WO2010005600A1 - Methods for identifying modulators of ion channels - Google Patents
Methods for identifying modulators of ion channels Download PDFInfo
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- WO2010005600A1 WO2010005600A1 PCT/US2009/030412 US2009030412W WO2010005600A1 WO 2010005600 A1 WO2010005600 A1 WO 2010005600A1 US 2009030412 W US2009030412 W US 2009030412W WO 2010005600 A1 WO2010005600 A1 WO 2010005600A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56988—HIV or HTLV
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- the therapeutic targeting of proteins involved in regulating the flux of ions into and out of a cell have dramatic effects on patient health.
- Testing compounds for their ability to modulate ion channel targets can be difficult and time consuming.
- the patch- clamp assay is an extremely sensitive assay for the biological action of ion channel modulators.
- the patch-clamp method is complicated and has a low throughput of test compounds.
- the methods of the current invention allow efficient high through put, label-free screening of parental (non-recombinant) cell lines without manipulation of the cells for specific response to test compounds that may be useful as drugs.
- the invention provides a method of identifying an antagonist or agonist of an ion channel.
- the method comprises applying cells to a first location and a second location on a surface of colorimetric resonant reflectance optical biosensor and applying a test reagent to the first location.
- a known ion channel antagonist or agonist of the cells is applied to the second location.
- a colorimetric resonant reflectance optical first peak wavelength value (PWV) for the first location is detected and a second PWV is detected for the second location. If the first and second PWVs are the same or similar, then the test reagent is an antagonist or agonist of an ion channel. If the first and second PWVs are different, then the test reagent is not an antagonist or agonist.
- the cells can be incubated for a period of time after their application to the first location; after the application of the test reagent to the first location; after the cells are applied to the second location, after the known ion channel antagonist or agonist is applied to the second location, or a combination thereof.
- the PWV can be detected using a scanner with a lens having a lower limit pixel size of about 2 micrometers to about 200 micrometers.
- the first location and second location on the surface of the colorimetric resonant reflectance optical biosensor can be an internal surface of a vessel selected from the group consisting of a microtiter well, microtiter plate, test tube, Petri dish, microfluidic channel, and microarray.
- the cells, test reagent, and ion channel antagonist or agonist may not comprise detection labels.
- the method can be performed at a temperature of about 25, 30, or 37 degrees Celsius.
- Another embodiment of the invention provides a method of identifying a modulator of an ion channel.
- the method comprises applying cells to a first location on a surface of a colorimetric resonant reflectance optical biosensor and applying a test reagent and a known ion channel antagonist or agonist of the cells to the first location.
- a first PWV is determined for the first location.
- Cells are applied to a second location on a surface of a colorimetric resonant reflectance optical biosensor and a known ion channel antagonist or agonist of the cells is applied to the second location.
- a second PWV is determined for the second location. If the first and second PWVs are different, then the test reagent is a modulator of an ion channel.
- the test reagent is not a modulator of an ion channel.
- the cells can be incubated for a period of time after their application to the first location; after the application of the test reagent to the first location; after the cells are applied to the second location, after the known ion channel antagonist or agonist is applied to the second location, or a combination thereof.
- the PWV can be detected using a scanner with a lens having a lower limit pixel size of about 2 micrometers to about 200 micrometers.
- the first location and second location on the surface of the colorimetric resonant reflectance optical biosensor can be an internal surface of a vessel selected from the group consisting of a microtiter well, microtiter plate, test tube, Petri dish, microfluidic channel, and microarray.
- the cells, test reagent, and ion channel antagonist or agonist may not comprise detection labels.
- the method can be performed at a temperature of about 25, 30, or 37 degrees Celsius.
- Even another embodiment of the invention provides a method for identifying a modulator of an ion channel.
- the method comprises applying cells to a first location on a surface of a colorimetric resonant reflectance optical biosensor and detecting a PWV for the first location.
- a test reagent is applied to the first location.
- a second PWV is detected for the first location.
- a first value is determined, wherein the first value is the difference between the first PWV and the second PWV.
- the first value is compared to a control test, wherein the control test comprises applying cells to a second location on a surface of a colorimetric resonant reflectance optical biosensor and detecting a third PWV for the second location.
- a known ion channel antagonist or agonist of the cells is applied to the second location.
- a fourth PWV for the second location is determined.
- a second value is determined, wherein the second value is the difference between the third PWV and the fourth PWV of the second location. If the first and second values are the same or similar, then the test reagent is a modulator of an ion channel. If the first and second values are different, then the test reagent is a not a modulator of an ion channel.
- Yet another embodiment of the invention provides a method of identifying a modulator of an ion channel.
- the method comprises applying cells to a first location on a surface of a colorimetric resonant reflectance optical biosensor and applying a test reagent to the first location.
- a first PWV is determined for the first location.
- a known ion channel antagonist or agonist of the cells is applied to the first location.
- a second PWV is determined for the first location.
- a first value is determined, wherein the first value is the difference between the first PWV and the second PWV.
- Cells are applied to a second location on a surface of a colorimetric resonant reflectance optical biosensor and a third PWV for the second location is determined.
- a known ion channel antagonist or agonist of the cells is applied to the second location.
- a fourth PWV for the second location is determined.
- a second value is determined, wherein the second value is the difference between the third PWV and the fourth PWV. If the first and second values are different, then the test reagent is a modulator of an ion channel. If the first and second values are the same or similar, then the test reagent is not a modulator of an ion channel.
- Still another embodiment of the invention provides a method of confirming that a test reagent is a modulator of an ion channel.
- the method comprises applying cells to a first location on a surface of a colorimetric resonant reflectance optical biosensor and detecting a first PWV for the first location.
- a known ion channel agonist or antagonist of the cells and a test reagent is applied to the first location.
- a second PWV is determined for the first location.
- a first value is determined, wherein the first value is the difference between the first PWV and the second PWV.
- Cells are applied to a second location on a surface of a colorimetric resonant reflectance optical biosensor and a third PWV is detected for the second location.
- a known ion channel agonist or antagonist of the cells is applied to the second location and a fourth PWV for the second location is determined.
- a second value is determined, wherein the second value is the difference between the third PWV and the fourth PWV. If the first and second values are different, then the test reagent is confirmed as a modulator of an ion channel. If the first and second values are the same or similar, then the test reagent is a not a modulator of an ion channel.
- Yet another embodiment of the invention provides a method of confirming that a test reagent is a modulator of an ion channel.
- the method comprises applying cells to a first location on a surface of a colorimetric resonant reflectance optical biosensor and applying a known ion channel agonist or antagonist of the cells and the test reagent to the first location.
- a first PWV for the first location is determined.
- Cells are applied to a second location on a surface of a colorimetric resonant reflectance optical biosensor and a known ion channel agonist or antagonist of the cells is applied to the second location.
- a second PWV for the second location is determined.
- Another embodiment of the invention provides a method of determining ion channel modulating properties of a test reagent.
- the method comprises applying cells to a surface of a colorimetric resonant reflectance optical biosensor; substantially blocking the functional activity of one or more first types of ion channels, wherein the activity of one or more second types of ion channels is not blocked; detecting a colorimetric resonant reflectance optical first peak wavelength value (PWV) for the cells; applying a test reagent to the cells; detecting a colorimetric resonant reflectance optical second PWV for the cells; and determining ion channel modulation properties of the test reagent based on a comparison of the first PWV and second PWV.
- PWV colorimetric resonant reflectance optical first peak wavelength value
- the functional activity of one or more types of ion channels can be substantially blocked by an antibody.
- the one or more first type and second type of ion channels can be voltage-gated sodium channels, voltage-gated calcium channels, potassium channels, inward-rectifier potassium channels, calcium-activated potassium channels, voltage-gated potassium channels, two pore domain potassium channels, transient receptor potential channels, cation channels of sperm, cyclic nucleotide gated channels, hyperpolaraization activated cyclic nucleotide gated channels, two pore channels, ligand gated channels, and light- gated channels.
- the cells can be incubated for a period of time after each step of the method.
- the surface of the colorimetric resonant reflectance optical biosensor can be an internal surface of a vessel selected from the group consisting of a microtiter well, microtiter plate, test tube, Petri dish, microfluidic channel, and microarray.
- the first and second PWVs can be detected using a scanner with a lens having a lower limit pixel size of about 2 micrometers to about 200 micrometers.
- the cells and test reagent may not comprise detection labels.
- the method can be performed at a temperature of about 2, 10, 15, 25, 30, or 37 degrees Celsius.
- the method can further comprising the steps of washing the cells; equilibrating the cells and optionally repeating the steps of the assay.
- Still another embodiment of the invention provides a method of identifying a modulator of an ion channel.
- the method comprises applying cells in a serum-free medium to a surface of a colorimetric resonant reflectance optical biosensor, wherein one or more extracellular matrix (ECM) ligands are immobilized to the surface of the biosensor; detecting a colorimetric resonant reflectance optical first peak wavelength value (PWV) for the cells; applying one or more test ion channel modulators to the surface of the biosensor; effecting a change in the activity of one or more ion channels of the cells; detecting a colorimetric resonant reflectance optical second PWV for the cells; and determining if the one or more test ion channel modulators modulated the one or more ion channels of the cells.
- the method can further comprise the steps of: washing the cells; equilibrating the cells; and optionally repeating the steps of the assay.
- Yet another embodiment of the invention provides a method of identifying a modulator of an ion channel.
- the method comprises applying cells in a serum-free medium to a surface of a colorimetric resonant reflectance optical biosensor, wherein one or more extracellular matrix (ECM) ligands are immobilized to the surface of the biosensor; applying one or more test ion channel modulators to the surface of the biosensor; effecting a change in one or more ion channels of the cells; detecting a colorimetric resonant reflectance optical first PWV for the cells; effecting a change in one or more ion channels of the cells; detecting a colorimetric resonant reflectance optical second PWV for the cells; and determining if the one or more test ion channel modulators modulated the one or more ion channels of the cells.
- the method can further comprising the steps of: washing the cells; equilibrating the cells; and optionally repeating the steps.
- the invention provides methods for identifying and confirming modulators of ion channels without the use of recombinant cell lines over-expressing the ion channel proteins or the use of detection labels.
- Figure 1 demonstrates the effect of removing potassium from CHO cells transfected with the human ether-a-go-go (hERG) potassium channel and from parental CHO cells.
- Figure 2 demonstrates the effect of cisapride and E4031 on CHO cells transfected with hERG.
- Figure 3 demonstrates the effect of cisapride and E4031 on parental CHO cells.
- ion channels include, e.g., voltage-gated sodium channels (that sense the transmembrane potential and open or close in response to depolarization or hyperpolarization), voltage-gated calcium channels, mechano- sensitive ion channels, potassium channels (such as, inward-rectifier potassium channels, calcium- activated potassium channels, voltage-gated potassium channels, and two pore domain potassium channels), transient receptor potential channels, cation channels of sperm, cyclic nucleotide gated channels (such as hyperpolaraization activated cyclic nucleotide gated channels)(that open in response to internal solutes and mediate cellular responses to second messengers), stretch activated channels (that open or close in response to mechanical force) two pore channels, ligand gated channels (which open in response to a specific ligand molecule on the external face of the membrane), G-protein gated channels (which open in response to a specific ligand molecule on the external face of the membrane), G-protein gated channels (which open in response to
- Ligand gated ion channels open in response to specific ligand molecules binding to the extracellular domain of the receptor protein. Ligand binding causes a conformational change in the structure of the channel protein that leads to the opening of the channel gate and subsequent ion flux across the plasma membrane.
- Examples of such channels include the G-protein coupled ion channels, anion-permeable ⁇ -aminobutyric acid-gated GABA A or c receptors, ionotropic glutamate-gated receptors, serotonin/5 -HT 3 receptor, ATP-gated P2X receptors, and cation-permeable "nicotinic" acetylcholine receptor.
- GPC G-protein coupled ion channels
- GCR G-protein coupled receptor
- G-Proteins allow the channel to open and ions are able to flow across the cell membrane. Because of the movement from the receptor to the ion, the speed of the channel opening is delayed, however the channel stays open for a longer time.
- GPC channels such as the GABA (subtypes A & C) and NMDA (antagonists), are likely therapeutic sites for the function of anesthetics for blocking of sensation, temporarily taken away muscle activation, and behavior modification.
- GABA molecules neurotransmitter, gamma- aminobutyric acid
- GABAc receptors Binding of GABA molecules (neurotransmitter, gamma- aminobutyric acid) to their binding sites in the extracellular part of GABA A and GABAc receptors triggers opening of a chloride ion-selective pore.
- the increased chloride conductance drives the membrane potential towards the reversal potential of the Cl " ion in neurons, inhibiting the firing of new action potentials or nerve impulses.
- Glutamate regulates ion channels and is the most abundant excitatory neurotransmitter in the mammalian nervous system. Nerve impulses trigger release of glutamate from the presynaptic cell. In the opposing post-synaptic cell, glutamate receptors, such as the NMDA (ligand gated ion channel) receptor, bind glutamate and are activated. Glutamate transporters are found in neuronal and glial membranes. They rapidly remove glutamate from the extracellular space. In brain injury or disease, they can work in reverse and excess glutamate can accumulate outside cells. This process causes calcium ions to enter cells via NMDA receptor channels, leading to neuronal damage and eventual cell death.
- NMDA ligand gated ion channel
- 5-HT 3 antagonists include ondansetron, granisetron, and tropisetron.
- P2X receptors are cation-permeable ligand gated ion channels that open in response to the binding of extracellular adenosine 5 '-triphosphate (ATP).
- ATP adenosine 5 '-triphosphate
- ATP binds to the P2X receptor and cause a conformational change in the structure of the ion channel that results in the opening of the ion-permeable pore.
- the different protein parts of this channel have been found responsible for regulating ATP binding, ion permeation, pore dilation and desensitization.
- Acetylcholine can open ligand gated sodium channels when it binds to acetylcholine receptors on skeletal muscle fibers.
- Inward rectifying potassum (K ir ) channels are found on multiple cell types. In cardiac myocytes K ir channels close upon depolarization, slowing membrane repolarization and helping maintain a more prolonged action potential. This type of inward-rectifier channel is distinct from delayed rectifier K + channels, which help re-polarize nerve and muscle cells after action potentials; and potassium leak channels, which provide much of the basis for the resting membrane potential.
- K 11 channels are involved in regulation of nitric oxide synthase.
- kidney cells K 11 export surplus potassium into collecting tubules for removal in the urine, or alternatively may be involved in the reuptake of potassium back into the body.
- G-protein activated IRKs (K ir 3) are important regulators. In pancreatic beta cellsKATP channels control insulin release.
- VDCC Calcium voltage gated ion channels
- VDCCs Activation of VDCCs allows Ca 2+ entry into the cell, which depending on the cell type, results in muscular contraction, excitation of neurons, up-regulation of gene expression, or release of hormones or neurotransmitters.
- Voltage-gated sodium channels control and set action potentials across cell membranes. When voltage-gated sodium channels open there is a change in the cell's membrane potential, and a small but significant number of Na + ions will move into the cell down their electrochemical gradient, thereby depolarizing the cell. Intracellular and extracellular blockers are known modulators of pharmacologic control of these ion channels. Alkaloid based toxins of plants and animals (puffer fish and blue-ringed octapus) act extracellularly on these channels and result in loss of neural activity. Intracellular blockage of these channels result in anesthetics, anti- arrhythmic and anti-convulsant agents.
- TRP transient receptor potential channels
- TRPC canonical
- TRPV vanilloid
- TRPA ankyrin
- TRPM melastatin
- TRPP polycystin
- TRPML mucolipin
- TRPN NOMPC
- Chemicals that can disrupt ion channel function include, e.g., lidocaine, novocaine, dedrotoxin, conotoxin, saxitoxin, iberiotoxin, heteropodatoxin, tetrodotoxin.
- Receptor tyrosine kinases, GPCRs, transient receptor potential channels, phopholipase C, signal transduction pathways (e.g. P13 kinases), cytokines, beta-arrestin pathway responses, cytoskeletal rearrangements, epigenetic signals can affect ion channel function.
- Genetic diseases that are caused by mutations in ion channel subunits or the proteins that regulate them include, e.g., alternating hemiplegia of childhood, Bartter syndrome, Brugada syndrome, congenital hyperinsulinism, cystic fibrosis, episodic ataxia, erythromelalgia, generalized epilepsy with febrile seizures, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, long QT syndrome, malignant hyperthermia, migraine, myasthenia gravis, myotonia congenita, neuromyotonia, familial hemiplegic migraine, spinocerebellar ataxia type 13, nonsyndromic deafness, paramyotonia congenita, potassium aggravated myotonias, periodic paralysis, retinitis pigmentosa, mucolipidosis type IV, Romano- Ward syndrome, short QT syndrome, and Timothy syndrome. Therefore, discovery of reagents that can modulate ion channels are of great interest
- One embodiment of the invention allows the direct detection of cell changes in response to ion channel regulators as they occur in real time with a colorimetric resonant reflectance biosensor and without the need to incorporate radiometric, colorimetric, or fluorescent labels.
- Changes in cells can be detected as the cells are probed with test reagents, agonists, and antagonists.
- the cellular changes can then be detected in real time using a high speed instruments such as the BIND ScannerTM (i.e., a colorimetric resonant reflectance biosensor system), and corresponding algorithms to quantify data. See, e.g., U.S. Pat. No. 6,951,715 and U.S. Pat. Publ. 2004/0151626.
- BIND ScannerTM i.e., a colorimetric resonant reflectance biosensor system
- Biosensors of the invention can be colorimetric resonant reflectance biosensors. See e.g., Cunningham et ah, "Colorimetric resonant reflection as a direct biochemical assay technique," Sensors and Actuators B, Volume 81, p. 316-328, Jan 5 2002; U.S. Pat. Publ. No. 2004/0091397.
- Colorimetric resonant biosensors are not surface plasmon resonant (SPR) biosensors.
- SPR biosensors have a thin metal layer, such as silver, gold, copper, aluminum, sodium, and indium. The metal must have conduction band electrons capable of resonating with light at a suitable wavelength.
- a SPR biosensor surface exposed to light must be pure metal. Oxides, sulfides and other films interfere with SPR.
- Colorimetric resonant biosensors do not have a metal layer, rather they have a dielectric coating of high refractive index material, such as TiO
- Grating-based waveguide biosensors are described in, e.g., U.S. Pat. No. 5,738,825.
- a grating-based waveguide biosensor comprises a waveguiding film and a diffraction grating that incouples an incident light field into the waveguiding film to generate a diffracted light field. A change in the effective refractive index of the waveguiding film is detected.
- a colorimetric resonant reflectance biosensor allows biochemical interactions to be measured on the biosensor's surface without the use of fluorescent tags, colorimetric labels or any other type of detection tag or detection label.
- a biosensor surface contains an optical structure that, when illuminated with collimated and/or white light, is designed to reflect only a narrow band of wavelengths ("a resonant grating effect"). The narrow wavelength band is described as a wavelength "peak.”
- the "peak wavelength value" (PWV) changes when materials, such as biological materials, are deposited or removed from the biosensor surface.
- a readout instrument is used to illuminate distinct locations on a biosensor surface with collimated and/or white light, and to collect reflected light. The collected light is gathered into a wavelength spectrometer for determination of a PWV.
- a biosensor can be incorporated into standard disposable laboratory items such as microtiter plates by bonding the structure (biosensor side up) into the bottom of a bottomless microtiter plate cartridge. Incorporation of a biosensor into common laboratory format cartridges is desirable for compatibility with existing microtiter plate handling equipment such as mixers, incubators, and liquid dispensing equipment. Colorimetric resonant reflectance biosensors can also be incorporated into, e.g., microfluidic, macrofluidic, or microarray devices (see, e.g., U.S. Pat. No. 7,033,819, U.S. Pat. No. 7,033,821).
- Colorimetric resonant reflectance biosensors can be used with well-know methodology in the art (see, e.g., Methods of Molecular Biology edited by Jun-Lin Guan, Vol. 294, Humana Press, Totowa, New Jersey) to monitor cell behavioral changes or the lack of these changes upon exposure to one or more extracellular reagents.
- Colorimetric resonant reflectance biosensors comprise subwavelength structured surfaces (SWS) and are an unconventional type of diffractive optic that can mimic the effect of thin-film coatings. (Peng & Morris, "Resonant scattering from two-dimensional gratings," J. Opt. Soc. Am. A, Vol. 13, No. 5, p.
- a SWS structure contains a one-dimensional, two-dimensional, or three dimensional grating in which the grating period is small compared to the wavelength of incident light so that no diffractive orders other than the reflected and transmitted zeroth orders are allowed to propagate. Propagation of guided modes in the lateral direction are not supported. Rather, the guided mode resonant effect occurs over a highly localized region of approximately 3 microns from the point that any photon enters the biosensor structure.
- the reflected or transmitted light of a colorimetric resonant reflectance biosensor can be modulated by the addition of molecules such as specific binding substances or binding partners or both to the upper surface of the biosensor.
- the added molecules increase the optical path length of incident radiation through the structure, and thus modify the wavelength at which maximum reflectance or transmittance will occur.
- a colorimetric resonant reflectance biosensor when illuminated with white and/or collimated light, is designed to reflect a single wavelength or a narrow band of wavelengths (a "resonant grating effect").
- Light can illuminate the biosensor from either the top or the bottom.
- mass is deposited on the surface of the biosensor, the reflected wavelength is shifted due to the change of the optical path of light that is shown on the biosensor.
- a detection system consists of, for example, a light source that illuminates a small spot of a biosensor at normal incidence through, for example, a fiber optic probe, and a spectrometer that collects the reflected light through, for example, a second fiber optic probe also at normal incidence.
- a colorimetric resonant reflectance biosensor comprises, e.g., an optical grating comprised of a high refractive index material, a substrate layer that supports the grating, and optionally one or more specific binding substances or linkers immobilized on the surface of the grating opposite of the substrate layer.
- the high refractive index material has a higher refractive index than a substrate layer. See, e.g., U.S. Pat. No. 7,094,595; U.S. Pat. No. 7,070,987.
- a cover layer covers the grating surface.
- the refractive index of the optical grating can be less than the refractive index of the optional cover layer.
- An optical grating is coated with a high refractive index dielectric film which can be comprised of a material that includes, for example, zinc sulfide, titanium dioxide, tantalum oxide, silicon nitride, and silicon dioxide.
- a cross-sectional profile of a grating with optical features can comprise any periodically repeating function, for example, a "square - wave.”
- An optical grating can also comprise a repeating pattern of shapes selected from the group consisting of lines (one-dimensional), squares, circles, ellipses, triangles, trapezoids, sinusoidal waves, ovals, rectangles, and hexagons.
- a colorimetric resonant reflectance biosensor of the invention can also comprise an optical grating comprised of, for example, plastic or epoxy, which is coated with a high refractive index material.
- Layer thicknesses i.e. cover layer, biological material, or an optical grating
- the grating period is selected to achieve resonance at a desired wavelength.
- Linear gratings (i.e., one dimensional gratings) have resonant characteristics where the illuminating light polarization is oriented perpendicular to the grating period.
- a colorimetric resonant reflection biosensor can also comprise, for example, a two-dimensional grating, e.g., a hexagonal array of holes or squares. Other shapes can be used as well.
- a linear grating has the same pitch (i.e. distance between regions of high and low refractive index), period, layer thicknesses, and material properties as a hexagonal array grating. However, light must be polarized perpendicular to the grating lines in order to be resonantly coupled into the optical structure.
- a polarizing filter oriented with its polarization axis perpendicular to the linear grating must be inserted between the illumination source and the biosensor surface. Because only a small portion of the illuminating light source is correctly polarized, a longer integration time is required to collect an equivalent amount of resonantly reflected light compared to a hexagonal grating.
- An optical grating can also comprise, for example, a "stepped" profile, in which high refractive index regions of a single, fixed height are embedded within a lower refractive index cover layer. The alternating regions of high and low refractive index provide an optical waveguide parallel to the top surface of the biosensor.
- a colorimetric resonant reflectance biosensor of the invention can further comprise a cover layer on the surface of an optical grating opposite of a substrate layer. Where a cover layer is present, the one or more specific binding substances are immobilized on the surface of the cover layer opposite of the grating.
- a cover layer comprises a material that has a lower refractive index than a material that comprises the grating.
- a cover layer can be comprised of, for example, glass (including spin-on glass (SOG)), epoxy, or plastic.
- various polymers that meet the refractive index requirement of a biosensor can be used for a cover layer.
- SOG can be used due to its favorable refractive index, ease of handling, and readiness of being activated with specific binding substances using the wealth of glass surface activation techniques.
- a grating structure of SiN/glass can directly be used as the sensing surface, the activation of which can be done using the same means as on a glass surface.
- Resonant reflection can also be obtained without a planarizing cover layer over an optical grating.
- a biosensor can contain only a substrate coated with a structured thin film layer of high refractive index material. Without the use of a planarizing cover layer, the surrounding medium (such as air or water) fills the grating. Therefore, specific binding substances are immobilized to the biosensor on all surfaces of an optical grating exposed to the specific binding substances, rather than only on an upper surface.
- a colorimetric resonant reflectance biosensor of the invention will be illuminated with white and/or collimated light that will contain light of every polarization angle. The orientation of the polarization angle with respect to repeating features in a biosensor grating will determine the resonance wavelength.
- a “linear grating” i.e., a one-dimensional grating
- a biosensor consisting of a set of repeating lines and spaces will have two optical polarizations that can generate separate resonant reflections.
- Light that is polarized perpendicularly to the lines is called “s-polarized,” while light that is polarized parallel to the lines is called “p-polarized.”
- s-polarized Light that is polarized perpendicularly to the lines
- p-polarized Both the s and p components of incident light exist simultaneously in an unfiltered illumination beam, and each generates a separate resonant signal.
- a biosensor can generally be designed to optimize the properties of only one polarization (the s-polarization), and the non-optimized polarization is easily removed by a polarizing filter.
- an alternate biosensor structure can be used that consists of a set of concentric rings.
- the difference between the inside diameter and the outside diameter of each concentric ring is equal to about one-half of a grating period.
- Each successive ring has an inside diameter that is about one grating period greater than the inside diameter of the previous ring.
- the concentric ring pattern extends to cover a single sensor location - such as an array spot or a microtiter plate well.
- Each separate microarray spot or microtiter plate well has a separate concentric ring pattern centered within it. All polarization directions of such a structure have the same cross- sectional profile.
- the concentric ring structure must be illuminated precisely on-center to preserve polarization independence.
- the grating period of a concentric ring structure is less than the wavelength of the resonantly reflected light.
- the grating period is about 0.01 micron to about 1 micron.
- the grating depth is about 0.01 to about 1 micron.
- an array of holes or posts are arranged to closely approximate the concentric circle structure described above without requiring the illumination beam to be centered upon any particular location of the grid.
- Such an array pattern is automatically generated by the optical interference of three laser beams incident on a surface from three directions at equal angles.
- the holes (or posts) are centered upon the corners of an array of closely packed hexagons.
- the holes or posts also occur in the center of each hexagon.
- Such a hexagonal grid of holes or posts has three polarization directions that "see" the same cross- sectional profile.
- the hexagonal grid structure therefore, provides equivalent resonant reflection spectra using light of any polarization angle. Thus, no polarizing filter is required to remove unwanted reflected signal components.
- the period of the holes or posts can be about 0.01 microns to about 1 micron and the depth or height can be about 0.01 microns to about 1 micron.
- a detection system can comprise a colorimetric resonant reflectance biosensor a light source that directs light to the colorimetric resonant reflectance biosensor, and a detector that detects light reflected from the biosensor.
- a filter it is possible to simplify the readout instrumentation by the application of a filter so that only positive results over a determined threshold trigger a detection.
- a colorimetric resonant reflectance biosensor of the invention By measuring the shift in resonant wavelength at each distinct location of a colorimetric resonant reflectance biosensor of the invention, it is possible to determine which distinct locations have, e.g., biological material deposited on them.
- the extent of the shift can be used to determine, e.g., the amount of binding partners in a test sample and the chemical affinity between one or more specific binding substances and the binding partners of the test sample.
- a colorimetric resonant reflectance biosensor can be illuminated two or more times.
- a first measurement can determine the reflectance spectra of one or more distinct locations of a biosensor with, e.g., no biological material on the biosensor or with cells on the surface.
- a second, third, fourth or additional measurements can determine the reflectance spectra after, e.g., one or more cells, test reagents, ion channel agonists, or ion channel antagonists are applied to a biosensor or after an incubation period or wash step.
- the difference in peak wavelength between these two or more measurements can be used to, e.g., determine the presence or amount of cells on the biosensor, or the activity of a test reagent, ion channel agonist, or ion channel antagonist on the cells. Additionally, this method of illumination can control for small imperfections in a surface of a biosensor that can result in regions with slight variations in the peak resonant wavelength. This method can also control for varying concentrations or density of cell matter on a biosensor.
- One or more cells can be immobilized on a biosensor by for example, physical adsorption or by chemical binding.
- the cells can be non-adherent cells or adherent cells.
- the cells can natively express one or more ion channels or one or more ion channel components.
- the cells can recombinantly express one or more ion channels or ion channel components.
- the cells can be mammalian cells, such as human cells.
- a cell can specifically bind to a biosensor surface via a specific binding substance such as a nucleic acid, peptide, protein solution, peptide solution, solutions containing compounds from a combinatorial chemical library, antigen, polyclonal antibody, monoclonal antibody, single chain antibody (scFv), F(ab) fragment, F(ab') 2 fragment, Fv fragment, small organic molecule, virus, polymer or biological sample, wherein the specific binding substance is immobilized to the surface of the biosensor and the binding partner is on the surface of the cell.
- a specific binding substance such as a nucleic acid, peptide, protein solution, peptide solution, solutions containing compounds from a combinatorial chemical library, antigen, polyclonal antibody, monoclonal antibody, single chain antibody (scFv), F(ab) fragment, F(ab') 2 fragment, Fv fragment, small organic molecule, virus, polymer or biological sample, wherein the specific binding substance is immobilized to the surface of the biosensor and
- Cells can be arranged in an array of one or more distinct locations on the biosensor surface, said surface residing within one or more wells of a multiwell plate and comprising one or more surfaces of the multiwell plate or microarray.
- the array of cells comprises one or more cells on the biosensor surface within a microwell plate such that a surface contains one or more distinct locations, each with a different cell or with a different amount of cells.
- an array can comprise 1, 10, 100, 1,000, 10,000 or 100,000 or greater distinct locations.
- each well of a multiwell plate or microarray can have within it an array of one or more distinct locations separate from the other wells of the multiwell plate, which allows multiple different samples to be processed on one multiwell plate.
- the array or arrays within any one well can be the same or different than the array or arrays found in any other microtiter wells of the same microtiter plate.
- Immobilization of a cell to a biosensor surface can be also be affected via binding to, for example, the following functional linkers: a nickel group, an amine group, an aldehyde group, an acid group, an alkane group, an alkene group, an alkyne group, an aromatic group, an alcohol group, an ether group, a ketone group, an ester group, an amide group, an amino acid group, a nitro group, a nitrile group, a carbohydrate group, a thiol group, an organic phosphate group, a lipid group, a phospholipid group or a steroid group.
- a nickel group an amine group, an aldehyde group, an acid group, an alkane group, an alkene group, an alkyne group, an aromatic group, an alcohol group, an ether group, a ketone group, an ester group, an amide group, an amino acid group, a nitro group, a nitrile group,
- a cell can be immobilized on the surface of a biosensor via physical adsorption, chemical binding, electrochemical binding, electrostatic binding, passive or active adhesion molecules, hydrophobic binding or hydrophilic binding, and immunocapture methods.
- Methods for coating surfaces to make them amenable to cell attachment and/or growth are well known by persons skilled in the art of cell culture and can be comprised of a coating for the biosensor with materials or their derivatives including but not limited to poly-D-lysine, fibronectin, actin, integrins, adherins, cadherins, collagen, human serum, fetal bovine serum, calf serum, laminin, or other materials.
- a biosensor can be coated with a linker such as, e.g., a nickel group, an amine group, an aldehyde group, an acid group, an alkane group, an alkene group, an alkyne group, an aromatic group, an alcohol group, an ether group, a ketone group, an ester group, an amide group, an amino acid group, a nitro group, a nitrile group, a carbohydrate group, a thiol group, an organic phosphate group, a lipid group, a phospholipid group or a steroid group.
- a linker such as, e.g., a nickel group, an amine group, an aldehyde group, an acid group, an alkane group, an alkene group, an alkyne group, an aromatic group, an alcohol group, an ether group, a ketone group, an ester group, an amide group, an amino acid group, a nitro group, a n
- an amine surface can be used to attach several types of linker molecules while an aldehyde surface can be used to bind proteins directly, without an additional linker.
- a nickel surface can be used to bind molecules that have an incorporated histidine (“his”) tag. Detection of "his-tagged" molecules with a nickel-activated surface is well known in the art (Whitesides, Anal. Chem. 68, 490, (1996)).
- Linkers and specific binding substances can be immobilized on the surface of a biosensor such that each well has the same linkers and/or specific binding substances immobilized therein.
- each well can contain a different combination of linkers and/or specific binding substances.
- a cell can specifically or non- specifically bind to a linker or specific binding substance immobilized on the surface of a biosensor.
- the surface of the biosensor can have no linker or specific binding substance and a cell can bind to the biosensor surface non- specifically.
- Immobilization of one or more specific binding substances or linker onto a biosensor is performed so that a specific binding substance or linker will not be washed away by rinsing procedures, and so that its binding to cells in a test sample is unimpeded by the biosensor surface.
- Several different types of surface chemistry strategies have been implemented for covalent attachment of specific binding substances to, for example, glass or polymers for use in various types of microarrays and biosensors.
- Surface preparation of a biosensor so that it contains the correct functional groups for binding one or more specific binding substances is an integral part of the biosensor manufacturing process.
- One or more specific cells can be attached to a biosensor surface by physical adsorption (i.e., without the use of chemical linkers) or by chemical binding (i.e., with the use of chemical linkers) as well as electrochemical binding, electrostatic binding, hydrophobic binding and hydrophilic binding. Chemical binding can generate stronger attachment of specific binding substances on a biosensor surface and provide defined orientation and conformation of the surface-bound molecules.
- Immobilization of specific binding substances to plastic, epoxy, or high refractive index material can be performed essentially as described for immobilization to glass. However, the acid wash step can be eliminated where such a treatment would damage the material to which the specific binding substances are immobilized.
- One embodiment of the invention provides a method of identifying a modulator of an ion channel.
- a modulator of an ion channel has an effect on the functional activity of an ion channel.
- An effect on the functional activity includes blocking or inhibiting the activity of the ion channel.
- Modulators can block, inhibit, enhance, or increase the functional activity of an ion channel. The blocking, inhibition, enhancement or increase may take place in the presence of, or in response to, a molecule that blocks the ion channel.
- Modulators can, for example, act on ion channels to change gating /ligand ion selectivity, modify the chemical signals for channel function (pH, osmolarity), change the potential function for gating activity
- altered polarizability or voltage dependence cause a physical blockage, cause a physical widening (altering folding function of channel control protein), modify refractory period, modify the mechanical force function (mechanical stretch) or temperature required for channel function.
- the methods of the invention can be used with cells that have not been recombinantly altered to express or over-express certain ion channels or recombinant cells, however, cells that recombinantly express or over-express ion channel proteins or subunits are not required by the methods of the invention.
- Cells can be applied to a first location on a surface of a colorimetric resonant reflectance optical biosensor.
- the cells can be immobilized to the biosensor or they can merely be grown on the surface of the biosensor.
- the cells can be incubated on the surface of the biosensor at any step of the methods of the invention for about, 1, 5, 10, 30 or more minutes or about 1, 2, 5, 24, 48, 36, or more hours.
- the assays can be performed at a temperature of about 2, 5, 10, 25, 30, or 37 degrees Celsius (or any range of temperatures between about 2 and about 37 degrees Celsius).
- the cells can be washed, equilibrated and used again to complete the same assay or a different assay. This is particularly advantageous where cells are expensive or difficult to handle.
- a method of identifying an antagonist or agonist of an ion channel comprises applying cells to a first location and a second location on a surface of colorimetric resonant reflectance optical biosensor.
- the cells can be applied to the second location at the same time the first set of cells are applied to the first location or at an earlier or later time. These cells can be the same type of cells or different cells as the set of cells applied to the first location.
- a test reagent is added to the first location before the cells are added to the first location, at the same time the cells are added to the first location, or after the cells are added to the first location.
- a known ion channel antagonist or agonist of the cells is added to the second location.
- the known ion channel agonist or antagonist is added to the second location before the cells are added to the second location, at the same time the cells are added to the second location, or after the cells are added to the second location.
- the use of known agonists or antagonists can determine the specificity of the ion channel.
- Antagonists and agonists including, e.g., specific toxins and other small molecules with known functions or known target can be used to determine the specificity of the test reagent/stimuli/modulator response on an ion channel.
- use of compounds that act at sites upstream or downstream of an ion channel can be used to determine the specificity of the response to a particular channel as well.
- the first and second values are the same or similar if they are within about 1 nm or less of each other. If the first and second PWVs are substantially different (that is the first and second PWVs differ by greater than about 1 nm, 2 nm, 3 nm, 4 nm or more), then the test reagent is not an antagonist or agonist.
- one of more additional PWVs may be determined before or after any of the steps or additions of the method and compared to any other PWV.
- a method of identifying an antagonist or agonist of an ion channel comprises applying cells to a first location.
- a PWV can be determined for the first location.
- a test reagent is applied to the first location.
- the cells and test reagent can be incubated for period of time if desired.
- a second PWV for the first location can be determined.
- a first value can be calculated, wherein the first value is the difference between the first PWV and the second PWV.
- the first value can be compared to a control test.
- the control test can comprise applying cells to a second location on a surface of a colorimetric resonant reflectance optical biosensor.
- the cells can be incubated for a period of time if desired.
- These cells can be applied to the second location at the same time the first set of cells are applied to the first location or at an earlier or later time. These cells can be the same type of cells or different cells as the set of cells applied to the first location.
- a third PWV for the second location can be detected.
- a known ion channel antagonist or agonist of the cells is applied to the second location.
- the cells can be incubated for a period of time if desired.
- a fourth PWV for the second location can be detected.
- a second value can be determined, wherein the second value can be the difference between the third PWV and the fourth PWV of the second location. If the first and second values are the same or similar, then the test reagent is a modulator of an ion channel.
- the first and second values are the same or similar if they are within about 1 nm or less of each other. If the first and second PWVs are substantially different (that is the first and second PWVs differ by greater than about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm or more), then the test reagent is not an antagonist or agonist. Because the label free biosensor method of interrogating the cells is not destructive to the cells, the cells may be treated more than one time to look for differences over a number of minutes, hours, or days. There are multitudes of known ion channel antagonists or agonists in the art.
- a small sampling of examples include calcium channel blockers (such as nisoldipine, nifedipine, nicardipine, bepridil, isradipine, nimodipine, felodipine, amlodipine, diltiazem, and verapamil), modulators of potassium ion channels (see, e.g., US 20050267089), Na + ion channel modulating compounds (see, e.g, US 6,576,791), see also, US 7,101,877; US 7,057,053, US 20070004718; US 20060199848, US 20060135536, US 20050192208, US 20050026993, US 20040029937, US 20030008906.
- calcium channel blockers such as nisoldipine, nifedipine, nicardipine, bepridil, isradipine, nimodipine, felodipine, amlodipine, diltiazem, and verapamil
- the first location and second location on the surface of the colorimetric resonant reflectance optical biosensor can be an internal surface of a vessel selected from the group consisting of a microtiter well, microtiter plate, test tube, Petri dish, microfluidic channel, and microarray.
- the cells, test reagent, and ion channel antagonist or agonist do not have to comprise detection labels in the assays of the invention.
- Another embodiment of the invention provides a method of identifying a modulator of an ion channel.
- Cells can be applied to a first location on a surface of a colorimetric resonant reflectance optical biosensor.
- a test reagent and a known ion channel antagonist or agonist of the cells is applied to the first location.
- the cells, test reagent and known ion channel antagonist or agonist of the cells can be added to the sensor surface in any order or at the same time.
- a first PWV for the first location can be determined.
- Cells can be applied to a second location on a surface of a colorimetric resonant reflectance optical biosensor. The cells can be applied to the second location at the same time the first set of cells are applied to the first location or at an earlier or later time.
- These cells can be the same type of cells or different cells as the set of cells applied to the first location.
- a known ion channel antagonist or agonist of the cells is applied to the second location.
- the cells and known ion channel antagonist or agonist of the cells can be applied to the biosensor surface in any order or at the same time.
- a second PWV can be determined for the second location. If the first and second PWVs are different, then the test reagent is a modulator of an ion channel.
- the first and second values are different if they are greater than about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, or more apart. If the first and second PWVs are the same or similar then the test reagent is not a modulator of an ion channel.
- the first and second values are the same or similar if they are within about 1 nm or less of each other.
- a method of identifying a modulator of an ion channel comprises applying cells to a first location on a surface of a colorimetric resonant reflectance optical biosensor, applying a test reagent to the first location, and detecting a first PWV for the first location.
- the cells and test reagent can be applied to the first location in any order or at the same time.
- a known ion channel antagonist or agonist of the cells is then applied to the first location and a second PWV for the first location can be detected.
- a first value is determined wherein the first value is the difference between the first PWV and the second PWV.
- the method can alternatively comprise applying cells to a first location on a surface of a colorimetric resonant reflectance optical biosensor, applying a known ion channel antagonist or agonist of the cells to the first location, and detecting a first PWV for the first location.
- the cells and known ion channel antagonist or agonist of the cells can be applied to the first location in any order or at the same time.
- a test reagent is then applied to the first location and a second PWV for the first location can be detected.
- a first value is determined wherein the first value is the difference between the first PWV and the second PWV.
- Cells are applied to a second location on a surface of a colorimetric resonant reflectance optical biosensor. These cells can be applied to the second location at the same time the first set of cells are applied to the first location or at an earlier or later time. These cells can be the same type of cells or different cells as the set of cells applied to the first location.
- a third PWV for the second location can be detected.
- a known ion channel antagonist or agonist of the cells is applied to the second location.
- a fourth PWV for the second location can be determined.
- a second value is determined, wherein the second value is the difference between the third PWV and the fourth PWV. If the first and second values are different, then the test reagent is a modulator of an ion channel.
- the first and second values are different if they are greater than about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, or more apart. If the first and second values are the same or similar, then the test reagent is not a modulator of an ion channel. The first and second values are the same or similar if they are within about 1 nm or less of each other.
- the cells can be incubated for a period of time after their application to the first location, after the application of the test reagent to the first location, after the application of the known ion channel agonist or antagonist to the first location, after the application of the cells to the second location, after the application of the known ion channel antagonist or agonist to the second location, or at any other point during the assay, or a combination thereof.
- the cells are incubated for a period of time after their application to a surface of the colorimetric resonant reflectance optical biosensor; after the application of the test reagent; after the application of the ion channel agonist or antagonist, or any other point during the assay, or a combination thereof.
- the label free biosensor method of interrogating the cells is not destructive to the cells, the cells may also be treated more than one time to look for differences over a number of minutes, hours, or days.
- Another embodiment of the invention provides a method of confirming that a test reagent is a modulator of an ion channel comprising applying cells to a first location on a surface of a colorimetric resonant reflectance optical biosensor, and optionally detecting a first PWV for the first location.
- a known ion channel agonist or antagonist of the cells is applied to the first location; and the test reagent is applied to the first location.
- the known ion channel agonist or antagonist and the test reagent can be applied to the first location at the same time or one may be applied to the first location before the other.
- a second PWV can be detected for the first location.
- a first value can be determined, wherein the first value is the difference between the first PWV and the second PWV.
- Cells are applied to a second location on a surface of a colorimetric resonant reflectance optical biosensor. These cells can be applied to the second location at the same time the first set of cells are applied to the first location or at an earlier or later time. These cells can be the same type of cells or different cells as the set of cells applied to the first location.
- a third PWV can be determined for the second location.
- a known ion channel agonist or antagonist of the cells is applied to the second location and a fourth PWV can be determined for the second location.
- the known ion channel agonist or antagonist can be the same as that used at the first location or a different known ion channel agonist or antagonist.
- a second value can be determined, wherein the second value is the difference between the third PWV and the fourth PWV. If the first and second values are different, then the test reagent is confirmed as a modulator of an ion channel. The first and second values are different if they are greater than about 1 nm, 2 nm, 3nm, 4 nm, 5 nm, or more apart.
- the cells can be incubated for a period of time after their application to the biosensor; after the application of the test reagent, after the application of the known ion channel agonist or antagonist, or at any other point in the assay, or a combination thereof.
- Agonists, antagonists and modulators can affect more than one ion channel. Therefore, it can be advantageous to inhibit the function of one or more first types of ion channels in order to determine the function of an added test reagent on one or more second types of ion channels.
- the first and second types of ion channels can be the same or different.
- one or more antibodies, proteins, small molecules, siRNAs, anti-sense RNAs, or other agents are used to inhibit some or all of the function of one or more types of ion channels.
- an antibody can be specific for one or more pore subunit proteins of an ion channel such that when the antibody specifically binds to the pore protein the ion channel ceases to function in part or substantially.
- a test regent such as an agonist, antagonist, ion channel modulator, or any other compound, can be added to the cells to determine the effect of the test reagent on the non-blocked types of ion channels.
- a test regent such as an agonist, antagonist, ion channel modulator, or any other compound, can be added to the cells to determine the effect of the test reagent on the non-blocked types of ion channels.
- One embodiment of the invention provides a method for screening a test reagent for modulation of ion channel activity. The method can comprise contacting cells that express one or more ion channels with an agent that can partially or substantially block one or more types of ion channels. The cells are contacted with a test reagent. Ion channel activity of one or more types of ion channels can be determined before and/or after contact between the cell and the test reagent and/or before and/or after contact between the cells and the blocking agent. An alteration in ion channel activity as determined by a shift in PW
- a test reagent can be any molecule that potentially modulates the activity of an ion channel or alters the expression of ion channel proteins within cells.
- a molecule can be, for example, a polypeptide, polynucleotide, antibody, small organic molecule, polysaccharide, fatty acid, steroid, purine, pyrimidine, or multivalent cation, among others.
- Small organic molecules can have a molecular weight of more than 100 and less than about 2,500 Daltons (D). Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D.
- one or more ECM ligands are coated on one or more surfaces of biosensor.
- One of more types of cells in serum-free medium are added to the surface of the biosensor.
- a serum- free medium contains about 5, 4, 3, 2, 1, 0.5% or less serum.
- a serum- free medium can comprise about 0% serum or about 0% to about 1% serum.
- a PWV can be determined after the addition of the cells to the biosensor.
- One or more potential or known modulators of ion channels can be added to the biosensor surface.
- a PWV can be determined after the addition of the one or more modulators.
- a change can be effected in the activity of one or more ion channels of the cells. For example, the cells can be depolarized.
- a change in the activity of one or more ion channels can be effected by depolarizing the cells.
- Depolarization is a decrease in the absolute value of a cell's membrane potential. Thus, changes where the membrane potential becomes less positive or less negative are both depolarizations.
- Depolarization can be caused by influx of cations, e.g. Na + through Na + channels, or Ca 2+ through Ca 2+ channels. Depolarization can be inhibited by outflux of K + through K + channels. Therefore, depolarization can be caused by manipulation of salt concentration, pressure and current. See, e.g., Boulton et al. (eds), The Neuronal Microenvironment, Springer, NY (1988); Arbib, The Handbook of brain theory and neural networks 2 nd Ed., MIT Press, Cambridge Mass. (2003).
- Cell medium can be removed, diluted or replaced with medium with a reduced concentration of an ion, such as potassium in order to "open" ion channels.
- the cells can be exposed to an ion channel opener, e.g. scorpion venom toxin.
- a PWV can be determined after the change in activity is effected in one or more ion channels (e.g., the addition of an ion channel opener).
- another change can be effected in the activity of one or more ion channels of the cells.
- the ion concentration can be increased or voltage can be applied to effect a depolarization of the ion channel.
- a PWV is then determined.
- One of more of the PWVs can be compared to control cells, such as cells that did not have change in activity effected in one or more ion channels of the cells (e.g., no addition of an ion channel opener), cells lacking the ion channel altogether, cells receiving a channel opener, but no modulator. These responses can be measured on separate locations of the biosensor, e.g., in different wells of a microplate.
- the PWVs can be compared to determine if the test ion channel modulators modulated the one or more ion channels of the cells. For example, where a test modulator of an ion channel is added to cells on a biosensor surface, a first PWV is taken, an ion channel opener is added to the cells, and a second PWV is taken, the first and second PWVs can be compared. Where the PWVs are substantially different, the test modulator has indeed modulated the ion channel. In general, resolution of differences of PWV as small as 5 pm can be detected; however, differences of greater than about 20 pm, 100 pm, 200 pm or more can also be detected. Alternatively, the PWVs can be compared to controls to determine if the test modulator has modulated the ion channels.
- a PWV reading can be taken constantly over the entire course or part of the assay.
- modulation of ion channels or lack thereof can be detected as it occurs, thus circumventing the need to incorporate radiometric, colorimetric, fluorescent labels or microscopy for evaluation.
- Changes in cells that occur due to modulation of ion channels can be expediently monitored in real time, in a label free manner.
- the BIND BiosensorTM, BIND ReaderTM, and BIND ScannerTM e.g., a colorimetric resonant reflectance biosensor system
- Methods of the invention are advantageous because they do not require labeling of cells or reagents for microscopic or colorimetric/fluorimetric evaluation, they allow for continuous, multiple independent readings of the same population of cells in real time, they are quick, they require minimal reagent usage (both volume and type), they do not require recombinant cells lines, they do not require mechanical manipulation of cells, and they do not require flowing the cells through a counting device.
- the label-free biosensor method is non-destructive to the cells so that a cell or a set of cells may be monitored continuously or discontinuously over a long period of time with or without treatment of known or test compounds.
- Methods of the invention allow for continuous monitoring or multiple independent readings of the same population of cells in real time over many days. Cellular changes can be quantified expediently and objectively over longer periods of time in a normal culturing environment (static with proper media). Methods of the invention can also be used synergistically with fluorescent labels to obtain additional, intracellular data from each cell or cell population.
- Cell changes can be monitored by taking a PWV for one location over several time periods.
- scans of a receptacle holding the cells e.g., a microtiter plate well, can be done over several time periods.
- a test reagent can be identified as a potential ion channel modulator by comparing its PWV pattern over time to a known ion channel modulator.
- the PWVs over time exhibit a particular pattern. For example, after an ion channel modulator is added to cells the PWV may increase for a particular period of time and then decrease.
- Test reagents that exhibit the same pattern of PWV values over time can be identified as a potential ion channel regulator or modulator.
- One or more cells can be applied to a location, such as a microtiter well on a surface of a colorimetric resonant reflectance optical biosensor.
- a receptacle refers to one container and not a collection of containers, e.g., a multiwell plate.
- a colorimetric resonant reflectance optical peak wavelength value (PWV) for the location is detected.
- the one or more cells can be incubated for a period of time (e.g., 1 second, 30 seconds, 1, 2, 5, 10, 20, 30, 45 minutes, 1, 2, 5, 10 or more hours).
- test reagents, ion channel agonists, and/or ion channel antagonists can be applied to the one or more cells.
- the colorimetric resonant reflectance optical PWV for the location can be detected for a second time. If a change in the cells occurs then the reflected wavelength of light is shifted as compared to a situation where no change occurs.
- the first PWV can be compared to the second PWV. A change in the PWV can indicate a change in the cells. PWVs over several time periods can be determined and compared.
- Cell changes at a biosensor location can be detected via the PWVs of the biosensor surface or monitored more generally using a microscope, digital camera, conventional camera, or other visualization apparatus, magnifying or non-magnifying, that utilizes lens- based optics or electronics-based charge coupled device (CCD) technology.
- a microscope, digital camera, conventional camera, or other visualization apparatus magnifying or non-magnifying, that utilizes lens- based optics or electronics-based charge coupled device (CCD) technology.
- CCD charge coupled device
- the changes in PWV can be determined using a BIND Reader®, Scanner, or Cartridge Reader.
- assays can be completed in about 0.0005 to 8 hours.
- the resolution of the lens of the scanner determining the PWV has an about 2 to about 200, about 2 to about 50, or about 2 to about 15 micrometer pixel size.
- Assays of the invention can be completed in less than about 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, or 8 hours. That is, cell changes in response to, for example, the added reagent can be determined in a time efficient manner.
- This protocol employs potassium free starvation buffer to depolarize cells and thereby open ion channels such that screening ion channel modulators is possible.
- Colorimetric resonant reflectance biosensor plates were washed with 1 X PBS for hydration baseline for 3 to 5 minutes. A PWV was determined for each of the wells. This PWV can be useful to make sure that the biosensor is stable (not drifting) and is uniform in signal across the wells.
- the plates were coated with 25.0 ⁇ l/well of fibronectin for 2 hrs at room temperature. The plates were blocked for 1 hour at room temperature or overnight at 4 0 C by addition of 25.0 ⁇ l ovalbumin or BSA in PBS to the wells previously coated with fibronectin.
- Starvation buffers were prepared with and without potassium.
- potassium- free starvation buffer the following reagents were used: 25 ml of 3M NaCl for 150 mM NaCl final; 10.0 ml for 2OmM HEPES; 1.0 ml for 2mM calcium chloride; 0.5 ml for 2mM magnesium chloride; 1.0 ml for 5mM D-(+)-glucose; up to 500.0 ml with H 2 O HPLC.
- Potassium starvation buffer is prepared the same as above, but with 2mM KCl added.
- Cells (CHO cells transfected with the human ether-a-go-go (hERG) potassium channel or CHO parental cells) were harvested for 5 - 10 minutes with 3.0 mL versene/flask. The versene was deactivated with 8.0 mL full media with FBS per flask. The biosensor plates were equilibrated to room temperature and previously added PBS was removed. The cells were gently spun down for 5 minutes at IK rpm. The media was aspirated and potassium- containing starvation buffer was added to the cell pellet. The cells were then diluted from their initial concentration to a concentration of 10 6 cells per ml for 20,000 cells per 20.0 ⁇ l.). A baseline PWV for each well of the biosensor plate was taken as a quality control step to check for cell adhesion uniformity following a sufficient amount of time for the cells to settle and become attached.
- hERG human ether-a-go-go
- 20.0 ⁇ l of potassium-containing starvation buffer was added to each well. A PWV was taken for each well. The average number of cells/well was determined. For seeding 2OK cells in 20.0 ⁇ l per well, cells were prepared at 10 6 cells/ml. 20.0 ⁇ l of cells were added to the well already containing 20.0 ⁇ l media/buffer.
- the seeded biosensor test plates were allowed to sit at room temperature for about 15 minutes, before being placing in the 37 0 C incubator for 120 minutes for settling, attachment, and cell health recovery.
- the plates were placed in the 3O 0 C incubator to aid protein refolding.
- PWVs can be obtained for the freshly seeded biosensor plates during this time to monitor cell adherence.
- the cells are first be pre-incubated with inhibitor in media containing potassium. Then the cells are observed in ion channel opening conditions such as with removal of extracellular potassium or by the addition of a compound such as mallotoxin, specific for opening K+ channels. Those cells pre-incubated with inhibitor in the presence of potassium should not open under potassium-free conditions (that normally causes electrostatic potential leading to pore opening) or with lower amounts of mallotoxin. In striking comparison, those cells not exposed to inhibitor should open and a change in PWV should result indicative of normal channel function.
- PWVs are taken for the biosensor plates for 3 to 5 minutes or until a stable read of less than 1-2 pm change/minute is attained.
- An appropriate volume of ligand or test reagent from the reagent plates was added to the biosensor plates.
- the cells were incubated for about 15-30 minutes.
- the entire volume of potassium-containing starvation buffer containing the inhibitor was replaced with the same volume of potassium- free starvation buffer containing the same final desired concentration of ligand or test reagent to the wells.
- PWVs were taken for each of the wells.
- Figure 1 demonstrates that CHO cells transfected with hERG swell faster and more than the parental CHO cells because they have more ion channels and sodium and water are able to move into the cell quicker than in the parental CHO cells.
- Figures 2A- C demonstrate the effect of two potassium channel inhibitors (Cisapride monohydrate and E4031) on the cells. The greater the concentration of the inhibitor, the less the swelling of the cells in response to the lack of potassium. The parental CHO lines show little to no effect in the presence of the inhibitors due to the lack of the hERG ion channels. See Figure 3.
Abstract
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EP09794839A EP2306902A4 (en) | 2008-07-11 | 2009-01-08 | Methods for identifying modulators of ion channels |
AU2009269037A AU2009269037A1 (en) | 2008-07-11 | 2009-01-08 | Methods for identifying modulators of ion channels |
JP2011517431A JP2011527573A (en) | 2008-07-11 | 2009-01-08 | Methods for identifying regulators of ion channels |
CA2730415A CA2730415A1 (en) | 2008-07-11 | 2009-01-08 | Methods for identifying modulators of ion channels |
CN2009801359199A CN102149327A (en) | 2008-07-11 | 2009-01-08 | Methods for identifying modulators of ion channels |
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PCT/US2008/069752 WO2009009718A1 (en) | 2007-07-11 | 2008-07-11 | Methods of identifying modulators of ion channels |
US12/171,475 US9778267B2 (en) | 2007-07-11 | 2008-07-11 | Methods for identifying modulators of ion channels |
US12/171,475 | 2008-07-11 | ||
USPCT/US2008/069752 | 2008-07-11 | ||
US12/335,433 US9134307B2 (en) | 2007-07-11 | 2008-12-15 | Method for determining ion channel modulating properties of a test reagent |
US12/335,433 | 2008-12-15 |
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Cited By (13)
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EP2171454A1 (en) * | 2007-07-11 | 2010-04-07 | SRU Biosystems, Inc. | Methods of identifying modulators of ion channels |
US7923239B2 (en) | 2000-10-30 | 2011-04-12 | Sru Biosystems, Inc. | Guided mode resonant filter biosensor using a linear grating surface structure |
US7927822B2 (en) | 2002-09-09 | 2011-04-19 | Sru Biosystems, Inc. | Methods for screening cells and antibodies |
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CA2730415A1 (en) | 2010-01-14 |
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CN102149327A (en) | 2011-08-10 |
EP2306902A4 (en) | 2011-08-10 |
EP2306902A1 (en) | 2011-04-13 |
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