WO2002077605A2 - Reentrant cavity bioassay for detecting molecular or cellular events - Google Patents

Abstract

A reentrant cavity bioassay includes a reentrant cavity and a sample retaining structure. The reentrant cavity includes a reentrant post extending from a first interior surface and terminating proximate to a second interior surface, the gap between the termination of the reentrant post and the second interior surface defining a detection region. The sample retaining structure is configured to retain the sample in the detection region, wherein at least a portion of the sample retaining structure is located within the detection region.

Description

REENTRANT CAVITY BIOASSAY FOR DETECTING MOLECULAR OR CELLULAR EVENTS

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. provisional application no. 60/277,810, filed March 21, 2001.

BACKGROUND OF THE INVENTION

[0002] The present invention relates generally to the structure and function of bioassay devices, and more particularly to a reentrant cavity bioassay operable to detect solution or solid phase based molecular events in an aqueous-based sample.

[0003] Virtually every area of biological science is in need of a system to determine the ability of molecules of interest to interact with other molecules. Likewise, the ability to detect the presence and/or physical and functional properties of biological molecules and cells on a small scale is highly desirable. Such molecular interactions, as well as the detection of functional and physical properties of these biological molecules and cells in an aqueous environment are referred to here as "molecular events" and "cellular events," respectively.

[0004] The need to detect molecular and cellular events ranges from the basic science research lab, where chemical messenger pathways are being mapped out and their functions correlated to disease processes, to pre-clinical investigations, where candidate drugs are being evaluated for potential in vivo effectiveness. The need to detect physical and functional properties is also present in these research areas, such as for functional analysis of a newly discovered protein or of a genetic (or synthetic) variant of a molecule of know biological importance. Other areas that benefit from a better understanding of molecular and cellular events include pharmaceutical research, military applications, veterinary, food, and environmental applications. In many of these cases, knowledge of the ability of a particular analyte to bind a target molecule is highly useful, as is information relating to the quality of that binding (e.g., affinity and on-off rate), and other functional information about new molecules and cells, particularly when information can be obtained from a small amount of sample.

[0005] In PCT publication numbers WOL99/39190 and WO 01/27610, the applicant described methods and apparatuses operable to detect and identify molecular events within a sample by applying an electromagnetic test signal to the sample and monitoring the signal response that results. The signal response is largely influenced by the dielectric properties (e.g., permittivity) of the molecular events occurring within the sample. Because most molecular and cellular events possess unique dielectric properties, each resulting signal response is correspondingly unique. It is this unique signal response that provides the basis for the detection and identification of molecular and cellular events in a sample.

[0006] The applicants note that within the field of electromagnetics, reentrant cavities have been used as a focusing means to concentrate an electromagnetic signal within a particular region (referred to here as a "detection region"). As the applied electromagnetic signal becomes more concentrated within the detection region, changes occurring within that region can be more sensitively detected. If a similar structure could be used to focus an electromagnetic signal to the detection region of a bioassay where molecular or cellular events are occurring, the sensitivity of the bioassay could be correspondingly improved. In addition, the structure would preferably be able to detect molecular or cellular events in aqueous-based environment native to the molecular or cellular events.

SUMMARY OF THE INVENTION

[0007] The applicant now provides a reentrant cavity bioassay configured to detect molecular or cellular events occurring within an aqueous-based sample. In one embodiment, the reentrant cavity bioassay includes a reentrant cavity and a sample retaining structure. The reentrant cavity includes a reentrant post extending from a first interior surface and terminating proximate to a second interior surface, the gap between the termination of the reentrant post and the second interior surface defining a detection region. The sample retaining structure is configured to retain the sample in the detection region, wherein at least a portion of the sample retaining structure is located within the detection region.

[0008] Other advantages and aspects of the invention will be apparent when considered in view of the following drawings and description.

BRIEF DESCRIPTION OF THE DRAWINGS [0009] Fig. 1 illustrates a first embodiment of a reentrant cavity bioassay in accordance with the present invention.

Fig. 2 illustrates a second embodiment of a reentrant cavity bioassay in accordance with the present invention. Fig. 3 A illustrates a serial array of reentrant cavity bioassays in accordance with the present invention.

Fig. 3B illustrates a parallel array of reentrant cavity bioassays in accordance with the present invention.

Fig. 4 illustrates a method for detecting and/or identifying molecular events occurring within a test sample using the reentrant cavity bioassay in accordance with an embodiment of the present invention.

For simplicity in description, identical components are labeled with the same numerals in the figures of this application.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS Definitions

[0010] As used herein, the term "molecular binding event" (sometimes shortened to "binding event" or "binding") refers to the interaction of a molecule of interest with another molecule. The term "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. Together, "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."

[0011] Examples of molecular 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.

[0012] 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).

[0013] Molecular structure is typically detected by comparing the signal obtained from a molecule of unknown structure and/or function to the signal obtained from a molecule of known structure and/or function. Molecular binding events are typically detected by comparing the signal obtained from a sample containing one of the potential binding partners (or the signals from two individual samples, each containing one of the potential binding partners) to the signal obtained from a sample containing both potential binding partners. Together, the detection of a "molecular binding event" or "molecular structure" is often referred to as "molecular detection."

[0014] The term "cellular event" refers in a similar manner to reactions and structural rearrangements occurring as a result of the activity of a living cell (which includes cell death). Examples of cellular events include opening and closing of ion channels, leakage of cell contents, passage of material across a membrane (whether by passive or active transport), activation and inactivation of cellular processes, as well as all other functions of living cells. Cellular events are commonly detected by comparing modulated signals obtained from two cells (or collection of cells) that differ in some fashion, for example by being in different environments (e.g., the effect of heat or an added cell stimulant) or that have different genetic structures (e.g., a normal versus a mutated or genetically modified cell). Morpholic changes are also cellular events. The same bioassay systems can be used for molecular and cellular events, differing only in the biological needs of the cells versus the molecules being tested. Accordingly, this specification often refers simply to molecular events (the more difficult of the two measurements under most circumstances) for simplicity, in order to avoid the awkwardness of continually referring to "molecular and/or cellular" events, detection, sample handling, etc., when referring to an apparatus that can be used to detect either molecular events or cellular events. When appropriate for discussion of a particular event, the event will be described as, for example, a cellular event, a molecular binding event, or a molecular structure determination. When used in a claim, "molecular event" does not include "cellular event" and both are specified if appropriate.

[0015] 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. Here 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. It will be recognized that local physiological conditions vary from place to place within cells and organisms and that artificial conditions designed to mimic such conditions can also vary considerably. For example, a binding event may occur between a protein and a ligand in a subcellular compartment in the presence of helper proteins and small molecules that affect binding. Such conditions may differ greatly from the physiological conditions in serum, exemplified by the artificial medium referred to as "normal phosphate buffered saline" or PBS. Preferred conditions of the invention will typically be aqueous solutions at a minimum, although some amounts of organic solvents, such as DMSO, may be present to assist solubility of some components being tested. An "aqueous solution" contains at least 50 wt.% water, preferably at least 80 wt.% water, more preferably at least 90 wt.% water, even more preferably at least 95 wt.% water. Other conditions, such as osmolarity, pH, temperature, and pressure, can and will vary considerably in order to mimic local conditions of the intracellular environment in which, for example, a binding event is taking place. The natural conditions in, for example, the cytosol of a cell and a lysosome of that cell, are quite different, and different artificial media would be used to mimic those conditions. Examples of artificial conditions designed to mimic natural ones for the study of various biological events and structures are replete in the literature. Many such artificial media are sold commercially, as exemplified by various scientific supply catalogues, such as the 2000/2001 issue of the Calbiochem General Catalogue, pages 81-82, which lists 60 commercially available buffers with pH values ranging from 3J3 to 9.24 typically used in biological investigations. Also see general references on the preparation of typical media, such as chapter 7 ("The Culture Environment") of Culture of Animal Cells: A Manual of Basic Techniques, Third Edition, R. Ian Freshney, Wiley-Liss, New York (1994).

[0016] As used herein, the term "test sample" refers to the material being investigated (the analyte) and the medium/buffer in which the analyte is found. The medium or buffer can included solid, liquid or gaseous phase materials; the principal component of most physiological media/buffers is water. Solid phase media can be comprised of naturally occurring or synthetic molecules including carbohydrates, proteins, oligonucleotides, SiO , GaAs, Au, or alternatively, any organic polymeric material, such as Nylon^®, Rayon^®, Dacryon^®, polypropylene, Teflon^®, neoprene, delrin or the like. Liquid phase media include those containing an aqueous, organic or other primary components, gels, gases, and emulsions. Exemplary media include celluloses, dextran derivatives, aqueous solution of d- PBS, Tris, deionized water, blood, cerebrospinal fluid, urine, saliva, water, and organic solvents.

[0017] As used herein, the term "electromagnetically coupled" refers to the transfer of electromagnetic energy between two objects, e.g., the reentrant post and molecular events occurring within the test sample. The two objects can be electromagnetically coupled when the objects are in direct contact, (e.g., molecular events occurring along the surface of a reentrant post), or when the objects are physically separated from each other (e.g., molecular events occurring within a sample flowing through a flow tube, the flow tube positioned within the detection region). As a modification, 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).

[0018] As used herein, the term "test signal" refers to an ac time-varying signal. In specific embodiments, the test signal is preferably at or above 1 MHz (lxl 0⁶ Hz) and at or below 1000 GHz (lxlO¹² Hz), such as 10 MHz, 20 MHz, 45 MHz, 100 MHz, 500 MHz, 1 GHz (lxlO⁹ Hz), 2 GHz, 5 GHz, 7.5 GHz, 10 GHz, 12 GHz, 15 GHz, 18 GHz, 20 GHz, 25 GHz, 30 GHz, 44 GHz, 60 GHz, 110 GHz, 200 GHz, 500 GHz, or 1000 GHz and range anywhere therebetween. A preferred region is from 10 MHz to 110 GHz, a more particularly from 45 MHz to 20 GHz. "Test signal" can refer to a range of frequencies rather than a single frequency, and such a range can be selected over any terminal frequencies, including frequency ranges bounded by the specific frequencies named in this paragraph. When referring to the detected range (or multiple) of modulated signals obtained after a range of frequencies has been coupled to a test sample, the term "spectrum" is sometimes used. An "incident test signal" is a test signal that originates from the signal source and is destined for the detection region for interaction with the sample. A "modulated test signal" is a test signal that has previously interacted with the test sample and is destined for a signal detector that can recover the modulation imparted by the signal interaction with the sample.

Reentrant Cavity Bioassay

[0019] Fig. 1 illustrates one embodiment of a reentrant cavity bioassay 100 in accordance with the present invention. The bioassay 100 includes the reentrant cavity assembly 120, a flow tube 130, and a probe assembly 140. This configuration is useful when detecting or identifying molecular events in "solution phase," i.e., when these events are moving or suspended in the test sample.

[0020] The reentrant cavity assembly 120 is configured to provide a high Q resonance at one or more frequencies. The cavity 120 includes a top member 122, a cavity body 123, a reentrant post 124, a probe access port 125 and a flow tube access port 126 located on the cavity floor 127. The top member 122 is removably attached to the cavity body 123, using, e.g., screws threadingly engaged into the cavity body 123. In the illustrated embodiment, the reentrant post 124 is a hollow tube having a top aperture 121 and a bottom aperture 128 located above the cavity floor 127. In another embodiment of the invention (further described below), the reentrant post 124 is solid. In a specific embodiment, the top member 120, cavity body 123, and reentrant post 124 are constructed from aluminum and the interior cavity has a radius of 25.4 mm and is 25.4 mm tall. The interior of the cavity may be gold-plated to increase the cavity's q-factor and enhance resonant frequency stability. The reentrant post 124 has an outer radius of 6.35 mm and extends down 24.28 mm from the top member 122, leaving a 1.18 mm gap between the post bottom 128 and the cavity floor 127. In this configuration the reentrant cavity exhibits a fundamental resonance at 1 JO GHz.

[0021] The detection region defines a region through which a large number of electric field lines will extend when a test signal is electromagnetically coupled into the cavity 120, and is generally formed between the closest proximate surfaces of the reentrant post and cavity interior. In the illustrated embodiment, the detection region 129 is formed longitudinally between the post bottom and the cavity floor 127 and measures approximately 150 μl (150 xl0^"9m³). The detection region 129 is not limited to any particular volume, but will typically measure smaller than 1 ml (1 x 10^"6 m³). Very small detection region volumes, such as 1 μl (1 x 10^"9 m³), 1 nl (1 x 10^"12 m³), or 1 pi (1 x 10^"15 m³) (or ranges between these volumes) may also be used to test binding ability of potential pharmaceutical compounds due to the small size and expense of the available samples. In an alternative embodiment, the detection region 129 can be formed circumferentially along the cavity sidewall, between it and the side of the reentrant post 124.

[0022] Molecular event detection and identification preferably occurs at the fundamental or higher order resonant frequencies of the cavity 120, as operation at these frequencies will provide the highest sensitivity within the detection region 129. The dielectric properties of molecular events introduced into the detection region 129 will operate to shift the cavity resonant frequency above or below its resonant frequency. Because many of molecular events have distinctive dielectric properties (e.g., permittivity), each will produce a unique frequency shift, thereby enabling their identification.

[0023] The sample is retained within the detection region 129 using a sample retaining structure, an embodiment of which is a flow tube 130. The flow tube 130 is fed through the top aperture 121 through the hollow body of the reentrant post 124, through the bottom aperture 128, and out of the aperture 126 located on the cavity floor 127. The flow tube 130 is preferably constructed of a material that exhibits low dielectric loss and high signal transparency at the desired test frequency, and which further preferably has a smooth, resilient surface morphology that inhibits analyte formation along the inner surface. In a specific embodiment, the flow tube is PTFE tube having an outer diameter of 1.07 mm and an inner diameter of 0.56 mm. In the aforementioned embodiment in which the detection region 129 is formed circumferentially around the cavity side wall, the flow tube is wound around the reentrant post.

[0024] In another embodiment, the sample retaining structure consists of an unenclosed substrate (e.g., a glass slide) positioned within the detection region (through a slot in the cavity's side wall, not shown). This arrangement is particularly useful for investigating cellular events occurring within a solution dispensed onto a slide. Other enclosed and unenclosed sample retaining structures (e.g., flow cells, microfluidic channels, capillaries, etc.) can be used in alternative embodiments under the present invention.

[0025] The probe assembly 140 is configured to electromagnetically couple, at one or more frequencies, an incident test signal into and recover a modulated signal from the cavity assembly 120. In one embodiment, the probe assembly 140 includes a first coaxial section 142 and a probe tip 144 extending into the interior of the reentrant cavity 123 via the probe access port 125. An ultra fine adjusting motor 145 is preferably used to move the probe tip 144 into and out of the cavity 123. The first coaxial section 142 is connected to a coaxial cable 146, that is in turn connected to the measurement instrument, a network analyzer in one embodiment. In a specific embodiment, the first coaxial section 142 consists of a 75 mm of coaxial cable and the probe tip 144 measures approximately 100.0 mm long. Various tip configurations may be used, for example, a monopole, a dipole, ground loop, spiral, or other antenna design capable of transmitting/receiving signals into/from the cavity assembly 120. The motorized assembly 145 consists of a picomotor assembly manufactured by New Focus, Inc. (Santa Clara, CA). The first coaxial section 142 consists of 5.0 mm O.D. cable manufactured by RF Labs (Stuart, FL.). Other probes structures may be used in alternative embodiments of the present invention.

[0026] In a modified embodiment of Fig 1 , the flow tube 130 enters through an aperture (not shown) in the cavity wall, extends along the cavity floor 127 below the post bottom 128 and exits through a second aperture (not shown) in the cavity side wall. In this embodiment, the reentrant post 124 may be solid. In another modification, the sample retaining structure consists of a flow cell located between the post bottom 128 and the cavity floor 127 which is supplied via a flow tube as described above. In still another modification, a second probe extends into the cavity body 123 and the first and second probed can be used to make a two-port (e.g., a S₂₁ s-parameter) measurement. In a fourth possible modification, the reentrant post 128 may have a varying radius along the vertical length and/or a tapered tip. These examples represent only a few of the possible variations of the present invention. Other modifications will be obvious in light of teachings in the arts of bioassay device architecture and high frequency circuit design.

[0027] Fig. 2 illustrates a second reentrant cavity bioassay 200 in accordance with the present invention. The bioassay 200 includes a reentrant cavity assembly 220, a sample vial 230, and a probe 240. This configuration is useful when detecting or identifying molecular events in "solid phase," i.e., when molecular events are immobilized on a surface.

[0028] Similar in construction to the reentrant cavity assembly 120 shown in Fig. 1 , the reentrant cavity assembly 220 includes a top member 222 and a cavity body 223. The top member 222 includes a sample aperture 221, a probe aperture 225, and a hollow reentrant post 224 that extends vertically into the cavity body 223 terminating proximate to the cavity floor 227. The region between the post bottom and cavity floor 227 represents the detection region 229 through which a large number of fields pass when a signal is electromagnetically coupled into the cavity 220.

[0029] The diameter of the top aperture 221 and inner diameter of the reentrant post 224 is of sufficient size to permit passage of the sample retaining structure, a sample vial 230 in the illustrated embodiment. The sample vial 230 is preferably constructed from a material that exhibits low dielectric loss, high signal transparency at the desired operational frequency and that has a surface morphology that is conducive to facilitate molecular binding thereto. Suitable materials include glass, PTFE, and the like. Further preferably, the bottom thickness of the sample vial is minimal, on the order of .1 to 10 mm. In a specific embodiment, the sample vial is an open well container of the type typically used in biological, chemical, or pharmaceutical applications. In another embodiment, the open well is sealed to minimize sample evaporation.

[0030] The sample vial 230 further includes a molecular binding region 232 formed along the bottom inner surface of the sample vial 230. The molecular binding region 232 is located within the detection region 229 and includes antiligands 232a that have a strong affinity to bind ligands 252 that are suspected of being contained within the supplied test sample 250. The formation of the ligand/anti-ligand complex within the high field density region 229 operates to shift the resonant frequency of the cavity 220, thereby enabling detection and identification of the binding events within the sample 250. The fabrication and makeup of the molecular binding region 232 can be diverse and is described in greater detail in the applicant's commonly owned, co-pending patent application entitled: "System and Methods for Detecting Molecular Binding Events, serial no. 09/365,578, filed August 2, 1999.

[0031] The probe 240 is operable to electromagnetically couple, at one or more frequencies, an incident test signal into, and recover the modulated signal from the cavity assembly 220. The probe 240 includes a coaxial cable 242 and a probe tip 244 consisting of the center conductor of the coaxial cable 242. In a specific embodiment, the probe tip 244 forms a loop and terminates onto the outer ground shield of the coaxial cable 242. The probe 240 extends into the cavity body 223 through the probe aperture 225 of the top member 222 in the illustrated embodiment, although in an alternative embodiment, the probe 240 may enter the cavity at other locations.

[0032] The probe assembly is rotatably attached to the top member 222 such that it can freely rotate relative to the cavity assembly 220. Tuning is performed by rotating the probe in either direction relative to the cavity assembly. In a specific embodiment, the cavity assembly 220 is constructed from aluminum and has interior dimensions of 50.8 mm diameter by 25.4 mm tall. The reentrant post 224 has an outer diameter of 6.35 mm and an inner diameter of 1.27 mm and extends 24.28 mm into the cavity body 223, terminating 1.12 mm from the cavity floor 227. The sample aperture 221 on the top member 222 is 1.27 mm in diameter and the sample vial 230 has an outer diameter of 1.27 mm and an inner diameter of 6.35 mm. The probe 240 is constructed from 6.35 mm semi-rigid type RG 401 coaxial cable and the probe tip 244 is approximately 125 mm long.

[0033] The bioassay 200 of Fig. 2 may also be modified in a variety of ways to provide solid phase detection and identification of immobilized molecular events. For example, the sample retaining structure may consist of a flow tube operable to transport beads or other structures that contain immobilized antiligands 232a. The antiligands 232a are selected to bind to a particular ligand 252 potentially contained within the sample 250. The beads can be mixed with the test sample either prior to transportation to the detection region 229, or within the detection region 229 in order to study the kinetics of the binding interaction. In another embodiment, antiligands 232a are immobilized on an interior stationary surface of a flow cell, microfluidic channel, or another similar sample retaining structure. In either of these embodiments, the reentrant post 224 may consist of a hollow structure (in which case the sample retaining structure may extend through it), or a solid structure (in which case the sample retaining structure will extend between the cavity floor 227 and post bottom 228). In still a further embodiment, cellular events may be monitored by depositing cells on a substrate, such as a glass slide, and positioning the slide within the detection region, for instance through slits (not shown) located in the cavity's side wall. In this embodiment, the reentrant post 224 can be either solid or hollow. Other modifications will be obvious in light of teachings in the arts of bioassay device architecture and high frequency circuit design.

Reentrant Cavity Bioassay Array

[0034] The reentrant cavity of the present invention may be employed in an array to detect and/or identify molecular events in a sample. The bioassay array may be configured serially, in which case a single sample retaining structure, such as a flow tube, is used to supply the sample to each of the cavities' detection regions. Alternatively, the bioassay array may be configured in a parallel arrangement where separate sample retaining structures supply sample to each of the cavities' detection regions.

[0035] Fig. 3 A illustrates a serial array of reentrant cavity bioassays in accordance with the present invention. Using this configuration, a sample plug can be interrogated at a number of different frequencies to provide the sample's dielectric properties over a full frequency spectrum. [0036] The serial array 320 includes several reentrant cavities 325 and a single flow tube 326 extending into each of the cavities' detection regions 329. In the illustrated array, the detection regions 329 are formed longitudinally between the post bottom and the cavity floor, and the reentrant posts are hollow permitting the passage of the flow tube 326 therethrough. Alternatively, the detection regions 329 can be formed circumferentially around the cavities' side wall, in which case the flow tube 326 may be wound around each of the cavities' reentrant posts. Sample plugs 327 are advanced through the flow tube 326 and to detection regions 329 by applying positive pressure to the top or negative pressure to the bottom of the flow tube 326.

[0037] In a specific embodiment, the array 320 employs reentrant cavities 325 having different fundamental resonant frequencies. This permits sample interrogation at different test frequencies as the sample is moved into the detection region of the various cavities 329. In another embodiment, the array 320 is composed of several reentrant cavities 329, each having the same fundamental resonant frequency but tuned (using the tunable probes described above) to a different higher-order cavity resonant frequency. As known in the art of waveguide structures and cavities, the reentrant cavity will exhibit several higher- order resonant frequencies, depending upon the dimensions of the reentrant cavity.

[0038] Fig. 3B illustrates a parallel array of reentrant cavity bioassays in accordance with the present invention. This array provides a high through-put testing platform for various applications, e.g., screening for particular compounds, binding events, or other molecular or cellular activity. This configuration can be used in combination with the aforementioned serial configuration 320 (Fig. 3A), in which case the illustrated reentrant cavities 352 would consist of a serial arrangement of cavities.

[0039] The parallel array 350 includes several reentrant cavities 352, each having a flow tube 356 extending into the cavity's detection region 359. An automated sample processor 355, such as model no. Biomek 2000 Laboratory Automation Workstation manufactured by Beckman Coulter, Inc. (Fullerton, CA.) supplies separate sample to each of the flow tubes 356. The supplied sample may consist of different compounds, for example, sample 1 is buffer; sample 2 is buffer + protein; sample 3 is buffer + ligand; and sample 4 is buffer + protein -f-ligand). In other embodiments, sample of the same composition but different temperature, pH, concentration, salt content etc may be supplied. Other variations of the sample composition will be evident to the reader.

[0040] As illustrated, the detection regions 359 are formed longitudinally between the post bottom and the cavity floor, and the reentrant posts are hollow permitting the passage

19 of the flow tube 356 therethrough. Alternatively, the detection regions 359 can be formed circumferentially around the cavities' side wall, in which case the flow tube 356 maybe wound around each of the cavities' reentrant posts. Sample plugs 357 are advanced through the flow tube 356 and to detection regions 359 by applying positive pressure to the top, or negative pressure to the bottom of the flow tube 356.

[0041] In a specific embodiment, the array 350 is composed of reentrant cavities 352 having the same fundamental frequency. This arrangement permits parallel interrogation of multiple samples at the same test frequency. These responses can be monitored for a known response indicating the occurrence of a particular molecular event within one of the tested samples. Alternatively (or in addition), each of the reentrant cavities may be designed to exhibit a unique fundamental resonant frequency (or the response taken at a unique high- order resonant frequency) in order to obtain a full frequency spectrum of the sample's dielectric properties.

Measurement Methodology

[0042] Fig. 4 illustrates a method for identifying molecular or cellular events occurring within a test sample using the reentrant cavity bioassay in accordance with one embodiment of the present invention. Initially at 402, the cavity assembly is designed to exhibit a resonant response at the desired frequency. The desired frequency is the frequency at or near which the sought molecular event will have dramatically varying dielectric properties. In a specific embodiment of this process, a three-dimensional CAD tool, such as HFSS available from Ansoft, Inc. (Pittsburgh, PA) is used to design the reentrant cavity and simulate its resonant response. Preferably, the three-dimensional CAD software is capable of modeling the sample retaining structure, such as the flow tube, positioned within the detection region of the reentrant cavity as the presence of this structure will vary the resonant frequency of the reentrant cavity.

[0043] After the reentrant cavity has been designed and assembled, a reference sample is supplied to the sample retaining structure and the reference sample fed to the detection region (process 404). In a specific embodiment, the reference sample may consist of the native environment or buffer solution in which the sought protein, cell, binding complex, or other molecular structure resides. In another embodiment, the reference sample may consist of the buffer plus the sought protein, cell, binding complex, or other molecular structure. The reader will appreciate that reference samples may consist of compositions under alternative embodiments of the present invention.

1 1 [0044] Subsequently at 406, the cavity assembly is tuned to a resonant frequency point. This process is accomplished by monitoring the amplitude of the returning signal (for instance, using a network analyzer to measure the input return loss of the probe) while adjusting the probe position until the amplitude of the returning signal reaches a minimum point. The position of the probe is adjusted, for instance, by inserting/extracting the probe into/out of the cavity assembly (Fig. 1), or by rotating the probe relative to the cavity assembly (Fig. 2). In the resonant condition, the cavity will exhibit a high degree of sensitivity in the change of the dielectric properties occurring within the detection region. As the cavity resonant frequency is obtained in the presence of the reference sample, the cavity is said to be "tuned to the buffer" or "buffer + protein," depending upon the composition of the reference sample. The resonant response is subsequently stored for later retrieval and comparison with the resonant response of the test sample, as described below. The process may be automated by monitoring the resonant response and adjusting the probe position until the minimum amplitude point is obtained.

[0045] Next at 408, the reference sample is removed from the detection region and the test sample supplied thereto. In the embodiment of Fig. 1 in which a flow tube is used, the reference and test samples may each comprise plug segments that are separated by air gaps, or other material such as detergent to remove any remaining components of the previous sample plug.

[0046] At 410, the resonant response is measured in the presence of the test sample. The test sample response is compared to the reference sample response and the measurements correlation are obtained. A close correlation is an indication of identity between the molecular event composition of the reference and test samples. For instance, if the reference sample is known to contain a particular protein, binding complex, ligand, cell, or other analyte, and the test and reference sample return loss responses exhibit a high degree of correlation (e.g., within ± .5 dB over frequency), it can be deduced that the test and reference samples have substantially similar molecular event compositions. A lower degree of correlation (e.g., within ± .75- 1.5 dB over frequency) can be indicative of molecular events of the same genus or family. Conversely, a low degree of correlation between the reference and test sample responses (e.g., when the test and reference sample return loss responses differ by more than ± 2.0 dB over frequency) is an indication of dissimilarity in the molecular event makeup between the reference and test samples. Experiments

[0047] An experiment was performed to compare the detection sensitivity of the reentrant cavity with the sensitivity of an open-ended resonant coaxial cable. Measurements were made to determine a change in the q-factor and resonant frequency per unit volume (ΔQ/vol. and Δf/vol.) for both the reentrant cavity and the open-ended coaxial resonator for the same samples. The tested reentrant cavity assembly include the reentrant cavity described in Fig. 1 and a PTFE flow tube having an I.D./O.D. of 0.56 mm/1.07 mm. The coaxial resonator consisted of an open-ended coaxial cable abutted to the aforementioned PTFE flow tube of 0.56mm/l .07mm I.D./O.D. Specific embodiments of the open-ended coaxial resonator are provide in applicant's co-pending application no. 09/687,456 entitled "System and method for detecting and identifying molecular events in a test sample," filed October 13, 2000 (Atty Dkt No. 12US). The measured samples included air, deionized water, and phosphate buffered saline (PBS). A sample plug volume of 12.0 μl was used in the flow tube used in the coaxial resonator assembly, and 1.2 μl was used in the flow tube of the reentrant cavity assembly.

Results

[0048] Table I illustrates the results of the sensitivity comparison between the reentrant cavity and coaxial resonator. These results indicate that the reentrant cavity is approximately five times more sensitive in measuring changes in Q-factor of the detector per unit volume of sample, and fifty-five times more sensitive in measuring changes in the resonant frequency of the detector per unit volume of sample. The increased sensitivity means that molecular events can be detected and identified with greater accuracy using less analyte material.

Table I

Sensitivity Comparison of Reentrant Cavity -v- Coaxial Resonator

Vol. Q_air Qdi Qpb_S Δf_di-_pbs [ΔQ_di-_Pbs]/vol. [Δf_di-p_bs]/vol.

Reentrant 1.2 μl 2000 200 70 44 MHz 108.3/μl 36.7 MHz/μl Cavity Coaxial 12 μl 700 500 250 8 MHz 20.8/μl 0.7 MHz/μl Resonator

1 * [0049] While the above is a complete description of possible embodiments of the invention, various alternatives, modifications, and equivalents can be used. Further, all publications and patent documents recited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication and patent document was so individually denoted.

[0050] Applicant's commonly-owned, concurrently filed application entitled "Pipette-loaded Bioassay Assembly for Detecting Molecular or Cellular Events," (Atty Dkt No. -22US) is herein incorporated by reference for all purposes.

[0051] The following commonly owned, co-pending applications are herein incorporated by reference in their entirety for all purposes:

Serial No. 09/243,194 entitled "Method and Apparatus for Detecting Molecular Binding Events, filed February 1, 1999 (Atty Dkt No. 19501-000200US);

Serial No. 09/365,578 entitled "Method and Apparatus for Detecting Molecular Binding Events," filed August 2, 1999 (Atty Dkt No. 19501-000210);

Serial No. 09/365,978 entitled "Test Systems and Sensors for Detecting Molecular Binding Events," filed August 2, 1999 (Atty Dkt No. 19501-000500);

Serial No. 09/365,581 entitled "Methods of Nucleic Acid Analysis," filed August 2, 1999 (Atty Dkt No. 19501-000600);

Serial No. 09/365,580 entitled "Methods for Analyzing Protein Binding Events," filed August 2, 1999 (Atty Dkt No. 19501-000700);

Serial No. 09/687,456 entitled "System and method for detecting and identifying molecular events in a test sample," filed October 13, 2000 (Atty Dkt No. -12US);

Serial No. 60/248,298 entitled "System and method for real-time detection of molecular interactions," filed November 13, 2000 (Atty Dkt No. -14P);

Serial No. 09/775,718 entitled "Bioassay device for detecting molecular events," filed February 1, 2001 (Atty Dkt No. -15US);

Serial No. 09/775,710 entitled "System and method for detecting and identifying molecular events in a test sample using a resonant test structure," filed February 1, 2001 (Atty Dkt No. -16US);

Serial No. 60/268,401 entitled "A system and method for characterizing the permittivity of molecular events," filed February 12, 2001 (Atty Dkt No. -17P);

Serial No. 60/275,022 entitled "Method for detecting molecular binding events using permittivity," filed March 12, 2001 (Atty Dkt No. -18P);

1 fi Serial No. 60/277,810 entitled "Bioassay device for detecting molecular events," filed March 21, 2001 (Atty Dkt No. -19P); and

Serial No.09/837,898 entitled "Method and Apparatus for Detection of Molecular Events Using Temperature Control of Detection Environment," filed April 18, 2001 (Atty DktNo. -20US).

Claims

WHAT IS CLAIMED IS:

1. A reentrant cavity bioassay operable to detect molecular or cellular events in an aqueous-based sample, the reentrant cavity bioassay comprising: a reentrant cavity having a reentrant post extending from a first interior surface and terminating proximate to a second interior surface, the gap between the termination of the reentrant post and the second interior surface defining a detection region; and a sample retaining structure configured to retain the aqueous- based sample in the detection region, wherein at least a portion of the sample retaining structure is located within the detection region.

2. The reentrant cavity bioassay of claim 1 , wherein the sample retaining structure comprises a flow tube extending through the detection region.

3. The reentrant cavity bioassay of claim 1 , wherein the sample retaining structure comprises a flow cell extending through the detection region.

4. The reentrant cavity bioassay of claim 1 , wherein the sample retaining structure comprises a substrate configured to retain cellular events within the detection region.

5. The reentrant cavity bioassay of claim 1 , wherein the reentrant post has a constant cross-sectional diameter along the longitudinal axis of the reentrant post

6. The reentrant cavity bioassay of claim 1 , wherein the reentrant post has a varying cross-sectional diameter along the longitudinal axis of the reentrant post.

7. The reentrant cavity bioassay of claim 1 , wherein the reentrant post comprises a hollow structure.

8. The reentrant cavity bioassay of claim 1 , wherein the detection region is formed longitudinally between the bottom of the reentrant post and the cavity floor.

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9. The reentrant cavity bioassay of claim 1 , wherein the detection region is formed circumferentially around the cavity sidewall and between the side of the reentrant post and the cavity sidewall.

10. The reentrant cavity bioassay of claim 1 , further comprising a molecular binding region disposed within said detection region.

11. The reentrant cavity bioassay of claim 1 , further comprising a first probe operable to electromagnetically couple an incident test signal into the reentrant cavity.

12. The reentrant cavity bioassay of claim 7, wherein the sample retaining structure comprises a flow tube extending through the hollow reentrant post and into the detection region.

13. The reentrant cavity bioassay of claim 7, wherein the sample retaining structure comprises a sample vial inserted into the hollow reentrant post and extending into the detection region.

14. The reentrant cavity bioassay of claim 7, wherein the sample retaining structure comprises a pipette tip inserted into the hollow reentrant post and extending into the detection region.

15. The reentrant cavity bioassay of claim 7, wherein the reentrant post has a constant cross-sectional outer diameter along the longitudinal axis of the reentrant post

16. The reentrant cavity bioassay of claim 7, wherein the reentrant post has a varying cross-sectional outer diameter along the longitudinal axis of the reentrant post.

17. The reentrant cavity bioassay of claim 11 , wherein the first probe is further configured to recover a modulated test signal from the reentrant cavity.

18. The reentrant cavity bioassay of claim 11 , further comprising a second probe configured to recover a modulated test signal from the reentrant cavity.

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19. The reentrant cavity bioassay of claim 15 , wherein the sample vial comprises a molecular binding region, the molecular binding region located within the detection region.

20. The reentrant cavity bioassay of claim 17, wherein the first probe is rotatably adjustable.

21. A reentrant cavity bioassay array operable to detect molecular or cellular events in an aqueous-based sample, the reentrant cavity bioassay array comprising: a plurality of reentrant cavities, each reentrant cavity having a reentrant post extending from a first interior surface and terminating proximate to a second interior surface, the gap between the termination of the reentrant post and the second interior surface defining a detection region; and a sample retaining structure extending into the detection region of each of the plurality of reentrant cavities, the sample retaining structure configured to retain the aqueous- based sample in the detection region.

22. The reentrant cavity bioassay array of claim 21 , wherein at least two of the plurality of reentrant cavities exhibit different fundamental resonant frequencies.

23. The reentrant cavity bioassay array of claim 21 , wherein the sample retaining structure comprises a single flow tube extending into the detection region of each of the plurality of reentrant cavities.

24. The reentrant cavity bioassay array of claim 21 , wherein the sample retaining structure comprises a respective plurality of flow tubes extending into the detection region of the plurality of reentrant cavities.

25. The reentrant cavity bioassay array of claim 21 , wherein the sample retaining structure comprises a respective plurality of substrates configured to retain cellular events within the detection region of the plurality of reentrant cavities.

26. The reentrant cavity bioassay array of claim 23, wherein the reentrant post comprises a hollow structure, wherein the detection region is formed longitudinally n between the bottom of the reentrant post and the cavity floor of the plurality of reentrant cavities, and wherein the flow tube extends through the hollow reentrant post into the detection region of the plurality of the reentrant cavities.

27. The reentrant cavity bioassay array of claim 23, further comprising a first probe operable to electromagnetically couple an incident test signal into the reentrant cavity.

28. The reentrant cavity bioassay array of claim 24, wherein the reentrant post comprises a hollow structure, wherein the detection region is formed longitudinally between the bottom of the reentrant post and the cavity floor, and wherein the flow tube extends through the hollow reenfrant post into the detection region.

29. The reentrant cavity bioassay array of claim 24, further comprising a first probe operable to electromagnetically couple an incident test signal into the reentrant cavity.

30. A method for detecting cellular or molecular events occurring within an aqueous-based sample, the method comprising: providing a reentrant cavity, the reentrant cavity comprising a reentrant cavity having a reentrant post extending from a first interior surface and terminating proximate to a second interior surface, the gap between the termination of the reentrant post and the second interior surface defining a detection region; and a sample retaining structure configured to retain the aqueous-based sample in the detection region, wherein at least a portion of the sample retaining structure is located within the detection region; supplying an aqueous-based reference sample to the detection region; tuning the reentrant cavity to the resonant frequency point while the aqueous- based reference sample is in the detection region and obtaining a response comprising a reference sample response; storing said reference sample response; removing the aqueous-based reference sample from the detection region; supplying an aqueous-based test sample to the detection region and obtaining a response comprising a test sample response; and comparing the test sample response to the reference sample response.

31. The method of claim 30, wherein supplying the aqueous-based reference and test samples comprises supplying the aqueous-based reference and test samples through a flow tube.

32. The method of claim 30, wherein tuning the reentrant cavity comprises rotating, relative to the reentrant cavity, a probe extending into the reentrant cavity.