WO2002086475A2 - Procede et appareil de detection d'evenements moleculaires au moyen de la regulation de temperature dans un environnement de detection - Google Patents

Procede et appareil de detection d'evenements moleculaires au moyen de la regulation de temperature dans un environnement de detection Download PDF

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Publication number
WO2002086475A2
WO2002086475A2 PCT/US2001/012694 US0112694W WO02086475A2 WO 2002086475 A2 WO2002086475 A2 WO 2002086475A2 US 0112694 W US0112694 W US 0112694W WO 02086475 A2 WO02086475 A2 WO 02086475A2
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signal
sample
ofthe
test
fluid
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PCT/US2001/012694
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WO2002086475A3 (fr
Inventor
Robert G. Chapman
John J. Hefti
Barrett J. Bartell
Mark A. Rhodes
Min Zhao
Tyler Palmer
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Signature Bioscience, Inc.
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Priority to PCT/US2001/012694 priority Critical patent/WO2002086475A2/fr
Publication of WO2002086475A2 publication Critical patent/WO2002086475A2/fr
Publication of WO2002086475A3 publication Critical patent/WO2002086475A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N22/00—Investigating or analysing materials by the use of microwaves or radio waves, i.e. electromagnetic waves with a wavelength of one millimetre or more

Definitions

  • SPR surface plasmon resonance
  • the apparatus employs a band pass filter including containment means formed to contain the polar solution and electrically dispose the polar solution as a dielectric element in the band pass filter; conducting means; a source of electrical current connected to said band pass filter; frequency variation means electrically connected to the electric voltage source to enable variation ofthe frequency at which current is applied to the band pass filter; and voltage sensing means electrically connected to sense the peak voltage passed by the band pass filter.
  • the method includes providing a band pass filter having a conducting microstrip, disposing a specimen solution between the conducting microstrip and the ground plane; applying an electric current to the band pass filter; varying the frequency ofthe current; and determining the center frequency ofthe band pass filter as the current is varied.
  • the permittivity of polar solution being investigated and thus the center frequency ofthe circuit in which the polar solution is an element are a function of temperature. Accordingly, the temperature ofthe polar solution in the microstrip circuit is controlled.
  • a temperature regulator (temperature control means) of unspecified structure performs this task.
  • a pump circulates polar solution between microstrip assembly and the temperature regulator in order to regulate the temperature ofthe polar solution at the measurement location.
  • McKee states that, in practice, maintaining the polar solution at a constant temperature is a difficult task, even with use of a temperature regulator (in fact, there is no description in the patent ofthe temperature range that the solution would be controlled to within, other than "room temperature” or the "fixed temperature” of a standard permittivity value obtained from a reference source; see, McKee, col. 6, line 8, and col. 7, lines 8-10).
  • McKee therefore proposes an alternative method for compensating for temperature drift by calibrating his microstrip circuit to detect the temperature of polar solution and then calculating permittivity from a formula derived for the particular instrument being used. This is done by measuring the center frequency for the circuit over a range of temperatures and determining an equation from these measurements to relate center frequency to temperature for a given solution.
  • the present invention provides a method for detecting a molecular event, comprising (1) applying an electromagnetic test signal in a frequency range from 1 MHz to 1000 GHz to a sample in which a molecular event is being detected, whereby the sample interacts with and modulates the test signal to produce a modulated test signal, and (2) detecting the modulated test signal, wherein the applying and detecting take place in a temperature-controlled environment, wherein the temperature-controlled environment comprises the sample, a radiating portion of a signal generating circuit, and a receiving portion of a signal detection circuit and wherein the applying and detecting take place in the environment at a temperature controlled to within + 0.5°C.
  • FIG. 1 illustrates a bioassay test system in accordance with one embodiment ofthe present invention.
  • Fig. 2 illustrates a first embodiment of a bioassay device, an open- ended coaxial resonant probe.
  • Fig. 3 illustrates a second embodiment ofthe bioassay device, a broadband microstrip detector.
  • Fig. 4 illustrates a third embodiment ofthe bioassay device, a waveguide magic-t coupler assembly.
  • Fig. 5 illustrates an embodiment of a coaxial probe integrated with a fluidic transport system in accordance with the present invention.
  • Fig. 6 illustrates a bioassay test system in which a flow tube is used to supply the sample to a coaxial probe in accordance with the present invention.
  • Fig. 7 illustrates a flow cell for use with the waveguide magic-t detector shown in Fig. 4 in accordance with the present invention.
  • Fig. 8 illustrates a temperature controlled bioassay test set in accordance with one embodiment ofthe invention.
  • Fig. 9A illustrates a simplified block diagram of a computer system operable to execute a software program designed to perform each ofthe described methods.
  • Fig. 9B illustrates the internal architecture ofthe computer system shown in Fig. 9A.
  • Fig. 10 illustrates
  • Fig. 11 illustrates
  • 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 ofthe 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 ofthe activity of a living cells (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 ofthe 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 ofthe intracellular environment in which, for example, a binding event is taking place.
  • analyte refers to a molecular entity whose presence, structure, binding ability, etc., is being detected or analyzed.
  • Suitable analytes for practice of this invention include, but are not limited to antibodies, antigens, nucleic acids (e.g. natural or synthetic DNA, RNA, gDNA, cDNA, mRNA, tRNA), lectins, sugars, glycoproteins, receptors and their cognate ligand (e.g.
  • growth factors and their associated receptors growth factors and their associated receptors, cytokines and their associated receptors, signaling molecules and their receptors
  • small molecules such as existing pharmaceuticals and drug candidates (either from natural products or synthetic analogues developed and stored in combinatorial libraries), metabolites, drugs of abuse and their metabolic by-products, co-factors such as vitamins and other naturally occurring and synthetic compounds, oxygen and other gases found in physiologic fluids, cells, phages, viruses, cellular constituents cell membranes and associated structures, other natural products found in plant and animal sources, and other partially or completely synthetic products.
  • ligand is commonly used herein to refer to any molecule for which there exists another molecule (i. e. an "antiligand”) that binds to the ligand, owing to a favorable (i.e., negative) change in free energy upon contact between the ligand and antiligand.
  • an antiligand i.e. an antiligand
  • ligand and antiligand both have this broad sense and can be used interchangeably.
  • ligand it is recognized that there is a general tendency in the field of biology to use the word "ligand” to refer to the smaller ofthe two binding partners that interact with each other, and this convention is followed whenever possible.
  • ligand/antiligand complex refers to the ligand bound to the antiligand.
  • the binding can be specific or non-specific, and the interacting ligand/antiligand complex are typically bonded to each other through noncovalent forces such as hydrogen bonds, Van der Waals interactions, or other types of molecular interactions.
  • the specified antiligand binds to its particular "target” and does not bind in an indistinguishable amount to other potential ligands present in the sample.
  • a cell surface receptor for a hormonal signal e.g., the estrogen receptor
  • a specific hormone e.g., estradiol
  • nucleic acid sequences that are completely complementary will hybridize to one another under preselected conditions such that other nucleic acids, even those different in sequence at the position of a single nucleotide, hybridize to a lesser extent.
  • the method ofthe invention can be applied to situations in which one ofthe members of a binding pair is immobilized on a surface while test compounds in solution contact the immobilized molecule (individually, in a mixture, or sequentially).
  • the term "antiligand” is usually used to refer to the molecule immobilized on the surface.
  • the antiligand for example, can be an antibody and the ligand can be a molecule such as an antigen that binds specifically to the antibody.
  • the antibody can be considered to be the ligand and the antigen considered to be the antiligand. Additionally, once an antiligand has bound to a ligand, the resulting antiligand/ligand complex can be considered an antiligand for the purposes of subsequent binding.
  • the terms "molecule" refers to a biological or chemical entity that exists in the form of a chemical molecule or molecules, as opposed to salts or other non-molecular forms of matter.
  • molecules are ofthe type referred to as organic molecules (compounds containing carbon atoms, among others, connected by covalent bonds), although some molecules do not contain carbon (including simple molecular gases such as molecular oxygen and more complex molecules such as some sulfur-based polymers).
  • the general term "molecule” includes numerous descriptive classes or groups of molecules, such as proteins, nucleic acids, carbohydrates, steroids, organic pharmaceuticals, receptors, antibodies, and lipids. When appropriate, one or more of these more descriptive terms (many of which, such as "protein,” themselves describe overlapping groups of compounds) will be used herein because of application ofthe method to a subgroup of molecules, without detracting from the intent to have such compounds be representative of both the general class "molecules" and the named subclass, such as proteins.
  • a "molecule” also includes bound complexes of individual molecules, such as those described below.
  • An ionic bond can be present in a primarily covalently bound molecule (such as in a salt of a carboxylic acid or a protein with a metal ion bound to its amino acid residues), and such molecules are still considered to be molecular structures.
  • salts e.g., sodium chloride
  • Such salts will participate in the overall dielectric response, but a molecular binding event or property can be detected in their presence.
  • a "molecular binding event” includes the binding of a molecule to an atom or ion, such as in a chelation process (e.g., interation of an iron ion with the heme moiety of hemoglobin).
  • binding partners refers to pairs (or larger groups; see below) of molecules that specifically contact (e.g. bind to) each other to form a bound complex.
  • a pair or other grouping typically consists of two or more molecules that are interacting with each other, usually by the formation of noncovalent bonds (such as dipole-dipole interactions, hydrogen bonding, or van der Waals interactions).
  • the time of interaction (sometimes referred to as the on-off time) can vary considerably, even for molecules that have similar binding affinities, as is well known in the art.
  • Biological binding partners need not be limited to pairs of single molecules.
  • a single ligand can be bound by the coordinated action of two or more anti-ligands, or a first antigen/antibody pair can be bound by a second antibody that is specific for the first antibody. Binding can occur with all binding components in solution or with one (or more) ofthe components attached to a surface and can include complex binding that involves the serial or simultaneous binding of three or more separate molecular entities.
  • complex binding examples include GPCR-ligand binding, followed by GPCR/G-protein binding; nuclear receptor/cofactor/ligand DNA binding; or a binding complex including chaperone proteins, along with a small-molecule ligand.
  • GPCR-ligand binding followed by GPCR/G-protein binding
  • nuclear receptor/cofactor/ligand DNA binding nuclear receptor/cofactor/ligand DNA binding
  • a binding complex including chaperone proteins, along with a small-molecule ligand include GPCR-ligand binding, followed by GPCR/G-protein binding; nuclear receptor/cofactor/ligand DNA binding; or a binding complex including chaperone proteins, along with a small-molecule ligand.
  • Other examples will be readily apparent to those skilled in the art.
  • the terms “isolated,” “purified,” and “biologically pure” refer to material which is substantially or essentially free from components that normally accompany it as found in its native state.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and, unless otherwise limited, encompasses such polymers that contain one or more analogs of natural nucleotides that can hybridize in a similar manner to naturally occurring nucleotides.
  • polypeptide As used herein, the terms "polypeptide,” “peptide,” and “protein” are generally used interchangeably to refer to a polymer of amino acid residues. These terms do not appear to have a consistent use in the art in reference to the size of molecules, although “polypeptide” is often used without regard to size, while “peptides” are smaller than “proteins.” Proteins are generally considered to be more complex than simple peptides and often contain material other than amino acids, such as polysaccharide chains. All of these terms apply to polymers containing amino acids in which one or more amino acid residue or peptide bond is an artificial chemical analogue of a corresponding naturally occurring amino acid or bond, as well as to naturally occurring amino acid polymers.
  • the term "enzyme” refers to a protein that acts as a catalyst and reduces the activation energy of a chemical reaction occurring between other compounds or of a chemical reaction in which one compound is broken apart into smaller compounds.
  • the compounds that undergo the reaction under the influence of the enzyme are referred to as "substrates.”
  • the enzyme is not a starting material or final product in the reaction, but is unchanged after the reaction is completed.
  • the terms "molecular binding layer” or “MBL” refers to a layer having at least one molecular structure (e.g., an analyte, antiligand, or a ligand/antiligand pair) that is electromagnetically coupled to the signal path.
  • the MBL is typically formed on a fixed surface in the detection region, although mobile surfaces, such as beads or cells, can easily be used along with appropriate fluid movement controls.
  • the molecular binding layer can consist of one or more ligands, antiligands, ligand/antiligand complexes, linkers, matrices of polymers and other materials, or other molecular structures described herein.
  • the molecular binding layer can be extremely diverse and can include one or more components, including matrix layers and/or insulating layers, that have one or more linking groups.
  • the MBL can be electromagnetically coupled to the signal path either via a direct or indirect physical connection or when the ligand is located proximate to, but physically separated from, the signal path.
  • the MBL can be formed on a derivatized surface, such as one having thiol linkers formed from biotinylated metals, all in accordance with standard practice in the art.
  • MBL molecular binding region
  • MLR molecular binding region
  • linking group refers to a chemical structure used to attach any two components to each other, often on the bioassay device.
  • the linking groups thus have a first binding portion that binds to one component, such as the conductive surface, and a second binding portion that binds to another component, such as the matrix or the antiligand.
  • the "components of said molecular event sufficient for said molecular event to occur” can vary greatly, depending on the particular molecular event being detected.
  • the quoted phrase can refer to a single protein when the structure of that protein is being investigated.
  • three or more components may be necessary for the binding event to occur (many more, for example, in even more complex binding situation, such as in the formation of a functional ribosome from its component parts).
  • One of ordinary skill in the molecular event under investigation can readily determine the minimum components sufficient for the molecular event to occur, either from prior knowledge or from the detection of a modulated signal that is indicative of binding.
  • thermal barrier is meant any physical material that acts to prevent or inhibit heat energy from being transmitted from one region to another, whether by conduction, convection, or radiation.
  • a material that transmits heat energy by one method e.g., conduction
  • a preferred thermal barrier has a total thermal conductivity of 50 mW/m-K or less. Examples include polyurethane foam, fiberglass, and acrylic plastic.
  • Temporal controller has its normal meaning and refers to any apparatus that acts to measure and maintain the temperature of a temperature-controlled environment within a desired range.
  • Thermal Gain describes the ability of a temperature-controlled environment or enclosure to isolate the sample, detector, and/or detection electronics from changes in the ambient temperature.
  • the ratio ofthe change in temperature that occurs in a given time outside the enclosure to the change in temperature that occurs in the same amount of time inside the enclosure is the thermal gain. For example, a 10-degree change in the ambient temperature with a corresponding 1 -degree change in temperature-controlled environment constitutes a thermal gain of 10.
  • solution refers to the resulting mixture formed from a first material (the “solvent,” which forms the bulk ofthe solution) in which a second material (the “solute”, such as a target ligand) resides primarily as separate molecules rather than as aggregates of molecules.
  • Solutions can exist in any ofthe solid, liquid or gaseous states. Solid solutions can be formed from “solvents” made of naturally occurring or synthetic molecules, including carbohydrates, proteins, and oligonucleotides, or of organic polymeric materials, such as nylon, rayon, dacron, polypropylene, teflon, neoprene, and delrin.
  • Liquid solutions include those containing an aqueous, organic or other liquid solvent, including gels, emulsions, and other viscous materials formed from liquids mixed with other substances.
  • Exemplary liquid solutions include those formed from celluloses, dextran derivatives, aqueous solution of d-PBS, Tris buffers, deionized water, blood, physiological buffer, cerebrospinal fluid, urine, saliva, water, and organic solvents, such as ethers or alcohols.
  • Gaseous solutions can consist of organic molecules as gases or vapors in air, nitrogen, hydrogen, or other gaseous solvents.
  • solution is used herein in many cases to refer to a mixture containing a target ligand and or antiligand that is being applied to a molecular binding surface. Another example of a solution is the sample that is being analyzed.
  • liquid solutions, particularly aqueous ones are preferred for the practice ofthe invention.
  • 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 2 , 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.
  • a biological sample is a sample of biological tissue or fluid that, in a healthy and/or pathological state, is to be assayed for the structure(s) or event(s) of interest.
  • biological samples include, but are not limited to, sputum, amniotic fluid, blood, blood cells (e.g., white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, pleural fluid, and cells from any of these sources.
  • Biological samples also include cells grown in cultures, both mammalian and others.
  • Biological samples further include sections of tissues such as frozen sections taken for histological purposes. Although a biological sample is often taken from a human patient, the meaning is not so limited.
  • the same assays can be used to detect a molecular event of interest in samples from any mammal, such as dogs, cats, sheep, cattle, and pigs, as well as samples from other animal species (e.g., birds, such as chickens or turkey) and plants (e.g. , ornamental plants and plants used as foods, such as corn or wheat).
  • the biological sample can be pretreated as necessary by dilution in an appropriate transporting medium solution or concentrated, if desired, and is still referred to as a "biological sample.” Any of a number of standard aqueous transporting medium solutions, employing one of a variety of transporting media, such as phosphate, Tris, or the like, preferably at physiological pH can be used.
  • pretreatment of a more general sample by dilution, extraction, etc.) once it is obtained from a source material do not prevent the material from being referred to as a sample.
  • fluid reservoir refers to any location, without regard to physical size or shape, in which the sample fluid is retained prior or subsequent to application ofthe sample fluid across the detection region.
  • Fluid reservoir can refer to a fluid droplet or layer formed on a flat surface and maintained at that location by inertia and/or surface tension.
  • chip designs commonly used in genomics in which a sample fluid is washed across the surface of a chip that has specific molecular probes (usually DNA fragments of know sequence) attached at known locations on the surface.
  • the "fluid reservoir,” however, can be and often is contained within physical walls that restrain movement ofthe fluid, such as vertical walls that constrain gravitational spreading (as in the side walls of test tube or microtitre plate), completely surrounding walls (as in a sealed container), or partially surrounding walls that direct and/or permit motion in a limited number of directions (such as the walls of a tube or other channel).
  • the last of these named possibilities is often referred to herein as a "fluid channel” and occurs commonly in situations were a fluid is being moved from one location to another (such as in a microfluidics chip) to allow interaction with other samples and/or solutions containing reagents or to allow multiple samples to be transported past a single detection region.
  • the term "signal path" refers to a transmission medium that supports the propagation of an electromagnetic signal at the desired frequency of operation.
  • the signal path consists of a signal plane/ground plane/dielectric substrate structure capable of supporting a transverse electromagnetic (TEM) signal.
  • TEM transverse electromagnetic
  • Exemplary embodiments of this signal path architecture include coaxial cable, microstrip, stripline, coplanar waveguide, slotline, and suspended substrate.
  • Other exemplary architectures include wire, printed circuit board traces, conductive or dielectric waveguide structures, and mutlipolar (e.g., quadrapolar, octapolar) transmission structures.
  • the signal path includes a single signal port that receives an incident test signal and from which a reflected modulate signal is recovered.
  • the signal path consists of two or more signal ports: at least one that receives an incident test signal and one that outputs the corresponding modulated test signal.
  • the term "detection region” refers to a region of the bioassay device over which the test sample and signal path are electromagnetically coupled.
  • the detection region may be realized in a variety of forms, e.g., an area within a fluid transport channel located proximate to an open-ended coaxial probe, an area of a flowcell located within a waveguide aperture, or a length of PTFE tubing vertically aligned between the transmission line and ground plane of a microstrip structure to name a few possibilities.
  • the detection region is not limited to any particular volume, but is typically less than 1 ml (1 x 10 "6 m 3 ).
  • Smaller 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) are preferable for many ofthe methods used for testing of binding ability of potential pharmaceutical compounds, because ofthe small size and expense ofthe available samples.
  • the term “electromagnetically coupled” refers to the transfer of electromagnetic energy between two objects, e.g., the signal path 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 in a MBL formed along the surface of a microstrip transmission line), or when the objects are physically separated from each other (e.g., molecular events occurring in solution within a sample that is separated from an open-ended coaxial probe by the wall of a PTFE tube).
  • 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 (1x10° 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 selected over any terminal frequencies, including frequency ranges bounded by the specific frequencies named in this paragraph.
  • the term “spectrum” is sometimes used.
  • radiating portion of a signal generating circuit is meant that portion of a signal path that launches a signal that couples to the sample in the detection region.
  • receiving portion of a signal detection circuit is meant that portion of a signal path that couples to and receives the modulated signal from the detection region ofthe sample.
  • the radiating and receiving portions can be part ofthe same circuit or parts of different circuits. When part of the same circuit, they can be identical (as shown by some ofthe specific embodiments that follow).
  • the term "bioassay device” refers to a structure that incorporates the radiating portion ofthe a signal generating circuit or the receiving portion of a signal receiving circuit.
  • a single structure e.g., a coaxial measurement probe
  • the bioassay device further includes a cavity, recessed area, enclosure, tube, flow cell, or other surface feature or structure that is configured to retain a volume of sample within the detection region ofthe bioassay device.
  • the bioassay device is not limited to any particular geometry or size, and is defined primarily by the architecture ofthe signal path and desired volume of the interrogated sample.
  • bioassay system is meant the overall apparatus, optionally including fluids and/or other materials used as consumables, in which the methods described herein are carried out.
  • the “bioassay system” refers to the bioassay device as described above, in combination with the components necessary to supply and recover the test signals to and from the bioassay device and to analyze the results therefrom.
  • These components can include test equipment (e.g., a network analyzer, vector voltmeter, signal generator, frequency counter, spectrum analyzer), control equipment (e.g., computers, temperature compensation circuitry and components), and sample handling components.
  • matrix refers to a layer of material on the bioassay device that is used as a spacer or to enhance surface area available for binding or to optimize orientation of molecules for enhanced binding, or to enhance any other property of binding so as to optimize the bio-assay device.
  • the matrix layer can be formed from carbohydrates such as dextran, poly amino acids, cross-linked or non-cross linked proteins, and the like.
  • the general techniques used with the present invention make use ofthe observation that molecules can be distinguished and their structural properties and binding abilities measured based upon their dielectric properties in a region ofthe electromagnetic spectrum not previously used to detect molecular events and/or by using techniques not previously applied to detection of molecular events.
  • These dielectric properties are observed by initially coupling a test signal to a test sample that includes an analyte of interest. The dielectric properties ofthe analyte modulate the test signal and produce a distinguishable signal response. This response can be recovered, stored, and used to detect and identify the molecule in other test samples.
  • interactions of other molecules with the first molecule can also be detected, as the test signal is further modified by the interaction of a second molecule with the first.
  • Detection and identification of molecule properties and of binding events can occur in the liquid, gas, or solid phase, but are preferably carried out in an aqueous physiological environment in order to identify properties ofthe molecule associated with its function in a biological environment.
  • the detector assemblies used with the present invention provide a measurement probe operable to couple a test signal to a test sample in which a molecular event is taking place.
  • the test sample is in a fluid reservoir, often a fluid channel or a well of a multiwell plate.
  • a portion of the fluid reservoir, referred to as the detection region, is illuminated with the test signal.
  • the dielectric properties ofthe molecules involved in the molecular event operate to modulate the test signal, providing a signal having a signal response that is different from the signal response that would be detected if the same test signal were applied to a sample, otherwise identical, that did not contain the molecular event.
  • the signal response is then recovered and provides information about one or more properties ofthe molecule or molecules involved in the molecular or cellular event under investigation.
  • the present invention provides a method for detecting a molecular event.
  • the method comprises coupling an electromagnetic test signal in a frequency range from 1 MHz to 1000 GHz to a sample in which a molecular event is being detected, whereby the sample interacts with and modulates the test signal to produce a modulated test signal.
  • the modulated test signal is detected and analyzed to detect the molecular event. It has been found that significant improvements are present when the coupling and detecting take place in a temperature-controlled environment, where the environment comprises the sample, a radiating portion of a signal generating circuit, and a receiving portion of a signal detection circuit. If only the sample (on the one hand) or the electronic components (on the other hand) are temperature controlled, signal analysis is difficult.
  • the applying and detecting take place in the environment at a temperature controlled to within + 0.5°C. Satisfactory results have been obtained in this range, whereas larger temperature ranges have given unsatisfactory results. However, control of temperature to an even greater extent is desired, in order to detect and analyze the signals of molecular events that have relatively smaller electromagnetic signatures. Accordingly, it is preferred to control the temperature ofthe environment to within + 0.05 °C or even more preferably to within + O.OTC, + 0.001°C, + 0.0001°C, or less.
  • Temperature control to within + 0.00001 °C is obtainable now in zero-gradient crystal ovens, as described in Karlquist et al., "The Theory of Zero-Gradient Crystal Ovens," 1997 IEEE International Frequency Control Symposium, pp. 898-908.
  • temperature control used to control frequency of standard electronic circuitry
  • the existing technology can readily be applied to the bioassay systems ofthe invention, now that a need has been demonstrated by the present invention to be appropriate for a system in which one is, for example, detecting binding of a ligand with an antiligand and the binding is measured without separating bound from unbound ligand.
  • a resonant probe comprising a first coaxial section comprising a longitudinally extending center conductor, a dielectric insulator disposed around the longitudinal axis ofthe center conductor, and an outer ground plane disposed around the longitudinal axis ofthe dielectric insulator, the first coaxial section having a probe head and a first gap end, the probe head comprising an open-end coaxial cross section; a second coaxial section comprising a longitudinally extending center conductor, a dielectric insulator disposed around the longitudinal axis ofthe center conductor, and an outer ground plane disposed around the longitudinal axis ofthe dielectric insulator, the second coaxial section having a second gap end and a connecting end, the gap end comprising a open-end coaxial cross section and the connecting
  • All of these parts ofthe electronic circuitry should be within the temperature-controlled environment, along with the sample.
  • Other examples of configurations that can be used to couple a signal to a sample and that should be included in the temperature- controlled environment are a resonant probe comprising a reentrant cavity, typically used to concentrate signal into the detection region ofthe sample.
  • Reentrant cavities are well known, as exemplified by Goodwin et al., "Reentrant radio-frequency resonator for automated phase-equilibria and dielectric measurements in fluids," Rev. Sci. Instrumen., 67 (12) 1996, pp. 4294-4303.
  • non-resonant probes such as a non-resonant coaxial probe or transmission line probe
  • performance of non-resonant probes is also improved by the use of a temperature-controlled environment as described herein.
  • the time period over which the temperature needs to be controlled depends on the timing ofthe coupling and detection operations. These depend on the particular instrument being used, and there are no limits on the timing, as long as temperature can be controlled during the relevant coupling and detection operations. These operations can be for a single sample or for a set of samples (containing any number of members) whose modulated signals are being compared and analyzed in order to determine whether a molecular event has taken place or not.
  • An example of comparison of signals of a set would be a background signal obtained on buffer, two test samples each containing one member of a potential binding pair, and a test sample containing the mixed potential binding-pair members.
  • Typical coupling and detecting operations take place over a time period of from 2 seconds to 2 minutes for an individual sample using the instruments available in the laboratories ofthe inventors, but these should not be taken as limitations on the invention, but as examples of typical operation, unless specifically recited in a claim.
  • coupling and detecting of all samples in a given set typically take place over a time period of from 1 minute to two hours.
  • the size ofthe temperature-controlled environment need not be large in most cases.
  • a detection operation is carried out on a sample in a fluid reservoir having a detection region with a volume of less than 1.0 mL.
  • Such operations are often carried out by (a) introducing a first sample into a fluid channel of a fluid transport system, the fluid transport system having a fluid movement controller and the fluid channel having a sample entry end, a detection region, and a sample exit end, the detection region having a volume of less than 1 mL; (b) causing the sample to move through the channel from the sample entry end toward the sample exit end under the control ofthe fluid controller; (c) applying a test signal of greater than 10 MHz and less than 1000 GHz to the detection region of the fluid channel; and (d) detecting a change in the test signal as a result of interaction ofthe test signal with the sample.
  • the fluid-handling operations are typical of those used in microfluidics operations and other laboratory techniques for manipulating small liquid samples.
  • other typical operations include (e) introducing a spacer material into the channel after the first test sample, (f) introducing a further test sample into the channel after the spacer material, (g) causing the further test sample to move through the channel under the control of the fluid controller, whereby a plurality of different test samples separated by spacer material is transported through the channel without intermixing different test samples, and (f) optionally repeating steps (c)-(d) for the further test sample.
  • the spacer material typically comprises a solution of ionic strength sufficiently high to be transported by electroosmotic pumping and the fluid movement controller utilizes electroosmotic pumping ofthe fluid.
  • Spacers are often a fluid that is substantially immiscible with the test samples and can comprise a gaseous bubble, with the fluid movement controller utilizing physical pumping ofthe fluid.
  • Microfluidic systems are often used to handle mixture operations, as well as to move samples from one location to another.
  • a typical mixing operation is carried out by providing a further fluid channel that intersects the first fluid channel in the fluidic transport system.
  • the system provides separate control of fluid movement in the second fluid channel, the second fluid channel containing a test compound or a series of test compounds, to be mixed with sample in the first fluid channel.
  • a test compound from the second fluid channel is mixed with a test sample in the first fluid channel sufficiently upstream from the test signal so that the test compound has time to bind with a molecular structure in a test sample in the first fluid channel before the test sample reaches the test signal.
  • a preferred embodiment is a method for detecting a molecular event in a test sample in a detection region of a fluid reservoir, the method comprising locating a measurement probe that exhibits a resonant signal response at a predefined frequency in a range from 10 MHz to 1000 GHz proximate to the detection region to electromagnetically couple a signal thereto; supplying a reference medium to the detection region; coupling a test signal to the detection region and recording a baseline signal response; supplying a test sample containing or suspected of containing the molecular event to the detection region; coupling a test signal to the detection region and obtaining a test sample response; determining the difference, if any, between the test sample response and the baseline response; and relating the difference to the molecular event, with temperature being controlled as described elsewhere herein.
  • Use of a measurement probe that exhibits an Si i resonant response is preferred in some embodiments.
  • a preferred technique for coupling a test signal to the detection region and obtaining a baseline signal response comprises generating an incident signal; coupling the incident signal to the detection region; recovering a reflected signal from the detection region; and comparing amplitude or phase ofthe incident signal to amplitude or phase ofthe reflected signal. Measurement operations need not be carried out concurrently, so in some embodiments sample measurements are made at different times, followed by comparing a later test sample response with the stored first test sample response.
  • a measurement probe comprises a first coaxial section comprising a longitudinally extending center conductor, a dielectric insulator disposed around the longitudinal axis ofthe center conductor, and an outer ground plane disposed around the longitudinal axis ofthe dielectric insulator, the first coaxial section having a probe head and a first gap end, the probe head comprising an open-end coaxial cross section; a second coaxial section comprising a longitudinally extending center conductor, a dielectric insulator disposed around the longitudinal axis ofthe center conductor, and an outer ground plane disposed around the longitudinal axis ofthe dielectric insulator, the second coaxial section having a second gap end and a connecting end, the gap end comprising a open-end coaxial cross section and the connecting end comprising a coaxial connector; and a tuning element adjustably
  • a bioassay system configured to detect a molecular event in a test sample, comprising thermal barriers forming boundaries of a temperature-controlled environment, a temperature controller operably connected to the temperature-controlled environment that controls temperature in the temperature-controlled environment to within + 0.5°C during a time period in which an environment immediately external to the temperature- controlled environment changes by + 5°C, a radiating portion of a signal generating circuit located in the environment, a sample container located in the environment and positioned to receive an electromagnetic test signal from the radiating portion ofthe signal generating circuit, whereby sample present in the sample container interacts with and modulates the test signal to produce a modulated test signal, and a receiving portion of a signal detection circuit located in the environment and positioned to receive the modulated signal, where the sample contains a solution or molecular binding layer containing components ofthe molecular event sufficient for the molecular event to occur.
  • the sample container can comprise a fluid reservoir, the fluid reservoir comprising a detection region having of volume of less than 1.0 mL.
  • the fluid reservoir comprising a detection region having of volume of less than 1.0 mL.
  • a signal circuit generally further contains a signal source operable to transmit an electromagnetic incident test signal to the radiating portion ofthe circuit.
  • the signal source is located outside the temperature- controlled environment.
  • Examples of a signal source and signal detector in a circuit include a vector network analyzer system, a scalar network analyzer system, or a time domain reflectometer, with the signal being generated outside the controlled- temperature environment and the signal being detected inside the controlled- temperature environment.
  • a particular signal detector can be used or not can be determined by whether or not the detector operates at a sufficiently high sensitivity to detect that a first modulated test signal is different from a second modulated test signal when the first modulated test signal is obtained while an aqueous sample containing 0.3 ⁇ g or less of fibrinogen is present in the detection region and the second modulated test signal is obtained while a second aqueous sample is present in the detection region, the second aqueous sample being identical to the first aqueous sample except that it does not contain any fibrinogen.
  • one aspect ofthe invention is a computer- readable storage medium containing information obtained by the methods as described herein.
  • Fig. 1 illustrates a bioassay test system 100 in accordance with one embodiment of the present invention.
  • the test system 100 includes a signal source 110a and a signal detector 190a connected to a first port 152 ofthe bioassay device 150.
  • the signal source and detector can be used to obtain a one-port (/. e. , a reflection) signal response.
  • the test system 100 may include a signal detector 190b connected a second port 158 ofthe bioassay device 150.
  • the signal source 110a and the signal detector 190b can be used to provide a two-port (i.e., a "through") signal response ofthe bioassay device 150.
  • a second signal source 110b may be further included to provide a reflection measurement capability at the second port 158 ofthe bioassay device 150.
  • the signal sources 110 are operable to generate and launch an electromagnetic signal 160 ("incident test signal") at one or more amplitudes and/or frequencies.
  • the signal detectors operate to recover the test signal after it has interacted with (i.e., after electromagnetically coupling to) the test sample in the bioassay device 150.
  • the signal source 110 and the signal detectors 190 are included within an automated network analyzer, such as model number 8510C from the Hewlett-Packard Company.
  • Other measurement systems such as vector voltmeters, scalar network analyzers, time domain reflectometers, and the like that use signal characteristics of incident, transmitted, and reflected signals to evaluate an object under test may be used in alternative embodiment under the present invention.
  • the sample handling assembly 130 includes a sample handling device 132 and a sample delivery apparatus 134.
  • the sample handling device 130 may include sample preparation, mixing, and storage functions that may be integrated on a micro-miniature scale using, for instance, a microfluidic platform.
  • the sample delivery apparatus 134 may consist of a tube, etched or photolithographcially formed channel or capillary, or other similar structure that delivers a volume of test sample to a location proximate to the signal path, such that the incident test signal propagating along the signal path will electromagnetically couple to the test sample. Specific embodiments ofthe sample handling and delivery structures are provided below.
  • the bioassay device 150 operates as a bioelectrical interface that detects molecular events occurring within the sample using electromagnetic signals.
  • the bioassay device 150 includes a signal path that is configured to support the propagation of electromagnetic signals over the desired frequency range. Electrical engineers will appreciate that the signal path may consist of a variety of different architectures, for instance a waveguide, transverse electromagnetic (TEM) mode structures such as coaxial cable, coplanar waveguide, stripline, microstrip, suspended substrate, and slotline, as well as other structures such as twisted pair, printed circuits, and the like. Specific embodiments ofthe signal path are illustrated below.
  • TEM transverse electromagnetic
  • An incident test signal 160 is generated by the signal source 110a and launched along the signal path where it electromagnetically couples from the signal path to the supplied test sample.
  • One or more signal characteristics (amplitude, phase, frequency, group delay, etc.) ofthe incident test signal 160 are modulated by its interaction with the sample.
  • a portion ofthe modulated signal 180 is reflected back along the signal path and recovered by the signal detector 190.
  • a portion of the modulated signal is transmitted through to the second port and recovered by the second signal detector 190b.
  • the modulation caused by the electromagnetically coupling may consist of a change in the amplitude, phase, frequency, group delay, or other signal parameters.
  • the modulated test signal 180 (and/or 170) is recovered and its signal characteristics (amplitude, phase, etc.) are compared to signal characteristics of the corresponding incident test signal 160.
  • changes in the amplitude and phase ofthe modulated reflected signal 180 relative to the incident test signal 160 are computed at each test frequency and a response plotted over the test frequencies as an s-parameter return loss response.
  • changes in the amplitude and phase ofthe modulated transmitted signal 170 relative to the incident test signal 160 are computed at each test frequency and a response plotted over the test frequencies as an s-parameter transmission loss response.
  • the signal responses may be used to compute other quantities to further characterize the test sample makeup. Quantities such as impedance, permeability, resonant frequency, and quality factor of resonant structures may also be either measured directly from the measurement system, or computed indirectly therefrom and used as a metric in characterizing the test sample.
  • Fig. 2 illustrates a first embodiment ofthe bioassay device 150 shown in Fig. 1, an open-ended coaxial resonant probe 250.
  • the resonant probe 250 includes a first coaxial section 251, a bracket 252, an attachment platform 253, contact rings 255, a tuning gap 256, a second coaxial section 257, a conductive ground tube 258, and a fluidics shelf 259.
  • the first coaxial section 251 is coupled to a signal source and a signal detector illustrated and described below.
  • the first and second coaxial sections consist of RG401 semi-rigid cable. Those of skill in the art will appreciate that other types of semi-rigid cable as well as other transmission structures can be used in alternative embodiments under the present invention.
  • the first coaxial section 251 extends into the gap area 254 near the bottom ofthe fluidics shelf 259.
  • Contact rings 255a and 255b can be optionally attached around the outer surface ofthe first coaxial section 251 to provide ground conductivity between the first coaxial section 251 and the inner surface ofthe ground tube 258.
  • the contact rings are highly conductive springs, although other structures can be used instead.
  • the outer surface ofthe first coaxial section 251 is brought into contact with the interior surface ofthe ground tube 258 (copper in one embodiment) to a sufficient degree, thereby obviating the need for the contact rings 255.
  • the second coaxial section 257 terminates in an open-end and has a length that is approximately one-half of a wavelength ( ⁇ /2) at the desired resonant frequency.
  • the first section 257 is approximately 4 inches, which corresponds to a resonant frequency of 1 GHz.
  • the test sample is supplied at/near the open-end ofthe second coaxial section 257 such that a signal propagating along the second section 257 is electromagnetically coupled to the test sample.
  • the test sample comes into direct contact with the open- end cross-section ofthe second section 257.
  • the test sample and open-end section are separated by an intervening layer, such as the outer diameter of a fluidic channel or tube.
  • the intervening layer is sufficiently signal transparent to permit electromagnetic coupling through the intervening layer to the test sample. Occurrence of a molecular event may be detected either in “solid phase” by using probes immobilized over the detection region surface to bind to predefined targets in the solution, or in “solution phase” in which mobile molecular events occur over the detection region.
  • the first and second coaxial sections 251 and 257 are separated by a tuning gap 256 that electrically operates to fine-tune the resonant response to the desired frequency.
  • the second coaxial section 257 is secured within the ground tube 258 within the fluidics shelf 259.
  • the first coaxial section 251 is inserted into the gap region 254, the outer surface ofthe first coaxial section 251 making electrical contact with the interior surface ofthe ground tube 258, thereby providing a continuous ground potential therebetween.
  • the tuning gap 256 formed between the first and second coaxial sections 251 and 257 is made either shorter or longer by moving the bracket 252 either up or down, respectively.
  • the position ofthe second coaxial section 257 within the conductive ground tube 258 can be adjustable, either alternatively or in addition to the first coaxial section 251.
  • the attachment platform 253 attaches to and holds stationary the fluidics shelf 259, allowing the bracket to either insert or remove the first coaxial section 251 therefrom.
  • the bracket 252 is motor driven and included within a precision motorized translational stage assembly available from the Newport Corporation of Irvine, California.
  • Fig. 3 illustrates a second embodiment ofthe bioassay device, a broadband microstrip detector.
  • the microstrip detector 300 includes top and bottom dielectric plates 310 and 320 and a flow tube 330 interposed therebetween.
  • Top and bottom dielectric plates 310 and 320 are preferably constructed from a material exhibiting a low loss tangent at the desired frequency of operation.
  • the dielectric plates 310 and 320 are each .030" thick of GML 1000 (available from Gil Technologies of Collierville, TN) having a relative dielectric constant of approximately 3.2.
  • flow tube 330 is constructed from a material having a low loss tangent and a smooth, resilient surface morphology that inhibits analyte formation along the inner surface and detection of molecular events occur in solution phase as they move along the detection length 340 ofthe device.
  • the flow tube 330 may include immobilized probes on the inner surface which are operable to capture predefined targets occurring within the test sample.
  • a PTFE tube having an ID of .015" and OD of .030" is used in the illustrated embodiment, although other materials and/or sizes may be used as well.
  • the top dielectric plate 310 includes a transmission line 312 deposited on the top surface and a channel 314 formed on the bottom surface.
  • the width of transmission line 312 is chosen to provide a predetermined characteristic impedance along the detection length 340 (further described below). The impedance calculation may take into account the varying dielectric constants and dimensions introduced by channels 314 and 324 and flow tube 330.
  • the transmission line 312 is typically formed from gold or copper.
  • the second dielectric plate 320 includes a channel 324 formed on the top surface and metallization deposited on the bottom surface.
  • the channel 324 is aligned with channel 314 to form a cavity within which the flow tube 330 extends.
  • the metallization 322 deposited on the bottom surface functions as the ground plane ofthe microstrip detector and will typically consist of a highly conductive material such as gold or copper.
  • Channels 314 and 324 are aligned to form a cavity that retains the flow tube 330 in a substantially vertically aligned position between the transmission line 312 and the ground plane 322. The flow tube is held between the transmission line 312 and the ground plane 322 along the detection length 340.
  • This configuration results in the passage of a significant number of field lines emanating from the transmission line through the flow tube (and accordingly, the test sample) before terminating on the ground plane 322.
  • the dielectric properties ofthe molecular events within the sample will modulate the signal propagating along the transmission line 312 (i.e., by altering the field lines setup between the transmission line 312 and ground plane 322), thereby providing a means to detect and identify the molecular events occurring in the test sample.
  • Fig. 4 illustrates a third embodiment ofthe bioassay device, a waveguide magic-t coupler assembly 400.
  • magic-t couplers can be configured to produce an output that represents the difference in the dielectric properties of two loads 442 and 452 connected to the coupler.
  • two loads are connected to the magic-t coupler, the first load 442 consisting of a reference sample in which a particular molecular event is known to be present or absent, and the second load 452 consisting of an unknown sample that is being interrogated for the presence ofthe particular molecular event.
  • a test signal at one or more frequencies is propagated into the ⁇ (sum) port and is electromagnetically coupled to the loads.
  • the resulting output signal at the ⁇ (delta) port represents a comparison between the dielectric properties ofthe two loads 442 and 452.
  • the waveguide magic-t coupler includes two load ports 444 and 454 consisting of waveguide apertures over which the load 452, consisting of a section of meandered tubing (PTFE in one embodiment) is positioned.
  • Tubing 452 is operable to transport the sample to, and contain it within, a cross sectional area across the waveguide aperture 454 where the incident test signal electromagnetically couples to the sample.
  • the magic-t assembly consists of an X-band magic-t coupler (available from Penn Engineering North Hollywood, CA.) and 0.020" ID PTFE tubing.
  • Fig. 5 illustrates an embodiment of a coaxial probe 250 (Fig. 2) integrated with a fluidic transport system 130 in accordance with one embodiment of the present invention.
  • the fluid transport system 130 includes a fluid channel 131 through which the test sample flows.
  • the fluid channel 131 can take on a variety of forms.
  • the fluid channel 131 is a Teflon® (polytetrafluoroethylene; PTFE) or other hard plastic or polymer tube (for example TEZELTM (ETFE) tube) operable to transport the test sample to and from the detection region 131.
  • the channel 131 consists of one or more etched channels (open or enclosed) in a microfluidic transport system, further described below.
  • Two or more channels can be used to provide a larger detection region 135 to improve detection sensitivity.
  • the fluid channel 131 is formed through well-known semiconductor processing techniques. Those of skill in the art will appreciate that other construction and architectures ofthe fluid channel 131 can be adapted to operate under the present invention.
  • the buffer can consist of a variety of solutions, gases, or other mediums depending upon the particular analyte therein.
  • Dulbecco's phosphate buffer saline (d-PBS) or a similar medium can be used as a buffer to provide an environment which resembles the biological molecule's natural environment.
  • d-PBS Dulbecco's phosphate buffer saline
  • other buffers such as DMSO, sodium phosphate (Na3PO4), MOPS, phosphate, citrate, glycine, Tris, autate, borate as well as others can be used in other embodiments under the present invention.
  • the fluid channel 131 includes a detection region 135 over which the coaxial probe 250 illuminates the sample.
  • Molecular event detection and/or identification can be accomplished in "solution phase” where the molecular events are free-flowing in the test sample as they move through the detection region, or alternatively in “solid phase,” in which probes are deposited or otherwise formed over the detection region and targeted molecular events attach thereto.
  • the area ofthe detection region 135 will be influenced by several factors including the architecture and material composition ofthe fluid channel 131, concentration of the molecular events occurring within the solution, desired detection time, the rate at which the test sample advances through the channel, and other factors as appreciable to those skilled in the art.
  • probes are formed within the detection region 135, the area of which will be influenced by binding surface chemistry, the material and morphology ofthe binding surface, and other factors appreciable to those skilled in the art.
  • Exemplary dimensions ofthe binding surface will be on the orders of 10 ⁇ 'm 2 to 10 "15 m 2 or any range within these limits. The larger numbers in this range are preferably achieved in a small volume by using a convoluted or porous surface. Smaller numbers within those listed will be more typical of microfluidic devices and systems fabricated using semiconductor processing technology.
  • the detection region 135 can alternatively be modified to accommodate other diagnostic applications, such as proteomics chips, known in the art.
  • the size or shape of detection region need only be such that signal propagation thereto and analyte passage therethrough are possible, subject to other constraints described herein.
  • the fluid controller 136 is connected to a reservoir 137. Fluid controller 136 uses fluid from the reservoir 137 to move the test sample through channel 131, which requires less test sample than simple pumping of sample alone through the channel.
  • a second reservoir 138 can be used to store a second analyte or test sample for mixture in the reservoir 137.
  • the fluid controller 136 can be further configured to rapidly mix the two test samples and supply the resulting mixture to the detection region 135.
  • This technique (known as stopped-flow kinetics in the art of fluidic movement systems) permits the operator to observe and record changes in the signal response as binding events occur between analytes ofthe two test samples. This data can also be used to determine the kinetics of binding events occurring between the analytes ofthe two samples.
  • the fluidics of conventional stopped-flow kinetic systems such as model no. Cary 50 available from Varian Australia Pty Ltd. of Victoria, Australia, can be adapted to operate with the present invention or integrated within the detector assembly 130. See www.hi- techsci.co.uk/scientific/index.html for additional information about stopped-flow fluidic systems.
  • fluid controller 136 fluid reservoirs 137 and 138 and other components associated with fluidic movement can comprise discrete components ofthe fluid transport system 130 or alternatively be partially or completely integrated.
  • Fig. 6 illustrates a bioassay test system in which a flow tube is used to supply the sample to a coaxial probe in accordance with the present invention.
  • the system includes a vector network analyzer model number HP 8714 available from Agilent Technologies, Inc. (formerly the Hewlett Packard Corporation), a computer, an open-ended coaxial measurement probe functioning as the bioassay device, and a length of PTFE tube (Cole-Parmer Instrument Company of Vernon Hills, IL) used as a fluid channel to transport the transporting medium and test sample to the detection region ofthe measurement probe.
  • HP 8714 available from Agilent Technologies, Inc. (formerly the Hewlett Packard Corporation)
  • HP 8714 available from Agilent Technologies, Inc. (formerly the Hewlett Packard Corporation)
  • a computer an open-ended coaxial measurement probe functioning as the bioassay device
  • a length of PTFE tube Cold-Parmer Instrument Company of Vernon Hills, IL
  • the PTFE tube (0.031" I.D., 0.063" O.D., wall 0.016") is located over the detection region of the measurement probe and is secured using a grooved top cover that was screwed into the shelf of the measurement probe.
  • the tubing extends from the measurement probe in two directions. One end ofthe tubing is connected to a syringe pump while the other end was immersed in the fluidic test sample to be analyzed.
  • the syringe pump provided negative pressure that was applied to pull the test sample through the tube and over the detection region.
  • the syringe pump aspirates fluid at a rate of ⁇ 0.05 mL/min. Further preferred is the introduction of air gaps between two test samples to prevent mixing.
  • Fig. 7 illustrates a flow cell 760 for use with the waveguide magic-t detector shown in Fig. 4.
  • the flow cell 760 is sized to fit into the waveguide aperture 754 located at the load ports and is constructed from acrylic ([poly]methylmethacrylate) in one embodiment.
  • the flow cell 760 includes a sample chamber 762 (holding 25 ⁇ l in one embodiment) and inlet/outlet needles 764, which are UV epoxied to the ends ofthe chamber 762.
  • the diameter of needles 764 is chosen to insert securely within a section of tubing (0.020" ID PTFE tube in one embodiment) which supplies the sample.
  • Fig. 8 illustrates a temperature controlled bioassay test set 800 in accordance with one embodiment ofthe invention.
  • the set-up 800 includes a temperature control system 810, a temperature-controlled chamber 850, and a bioassay assembly 860.
  • the temperature control system 810 includes a temperature controller 811, a Resistance Temperature Detector (RTD) 812, and a fan and heating assembly 816.
  • the temperature controller 811 includes a panel for entering in a desired RTD temperature and a readout displaying the current RTD temperature.
  • the RTD 812 is connected to the input port ofthe controller 811 and is located inside the chamber 850 to monitor the interior temperature.
  • the fan and heating assembly 816 is connected to the controller's output port and used to heat or cool the chamber 850 responsive to the desired input temperature.
  • the temperature controller 811 is model no. CN76000 (Omega Engineering, Inc., Stamford, CT.) and the heating and cooling assembly 816 is model no. 18TP-1-10 (Payne Engineering, Scott Depot, WV.).
  • the temperature-controlled chamber 850 includes air intake/exhaust nozzles 851, an airflow diverter 852, support shelf 853 and support posts 854.
  • the air intake nozzle 851 is physically separated from the fan and heating assembly 816 by a gap in order to provide the chamber 850 mechanical isolation from vibrations created by the fan and heating assembly 816.
  • the airflow diverter 852 functions to redirect and circulate the incoming airflow through the chamber 850.
  • a support shelf 853 configured to support the bioassay assembly 860 is elevated by support posts 854.
  • the outer walls ofthe chamber 850, air nozzles 851, flow diverter 852, and support posts 854 are constructed from Acrylic and the chamber measures approximate 10" deep, 11.5" high and 7" wide.
  • the support shelf is fabricated from aluminum in one embodiment.
  • the bioassay assembly includes a first coaxial section 861, a second coaxial section 862, a flow cell 863, feed tube sections 864, a cap plate 865, a tuning assembly 866, and a coaxial cable 867.
  • the first coaxial section 861 includes an open-ended cross section over which a flow cell 863 is positioned.
  • the flow cell 863 is preferably constructed from a material that is substantially transparent (i. e. has low signal loss) to the applied test signal.
  • Feed tubes 864 (PTFE in one embodiment) are connected to the flow cell 863 and configured supply the sample thereto.
  • the cap plate 865 serves to retain the flow tube sections 864 connected to the flow cell 863 and to align the flow cell 863 over the open-ended portion ofthe first coaxial section 861.
  • the cap plate 865 may include a center bore for accepting a small container such as an open well.
  • the length ofthe first coaxial section 861 is selected to be approximately one-half of one wavelength ( ⁇ /2) long at the desired resonant frequency.
  • the tuning assembly 866 includes a bracket 866a which has a hollow gap region formed between the first and second coaxial sections 861 and 862.
  • the tuning assembly 866 is operable to adjustably move the second coaxial section 862 into and out ofthe hollow region within bracket 866a.
  • the second coaxial section 862 is connected to the coaxial cable 867, which is in turn connected to the measurement system, a network analyzer in one embodiment ofthe present invention.
  • the apparatuses and sub-assemblies described herein can used to provide information about numerous properties of a test sample, such as the detection and identification of molecular binding events, analyte concentrations, changes in dielectric properties ofthe bulk test sample, classification of detected binding events, and the like.
  • Preferred methods involve detection of molecular events, and the precise temperature controls described here greatly improve the reliability of such measurements.
  • an apparatus ofthe invention can be used for other purposes as well, as the accuracy of permittivity measurements is increased by the methods and apparatuses described herein, regardless of their intended use. Based upon the described methods and structures, modifications and additional uses will be apparent to those skilled in the art.
  • the present invention can be used in methods that identify substructures or binding events involving analytes, for example proteins.
  • the signal responses of a large number of known proteins can be determined and stored.
  • the dielectric properties ofthe system can be measured and the dielectric properties ofthe signal used to identify the protein's properties. Because each protein's fingerprint response is stored, the unknown response can be compared with the stored responses and pattern recognition can be used to identify the unknown protein.
  • the invention can be used in a parallel assay format.
  • the device in such a format will have multiple addressable channels, each of which can be interrogated separately.
  • responses at each site will be measured and characterized.
  • a device of this type can be used to measure and/or identify the presence of specific nucleic acid sequences in a test sample by attaching a unique nucleic sequence as the antiligand to the detection region or a part thereof.
  • complementary sequences will bind to appropriate sites. The response at each site will indicate whether a sequence has bound.
  • Such measurement will also indicate whether the bound sequence is a perfect match with the antiligand sequence or if there are one or multiple mismatches. See, for example, U.S. Application Serial No. 09/365,581 (from the laboratories ofthe present inventors), which describes this method in detail.
  • This embodiment can also be used to identify proteins and classes of proteins, by analyzing signals obtained from a particular sample and comparing that signal to signals obtained from a collection of known proteins.
  • the present invention can be used as part of a technique that generates a standard curve or titration curve that would be used subsequently to determine the unknown concentration of a particular analyte or ligand binding curve.
  • an antibody could be attached to the detection region.
  • the device could be exposed to several different concentrations ofthe analyte and the response for each concentration measured.
  • Such a curve is also known to those skilled in the art as a dose-response curve.
  • An unknown test sample can be exposed to the device and the response measured. Its response can be compared with the standard curve to determine the concentration ofthe analyte in the unknown test sample.
  • binding curves of different ligands can be compared to determine which of several different ligands has the highest (or lowest) affinity constant for binding to a particular protein or other molecule.
  • this invention can be used with embodiments that calibrate for losses due to aging and other stability issues.
  • one can measure the amount of active antibody in a test sample. The signal response is compared to standard signals for samples of known activity in order to determine the activity ofthe unknown.
  • the present invention enables the detection ofthe presence of a molecular structure or of molecular binding events in the detection region ofthe detection system.
  • Detectable binding events include primary, secondary, and higher- order binding events. For instance, mixing of two test solutions can lead to binding between ligand/antiligand pairs, or to simple mixing without binding if the two components have no affinity for each other.
  • a solution is provided which contains a test molecule or molecular structure.
  • a test signal is propagated along the signal path and coupled to the sample. Alternatively, the test signal can be launched during or shortly after a mixing operation in order to observe in real time the signal response occurring as a result of binding events. The test signal is recovered, the response of which indicates detection ofthe analyte, substructure, or binding event.
  • the dielectric property of a test sample induce numerous signal responses, each of which can be indicative of molecular binding (with appropriate signal analysis). For instance, the dispersive properties ofthe test sample can vary dramatically over frequency. In this instance, the test signal response will exhibit large changes in the amplitude and/or phase response over frequency when molecular events occur in the detection region, thereby providing a means for detecting molecular binding events or other time dependent events after the mixing of test samples.
  • the dielectric relaxation properties of the test sample in the detection region will vary as a function of pulse period ofthe input signal.
  • the test signal response will indicate a change in the amount of power absorbed, or change in some other parameter ofthe test signal like phase or amplitude, at or near a particular pulse period.
  • binding events can be detected.
  • Other quantities such characteristic impedances, propagation speed, amplitude, phase, dispersion, loss, permittivity, susceptibility, frequency, and dielectric constant are also possible indicators of molecular presence or binding events.
  • Important information regarding molecular properties can also be determined by measuring signals, such as these, during changes in the environment ofthe molecular structure being detected (such as changes in pH or ionic strength).
  • the above-described method can be used to detect the primary binding of an antiligand and ligand. Similarly, the process can also be used to detect secondary binding of a ligand to an antiligand.
  • the method not limited to detection of primary or secondary binding events occurring along the signal path. Indeed, tertiary, and higher-order binding events occurring either along the signal path or suspended in solution can be detected using this method.
  • a primary binding event is detected and the signal response measured, as described herein.
  • the primary binding event signal response is stored and used as a baseline response.
  • a second molecular solution is added to the assay device. Detection steps are repeated to obtain a second signal response.
  • the second signal response and the baseline response are compared. Little or no change indicates that the two signal responses are very close, indicating that the structural and dielectric properties ofthe test sample have not been altered by the addition ofthe molecules within the new solution. In this case, secondary binding has not occurred to a significant degree. If the comparison results in a change outside of a predetermined range, the structure and/or dielectric properties ofthe test sample have been altered, thereby indicating secondary binding events.
  • Quantities which can be used to indicate secondary binding events will parallel the aforementioned quantities, e.g., amplitude, phase, frequency, dispersion, loss, permittivity, susceptibility, impedance, propagation speed, dielectric constant as well as other factors. Tertiary or high-order binding events can be detected using this approach.
  • an alternative method of detecting secondary or higher order binding events does not required prior knowledge ofthe specific primary binding event.
  • the assay device is designed in the assay development stage to operate with known parameters, so that whenever a pre-defined change in one of these parameters is detected, for example at the point-of-use, the binding event or events are then known to have occurred.
  • the pre-measurement of a primary binding event is not necessary, as the initial characterization has already been done either at the time of fabrication or at the time of design.
  • Secondary binding events can also be achieved by detecting changes in the structure ofthe primary molecules structure. When a molecule becomes bound, it undergoes conformational and other changes in its molecular structure relative to its unbound state. These changes affect the primary binding molecule's dielectric properties as well as inducing changes in the surrounding solution, the variation of which can be detected as described above.
  • Quantities that can be monitored to indicate a change in the dielectric properties ofthe primary bound molecule include the aforementioned quantities, e.g., amplitude, phase, frequency, dispersion, loss, permittivity, susceptibility, impedance, propagation speed, and dielectric constant, as well as other factors.
  • the detection systems described herein can also be used to measure the dielectric changes ofthe test sample as a result changes in temperature, pH, ionic strength and the like.
  • the process closely parallels the disclosed method for identifying binding events, the exception being that the method allows for the detection and quantitation of changes in dielectric properties ofthe test sample without reference to a binding event.
  • the process begins when a solution having an initial dielectric property is added to the detector assembly.
  • the signal response is measured and recorded, as previously described.
  • a second measurement is made and a second signal response is recorded.
  • a comparison is then made between the first and second signals to determine whether the two signals correlate within a predefined range. If so, the properties ofthe solution are deemed to not have undergone any dielectric changes.
  • the signal responses do not correlate within a predefined range, at least dielectric property ofthe solution will have undergone a change.
  • the change in dielectric properties can be quantitated.
  • the second signal is stored and correlated to a known signal response. The closest correlated response will identify the dielectric property ofthe solution and the first signal response can be correlated to the initial value ofthe dielectric property, the difference of which can be used to determine the amount by which the identified dielectric property has been altered.
  • the comparison can be performed using one or more data points to determine the correlation between one or more stored responses, and can involve the use of pattern recognition software or similar means to determine the correlation.
  • the process can be used to identify an individual structure/substructure, as well as primary, secondary or higher-order bound molecular structures.
  • the process proceeds as shown in section D immediately above, except that a number of molecular sub-structures are measured and their responses stored. Each stored signal response will correspond to one or more sub-structures. The process continues until a sufficient number or structures have been detected and characterized to identify the unknown compound. Once a sufficient number of correlations occur, it is then possible to classify the unknown molecular structure.
  • a detector assembly can be provided which has multiple addressable parallel channels, each of which has a antiligand for a specific ligand sub-structure bound in the detection region.
  • the presence of particular sub-structures is detected by the binding of each to its respective antiligand and subsequent characterization. In one embodiment, this step is performed as described above, but other variations can be carried out as well.
  • each ofthe binding events is then characterized by identification of qualities such as affinity, kinetics, and spectral response. A correlation is then made between the known and unknown responses.
  • the ligand is identified as the ligand corresponding to the known response. If the sub-structures exhibit both correlated and uncorrelated responses, the correlated responses can be used to construct a more general classification ofthe unknown ligand. This process can be used to identify any molecular structure, for example proteins, which occur within the same class or have re-occurring structural homologies.
  • Specific binding can be distinguished from non-specific binding by the spectral fingerprint ofthe binding events.
  • any two binding events such as the binding of one molecular structure on separate occasions with two similar but different molecular partners, can generally be distinguished by the spectral fingerprints ofthe two binding events.
  • a given binding event of interest such as antibody binding to antigen
  • a broad spectral study is then carried out to see when in the spectrum the strongest responses are found.
  • the assay is then repeated in the solutions typically found in the dedicated applications, for example whole blood, to determine what effects non-specific binding has on the response.
  • any given protein can be characterized by determining both the presence and absence of certain sub-structures as well as the dielectric properties of the protein itself. Further refinements to this classification strategy can include looking at temperature, pH, ionic strength, as well as other environmental effects on the above-mentioned properties.
  • Nucleic acids can also be characterized by following a similar paradigm.
  • a given gene can be known to have a certain base pair sequence. Often times in nature there will be small variations in this sequence.
  • the gene which codes for a chloride ion transport channel in many cell membranes there are common single base-pair mutations, or changes. Such changes lead to a disease called cystic fibrosis in humans.
  • cystic fibrosis in humans.
  • Such variations are often called polymorphisms, and such polymorphisms are currently detected by forming complementary strands for each ofthe known polymorphisms.
  • any given gene can take the form of any one of hundreds or even thousands of polymorphisms, it is often an arduous task to generate complementary strands for each polymorphism.
  • non-complementary binding or hybridization can be detected and distinguished by measuring many ofthe same physical properties as were described in the previous paragraph:
  • the dielectric properties ofthe hybridization event can be characterized and correlated to known data, thereby determining the type of hybridization which has occurred — either complete or incomplete.
  • hundreds of different polymorphisms (as ligands) can be detected by the characterization ofthe binding event.
  • a molecule that binds and yields a similar dielectric response is then known to have a similar effect on the receptor as the known agonist, and therefore will have a much higher probability of being an agonist.
  • This paradigm can be used to characterize virtually any type of target-receptor binding event of interest, and represents a significant improvement over current detection strategies which determine only if a binding event has occurred or not.
  • Those of skill in the art will readily appreciate that there are many other classes of binding events in which the present invention can be applied.
  • sub-structures which can be used in the above method include: Protein secondary and tertiary structures, such as alpha-helices, beta- sheets, triple helices, domains, barrel structures, beta-turns, and various symmetry groups found in quaternary structures such as C symmetry, C 3 symmetry, C 4 symmetry, D 2 symmetry, cubic symmetry, and icosahedral symmetry.
  • Protein secondary and tertiary structures such as alpha-helices, beta- sheets, triple helices, domains, barrel structures, beta-turns, and various symmetry groups found in quaternary structures such as C symmetry, C 3 symmetry, C 4 symmetry, D 2 symmetry, cubic symmetry, and icosahedral symmetry.
  • Sub-structures of nucleic acids which can be analyzed include: sequence homologies and sequence polymorphisms, A, B and Z forms of DNA, single and double strand forms, supercoiling forms, anticodon loops, D loops, and T ⁇ C loops in tRNA, as well as different classes of tRNA molecules.
  • sequence homologies and sequence polymorphisms A, B and Z forms of DNA, single and double strand forms, supercoiling forms, anticodon loops, D loops, and T ⁇ C loops in tRNA, as well as different classes of tRNA molecules.
  • the detector assemblies described herein can also be used to quantitate the concentrations of analytes.
  • initially anti-ligands are chosen having the appropriate binding properties, such as binding affinity or kinetics, for the measured analyte. These properties are selected such that the anti-ligand's equilibrium constant is near the center of its linear operating region. For applications where the range of concentration is too wide for the use of a single antiligand, several anti-ligands can be used with differing affinities and/or linear operating ranges, thereby yielding a value for the concentration over a much wider range.
  • the anti-ligands are added or attached to the detector assembly or chip and the device is connected to the measurement system.
  • a decision is then made as to whether the response requires characterization for maximum specificity. If so, a spectral analysis is performed in which the frequency or frequencies where analyte binding has maximal effect on the signal are determined, the regions where the non-specific binding has maximal effect are determined, and the response due to analyte binding is determined. If characterization is not required, or if so, after its completion, the device is calibrated. This step is performed in one embodiment by supplying a known concentration of ligands to the detector assembly and measuring the resulting response.
  • a test sample can be chosen with a different concentration, and the response against this concentration can be measured. Subsequently, an extrapolation algorithm is generated by recording the calibration points from the foregoing response. Next, a test sample of unknown analyte concentration is measured. This step is accomplished in one embodiment by supplying the unknown test sample to the detector assembly, correlating the response to the titration algorithm, and determining therefrom the analyte concentration.
  • a detector assembly In the event that a given detector assembly is either pre- calibrated, or calibrated by design, the only step required is to mix the binding pairs and measure the response.
  • a detector assembly can be realized in many different ways. For example, some circuit parameter, such as impedance or characteristic frequency of a resonant circuit, can be designed to change in a pre-determined way when the binding event occurs, and the amount by which the parameter changes can further be designed to have a dose-response. Thus, a measurement ofthe circuit parameter will, when analyzed via a suitable algorithm, immediately yield a quantitative value for the concentration of a given analyte or ligand.
  • the detector assembly possess a self-diagnostic capability and thus a point-of-use quality control and assurance.
  • a particular antiligand (primary binding species) will act as an antiligand for some ligand (the secondarily binding species) of interest in the solution.
  • the primary binding species can be attached at the point of fabrication, and the secondary binding species can be attached at the point-of-use.
  • variations in fabrication especially the attachment ofthe primary species — will cause variations in the ability ofthe device to bind its specific ligand.
  • the amount of ligand bound will be in direct proportion to the amount of antiligand bound, thus a ratiometic measurement ofthe two is possible.
  • a molecular binding surface is formed along the signal path by binding the appropriate antibody at various concentrations and characterizing the resulting response for each of these concentrations, yielding some value "x" for each concentration.
  • a similar titration curve is generated for the ligand by measuring the antibody/ligand binding response for several different concentrations of ligand, and a ligand titration curve is pre-determined.
  • a scale factor A is generated by taking the ratio of responses of antibody binding to ligand binding.
  • the uncalibrated assay is then first probed to determine the amount of bound antibody "x" and the scale factor "y” resulting therefrom.
  • the ligand is then applied to the assay and the response is measured, and the response and predetermined titration curve are scaled by the scale factor "y" to determine unknown concentration.
  • the process can also be modified to allow quantitating the amount of binding in the solution.
  • the binding surface ofthe detector assembly includes antiligands having a predefined affinity and ligand specificity.
  • the solution is subsequently applied to the device, and a response is measured.
  • the signal response will be proportional to the amount ofthe ligand that has bound.
  • a titration of any given ligand can be carried out by choosing an antiligand with an appropriate linear operating range — the range in which the equilibrium constant is within a couple of log units ofthe desired range of concentrations to be detected.
  • the same ratiometic analysis as described above can be applied to yield a robust and precise quantitative assay with internal controls and calibration necessary to insure reliability.
  • Fig. 9A illustrates a simplified block diagram of a computer system 910 operable to execute a software program designed to perform each ofthe described methods.
  • the computer system 900 includes a monitor 914, screen 912, cabinet 918, and keyboard 934.
  • a mouse (not shown), light pen, or other I/O interface, such as virtual reality interfaces can also be included for providing I/O commands.
  • Cabinet 918 houses a CD-ROM drive 916, a hard drive (not shown) or other storage data mediums which can be utilized to store and retrieve digital data and software programs incorporating the present method, and the like.
  • CD- ROM 916 is shown as the removable media, other removable tangible media including floppy disks, tape, and flash memory can be utilized.
  • Cabinet 918 also houses familiar computer components (not shown) such as a processor, memory, and the like.
  • Fig. 9B illustrates the internal architecture ofthe computer system 910.
  • the computer system 910 includes monitor 914 which optionally is interactive with the I/O controller 924.
  • Computer system 910 further includes subsystems such as system memory 926, central processor 928, speaker 930, removable disk 932, keyboard 934, fixed disk 936, and network interface 938.
  • Other computer systems suitable for use with the described method can include additional or fewer subsystems.
  • another computer system could include more than one processor 928 (i.e., a multi-processor system) for processing the digital data.
  • Arrows such as 940 represent the system bus architecture of computer system 910. However, these arrows 940 are illustrative of any interconnection scheme serving to link the subsystems.
  • a local bus could be utilized to connect the central processor 928 to the system memory 926.
  • Computer system 910 shown in Fig. 9A is but an example of a computer system suitable for use with the present invention. Other configurations of subsystems suitable for use with the present invention will be readily apparent to of skill in the art. VII. Experiment
  • Bovine serum albumin (BSA) A2153, Human Serum albumin (HSA) A1653, lysozyme from chicken egg white L6876, myoglobin from horse skeletal muscle M0630, ovalbumin A5503, and ribonuclease A (RNase A) from bovine pancreas R5503 were purchased from Sigma (St. Louis, MO).
  • the sodium phosphate buffer 25 mM phosphate, 0.05% v/v NP-40 surfactant, pH 7.7 was freshly prepared in 18 mega Ohm water.
  • the instrumental setup included an Agilent 8714ET RF network analyzer, a Dell personal computer running custom Labview software for recording data, a Newport isolation table with a mounted coaxial resonating detector, a Pico motor from New Focus, a custom fluidic flowcell mounted to the detector (the flowcell is made of poly(etherimide) and has a 0.030 inch internal diameter channel with a thin 0.007 inch bottom) and a temperature controlling apparatus, which housed the detector.
  • the temperature controlling apparatus and the coaxial resonator are described above.
  • Phosphate buffer was loaded into position in the fluidic flowcell by aspiration (150 ⁇ L volume was used) and the resonating detector was tuned to the resonant frequncy point by adjustment ofthe resonators gap size using the Pico motor. Data was collected at 401 point resolution using a system bandwidth of 15 Hz and a power level of 0 dBm. Two spans 1 MHz and 200 KHz are recorded for purposes of calculating the permitivity ofthe test samples. The experiment was performed once while using temperature controlling apparatus (with Temperature Control) and a second time after removing the temperature controlling apparatus (without Temperature Control).
  • the signal for each test sample was recorded 1 minute after it was position in the fluidic flowcell ofthe apparatus. The 1 minute waiting period was determined to be sufficient to allow for equilibration ofthe sample to the temperature ofthe detecting apparatus.
  • the signal was measured for six protein solutions each of which flanked by a measurement ofthe phosphate buffer, which served as the reference for this experiment. The series of measurements were repeated four times in series. The same buffer and protein solutions were used for the experiment with and without temperature control. Included in each experiment but not shown are the signals for two calibration solutions, which are used for the purpose of calculating the permitivity ofthe test samples.

Abstract

L'invention concerne un procédé de détection d'un événement moléculaire consistant à (1) appliquer un signal de test électromagnétique à un échantillon dans lequel un événement moléculaire est détecté. Ainsi, l'échantillon interagit avec et module le signal de test en vue de produire un signal de test modulé afin de générer un signal de test modulé et (2) détecter le signal de test modulé, dans lequel l'appareil et la détection se déroulent dans un environnement à température régulée, dans lequel ledit environnement contient l'échantillon, une partie émettant des rayonnements d'un circuit de génération de signal et une partie réceptrice d'un circuit de détection de signal et dans lequel l'application et la détection se déroulent dans l'environnement à une température régulée dans une marge comprise entre ? 0,5 °C.
PCT/US2001/012694 2001-04-18 2001-04-18 Procede et appareil de detection d'evenements moleculaires au moyen de la regulation de temperature dans un environnement de detection WO2002086475A2 (fr)

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WO2003016887A2 (fr) * 2001-08-13 2003-02-27 Signature Bioscience, Inc. Procede d'analyse d'evenements cellulaires
GB2433603A (en) * 2005-12-14 2007-06-27 Agilent Technologies Inc Microwave spectroscopy probe
CN105606535A (zh) * 2016-02-19 2016-05-25 清华大学 表面等离子体共振传感芯片及细胞响应检测系统和方法
CN113906289A (zh) * 2019-03-28 2022-01-07 西澳大学 直接检测固体形成的装置

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US4327180A (en) * 1979-09-14 1982-04-27 Board Of Governors, Wayne State Univ. Method and apparatus for electromagnetic radiation of biological material
US5960160A (en) * 1992-03-27 1999-09-28 Abbott Laboratories Liquid heater assembly with a pair temperature controlled electric heating elements and a coiled tube therebetween

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US4327180A (en) * 1979-09-14 1982-04-27 Board Of Governors, Wayne State Univ. Method and apparatus for electromagnetic radiation of biological material
US5960160A (en) * 1992-03-27 1999-09-28 Abbott Laboratories Liquid heater assembly with a pair temperature controlled electric heating elements and a coiled tube therebetween

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003016887A2 (fr) * 2001-08-13 2003-02-27 Signature Bioscience, Inc. Procede d'analyse d'evenements cellulaires
WO2003016887A3 (fr) * 2001-08-13 2004-02-12 Signature Bioscience Inc Procede d'analyse d'evenements cellulaires
GB2433603A (en) * 2005-12-14 2007-06-27 Agilent Technologies Inc Microwave spectroscopy probe
US7532015B2 (en) 2005-12-14 2009-05-12 Agilent Technologies, Inc. Microwave spectroscopy probe
CN105606535A (zh) * 2016-02-19 2016-05-25 清华大学 表面等离子体共振传感芯片及细胞响应检测系统和方法
CN113906289A (zh) * 2019-03-28 2022-01-07 西澳大学 直接检测固体形成的装置

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