Nothing Special   »   [go: up one dir, main page]

WO2010082860A1 - Procédé et dispositif de détection, basée sur les nanopores, des interactions protéine/protéine dans des molécules uniques - Google Patents

Procédé et dispositif de détection, basée sur les nanopores, des interactions protéine/protéine dans des molécules uniques Download PDF

Info

Publication number
WO2010082860A1
WO2010082860A1 PCT/PT2009/000005 PT2009000005W WO2010082860A1 WO 2010082860 A1 WO2010082860 A1 WO 2010082860A1 PT 2009000005 W PT2009000005 W PT 2009000005W WO 2010082860 A1 WO2010082860 A1 WO 2010082860A1
Authority
WO
WIPO (PCT)
Prior art keywords
nanopore
protein
polymer
molecule
complex
Prior art date
Application number
PCT/PT2009/000005
Other languages
English (en)
Inventor
Yann Astier
José BRAGANÇA
Original Assignee
Instituto De Biologia Experimental E Tecnologia (Ibet)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Instituto De Biologia Experimental E Tecnologia (Ibet) filed Critical Instituto De Biologia Experimental E Tecnologia (Ibet)
Priority to PCT/PT2009/000005 priority Critical patent/WO2010082860A1/fr
Publication of WO2010082860A1 publication Critical patent/WO2010082860A1/fr

Links

Classifications

    • 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
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures

Definitions

  • the present invention relates to a method and a device for nanopore based single-molecule protein/protein interaction detection. applied to bio, and chemical sensing.
  • US2008187915 discloses a method for controlling a position of a linear charged polymer inside a nanopore, comprising the steps of: using electrostatic control to position a linear charged polymer inside a nanopore; and creating an electrostatic potential well inside the nanopore, wherein the electrostatic potential well controls a position of the linear charged polymer inside the nanopore.
  • the linear charged polymer may comprise DNA
  • the performing one or more characterization activities may comprise DNA sequencing.
  • EP1956367 discloses a method for determining the duration of interaction of a polymer with a nanopore, the method comprising the steps of: (a) providing a structure comprinsing a nanopore connecting a first pool and a second pool, wherein a copolymer to be analyzed is placed in the first pool; b) causing the polymer to traverse the ion permeable passage; and c) measuring the duration of step (b) . The measuring detects an electrical property of the structure, while the polymer traverses the nanopore.
  • the structure may comprise a lipid bilayer.
  • the structure futher comprises a molecular motor, which may comprise a DNA polymerase, a RNA polymerase, an exonuclease, or a ribosome.
  • the structure may further comprise a DNA binding protein.
  • the polymer may comprise a polynucleotide.
  • the nanopore has diameter of about 1,0 to 4,0 nanometers or 0,5 to 2 nanometers.
  • US2007042366 (Al) relates to devices and systems embodying one or more solid-state nanopores that can be used to sense and/or characterize single macromolecules as well as sequencing DNA or RNA.
  • the devices and systems of the invention can be used in a variety of applications involving, but not limited to single-molecule biophysics, molecular biology, and biochemistry.
  • nanopore devices and systems of the invention are contemplated for use as a molecular comb to probe the secondary structure of RNA molecules, for use in detecting biological warfare agents, and contaminants/pollutants in air and/or water.
  • a device including a member of an insulating material, wherein the insulating member is configured and arranged so as to include a through aperture comprising a nanopore therein.
  • the through aperture comprises a plurality of crystals that have been cleaved to form atomically sharp edges, which crystals are arranged in fixed relation while forming the insulating material.
  • the crystal edges cross each other at a predetermined angle, more particularly an angle of about 90 degrees. It is within the scope of the invention, however, for the crystal edges to be crossed each other at an angle of less than or more than 90 degrees thereby forming nanopores having different cross-sections or different crosssectional shapes.
  • the crossing cleaved crystal edges essentially form or define an area that is small enough that the molecules making up the insulating material cannot enter into this area.
  • the molecules of the insulating material should be oriented with respect to the cleaved edges and thus forced to make a contour around the crossing point, thereby leaving a small hole comprising the through aperture therein.
  • the insulating material is a material that is characterized as being flowable and which solidifies at the end of the process for forming the nanopore as well as during normal operating conditions
  • the insulating member is made from a curable polymer such as liquid PDMS (poly-dimethylsiloxane) , polystyrene or PMMA and GaAs crystals are used to form the nanopore through aperture.
  • PDMS poly-dimethylsiloxane
  • PMMA polystyrene
  • GaAs crystals are used to form the nanopore through aperture.
  • the curable polymer liquid PDMS (poly-dimethylsiloxane) is poured into the region of the cutting edges and cured while the two crystal edges are held fixed. As indicated herein, at the crossing point between the two edges, the distance between the edges is so small that no molecules of the polymer can enter this region.
  • the molecules of the polymer also are advantageously oriented parallel to the respective cleaved edges and be forced to make a contour around the crossing point, leaving a small hole. In such a methodology, the width and the length of the nanopore are controlled by the distance between the two edges and the diameter of polymers. Following curing or formation of the insulating member in its final form, the crystals are removed.
  • nanopore device of the present invention to perform any of a number of analytical processes including but not limited to characterizing biomolecules, sequencing DNA and determining RNA secondary structures.
  • Such methods include providing an insulating member as herein above described including a nanopore, wherein a diameter and length of the nanopore are defined by the sharp edges of cleaved crystals that are maintained in fixed relation during the formation of the insulating member and locating the insulating member so as to be disposed between two ionic reservoirs.
  • Such methods further include passing the biomolecules or DNA through the nanopore and characterizing the biomolecule or the DNA based on changes in ionic current or other physical parameter.
  • the method further includes operably coupling one end of the RNA to an optical tweezer and measuring a force at said one end as the RNA molecule is pulled hrough the nanopore.
  • WO 2008124107 (Al) provides for devices and methods that can detect and control an individual polymer in a mixture is acted upon by another compound, for example, an enzyme, in a nanopore. The devices and methods are also used to determine rapidly ( ⁇ >50 Hz) the nucleotide base sequence of a polynucleotide under feedback control or using signals generated by the interactions between the polynucleotide and the nanopore.
  • the invention is of particular use in the fields of molecular biology, structural biology, cell biology, molecular switches, molecular circuits, and molecular computational devices, and the manufacture thereof.
  • the subject devices comprise cis and trans chambers connected by an electrical communication means.
  • the cis and trans chambers are separated by a thin film comprising at least one pore or channel.
  • the devices further comprise a means for applying an electric field between the cis and the trans chambers.
  • the pore or channel is shaped and sized having dimensions suitable for passaging a polymer. In one preferred embodiment the pore or channel accommodates a part but not all of the polymer.
  • the polymer is a polynucleotide. In an alternative preferred embodiment, the polymer is a polypeptide.
  • Other polymers provided by the invention include polypeptides, phospholipids, polysaccharides, and polyketides.
  • the thin film further comprises a compound having a binding affinity for the polymer.
  • the compound is a channel.
  • the channel has biological activity.
  • the compound comprises the pore.
  • the compound comprises enzyme activity.
  • the enzyme activity can be, for example, but not limited to, enzyme activity of proteases, kinases, phosphatases, hydrolases, oxidoreductases, isomerases, transferases, methylases, acetylases, ligases, lyases, and the like.
  • the enzyme activity can be enzyme activity of DNA polymerase, RNA polymerase, endonuclease, exonuclease, DNA ligase, DNase, uracil-DNA glycosidase, ribosomes, kinase, phosphatase, methylase, acetylase, or the like.
  • the pore is sized and shaped to allow passage of an activator.
  • the pore is sized and shaped to allow passage of a cofactor.
  • the pore or channel is a pore molecule or a channel molecule and comprises a biological molecule, or a synthetic modified molecule, or altered biological molecule, or a combination thereof.
  • the biological molecule is [alpha] -hemolysin.
  • the compound comprises non-enzyme biological activity.
  • the compound can have antigenic activity.
  • the compound can have selective binding properties whereby the polymer binds to the compound under a particular controlled environmental condition, but not when the environmental conditions are changed. Such conditions can be, for example, but not limited to, change in [H ⁇ +>] , change in environmental temperature, change in stringency, change in hydrophobicity, change in hydrophilicity, or the like.
  • the invention provides a compound, wherein the compound further comprises a linker molecule.
  • the thin film comprises a plurality of pores.
  • the device comprises a plurality of electrodes.
  • the invention provides a method for controlling binding of an enzyme to a polymer, the method comprising: providing two separate, adjacent pools of a medium and an interface between the two pools, the interface having a channel so dimensioned as to allow sequential monomer-by-monomer passage from one pool to the other pool of only one polymer at a time; providing an enzyme having binding activity to a polymer; introducing the polymer into one of the two pools; introducing the enzyme into one of the two pools; applying a potential difference between the two pools, thereby creating a first polarity; reversing the potential difference a first time, thereby creating a second polarity; reversing the potential difference a second time to create the first polarity, thereby controlling the binding of the enzyme to the polymer.
  • the medium is electrically conductive. In a more preferred embodiment, the medium is an aqueous solution. In another preferred embodiment, the method further comprises the steps of measuring the electrical current between the two pools; comparing the electrical current value (I,) obtained at the first time the first polarity was induced with the electrical current value (12) obtained at the time the second time the first polarity was induced; and determining the difference between Il and 12 thereby obtaining a difference value dl.
  • the method further comprises the steps of measuring the electrical current between the two pools; comparing the electrical current value (I) obtained at the first time the first polarity was induced with the electrical current value (12) obtained at a later time and determining the difference between Il and 12 thereby obtaining a difference value dl .
  • the method further comprises the steps of providing reagents that initiate enzyme activity; introducing the reagents to the pool comprising the polynucleotide complex; and incubating the pool at a suitable temperature.
  • the invention provides a method for controlling binding of an enzyme to a partially doublestranded polynucleotide complex, the method comprising: providing two separate, adjacent pools of a medium and an interface between the two pools, the interface having a channel so dimensioned as to allow sequential monomer-by-monomer passage from one pool to the other pool of only one polynucleotide at a time; providing an enzyme having binding activity to a partially double-stranded polynucleotide complex; providing a polynucleotide .
  • the complex comprising a first polynucleotide and a second polynucleotide, wherein a portion of the polynucleotide complex is double-stranded, and wherein the first polynucleotide further comprises a moiety that is incompatible with the second polynucleotide; introducing the polynucleotide complex into one of the two pools; introducing the enzyme into one of the two pools; applying apotential difference between the two pools, thereby creating a first polarity; reversing the potential difference a first time, thereby creating a second polarity; reversing the potential difference a second time to create the first polarity, thereby controlling the binding of the enzyme to the partially double-stranded polynucleotide complex.
  • the method further comprises the steps of measuring the electrical current between the two pools; comparing the electrical current value obtained at the first time the first polarity was induced with the electrical current value obtained at the time the second time the first polarity was induced.
  • the method further comprises the steps of measuring the electrical current between the two pools; comparing the electrical current value obtained at the first time the first polarity was induced with the electrical current value obtained at a later time.
  • the method further comprises the steps of providing at least one reagent that initiates enzyme activity; introducing the reagent to the pool comprising the polynucleotide complex; and incubating the pool at a suitable temperature.
  • WO2008124706 discloses a system for recognition of a translocating polymeric target molecule, which includes a device having at least one constriction that is sized to permit translocation of only a single copy of the molecule.
  • a pair of spaced apart sensing electrodes borders the constriction, which may be a nanopore.
  • the first electrode is connected to a first affinity element and the second electrode is connected to a second affinity element.
  • Each affinity element may be connected to its corresponding electrode via one or more intermediary compounds, such as a linker molecule and/or an electrode attachment molecule.
  • the first and second affinity elements are configured to temporarily form hydrogen bonds with first and second portions of the target molecule as the latter passes through the constriction.
  • the electrodes, affinity elements and first and second portions of the target molecule complete an electrical circuit and allow a measurable electrical current to pass between the first and second electrodes.
  • the time-varying nature of this electrical current, and the specific affinity elements employed, allow one to characterize the target molecule. All these patents focus on the nanopore based detection and characterisation of a polymer that is caught or is threading inside a nanopore.
  • the problem to be solved by the present invention is to provide a physical measurement to detect the interaction between two nanosized biological molecules at the single molecule level.
  • This technology has the capacity to detect picomolar to nanomolar concentrations of analyte with 100% fiability.
  • the need for this technology is driven by the demand for Point of Care biological sensors that can assess blood levels near the patient without need for a laboratory treatment of the samples. Also a growing demand for trace level detectors of improvised explosive devices, and biological threats can be satisfied by the present invention.
  • the solution is based on that the present inventors have identified that by adding an electrostatically charged polymer chain or polymer chain complex to a molecule by chemical or genetic methods greatly increases the electrophoretic migration of the molecule.
  • the charged polymer acts as an electrophoretic migration motor and is used to drive the complex into a nanopore by applying a potential field.
  • the value of the electric field required to hold the charged linear polymer in the nanopore is dependent on the size, mass, density and charge of the compound attached to the polymer.
  • a first aspect of the invention relates to a method for nanopore based single-molecule protein/protein interaction detection characterized in that it comprises the steps of a) chemically or genetically modifying a compound by adding an electrostatically charged polymer chain or polymer chain complex; b) applying a potential field for driving the polymer modified compound into a nanopore; c) gradually deacreasing the potential until the polymer exits the nanopore; d) determining the value potential at which the polymer chain vacates the nanopore.
  • Another aspect of the invention is related to a device for nanopore based single-molecule protein/protein interaction detection characterized in that it comprises two chambers of aqueous electrolyte separated by a membrane; a nanopore inserted in the membrane, wherein the nanopore connects the two chambers; a control unit to perform trans-membrane ionic current measurements for monitoring the the change in ion conductance of a single nanopore when a polymer modified compound interacts with the nanopore inner channel conductance.
  • the single molecule character of the measurement affords a very high sensivity (10-100 nM) and a great fiability of the result by numerous repeats of the measurement on the same sample.
  • the present invention focuses on the reactions involving an analyte attached to the polymer taking place outside the nanopore.
  • Figure 1 A charged polymer labeled molecule is held at the entrance of the nanopore ⁇ HL.
  • the polymer threads through the nanopore under the effect of an electric field.
  • the model was obtained by clipping the whole protein structure from its published pdb file in MacPyMol.
  • complex 1 is a biotinylated ssDNA complexed with a streptavidin
  • complex 2 is a biotinylated ssDNA complexed with a streptavidin, and its anti-streptavidin antibody (not at scale)
  • complex 3 is a biotinylated ssDNA complexed with a streptavidin attached to a 50 run diameter polystyrene bead (not at scale) .
  • FIG. 3 A-ionic current recording of a single ⁇ HL pore (black) .
  • the experiment is carried out at pH 8.1 Tris-HCl 0.1M, 2M KCl.
  • a drop in current intensity is labeled El when the complex is captured by the nanopore, while voltage (dotted) is increasing, and a rise in current intensity labeled E2 is observed when the complex is released, while voltage is decreased.
  • C- show the same potential scan in the presence of 90 nM complex 2 in the cis chamber.
  • D- shows a typical trace in the presence of 100 nM complex 3 in the cis chamber.
  • Figure 4 - A- shows a current trace from a 4 nm diameter Si3N4 based nanopore at 100 mV in pH 8.1 Tris-HCl 2M KCl.
  • B- is the trace from the same nanopore once complex 1 is captured in the artificial pore.
  • Molecules will migrate under an electric field according to their electrochemical properties.
  • the shape of the molecule is also known to play a role in the capacity of the protein to migrate.
  • the charge and density of molecules are used to separate molecules from a mix under an applied potential.
  • the charged polymer acts as an electrophoretic migration motor.
  • the present invention combines the use of a charged polymer to drive a target molecule to the entrance of a nanopore, for the charged polymer to thread through the pore, and the molecule attached to the polymer to be held at the entrance of the nanopore as a dumbbell.
  • Trans-membrane ionic current measurements can be used to monitor the ion conductance of a single nanopore. Dissolved analytes can be detected if they enter and interact with the nanopore inner channel by altering its conductance. Depending on the pore dimensions, single molecules can be detected in this way.
  • nanopores are an emerging class of single-molecule sensors.
  • a large drop in ionic conductivity is observed.
  • the physical properties of the molecule attached at the end of the polymer can be probed by the electric force required to keep the polymer threaded through the pore.
  • the field potential can be scanned to assess at which potential the complex is no longer held inside the pore, when the electric force applied to the polymer charges is too weak to hold the complex in the nanopore, the polymer comes out of the nanopore, and the original pore conductance is restored.
  • compounds such as but not limited to: chemical molecule, protein, protein complex, nanoparticle, oligosaccharides, antibodies or or antibody fragment AKA Fab are chemically or genetically modified by adding an electrostatically charged polymer chain or polymer chain complex.
  • the charges on the polymer chain or polymer chain complex are used to drive the complex into a nanopore by applying a potential field.
  • the nanopore diameter is superior to the polymer chain diameter, and inferior to the diameter of the analyte or combination of analytes attached at the end of the chain.
  • the ionic current flowing through the pore reveals whether the nanopore is open, or if a polymer is engaged and obstructs the ionic flow through the nanopore.
  • the potential is decreased gradually until the polymer exitss the nanopore.
  • the value of the potential at which the polymer chain vacates the pore is related to the size, density and charge of the analyte the polymer chain is attached to.
  • the inventors have shown how an individual streptavidin attached to a biotinylated 60 bases single stranded DNA can be held with the ssDNA threaded through the ⁇ -Hemolysin nanopore at a potential below 10 mV.
  • an anti-streptavidin antibody binds to the streptavidin, the same complex will exit from the pore below 70 mV.
  • the streptavidin is attached to a 50 nm diameter polystyrene nanobead, the complex escapes the pore below 30 mV.
  • the bacterial pore-forming toxin staphylococcal ⁇ - hemolysin is an ideal system to work at the single molecule level, given its ability to insert into a suspended lipid bilayer separating two ionic solutions.
  • a high-resolution three-dimensional structure of the pore is available ( Figure 1) . From top to bottom, the pore entrance (2.9 ran) opens into an inner cavity (4,5 niti diameter) followed by a constriction (1,5 nin) , and the trans-membrane barrel (2 nm diameter) .
  • Solid-state nanopores can be fabricated in thin Si3N 4 and Si ⁇ 2 membranes, using either an Ar+ beam 16 ' 71 or an electron beam (e-beam) in a transmission electron microscope (TEM) .
  • Solid-state nanopores size can be tuned with nanometer precision. They exhibit an increased mechanical, chemical, and electrical stability, and can be exposed to a wide range of potential, temperature, and solvents. In addition to ion current measurements, they can be integrated into devices compatible with single molecule sensing optical methods.
  • ⁇ HL is a heptameric protein pore ( Figure 1) , for which the
  • ⁇ HL X-ray structure was resolved. 191
  • the ability of ⁇ HL to insert into an insulating lipid bilayer separating two chambers of aqueous electrolyte has made it a useful system to measure the ionic current that passes through the pore under an applied potential.' 10 ' u)
  • an analyte interacts with a binding site within the pore, a significant change in the pore conductivity is observed.
  • the extent and duration of the current block from each binding event help reveal the identity of the analyte, while the frequency of the binding events reveal the analyte concentration.
  • ⁇ HL is very amenable to genetic modifications.
  • ⁇ HL bind molecular adapters ( ⁇ , ⁇ , and ⁇ -cyclodextrins) affords great precision and control over atomic scale modifications that can be engineered in the nanopore.
  • Chemical and bio-molecular engineering of ⁇ HL permit the stochastic sensing of molecules by tailoring internal analyte binding sites. 1111 Detection, quantification, and characterization of ssRNA and ssDNA were described, and lead to "Nanopore Force Spectroscopy”.
  • [12 ⁇ 161 Single-base mismatch in a short polynucleotide strand, toxic metal ions, drugs, enantiomers, TNT, and nucleotides have also been specifically detected.
  • the complexes captured in the ⁇ HL nanopore were:
  • Complex 1 is a biotinylated ssDNA complexed with a streptavidin
  • Complex 2 is a biotinylated ssDNA complexed with a streptavidin, and its anti-streptavidin antibody (not at scale) ;
  • Complex 3 is a biotinylated ssDNA complexed with a streptavidin attached to a 50 nm diameter polystyrene bead (not at scale) .
  • Each construct was designed to have 60 negative charges on the polymer chain, and different nanocomplex densities tethered to the charged polymer.
  • Figure 3 shows the conductance recording of a single ⁇ HL nanopore, when the potential is ramped from 0 to 200 mV and back down to 0 mV at 40 mV per second in 2M KCl.
  • the capture and release of complexes 1 to 3 depend on the nature of the protein, or molecular assembly attached to the DNA.
  • the control shows the pore conductance behaviour when no polymer modified analyte is present in the solution.
  • (B) shows the capture of complex 1 (El) and its release (E2) .
  • C shows the capture and release of complex 2, and D, that complex 3.
  • Complex 1 is very stable when captured by the nanopore, and its release is only ever observed when the field potential is lowered below 10 mV (100% of E2 events occur below 10 mV; n>1000) .
  • Complex 2 is the least stable inside the pore, and is systematically seen to exit the pore between 140 and 70 mV(95% of releases (E2) occur by 70 mV; n>150) .
  • Complex 3 is more stable than complex 2, and exits the pore between 40 and 20 mV in 100% of cases (n>150) .
  • Reagents were obtained as follows: 1,2- diphytanoylsn-glycero-3-phosphocholine (Avanti Polar Lipids); pentane (Fluka) ; hexadecane (99+%, Sigma-Aldrich); Trizma base (99.9%, Sigma-Aldrich); concentrated HCl (analytical reagent grade, Fisher Scientific) ; potassium chloride (99%, Panreac) ; GoId(III) chloride trihydrate (99.9+%, Sigma-Aldrich); Sodium borohydride (ReagentPlus ® , 99%, Sigma-Aldrich); Sodium 3- mercapto-1-propanesulfonate (MPSA), (technical grade, 90%, Sigma-Aldrich); 1-Octanethiol, 98.5+%, Sigma-Aldrich); Streptavidin, 99%, Sigma-Aldrich. Mouse monoclonal [S10D4] to Str
  • a bilayer of 1, 2-diphytanoyl-sn- glycero-3-phosphocholine (Avanti Polar Lipids) was formed on an aperture 100-150 ⁇ m in diameter in a polycarbonate film (20- ⁇ m thickness from Goodfellow, Malvern, PA) that divided a planar bilayer chamber into two compartments, cis and trans. Both compartments contained 0.4 mL of buffer.
  • Controls ssDNA-biotin were added separately to the trans compartment, and no current block was observed under positive potential.
  • the pdb file of the heptameric D-HL pore (PDB file 7AHL) was opened in MacPyMol (2006 DeLano Scientific LLC) .
  • the model of ⁇ -HL is displayed in MacPyMol as an orange surface representation, the clipping command was used to bring the clipping plane where ⁇ -HL is cut in its middle.
  • Artificial nanopores Si 3 N 4 based artificial pores can be reproducibly manufactured down to 2 nm in diameter by focused electron beam perforation of a 30 nm thick film. These pores can be chemically treated to display hydrophilic properties, and become permeable to aqueous solutions. TEM tomography studies of these artificial nanopores revealed an hourglass nanopore profile. Besides aqueous media, Si 3 N 4 pores are very stable in organic solvents, and ionic liquids.
  • the present invention shows how nanopores can help reveal the nature of a nano-complex tethered to a charged polymer.
  • the single molecule character of the measurements affords a very high sensitivity (10-100 nM) and a great fiability of the result by numerous repeats of the measurement on the same sample.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Nanotechnology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé et un dispositif de détection, basée sur les nanopores, des interactions protéine/protéine dans des molécules uniques. L'invention combine l'utilisation d'un polymère chargé pour conduire la molécule cible jusqu'à l'entrée d'un nanopore, afin que le polymère chargé passe à travers le pore, et que la molécule fixée au polymère soit maintenue à l'entrée du nanopore comme un haltère. Il est possible d'utiliser des mesures du courant ionique transmembranaire pour surveiller la conductance ionique d'un seul nanopore. Il est possible de détecter les analytes dissous s'ils entrent dans le canal interne des nanopores et interagissent avec celui-ci par modification de sa conductance. Selon la dimension des pores, les molécules uniques peuvent être détectées de cette manière.
PCT/PT2009/000005 2009-01-19 2009-01-19 Procédé et dispositif de détection, basée sur les nanopores, des interactions protéine/protéine dans des molécules uniques WO2010082860A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/PT2009/000005 WO2010082860A1 (fr) 2009-01-19 2009-01-19 Procédé et dispositif de détection, basée sur les nanopores, des interactions protéine/protéine dans des molécules uniques

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/PT2009/000005 WO2010082860A1 (fr) 2009-01-19 2009-01-19 Procédé et dispositif de détection, basée sur les nanopores, des interactions protéine/protéine dans des molécules uniques

Publications (1)

Publication Number Publication Date
WO2010082860A1 true WO2010082860A1 (fr) 2010-07-22

Family

ID=40957598

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/PT2009/000005 WO2010082860A1 (fr) 2009-01-19 2009-01-19 Procédé et dispositif de détection, basée sur les nanopores, des interactions protéine/protéine dans des molécules uniques

Country Status (1)

Country Link
WO (1) WO2010082860A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013012881A3 (fr) * 2011-07-20 2013-03-28 The Regents Of The University Of California Dispositif à deux pores
EP2880186A1 (fr) * 2012-08-03 2015-06-10 University of Washington through its Center for Commercialization Compositions et procédés pour améliorer le séquençage de nanopores
EP2814939A4 (fr) * 2012-02-16 2016-02-24 Univ California Détecteur de nanopore pour la translocation de protéine à médiation par enzyme
JP2017106933A (ja) * 2012-02-16 2017-06-15 ジニア テクノロジーズ， インコーポレイテッド ナノ細孔センサーとともに使用するための二重層を作製するための方法
US20180209953A1 (en) * 2013-03-05 2018-07-26 Arizona Board Of Regents On Behalf Of Arizona State University Translocation of a polymer through a nanopore
CN108474760A (zh) * 2015-11-23 2018-08-31 双孔人公司 用于追踪和检测的靶修饰
WO2020105318A1 (fr) * 2018-11-21 2020-05-28 株式会社日立製作所 Dispositif d'analyse de biomolécules et procédé d'analyse de biomolécules
WO2022140655A1 (fr) * 2020-12-23 2022-06-30 The Trustees Of Columbia University In The City Of New York Immunoessais par nanopores multiplexes électroniques à molécule unique permettant la détection de biomarqueurs
US12105079B2 (en) 2018-09-11 2024-10-01 Rijksuniversiteit Groningen Biological nanopores having tunable pore diameters and uses thereof as analytical tools

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000034527A2 (fr) * 1998-12-11 2000-06-15 The Regents Of The University Of California Codes a barres moleculaires cibles et procedes d'utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000034527A2 (fr) * 1998-12-11 2000-06-15 The Regents Of The University Of California Codes a barres moleculaires cibles et procedes d'utilisation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHANDLER EMILY L ET AL: "Membrane surface dynamics of DNA-threaded nanopores revealed by simultaneous single-molecule optical and ensemble electrical recording.", LANGMUIR : THE ACS JOURNAL OF SURFACES AND COLLOIDS 3 FEB 2004, vol. 20, no. 3, 3 February 2004 (2004-02-03), pages 898 - 905, XP007909662, ISSN: 0743-7463 *
GERSHOW MARC ET AL: "Recapturing and trapping single molecules with a solid-state nanopore", NATURE NANOTECHNOLOGY, NATURE PUBLISHING GROUP, GB, vol. 2, no. 12, 1 December 2007 (2007-12-01), pages 775 - 779, XP007907683, ISSN: 1748-3395 *
WINTERS-HILT S: "The [alpha]-Hemolysin nanopore transduction detector - Single-molecule binding studies and immunological screening of antibodies and aptamers", BMC BIOINFORMATICS 20071101 GB, vol. 8, no. SUPPL. 7, 1 November 2007 (2007-11-01), XP021027690, ISSN: 1471-2105 *

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013012881A3 (fr) * 2011-07-20 2013-03-28 The Regents Of The University Of California Dispositif à deux pores
AU2012284137B2 (en) * 2011-07-20 2013-11-28 The Regents Of The University Of California Dual-pore device
US8961763B2 (en) 2011-07-20 2015-02-24 The Regents Of The University Of California Dual-pore device
CN103328973A (zh) * 2011-07-20 2013-09-25 加利福尼亚大学董事会 双孔装置
US9863912B2 (en) 2011-07-20 2018-01-09 The Regents Of The University Of California Dual-pore device
CN103328973B (zh) * 2011-07-20 2015-04-01 加利福尼亚大学董事会 双孔装置
AU2012284137C1 (en) * 2011-07-20 2017-02-02 The Regents Of The University Of California Dual-pore device
JP2017106933A (ja) * 2012-02-16 2017-06-15 ジニア テクノロジーズ， インコーポレイテッド ナノ細孔センサーとともに使用するための二重層を作製するための方法
EP2814939A4 (fr) * 2012-02-16 2016-02-24 Univ California Détecteur de nanopore pour la translocation de protéine à médiation par enzyme
US11339365B2 (en) 2012-02-16 2022-05-24 The Regents Of The University Of California Nanopore sensor for enzyme-mediated protein translocation
EP2880186A4 (fr) * 2012-08-03 2016-04-27 Univ Washington Ct Commerciali Compositions et procédés pour améliorer le séquençage de nanopores
US20150191782A1 (en) * 2012-08-03 2015-07-09 University Of Washington Through Its Center For Commercialization Compositions and methods for improving nanopore sequencing
US10858700B2 (en) 2012-08-03 2020-12-08 University Of Washington Through Its Center For Commercialization Compositions and methods for improving nanopore sequencing
US11821033B2 (en) 2012-08-03 2023-11-21 University Of Washington Compositions and methods for improving nanopore sequencing
EP2880186A1 (fr) * 2012-08-03 2015-06-10 University of Washington through its Center for Commercialization Compositions et procédés pour améliorer le séquençage de nanopores
EP3511424A1 (fr) * 2012-08-03 2019-07-17 University Of Washington Through Its Center For Commercialization Compositions et procédés pour améliorer le séquençage des nanopores
US20180209953A1 (en) * 2013-03-05 2018-07-26 Arizona Board Of Regents On Behalf Of Arizona State University Translocation of a polymer through a nanopore
US10267785B2 (en) * 2013-03-05 2019-04-23 Arizona Board Of Regents On Behalf Of Arizona State University Translocation of a polymer through a nanopore
EP3380834A4 (fr) * 2015-11-23 2019-08-21 Ontera Inc. Modification de cible pour le suivi et la détection
CN108474760A (zh) * 2015-11-23 2018-08-31 双孔人公司 用于追踪和检测的靶修饰
US12105079B2 (en) 2018-09-11 2024-10-01 Rijksuniversiteit Groningen Biological nanopores having tunable pore diameters and uses thereof as analytical tools
WO2020105318A1 (fr) * 2018-11-21 2020-05-28 株式会社日立製作所 Dispositif d'analyse de biomolécules et procédé d'analyse de biomolécules
JP2020085578A (ja) * 2018-11-21 2020-06-04 株式会社日立製作所 生体分子分析装置及び生体分子分析方法
JP7132100B2 (ja) 2018-11-21 2022-09-06 株式会社日立製作所 生体分子分析装置及び生体分子分析方法
WO2022140655A1 (fr) * 2020-12-23 2022-06-30 The Trustees Of Columbia University In The City Of New York Immunoessais par nanopores multiplexes électroniques à molécule unique permettant la détection de biomarqueurs

Similar Documents

Publication Publication Date Title
Davenport et al. The role of pore geometry in single nanoparticle detection
US7777505B2 (en) Nanopore platforms for ion channel recordings and single molecule detection and analysis
Holden et al. Resistive pulse analysis of microgel deformation during nanopore translocation
WO2010082860A1 (fr) Procédé et dispositif de détection, basée sur les nanopores, des interactions protéine/protéine dans des molécules uniques
Nakane et al. Nanopore sensors for nucleic acid analysis
Rhee et al. Nanopore sequencing technology: nanopore preparations
Gu et al. Single molecule sensing by nanopores and nanopore devices
Oukhaled et al. Dynamics of completely unfolded and native proteins through solid-state nanopores as a function of electric driving force
Sexton et al. An adsorption-based model for pulse duration in resistive-pulse protein sensing
Howorka et al. Nanopore analytics: sensing of single molecules
Uram et al. Noise and bandwidth of current recordings from submicrometer pores and nanopores
Wang et al. Nanopore stochastic detection: diversity, sensitivity, and beyond
Actis et al. Functionalized nanopipettes: toward label-free, single cell biosensors
Blundell et al. Emergence of tunable resistive pulse sensing as a biosensor
Shoji et al. Spatially resolved chemical detection with a nanoneedle-probe-supported biological nanopore
Wu et al. Translocation of rigid rod-shaped virus through various solid-state nanopores
Gong et al. Label-free in-flow detection of single DNA molecules using glass nanopipettes
Ding et al. Internal vs fishhook hairpin DNA: unzipping locations and mechanisms in the α-hemolysin nanopore
Jeon et al. Electrostatic control of polymer translocation speed through α-hemolysin protein pore
Shi et al. Dynamics of a molecular plug docked onto a solid-state nanopore
Schmidt Membrane platforms for biological nanopore sensing and sequencing
Zhou et al. Ion channel probes for scanning ion conductance microscopy
Jiang et al. Nanopore-based sensing and analysis: beyond the resistive-pulse method
Shoji et al. Recessed Ag/AgCl microelectrode-supported lipid bilayer for nanopore sensing
Gibb et al. Single molecule ionic current sensing in segmented flow microfluidics

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09788426

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09788426

Country of ref document: EP

Kind code of ref document: A1