having all three biotin sites bound by Neutravidin; and (d) corresponding current traces as measured in the chamber above the pore in a nanopore device fabricated in accordance with the present disclosure.
• the current traces depict representative translocation events in the recorded current from separate nanopore experiments with DNA alone, Neutravidin alone, and DNA/PNA/Neutravidin complexes.
• the deeper and longer event pattern in the D/P/N experiment is identified as a DNA/PNA/Neutravidin event and is clearly distinguished from DNA alone or Neutravidin alone events.
• FIGs. 17(a)-(c) illustrate: (a) a scatter plot of the current shift vs. the duration (
• D/P/N subpopulations overlap with the N and D control experiment populations, with most events in the DPN experiment matching unbound N event characteristics; (b) a horizontal probability histogram of
• the inset shows a histogram for a subset of 578 DPN events with t D > 0.08 ms, which attempts to trim out non-DNA events from the D/P/N data set (from controls, 8% of N events and 54% of D events have t D > 0.08 ms).
• DPN events have significant spread in
• MSA DNA/PNA/Neutravidin Gel Shift Experiments
• an electrode includes a plurality of electrodes, including mixtures thereof.
• the term “comprising” is intended to mean that the systems, devices, and methods include the recited components or steps, but not excluding others. "Consisting essentially of when used to define systems, devices, and methods, shall mean excluding other components or steps of any essential significance to the combination. "Consisting of shall mean excluding other components or steps. Embodiments defined by each of these transition terms are within the scope of this invention.
• a device comprising a pore that separates an interior space shall refer to a device having a pore that comprises an opening within a structure, the structure separating an interior space into more than one volume or chamber.
• the present disclosure provides methods and systems for molecular detection and quantitation.
• the methods and systems can also be configured to measure the affinity of a molecule binding with another molecule.
• FIG. 1 provides an illustration of one embodiment of the disclosed methods and systems. More specifically, the system includes a ligand 104 that is capable of binding to a target molecule 105 to be detected or quantitated.
• the ligand 104 can be part of, or be linked to, a binding moiety (referred to as "binding domain") 103 that is capable of binding to a specific binding motif 101 on a polymer scaffold 109.
• binding domain a binding moiety
• both components of the fusion molecule 102 bind to their respective targets (e.g., target molecule 105 and binding motif 101 , respectively) with high affinity and specificity.
• the fusion molecule 102 binds, on one end, to a polymer scaffold (or simply, "polymer”) 109 through the specific recognition and binding between the binding motif 101 and the binding domain 103, and on the other end, to the target molecule 105 by virtue of the interaction between the ligand 104 and the target molecule 105.
• a polymer scaffold or simply, "polymer”
• Such bindings cause the formation of a complex that includes the polymer 109, the fusion molecule 102 and the target molecule 105.
• the formed complex can be detected using a device 108 that includes a nanopore (or simply, pore) 107, and a sensor.
• the pore 107 is a nano-scale or micro-scale opening in a structure separating two volumes.
• the sensor 107 may be positioned within or adjacent the pore 107 or elsewhere within the two volumes.
• the sensor is configured to identify objects passing through the pore 107. For example, in some embodiments, the sensor identifies objects passing through the pore 107 by detecting a change in a measurable parameter, wherein the change is indicative of an object passing through the pore 107.
• This device is referred throughout as a
• the nanopore device 108 includes means, such as electrodes connected to power sources, for moving the polymer 109 from one volume to another, across the pore 107.
• means such as electrodes connected to power sources, for moving the polymer 109 from one volume to another, across the pore 107.
• the polymer 109 can be charged or be modified to contain charges, one example of such means generates a potential or voltage across the pore 107 to facilitate and control the movement of the polymer 109.
• the sensor comprises a pair of electrodes, which are configured to both detect the passage of objects, and provide a voltage, across the pore 107.
• a voltage-clamp or a patch-clamp is used to
• the nanopore device 108 can be configured to pass the polymer 109 through the pore 107.
• the binding motif 101 is within the pore or adjacent to the pore 107, the binding status of the motif 101 can be detected by the sensor.
• binding status of a binding motif refers to whether the binding motif is bound to a fusion molecule with a corresponding binding domain, and whether the fusion molecule is also bound to a target molecule.
• the binding status can be one of three potential statuses: (i) the binding motif is free and not bound to a fusion molecule (see 305 in FIG. 3); (ii) the binding motif is bound to a fusion molecule that does not bind to a target molecule (see 306 in FIG. 3); or (iii) the binding motif is bound to a fusion molecule that is bound to a target molecule (see 307 in FIG. 3).
• Detection of the binding status of a binding motif can be carried out by various methods.
• the different sizes of the binding motif at each status when the binding motif passes through the pore, the different sizes result in different electrical currents across the pore.
• the measured current signals 301, when 305, 306, and 307 pass through the pore are signals 302, 303, and 304, respectively. All three event types are subjected to current attenuation when KCI concentrations are greater than 0.4 M, causing a reduction in the positive current flow.
• the three signals 302, 303, and 304 can be differentiated from one another by the amount of the current shift (height) and/or the duration of the current shift (width), or by any other feature in the signal that differentiates the three event types. It may also be that 304 is commonly different than 302 and 303, but that 302 and 303 are not commonly different from each other, in which case, robust detection of the biomarker bound to the passing molecule can still be accomplished.
• the measured current signals 308, when 312, 313, and 314 pass through the pore are signals 309, 310, and 311 , respectively.
• the signal 309 can be differentiated from 310 and 311 by the event amplitude direction (polarity) relative to the open channel baseline current level (308), in addition to the three signals commonly having different amounts of the current shift (height) and/or the duration of the current shift (width), or by any other feature in the signal that differentiates the three event types.
• the event amplitude direction polarity
• the open channel baseline current level 308
• the negative measured current signals 315, when 319, 320, and 321 pass through the pore are signals 316, 317, and 318, respectively.
• the signals 316, 317, and 318 have the opposite polarity since the applied voltage has the opposite (negative) polarity.
• the sensor comprises electrodes, which are connected to power sources and can detect the current. Either one or both of the electrodes, therefore, serve as a "sensor.”
• a voltage-clamp or a patch-clamp is used to simultaneously supply a voltage across the pore and measure the current through the pore.
• an agent 106 as shown in FIG. 1 is added to the complex to aid detection.
• This agent is capable of binding to the target molecule or the ligand/target molecule complex.
• the agent includes a charge, either negative or positive, to facilitate detection.
• the agent adds size to facilitate detection.
• the agent includes a detectable label, such as a fluorophore.
• an identification of status (iii) indicates that a polymer-fusion molecule-target molecule complex has formed. In other words, the target molecule is detected.
• the present disclosure also provides, in some aspects, methods and systems for detecting, quantitating, and measuring particles such as proteins, protein aggregates, oligomers, or protein/DNA complexes, or cells and microorganisms, including viruses, bacteria, and cellular aggregates.
• the pore within the structure that separates the device into two volumes has a size that allows particles, such as viruses, bacteria, cells, or cellular aggregates, to pass through.
• a ligand that is capable of binding to a target particle to be detected or quantitated can be included in the solution in the nanopore device such that the ligand can bind to the unique target particle and the polymer scaffold through a binding domain and a binding motif to form a complex.
• Many such particles have unique markers on their surfaces that can be specifically recognized by a ligand. For instance, tumor cells can have tumor antigens expressed on the cell surface, and bacterial cells can have endotoxins attached on the cell membrane.
• the binding status of the complex within or adjacent to the pore can be detected such that the target microorganisms bound to the ligands can be identified using methods similar to the molecular detection methods described elsewhere in the disclosure.
• a polymer scaffold suitable for use in the present technology is a scaffold that can be loaded into a nanopore device and passed through the pore from one end to the other.
• Non-limiting examples of polymers include nucleic acids, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or peptide nucleic acid (PNA), dendrimers, and linearized proteins or peptides.
• the DNA or RNA can be single-stranded or double-stranded, or can be a DNA/RNA hybrid molecule.
• the polymer is synthetic or chemically modified.
• Chemical modification can help to stabilize the polymer, add charges to the polymer to increase mobility, maintain linearity, or add or modify the binding specificity.
• the chemical modification is acetylation, methylation, summolation, oxidation, phosphorylation, glycosylation, or the addition of biotin.
• the polymer is electrically charged.
• DNA, RNA, PNA and proteins are typically charged under physiological conditions.
• Such polymers can be further modified to increase or decrease the carried charge.
• Other polymers can be modified to introduce charges. Charges on the polymer can be useful for driving the polymer to pass through the pore of a nanopore device. For instance, a charged polymer can move across the pore by virtue of an application of voltage across the pore.
• the charges when charges are introduced to the polymer, the charges can be added at the ends of the polymer. In some aspects, the charges are spread over the polymer.
• each unit of the charged polymer is charged at the pH selected.
• the charged polymer includes sufficient charged units to be pulled into and through the pore by electrostatic forces.
• a peptide containing sufficient entities can be charged at a selected pH (lysine, aspartic acid, glutamic acid, etc.) so as to be used in the devices and methods described herein.
• a co-polymer comprising methacrylic acid and ethylene is a charged polymer for the purposes of this invention if there is sufficient charged carboxylate groups of the methacrylic acid residue to be used in the devices and methods described herein.
• the charged polymer includes one or more charged units at or close to one terminus of the polymer.
• the charged polymer includes one or more charged units at or close to both termini of the polymer.
• One co-polymer example is a DNA wrapped around protein (e.g. DNA/nucleosome).
• Another example of a co-polymer is a linearized protein conjugated to DNA at the N- and C- terminus.
• a binding motif can be a nucleotide or peptide sequence that is recognizable by a binding domain.
• the binding domain is a peptide sequence forming a functional portion of a protein, although the binding domain does not have to be a protein.
• sequences motifs
• the binding motif includes a chemical modification that causes or facilitates recognition and binding by a binding domain.
• methylated DNA sequences can be recognized by transcription factors, DNA methyltransferases or methylation repair enzymes.
• biotin may be incorporated into, and recognized by, avidin family members. In such
• biotin forms the binding motif and avidin or an avidin family member is the binding domain.
• Molecules in particular proteins, that are capable of specifically recognizing nucleotide binding motifs are known in the art.
• protein domains such as helix-turn-helix, a zinc finger, a leucine zipper, a winged helix, a winged helix turn helix, a helix-loop-helix and an HMG-box, are known to be able to bind to nucleotide sequences.
• the binding domains can be locked nucleic acids (LNAs), Protein Nucleic Acids of all types (e.g. bisPNAs, gamma-PNAs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPRs), or aptamers (e.g., DNA, RNA, protein, or
• the binding domains are one or more of DNA binding proteins (e.g., zinc finger proteins), antibody fragments (Fab), chemically synthesized binders (e.g., PNA, LNA, TALENS, or CRISPR), or a chemical modification (i.e., reactive moieties) in the synthetic polymer (e.g., thiolate, biotin, amines, carboxylates).
• DNA binding proteins e.g., zinc finger proteins
• Fab antibody fragments
• chemically synthesized binders e.g., PNA, LNA, TALENS, or CRISPR
• a chemical modification i.e., reactive moieties
• a target molecule or particle is detected or quantitated by virtue of its binding to a ligand in a fusion molecule that binds to a polymer scaffold.
• a target molecule or particle and a corresponding binding ligand can recognize and bind each other.
• there can be surface molecules or markers suitable for a ligand to bind therefore the marker and the ligand form a binding pair).
• binding pairs that enable binding between a target molecule or a molecule on a particle include, but are not limited to, antigen/antibody (or antibody fragment); hormone, neurotransmitter, cytokine, growth factor or cell recognition molecule/receptor; and ion or element/chelate agent or ion binding protein, such as a calmodulin.
• the binding pairs can also be single-stranded nucleic acids having complementary sequences, enzymes and substrates, members of protein complex that bind each other, enzymes and cofactors, enzymes and one or more of their inhibitors (allosteric or otherwise), nucleic acid/protein, or cells or proteins detectable by cysteine-constrained peptides.
• the ligand is a protein, protein scaffold, peptide, aptamer (DNA or protein), nucleic acid (DNA or RNA), antibody fragment (Fab), chemically synthesized molecule, chemically reactive functional group or any other suitable structure that forms a binding pair with a target molecule.
• any target molecule in need of detection or quantitation such as proteins, peptides, nucleic acids, chemical compounds, ions, and elements, can find a corresponding binding ligand.
• an antibody or a complementary sequence, or an aptamer can be readily prepared.
• binding ligands can be readily found or prepared for particles, such as protein complexes and protein aggregates, protein/nucleic acid complexes, fragmented or fully assembled viruses, bacteria, cells, and cellular aggregates.
• a "fusion molecule” is intended to mean a molecule or complex that contains two functional regions, a binding domain and a ligand.
• the binding domain is capable of binding to a binding motif on a polymer scaffold, and the ligand is capable of binding to a target molecule.
• the fusion molecule is prepared by linking the two regions with a bond or force.
• a bond and force can be, for instance, a covalent bond, a hydrogen bond, an ionic bond, a metallic bond, van der Walls force, hydrophobic interaction, or planar stacking interaction.
• the fusion molecule such as a fusion protein, can be expressed as a single molecule from a recombinant coding nucleotide.
• the fusion molecule is a natural molecule having a binding domain and a ligand suitable for use in the present technology.
• the components may be connected via chemical coupling through functionalized linkers such as free amine, carboxylate coupling, thiolate, hydrazide, or azide (click) chemistry or the binding domain and the ligand may form one continuous transcript.
• functionalized linkers such as free amine, carboxylate coupling, thiolate, hydrazide, or azide (click) chemistry
• FIG. 2 illustrates a more specific embodiment of the system shown in FIG. 1.
• the fusion molecule is a chimeric protein that includes a zinc finger protein or domain 202 and a human immunodeficiency virus (HIV) envelop protein 203.
• the zinc finger protein 202 can bind to a suitable nucleotide sequence on the polymer scaffold, a double-stranded DNA 201 ;
• the HIV envelop protein 203 can bind to an anti-HIV antibody 204 which can be present in a biological sample (e.g., a blood sample from a patient) for detection.
• a biological sample e.g., a blood sample from a patient
• the nanopore device 206 can detect whether a fusion molecule is bound to the DNA 201 and whether the bound fusion molecule binds to an anti-HIV antibody 204. Measurement of Affinity of Binding
• the present technology can be used also for measuring the binding affinity between two molecules and to determine other binding dynamics. For instance, after the binding motif passes through the pore of a nanopore device, the device can be reconfigured to reverse the moving direction of the polymer scaffold (as described below) such that the binding motif can pass through the pore again.
• changing the condition can be one or more of removing the target molecule from the sample, adding an agent that competes with the target molecule or the ligand for binding, and changing the pH, salt, or temperature.
• the binding motif may be passed through the pore again. Therefore, whether the target molecule is still bound to the fusion molecule can be detected to determine how the changed conditions impact the binding.
• the binding motif is in the pore, it is retained there while the conditions are changed, and thus the impact of the changed conditions can be measured in situ.
• the polymer scaffold can include multiple binding motifs and each of the binding motifs can bind to a fusion molecule that can bind to one or more specific a target molecule(s) or particle(s). While each binding motif passes through the pore, the conditions of the sample can be changed, allowing detection of changed binding between the ligand and the target molecule or particle on a continued basis.
• a polymer scaffold can include multiple types of binding motifs, each having different corresponding binding domains.
• a sample can include multiple types of fusion molecules, each type including one of the different corresponding binding domains and a ligand for a different target molecule or target microorganism.
• An additional method of multiplexing includes assaying a collection of different scaffold molecules during a test, with each different scaffold associating with different fusion molecule(s). To determine what target molecules are in solution, scaffolds of the same type are labeled such that the sensor can identify what fusion molecule will bind to that particular scaffold. This can be accomplished, for example, by barcoding each type of scaffold with polyethylene glycol molecules of varying lengths or sizes.
• a single polymer scaffold can be used to detect multiple types of target molecules or target microorganisms (e.g. bacterium or virus), or target cells (e.g. circulating tumor cells).
• FIG. 4 illustrates such a method.
• a double- stranded DNA 403 is used as the polymer scaffold, the double-stranded DNA 403 including multiple binding motifs: two copies of a first binding motif 404, two copies of a second binding motif 405, and one copy of a third binding motif 406.
• the present technology can simultaneously detect multiple different target molecules or target microstructure (e.g., aggregates, microorganisms, or cells). Further, by determining how many copies of binding motifs are bound to the target molecules or target microorganisms, and by tuning conditions that impact the bindings, the system can obtain more detailed binding dynamic information.
• target molecules or target microstructure e.g., aggregates, microorganisms, or cells.
• a nanopore device includes at least a pore that forms an opening in a structure separating an interior space of the device into two volumes, and at least a sensor configured to identify objects (for example, by detecting changes in parameters indicative of objects) passing through the pore.
• the pore(s) in the nanopore device are of a nano scale or micro scale. In one aspect, each pore has a size that allows a small or large molecule or
• each pore is at least about 1 nm in diameter.
• each pore is at least about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 1 1 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm in diameter.
• the pore is no more than about 100 nm in diameter.
• the pore is no more than about 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, or 10 nm in diameter.
• each pore is at least about 100 nm, 200 nm, 500 nm, 1000 nm, 2000 nm, 3000 nm, 5000 nm, 10000 nm, 20000 nm, or 30000 nm in diameter. In one aspect, the pore is no more than about 100000 nm in diameter. Alternatively, the pore is no more than about 50000 nm, 40000 nm, 30000 nm, 20000 nm, 10000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm, or 1000 nm in diameter.
• the pore has a diameter that is between about 1 nm and about 100 nm, or alternatively between about 2 nm and about 80 nm, or between about 3 nm and about 70 nm, or between about 4 nm and about 60 nm, or between about 5 nm and about 50 nm, or between about 10 nm and about 40 nm, or between about 15 nm and about 30 nm.
• the pore(s) in the nanopore device are of a larger scale for detecting large microorganisms or cells.
• each pore has a size that allows a large cell or microorganism to pass.
• each pore is at least about 100 nm in diameter.
• each pore is at least about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1 100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2500 nm, 3000 nm, 3500 nm, 4000 nm, 4500 nm, or 5000 nm in diameter.
• the pore is no more than about 100,000 nm in diameter.
• the pore is no more than about 90,000 nm, 80,000 nm, 70,000 nm, 60,000 nm, 50,000 nm, 40,000 nm, 30,000 nm, 20,000 nm, 10,000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm, or 1000 nm in diameter.
• the pore has a diameter that is between about 100 nm and about 10000 nm, or alternatively between about 200 nm and about 9000 nm, or between about 300 nm and about 8000 nm, or between about 400 nm and about 7000 nm, or between about 500 nm and about 6000 nm, or between about 1000 nm and about 5000 nm, or between about 1500 nm and about 3000 nm.
• the nanopore device further includes means to move a polymer scaffold across the pore and/or means to identify objects that pass through the pore. Further details are provided below, described in the context of a two-pore device.
• a two-pore device can be more easily configured to provide good control of speed and direction of the movement of the polymer across the pores.
• the nanopore device includes a plurality of chambers, each chamber in communication with an adjacent chamber through at least one pore. Among these pores, two pores, namely a first pore and a second pore, are placed so as to allow at least a portion of a polymer to move out of the first pore and into the second pore. Further, the device includes a sensor capable of identifying the polymer during the movement. In one aspect, the identification entails identifying individual components of the polymer. In another aspect, the identification entails identifying fusion molecules and/or target molecules bound to the polymer. When a single sensor is employed, the single sensor may include two electrodes placed at both ends of a pore to measure an ionic current across the pore. In another embodiment, the single sensor comprises a component other than electrodes.
• the device includes three chambers connected through two pores.
• Devices with more than three chambers can be readily designed to include one or more additional chambers on either side of a three-chamber device, or between any two of the three chambers.
• more than two pores can be included in the device to connect the chambers.
• the device further includes means to move a polymer from one chamber to another.
• the movement results in loading the polymer across both the first pore and the second pore at the same time.
• the means further enables the movement of the polymer, through both pores, in the same direction.
• each of the chambers can contain an electrode for connecting to a power supply so that a separate voltage can be applied across each of the pores between the chambers.
• a device comprising an upper chamber, a middle chamber and a lower chamber, wherein the upper chamber is in communication with the middle chamber through a first pore, and the middle chamber is in communication with the lower chamber through a second pore.
• a device may have any of the dimensions or other characteristics previously disclosed in U.S. Publ. No. 2013-0233709, entitled Dual- Pore Device, which is herein incorporated by reference in its entirety.
• the device includes an upper chamber 505 (Chamber A), a middle chamber 504 (Chamber B), and a lower chamber 503 (Chamber C).
• the chambers are separated by two separating layers or membranes (501 and 502) each having a separate pore (511 or 512). Further, each chamber contains an electrode (521 , 522 or 523) for connecting to a power supply.
• the annotation of upper, middle and lower chamber is in relative terms and does not indicate that, for instance, the upper chamber is placed above the middle or lower chamber relative to the ground, or vice versa.
• each of the pores 511 and 512 independently has a size that allows a small or large molecule or microorganism to pass.
• each pore is at least about 1 nm in diameter.
• each pore is at least about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm in diameter.
• the pore is no more than about 100 nm in diameter.
• the pore is no more than about 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, or 10 nm in diameter.
• the pore has a diameter that is between about 1 nm and about 100 nm, or alternatively between about 2 nm and about 80 nm, or between about 3 nm and about 70 nm, or between about 4 nm and about 60 nm, or between about 5 nm and about 50 nm, or between about 10 nm and about 40 nm, or between about 15 nm and about 30 nm.
• each pore is at least about 100 nm, 200 nm, 500 nm, 1000 nm, 2000 nm, 3000 nm, 5000 nm, 10000 nm, 20000 nm, or 30000 nm in diameter. In one aspect, each pore is 50,000 nm to 100,000 nm in diameter. In one aspect, the pore is no more than about 100000 nm in diameter.
• the pore is no more than about 50000 nm, 40000 nm, 30000 nm, 20000 nm, 10000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm, or 1000 nm in diameter.
• the pore has a substantially round shape.
• substantially round refers to a shape that is at least about 80 or 90% in the form of a cylinder.
• the pore is square, rectangular, triangular, oval, or hexangular in shape.
• Each of the pores 511 and 512 independently has a depth (i.e., a length of the pore extending between two adjacent volumes).
• each pore has a depth that is least about 0.3 nm.
• each pore has a depth that is at least about 0.6 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 1 1 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, or 90 nm.
• each pore has a depth that is no more than about 100 nm.
• the depth is no more than about 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, or 10 nm.
• the pore has a depth that is between about 1 nm and about 100 nm, or alternatively, between about 2 nm and about 80 nm, or between about 3 nm and about 70 nm, or between about 4 nm and about 60 nm, or between about 5 nm and about 50 nm, or between about 10 nm and about 40 nm, or between about 15 nm and about 30 nm.
• the nanopore extends through a membrane.
• the pore may be a protein channel inserted in a lipid bilayer membrane or it may be engineered by drilling, etching, or otherwise forming the pore through a solid-state substrate such as silicon dioxide, silicon nitride, grapheme, or layers formed of combinations of these or other materials.
• the length or depth of the nanopore is sufficiently large so as to form a channel connecting two otherwise separate volumes.
• the depth of each pore is greater than 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, or 900 nm. In some aspects, the depth of each pore is no more than 2000 nm or 1000 nm.
• the pores are spaced apart at a distance that is between about 10 nm and about 1000 nm. In some aspects, the distance between the pores is greater than 1000 nm, 2000 nm, 3000 nm, 4000 nm, 5000 nm, 6000 nm, 7000 nm, 8000 nm, or 9000 nm. In some aspects, the pores are spaced no more than 30000 nm, 20000 nm, or 10000 nm apart.
• the distance is at least about 10 nm, or alternatively, at least about 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, or 300 nm. In another aspect, the distance is no more than about 1000 nm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 250 nm, 200 nm, 150 nm, or 100 nm.
• the distance between the pores is between about 20 nm and about 800 nm, between about 30 nm and about 700 nm, between about 40 nm and about 500 nm, or between about 50 nm and about 300 nm.
• the two pores can be arranged in any position so long as they allow fluid communication between the chambers and have the prescribed size and distance between them.
• the pores are placed so that there is no direct blockage between them.
• the pores are substantially coaxial, as illustrated in FIG. 5(1).
• the device through the electrodes 521 , 522, and 523 in the chambers 503, 504, and 505, respectively, is connected to one or more power supplies.
• the power supply includes a voltage-clamp or a patch-clamp, which can supply a voltage across each pore and measure the current through each pore independently.
• the power supply and the electrode configuration can set the middle chamber to a common ground for both power supplies.
• the power supply or supplies are configured to apply a first voltage Vi between the upper chamber 505 (Chamber A) and the middle chamber 504 (Chamber B), and a second voltage V 2 between the middle chamber 504 and the lower chamber 503 (Chamber C).
• the first voltage Vi and the second voltage V 2 are independently adjustable.
• the middle chamber is adjusted to be a ground relative to the two voltages.
• the middle chamber comprises a medium for providing conductance between each of the pores and the electrode in the middle chamber.
• the middle chamber includes a medium for providing a resistance between each of the pores and the electrode in the middle chamber. Keeping such a resistance sufficiently small relative to the nanopore resistances is useful for decoupling the two voltages and currents across the pores, which is helpful for the independent adjustment of the voltages.
• Adjustment of the voltages can be used to control the movement of charged particles in the chambers. For instance, when both voltages are set in the same polarity, a properly charged particle can be moved from the upper chamber to the middle chamber and to the lower chamber, or the other way around, sequentially. In some aspects, when the two voltages are set to opposite polarity, a charged particle can be moved from either the upper or the lower chamber to the middle chamber and kept there.
• the adjustment of the voltages in the device can be particularly useful for controlling the movement of a large molecule, such as a charged polymer, that is long enough to cross both pores at the same time.
• a large molecule such as a charged polymer
• the direction and the speed of the movement of the molecule can be controlled by the relative magnitude and polarity of the voltages as described below.
• the device can contain materials suitable for holding liquid samples, in particular, biological samples, and/or materials suitable for nanofabrication.
• materials include dielectric materials such as, but not limited to, silicon, silicon nitride, silicon dioxide, graphene, carbon nanotubes, Ti0 2 , Hf0 2 , Al 2 03, or other metallic layers, or any combination of these materials.
• a single sheet of graphene membrane of about 0.3 nm thick can be used as the pore- bearing membrane.
• Devices that are microfluidic and that house two-pore microfluidic chip implementations can be made by a variety of means and methods.
• a microfluidic chip comprised of two parallel membranes both membranes can be simultaneously drilled by a single beam to form two concentric pores, though using different beams on each side of the membranes is also possible in concert with any suitable alignment technique.
• the housing ensures sealed separation of Chambers A- C.
• the housing would provide minimal access resistance between the voltage electrodes 521 , 522, and 523 and the nanopores 511 and 512, to ensure that each voltage is applied principally across each pore.
• the device includes a microfluidic chip (labeled as "Dual-core chip") is comprised of two parallel membranes connected by spacers. Each membrane contains a pore drilled by a single beam through the center of the membrane. Further, the device preferably has a Teflon ® housing for the chip. The housing ensures sealed separation of Chambers A-C and provides minimal access resistance for the electrode to ensure that each voltage is applied principally across each pore.
• the pore-bearing membranes can be made with
• TEM transmission electron microscopy
• a focused electron or ion beam can be used to drill pores through the membranes, naturally aligning them.
• the pores can also be sculpted (shrunk) to smaller sizes by applying a correct beam focusing to each layer.
• Any single nanopore drilling method can also be used to drill the pair of pores in the two membranes, with consideration to the drill depth possible for a given method and the thickness of the membranes. Predrilling a micro-pore to a prescribed depth and then a nanopore through the remainder of the membranes is also possible to further refine the membrane thickness.
• the insertion of biological nanopores into solid-state nanopores to form a hybrid pore can be used in either or both pores in the two-pore method.
• the biological pore can increase the sensitivity of the ionic current measurements, and is useful when only single-stranded polynucleotides are to be captured and controlled in the two-pore device, e.g., for sequencing.
• One example concerns a charged polymer scaffold, such as a DNA, having a length that is longer than the combined distance that includes the depth of both pores plus the distance between the two pores.
• a 1000 bp dsDNA is about 340 nm in length, and would be substantially longer than the 40 nm spanned by two 10 nm-deep pores separated by 20 nm.
• the polynucleotide is loaded into either the upper or the lower chamber. By virtue of its negative charge under a physiological condition at a pH of about 7.4, the polynucleotide can be moved across a pore on which a voltage is applied.
• two voltages in the same polarity and at the same or similar magnitudes, are applied to the pores to move the polynucleotide across both pores sequentially.
• one or both of the voltages can be changed. Since the distance between the two pores is selected to be shorter than the length of the polynucleotide, when the polynucleotide reaches the second pore, it is also in the first pore. A prompt change of polarity of the voltage at the first pore, therefore, will generate a force that pulls the polynucleotide away from the second pore as illustrated in FIG. 5(lll).
• the polynucleotide will continue crossing both pores towards the second pore, but at a lower speed.
• speed and direction of the movement of the polynucleotide can be controlled by the polarities and magnitudes of both voltages. As will be further described below, such a fine control of movement has broad applications.
• a method for controlling the movement of a charged polymer through a nanopore device entails (a) loading a sample comprising a charged polymer in one of the upper chamber, middle chamber or lower chamber of the device of any of the above embodiments, wherein the device is connected to one or more power supplies for providing a first voltage between the upper chamber and the middle chamber, and a second voltage between the middle chamber and the lower chamber; (b) setting an initial first voltage and an initial second voltage so that the polymer moves between the chambers, thereby locating the polymer across both the first and second pores; and (c) adjusting the first voltage and the second voltage so that both voltages generate force to pull the charged polymer away from the middle chamber (voltage-competition mode), wherein the two voltages are different in magnitude, under controlled conditions, so that the charged polymer moves across both pores in either direction and in a controlled manner.
• the relative force exerted by each voltage at each pore is to be determined for each two-pore device used, and this can be done with calibration experiments by observing the influence of different voltage values on the motion of the polynucleotide, which can be measured by sensing known-location and detectable features in the polynucleotide, with examples of such features detailed later in this disclosure. If the forces are equivalent at each common voltage, for example, then using the same voltage value at each pore (with common polarity in upper and lower chambers relative to grounded middle chamber) creates a zero net motion in the absence of thermal agitation (the presence and influence of Brownian motion is discussed below).
• the sample containing the charged polymer is loaded into the upper chamber and the initial first voltage is set to pull the charged polymer from the upper chamber to the middle chamber and the initial second voltage is set to pull the polymer from the middle chamber to the lower chamber.
• the sample can be initially loaded into the lower chamber, and the charged polymer can be pulled to the middle and the upper chambers.
• the sample containing the charged polymer is loaded into the middle chamber; the initial first voltage is set to pull the charged polymer from the middle chamber to the upper chamber; and the initial second voltage is set to pull the charged polymer from the middle chamber to the lower chamber.
• the adjusted first voltage and second voltage at step (c) are about 10 times to about 10,000 times as high, in magnitude, as the
• the magnitude of the voltages is no more than about 10000 times, 9000 times, 8000 times, 7000 times, 6000 times, 5000 times, 4000 times, 3000 times, 2000 times, 1000 times, 500 times, 400 times, 300 times, 200 times, or 100 times as high as the difference/differential between them.
• real-time or on-line adjustments to the first voltage and the second voltage at step (c) are performed by active control or feedback control using dedicated hardware and software, at clock rates up to hundreds of megahertz.
• Automated control of the first or second or both voltages is based on feedback of the first or second or both ionic current measurements.
• the nanopore device further includes one or more sensors to carry out the identification of the binding status of the binding motifs.
• the sensors used in the device can be any sensor suitable for identifying a molecule or particle, such as a polymer.
• a sensor can be configured to identify the polymer by measuring a current, a voltage, a pH value, an optical feature, or residence time associated with the polymer.
• the sensor may be configured to identify one or more individual components of the polymer or one or more components bound to the polymer.
• the sensor may be formed of any component configured to detect a change in a measurable parameter where the change is indicative of the polymer, a component of the polymer, or preferably, a component bound to the polymer.
• the senor includes a pair of electrodes placed at two sides of a pore to measure an ionic current across the pore when a molecule or particle, in particular a polymer, moves through the pore.
• the ionic current across the pore changes measurably when a polymer segment passing through the pore is bound to a fusion molecule and/or fusion molecule-target molecule complex. Such changes in current may vary in predictable, measurable ways corresponding with, for example, the presence, absence, and/or size of the fusion molecules and target molecules present.
• the senor measures an optical feature of the polymer, a component (or unit) of the polymer, or a component bound to the polymer.
• One example of such measurement includes the identification of an absorption band unique to a particular unit by infrared (or ultraviolet) spectroscopy.
• the size of the component can be correlated to the specific component based on the length of time it takes to pass through the sensing device.
• the senor can include an enzyme distal to the sensing device, where the enzyme is capable of separating the terminal unit of the polymer from the penultimate unit, thereby providing for a single molecular unit of the polymer.
• the single molecule such as a single nucleotide or an amino acid, can then translocate through the pore and may or may not be detected.
• the enzyme encounters a bound target molecule, the enzyme will not be able to cleave the penultimate unit, and therefore will become stalled or will skip to the next available cleavage sites, thus releasing a fragment that has a comparable size difference from a single unit and is thus detectable.
• Detection can be done with sensors as described in this application or detected with methods such as mass spectrometry. Methods for measuring such units are known in the art and include those developed by Cal Tech (see, e.g., spectrum. ieee.org/tech-talk/at-work/test-and-measurement/a-scale-for-weighing- single-molecules). The results of such analysis can be compared to those of the sensing device to confirm the correctness of the analysis.
• the senor is functionalized with reagents that form distinct non-covalent bonds with each association site or each associated target molecule.
• the gap is large enough to allow effective measuring. For instance, when a sensor is functionalized with reagents to detect a feature on DNA that is 5 nm on a dsDNA scaffold, a 7.5 nm gap can be used, because DNA is 2.5 nm wide.
• Tunnel sensing with a functionalized sensor is termed "recognition tunneling.” Using current technology, a Scanning Tunneling Microscope (STM) with recognition tunneling identifies a DNA base flanked by other bases in a short DNA oligomer.
• STM Scanning Tunneling Microscope
• recognition tunneling can provide a "universal reader" designed to hydrogen-bond in a unique orientation to molecules that a user desires to be detected. Most reported is the identification of nucleic acids; however, it is herein modified to be employed to detect target molecules on a scaffold.
• a limitation with the conventional recognition tunneling is that it can detect only freely diffusing molecules that randomly bind in the gap, or that happen to be in the gap during microscope motion, with no method of explicit capture in the gap.
• the collective drawbacks of the STM setup can be eliminated by incorporating the recognition reagent, optimized for sensitivity, within an electrode tunneling gap in a nanopore channel.
• the senor includes surface modification by a reagent.
• the reagent is capable of forming a non-covalent bond with an association site or an attached target molecule.
• the bond is a hydrogen bond.
• the reagent include 4- mercaptobenzamide and 1 -H-lmidazole-2-carboxamide.
• the methods of the present technology can provide DNA delivery rate control for one or more recognition tunneling sites, each positioned in one or both of the nanopore channels, and voltage control can ensure that each target molecule resides in each site for a sufficient duration for robust identification.
• Sensors in the devices and methods of the present disclosure can comprise gold, platinum, graphene, or carbon, or other suitable materials.
• the sensor includes parts made of graphene.
• Graphene can act as a conductor and an insulator, thus tunneling currents through the graphene and across the nanopore can sequence the translocating DNA.
• the tunnel gap has a width from about 1 nm to about 20 nm.
• the width of the gap is at least about 1 nm, or alternatively, at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 12, or 15 nm.
• the width of the gap is not greater than about 20 nm, or alternatively, not greater than about 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, or 2 nm.
• the width is between about 1 nm and about 15 nm, between about 1 nm and about 10 nm, between about 2 nm and about 10 nm, between about 2.5 nm and about 10 nm, or between about 2.5 nm and about 5 nm.
• the tunnel gap is suitable for detecting micro-sized particles (e.g., viruses, bacteria, and/or cells) and has a width from about 1000 nm to about 100,000 nm. In some embodiments, the width of the gap is between about 10,000 nm and 80,000 nm or between about 20,000 nm and 50,000 nm. In another embodiment, the width of the gap is between about 50,000 nm and 100,000 nm.
• micro-sized particles e.g., viruses, bacteria, and/or cells
• the width of the gap is not greater than about 100,000 nm, 90,000 nm, 80,000 nm, 70,000 nm, 60,000 nm, 50,000 nm, 40,000 nm, 30,000 nm, 20,000 nm, 10,000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm, or 1000 nm.
• the senor is an electric sensor. In some embodiments, the sensor is an electric sensor.
• the senor detects a fluorescent detection means when the target molecule or the detectable label passing through has a unique fluorescent signature.
• a radiation source at the outlet of the pore can be used to detect that signature.
• the example section begins by first pointing out principal reasons to use a polymer scaffold and fusion molecules in biomarker detection.
• a primary reason is that a biomarker alone, below a certain size threshold, is undetectable with a nanopore, as shown for proteins of varying sizes in Calin Plesa, Stefan W.
• Nanopore instruments use a sensitive voltage-clamp amplifier to apply a voltage V across the pore while measuring the ionic current l 0 through the open pore (FIG. 6a).
• a single charged molecule such as a double-stranded DNA (dsDNA)
• dsDNA double-stranded DNA
• FIG. 6b electrophoresis
• distributions of the events (FIG. 6c) are analyzed to characterize the corresponding molecule.
• nanopores provide a simple, label-free, purely electrical single- molecule method for biomolecular sensing.
• the single nanopore fabricated in silicon nitride (SiN) substrate is a 40 nm diameter pore in 100 nm thick SiN membrane (FIG. 6a).
• the representative current trace shows a blockade event caused by a 5.6 kb dsDNA passing in a single file manner (unfolded) through an 1 1 nm diameter nanopore in 10 nm thick SiN at 200 mV and 1 M KCI.
• the scatter plot shows
• VspR protein is a 90 kDa protein from V. cholerae that binds directly to dsDNA with high micromolar affinity (see reference: Yildiz, Fitnat H., Nadia A. Dolganov, and Gary K. Schoolnik. "VpsR, a Member of the Response Regulators of the Two-Component Regulatory Systems, Is Required for Expression of Biosynthesis Genes and EPSETr-Associated Phenotypes in Vibrio cholerae 01 El Tor.” Journal of bacteriology 183, no. 5 (2001 ): 1716-1726).
• VspR acts as the fusion molecule with a site-specific DNA binding domain, and a ligand specific binding site that can be engineered for the purpose of detecting a variety of targets, including antibodies or sugars.
• the scaffold contains 10 VspR specific binding sites (FIG. 8).
• FCI VspR specific binding sites
• the 5.631 kb DNA scaffold contains 10 total VspR binding sites: 5 of one sequence (14 base pairs), 3 of a different sequence (18 base pairs), and 2 of a third sequence (27 bp).
• the three different sequences may not bind VspR with equal affinity.
• VspR protein concentration is 18 nM in the recording buffer, and 180 nM during labeling (binding step). This results in 18x excess of VspR protein to binding sites on DNA.
• the experiment was run at pH 8.0 (pi of VspR protein is 5.8). Taking Kd and DNA concentration into account, only 0.1 -1 % of DNA should be fully occupied by VspR, with a larger percentage partially occupied, and some unknown remaining percentage of DNA entirely unbound. There is also free VspR protein in solution during the nanopore experiment.
• VspR concentration was 18 nM (1.6 mg/L), 10 nM binding sites.
• the scaffold concentration was 1 nM resulting in capture every 6.6 seconds. From this, the theoretical sensitivity using a 10 ul sample is 116 pM (0.01 mg/ml).
• the pore size is 15 nm in diameter and length.
• the voltage is -100 mV, and note that negative voltages create negative currents, so upward shifts correspond to attenuation events, as shown for the VspR-bound DNA event (FIG. 9b), whereas downward shifts create positive shifts as shown for the unbound DNA scaffold event (FIG. 9a).
• FIG. 10 shows ten more representative current attenuation events consistent with the VspR-bound scaffold passing through the pore. There were 90 such events over 10 minutes of recording, corresponding to 1 VspR-bound event every 6.6 seconds. Events were attenuations of 50 to 150 pA in amplitude and 0.2 to 2 milliseconds in duration. As stated, downward events correspond to current enhancement events and upward events correspond to current attenuation events in FIG. 9-10, and this shift direction is preserved even though the baseline is zeroed for display purposes.
• RecA comprises the elements of a fusion molecule, and this example demonstrates the ability to use these elements to detect a target biomarker.
• the fusion molecule consists of the portion of RecA that binds DNA (i.e. the DNA binding domain) and the portion of RecA (epitope) that baits the biomarker (anti-RecA antibody). DNA and RecA experiments were performed first in the absence and then in the presence of anti-RecA antibody.
• FIG. 13a and FIG. 13c show on the vertical axis the maximum and mean current shift, respectively, normalized by voltage, and the event duration on the horizontal axis.
• RecA antibody In separate experiments, to demonstrate detection of a target antibody, RecA antibody was used.
• the DNA/RecA reagent binds an antibody biomarker creating a DNA/RecA/Ab complex by incubating one nanomolar DNA/RecA for 30 mins with either an anti-RecA monoclonal antibody (ARM 191 , Fisher Scientific) or polyclonal RecA anti-serum (gift from Prof. Ken Knight, Ph. D., UMass Medical School), at a 1 : 10000 dilution.
• Electrophoretic mobility shift assays 5% TBE polyacrylamide gel in 1x TBE buffer, are used to test the DNA/RecA and DNA/RecA/Ab complexes by comparing migration of complexes to DNA only or the proper controls.
• FIG. 14a shows a clear shift for DNA/RecA/mAb above DNA/RecA, which is in turn well above the unbound 5.6 kb dsDNA scaffold.
• This complex was tested experimentally with a nanopore. Specifically, 0.1 nM DNA was added to the chamber above the pore, and after 10 minutes of recording, 1.25 nM DNA/RecA was added. After another period of recording, 1 .25 nM DNA/RecA/mAb was added.
• FIG. 14b a new multi-level event type was observed (FIG. 14b) that did not match event patterns characteristic of the other two complex types (DNA, DNA/RecA).
• the ⁇ / vs. t D distributions of events recorded during each phase of the experiment show that RecA-bound DNA events have longer durations t D , and 3 times as many events had a mean amplitude shift ⁇ / greater than 0.6 nA after DNA/RecA/mAb was added.
• a simple criteria for tagging events in this data set as also being Ab-bound is ( ⁇ /, t D ) > (0.6 nA, 0.2 ms).
• Identifying a best signature that is almost absent in unbound DNA events, but is present in a significant fraction of RecA-bound events (with or without antibody also bound to DNA/RecA), is useful for detection of the presence of RecA-bound DNA complexes in solution above the nanopore.
• antibody detection we take this a step further, and aim to identify a best signature that is almost absent in unbound DNA and RecA-bound DNA event types, but is present in a significant fraction of RecA-bound events with antibody also bound to DNA/RecA. This provides a criterion for detection of the presence of RecA-bound DNA complexes in solution above the nanopore.
• these DNA and RecA and RecA-antibody experiments are done with a positive voltage with KCI concentration above 0.4 M, we see that the event patterns in FIG. 14b are comparable to the idealized patterns in FIG. 3A.
• Example 4 Fusion Molecules comprising PNA and biotin for target protein detection
• FIG. 15b Our data (FIG. 16) shows that the DNA/PNA/Neut complexes cause event signatures that are detectable above other background event types (unbound DNA alone, Neutravidin alone, PNA/Neutravidin alone) and can therefore be tagged as fully assembled (i.e. DNA/PNA/Neutravidin) events.
• DNA/PNA/Neutravidin the fully assembled DNA/PNA/Neutravidin complex that acts as the scaffold+fusion molecule.
• DNA/PNA/Neutravidin complexes can be detected with a nanopore.
• the fusion molecule contains two separate domains, one that binds a unique DNA sequence and another that binds to an anti-Neutravidin antibody.
• the DNA binding domain is a protein nucleic acid molecule (PNA) that binds to the unique sequence (GAAAGTGAAAGT, uSeql ) that is repeated 25 times throughout the scaffold (FIG. 15b).
• PNA molecules are similar to oligonucleotides having
• A,T,C,G bases which are capable of pairing with their complementary sequences, but instead of a phosphate backbone like typical oligonucleotides, the backbone is protein. This eliminates the negative charge provided by the phosphate backbone, and thus, PNA molecules can incorporate into dsDNA by displacing the complementary DNA strand, making a new DNA/PNA hybrid for the short stretch that encompasses the PNA molecule.
• the PNA used in the experiment had the sequence GAA * AGT * GAA * AGT where the * indicates that a biotin was incorporated into the PNA backbone at the gamma position by coupling to a Lysine amino acid, and thus, each PNA has three biotin molecules (PNABio).
• a 60 nM scaffold is heated to 95C for 2 minutes, cooled to 60C and incubated with a 10x excess of PNA to possible binding sites in 15 mM NaCI for 1 hr and then cooled to 4C.
• the excess PNA is dialyzed out (20k MWCO, Thermo Scientific) for 2 hrs against 10 mM Tris pH 8.0.
• This DNA/PNA complex is then labeled with a 10 fold excess Neutravidin protein (Pierce/Thermo Scientific) to possible biotin sites (assuming a 60% reduction of PNA during dialysis).
• the reaction is electrophoresed as described above to assess purity, concentration, and potential aggregation.
• This reagent, DNA/PNA/Neutravidin (D/P/N) is stored at -20C until use.
• FIGs. 17a-b show data comparing ⁇ vs. tD distributions from three separate experiments: DNA alone, Neutravidin alone, and D/P/N reagents.
• events in the D/P/N experiment are most likely attributed to D/P/N complexes (FIG. 16d), providing a simple criteria for tagging events as fusion molecule bound (i.e., scaffold with PNA and Neutravidin bound). Specifically, we can flag an event as being "fusion-molecule bound” if

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