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
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
  • B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
  • B05D1/00—Processes for applying liquids or other fluent materials
  • B05D1/007—Processes for applying liquids or other fluent materials using an electrostatic field
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
  • C08J7/04—Coating
  • C08J7/0427—Coating with only one layer of a composition containing a polymer binder
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6816—Hybridisation assays characterised by the detection means
  • C12Q1/6825—Nucleic acid detection involving sensors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44704—Details; Accessories
  • G01N27/44747—Composition of gel or of carrier mixture
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44756—Apparatus specially adapted therefor
  • G01N27/44791—Microapparatus
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J2309/00—Characterised by the use of homopolymers or copolymers of conjugated diene hydrocarbons
  • C08J2309/02—Copolymers with acrylonitrile
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J2477/00—Characterised by the use of polyamides obtained by reactions forming a carboxylic amide link in the main chain; Derivatives of such polymers
  • C08J2477/02—Polyamides derived from omega-amino carboxylic acids or from lactams thereof

Definitions

• compositions, compounds, processes, and methods of use of 3D porous coating(s) on or near a nanopore(s) for analysis or detection of biologies such as nucleic acids, proteins, protein-nucleic acid complexes, small molecule-biological complexes, and/or polymer-biological complexes.
• the invention is based, at least in part, on the discovery that: 1) membranes or thin solid- state, polymeric, lipid, or solid-like films containing nanopores can be coated with fibers, gels, or other compositions to create a 3D porous structure above or below the membrane or both; 2) one or many nanopores in a given membrane can be coated to create a multiplexed system; 3) the coated nanopore alters the rate at which a biologic transits or translocates through the nanopore compared to a bare membrane; 4) the coating can be modified or derivatized to change its hydrophilicity/ hydrophobicity; 5) the coating can be modified or derivatized to introduce one or more targeting moiety that captures or binds an analyte for subsequent translocation through the nanopore; and 6) the act of translocating through the nanopore enables identification of the biologic as a change in electrical conductivity, fluid flow, refractive index, as measured by either electrical or optical means or both.
• One aspect of this invention is the use of the coated nanopores for detecting a specific nucleic acid sequence (e.g., DNA, RNA, mRNA, miRNA, etc) or protein (e.g., hemoglobin, insulin, antibody, etc).
• a further aspect of this invention is the use of the coated nanopores for sequencing nucleic acids or proteins.
• An additional aspect of this invention is the use of the coated nanopores to size nucleic acids or proteins for genomic, transcriptomic, or proteomic analysis.
• Another aspect of the coating is the use of stimuli responsive materials (heat, light, pH, redox, enzymatic, magnetic, etc.) to affect the properties of the coating.
• compositions, compounds, processes, and methods of use of 3D porous coated nanopores for analysis or detection of biologies Provided herein are compositions, compounds, processes, and methods of use of 3D porous coated nanopores for analysis or detection of biologies.
• the 3D coating provides a high surface area for interaction with the
• FIGS. 1A-1D demonstrates that the avidin functionalized electrospun mesh can be characterized with biotinylated fluorescein.
• FIG. 1A is a schematic diagram of the experiment illustrating biotinylated fluorescein binding the avidin coated mesh.
• FIG. IB shows a confocal microscopy image of a mesh coated in 1/10,000 vs. 1/100 equivalents avidin to carboxylic acid on the mesh surface.
• FIG. 1C is the fluorescence intensity increase with increasing amounts of avidin bound to the mesh surface. An adsorption control is shown (red square) which highlights the need for EDC coupling to covalently link the avidin to the fiber surface.
• FIG. ID is a biotin- fluorescein titration curve to quantify the avidin loading on the 1/250 equivalent mesh surface. The fluorescent signal begins to drop when there is no longer enough biotin- fluorescein to bind all of the biotin binding sites.
• FIG. 2 depicts scanning electron microscopy of poly(oxanorbornene-dicarboxamine- butyl) electrospun meshes fabricated using different solvent systems.
• FIG. 3 depicts the native chemical ligation chemistry utilized as a stimulus for the stimuli responsive separation of biotin and antibody (IgG).
• FIG. 4 demonstrates that a UV dose dependent wetting profile was observed with smaller UV doses wetting more slowly over time compared to larger UV doses (5.4 J/cm2 vs. 10.8 J/cm2 for 30 minutes and 60 minutes of UV exposure, respectively).
• the ACA decreased substantially over 600 seconds compared to the unexposed control meshes (ACA -20° vs -135°).
• Doubling the UV exposure time to 30 minutes resulted in more consistent ACAs and a fully wetted surface (ACA -0°) within 300 seconds.
• Maximum wetting rates were achieved with UV exposure times greater than 60 minutes where the meshes fully wetted within 150 seconds.
• FIGS. 5A-5C depict cell patterning on stimuli responsive polymer using ultraviolet light activated hydrophobic doping agent.
• the utility of the poly(glycerol 12-(l-(2- nitrophenyl)ethoxy)-12-oxododecanoic acid-co-caprolactone) photoinduced wetting method to print 3D hydrophilic cavities surrounded by hydrophobic regions for controlled cell patterning was evaluated.
• a circular photo mask (1590 ⁇ in diameter) was used to create 3D hydrophilic cavities of various depths within the hydrophobic bulk material by varying the UV exposure time (FIG. 5A).
• the aqueous CT contrast agent solution was restricted to the surface of the hydrophobic mesh.
• the aqueous solution penetrated into the cavities formed via photolysis.
• a linear relationship between the UV exposure time and the depth of the cavities was determined (FIG. 5B).
• a 150 ⁇ thick mesh and a 1590 ⁇ in diameter photomask was used to selectively expose a small circular region of the mesh to UV light and create a hydrophilic region.
• FIG. 6 depicts Tunneling Electron Microscope (TEM) image of nanopore taken after drilling.
• TEM Tunneling Electron Microscope
• FIG. 7 depicts the nanopore-nanofiber mesh (NP-NFM) sensor Top (right to left): Panels show a scanning electron micrograph of the NFM, a close-up schematic of the mesh near the nanopore with DNA translocating, and a transmission electron micrograph of the nanopore. Bottom: Schematic depiction of a nanopore sensor coated with an electrospun polymeric nano fiber mesh (NFM).
• NP-NFM nanopore-nanofiber mesh
• FIG. 8 depicts nanopore chips before and after electrospinning. Images of nanopore chips before (left) and after (right) fabrication, showing parallelization of electrospinning technique. Up to 50 chips may be spun at once using our current apparatus.
• FIGS. 9A-9B Contact angle characterization of NFM hydrophobicity.
• FIG. 9A A 4 ul droplet is placed on the mesh and measured according to the angle formed at the interface of the droplet with the surface.
• FIG. 9B Contact angle as a function of mesh composition. Please note that Fig. 9B presents the same data as shown in Fig. 4A (now presented as Fig. 24A) of the priority provisional application.
• FIG. 10 depicts SEM images of three selected NFM copolymer blends with a constant PCL weight %.
• FIG. 11 depicts SEM images of a 7:3 PCL:PGC-C18 NP-NFM devices at 100X, ⁇ , ⁇ , ⁇ , ⁇ , and 38J70X.
• the scale bars are 100 ⁇ , 10 ⁇ , 1 ⁇ , and 200 nm for the 100X, 1,000X, 10,000X, and 38J70X images, respectively.
• All NFM copolymer blends produce similar fiber diameters and bead morphology. Both micrometer and nanometer scale texture is produced by the NFMs allowing for the enhanced hydrophobicity.
• the SiN membrane is visible below approximately 3-4 layers of nanofibers (10,000X image) making the NFM approximately 1-2 ⁇ thick.
• FIG. 12 depicts I-V curves for coated and uncoated nanopores.
• FIG. 13 depicts current traces for DNA in an NP-NFM at 500 mV.
• These continuous current recordings for an NP-NFM (8:2 PCL:PGC-C 18) initially show a clean pore with a steady open pore current of ⁇ 4.5 nA. After adding 1000 bp DNA, transient drops in current indicate the passage of individual molecules through the nanopore. The DNA was rinsed out with a lOx wash, returning the current trace to its original clean and open state. Data was collected at 500 mV in a 1M: 1M KC1 buffer, pH 7.5. Current was recorded at 250 kHz and filtered at 100 kHz.
• FIGS. 14A-14B depicts a comparison of translocations in bare pore vs. NP-NFM.
• FIG. 14B depicts event diagrams for the corresponding full data sets and histograms of translocation time for same bare and coated pore with exponential decay to characterize translocation time. Coated nanopore requires a double exponential decay fit to accurately capture the distribution.
• FIG. 15 depict current blockage level for NP-NFM with varying chemical composition.
• Current blockage level IB for: Bare pore, PCL only, 9: 1 , 8:2, 7:3, 6:4, and 5:5 PCL:PGC-C18 blends. All data are for 1000 bp dsDNA in 4-4.5 nm nanopores at 300 mV (error bar: ⁇ ⁇ 95% fit confidence interval for Gaussian current level fits. Red, green, and blue pores correspond to the same experiments used in FIG. 12).
• FIG. 16 depict relative slowing factor for translocations in NP-NFM of various chemical composition.
• FIGS. 17A-17B depict event diagram for translocations of 0.5-20 kbp DNA.
• FIG 17A depicts an event diagram for five lengths of dsDNA (0.5, 1, 5, 10, and 20 kbp) translocating through a 6 nm nanopore coated with 7:3 PCL:PGC-C18. Ib is normalized for clarity.
• FIG 17B depicts a characteristic translocation time ⁇ for each DNA length (error bar: ⁇ ⁇ 95% fit confidence interval). Dotted line is a guide to the eye.
• FIGS. 18A-18B depicts translocations of 1 -20 kbp DNA in a bare nanopore.
• FIG. 18A depicts an event diagram for translocation of 1 kbp, 5 kbp, and 10 kbp DNA in a bare 6 nm diameter nanopore at 500 mV.
• FIG. 18B depicts a relative ⁇ for 1, 5, and 10 kbp in the 7:3 PCL:PGC-C18 coated nanopore, normalized by this bare pore data. Although a slight increase in this retardation factor is observed with increasing length, there is little or no increase within the fit error (error bars for 1 kbp are smaller than marker).
• FIG. 19 depicts on the left, sample nanopore length profiles for short DNA.
• FIG. 20 depicts a calibration of avidin translocation rates as a function of sample concentration through a 5 nm solid-state nanopore. Avidin is detectable down to ⁇ 10 pM.
• FIG. 21 depicts a schematic diagram of the proof of concept capture and release nanopore-nanofiber mesh protein detection assay.
• a biotinylated nanofiber mesh is coated in avidin which is then linked to a biotinylated capture anti-mouse antibody.
• the target antibody then binds followed by the second anti-mouse antibody which is functionalized to release avidin into solution upon the addition of a stimulus.
• This released avidin is detected in a nanopore only if the target molecule is present and it is released in an amount directly related to the amount of captured analyte.
• FIG. 22 depicts a calibration curve for detecting 20 kg/mol poly(acrylic acid) in a 5 nm solid-state nanopore. The event rate increases with increasing concentration of the polyelectrolyte.
• FIG. 23A depicts a schematic of voltage-clamp acquisition for a solid-state nanopore chip (not to scale). Measurements typically performed in 1M KC1 with 300 mV applied.
• FIG. 23B depicts a sample current trace for 1200 bp ds DNA passing through a nanopore showing current blockage IBL and dwell time tD.
• FIG. 23C depicts a synthetic scheme of PGC functionalized with a hydrophobic side chain.
• FIG. 23D is similar to Fig. 7 and depicts a cartoon representation of the nanopore-nano fiber mesh (NP-NFM) device.
• a nanopore sensor (gold) is coated with an electrospun polymeric nanofiber mesh (NFM). A DNA molecule threading through the pore is shown.
• Insets show electron micrographs of the NFM (left) and nanopore (right).
• FIG. 24B shows Scanning Electron Micrographs of a continuous nonwoven electrospun polymeric mesh at low and high magnification, respectively. Both the mesh density and the fiber diameter may be controlled independently to vary mesh properties. Current versus voltage curves measured for an uncoated nanopore and two NP-NFM devices (PCL only; 70:30 PCL:PGC). The identical conductance for all conditions indicates that the NFM coating does not alter the electrical properties of the nanopore.
• FIG. 25A shows a time of translocation PDF for NP-NFM devices with varying hydrophobicities.
• FIG. 26 shows translocation time PDF for varying lengths of DNA in a PCL-coated NP-NFM sensor.
• the distribution looks more Gaussian than for shorter DNA, and therefore can be modeled by the actual mean rather than a characteristic decay rate for the distribution tail.
• FIG. 27 shows current versus voltage curves measured for an uncoated (blue) and two NP-NFM devices (PCL only, green; 70:30 PCL:PGC, red). The identical conductance for all conditions indicates that the NFM coating does not alter the electrical properties of the nanopore
• the disclosure provides an article comprising: (i) a substrate having a first surface and as second surface; (ii) at least one nanopore extending through the substrate, thus forming a channel connecting from the first surface to the second surface of the substrate, wherein the nanopore has a first opening that opens to the first surface of the substrate and a second opening that opens to the second surface of the substrate; and (iii) a porous coating on at least one of the first or second surface of the substrate.
• the substrate can be configured to include more than one nanopore, or an array of nanopores. Each individual nanopore can be enclosed in an individual chamber and such individual chambers can be arranged in an array format.
• the porous coating can alter the rate at which a molecule transits or translocates through the nanopore compared to when no porous coating is present.
• the substrate is a membrane or thin solid-state, polymeric, lipid, or solid-like film.
• the substrate can comprise any suitable material.
• the substrate can be of a pure substance, a mixture or a composite.
• the substrate can comprise a semiconductor material, polymer material, lipid, quartz, glass, and the like.
• the substrate can be made of, for example, glass, Si, S1O 2 , SIN 4 , quartz, alumina, nitrindes, metals, polymers, or any combinations thereof.
• the substrate comprises an insulating material.
• Exemplary insulating materials include, but are not limited to, SIN, S1O 2 , AI 2 O3, T1O 2 , BaTl(3 ⁇ 4, PbTi0 3 , and the like.
• the substrate can comprise a semiconductor material, such as, but not limited to, silicon (Si), SiN, germanium (Ge), GaAs, GaN, and the like.
• a preferred material is SiN.
• the substrate can be of any desired thickness. In some embodiments of the various aspects disclosed herein, thickness of the substrate can range from about 5 nm to about 1000 nm.
• the substrate is electrically insulating, i.e., is an electrical insulator.
• the substrate comprises an opening defining a nanopore of a suitable size.
• the substrate comprises a first surface and a second surface.
• the first and the second surfaces can be arranged substantially parallel to one another.
• the nanopore forms a passage through the substrate from the first surface to the second surface.
• the nanopore has a first opening on the first surface of the substrate and a second opening on the second surface of the substrate.
• the openings defining the nanopore can be of any suitable shape, but are typically approximately circular.
• the substrate is referred to herein as having a "first" and "second" surface.
• Such references are merely for the purposes of explaining and illustrating the invention and various embodiments thereof. These references are not intended to imply any particular orientation in use, and are not intended to limit the invention in any other manner.
• the substrate comprising the one or more nanopores can divide a volume into two separate compartments, each of which can contain different types and/or concentrations of analytes.
• One or more nanopore(s) is the only passage between these two compartments.
• an electric field develops across the nanopore.
• the applied electric field acts as a force on charged molecules and ions inside the nanopore.
• nanopore in a substrate coated with a porous coating have the same conductivity as a those in lacking that porous coating but translocation dynamics are dependent upon the properties of the applied porous coating.
• characteristic translocation time for porous coating depends upon hydrophobicity of the coating. By optimizing the hydrophobicity of the coating, inventors demonstrated a retardation factor for the porous coating as compared to translocation through a bare nanopore from ⁇ 2 to more than two orders of magnitude.
• nanopore refers to a nanometer sized opening in the substrate, i.e., pores have a pore size in the nanometer range.
• the nanopore extends through the substrate and forms a channel connecting the first surface to the second surface, wherein the nanopore has a first opening that opens to the first surface of the substrate and a second opening that opens to the second surface of the membrane
• pore size refers to a diameter or an effective diameter of the cross-sections of the pores.
• the term “pore size” can also refer to an average diameter or an average effective diameter of the cross-sections of the pores, based on the measurements of a plurality of pores.
• the effective diameter of a cross-section that is not circular equals the diameter of a circular cross-section that has the same cross-sectional area as that of the non-circular cross- section.
• a “nanopore” includes a nanometer opening in solid state, polymeric, lipid, or alpha-hemolysin structure containing pores of 2 to 10000 nm in diameter.
• the term "size" as used herein refers to the mode of a size distribution of pores, i.e., the value that occurs most frequently in the size distribution.
• the pore size distribution of the nanopores can range from about 2 nm to about 10,000 nm.
• the nanopore diameter can range from about 5 nm to about 1000 nm, from 5 nm to about 500 nm.
• the nanopore diameter can range from about 2 nm to about 50 nm.
• the nanopore diameter can range from about 5 nm to about 20 nm. In one particular embodiment, the nanopore diameter is about 5 nm.
• a preferred nanopore size is between 4 and 10 nm.
• nanopores are nanometers to micrometers in width, it is possible that many thousands of them can be created on a single substrate such as a silicon micro-chip semiconductor wafer. Accordingly, a single substrate can hold hundreds or thousands of nanopores to simultaneously detect many different biological and/or chemical materials.
• nanopore is not intended that the term "nanopore” be limited to circular geometries, as openings having multi-sided geometries are also suitable for providing a nanopore.
• nanopores are not limited to cylindrical surface geometries and/or circular cross- sections.
• a nanopore in the various embodiments of the various aspects disclosed herein can have a cylindrical or non-cylindrical surface geometry and/or a circular or non-circular cross-section. In some embodiments, the nanopore has a cylindrical surface geometry or cross-section.
• the nanopore can, optionally, taper from the first surface of the substrate to the second surface of the substrate.
• the nanopore can have a diameter at the first surface of the substrate that is smaller or larger than the diameter of the nanopore at the second surface of the substrate.
• the nanopore has a diameter at the first surface of the substrate that is substantially similar to the diameter at the second surface of the substrate.
• porous coating is discussed as being a single layer, it is to be understood that the porous coating layer can comprise more than one (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more layers). Further, description of porous coating as disclosed herein can apply to each of the individual layers of the coating. Moreover, when two or more layers are present in the porous coating, all the layers can be same, all different, or some same and some different. The differences can be based on the components of the layers, thickness of the layers, porosity of the layers, chemical properties, e.g., hydrophobicity or hydrophilicity of the layers, and the like.
• the porous coating can comprise any suitable material through which a molecule of interest, for example, a target molecule or analyte molecule, can pass, but which will reduce the translocation speed of the molecule passing through the nanopore as compared to the
• the porous coating material can be hydrophilic, hydrophobic, anionic, cationic, or any combinations thereof.
• the porous coating can be in the form of gels, hydrogels, fibers, nanofibers, nanoparticles, meshes, mats, 3D-scaffolds, and the like.
• the porous coating is a non-woven mesh or mat.
• the porous coating comprises electrospun fibers.
• hydrogel refers to a three-dimensional polymeric structure that is insoluble in water but which is capable of absorbing and retaining large quantities of water to form a stable, often soft and pliable, structure.
• water can penetrate in between the polymer chains of the polymer network, subsequently causing swelling and the formation of a hydrogel.
• the coating layer comprises two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) different materials.
• ratio of the different materials can be varied to optimize one or more desired properties of the porous coating. Ratio between any two different materials can range from 99: 1 to 1 :99.
• the porous coating layer comprises an oligomeric or polymeric material.
• the porous coating layer can be composed of a linear, comb, branched, or dendritic oligomer or polymer.
• the porous coating material comprises a reactive functional group.
• reactive functional group refers to a functional group that allows covalent linkage of a molecule of interest to the porous coating, for example, linking of a targeting moiety to the porous coating layer. Targeting moieties are described in more detail elsewhere in the disclosure.
• exemplary reactive functional groups include, but are not limited to, hydroxyls or alcohols, amines, azides, alkynes, alkenes, NHS, MAL, thiols, thials, sulfinos, acids, carboxylic acids, and the like.
• the porous coating comprises an oligomer or polymer
• each Q is independently selected from O, S, Se, and NH;
• Q' is independently selected from O, S, Se, or NH;
• G' is each independently selected from the following structures:
• R'i is selected from among a hydrogen, straight or branched alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl or arylalkyl chain of 1-50 carbons, wherein each alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl, fluorocarbon, or arylalkyl chain is optionally substituted internally or terminally by one or more hydroxyl, hydroxyether, carboxyl, carboxyester, carboxyamide, amino, mono- or di-substituted amino, thiol, thioester, sulfate, phosphate, phosphonate, or halogen substituents; or
• R'i is selected from among poly(ethylene glycol), poly(ethylene oxide),
• poly(hydroxyacid) a carbohydrate, a protein, a polypeptide, an amino acid, a nucleic acid, a nucleotide, a polynucleotide, any DNA or RNA segment, a lipid, a polysaccharide, an antibody, a pharmaceutical agent, or any epitope for a biological receptor; or
• R'i is selected from among a photocrosslinkable or ionically crosslinkable group
• R'2 is selected from among hydrogen, a straight or branched alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl, fluorocarbon, or arylalkyl chain of 1-50 carbons, wherein each alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl or arylalkyl chain is optionally substituted internally or terminally by one or more hydroxyl, hydroxyether, carboxyl, carboxyester, carboxyamide, amino, mono- or di-substituted amino, thiol, thioester, sulfate, phosphate, phosphonate, or halogen substituents;
• n, a, or b are each independently selected from an integer of 1 -1000;
• each polymeric terminal group is selected from among amines, thiols, amides,
• phosphates sulphates, hydroxides, metals, alkanes, alkenes and alkynes.
• the oligomer or polymer represented by one of the above-noted formulas can be a linear, comb, branched, or dendritic oligomer or polymer.
• the oligomer or polymer represented by one of the above-noted formulas can also comprise one or more reactive functional groups.
• the porous coating layer comprises poly(caprolactone). In some embodiments, the porous coating layer comprises poly(e-caprolactone) (PCL).
• the porous coating layer can comprise modified or unmodified poly(glycerol-co-e- caprolactone) co-polymer.
• the free hydroxyl group in the glycerol monomer can be modified with hydrophobic, hydrophilic, cationic and/or anion groups.
• modifications of the poly(glycerol-co-e-caprolactone) co-polymer can be varied to optimize one or more desired properties of the co-polymer and consequently those of the porous coating. Desired properties can include, but are not limited to, hydrophobicity, hydrophilicity, ion density, cationic charge, anion charge, and the like.
• ratio of caprolactone monomers to glycerol monomers can range from about 100: 1 to about 1 : 100.
• ratio of caprolactone monomers to glycerol monomers can be from about 90: 10 to about 90: 10.
• ratio of caprolactone monomers to glycerol monomers can be from 95:5 to about 55:45.
• ratio of caprolactone monomers to glycerol monomers can be about 90: 10, about 80:20, about 70:30, about 60:40, or about 50:50.
• either the caprolactone or the glycerol monomers can be present in a higher amount than the other.
• the porous coating comprises one or more of the following oligomers or polymers:
• Ratio between two different oligomers and/or polymers in the coating can range from about 1000: 1 to about 1 : 1000. In some embodiments of the various aspect disclosed herein, the ratio between two different oligomers and/or polymers can range from about 500: 1 to about 1 :500, from about 250: 1 to about 1 :250, or from about 100: 1 to about 1 : 100. In some embodiments, the ratio between two different oligomers and/or polymers can range from about 95:5 to about 45:55. In some embodiments of the various aspects disclosed herein, the ratio between two different oligomers and/or polymers can be about 90: 10, about 70:30, or about 50:50.
• the porous coating layer can comprise two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) different oligomers or polymers.
• the porous coating can comprise poly(e-caprolactone) and the modified or unmodified poly(glycerol-co-e-caprolactone) co-polymer.
• the poly(e-caprolactone) and the modified or unmodified poly(glycerol-co-e-caprolactone) co-polymer can be present in any desired ratio in the porous coating.
• ratio of poly(6-caprolactone) to the modified or unmodified poly(glycerol- co-s-caprolactone) co-polymer can range from about 1000: 1 to about 1 : 1000.
• the ratio of poly(e-caprolactone) to the modified or unmodified poly(glycerol-co-e-caprolactone) co-polymer can range from about 500: 1 to about 1 :500, from about 250: 1 to about 1 :250, from about 100: 1 to about 1 : 100.
• the ratio of poly(e-caprolactone) to the modified or unmodified poly(glycerol-co- ⁇ -caprolactone) co-polymer can range from about 95:5 to about 45:55. Either the poly(e- caprolactone) or the modified or unmodified poly(glycerol-co-e-caprolactone) co-polymer can be present in a higher amount than the other.
• the preferred ratios for PCL: PGC (modified with a stearic acid) are 9: 1 and 8:2.
• ratio of poly(e- caprolactone) to the modified or unmodified poly(glycerol-co-e-caprolactone) co-polymer is about 90: 10, about 70:30, or about 50:50.
• ratio described in the disclosure can be based on weight, mole or volume.
• ratio can be weight/weight, mole/mole or volume/volume.
• the modified poly(glycerol-co-e-caprolactone) co-polymer is functionalized or modified with a lipid.
• a lipid can be linked to a hydroxyl group of a glycerol monomer in the PGC.
• a lipid for functionalizing or modifying poly(glycerol-co-e- caprolactone) co-polymer can be selected from the group consisting of fatty acids, fatty alcohols, glycerolipids (e.g., monoglycerides, diglycerides, and triglycerides), phospholipids,
• the lipid can be selected from the group consisting of 1,3-Propanediol Dicaprylate/Dicaprate; 10-undecenoic acid; 1 -dotriacontanol; 1- heptacosanol; 1-nonacosanol; 2-ethyl hexanol; Androstanes; Arachidic acid; Arachidonic acid; arachidyl alcohol; Behenic acid; behenyl alcohol; Capmul MCM CIO; Capric acid; capric alcohol; capryl alcohol; Caprylic acid; Caprylic/Capric Acid Ester of Saturated Fatty Alcohol C12-C18; Caprylic/Capric Triglyceride; Caprylic/Capric Triglyceride; Ceramide
• phosphorylcholine Sphingomyelin, SPH
• Ceramide phosphorylethanolamine Sphingomyelin, Cer-PE
• Ceramide phosphorylglycerol Ceroplastic acid; Cerotic acid; Cerotic acid; ceryl alcohol; Cetearyl alcohol; Ceteth-10; cetyl alcohol; Cholanes; Cholestanes; cholesterol; cis-l l- eicosenoic acid; cis-1 1-octadecenoic acid; cis-13-docosenoic acid; cluytyl alcohol; coenzyme Q10 (CoQIO); Dihomo-y-linolenic; Docosahexaenoic acid; egg lecithin; Eicosapentaenoic acid; Eicosenoic acid; Elaidic acid; elaidolinolenyl alcohol; elaidolinoleyl alcohol; elaidyl alcohol; Erucic acid; erucyl alcohol; Estra
• undecylenic acid Undecylic acid; Vaccenic acid; a-Linolenic acid; ⁇ -Linolenic acid; a fatty acid salt of 10-undecenoic acid, adapalene, arachidic acid, arachidonic acid, behenic acid, butyric acid, capric acid, caprylic acid, cerotic acid, cis-1 1 -eicosenoic acid, cis-11-octadecenoic acid, cis- 13-docosenoic acid, docosahexaenoic acid, eicosapentaenoic acid, elaidic acid, erucic acid, heneicosylic acid, heptacosylic acid, heptadecanoic acid, isostearic acid, lauric acid, lignoceric acid, linoelaidic acid, linoleic acid, montanic acid, myristic acid, myristoleic
• zinc recinoleate sapienic acid, stearic acid, tricosylic acid, tridecylic acid, undecylenic acid, undecylic acid, vaccenic acid, valeric acid, a-linolenic acid, or ⁇ -linolenic acid; paraffin; and any combinations thereof.
• the lipid can be functionalized to provide an anionic or cationic functional group on the modified PGC.
• the lipid can be modified to comprise an amino group, a hydroxyl group, a thiol group, a carbocyclic acid group, and the like.
• the modified poly(glycerol-co-e-caprolactone) co-polymer comprises an aryl or heteroaryl group linked to a hydroxyl group of a glycerol monomer in the PGC.
• the linkage can be via a linker.
• the modified poly(glycerol-co-e- caprolactone) co-polymer comprises an optionally substituted benzyl group to a hydroxyl group of a glycerol monomer in the PGC.
• the poly(glycerol-co-e-caprolactone) functionalized with a stearic acid is a preferred composition.
• the porous coating comprises collagen or polyacrylamide. In some embodiments, the porous coating is gel or hydrogel and comprises collagen or
• the thickness and porosity of the porous coating can be selected according to the desired degree of reduction of the translocation speed of target molecules to be detected.
• the porous coating has a thickness sufficient to reduce the translocation speed of an analyte molecule (e.g., nucleic acid or protein) passing through the nanopore as compared to the translocation speed in the absence of the porous coating, but not so thick as to prevent translocation of the analyte molecule.
• the thickness of the porous coating can range from nm to hundreds of micrometers.
• the porous coating layer has a thickness of about 1 nm to about 1000 nm.
• the porous coating covers one or both openings of the nanopore.
• Additional molecules or components can be present in the porous coating layer.
• additional components present in the porous coating layer can modulate the interaction of the analyte molecules with the coating.
• Such additional molecules or components can be covalently or non-covalently liked to the coating.
• the coating layer further comprises a denaturation agent.
• denaturation agent means a compound or composition that inhibits or reduces binding of the analyte molecule to the coating or a targeting entity present in the coating.
• denaturation agents include, but are not limited to, guanidinium chloride, urea, trichloroacetic acid, sulfosalicylic acid, and the like.
• the denaturation agent can be encapsulated in the porous coating or covalently linked with the porous coating.
• the denaturation agent can be present in the porous coating without being covalently linked to another component of the coating or the denaturation agent can be linked to another component of the porous coating.
• the denaturation agent is covalently linked to an oligomer/polymer of the porous coating. In some embodiments, the denaturation agent is covalently linked to an
• the porous coating can include a target binding moiety or analyte binding moiety.
• a target binding moiety means a molecule that binds or interacts with an analyte molecule or an analyte capture probe.
• the target binding moiety has enhanced or specific binding affinity for a selected analyte or analyte capture probe.
• the porous coating comprises two or more (e.g., three, four, five, six, seven, eight, nine, ten or more) different target binding moieties for capturing different analytes. Without wishing to be bound by a theory, this can allow analysis of multiple analytes in a multiplex format.
• the target binding moiety binds to a capture probe which binds to the analyte.
• capture probe refers to a molecule that binds or interacts with an analyte molecule and the target binding moiety binds or interacts with the capture probe.
• the target binding moiety can be encapsulated in the porous coating or covalently linked with the porous coating.
• the target binding moiety can be present in the porous coating without being covalently linked to another component of the coating or the target binding moiety can be linked to another component of the porous coating.
• the target binding moiety is covalently linked to an oligomer/polymer of the porous coating.
• the target binding moiety is covalently linked to an oligomer/polymer of the porous coating via a linker.
• the target binding moiety or the capture probe can comprise a wide variety of molecules. Such molecules can include naturally occurring molecules, or recombinant or synthetic molecules.
• the target binding moiety and/or the capture probe is selected from the group consisting of antibodies, Fab fragments, scFv, aptamers, nucleic acids, proteins, peptides, other appropriate affinity molecule, and any combinations thereof.
• the target binding moiety and/or the capture probe is a nucleic acid.
• the target binding moiety and/or the capture probe is a single stranded oligonucleotide. For example, a single stranded oligonucleotide having a nucleotide sequence substantially complementary to nucleic acid analyte molecule of interest.
• linker means an organic moiety that connects two parts of a compound.
• Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NH, C(O), C(0)NH, SO, SO 2 , SO 2 NH or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkyn
• alkynylheteroarylalkenyl alkynylheteroarylalkynyl, alkylheterocyclylalkyl,
• alkylheterocyclylalkenyl alkylhererocyclylalkynyl, alkenylheterocyclylalkyl,
• alkenylheterocyclylalkenyl alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,
• linker and spacer are used interchangeably herein.
• the linker can comprise any combinations of the above. Accordingly, in some embodiments, the linker can comprise hydrocarbons, amino acids, peptides, polyethylene glycol of various lengths, cyclodextrins, and derivatives and any combinations thereof.
• the linker can be a branched linker.
• the branch point of the branched linker can be at least trivalent, but can be a tetravalent, pentavalent or hexavalent atom, or a group presenting such multiple valencies.
• the branchpoint is -N, - N(Q)-C, -O-C, -S-C, -SS-C, -C(0)N(Q)-C, -OC(0)N(Q)-C, -N(Q)C(0)-C, or -N(Q)C(0)0-C; wherein Q is independently for each occurrence H or optionally substituted alkyl.
• the branch point is glycerol or derivative thereof, and normal chain sugars such as monosaccharides and polysaccharides.
• a branched linker can be used to connect two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) molecules of interest (which can be same or different) to one affinity ligand; two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) affinity ligands (which can be same or different) to one molecule of interest; or two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) molecules of interest (which can be same or different) to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) affinity ligands (which can be same or different).
• the linker comprises at least one cleavable linking group.
• a cleavable linking group is one which is sufficiently stable, but which can be cleaved to release the two parts the linker is holding together.
• the cleavable linking group is cleaved at least 10 times or more, preferably at least 100 times faster in the target system or under a first reference condition (which can, e.g., be selected to mimic or represent conditions for binding of analyte molecule to targeting moiety) than under a second reference condition (which can, e.g., be selected to mimic or represent conditions for releasing the analyte molecule from the targeting moiety or releasing the analyte molecule from the targeting moiety.
• a first reference condition which can, e.g., be selected to mimic or represent conditions for binding of analyte molecule to targeting moiety
• a second reference condition which can, e.g., be selected to mimic or represent conditions for releasing the analyte molecule
• Cleavable linking groups are susceptible to cleavage agents, e.g., pH, light, redox potential or the presence of degradative molecules.
• degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans that can degrade a redox cleavable linking group by reduction; esterases; amidases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific) and proteases, and phosphatases.
• the cleavable linking group can comprise esters, peptides, carbamates, acid-labile, reduction-labile, oxidation-labile, disulfides, thiolesters, and modifications thereof.
• a preferred example are thiolester linkages which are cleaved via a thioester-thiol exchange reaction.
• a linker can include a cleavable linking group that is cleavable by a particular enzyme.
• the type of cleavable linking group incorporated into a linker can depend on the cell to be targeted.
• cleavable linking group is cleaved at least 1.25, 1.5, 1.75, 2, 3, 4, 5, 10, 25, 50, or 100 times faster in the presence of the enzyme as compared to in the absence of the enzyme.
• the cleavable linking group is cleaved by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% in the absence of the enzyme as compared to in the presence of the enzyme.
• Exemplary cleavable linking groups include, but are not limited to, redox cleavable linking groups (e.g., -S-S- and -C(R) 2 -S-S-, wherein R is H or Ci-C6 alkyl and at least one R is C 1 -C5 alkyl such as CH3 or CH 2 CH3); phosphate-based cleavable linking groups (e.g., -O- P(0)(OR)-0-, -0-P(S)(OR)-0-, -0-P(S)(SR)-0-, -S-P(0)(OR)-0-, -0-P(0)(OR)-S-, -S- P(0)(OR)-S-, -0-P(S)(ORk)-S-, -S-P(S)(OR)-0-, -0-P(0)(R)-0-, -0-P(S)(R)-0-, -S-P(0)(R)-0-, -S-
• a peptide based cleavable linking group comprises two or more amino acids.
• the peptide-based cleavage linkage comprises the amino acid sequence that is the substrate for a peptidase or a protease found in cells.
• an acid cleavable linking group is cleaveable in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid.
• the linker comprises an acid labile group, e.g., hydrazone or carbamate.
• affinity binding pair or "binding pair” refers to first and second molecules that specifically bind to each other. One member of the binding pair is conjugated with first part to be linked while the second member is conjugated with the second part to be linked.
• specific binding refers to binding of the first member of the binding pair to the second member of the binding pair with greater affinity and specificity than to other molecules.
• Exemplary binding pairs include any haptenic or antigenic compound in combination with a corresponding antibody or binding portion or fragment thereof (e.g., digoxigenin and anti- digoxigenin; mouse immunoglobulin and goat antimouse immunoglobulin) and
• nonimmunological binding pairs e.g., biotin-avidin, biotin-streptavidin, biotin-neutravidin, hormone [e.g., thyroxine and cortisol-hormone binding protein, receptor-receptor agonist, receptor-receptor antagonist (e.g., acetylcholine receptor-acetylcholine or an analog thereof), IgG-protein A, IgG-protein G, IgG-synthesized protein AG, lectin-carbohydrate, enzyme-enzyme co factor, enzyme-enzyme inhibitor, and complementary oligonucleoitde pairs capable of forming nucleic acid duplexes), and the like.
• the binding pair can also include a first molecule which is negatively charged and a second molecule which is positively charged.
• binding pair conjugation is the biotin-avidin, biotin- streptavidin or biotin-neutravidin conjugation.
• one of the molecule or the peptide is biotinylated and the other is conjugated with avidin or streptavidin.
• Many commercial kits are also available for biotinylating molecules, such as proteins.
• Another example of using binding pair conjugation is the biotin-sandwich method. See, e.g., example Davis et al., Proc. Natl. Acad. Sci. USA, 103: 8155-60 (2006).
• the two molecules to be conjugated together are biotinylated and then conjugated together using at least one tetravalent avidin-like molecule (e.g., avidin, streptavidin, or neutravidin) as a linker.
• the disclosure also provides a method of preparing the article comprising the substrate comprising a nanopore and a porous coating on at least one surface of the substrate.
• the method comprises preparing a substrate with a nanopore and depositing or polymerizing the porous coating on at least one surface (e.g., the first surface, the second surface, or both the first and second surfaces) of the substrate.
• the method comprises: (i) forming an opening defining a nanopore in a substrate; and (ii) forming porous coating layer on at least one surface (e.g., the first surface, the second surface, or both the first and second surfaces) of the substrate.
• the nanopore can be formed in the substrate by any suitable method, such as by using a laser drill or by an etching method or the like. When the nanopore is to have a tapered shape, a selective etching method is particularly suitable.
• the nanopore can be formed using a TEM, a SEM, ort the like.
• the nanopore can be formed using an electron beam, a focused ion beam, a neutron beam, an alpha-ray, a beta-ray, an X-ray, a ⁇ -ray, or the like, which is emitted from a TEM, a SEM, or the like.
• nanopores can be manufactured by Focused Ion Beam (FIB) drilling on a variety of substrates such as glass and polymeric materials. See, for example, Storm A. J. et al., Nature Materials 2003), 2, 537-540 and Siwy, Z. & Fulinski, A. Phys. Rev. Lett. (2002), 89, 198103-198106, contents of both which are incorporated herein by reference in their entireties.
• the fabrication method using FIB on polymeric and glass substrates is identical to the fabrication technique when using FIB on carbon or semiconductor substrates. Fabrication of nanopores using carbon and semiconductors (e.g., silicon) can be undertaken by FIB or electrochemical etching.
• the porous coating can be deposited on the substrate in any manner suitable for forming a thin film, such as by a coating or depositing method.
• Methods of coating or depositing thin films on the surface of a substrate are known in the art.
• material of the porous coating e.g., an oligomer/polymer
• the porous coating material can be polymerized or gelled on the surface of the substrate.
• a composition comprising polymerizable monomers i.e. a polymerizable composition
• the monomers can be polymerized using any means available to one skill in the art for
• polymerization For example, monomers can be polymerized using radical polymerization, cationic polymerization, anionic polymerization, reversible addition-fragmentation chain transfer (RAFT) polymerization, atom-transfer radical (ATR) polymerization, or any combinations thereof.
• RAFT reversible addition-fragmentation chain transfer
• ATR atom-transfer radical
• polymerization can occur spontaneously after forming the coating layer on the surface.
• the polymerization or gelling can be via mixing, heat, light or chemical induction.
• polymerization can be initiated using a light source.
• the light source can emit light radially or non-radially.
• Useful light sources include, but are not limited to, lamps, fiber optics devices, lasers, etc. ...
• light can be applied for a period of seconds to several minutes or hours. For example, the light can be applied for about 10 seconds to about 5 minutes.
• the light source can allow variation of the wavelength of light and/ or the intensity of the light. Light of any wavelength can be used based on the monomers utilized.
• polymerization can be initiated using UV light (200-500 nm). In certain embodiments, long UV rays can be used. In other embodiments, short UV rays can be used.
• polymerization can be initiated using visible light (400-800 nm). In certain embodiments, polymerization can be initiated using blue light (420-500 nm). In certain embodiments, polymerization can be initiated using green light (500-575 nm). In some embodiments, polymerization can be initiated using IR light (800-2500 nm).
• the output of light can be controlled to provide greater control over the polymerization reaction. Control over the reaction in turn results in control over the characteristics and/or properties of the resulting polymer. In certain embodiments, the intensity of light ranges from about 500 to about 10 6 ⁇ ; ⁇ 2 .
• the intensity of light is about 4000, about 5000, about 6000, about 7000, about 8000, or about 9000 ⁇ W/cm 2 . In some embodiments, the intensity of light is about 200,000-500,000 ⁇ W/cm 2 .
• the composition can further comprise one or a combination of two or more photo-initiators.
• Photo-initiators produce reactive free radical species that initiate the crosshnking and/or polymerization of monomers upon exposure to light. Any photo-initiator can be used in the crosshnking and/or polymerization reaction.
• Photoinitiated polymerizations and photo-initiators are discussed in detail in Rabek, Mechanisms of Photophysical Processes and Photochemical Reactions in Polymers, New York: Wiley & Sons, 1987; Fouassier, Photoinitiation, Photopolymerization, and Photocuring, Cincinnati, Ohio: Hanser/Gardner; Fisheretal., 2001 , Annu.
• a photo- initiator can be designed to produce free radicals at any wavelength of light.
• a photo-initiator can be designed to work using UV light (200-500 nm).
• a photo-initiator is designed to work using visible light (400-800 nm).
• a photo-initiator is designed to work using blue light (420-500 nm).
• a photo-initiator is designed to work using green light (500-575 nm).
• the photo-initiator is designed to work using IR light (800-2500 nm).
• acylphosphineoxide a sulfur-containing compound, a quinone.
• exemplary photo-initiators include, but are not limited to, acetophenone; anisoin; anthraquinone; anthraquinone-2-sulfonic acid, sodium salt monohydrate; (benzene) tricarbonylchromium; 4-(boc-aminomethyl)phenyl isothiocyanate; benzin; benzoin; benzoin ethyl ether; benzoin isobutyl ether; benzoin methyl ether; benzoic acid; benzophenyl-hydroxycyclohexyl phenyl ketone; 3,3',4,4'- benzophenonetetracarboxylic dianhydride; 4- benzoylbiphenyl; 2-benzyl-2-(dimethylamino)-4'- morpholinobutyrophenone; 4,4'-bis(diefhylamino)benzophen
• dibenzosuberenone 2,2-diefhoxyacetophenone; 4,4'-dihydroxybenzophenone; 2,2-dimethoxy2- phenylacetophenone; 4-(dimethylamino)benzophenone; 4,4'-dimethylbenzyl; 2,5- dimethylbenzophenone; 3 ,4-dimethylbenzophenone; diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide; 2-hydroxy-2-methylpropiophenone; 4'-ethoxyacetophenone; 2-ethylanthraquinone;
• ferrocene 3 '-hydroxy acetophenone; 4'-hydroxyacetophenone; 3 -hydroxybenzophenone; 4- hydroxybenzophenone; 1 -hydroxycyclohexyl phenyl ketone; 2-hydroxy-2-methylpropiophenone; 2-methylbenzophenone; 3-methylbenzophenone; methybenzoylformate; 2-methyl-4'- (methylthio)-2-morpholinopropiophenone; 9, 10-phenanthrenequinone; 4'-phenoxyacetophenone; thioxanthen-9-one; triarylsulfonium hexafluoroantimonate salts; triarylsulfonium
• the photo-initiator is acetophenone; diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide; 4,4'- dimethoxybenzoin; anthraquinone; anthraquinone-2-sulfonic acid; benzene-chromium(O) tricarbonyl; 4-(boc-aminomethyl)phenyl isothiocyanate; benzil; benzoin; benzoin ethyl ether; benzoin isobutyl ether; benzoin methyl ether; benzophenone; benzoic acid; benzophenone/ 1 hydroxycyclohexyl phenyl ketone, 50/50 blend; benzophenone-3,3',4,4'-tetracarboxylic dianhydride; 4-benzoylbiphenyl; 2 -benzyl -2 -(dimethyl amino) -4' morpholinobutyrophenone; 4,4'-
• the free radical initiator is selected from the group consisting of benzophenone, benzyl dimethyl ketal, 2-hydroxy-2-methyl-phenylpropanone; 2,4,6- trimethylbenzoyldiphenyl phosphine oxide; 2,4,6-trimethyl benzophenone; oligo(2-hydroxy-2- methyl-1 (4-(l-methylvinyl)phenyl)propanone and 4-methylbenzophenone.
• benzophenone benzyl dimethyl ketal, 2-hydroxy-2-methyl-phenylpropanone
• 2,4,6- trimethylbenzoyldiphenyl phosphine oxide 2,4,6-trimethyl benzophenone
• oligo(2-hydroxy-2- methyl-1 (4-(l-methylvinyl)phenyl)propanone and 4-methylbenzophenone is selected from the group consisting of benzophenone, benzyl dimethyl ketal, 2-hydroxy-2-methyl-phenylpropanone; 2,4,6- trimethylbenzoyldiphen
• the photo-initiator is dimethoxy-2-phenyl-acetophenone (DMPA), a titanocene, 2- hydroxy-l-(4(hydroxyethoxy)phenyl)-2-methyl-l -propanone, Igracure.
• DMPA dimethoxy-2-phenyl-acetophenone
• the initator is 2-hydroxy-l-(4-(hydroxyethoxy) phenyl)-2-methyl- 1 -propanone (Irgacure 2959, CIBA Chemicals).
• An initiator of a cationic or anionic crosslinking and/or polymerization process can be used.
• any chromophore or a compound having a plurality of conjugated pi bonds that can be excited by light and can promote an electron from a ground state to an excited state, thus rendering the electron capable of being transferred can be used as an initiator for the polymerization process.
• Exemplary photo-initiators of cationic crosslinking and/or polymerization include, but are not limited to, titanium tetrachloride, vanadium tetrachloride, bis(cyclopentadienyl)titanium dichloride, ferrocene, cyclopentadienyl manganese tricarbonyl, manganese decacarbonyl, diazonium salts, diaryliodonium salts (e.g., 3,3'-dinitrodiphenyliodonium hexafluoroarsenate, diphenyliodonium fluoroborate, 4-methoxydiphenyliodonium fluoroborate) and triarylsulfonium salts.
• titanium tetrachloride vanadium tetrachloride
• bis(cyclopentadienyl)titanium dichloride bis(cyclopentadienyl)titanium dichloride
• ferrocene
• photo-initiators are utilized at concentrations ranging between approximately 0.0005% w/v and 5.0% w/v.
• photo-initiators can be utilized at concentrations of about 0.005% w/v, about 0.01% w/v, about 0.025% w/v, about 0.05% w/v, about 0.075% w/v, about 0.1 % w/w, about 0.125% w/v, about 0.25% w/v, about 0.5% w/v, about 0.75% w/v, about 1 % w/v, about 1.125% w/v, about 1.25% w/v, about 1.5% w/v, about 1.75% w/v, about 2% w/v, about 2.125% w/v, about 2.25% w/v, about 2.5% w/v, about 2.75% w/v, about 3%o w/v, about 3.125% w/v, about 3.25% w/
• the polymerizable composition further comprises a co- initiator.
• the co-initiator is an amine.
• a co-initiator is exogenously added.
• a co-initiator is not exogenously added, as a reactant molecule already participating in the polymerization serves a secondary role of co- initiator.
• the co-initiator is selected from the group consisting of triethanolamine, N-methyl-N,N-diethanolamine, N-ethyl-N,N-diethanolamine, an ester of dimethylaminobenzoic acid, 2,6-diisopropyl-N,N-dimethylaniline, 2-benzyl-2-(dimethylamino)- 4'-morpholinobutyrophenone, carbon tetrabromide, [4-[(2- hydroxytetradecyl)oxy]phenyl]phenyliodonium hexafluoroantimonate, Ethylenediamine- N,N,N',N'-tetra(2-propanol), 1 ,4-dimethylpiperazine, Tribenzylamine, diazabicyclo[2,2,2]octane, N-phenyldiethanolamine, allylthiourea, 4-(Dimethylamino)benzalde
• a monomer in the polymerization can serve the role of co-initator (e.g. an amine-containing monomer), and the primary photoinitiator can also serve the role of its own co-initiator (e.g. if the photoinitiator contains an amine, one molecule gets excited by light, and another molecule's amine takes part in co-initiation).
• co-initator e.g. an amine-containing monomer
• the primary photoinitiator can also serve the role of its own co-initiator (e.g. if the photoinitiator contains an amine, one molecule gets excited by light, and another molecule's amine takes part in co-initiation).
• the polymerizable composition further comprises an accelerant, e.g., a polymerization accelerant.
• an accelerant e.g., a polymerization accelerant.
• polymerization reaction refers to a compound that can assist the polymerization of polymerizable material following initiation of the reaction.
• an accelerator will promote completion of the polymerization reaction and/or increase the rate that the polymerizable material becomes incorporated into a polymerized product.
• Compounds with an N-vinyl group can serve as accelerants in the compositions, polymers, and methods disclosed herein.
• accelerant is N-vinyl pyrrolidone.
• Other exemplary accelerants are described in, for example, PCT Publication No. WO2005054304 and PCT Application No. PCT/US2004/038053
• accelerants are utilized at concentrations ranging between approximately 0.005% w/v and 5.0% w/v.
• accelerant can be utilized at concentrations of about 0.005% w/v, about 0.01% w/v, about 0.025% w/v, about 0.05%» w/v, about 0.075% w/v, about 0.1 % w/w, about 0.125% w/v, about 0.25% w/v, about 0.5% w/v, about 0.75% w/v, about 1 % w/v, about 1.125% w/v, about 1.25% w/v, about 1.5% w/v, about 1.75% w/v, about 2% w/v, about 2.125% w/v, about 2.25% w/v, about 2.5% w/v, about 2.75% w/v, about 3% w/v, about 3.125% w/v, about 3.25% w/v,
• Nanopores have emerged in recent years as versatile single-molecule detectors. The sensing principle is based on transient interruptions in the ion-current of an electrolyte, induced by the entry, transport, and exit of a particular analyte from the pore. A distinguishing feature of nanopores is that they can be used to analyze not only small molecules, but also long
• biopolymers such as DNA and RNA, with resolution on the order of the nanopore length (several nm).
• a well-studied system involves the lipid-embedded a-hemolysin (a-HL) protein pore, which can accommodate various types of biopolymers.
• a-HL lipid-embedded a-hemolysin
• a-HL has been used extensively to discriminate between DNA and RNA sequences, to study DNA unzipping kinetics, orientation of entry, DNA-protein interactions, and peptide transport.
• An important outcome of these studies has been the realization that threaded biopolymer dynamics is governed by the biopolymer's interactions with the nanopore walls.
• Nanopores incorporated in thin solid-state inorganic membranes are highly promising materials, since the nanopore volume can be reduced to a few nm in all dimensions, on par with biological membrane channels.
• planar geometry permits high-resolution fabrication and characterization. Further, the fabrication of high-density nanopore arrays is possible, setting the stage for high-throughput bimolecular analysis, in particular ultra- fast DNA or protein sequencing.
• Coated nanopores in thin solid-state inorganic membranes enable a broad range of nanopore sensing applications. Because a variety of coatings may be used, as suitable for each sensing application, the detection mechanism is not limited to electrical detection only. Optical detection mechanisms can be preferable for certain embodiments.
• the present technology is highly scalable, with both optically- and electrically-addressable nanopore array assemblies enabling detection over a surface area. Without limitations, by controlling the properties of the porous coating, the characteristics of the nanopore can be refined for a variety of applications.
• Ion current sensing for individual nanopores and nanopore arrays typically uses a potassium chloride or other electrolyte solution (salt solution).
• a substrate comprising a nanopore separates two reservoirs of ionic solution. When voltage is applied across the two reservoirs, the potential drop almost entirely occurs at the nanopore. Therefore the ionic conductance or resistance between the two reservoirs is also the conductance or resistance of the nanopore.
• the nanopore conductance transiently drops when a molecule (e.g. DNA or protein) enters and exits the nanopore, allowing its detection.
• a molecule e.g. DNA or protein
• This detection scheme can be parallelized using an array of nanopores with individual electrodes situated at each chamber. The individual electrodes are then uniquely addressable using techniques well-known in the semiconductor industry.
• Nanopore surfaces may be chemically functionalized with fluorescent molecules.
• a voltage is used to drive molecules through the nanopores, while a microscope is used to sense light output from each nanopore in the membrane.
• the nanopore (or array of nanopores) is assembled in a cell containing a transparent window allowing optical probing of the membrane, while fluorescent molecules are detected as they occupy the pore.
• creating chemically-modified nanopores entails introducing fluorescent molecules only at the pore (as opposed to over an area of the membrane) by performing two complementary reactions at opposite sides of the membrane.
• each pore in the array can be either uniform or varying (for example, a gradient of size and shape across a portion of the membrane).
• the location of each pore in the array is specified during the fabrication process so that each pore has a known location.
• the pores can be optically detected using fluorescent molecules.
• the spacing between pores is chosen so that optical probing would have sufficient resolution to address each pore (e.g., approximately 500 nm spacing between adjacent pores).
• analyte is a broad term and is used in its ordinary sense and refers, without limitation, to any compound or composition the identity, presence or concentration of which is sought in a sample.
• an analyte can be a nucleic acid, an amino acid, peptide, protein, growth factor, saccharide, or a molecule produced by a cell.
• the analyte includes a biopolymer. Exemplary biopolymers include, but are not limited to, single- stranded DNA, double-stranded DNA, RNA, a nucleic acid polypeptide, a protein, a peptide, and the like.
• a method for characterizing an analyte includes receiving the analyte through the nanopore, and detecting variations in current flow through nanopore.
• the variations in current correspond to translocation of the analyte through the nanopore.
• the coated nanopore substrate disclosed herein can be used to distinguish between single-stranded and double-stranded nucleic acids.
• the coated nanopore substrate disclosed herein can be used to determine interactions of different molecules with each other, for example, nucleic acid/nucleic acid interactions, nucleic acid/protein interactions, protein/protein interactions and the like.
• the nanopore current varies with length of the analyte, e.g., length of a nucleic acid or protein analyte.
• Nanopore detection techniques have been used for biomolecule detection.
• various nanopore sequencing methods have been proposed.
• Bezrukov, Vodyanoy and Parsegian showed that one can use a biological nanopore as a Coulter counter to count individual molecules (Counting polymers moving through a single ion channel, Nature 370, 279-281 (1994) incorporated, herein, by reference).
• Kasianowicz, Brandin, Branton and Deamer proposed an ambitious idea for ultrafast single-molecule sequencing of single-stranded DNA molecules using nanopore ionic conductance as a sensing mechanism (Characterization of individual polynucleotide molecules using a membrane channel, Proc. Nat. Acad. Sci.
• U.S. Pat. No. 6,428,959 describes methods for determining the presence of double-stranded nucleic acids in a sample.
• nucleic acids present in a fluid sample are translocated through a nanopore, e.g., by application of an electric field to the fluid sample.
• the current amplitude through the nanopore is monitored during the translocation process and changes in the amplitude are related to the passage of single- or double-stranded molecules through the nanopore.
• Those methods find use in a variety of applications in which the detection of the presence of double- stranded nucleic acids in a sample is desired.
• the article comprising the porous coating coated substrate disclosed herein can be used for, but is not limited to, to sequence nucleic acids; to sequence proteins; to detect a nucleic acid sequence; to detect a protein to detect a protein to nucleic acid interaction(s); to detect protein to protein interaction(s); to detect nucleic acid to nucleic acid interaction(s); to determine the length of a nucleic acid sequence, e.g., for genomic or transcriptomic analysis for DNA or RNA, respectively; to determine the length of an amino acid sequence for proteomic analysis; or any combinations thereof.
• the disclosure provides a method for identifying an analyte using a porous coating coated substrate disclosed herein.
• the method comprising exposing the coated substrate to a solution comprising the analyte of interest, wherein the porous coating comprises a analyte binding moiety; allowing the analyte to bind with the analyte binding moiety; optionally washing any unbound analyte; releasing the bound analyte from the analyte binding moiety; and detecting variation in current flow through the nanopore, wherein the variation in current correspond to translocation of the analyte through the nanopore.
• Any method or reagent available to one of skill in the art for releasing two bound molecules from each other can be used. Some examples include, but are not limited to, changes in pH, addition of thiols, change in ionic strength, heating, enzymatic degradation, chemical treatments, and the like. Alternatively, a reporter species can be measured which is directly related to the analyte of interest.
• the analyte is a nucleic acid.
• the disclosure provides a method for identifying a nucleic acid sequence using a porous coating coated substrate disclosed herein.
• the method comprising exposing the coated substrate to a solution comprising the nucleic acid of interest, wherein the porous coating comprises a nucleic acid binding moiety, for example an oligonucleotide comprising a nucleotide sequence substantially complimentary to at least part of the nucleotide sequence of the nucleic acid of interest; allowing the nucleic acid of interest to bind with the nucleic acid binding moiety;
• the disclosure provides a method for identifying an analyte using a porous coating coated substrate disclosed herein.
• the method comprising exposing the coated substrate to a solution comprising the analyte of interest, wherein the porous coating comprises a target binding moiety; allowing the analyte to bind with the target binding moiety; optionally washing any unbound analyte; releasing the analyte/target binding moiety as a complex; and detecting variation in current flow through the nanopore, wherein the variation in current correspond to translocation of the analyte/target binding moiety complex through the nanopore. Any method or reagent available to one of skill in the art for releasing two bound molecules from each other can be used.
• translocation time and/or dynamics for analyte/target binding moiety complex are different from the translocation time and/or dynamics of unbound analyte and the target binding moiety.
• the analyte/target binding moiety complex can be released by exposing the substrate to a condition under which the cleavable group is cleaved.
• the analyte is a nucleic acid and the method comprises:
• the porous coating comprises a nucleic acid binding moiety, for example an oligonucleotide comprising a nucleotide sequence substantially complimentary to at least part of the nucleotide sequence of the nucleic acid of interest; allowing the nucleic acid of interest to bind with the nucleic acid binding moiety; optionally washing any unbound nucleic acid of interest; releasing the nucleic acid and the nucleic acid binding moiety as a complex from the porous coating interest; and detecting variation in current flow through the nanopore, wherein the variation in current correspond to translocation of the nucleic acid bound to the nucleic acid binding moiety through the nanopore.
• a nucleic acid binding moiety for example an oligonucleotide comprising a nucleotide sequence substantially complimentary to at least part of the nucleotide sequence of the nucleic acid of interest
• a nucleic acid has translocation time and/or dynamics, i.e., variations in current, that are different when the nucleic acid is bound to a nucleic acid binding moiety compared to when not bound to a nucleic acid binding moiety.
• the method comprises: preparing an analyte/capture probe complex; exposing the coated substrate to a solution comprising the analyte/capture probe complex; allowing the capture probe in the analyte/capture probe complex to bind with the porous coating; optionally washing any unbound analyte/capture probe complex; releasing the analyte from the porous coating bound analyte/capture probe complex; and detecting variation in current flow through the nanopore, wherein the variation in current correspond to translocation of the analyte through the nanopore.
• the capture probe comprises a reactive functional group for linking the capture probe to the porous coating.
• the porous coating comprises a reactive function group for binding the capture probe.
• the method comprises: preparing a nucleic acid/capture probe, wherein the capture probe is a nucleic acid binding moiety, for example an oligonucleotide comprising a nucleotide sequence substantially complimentary to at least part of the nucleotide sequence of the nucleic acid of interest; exposing the coated substrate to a solution comprising the nucleic acid/capture probe complex; allowing the capture probe in the complex to bind with the porous coating; optionally washing any unbound nucleic acid/capture probe complex; releasing the nucleic acid from the porous coating bound nucleic acid/capture probe complex; and detecting variation in current flow through the nanopore, wherein the variation in current flow correspond to translocation of the nucleic acid through the nanopore.
• the capture probe is an nucleic acid.
• the method comprises: preparing an analyte/capture probe complex; exposing the coated substrate to a solution comprising the analyte/capture probe complex, wherein the porous coating comprises a target binding moiety that binds or interacts with the analyte/capture probe complex; allowing the analyte/capture probe complex to bind with the target binding moiety in the porous coating; optionally washing any unbound analyte/capture probe complex; releasing the analyte from the target binding moiety bound analyte/capture probe complex; and detecting variation in current flow through the nanopore, wherein the variation in current correspond to translocation of the analyte through the nanopore.
• the porous coating comprises two or more (e.g., three, four, five, six, seven, eight, nine, ten or more) different target binding moieties for capturing different analytes.
• this can allow analysis of multiple analytes in a multiplex format.
• Analyte characterization methods using substrates comprising porous coating with more than one target binding moieties can be used for analysis of multiple analytes in multiplex format.
• bound analytes and/or analyte/capture probe complexes can be released in a sequential manner thereby allowing characterization of multiple analytes.
• the disclosure also provides a method for sizing the length of an analyte.
• the method comprises: exposing the coated substrate to a solution comprising the analyte of interest; and detecting variation in current flow through the nanopore, wherein the variation in current correspond to length of the analyte.
• the analyte is a nucleic acid (single-stranded or double- stranded) and the method comprises: exposing the coated substrate to a solution comprising the nucleic acid; and detecting variation in current flow through the nanopore, wherein the variation in current corresponds to length of the nucleic acid.
• the analyte is a protein and the method comprises: exposing the coated substrate to a solution comprising the protein; and detecting variation in current flow through the nanopore, wherein the variation in current corresponds to length of the nucleic acid.
• the substrate comprising the 3D porous coating can also be used for detecting synthetic polyelectrolytes like PAA (polyacrylic acid).
• PAA polyacrylic acid
• identifying/characterizing analytes such as nucleic acids and proteins, can also be used for detection/characterization of polyelectrolytes, including but not limited to, anionic
• An article comprising: (i) a substrate having a first surface and as second surface; (ii) at least one nanopore extending through the substrate, thus forming a channel connecting from the first surface to the second surface of the substrate, wherein the nanopore has a first opening that opens to the first surface of the substrate and a second opening that opens to the second surface of the substrate; and (iii) a porous coating on at least one of the first or second surface of the substrate.
• the substrate comprises Si, S1O 2 , S1N 4 , quartz, alumina, nitrides, metals, polymers, or any combinations thereof.
• nanopore opening is from about 2 nm to about 10,000 nm.
• 3D porous coating comprises nanofibers or nanoparticles.
• the 3D porous coating is a gel or hydrogel.
• the 3D porous coating comprises collagen or polyacrylamide.
• the target binding moiety is selected from the group consisting of antibodies, Fab fragments, scFv, aptamers, nucleic acids, proteins, peptides, other affinity molecule, and any combinations thereof.
• the target binding moiety is a nucleic acid.
• the nucleic acid is single-stranded DNA, double-stranded DNA, RNA, mRNA, miRNA, or pre-miRNA.
• the target binding moiety is selected from antibodies, Fab fragments, scFv, and aptamers.
• the denaturing agent is selected from the group consisting of guanidinium chloride, urea, trichloroacetic acid, sulfosalicylic acid, and any combinations thereof.
• the 3D porous coating comprises a linear, comb, branched, or dendritic oligomer or polymer.
• the oligomer or polymer further comprises a reactive functional group.
• the reactive group is selected from the group consisting of hydroxyl, alcohols, amines, azides, alkynes, alkenes, NHS, MAL, thiols, thials, sulfinos, acids, carboxylic acids, and any combinations thereof.
• each Q' is independently selected from O, S, Se, or NH;
• each G' is independently selected from the following structures:
• R'i is selected from among a hydrogen, straight or branched alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl or arylalkyl chain of 1 -50 carbons, wherein each alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl, fluorocarbon, or arylalkyl chain is optionally substituted internally or terminally by one or more hydroxyl, hydroxyether, carboxyl, carboxyester, carboxyamide, amino, mono- or di-substituted amino, thiol, thioester, sulfate, phosphate, phosphonate, or halogen substituents; or
• R'i is selected from among poly(ethylene glycol), poly(ethylene oxide),
• poly(hydroxyacid) a carbohydrate, a protein, a polypeptide, an amino acid, a nucleic acid, a nucleotide, a polynucleotide, any DNA or RNA segment, a lipid, a polysaccharide, an antibody, a pharmaceutical agent, or any epitope for a biological receptor; or
• R'i is selected from among a photocrosslinkable or ionically crosslinkable group
• '2 is selected from among hydrogen, a straight or branched alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl, fluorocarbon, or arylalkyl chain of 1- 50 carbons, wherein each alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl or arylalkyl chain is optionally substituted internally or terminally by one or more hydroxyl, hydroxyether, carboxyl, carboxyester, carboxyamide, amino, mono- or di-substituted amino, thiol, thioester, sulfate, phosphate, phosphonate, or halogen substituents;
• n, a, or b are each independently selected from an integer of 1-1000;
• each polymeric terminal group is selected from among amines, thiols, amides,
• phosphates sulphates, hydroxides, metals, alkanes, alkenes and alkynes.
• copolymer is a modified or unmodified poly(glycerol-co-e-caprolactone) co-polymer.
• poly(glycerol-co-e-caprolactone) copolymer is modified to comprise at least one group selected from lipids, hydrophobic groups, hydrophilic groups, cationic groups, anion groups, and any combinations thereof.
• the 3D porous coating comprises at least one oligomer or polymer selected from the group consisting of:
• Q is independently selected from among O, S, Se, or NH;
• G' is each independently selected from among the following structures:
• R is selected from among a hydrogen, straight or branched alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl or arylalkyl chain of 1-50 carbons, wherein each alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl, fluorocarbon, or arylalkyl chain is optionally substituted internally or terminally by one or more hydroxyl, hydroxyether, carboxyl, carboxyester, carboxyamide, amino, mono- or di-substituted amino, thiol, thioester, sulfate, phosphate, phosphonate, or halogen substituents; or
• R is selected from among poly(ethylene glycol), poly(ethylene oxide), poly(hydroxyacid)), a carbohydrate, a protein, a polypeptide, an amino acid, a nucleic acid, a nucleotide, a polynucleotide, any DNA or RNA segment, a lipid, a polysaccharide, an antibody, a pharmaceutical agent, or any epitope for a biological receptor; or
• Ris selected from among a photocrosslinkable or ionically crosslinkable group; n is independently selected from an integer of 1-1000;
• each polymeric terminal group is selected from among amines, thiols, amides, phosphates, sulphates, hydroxides, metals, alkanes, alkenes and alkynes.
• a method of preparing an article comprising a substrate comprising at least one nanopore and a 3D porous coating on at least one surface of the substrate, the method comprising:
• said polymerization comprises radical polymerization, cationic polymerization, anionic polymerization, reversible addition- fragmentation chain transfer (RAFT) polymerization, atom-transfer radical (ATR) polymerization, or any combinations thereof.
• RAFT reversible addition- fragmentation chain transfer
• ATR atom-transfer radical
• each Q' is independently selected from O, S, Se, or NH;
• each G' is independently selected from the following structures:
• Q' Q' 'i is selected from among a hydrogen, straight or branched alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl or arylalkyl chain of 1-50 carbons, wherein each alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl, fluorocarbon, or arylalkyl chain is optionally substituted internally or terminally by one or more hydroxyl, hydroxyether, carboxyl, carboxyester, carboxyamide, amino, mono- or di-substituted amino, thiol, thioester, sulfate, phosphate, phosphonate, or halogen substituents; or
• R'i is selected from among poly(ethylene glycol), poly(ethylene oxide),
• poly(hydroxyacid) a carbohydrate, a protein, a polypeptide, an amino acid, a nucleic acid, a nucleotide, a polynucleotide, any DNA or RNA segment, a lipid, a polysaccharide, an antibody, a pharmaceutical agent, or any epitope for a biological receptor; or
• R'i is selected from among a photocrosslinkable or ionically crosslinkable group
• '2 is selected from among hydrogen, a straight or branched alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl, fluorocarbon, or arylalkyl chain of 1- 50 carbons, wherein each alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl or arylalkyl chain is optionally substituted internally or terminally by one or more hydroxyl, hydroxyether, carboxyl, carboxyester, carboxyamide, amino, mono- or di-substituted amino, thiol, thioester, sulfate, phosphate, phosphonate, or halogen substituents;
• n, a, or b are each independently selected from an integer of 1-1000;
• each polymeric terminal group is selected from among amines, thiols, amides,
• phosphates sulphates, hydroxides, metals, alkanes, alkenes and alkynes.
• oligomer or polymer is a linear, comb, branched, or dendritic oligomer or polymer represented by one of the following formulas:
• Q is independently selected from among O, S, Se, or NH;
• G' is each independently selected from among the following structures:
• R is selected from among a hydrogen, straight or branched alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl or arylalkyl chain of 1-50 carbons, wherein each alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl, fluorocarbon, or arylalkyl chain is optionally substituted internally or terminally by one or more hydroxyl, hydroxyether, carboxyl, carboxyester, carboxyamide, amino, mono- or di-substituted amino, thiol, thioester, sulfate, phosphate, phosphonate, or halogen substituents; or
• R is selected from among poly(ethylene glycol), poly(ethylene oxide), poly(hydroxyacid)), a carbohydrate, a protein, a polypeptide, an amino acid, a nucleic acid, a nucleotide, a polynucleotide, any DNA or RNA segment, a lipid, a polysaccharide, an antibody, a pharmaceutical agent, or any epitope for a biological receptor; or
• Ris selected from among a photocrosslinkable or ionically crosslinkable group; n is independently selected from an integer of 1-1000;
• each polymeric terminal group is selected from among amines, thiols, amides, phosphates, sulphates, hydroxides, metals, alkanes, alkenes and alkynes.
• biopolymer is a nucleic acid or a protein.
• detecting variations in current flow comprises detecting an open nanopore current and a blocked nanopore current, the blocked nanopore current varying with respect to analyte translocation through the nanopore.
• analyte comprises a biopolymer and the blocked nanopore current varies with respect to sequence of the biopolymer.
• characterization or identifying comprises: (i) sequencing a nucleic acid; (ii) detecting a nucleic acid sequence; (iii) detecting a protein; (iv) detecting protein to nucleic acid interaction(s); (v) detecting protein to protein interaction(s); (vi) detecting nucleic acid to nucleic acid interaction(s); (vii) determining length of a nucleic acid sequence; (viii) determining the length of an amino acid sequence for proteomic analysis; or (ix) any combinations of (i)-(viii).
• a method for identifying a nucleic acid sequence comprising:
• a method for identifying a nucleic acid sequence comprising:
• a method for identifying a nucleic acid sequence comprising:
• the 3D porous coating comprises two or more different target binding moieties and each different target binding moiety capturing a different analyte, thereby allowing multiplex characterization of two or more analytes.
• the 3D porous coating comprises two or more different target binding moieties, each different target binding moiety capturing a different analyte, and said different captured analytes being released sequentially, thereby allowing multiplex characterization of said analytes.
• a method for determining the length of a nucleic acid sequence comprising:
• a method for identifying a protein comprising:
• a method for identifying a protein comprising:
• a method for identifying or characterizing an analyte comprising: (i) exposing an article of any of paragraphs 1-34 to a solution comprising a molecule comprising a first target binding moiety, wherein the 3D porous coating comprises a second target binding moiety, wherein the second target binding moiety binds to the molecule comprising the first target binding moiety;
• step (iv) exposing the article from step (iii) to a solution comprising the analyte of interest;
• step (vii) exposing the article from step (v) to a solution comprising a molecule comprising a third target binding moiety, wherein the third target binding moiety binds to the bound analyte;
• step (x) exposing the article from step (viii) to a solution a reporter molecule, wherein the reporter molecule binds to the molecule comprising the third target binding moiety;
• step (xiv) exposing the article from step (xiii) to a voltage potential, thereby drawing the reporter molecule through the nanopore and allowing for single molecule detection of the reporter molecule if the analyte of interest was captured by the 3D coating.
• a method for determining the concentration of a scale inhibitor in an oil sample comprising:
• a method for detecting or identifying an anionic polyelectrolyte in a sample comprising:
• polyelectrolyte is polyacrylic acid (PAA)
• a 3D porous coated membrane or thin solid-state, polymeric, lipid, or solid-like film containing nanopore composition comprising: a) nanopore(s) and 2) a 3D porous coating(s).
• composition of paragraph 1 wherein the coating is a fiber such as an electrospun polymer.
• composition of paragraph 1 wherein the coating is a gel or hydrogel such as a collagen gel or polyacrylamide gel.
• composition of paragraph 1 wherein the coating is assembled spheres such as an electrosprayed polymer.
• a 3D porous coated nanopore composition of paragraphs 1-5 comprising: a)
• a 3D porous coated nanopore composition of paragraphs 1-5 comprising: a)
• a 3D porous coated nanopore composition of paragraphs 1-5 comprising: a)
• a denaturation agent such as a guanidinium chloride, urea, trichloroacetic acid, or sulfosalicylic acid that is non-covalently or covalently bound to the coating.
• a 3D porous coated nanopore composition comprising: a) nanopore(s) and 2) one or more coatings.
• composition of paragraphs 1 or 8 Use of the composition of paragraphs 1 or 8 to detect a protein to nucleic acid interaction (s) 14. Use of the composition of paragraphs 1 or 8 to detect a protein to protein interaction (s)
• composition of paragraphs 1 or 8 Use of the composition of paragraphs 1 or 8 to determine the length of a nucleic acid sequence for genomic or transcriptomic analysis for DNA or RNA, respectively.
• a method of making a 3-dimensional coated nanopore composition of paragraphs 1-5 comprising the steps of:
• a method of making a 3-dimensional coated nanopore composition comprising the steps of:
• Q' is independently selected from among O, S, Se, or NH;
• G' is each independently selected from among the following structures:
• 'i is selected from among a hydrogen, straight or branched alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl or arylalkyl chain of 1-50 carbons, wherein each alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl, fluorocarbon, or arylalkyl chain is optionally substituted internally or terminally by one or more hydroxyl, hydroxyether, carboxyl, carboxyester, carboxyamide, amino, mono- or di-substituted amino, thiol, thioester, sulfate, phosphate, phosphonate, or halogen substituents; or
• R'i is selected from among poly(ethylene glycol), poly(ethylene oxide), poly(hydroxyacid)), a carbohydrate, a protein, a polypeptide, an amino acid, a nucleic acid, a nucleotide, a polynucleotide, any DNA or RNA segment, a lipid, a polysaccharide, an antibody, a pharmaceutical agent, or any epitope for a biological receptor; or
• R'i is selected from among a photocrosslinkable or ionically crosslinkable group
• R' 2 is selected from among hydrogen, a straight or branched alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl, fluorocarbon, or arylalkyl chain of 1-50 carbons, wherein each alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl or arylalkyl chain is optionally substituted internally or terminally by one or more hydroxyl, hydroxyether, carboxyl, carboxyester, carboxyamide, amino, mono- or di-substituted amino, thiol, thioester, sulfate, phosphate, phosphonate, or halogen substituents;
• n, a, or b are each independently selected from an integer of 1 - 1000;
• each polymeric terminal group is selected from among amines, thiols, amides, phosphates, sulphates, hydroxides, metals, alkanes, alkenes and alkynes.
• the polymer is a linear, comb, branched, or dendritic oligomer or polymer has a alcohol, acid, amine, azide, alkyne, alkene, NHS, MAL, or other such groups so that a targeting moiety to capture a biologic can be attached.
• a method of identifying a nucleic acid sequence using a coated nanopore composition comprising the steps of: (a) preparing the 3D coated nanopore with the target sequence incorporated within,
• the 3D coated nanopore is exposed to pH, a thiol containing molecule, heat, or chemical treatment such that the target & analyte nucleic acid are released as double strand,
• the double strand nucleic acid containing the analyte of interest is detected via translocation through the NP.
• the target strand released, not containing the captured analyte, is discernable from the target/capture analyte duplex via P sensing.
• a method of identifying a nucleic acid sequence using a coated nanopore composition comprising the steps of:
• the captured analyte nucleic acid is detected via translocation through the NP.
• a method of identifying a nucleic acid sequence using a coated nanopore composition comprising the steps of:
• the capture probe binds to 3D coating and the 3D coated nanopore is washed to remove unbound nucleic acid and other biologies
• the 3D coated nanopore is exposed to pH, a change in ionic strength, heat, or chemical treatment such that the captured analyte nucleic acid is released,
• the captured analyte nucleic acid is detected via translocation through the NP.
• composition the method comprising the paragraphs 22-24 wherein more than one capture probe is on the coating allowing for multiplex sensing.
• composition the method comprising the paragraphs 22-24 wherein more than one capture probe is on the coating allowing for multiplex sensing wherein the each targeted analyte is released sequentially for detection and identification.
• a method of sizing the length of a nucleic acid sequence using a coated nanopore composition comprising the steps of:
• the nucleic acid is detected via translocation through the NP and the time of translocation is related to the length of the nucleic acid.
• a method of identifying a protein using a coated nanopore composition comprising the steps of:
• the 3D coated nanopore is exposed to pH, a thiol containing molecule, heat, or chemical treatment such that the target and/or analyte protein are released,
• the analyte of interest is detected via translocation through the NP.
• a method of identifying a target protein using a coated nanopore composition comprising the steps of:
• step (b) exposing said 3D coated nanopore to an aqueous solution containing a molecule that binds the bio-recognition site in step (a) (e.g. biotin) which tethers the mesh to a second bio- recognition site (e.g. antibody, aptamer, etc.) specific for the analyte (protein) of interest (e.g. Biotinylated antibody)
• a molecule that binds the bio-recognition site in step (a) e.g. biotin
• a second bio- recognition site e.g. antibody, aptamer, etc.
• analyte (protein) of interest e.g. Biotinylated antibody
• step (c) expose said 3D coated nanopore to an aqueous solution which saturates the bio- recognition sites from step (a) (e.g. excess free biotin).
• step (d) expose said 3D coated nanopore to an aqueous solution of the analyte (e.g. protein) of interest allowing the analyte to bind to the second bio-recognition site from step (b)
• analyte e.g. protein
• the 3D coated nanopore is washed to remove unbound protein, other biologies, and any unbound molecules
• the 3D coated nanopore is exposed to an aqueous solution of a third bio-recognition site specific for the analyte of interest which is tethered to a stimuli responsive (heat, chemical, light, etc.) release unit capable of binding a reporter molecule (e.g. avidin) detectable in a nanopore
• a stimuli responsive (heat, chemical, light, etc.) release unit capable of binding a reporter molecule (e.g. avidin) detectable in a nanopore
• the 3D coated nanopore is exposed to an aqueous solution of the reporter molecule (e.g. avidin), in excess, which binds the tether (e.g. biotin) of the bio-recognition site in step (f)
• the reporter molecule e.g. avidin
• the tether e.g. biotin
• step (j) the 3D coated nanopore is exposed to the release stimulus from step (f) releasing the reporter molecule (e.g. avidin) into solution
• the 3D coated nanopore is exposed to a voltage potential drawing the reporter molecule through the pore allowing for single molecule detection of the reporter molecule (e.g. avidin) only if the analyte of interest was captured by the 3D coating
• Q' is independently selected from among O, S, Se, or NH;
• G' is each independently selected from among the following structures:
• R'i is selected from among a hydrogen, straight or branched alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl or arylalkyl chain of 1-50 carbons, wherein each alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl, fluorocarbon, or arylalkyl chain is optionally substituted internally or terminally by one or more hydroxyl, hydroixyether, carboxyl, carboxyester, carboxyamide, amino, mono- or di-substituted amino, thiol, thioester, sulfate, phosphate, phosphonate, or halogen substituents; or
• R'i is selected from among poly(ethylene glycol), poly(ethylene oxide),
• poly(hydroxyacid) a carbohydrate, a protein, a polypeptide, an amino acid, a nucleic acid, a nucleotide, a polynucleotide, any DNA or RNA segment, a lipid, a polysaccharide, an antibody, a pharmaceutical agent, or any epitope for a biological receptor; or
• R'i is selected from among a photocrosslinkable or ionically crosslinkable group
• R'2 is selected from among hydrogen, a straight or branched alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl, fluorocarbon, or arylalkyl chain of 1-50 carbons, wherein each alkyl, cycloalkyl, aryl, olefin, silyl, alkylsilyl, arylsilyl, alkylaryl or arylalkyl chain is optionally substituted internally or terminally by one or more hydroxyl, hydroxyether, carboxyl, carboxyester, carboxyamide, amino, mono- or di-substituted amino, thiol, thioester, sulfate, phosphate, phosphonate, or halogen substituents;
• n, a, or b are each independently selected from an integer of 1-1000; each polymeric terminal group is selected from among amines, thiols, amides, phosphates, sulphates, hydroxides, metals, alkanes, alkenes and alkynes.
• the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not.
• the use of “comprising” indicates inclusion rather than limitation. Accordingly, the terms “comprising” means “including principally, but not necessary solely”.
• variation of the word “comprising”, such as “comprise” and “comprises” have correspondingly the same meanings.
• the term “consisting essentially of means “including principally, but not necessary solely at least one", and as such, is intended to mean a “selection of one or more, and in any combination”. Stated another way, the term “consisting essentially of means that an element can be added, subtracted or substituted without materially affecting the novel characteristics of the invention. This applies equally to steps within a described method as well as compositions and components therein.
• “decrease” , “reduced”, “reduction” , “decrease” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount.
• “reduced”, “reduction” or “decrease” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%), or at least about 80%, or at least about 90%> or up to and including a 100% decrease (e.g. absent level as compared to a reference sample), or any decrease between 10- 100%» as compared to a reference level.
• the terms “increased” 'increase” or “enhance” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased”, “increase” or “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%», or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%), or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
• the term "statistically significant” or “significantly” refers to statistical significance and generally means at least two standard deviations (2SD) away from a reference level.
• the term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true.
• the terms “essentially” and “substantially” means a proportion of at least about 60%, or preferably at least about 70%) or at least about 80%, or at least about 90%, at least about 95%), at least about 97% or at least about 99% or more, or any integer between 70%» and 100%.
• the term “essentially” means a proportion of at least about 90%), at least about 95%, at least about 98%, at least about 99% or more, or any integer between 90%) and 100%».
• the term “essentially” can include 100%.
• aliphatic includes both saturated and unsaturated, straight chain (i.e., unbranched) or branched aliphatic hydrocarbons, which are optionally substituted with one or more functional groups, as defined below.
• aliphatic is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl moieties.
• alkyl includes straight and branched alkyl groups.
• alkyl encompass both substituted and unsubstituted groups.
• lower alkyl is used to indicate those alkyl groups (substituted, unsubstituted, branched or unbranched) having 1-6 carbon atoms.
• the alkyl, alkenyl and alkynyl groups employed in the invention contain 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1 - 10 aliphatic carbon atoms.
• the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1 -8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1 -6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1 -4 carbon atoms.
• Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl; sec-pentyl, isopentyl, tert-pentyl, n-hexyl, sec-hexyl, moieties and the like, which again, may bear one or more substituents, as previously defined.
• Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl, and the like.
• Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl and the like.
• alicyclic refers to compounds which combine the properties of aliphatic and cyclic compounds and include but are not limited to cyclic, or polycyclic aliphatic hydrocarbons and bridged cycloalkyl compounds, which are optionally substituted with one or more functional groups, as defined below.
• alicyclic is intended herein to include, but is not limited to, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties, which are optionally substituted with one or more functional groups.
• Illustrative alicyclic groups thus include, but are not limited to, for example, cyclopropyl, --CE -cyclopropyl, cyclobutyl, ⁇ CH 2 -cyclobutyl, cyclopentyl, --CH 2 - cyclopentyl-n, cyclohexyl, ⁇ CH 2 -cyclohexyl, cyclohexenylethyl, cyclohexanylethyl, norborbyl moieties and the like, which again, may bear one or more substituents.
• heteroaliphatic refers to aliphatic moieties in which one or more carbon atoms in the main chain have been substituted with an heteroatom.
• a heteroaliphatic group refers to an aliphatic chain which contains one or more oxygen sulfur, nitrogen, phosphorus or silicon atoms, e.g., in place of carbon atoms.
• Heteroaliphatic moieties may be saturated or unsaturated, branched or linear (i.e., unbranched), and substituted or unsubstituted.
• heteroalicyclic refers to compounds which combine the properties of heteroaliphatic and the cyclic compounds and include but are not limited to saturated and unsaturated mono- or polycyclic heterocycles such as morpholino, pyrrolidinyl, furanyl, thiofuranyl, pyrrolyl, etc, which are optionally substituted with one or more functional groups.
• Substituents include, but are not limited to, any of the substituents mentioned below, i.e., the substituents recited below resulting in the formation of a stable compound.
• alkyl refers to saturated, straight- or branched-chain hydrocarbon radicals derived from a hydrocarbon moiety containing between one and twenty carbon atoms by removal of a single hydrogen atom, which alkyl groups are optionally substituted with one or more functional groups.
• Substituents include, but are not limited to, any of the substituents mentioned below, i.e., the substituents recited below resulting in the formation of a stable compound.
• alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n- undecyl, and dodecyl.
• alkoxy refers to an alkyl group, as previously defined, attached to the parent molecular moiety through an oxygen atom. Examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy, and n- hexoxy.
• alkenyl denotes a monovalent group derived from a hydrocarbon moiety having at least one carbon-carbon double bond, which alkenyl group is optionally is substituted with one or more functional groups. In certain embodiments, an alkenyl group contains between one and twenty carbon atoms. Substituents include, but are not limited to, any of the substituents mentioned below, i.e., the substituents recited below resulting in the formation of a stable compound. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l- yl, and the like.
• alkynyl refers to a monovalent group derived from a hydrocarbon having at least one carbon-carbon triple bond, which alkynyl group is optionally substituted. In certain embodiments, an alkynyl group contains between one and twenty carbon atoms. Substituents include, but are not limited to, any of the substituents mentioned below, i.e., the substituents recited below resulting in the formation of a stable compound. Representative alkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.
• amine refers to one, two, or three alkyl groups, as previously defined, attached to the parent molecular moiety through a nitrogen atom.
• alkylamino refers to a group having the structure --NHR' wherein R' is an alkyl group, as previously defined; and the term “dialkylamino” refers to a group having the structure—NR'R", wherein R' and R" are each independently selected from the group consisting of alkyl groups.
• trimkylamino refers to a group having the structure ⁇ NR'R"R"', wherein R', R", and R'" are each independently selected from the group consisting of alkyl groups.
• R', R", and/or R'" taken together may optionally be -CH 2 ).sub.k— where k is an integer from 2 to 6.
• amino groups include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, iso-propylamino, piperidino, trimethylamino, and propylamino.
• aryl refers to stable mono- or polycyclic, unsaturated moieties having preferably 3-14 carbon atoms, each of which may be substituted or unsubstituted. Substituents include, but are not limited to, any of the substituents mentioned below, i.e., the substituents recited below resulting in the formation of a stable compound.
• aryl may refer to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl and the like.
• heteroaryl refers to a stable heterocyclic or
• polyheterocyclic, unsaturated radical having from five to ten ring atoms of which one ring atom is selected from S, O and N; zero, one or two ring atoms are additional heteroatoms independently selected from S, O and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms.
• Heteroaryl moieties may be substituted or unsubstituted. Substituents include, but are not limited to, any of the substituents mentioned below, i.e., the substituents recited below resulting in the formation of a stable compound.
• heteroaryl nuclei examples include pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.
• aryl and heteroaryl moieties may be attached via an aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, alkyl or heteroalkyl moiety and thus also include -aliphatic)aryl, -(heteroaliphatic)aryl, -(aliphatic)heteroaryl, -(heteroa- liphatic)heteroaryl, -alkyl)aryl, -(heteroalkyl)aryl, -(heteroalkyl)aryl, and -heteroalkyl)-heteroaryl moieties.
• aryl or heteroaryl and “aryl, heteroaryl, - (aliphatic)aryl, -(heteroaliphatic)aryl, -(aliphatic)heteroaryl, -(heteroaliphatic)heteroaryl, - (alkyl)aryl, -(heteroalkyl)aryl, -(heteroalkyl)aryl, and -(heteroalkyl)heteroaryl” are
• carboxylic acid refers to a group of formula -CO 2 H.
• halo refers to an atom selected from fluorine, chlorine, bromine, and iodine.
• methylol refers to an alcohol group of structure -CH 2 OH.
• hydroxyalkyl refers to an alkyl group, as defined above, bearing at least one OH group.
• mercaptoalkyl refers to an alkyl group, as defined above, bearing at least one SH group.
• heterocyclic refers to a non-aromatic partially unsaturated or fully saturated 3- to 10-membered ring system, which includes single rings of 3 to 8 atoms in size and bi- and tri-cyclic ring systems which may include aromatic six-membered aryl or aromatic heterocyclic groups fused to a non-aromatic ring.
• Heterocyclic moieties may be substituted or unsubstituted.
• Substituents include, but are not limited to, any of the substituents mentioned below, i.e., the substituents recited below resulting in the formation of a stable compound.
• Heterocyclic rings include those having from one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
• acyl groups include aldehydes, ketones, carboxylic acids, acyl halides, anhydrides, thioesters, amides, urea, carbamate, and carboxylic esters.
• hydrocarbon refers to any chemical group comprising hydrogen and carbon.
• the hydrocarbon may be substituted or unsubstituted.
• the hydrocarbon may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic.
• Illustrative hydrocarbons include, for example, methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, methoxy, diethylamino, and the like.
• fluorocarbon refers to any chemical group comprising more fluorine atoms than hydrogen atoms attached to carbons.
• the fluorocarbon may be substituted or unsubstituted.
• a fluorocarbon may be saturated, unsaturated, branched, unbranched, cyclic, polycyclic or heterocyclic.
• substituted refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
• substituted is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
• Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms.
• substituents include, but are not limited to aliphatic; alicyclic; heteroaliphatic; heteroalicyclic; aryl; heteroaryl; alkylaryl;
• alkylheteroaryl alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;
• R.sub.x independently includes, but is not limited to, H, aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl, wherein any of the aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, alkylaryl, or alkylheteroaryl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any
• nucleic acid or “oligonucleotide” or “polynucleotide” refers to a polymer or an oligomer of nucleotide or nucleoside monomers consisting of nucleobases, sugars and intersugar linkages.
• oligonucleotide also includes polymers or oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.
• the nucleic acids can be single-stranded or double-stranded.
• a single-stranded nucleic acid can have double-stranded regions and a double-stranded nucleic acid can have single-stranded regions.
• the depiction of a single strand also defines the sequence of the complementary strand.
• a nucleic acid also encompasses the complementary strand of a depicted single strand.
• many variants of a nucleic acid can be used for the same purpose as a given nucleic acid.
• a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.
• a single strand provides a probe that can hybridize to the target sequence under stringent hybridization conditions.
• a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.
• the nucleic acids can comprise any oligonucleotide modification known in the art.
• the modification is selected from the group consisting of sugar modificationa, non-phosphodiester inter-sugar (or inter-nucleoside) linkages, backbone replacements, nucleobase modifications, and any combinations thereof.
• the nucleic acid can comprise from 2 to thousands of nucleotides.
• the nucleic acid when the target binding moiety or the capture probe is a nucleic acid, can range from about 6 to 100 nucleotides in length.
• the nucleic acid can range in length from about 10 to about 50 nucleotides, from about 10 to about 35 nucleotides, from about 15 to about 30 nucleotides, from about 20 to about 30 nucleotides in length.
• nucleic acid is from about 8 to about 39 nucleotides in length.
• the nucleic acid is 6 to 25 nucleotides in length (e.g. , 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 18, 19, 20, 21 , 22, 23, or 24 nucleotides in length.
• the nucleic acid can be completely DNA, completely RNA, or comprise both RNA and DNA nucleotides. It is to be understood that when the nucleic acid is completely DNA, RNA, or a mix of both, the nucleic acid can comprise one or more of the modifications described herein.
• the analyte is a nucleic acid and the target binding moiety or the capture probe is specifically hybridizable or complementary with the nucleic acid analyte.
• specifically hybridizable and “complementary” is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non- traditional types. Determination of binding free energies for nucleic acid molecules, i.e., complementarity, is well known in the art (see, e.g., Turner et al, 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al, 1986, Proc. Nat. Acad. Sci.
• a percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" or 100%
• complementarity means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. Less than perfect complementarity refers to the situation in which some, but not all, nucleoside units of two strands can hydrogen bond with each other. "Substantial complementarity" refers to
• polynucleotide strands exhibiting 90% or greater complementarity, excluding regions of the polynucleotide strands that are selected so as to be noncomplementary. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the target binding moiety and or the capture probe to non-target analytes.
• the non-target analytes typically differ by at least two (e.g. two, three, four, five, six, seven, eight, nine, ten or more) nucleotides.
• proteins As used herein, the terms “proteins,” “polypeptide” and “peptides” are used interchangeably herein to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
• modified amino acids e.g., phosphorylated, glycated, etc.
• amino acid analogs regardless of its size or function.
• peptide refers to peptides, polypeptides, proteins and fragments of proteins, unless otherwise noted.
• protein and “peptide” are used interchangeably herein when referring to a gene product and fragments thereof.
• exemplary peptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
• antibody refers to an intact
• antibody-like molecules such as fragments of the antibodies, e.g., antigen-binding fragments.
• Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
• Antigen-binding fragments include, inter alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. Linear antibodies are also included for the purposes described herein.
• Fab, Fc, pFc', F(ab') 2 and Fv are employed with standard immunological meanings (Klein, Immunology (John Wiley, New York, N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations of Modern Immunology (Wiley & Sons, Inc., New York); and Roitt, I. (1991) Essential Immunology, 7th Ed., (Blackwell Scientific Publications, Oxford)).
• Antibodies or antigen-binding fragments specific for various antigens are available commercially from vendors such as R&D Systems, BD Biosciences, e-Biosciences and Miltenyi, or can be raised against these cell-surface markers by methods known to those skilled in the art.
• Nanopores pores of nanometer dimensions in an electrically insulating membrane, have shown promise for use in a variety of sensing applications, including single molecule detection.
• the nanopores used in such applications can be biological protein channels in a lipid bilayer or a pore in a solid-state membrane.
• Solid-state nanopores are generally made in silicon compound membranes, one of the most common being silicon nitride.
• Solid-state nanopores can be manufactured with several techniques including ion-beam sculpting of silicon nitride, and using electron-beam lithography and wet etching in crystalline silicon followed by oxidation.
• nanopores in single-molecule detection employs a detection principle based on monitoring the ionic current of an electrolyte solution passing through the nanopore as a voltage is applied across the membrane.
• a detection principle based on monitoring the ionic current of an electrolyte solution passing through the nanopore as a voltage is applied across the membrane.
• passage of molecules causes interruptions in the open pore current level.
• the temporal variation in current levels leads to a translocation event pulse.
• Nanopore detection techniques have been used for biomolecule detection.
• various nanopore sequencing methods have been proposed.
• Bezrukov, Vodyanoy and Parsegian showed that one can use a biological nanopore as a Coulter counter to count individual molecules (Counting polymers moving through a single ion channel, Nature 370, 279 - 281 (1994) incorporated, herein, by reference).
• Kasianowicz, Brandin, Branton and Deamer proposed ultrafast single-molecule sequencing of single- stranded DNA molecules using nanopore ionic conductance as a sensing mechanism (Characterization of individual
• nucleic acids present in a fluid sample are translocated through a nanopore, e.g., by application of an electric field to the fluid sample.
• the current amplitude through the nanopore is monitored during the translocation process and changes in the amplitude are related to the passage of single- or double-stranded molecules through the nanopore.
• a silicon nitride membrane provides a robust scaffold to chemical and physical modify a solid state nanopore.
• researchers have employed a variety of techniques to modify translocation behavior in solid-state nanopores. Changing pore geometry affects the amount of interaction between a molecule and the nanopore wall, and determines the upper size limit for molecules that may gain access to the pore.
• 1"4 Nanopores have been drilled in a range of membrane materials, including polymers, glass, silicon dioxide, and graphene, which provide a range of interactions with charged biopolymers. 5"9 Some researchers seek to control interactions by functionalizing the nanopore surface with a layer of organic molecules whose charge and hydrophobicity may be chemically tuned. 10 11 Other researchers have functionalized the mouths of nanopores with individual enzymes to directly regulate translocation speeds. 12"13
• Nanopore surfaces have been modified to discriminate specific "target” sequences of nucleic acids over non-specific sequences using tethered complementary “probe” sequences.
• Several groups have modified either solid state nanopores or a-hemolysin nanopores with probe sequences to detect target sequences by monitoring the characteristic changes in current associated with a sequences that hybridizes with a capture probe versus sequences that are non- complementary.
• 14"18 Mussi et al. functionalized large nanopores, 20-80 nm in diameter, with single stranded DNA probes to reduce the size of the nanopore by about 15.3 nm using 45-mer oligonucleotides.
• Complementary target DNA hybridizes with these probes in the nanopore space resulting in an altered current profile when the target sequence is translocating through the nanopore.
• Howorka et al. covalently linked a single-stranded DNA probe to the lumen of an a-hemolysin nanopore to detect single nucleotide polymorphisms based off of the characteristic translocation time of the target sequence.
• Translocations associated with a perfectly complementary target sequence and a sequence with a single base mismatch had significantly reduced translocation times where single base mismatches caused 6.5-75 times slower translocations if the mismatch was located at the end or in the middle of the sequence, respectively.
• Nanopores can be used to discriminate double-stranded nucleic acids from single- stranded nucleic acids or if a nucleic acid is bound to a protein or not based off of the characteristics of the reduced current as the molecule translocates through the nanopore. This feature has been utilized to discriminate target single-stranded nucleic acids bound to a complementary probe in solution. Several groups have used this strategy to identify the translocation of a target sequence by either discriminating double-stranded versus single-stranded nucleic acid current amplitudes or observing events associated the unzipping of a double-stranded nucleic acids (i.e.
• Length Profiling The translocation time is directly related to analyte length as it traverse small nanopores because it forces the analyte to translocate in a linear fashion.
• a nanopore may be used to size analyte.
• the translocation of an analyte through a bare nanopore is very fast and thus the invention describes a method to slow that rate enable discrimation of analyte sizes.
• MicroRNAs are a class of short (- 18-24 nucleotides) noncoding RNAs that regulate gene expression at the post-transcriptional level 2 . Depending on the degree of homology to their target sequences, miRNA binding induces either translational repression or cleavage of target mRNAs 2 . As powerful gene regulators, miRNAs play important roles in development, cell differentiation, and regulation of cell cycle, apoptosis and signaling pathways. Aberrant expression of miRNAs has been found in all types of tumors 4 ' 5 ; the different cancer types have distinct miRNA expression profiles 6 .
• Specific miRNAs including some miRNA families containing a few single nucleotide polymorphisms, are constantly released from the primary tumor into blood stream and are present in an incredibly stabile form. Recent studies demonstrated that circulating miRNAs are enveloped inside exosomal vesicles and can be transferable and functional in the recipient cells. Thus, detection of tumor specific circulating miRNAs provides a powerful tool for early diagnosis, staging, and monitoring of cancer cells 10 .
• MiRNA detection Several technologies including reverse transcription real-time PCR (RT-qPCR) and microarray for miRNA detection have been developed . Each technology has its own advantages, but limitations include requiring enzymatic amplification, target labeling (enzymatic or fluorescent), and semi-quantitative results 14 . In particular the short miRNA sequences make it difficult to selectively design the primers or probes, resulting in cross- hybridization and low selectivity. This is especially true when the miRNAs contain a few or a single nucleotide difference in a miRNA family.
• Biosensors designed to detect the presence of a specific protein of interest often compromise sensitivity for specificity or vice versa.
• Traditional methods such as the enzyme linked immunosorbent assay (ELISA) rely on antibody recognition of an analyte of interest followed by an enzymatic process to amplify a colorimetric signal to indirectly quantify the concentration of a target protein.
• ELISA enzyme linked immunosorbent assay
• Many amplification methods result in compromised specificity to enhance the sensitivity of the biosensor often resulting in suboptimal false positive rates.
• Newermethods such as using fluorescent molecules or quantum dots attempt to improve this technology by eliminating the need for amplification. However, these methods suffer from reduced sensitivity compared to amplification methods producing suboptimal false negative rates.
• this invention addresses this need by coupling a highly sensitive single- moleucle detection system with a highly specific capture surface.
• Scale Inhibitors An additional embodiment of this application is the use of a nanopore to measure the concentration of a scale inhibitor.
• Scale inhibitors are chemicals used to prevent the formation of inorganic precipitates on equipment, for example in drilling and pumping oil wells. Formation of mineral scales, such as calcium carbonate, calcium sulfate, calcium phosphate, magnesium silicate, and silica compounds, can increase corrosion rate, restrict flow, and otherwise interfere with operation of equipment.
• Many scale inhibitors are charged polymers, such as polyacrylic acid, polymaleic acid, polycarboxylic acid, etc. These polymers generally interfere with the growth of a crystal lattice, preventing or reversing scale formation.
• the resulting polymer formed either a viscous oil or white solid precipitate depending on the carbonate content of the copolymer.
• Copolymers were formed with the following carbonate mole fractions: 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 1.00.
• Table 1 below indicates the composition, molecular weight, and thermal data of the different copolymers, which are illustrated by structural formulas below the table.
• CL caprolactone
• CG carbonate of glycerol f cg mole percent carbonate monomer in polymerization feed
• F cg mole percent carbonate monomer in copolymer
• M n number average molecular weight
• PDI polydispersity index
• T g glass transition temperature
• T c
• the DCU was filtered and the solvent evaporated.
• the product was dissolved in dichloromethane (10 mL) and precipitated in cold methanol.
• the solvent was decanted and subsequently dried by evaporation (85% yield).
• Addition of the amine side chain was determined by the presence of the methylene group nearest the Fmoc protecting group, as well as the Fmoc protecting group itself, with peaks in the 3 ⁇ 4 NMR spectrum at 3.10-3.19 (m, 2H, OCH 2 ), and 4.45 (s, 2H, PhCH 2 ), 7.24-7.38 (m, 5H, aromatic), respectively.
• a primary alcohol-derivitized copolymer poly(6-hydroxy-hexanoic acid 2-oxo- 1 ,3- dioxan-5-yl ester-co-e-caprolactone) was synthesized using the following steps.
• a carboxylic acid-derivitized copolymer poly(hexanedioic acid mono-(2-oxo- 1 ,3- dioxan-5-yl) ester-co-e-caprolactone) was synthesized using the following steps.
• a carboxylic acid-derivitized copolymer poly(glycerol 12-(l-(2-nitrophenyl)ethoxy)- 12-oxododecanoic acid-co-e-capro lactone) was synthesized using the following steps.
• a carboxylic acid-derivitized copolymer poly(glycerol 6-(((l-(5-methoxy-2-nitro-4- (2-(stearoyloxy)ethoxy)phenyl)ethoxy)carbonyl)amino)hexanoic acid -co-e-caprolactone) was synthesized using the following steps.
• a silica gel column was used to purify the product using a 3 : 1 hexane:ethyl acetate mixture. The product was then precipitated in diethyl ether and filtered to afford 2-(2-methoxy-5-nitro-4-(l -(((4-nitrophenoxy)carbonyl)oxy)ethyl)phenoxy)ethyl stearate in 42.9% yield.
• 6-aminocaproic acid (0.99 g, 7.6 mmol) was added to triethylamine (0.764 g, 7.6 mmol) and dissolved in 2: 1 THF and combined with the previous THF mixture to make a 4: 1 THF:water mixture.
• the reaction was mixed for 18 hours at room temperature.
• the THF was concentrated and the reaction was extracted using DCM.
• the organic phase was washed with concentrated sodium chloride and brine.
• a silica gel column was used to purify the product (gradient: 4: 1 , 3 : 1 , 2: 1 , 1 : 1 , 1 :4 hexanes:ethyl acetate) to afford 6-(((l -(5-methoxy-2-nitro-4-(2- (stearoyloxy)ethoxy)phenyl)ethoxy)carbonyl)amino)hexanoic acid in 71.4% yield.
• DCM ⁇ , ⁇ '-dicyclohexylcarbodiimide
• poly(glycerol 6-(((l-(5-methoxy-2-nitro-4- (2-(stearoyloxy)ethoxy)phenyl)ethoxy)carbonyl)amino)hexanoic acid -co-e-caprolactone) as a white solid in 69.5% yield.
• poly(glycerol-co-e-caprolactone) (0.034 g, 0.059 mmol), and cat. 4-dimethylaminopyridine (DMAP) in dry DCM (3.0 mL).
• DCM dimethylaminopyridine
• DCC N,N'-dicyclohexylcarbodiimide
• the reaction was stirred overnight at room temperature.
• the solution was filtered to remove the N,N'dicyclohexylurea, a byproduct of the reaction, and then rotovaped so that the DCM was 1 mL.
• the polymer was precipitated in 30 mL of diethyl ether and filtered and washed with diethyl ether.
• the excess COOH-PEG34oo-Maleimide was dissolved in water and the polymer was centrifuged to the bottom of a tube to allow the water to be removed. The polymer was isolated and dried under high vacuum for 18 hours to afford the poly(glycerol-PEG34oo-Maleimide-co-e-caprolactone) product in 15% yield.
• GPC the polymer weight increased proportional to 15% of the hydroxyl groups being functionalized with the COOH-PEG34oo-Maleimide side chain.
• Butanedioic acid monobenzyl ester (0.184 g, 0.78 mmol), poly(5-hydroxy-l,3-dioxan-2-one-co- ⁇ -caprolactone) (1.5 g, 2.6 mmol, 22 mol % carbonate), DCC (0.129 g, 0.63 mmol), and DMAP (0.032 g, 0.26 mmol) were dissolved in dichloromethane (20 niL). The solution was stirred at RT for 18 h. The DCU was filtered and the solvent evaporated. The product was dissolved in dichloromethane (10 mL) and precipitated in cold methanol. The solvent was decanted and subsequently dried by evaporation (83% yield). Addition of the carboxylic acid side chain was determined by the presence of the benzyl protecting group, with peaks in the 3 ⁇ 4 NMR spectrum at 5.06 -7.33 (m, 5H, aromatic).
• R -H, -OH, saturated alliphatic chain, biotin, ol ethylene-glycol), aromatic group
• Electrospinning of poly(oxanorbornene) derivatives Poly(oxanorbornene) derivatives were dissolved in various electrospinning solvent systems (Chloroform:Methanol, Chloroform:Dimethylformamide, Hexafluoro-2-propanol, etc.) producing various fiber morphologies. The working distance, voltage, and needle gauge were adjusted to produce consistent fiber morphology for each solvent system.
• EXAMPLE 15 Stimuli responsive release unit: Native chemical ligation
• a stimuli responsive tether was synthesized to connect a maleimide group to a biotin group through a thiolester linkage which is susceptible to native chemical ligation in the presence of cysteine methylester.
• This tether can be utilized in example 33 as the release unit responsible for the stimuli responsive detection of the presence of a target analyte captured onto a nano fiber mesh surface using a released reporter molecule detected using a nanopore.
• Biotin-Thiolester-PEGiwo-Maleimide 28 mg
• commercially available Amine-PEG-Maleimide 3,400 g/mol
• Amine-PEG-Maleimide 3,400 g/mol
• the DMF was removed under high vacuum and the product was dissolved in water.
• the aqueous solution was dialyzed using 1000 molecular weight cutoff dialysis tubing placed in deionized water for 3 days (yield: 73.3%). 3 ⁇ 4 NMR and 13 C NMR were performed and are consistent with the structure.
• Non-woven polymer meshes and blends were prepared using an electrospinning apparatus. Solutions of polycaprolactone were prepared (20 w/v %) in a 5: 1
• the monomer ratio in the final polymer was about 80 mol % capro lactone and the molecular weight was about 10,000 Da.
• the molecular weight for the poly(caprolactone) was between 70,000-90,000 Da.
• the resulting meshes are 300 ⁇ thick, with an average fiber size of «7 ⁇ .
• the wettability of the meshes was assessed using static contact angle measurements, where electrospun PCL meshes doped with PGC-C18 asymptotically approach 153° with 50 wt% doping.
• Melted electrospun meshes were prepared by treating meshes at 80°C for 1 minute followed by quenching to collapse the porous structure on itself. This procedure was done quickly to prevent phase separation of PCL and PGC-C18, which was confirmed by differential scanning calorimetry (DSC) and consistent with their similar structures. Electrospun meshes and melted electrospun meshes for PCL and 10% doped PGC-C18 PCL were compared using SEM and showed that the melted meshes have a comparably smooth surface.
• Electrospun fibers showed a finite surface roughness (RMS ⁇ 50 nm) with consistent RMS values between fibers with different PGC-C18 doping concentrations. This finite roughness indicates that both intrafiber and interfiber roughness may contribute to high apparent contact angles.
• Electrospun mesh surfaces with ⁇ 25% PGC-C18 doping could be pushed into the stable Wenzel regime by dropping the water droplet used in contact angle measurements from 2 feet.
• Electrospun meshes with >25% PGC-C18 doping could not be pushed into the Wenzel regime in this way, indicating that 25% doping is an approximate boundary condition for the Wenzel-to- Cassie state transition.
• Solvent-cast poly(caprolactone) films were prepared containing 0 - 75 wt % poly(glycerol monostearate-co-caprolactone). The polymers were co-dissolved in
• This 3D superhydrophobic electrosprayed coating technique is a substrate generic approach to coat structurally and compositionally different materials such as collagen, cotton fabric, nitrile rubber, and aluminum foil. After electrospraying onto these surfaces, the resultant contact angle of all four surfaces is >167° (hysteresis ⁇ 7°), whereas the uncoated portions of the material are easily and quickly wetted. Materials which are electrically insulating, such as glass, can be coated with the use of conductive copper tape near the material surface to ground the current used in the electrospraying process.
• EXAMPLE 19 Layer-by-layer poly(caprolactone) meshes and poly(caprolactone) meshes.
• EXAMPLE 20 Stimuli responsive polymer wettability using ultraviolet light activated hydrophobic doping agent: Hydrophobic to hydrophilic transformation
• a poly(glycerol-co-e-caprolactone) (1 :4) (PGC) polymer was synthesized following a previously published protocol 23 , and 12-(l-(2-nitrophenyl)ethoxy)-12-oxododecanoic acid (CI 2- NPE) was attached to the free hydroxyl of the PGC through an ester linkage using a DCC coupling method.
• PCL poly(e-caprolactone)
• the resulting polymer solution at 10% by weight, was electrospun using the following parameters: the solution was flowed through a 20 gauge needle at 3 mL/hour and a 10 kV potential was applied between the needle and a rotating and translating collecting drum 10 cm away. 24"25
• the electrospun mesh was analyzed using a Zeiss SUPRA 55VP field emission scanning electron microscope (SEM) to identify micrometer ( ⁇ 3-5 ⁇ beads) and nanometer (fiber diameters -100
• EXAMPLE 21 Cell patterning on stimuli responsive polymer using ultraviolet light activated hydrophobic doping agent: Hydrophobic to hydrophilic transformation
• hydrophilic regions were analyzed by applying a dilute solution of a water soluble CT contrast agent (Visipaque, 80 mg of iodine/mL, GE Healthcare) to the surface of the meshes and using an X-ray CT scanner to measure the water penetration into the meshes.
• a water soluble CT contrast agent Visipaque, 80 mg of iodine/mL, GE Healthcare
• X-ray CT scanner to measure the water penetration into the meshes.
• Figures 5A and 5B if the mesh was not exposed to UV light the aqueous CT contrast agent solution was restricted to the surface of the hydrophobic mesh. In contrast, the aqueous solution penetrated into the cavities formed via photolysis. A linear relationship between the UV exposure time and the depth of the cavities was determined.
• One application for a material with tunable 3D hydrophobicity is selective cell patterning. Specifically, we determine whether a human breast cancer cell line (MCF7) would selectively adhere on either the hydrophobic or hydrophilic regions of the UV active meshes. As with the previous ⁇ experiments, we used a 150 ⁇ thick mesh and a 1590 ⁇ in diameter photomask to selectively expose a small circular region of the mesh to UV light and create a hydrophilic region.
• MCF7 human breast cancer cell line
• Nanopore chips are fabricated from a ⁇ 1,0,0> single-crystal silicon wafer through- etched to leave a thin ( ⁇ 20 nm) freestanding silicon nitride (SiN) membrane supported by a small (5 mm x 5 mm x 0.35 mm) silicon chip.
• SiN silicon nitride
• a nanopore is drilled through the SiN using a highly focused transmission electron microscope beam (10 8 - 10 9 e /nm 2 ) to sputter away material from the thin membrane according our previously published method.
• a sample image of a pore is shown in Figure 6. Nanopores are drilled and cleaned prior to electrospinning.
• EXAMPLE 25 Electrical characterization of a nanopoi e-nanofibei mesh sensor
• NP chips were sealed in a custom-built flow cell permitting a low-noise recording of the ion current flowing through the pore.
• Nanopore chips are assembled in a Teflon cell and PDMS is used to seal the edges of the chip to prevent current leakage.Reservoirs on each side of the membrane are filled with an electrolyte buffer (1M KCl, 10 mM Tris-HCl) and all bubbles are removed manually. The NFM coating may be hydrated using 5% ethanol, if necessary, which may then be rinsed out with a lOx buffer exchange.
• An Axon 200B amplifier is used to apply a voltage clamp (-300 mV) across the membrane via Ag/AgCl electrodes, and the resulting current is measured. 35
• FIG. 12 shows I-V curves for the same three nanopores (bare nanopore, a PCL-only-coated nanopore, and a 7:3 PCL:PGC-C18-coated nanopore) between -500 mV and +500 mV relative to the grounded cis chamber.
• the conductance of the nanopore ⁇ 9 nS does not appear to be affected by either NFM.
• EXAMPLE 26 DNA translocations and sample exchange in a nanopore-nanofiber mesh sensor
• the NP-NFM did not exhibit detectable non-specific binding that might complicate sample change via serial dilution and buffer replacement.
• Figure 13 shows a continuous current trace of DNA added to the cis side of an NP- NFM device with an 8:2 PCL:PGC-C18 electrospun mesh.
• a clean current trace is obtained from a 4 nm nanopore.
• Adding ⁇ lnM 1000 bp DNA into the cis chamber induces transient blockades in the ionic current corresponding to translocations of DNA from the grounded cis side to the positively biased trans side of the membrane. 35
• the nanopore current was returned to its original clean state, with no transient blockades.
• the NP-NFM can detect the presence of DNA in the same manner and at the same concentrations as a bare nanopore. Additionally, the NP-NFM may be cleared of one sample using the same wash protocol as for a bare nanopore, confirming that multiple samples may be used in succession in the NP-NFM without cross-contamination.
• EXAMPLE 27 Translocation of dsDNA through an NP-NFM
• NP-NFM detects transient blockades as well as a bare nanopore, we observe that a novel finding. Specifically, we note that while the current blockage level appears to be unaffected by the presence of an NFM, the overall translocation times are significantly longer.
• Figure 14A shows sample translocations collected for a bare nanopore (blue) and the same nanopore coated with a 7:3 PCL:PGC-C 18 NFM (red). These events are characterized by duration (tj) and relative current blockage level (3 ⁇ 4). Thousands of translocations were collected for each nanopore condition. Current levels for individual events are determined using Gaussian fits to all-points histograms. Overall open pore current, conductance changes, blockage levels, and so forth are fits to ensemble histograms. The overall population characteristics may be observed on the event diagram (scatter plot).
• Dwell time is characterized in Figure 14B: distributions for translocation time, t T , represent the tail of a Poisson-like distribution and are characterized by the timescale of an exponential decay fit. Where multiple populations could be distinguished, this fit used two terms, one for "normal” translocations, and one for “slowed” translocations, weighted for counting error. A typical r 2 value is 0.9 or higher for both types of fits.
• Figure 14 shows a broader spread in the DNA
• the dwell- time of a large fraction of the events falls between 0.5 ms and 10 ms ( Figure 14B); a range which exceeds the typical translocation time of the same uncoated pore by roughly an order of magnitude.
• EXAMPLE 28 Current blockage level during translocation of dsDNA does not depend on mesh composition
• Figure 17 shows a clear pattern of longer translocation times with larger DNA molecules. While we did not make an attempt to discriminate collision events (fast events that involve unsuccessful threading of the DNA into the pore) from true translocations, the overall trend of the translocation time is clear and consistent for all lengths. As before, we numerically characterized the translocation dwell-time distributions using exponential tail fits. These results are shown in Figure 17B, indicating mean translocation speeds of roughly 0.4-0.7 ⁇ 8/ ⁇ 8 ⁇ , which is 20-35x slower than for an uncoated pore under the same conditions (see Figure 18). A monotonic growth in the characteristic translocation time as a function of length is observed for DNA in the presence of the NFM coating.
• Avidin is the release unit for the protein detection assay which resembles an ELISE assay (NP-ELISA assay). The presence of the analyte is detected by translocations of avidin through the nanopore. The quantity of analyte is directly related to the quantity (concentration) of released avidin, which is detectable from -10 pM over ⁇ 6 orders of magnitude.
• Figure 20 depicts a typical calibration curve for avidin in a solid state nanopore. The line is to guide the eye.
• EXAMPLE 33 Target protein detection via capture of target protein onto a nanofiber mesh and controlled release of a reporter molecule for nanopore detection
• NP-NFM nanopore-nano fiber mesh
• the mesh is then washed followed by the addition of the analyte at varying concentrations.
• the mesh is washed followed by the addition of a second anti-mouse antibody which is tethered to a biotin through the stimuli responsive thiol-ester described in example 15.
• the mesh is washed and then exposed to excess Avidin which will bind any free biotin tethered to the second anti-mouse antibody.
• the mesh is washed and then exposed to the stimulus which in this case is 1000 equivalents of cysteine methyl ester which undergoes a thiol- thiolester exchange separating the antibody from the avidin.
• the free avidin in solution is then free to be detected on the nanopore. See example 32 for avidin detection in a nanopore.
• Polyacrylic acid is a common scale inhibitor.
• a nanopore sensing device can detect translocations of polyacrylic acid (20,000 Da MW) down to single nanomolar concentrations, more than an order of magnitude more sensitive than current sensing techniques for scale inhibitors. This technique could be used, for example, to quantify the amount of scale inhibitor present in an oil well and determine the proper timing for performing squeeze treatments.
• micropattern cells on common culture substrates by tuning substrate wettability Tissue Eng. 2004, 10, 865-872.

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