WO2002077605A2 - Dispositif de biodosage a cavite rentrante servant a detecter des evenements moleculaires ou cellulaires - Google Patents

Dispositif de biodosage a cavite rentrante servant a detecter des evenements moleculaires ou cellulaires Download PDF

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Publication number
WO2002077605A2
WO2002077605A2 PCT/US2002/008368 US0208368W WO02077605A2 WO 2002077605 A2 WO2002077605 A2 WO 2002077605A2 US 0208368 W US0208368 W US 0208368W WO 02077605 A2 WO02077605 A2 WO 02077605A2
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WIPO (PCT)
Prior art keywords
reentrant
cavity
bioassay
sample
detection region
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PCT/US2002/008368
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English (en)
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WO2002077605A3 (fr
Inventor
Barrett J. Bartell
Joseph A. Heanue
Mark A. Rhodes
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Signature Bioscience, Inc.
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Application filed by Signature Bioscience, Inc. filed Critical Signature Bioscience, Inc.
Priority to AU2002305063A priority Critical patent/AU2002305063A1/en
Publication of WO2002077605A2 publication Critical patent/WO2002077605A2/fr
Publication of WO2002077605A3 publication Critical patent/WO2002077605A3/fr

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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
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Definitions

  • the present invention relates generally to the structure and function of bioassay devices, and more particularly to a reentrant cavity bioassay operable to detect solution or solid phase based molecular events in an aqueous-based sample.
  • the need to detect molecular and cellular events ranges from the basic science research lab, where chemical messenger pathways are being mapped out and their functions correlated to disease processes, to pre-clinical investigations, where candidate drugs are being evaluated for potential in vivo effectiveness.
  • the need to detect physical and functional properties is also present in these research areas, such as for functional analysis of a newly discovered protein or of a genetic (or synthetic) variant of a molecule of know biological importance.
  • Other areas that benefit from a better understanding of molecular and cellular events include pharmaceutical research, military applications, veterinary, food, and environmental applications.
  • knowledge of the ability of a particular analyte to bind a target molecule is highly useful, as is information relating to the quality of that binding (e.g., affinity and on-off rate), and other functional information about new molecules and cells, particularly when information can be obtained from a small amount of sample.
  • reentrant cavities have been used as a focusing means to concentrate an electromagnetic signal within a particular region (referred to here as a "detection region"). As the applied electromagnetic signal becomes more concentrated within the detection region, changes occurring within that region can be more sensitively detected. If a similar structure could be used to focus an electromagnetic signal to the detection region of a bioassay where molecular or cellular events are occurring, the sensitivity of the bioassay could be correspondingly improved. In addition, the structure would preferably be able to detect molecular or cellular events in aqueous-based environment native to the molecular or cellular events.
  • the reentrant cavity bioassay includes a reentrant cavity and a sample retaining structure.
  • the reentrant cavity includes a reentrant post extending from a first interior surface and terminating proximate to a second interior surface, the gap between the termination of the reentrant post and the second interior surface defining a detection region.
  • the sample retaining structure is configured to retain the sample in the detection region, wherein at least a portion of the sample retaining structure is located within the detection region.
  • FIG. 1 illustrates a first embodiment of a reentrant cavity bioassay in accordance with the present invention.
  • Fig. 2 illustrates a second embodiment of a reentrant cavity bioassay in accordance with the present invention.
  • Fig. 3 A illustrates a serial array of reentrant cavity bioassays in accordance with the present invention.
  • Fig. 3B illustrates a parallel array of reentrant cavity bioassays in accordance with the present invention.
  • Fig. 4 illustrates a method for detecting and/or identifying molecular events occurring within a test sample using the reentrant cavity bioassay in accordance with an embodiment of the present invention.
  • molecular binding event refers to the interaction of a molecule of interest with another molecule.
  • molecular structure refers to all structural properties of molecules of interest, including the presence of specific molecular substructures (such as alpha helix regions, beta sheets, immunoglobulin domains, and other types of molecular substructures), as well as how the molecule changes its overall physical structure via interaction with other molecules (such as by bending or folding motions), including the molecule's interaction with its own solvation shell while in solution.
  • molecular structures and “molecular binding events” are referred to as “molecular events.”
  • the simple presence of a molecule of interest in the region where detection/analysis is taking place is not considered to be a “molecular event,” but is referred to as a "presence.”
  • binding events are (1) simple, non-covalent binding, such as occurs between a ligand and its antiligand, and (2) temporary covalent bond formation, such as often occurs when an enzyme is reacting with its substrate. More specific examples of binding events of interest include, but are not limited to, ligand/receptor, antigen/antibody, enzyme/substrate, DNA/DNA, DNA/RNA, RNA/RNA, nucleic acid mismatches, complementary nucleic acids and nucleic acid/proteins. Binding events can occur as primary, secondary, or higher order binding events.
  • a primary binding event is defined as a first molecule binding (specifically or non-specifically) to an entity of any type, whether an independent molecule or a material that is part of a first surface, typically a surface within the detection region, to form a first molecular interaction complex.
  • a secondary binding event is defined as a second molecule binding (specifically or non- specifically) to the first molecular interaction complex.
  • a tertiary binding event is defined as a third molecule binding (specifically or non-specifically) to the second molecular interaction complex, and so on for higher order binding events.
  • Examples of relevant molecular structures are the presence of a physical substructure (e.g., presence of an alpha helix, a beta sheet, a catalytic active site, a binding region, or a seven-trans-membrane protein structure in a molecule) or a structure relating to some functional capability (e.g., ability to function as an antibody, to transport a particular ligand, to function as an ion channel (or component thereof), or to function as a signal transducer).
  • a physical substructure e.g., presence of an alpha helix, a beta sheet, a catalytic active site, a binding region, or a seven-trans-membrane protein structure in a molecule
  • some functional capability e.g., ability to function as an antibody, to transport a particular ligand, to function as an ion channel (or component thereof), or to function as a signal transducer.
  • Molecular structure is typically detected by comparing the signal obtained from a molecule of unknown structure and/or function to the signal obtained from a molecule of known structure and/or function.
  • Molecular binding events are typically detected by comparing the signal obtained from a sample containing one of the potential binding partners (or the signals from two individual samples, each containing one of the potential binding partners) to the signal obtained from a sample containing both potential binding partners.
  • the detection of a "molecular binding event" or "molecular structure” is often referred to as "molecular detection.”
  • cellular event refers in a similar manner to reactions and structural rearrangements occurring as a result of the activity of a living cell (which includes cell death). Examples of cellular events include opening and closing of ion channels, leakage of cell contents, passage of material across a membrane (whether by passive or active transport), activation and inactivation of cellular processes, as well as all other functions of living cells. Cellular events are commonly detected by comparing modulated signals obtained from two cells (or collection of cells) that differ in some fashion, for example by being in different environments (e.g., the effect of heat or an added cell stimulant) or that have different genetic structures (e.g., a normal versus a mutated or genetically modified cell). Morpholic changes are also cellular events.
  • the methodology and apparatuses described herein are primarily of interest to detect and predict molecular and cellular events of biological and pharmaceutical importance that occur in physiological situations (such as in a cellular or subcellular membrane or in the cytosol of a cell). Accordingly, structural properties of molecules or interactions of molecules with each other under conditions that are not identical or similar to physiological conditions are of less interest. For example, formation of a complex of individual molecules under non-physiological conditions, such as would be present in the vacuum field of an electron microscope or in gaseous phase mixtures, would not be considered to be a preferred "molecular binding event," as this term is used herein.
  • preferred molecular events and properties are those that exist under "physiological conditions,” such as would be present in a natural cellular or intercellular environment, or in an artificial environment, such as in an aqueous buffer, designed to mimic a physiological condition.
  • physiological conditions vary from place to place within cells and organisms and that artificial conditions designed to mimic such conditions can also vary considerably.
  • a binding event may occur between a protein and a ligand in a subcellular compartment in the presence of helper proteins and small molecules that affect binding.
  • Such conditions may differ greatly from the physiological conditions in serum, exemplified by the artificial medium referred to as "normal phosphate buffered saline" or PBS.
  • Preferred conditions of the invention will typically be aqueous solutions at a minimum, although some amounts of organic solvents, such as DMSO, may be present to assist solubility of some components being tested.
  • An "aqueous solution” contains at least 50 wt.% water, preferably at least 80 wt.% water, more preferably at least 90 wt.% water, even more preferably at least 95 wt.% water.
  • Other conditions such as osmolarity, pH, temperature, and pressure, can and will vary considerably in order to mimic local conditions of the intracellular environment in which, for example, a binding event is taking place.
  • test sample refers to the material being investigated (the analyte) and the medium/buffer in which the analyte is found.
  • the medium or buffer can included solid, liquid or gaseous phase materials; the principal component of most physiological media/buffers is water.
  • Solid phase media can be comprised of naturally occurring or synthetic molecules including carbohydrates, proteins, oligonucleotides, SiO , GaAs, Au, or alternatively, any organic polymeric material, such as Nylon ® , Rayon ® , Dacryon ® , polypropylene, Teflon ® , neoprene, delrin or the like.
  • Liquid phase media include those containing an aqueous, organic or other primary components, gels, gases, and emulsions.
  • Exemplary media include celluloses, dextran derivatives, aqueous solution of d- PBS, Tris, deionized water, blood, cerebrospinal fluid, urine, saliva, water, and organic solvents.
  • the term “electromagnetically coupled” refers to the transfer of electromagnetic energy between two objects, e.g., the reentrant post and molecular events occurring within the test sample.
  • the two objects can be electromagnetically coupled when the objects are in direct contact, (e.g., molecular events occurring along the surface of a reentrant post), or when the objects are physically separated from each other (e.g., molecular events occurring within a sample flowing through a flow tube, the flow tube positioned within the detection region).
  • the term “electromagnetically couples” will indicate the interaction of an electromagnetic signal (e.g., the incident test signal) with an object (e.g., molecular events occurring within the test sample).
  • test signal refers to an ac time-varying signal.
  • the test signal is preferably at or above 1 MHz (lxl 0 6 Hz) and at or below 1000 GHz (lxlO 12 Hz), such as 10 MHz, 20 MHz, 45 MHz, 100 MHz, 500 MHz, 1 GHz (lxlO 9 Hz), 2 GHz, 5 GHz, 7.5 GHz, 10 GHz, 12 GHz, 15 GHz, 18 GHz, 20 GHz, 25 GHz, 30 GHz, 44 GHz, 60 GHz, 110 GHz, 200 GHz, 500 GHz, or 1000 GHz and range anywhere therebetween.
  • a preferred region is from 10 MHz to 110 GHz, a more particularly from 45 MHz to 20 GHz.
  • Test signal can refer to a range of frequencies rather than a single frequency, and such a range can be selected over any terminal frequencies, including frequency ranges bounded by the specific frequencies named in this paragraph.
  • the term “spectrum” is sometimes used.
  • An “incident test signal” is a test signal that originates from the signal source and is destined for the detection region for interaction with the sample.
  • a “modulated test signal” is a test signal that has previously interacted with the test sample and is destined for a signal detector that can recover the modulation imparted by the signal interaction with the sample.
  • Fig. 1 illustrates one embodiment of a reentrant cavity bioassay 100 in accordance with the present invention.
  • the bioassay 100 includes the reentrant cavity assembly 120, a flow tube 130, and a probe assembly 140. This configuration is useful when detecting or identifying molecular events in "solution phase," i.e., when these events are moving or suspended in the test sample.
  • the reentrant cavity assembly 120 is configured to provide a high Q resonance at one or more frequencies.
  • the cavity 120 includes a top member 122, a cavity body 123, a reentrant post 124, a probe access port 125 and a flow tube access port 126 located on the cavity floor 127.
  • the top member 122 is removably attached to the cavity body 123, using, e.g., screws threadingly engaged into the cavity body 123.
  • the reentrant post 124 is a hollow tube having a top aperture 121 and a bottom aperture 128 located above the cavity floor 127.
  • the reentrant post 124 is solid.
  • the top member 120, cavity body 123, and reentrant post 124 are constructed from aluminum and the interior cavity has a radius of 25.4 mm and is 25.4 mm tall.
  • the interior of the cavity may be gold-plated to increase the cavity's q-factor and enhance resonant frequency stability.
  • the reentrant post 124 has an outer radius of 6.35 mm and extends down 24.28 mm from the top member 122, leaving a 1.18 mm gap between the post bottom 128 and the cavity floor 127. In this configuration the reentrant cavity exhibits a fundamental resonance at 1 JO GHz.
  • the detection region defines a region through which a large number of electric field lines will extend when a test signal is electromagnetically coupled into the cavity 120, and is generally formed between the closest proximate surfaces of the reentrant post and cavity interior.
  • the detection region 129 is formed longitudinally between the post bottom and the cavity floor 127 and measures approximately 150 ⁇ l (150 xl0 "9 m 3 ).
  • the detection region 129 is not limited to any particular volume, but will typically measure smaller than 1 ml (1 x 10 "6 m 3 ).
  • Very small detection region volumes such as 1 ⁇ l (1 x 10 "9 m 3 ), 1 nl (1 x 10 "12 m 3 ), or 1 pi (1 x 10 "15 m 3 ) (or ranges between these volumes) may also be used to test binding ability of potential pharmaceutical compounds due to the small size and expense of the available samples.
  • the detection region 129 can be formed circumferentially along the cavity sidewall, between it and the side of the reentrant post 124.
  • Molecular event detection and identification preferably occurs at the fundamental or higher order resonant frequencies of the cavity 120, as operation at these frequencies will provide the highest sensitivity within the detection region 129.
  • the dielectric properties of molecular events introduced into the detection region 129 will operate to shift the cavity resonant frequency above or below its resonant frequency. Because many of molecular events have distinctive dielectric properties (e.g., permittivity), each will produce a unique frequency shift, thereby enabling their identification.
  • the sample is retained within the detection region 129 using a sample retaining structure, an embodiment of which is a flow tube 130.
  • the flow tube 130 is fed through the top aperture 121 through the hollow body of the reentrant post 124, through the bottom aperture 128, and out of the aperture 126 located on the cavity floor 127.
  • the flow tube 130 is preferably constructed of a material that exhibits low dielectric loss and high signal transparency at the desired test frequency, and which further preferably has a smooth, resilient surface morphology that inhibits analyte formation along the inner surface.
  • the flow tube is PTFE tube having an outer diameter of 1.07 mm and an inner diameter of 0.56 mm.
  • the detection region 129 is formed circumferentially around the cavity side wall, the flow tube is wound around the reentrant post.
  • the sample retaining structure consists of an unenclosed substrate (e.g., a glass slide) positioned within the detection region (through a slot in the cavity's side wall, not shown). This arrangement is particularly useful for investigating cellular events occurring within a solution dispensed onto a slide.
  • Other enclosed and unenclosed sample retaining structures e.g., flow cells, microfluidic channels, capillaries, etc.
  • flow cells e.g., flow cells, microfluidic channels, capillaries, etc.
  • the probe assembly 140 is configured to electromagnetically couple, at one or more frequencies, an incident test signal into and recover a modulated signal from the cavity assembly 120.
  • the probe assembly 140 includes a first coaxial section 142 and a probe tip 144 extending into the interior of the reentrant cavity 123 via the probe access port 125.
  • An ultra fine adjusting motor 145 is preferably used to move the probe tip 144 into and out of the cavity 123.
  • the first coaxial section 142 is connected to a coaxial cable 146, that is in turn connected to the measurement instrument, a network analyzer in one embodiment.
  • the first coaxial section 142 consists of a 75 mm of coaxial cable and the probe tip 144 measures approximately 100.0 mm long.
  • Various tip configurations may be used, for example, a monopole, a dipole, ground loop, spiral, or other antenna design capable of transmitting/receiving signals into/from the cavity assembly 120.
  • the motorized assembly 145 consists of a picomotor assembly manufactured by New Focus, Inc. (Santa Clara, CA).
  • the first coaxial section 142 consists of 5.0 mm O.D. cable manufactured by RF Labs (Stuart, FL.). Other probes structures may be used in alternative embodiments of the present invention.
  • the flow tube 130 enters through an aperture (not shown) in the cavity wall, extends along the cavity floor 127 below the post bottom 128 and exits through a second aperture (not shown) in the cavity side wall.
  • the reentrant post 124 may be solid.
  • the sample retaining structure consists of a flow cell located between the post bottom 128 and the cavity floor 127 which is supplied via a flow tube as described above.
  • a second probe extends into the cavity body 123 and the first and second probed can be used to make a two-port (e.g., a S 21 s-parameter) measurement.
  • the reentrant post 128 may have a varying radius along the vertical length and/or a tapered tip.
  • FIG. 2 illustrates a second reentrant cavity bioassay 200 in accordance with the present invention.
  • the bioassay 200 includes a reentrant cavity assembly 220, a sample vial 230, and a probe 240. This configuration is useful when detecting or identifying molecular events in "solid phase," i.e., when molecular events are immobilized on a surface.
  • the reentrant cavity assembly 220 includes a top member 222 and a cavity body 223.
  • the top member 222 includes a sample aperture 221, a probe aperture 225, and a hollow reentrant post 224 that extends vertically into the cavity body 223 terminating proximate to the cavity floor 227.
  • the region between the post bottom and cavity floor 227 represents the detection region 229 through which a large number of fields pass when a signal is electromagnetically coupled into the cavity 220.
  • the diameter of the top aperture 221 and inner diameter of the reentrant post 224 is of sufficient size to permit passage of the sample retaining structure, a sample vial 230 in the illustrated embodiment.
  • the sample vial 230 is preferably constructed from a material that exhibits low dielectric loss, high signal transparency at the desired operational frequency and that has a surface morphology that is conducive to facilitate molecular binding thereto. Suitable materials include glass, PTFE, and the like. Further preferably, the bottom thickness of the sample vial is minimal, on the order of .1 to 10 mm.
  • the sample vial is an open well container of the type typically used in biological, chemical, or pharmaceutical applications. In another embodiment, the open well is sealed to minimize sample evaporation.
  • the sample vial 230 further includes a molecular binding region 232 formed along the bottom inner surface of the sample vial 230.
  • the molecular binding region 232 is located within the detection region 229 and includes antiligands 232a that have a strong affinity to bind ligands 252 that are suspected of being contained within the supplied test sample 250.
  • the formation of the ligand/anti-ligand complex within the high field density region 229 operates to shift the resonant frequency of the cavity 220, thereby enabling detection and identification of the binding events within the sample 250.
  • the probe 240 is operable to electromagnetically couple, at one or more frequencies, an incident test signal into, and recover the modulated signal from the cavity assembly 220.
  • the probe 240 includes a coaxial cable 242 and a probe tip 244 consisting of the center conductor of the coaxial cable 242.
  • the probe tip 244 forms a loop and terminates onto the outer ground shield of the coaxial cable 242.
  • the probe 240 extends into the cavity body 223 through the probe aperture 225 of the top member 222 in the illustrated embodiment, although in an alternative embodiment, the probe 240 may enter the cavity at other locations.
  • the probe assembly is rotatably attached to the top member 222 such that it can freely rotate relative to the cavity assembly 220. Tuning is performed by rotating the probe in either direction relative to the cavity assembly.
  • the cavity assembly 220 is constructed from aluminum and has interior dimensions of 50.8 mm diameter by 25.4 mm tall.
  • the reentrant post 224 has an outer diameter of 6.35 mm and an inner diameter of 1.27 mm and extends 24.28 mm into the cavity body 223, terminating 1.12 mm from the cavity floor 227.
  • the sample aperture 221 on the top member 222 is 1.27 mm in diameter and the sample vial 230 has an outer diameter of 1.27 mm and an inner diameter of 6.35 mm.
  • the probe 240 is constructed from 6.35 mm semi-rigid type RG 401 coaxial cable and the probe tip 244 is approximately 125 mm long.
  • the bioassay 200 of Fig. 2 may also be modified in a variety of ways to provide solid phase detection and identification of immobilized molecular events.
  • the sample retaining structure may consist of a flow tube operable to transport beads or other structures that contain immobilized antiligands 232a.
  • the antiligands 232a are selected to bind to a particular ligand 252 potentially contained within the sample 250.
  • the beads can be mixed with the test sample either prior to transportation to the detection region 229, or within the detection region 229 in order to study the kinetics of the binding interaction.
  • antiligands 232a are immobilized on an interior stationary surface of a flow cell, microfluidic channel, or another similar sample retaining structure.
  • the reentrant post 224 may consist of a hollow structure (in which case the sample retaining structure may extend through it), or a solid structure (in which case the sample retaining structure will extend between the cavity floor 227 and post bottom 228).
  • cellular events may be monitored by depositing cells on a substrate, such as a glass slide, and positioning the slide within the detection region, for instance through slits (not shown) located in the cavity's side wall.
  • the reentrant post 224 can be either solid or hollow.
  • the reentrant cavity of the present invention may be employed in an array to detect and/or identify molecular events in a sample.
  • the bioassay array may be configured serially, in which case a single sample retaining structure, such as a flow tube, is used to supply the sample to each of the cavities' detection regions.
  • the bioassay array may be configured in a parallel arrangement where separate sample retaining structures supply sample to each of the cavities' detection regions.
  • Fig. 3 A illustrates a serial array of reentrant cavity bioassays in accordance with the present invention. Using this configuration, a sample plug can be interrogated at a number of different frequencies to provide the sample's dielectric properties over a full frequency spectrum.
  • the serial array 320 includes several reentrant cavities 325 and a single flow tube 326 extending into each of the cavities' detection regions 329. In the illustrated array, the detection regions 329 are formed longitudinally between the post bottom and the cavity floor, and the reentrant posts are hollow permitting the passage of the flow tube 326 therethrough.
  • the detection regions 329 can be formed circumferentially around the cavities' side wall, in which case the flow tube 326 may be wound around each of the cavities' reentrant posts. Sample plugs 327 are advanced through the flow tube 326 and to detection regions 329 by applying positive pressure to the top or negative pressure to the bottom of the flow tube 326.
  • the array 320 employs reentrant cavities 325 having different fundamental resonant frequencies. This permits sample interrogation at different test frequencies as the sample is moved into the detection region of the various cavities 329.
  • the array 320 is composed of several reentrant cavities 329, each having the same fundamental resonant frequency but tuned (using the tunable probes described above) to a different higher-order cavity resonant frequency. As known in the art of waveguide structures and cavities, the reentrant cavity will exhibit several higher- order resonant frequencies, depending upon the dimensions of the reentrant cavity.
  • Fig. 3B illustrates a parallel array of reentrant cavity bioassays in accordance with the present invention.
  • This array provides a high through-put testing platform for various applications, e.g., screening for particular compounds, binding events, or other molecular or cellular activity.
  • This configuration can be used in combination with the aforementioned serial configuration 320 (Fig. 3A), in which case the illustrated reentrant cavities 352 would consist of a serial arrangement of cavities.
  • the parallel array 350 includes several reentrant cavities 352, each having a flow tube 356 extending into the cavity's detection region 359.
  • the supplied sample may consist of different compounds, for example, sample 1 is buffer; sample 2 is buffer + protein; sample 3 is buffer + ligand; and sample 4 is buffer + protein -f-ligand).
  • sample of the same composition but different temperature, pH, concentration, salt content etc may be supplied. Other variations of the sample composition will be evident to the reader.
  • the detection regions 359 are formed longitudinally between the post bottom and the cavity floor, and the reentrant posts are hollow permitting the passage
  • the detection regions 359 can be formed circumferentially around the cavities' side wall, in which case the flow tube 356 maybe wound around each of the cavities' reentrant posts. Sample plugs 357 are advanced through the flow tube 356 and to detection regions 359 by applying positive pressure to the top, or negative pressure to the bottom of the flow tube 356.
  • the array 350 is composed of reentrant cavities 352 having the same fundamental frequency. This arrangement permits parallel interrogation of multiple samples at the same test frequency. These responses can be monitored for a known response indicating the occurrence of a particular molecular event within one of the tested samples.
  • each of the reentrant cavities may be designed to exhibit a unique fundamental resonant frequency (or the response taken at a unique high- order resonant frequency) in order to obtain a full frequency spectrum of the sample's dielectric properties.
  • Fig. 4 illustrates a method for identifying molecular or cellular events occurring within a test sample using the reentrant cavity bioassay in accordance with one embodiment of the present invention.
  • the cavity assembly is designed to exhibit a resonant response at the desired frequency.
  • the desired frequency is the frequency at or near which the sought molecular event will have dramatically varying dielectric properties.
  • a three-dimensional CAD tool such as HFSS available from Ansoft, Inc. (Pittsburgh, PA) is used to design the reentrant cavity and simulate its resonant response.
  • the three-dimensional CAD software is capable of modeling the sample retaining structure, such as the flow tube, positioned within the detection region of the reentrant cavity as the presence of this structure will vary the resonant frequency of the reentrant cavity.
  • a reference sample is supplied to the sample retaining structure and the reference sample fed to the detection region (process 404).
  • the reference sample may consist of the native environment or buffer solution in which the sought protein, cell, binding complex, or other molecular structure resides.
  • the reference sample may consist of the buffer plus the sought protein, cell, binding complex, or other molecular structure.
  • reference samples may consist of compositions under alternative embodiments of the present invention.
  • the cavity assembly is tuned to a resonant frequency point. This process is accomplished by monitoring the amplitude of the returning signal (for instance, using a network analyzer to measure the input return loss of the probe) while adjusting the probe position until the amplitude of the returning signal reaches a minimum point.
  • the position of the probe is adjusted, for instance, by inserting/extracting the probe into/out of the cavity assembly (Fig. 1), or by rotating the probe relative to the cavity assembly (Fig. 2).
  • the cavity will exhibit a high degree of sensitivity in the change of the dielectric properties occurring within the detection region.
  • the cavity resonant frequency is obtained in the presence of the reference sample, the cavity is said to be "tuned to the buffer” or “buffer + protein,” depending upon the composition of the reference sample.
  • the resonant response is subsequently stored for later retrieval and comparison with the resonant response of the test sample, as described below.
  • the process may be automated by monitoring the resonant response and adjusting the probe position until the minimum amplitude point is obtained.
  • the reference sample is removed from the detection region and the test sample supplied thereto.
  • the reference and test samples may each comprise plug segments that are separated by air gaps, or other material such as detergent to remove any remaining components of the previous sample plug.
  • the resonant response is measured in the presence of the test sample.
  • the test sample response is compared to the reference sample response and the measurements correlation are obtained.
  • a close correlation is an indication of identity between the molecular event composition of the reference and test samples. For instance, if the reference sample is known to contain a particular protein, binding complex, ligand, cell, or other analyte, and the test and reference sample return loss responses exhibit a high degree of correlation (e.g., within ⁇ .5 dB over frequency), it can be deduced that the test and reference samples have substantially similar molecular event compositions.
  • a lower degree of correlation (e.g., within ⁇ .75- 1.5 dB over frequency) can be indicative of molecular events of the same genus or family.
  • a low degree of correlation between the reference and test sample responses (e.g., when the test and reference sample return loss responses differ by more than ⁇ 2.0 dB over frequency) is an indication of dissimilarity in the molecular event makeup between the reference and test samples.
  • the tested reentrant cavity assembly include the reentrant cavity described in Fig. 1 and a PTFE flow tube having an I.D./O.D. of 0.56 mm/1.07 mm.
  • the coaxial resonator consisted of an open-ended coaxial cable abutted to the aforementioned PTFE flow tube of 0.56mm/l .07mm I.D./O.D.
  • Specific embodiments of the open-ended coaxial resonator are provide in applicant's co-pending application no. 09/687,456 entitled “System and method for detecting and identifying molecular events in a test sample," filed October 13, 2000 (Atty Dkt No. 12US).
  • the measured samples included air, deionized water, and phosphate buffered saline (PBS).
  • a sample plug volume of 12.0 ⁇ l was used in the flow tube used in the coaxial resonator assembly, and 1.2 ⁇ l was used in the flow tube of the reentrant cavity assembly.
  • Table I illustrates the results of the sensitivity comparison between the reentrant cavity and coaxial resonator. These results indicate that the reentrant cavity is approximately five times more sensitive in measuring changes in Q-factor of the detector per unit volume of sample, and fifty-five times more sensitive in measuring changes in the resonant frequency of the detector per unit volume of sample.
  • the increased sensitivity means that molecular events can be detected and identified with greater accuracy using less analyte material.

Abstract

Dispositif de biodosage à cavité rentrante comprenant une cavité rentrante et une structure retenant un spécimen. Cette cavité rentrante présente une tige rentrante s'étendant depuis une première surface intérieure et se terminant à proximité d'une deuxième surface intérieure, l'espace entre l'extrémité de cette tige rentrante et la deuxième surface intérieure définissant une zone de détection. La structure retenant le spécimen est conçue pour retenir le spécimen dans la zone de détection, au moins une partie de cette structure étant située à l'intérieur de cette zone de détection.
PCT/US2002/008368 2001-03-21 2002-03-18 Dispositif de biodosage a cavite rentrante servant a detecter des evenements moleculaires ou cellulaires WO2002077605A2 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999039190A1 (fr) * 1998-02-02 1999-08-05 Signature Bioscience Inc. Procede et appareil de detection d'evenements de liaison moleculaire
WO2001009606A1 (fr) * 1999-08-02 2001-02-08 Signature Bioscience, Inc. Methodes d'analyse d'evenements de liaisons de proteines
WO2002101367A2 (fr) * 2001-06-12 2002-12-19 Signature Bioscience, Inc. Ensemble pour dosage biologique charge par pipette pour la detection d'evenements moleculaires et cellulaires

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63210757A (ja) * 1987-02-27 1988-09-01 Nippon Glass Fiber Co Ltd 非電導性繊維中の導電性物質を検出する為の装置及び方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999039190A1 (fr) * 1998-02-02 1999-08-05 Signature Bioscience Inc. Procede et appareil de detection d'evenements de liaison moleculaire
WO2001009606A1 (fr) * 1999-08-02 2001-02-08 Signature Bioscience, Inc. Methodes d'analyse d'evenements de liaisons de proteines
WO2002101367A2 (fr) * 2001-06-12 2002-12-19 Signature Bioscience, Inc. Ensemble pour dosage biologique charge par pipette pour la detection d'evenements moleculaires et cellulaires

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
EBBE NYFORS ET AL: "INDUSTRIAL MICROWAVE SENSORS" 1989 , ARTECH HOUSE , BOSTON, LONDON XP002247222 ISBN: 0-89006-397-4 page 151, last paragraph; figure 3.13 page 154, paragraph 1 *
MCEUEN A ET AL: "Temperature controlled cavity applicator for radio-frequency wave irradiation of mammalian cells in vitro" SYMPOSIUM ON ELECTROMAGNETIC FIELDS IN BIOLOGICAL SYSTEMS, OTTAWA, ONT., CANADA, 27-30 JUNE 1978, pages 113-121, XP009013200 1979, Edmonton, Alta., Canada, Internat. Microwave Power Inst, Canada *
PATENT ABSTRACTS OF JAPAN vol. 013, no. 001 (P-808), 6 January 1989 (1989-01-06) -& JP 63 210757 A (NIPPON GLASS FIBER CO LTD), 1 September 1988 (1988-09-01) *
WEIGUO XI ET AL: "FIELD ANALYSIS OF NEW COAXIAL DIELECTROMETER" IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES, IEEE INC. NEW YORK, US, vol. 40, no. 10, 1 October 1992 (1992-10-01), pages 1927-1934, XP000307040 ISSN: 0018-9480 *

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WO2002077605A3 (fr) 2003-11-06

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