KR20020071853A - System and method for detecting and identifying molecular events in a test sample - Google Patents

Abstract

The present invention relates to a molecular detection system for detecting the presence or absence of molecular events in a test sample, the system comprising a fluid storage vessel, a signal source, a measurement probe, and a signal detection unit. The measurement probe includes a probe head and a connection end. The probe head is configured to electromagnetically couple an incident test signal to a test sample within the detection region of the fluid storage vessel. The interaction of the incident test signal with the test sample generates a modulated test signal, and at least a portion of the modulated test signal is retrieved by the probe head. The system further comprises a signal detector coupled to the connection end of the measurement probe and configured to recover the modulated test signal.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a system and method for detecting and identifying molecular events in test samples.

Virtually all biologic fields require systems that measure the ability of molecules of interest to interact with other molecules. Similarly, there is a great need for the ability to detect the presence and / or physical and functional properties of small sized biological molecules. Hereinafter, the functional and physical properties of molecules as well as molecular interactions are referred to as molecular events. The need for molecular event detection extends from basic scientific research to investigate chemical messenger pathways and their function in relation to disease processes, to pre-clinical research to evaluate candidate drugs for potential in vivo efficacy , Is required. The need to detect physical and functional properties also exists in research areas such as functional analysis of newly discovered proteins or functional analysis of genetic (or synthetic) modifications of known biological importance molecules. Other areas where it is desirable to better understand molecular events include pharmacy research, military applications, veterinary medicine, food and beverage, and the environment. In all of these cases, in particular when it is necessary to obtain information from a small amount of sample, the knowledge of the ability of a particular analyte to bind to the target molecule, the properties of such binding (e.g., affinity and on- On-off rate, and other functional information about new molecules are very useful.

Numerous methodologies such as enzyme immunoassay (ELISA), radioimmunoassay (RIA), various fluorescence measurements, nuclear magnetic resonance (NMR) spectroscopy, colorimetric assays and many more specialized assays have recently become available in these areas . Most of these measurement techniques require special preparation, purification, or amplification of the sample being tested. A detectable signal is needed to detect the binding event between the ligand and the antiligand, for example, the presence or extent of binding. Usually, the signals are provided by a label attached to the ligand or anti-ligand. When there is an appropriate label for detectable signals, the physical or chemical effects that produce them include radiation, fluorescence, chemiluminescence, phosphorescence, and enzymatic activity, and designate some of them. The label is then detected by a spectroscopic, radioactive, or optical tracking method.

Unfortunately, in many cases, it is difficult or even impossible to label all or any of the molecules required for a particular assay. Also, due to a number of reasons, including spatial steric effects, the presence of a label in a conventional manner may not be able to recognize molecules between two molecules. In addition, none of these labeling approaches can measure active-site binding to receptors, for example, as non-active, such as allosteric binding, by not measuring the exact nature of the binding event, - It can not distinguish from the site combination and therefore does not get functional information through the current detection methodology. In general, there is a limit to the specificity and sensitivity in most measurement systems. Cell debris and non-specific binding often cause noise in the measurement and make it difficult or impossible to obtain accurate and useful information. As noted above, the buried systems are too complex to attach labels to all relevant analytes or to perform precise optical measurements. Thus, practical, economical, and universal detection techniques that can directly monitor the presence of analytes such as binding event types, functions and extent actually occurring within a given system, without labeling and in real time, .

In particular, biomedical systems can be widely applied to various water-based or fluid-based physiological systems such as nucleic acid binding, protein-protein interactions, and small molecule binding, It requires improved general infrastructure skills. Ideally, the assay should not require highly specific probes, such as specific antibodies or precisely complementary nucleic acid probes. Such as whole blood or cytosolic mixtures, and other natural developmental systems. In some applications it may be desirable to determine whether a given mixture is bound to the active site as an agonist or antagonist on a particular drug acceptor or whether a given mixture is bound to an allosteric site and a binding event has occurred It should be able to provide information about the nature of the binding event, such as not acting as a simple marker. In many applications, the technique must be extremely small and parallel, so that complex biochemical pathways can be revealed, or very few, and a large number of binding mixtures can be used in drug selection protocols. In many applications, it is necessary to be able to monitor a complex series of reactions in real time, so that accurate kinetic and affinity information can be obtained almost instantaneously. Perhaps most importantly, for most commercial applications, there are few sample preparation steps, fewer electronic components, fewer disposable parts such as biochip surface chips used for measurement and discarded, .

Recent trends in the biological and biochemical field are in the miniaturization of measurements and sometimes measurements are made on a " chip " which is a device of the size used in the field of electronics (the name is derived from the field). Among the so-called chip devices are chip devices (also referred to as array technology) that perform multiple simultaneous analyzes on a chip, and chip devices that perform analysis by transferring fluids onto a chip across an analysis site (microfluidics ) Technology). Recently, many companies have realized using this technology. In the field of array technology, there are Affymetrix in Sandaklara, Calif., Incyte Pharmaceuticals Inc in Palo Alto, California, and Human Genome Sciences in Maryland Rockville, California. Lt; RTI ID = 0.0 > Aclara < / RTI > BioSciences Inc., Mountain View, CA, Calif. . Many patents have been granted in these two fields, for example, USPN 6,033, 546 entitled " Apparatus and Method for Performing Microfluidic Operations for Chemical Analysis and Synthesis ", " Methods of Moving Molecules by Applying Multiple Electric Fields USPN 5,126,022, entitled " Device and Device, " USPN 6,004,755 entitled " Mass Microarray Composite Measurements ", USPN 5,874,219 entitled " USPN 5,593,839 entitled " Computer Engineering System for Designing a Graphical Mask ". A large number of different array configurations and fabrication methods are known to those skilled in the art and are described, for example, in U.S. Patent Nos. 5,445,934; 5,532,128; 5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734; And 5,700,637, all of which are incorporated herein by reference. In the field of microfluidics, for example, US Pat. No. 6,012,902 (with name) micropumps; 6,011,252, METHOD AND APPARATUS FOR DETECTING LOW LIGHT LEVELS; 6,001,231, A method and system for controlling and monitoring flow rate in a microfluidic system; 5,989,402, controller / detector interface for microfluidic systems; 5,976,336, a microfluidic device with improved channel geometry; 5,972,187, electropiator and compensation means for electrophoretic bias; 5,965,410, current for controlling fluid parameters in a microchannel; 5,965,001, Variable control of electroosmotic and / or electromotive force within a fluid-containing structure via electrical force; 5,964,995, Method and system for enhanced fluid transfer; 5,959,291, Method and apparatus for low power signal measurement; 5,958,694, Apparatus and method for sequencing nucleic acids in a microfluidic system; 5,958,203, an electropiator and compensation means for the electrophoretic bias; 5,957,579, a microfluidic system with variable channel size; 5,955,028, Analysis system and method; 5,948,227, Methods and systems for electrophoretic molecular classification; 5,942,443, High Power Selective Measurement System in Microfluidics; 5,885, 470, controlled fluid transfer in a microfabricated polymer substrate; 5,882,465, Method of manufacturing microfluidic device; 5,880,071, an electropiator and compensating means for electric transfer bias; 5,876,675, Microfluidic devices and systems; 5,869,004, Method and apparatus for self-concentration and / or dilution of material in a microfluidic system; 5,852,495 Fourier detection of species moving in the microchannel; 5,842,787, a microfluidic system with variable channel size; 5,800,690, Variable control of electro-osmotic and / or electromigration forces in fluid-containing structures via electrical force; 5,779,868, an electropiator and compensation means for an electrophoretic bias; And 5,699,157 Fourier detection of species moving within the microchannel, the above patents being incorporated herein by reference. Publications representing current state of the art in the industry as a number of other publications including other patents have been described as a list of prior art cited within the specifications of the patents.

A series of individual wells (e.g., microtitre plates and other types of multiple well plates) that hold samples for each assay, or individual wells (e.g., ) Have also been developed. Microtiter plates having 96 wells are generally available, and the plates may have a different number of wells, such as a 384-well plate. Various automation devices have been developed to manipulate and analyze the contents of each container, regardless of whether each container, a multiple well plate, or multiple plates are handled simultaneously. There are other multi-well systems (in addition to conventional microtiter plate systems). For example, US Pat. No. 6,033,911, which shows a system designed to manipulate and perform measurements in various types of containers for each sample, an automated measurement device; 6,024,920, fine plate scanning read head; 5,993,746, plate holder; No. 5,998,236, a multiple syringe pump with a liquid handling machine; 5,985,214, A method and apparatus for rapidly identifying useful chemicals in a liquid sample; 5,976,470, sample cleaning station assembly; 5,972,295, Automated Analytical Apparatus; 5,968,731, Automated testing equipment for biological specimens; And 5,952,240, intermittent matrix plate placement devices. Publications representing current state of the art in the industry as a number of other publications including other patents have been described as a list of prior art cited within the specifications of the patents.

Among the historical approaches used to study biochemical systems, there are several types of dielectric measurements. In the 1950s, experiments were conducted to measure the dielectric properties of biological tissues using techniques for measuring the dielectric properties of known materials at that time. Since then, there have been several approaches to implementing such measurements, including time domain techniques such as frequency domain measurement methods and Time Domain Dielectric Spectroscopy. In these approaches, experiments were conducted using various types of coaxial transmission lines or other transmission lines and structures for typical applications in the dielectric characterization of materials. This includes studies investigating the use and relevance of the dielectric properties of a wide range of biological systems. From tissue samples taken from various mammalian organs, attention has been drawn to cellular and subcellular systems, including cell membranes and cell organelles. Most recently, there have been attempts to miniaturize the above-described techniques for sensing changes in the dielectric properties of molecular systems in array conditions (see, for example, USP 5,653,939; 5,627,322 and 5,846,708). Typically, they use a biological sample-tissue, cell system, molecular system, such as a side-by-side or a series of elements in an electrical circuit topology.

There are a number of techniques for large scale field probes to sense radiation from an object and to measure local material properties, such as IEEE Transaction on Microwave Theory and Techniques, 38 8-14 Non-invasive electrical characterization of materials at microwave frequencies using open-end axial lines such as those described in Misra et al., 1990 (1990): Noninvasive electrical characterization of materials at microwave frequencies using anopen quot; Measurement of high-temperature complex permittivity of a composite material using an open-end waveguide ", Journal of Electromagnetic Waves and Applications, 6, 1259-75 (1992); "Design and performance of a non-contact probe for measurement on a high frequency flat circuit" by Osofsky and Schwarz, IEEE Transaction on Microwave Theory and Techniques, 40 1701-8 (1992); "Theoretical and Experimental Investigation of Microwave Dielectric Constant Measurements Using an Open-End Elliptic Coaxial Probe", Xu, Ghannouchi, et al., IEEE Transaction on Microwave Theory and Techniques, 40 143-50 (1992); Jiang Wong, " Open-Ended Coaxial-Line Technology for Measurement of Microwave Dielectric Constants for Low-Loss Solids and Liquids " in Review of Scientific Instruments, 64 1614-21 (1993); Jiang Wong, " Measurement of Microwave Dielectric Constants for Finite Thickness Low-loss Samples Using Open-Ended Coaxial-Line Probes " in Review of Scientific Instruments, 64 1622-6 (1993) . There are publications on electron microscopy using radio frequency or microwave signals in addition to electron current in a microscope tip, for example, Keilmann, Keilmann, published in Optic Communications 129 15-18 (1996) , van der Weide et al., "Extreme sub-wavelength resolution using a scanning radio-frequency transmission microscope"; Field-scanning microwave microscope with a resolution of 100.mu.m, such as Vlahacos, Black et al., Applied Physics Letters, 69 3272-4 (1996); There is a " scanning tip microwave close-field microscope " of Wei, Xiang et al., Applied Physics Letters, 68 2506-8 (1996). Thus, it is known to use probes (both waveguides and coaxial probes) to test the properties of dielectric materials and is commonly used in other technical fields such as microscopic image collection and dielectric measurements of insulators and other materials in the electronics industry .

One form of relatively large-scale equipment used to analyze and control chemical processes, such as fermentation, was first used in the oil processing and punching industries. For example, USP 5,025,222 relates to a system and method for monitoring conditions in fluid media. The fluid medium flow flows to the UHF or microwave oscillator through a fluid vessel that is electrically constructed as a transmission line segment and is electrically coupled to a load. The oscillator is not isolated from the load and is freerunning at a selected starting frequency to provide a particularly strong transition of the dielectric constant of the fluid medium as the chemical reaction proceeds. Preferably, to measure the progress of the reaction, the insertion loss and frequency of the oscillator are monitored. Two recent patents related to this, namely systems, methods and probes for electronic monitoring and characterization using a single-ended coupling of a load-pulled oscillator to a system under test are disclosed in US Pat. No. 5,748,022 And 5,966,017. However, even though the purpose of these patents is in the detection of chemical species produced by fermentation (and other processes), none of the patents are considered to be specially considered for molecular characterization or for molecular binding.

Thus, additional development of molecular event detection methods that do not require labels such as fluorophore or radioactive isotopes, are quantitative and qualitative, specific to the molecule of interest, very sensitive, and relatively simple to implement . The present invention fulfills the above and other requirements, as described below.

This application claims the benefit of U.S. Provisional Application No. 60 / 159,175 entitled " Method of Utilizing a Dielectric Interface for the Detection of Molecular Interactions ", filed on October 13, 1999, Quot; Method and Apparatus for Detecting the Presence of Molecules in Molecules ", 60 / 191,702.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 a shows an integrated detector in accordance with an embodiment of the present invention.

1B illustrates a fluid delivery system in accordance with an embodiment of the present invention.

FIG. 2A is a view of a measurement probe according to an embodiment of the present invention; FIG.

2B is a top view of a coaxial probe according to one embodiment of the present invention.

2C is a sectional view of a second embodiment of a measurement probe according to the invention;

2d is a plan view of the measuring probe shown in Fig. 2c. Fig.

FIG. 2E illustrates a non-resonant coaxial measurement probe according to one embodiment of the present invention. FIG.

Figure 3a is a diagram of a molecular detection system in accordance with an embodiment of the present invention.

FIG. 3B illustrates a method of detecting an analyte according to an embodiment of the present invention. FIG.

3C is a graph illustrating an exemplary analyzed signal response in accordance with one embodiment of the present invention.

3D is a flow chart illustrating a method for detecting molecular binding events occurring in a test sample according to one embodiment of the present invention.

FIG. 3E illustrates two possible embodiments of a baseline buffer response, a first test sample response, an embodiment of a second test sample response, and a third sample signal response corresponding to a combining and non-combining condition.

Figure 3f shows a second method for detecting molecular binding events taking place in a test sample according to the present invention.

Figure 3g illustrates an embodiment of first, second and third sample responses indicating that there is no binding event using the method of Figure 3f in accordance with the present invention;

Figure 4a illustrates a second embodiment of a molecular detection system according to the present invention.

Figure 4b shows a third embodiment of a molecular detection system according to the present invention.

5A illustrates one embodiment of a computer system that can be operated to implement a software program designed to execute each of the methods described above in accordance with the present invention.

Figure 5B illustrates the internal structure of the computer system shown in Figure 5A in accordance with one embodiment of the present invention.

FIG. 6A is a diagram of a molecular detection system implemented in a particular experiment in accordance with the present invention. FIG.

FIGS. 6B-6G are graphs illustrating S ₁₁ signal responses generated upon detection of a particular analyte according to the present invention. FIG.

Figure 6h is a graph depicting S ₁₁ signal responses made upon detection of different concentration levels of NaCl in accordance with the present invention.

Figures 6i-6j are graphs illustrating S ₁₁ signal responses made upon detection of a specific molecular binding event using the method of Figure 3d according to the present invention.

6k-6l are graphs illustrating S ₁₁ signal responses made upon detection of specific molecular binding events using the method of Figure 3f according to the present invention.

Figure 6m is a graph showing a dose response curve made in accordance with the present invention.

The initial use in the laboratory of the present invention was directed to a measurement technique for detecting changes in the dielectric properties of the molecular structure using molecular coupled regions coupled along a single pathway. This application presents many of the general principles used in the present invention. For example, reference may be made to PCT WO 99/39190, published Aug. 5, 1999, and assigned to the present invention. The present invention differs in that the general purpose is to detect molecular events in closed fluid channels, such as in fluid storage vessels, such as in microtiter plates, or in microfluidic devices. In this case, as described below, in order to avoid contamination of the probe, the samples are typically separated from the electromagnetic probe by an air gap and / or dielectric material present in the wall of the fluid container.

Roughly speaking, the present invention is based on the discovery and characterization of the molecular structure as a self-molecular structure in a molecular bonding event or as a partner, in particular in an aqueous state, using the dielectric properties of the molecular structure and interaction with the illumination signal The present invention also provides a method and system for measuring a single port. The systems and methods of the present invention also allow identification of molecular structures and / or bonds with other molecules in a continuous fluid stream, thus enabling automation of the process. Because the molecules bind to each other in an aqueous environment of physiological conditions, the method of the present invention can be used for various sorting processes such as molecular identification in the biological and pharmaceutical field. By the method and apparatus of the present invention, it is possible to detect without having to maintain a label on one of the binding partners, so that it is not necessary to calculate the change of binding by the label, making it particularly suitable for detecting the intended drug substance do.

In a first embodiment of the present invention, a method for detecting a molecular structure in an aqueous sample is disclosed. The method includes the steps of: (a) introducing a first sample into a fluid channel of a fluid delivery system having a fluid channel having a sample inlet end, a detection region, and a sample discharge end; (b) moving the sample through the channel from the sample inlet end to the sample outlet end under the control of the fluid control; (c) applying a test signal of 10 MHz to 1000 GHz to the detection region of the fluid channel; And (d) detecting a test signal change as a result of interaction of the test signal and the sample. Non-limiting examples of fluid movement control include movement of liquid under the control of an electric field such as electrophoretic pumping as well as surface effects (capillary), gravity, inertial effects (e.g., tilting or spinning operations) And a mechanical pump. A frequency (or, in some cases, a continuous frequency such as multiple frequency sets or spectra) is selected to produce a detection electromagnetic field that differs from the detection electromagnetic field generated when the sample comprising the molecular structure does not include a molecular structure.

In another embodiment, the method further comprises: (e) introducing a spacer material into the channel after the first sample; (f) introducing an additional sample into the channel after the spacing material; (g) moving the additional sample through the channel under the control of the fluid control, so that a plurality of disparate samples separated by the spacing material are not mixed with each other; And (f) repeating steps (c) - (d) for an additional sample. In this way, a series of samples is provided to a single detector, which can significantly simplify the design of the detection system and greatly improve regeneration as well as easier design of the one-time analysis chip.

The above-described method can be executed in various apparatuses. In one embodiment of the present invention, a molecular detection system for detecting whether a molecular event is present in a test sample includes a fluid storage vessel, a signal source, a measurement probe, and a signal detector. The fluid storage vessel may be a separate vessel (open or closed) or a channel including a sample inlet end, a detection zone, and an outlet end. The signal source is operated to deliver an incident test signal. The measurement probe is coupled to the signal source and includes a probe head and a connection end. The probe head is configured to electromagnetically couple an incident test signal to a test sample in a detection region. The interaction of the incident test signal with the test sample generates a modulated test signal and the probe head is configured to recover at least a portion of the modulated signal. The system further includes a signal detector coupled to the connection end of the measurement probe, wherein the signal source is configured to recover the modulated test signal.

Thus, in general, the present invention relates to a method and apparatus for measuring electromagnetic properties of a molecule, such as an electromagnetic signal, typically from about 1 MHz to 1000 GHz, interacting with molecular events in a fluid storage vessel to determine the structural and functional properties of the molecule itself, And measuring the characteristics of the structure.

The features and advantages of the present invention will be better understood with reference to the following more detailed description and the accompanying drawings.

Contents

Ⅰ. Term Definition

Ⅱ. summary

Ⅲ. Detector assembly

IV. Exemplary molecular detection systems

Ⅴ. Exemplary Uses

VI. Software execution

VII. Experiment

Ⅰ. Term Definition

As used herein, the term " molecular binding event " (often abbreviated as " binding event " or " binding ") refers to the interaction of an interest molecule with another molecule. The term " molecular structure " refers not only to a specific molecular substructure (such as an alpha helical region, a beta sheet, an immunoglobulin region, or any other type of molecular substructure), but also to a solvated shell including all of the molecules' interaction with other molecules (such as bending or folding movements), including the interaction between the molecule and the solvation shell . &Quot; Molecular structure " and " molecular binding event " all refer to a " molecular event ". The mere presence of a molecule of interest within the region in which detection / analysis is performed is not referred to as a " molecular event "

Examples of molecular binding events include, but are not limited to, (1) simple, non-covalent bonds between ligands and their ligands, and (2) transient covalent bond formation, as often occurs when the enzyme is reacting with a substrate have. DNA / DNA, DNA / RNA, RNA / RNA, nucleic acid mismatch, complement nucleic acids, and nucleic acid / protein are more specific examples of related binding events. However, the present invention is not limited to these examples. A join event can occur as a primary, secondary, or higher-level join event. The primary binding event is the binding of the first molecule (specific or non-specific) to any type of entity, typically a substance that is part of the first surface that will be the surface within the detection region, or a separate molecule, ≪ / RTI > interacting complexes. The secondary binding event is defined as a second molecular binding (specific or non-specific) to the first molecular interaction complex. The tertiary binding event is defined as a third molecular binding (specific or non-specific) to the second molecular interaction complex, as is a higher dimensional binding event.

Examples of related molecular structures include, but are not limited to, the presence of a physical substructure (e. G., An alpha-helical region, a beta sheet, an immunoglobulin region, a catalytic activation site, a binding region, or a seven- trans- membrane The ability to act as an antibody, the ability to transport a particular ligand, the ability to act as an ion channel (or a component of its ion channel), or as a signal transduction moiety Ability to act).

Typically, structural features are detected by comparing signals obtained from molecules of unknown structure and / or action with signals obtained from molecules of known structure and / or action. Typically, the molecular binding event involves a signal obtained from a sample comprising one of the potential binding partners (or a signal from two separate samples each having one potential binding partner) with two potential binding partners To the signal obtained from the sample. Detection of " molecular binding events " or " molecular structures " are all often referred to as " molecular detection ".

The methodologies and devices described herein are primarily concerned with detecting and predicting the biological and pharmacological significance of molecular events that occur in a physiological condition, such as within a cell membrane or subcellular membrane or within the cellular cytoplasm. Thus, less attention is paid to the structural properties of molecules or interactions between molecules under conditions similar to, and not identical to, physiological conditions. For example, complex formation of each molecule under non-physiological conditions, such as in a vacuum field of an electron microscope, is not considered a preferred " molecular binding event " as used herein. Here, the preferred molecular events and properties exist under "physiological conditions" that exist in an artificial environment such as an aqueous buffer designed to simulate natural cell or intercellular environment, or physiological conditions. It will be appreciated that local physiological conditions will vary from location to location in cells and tissues, and will also vary significantly under artificial conditions that simulate such conditions. For example, a binding event will occur between the protein and ligand in the lower cell membrane in the presence of small molecules and helper proteins that affect binding. Such conditions will be significantly different from physiological conditions in the serum, such as, for example, " regulated phosphate saline " or an artificial medium referred to as PBS. Typically, the preferred conditions of the present invention will be aqueous solutions with a slight but minimal amount of organic solvent, such as DMSO, which may be present to aid dissolution of some of the components being tested. The "aqueous solution" comprises at least 50 wt%, preferably at least 80 wt%, most preferably at least 90 wt% water, more preferably at least 95 wt% water. Other conditions such as osmolality, pH, temperature, and pressure can be significantly altered to simulate the local conditions of, for example, the intercellular environment in which the binding event takes place. For example, the cytoplasm of a cell and the natural conditions in the lysosome of that cell are quite different from each other, and different artificial media will be used to simulate such conditions. Examples of artificial conditions designed to mimic natural things to study various biological events and structures are well documented in the literature. A number of such artificial media are commercially available, for example, Calbiochem General Catalog 81, published 2000/2001, which lists 60 commercially available buffers at pH 3.73 to 9.24, 82 to page 82 of the science-related product catalog. A general record relating to the manufacture of conventional media is described in Chapter 7 ("The Culture Environment") of R. Ian Freshney, Wiley-Liss, New York (1994) 3rd edition, Culture of Animal Cells: A Manual of Basic Techniques You can refer to the same record.

As used herein, the term " analyte " refers to a molecular entity in which the presence, structure, binding capacity, etc. are detected and analyzed. Substantially suitable analytes of the present invention include antibodies, antigens, nucleic acids (e. G., Natural or synthetic DNA, RNA, gDNA, cDNA, mRNA, tRNA), lectins, sugars, glycoproteins, (Such as growth factors and associated receptors, cytokines and related receptors, signaling molecules and receptors), existing drugs and drug components (developed and recorded in a combinatorial library) Natural products or similar synthetic substances), auxiliary metabolites such as metabolites, misuse drugs and their metabolites by-products, vitamins and other naturally occurring compounds and compounds, physiological fluids, cells, cell components, cell membranes and related structures Oxygen and other gases found in plants, other natural products found in plants and animals, and other partially and totally synthesized products.

Although most of the measurements described herein are made for one molecule or a pair of molecules in solution, it is preferred that at the site of the channel receiving the electromagnetic radiation while the test mixture is flowing through the immobile molecule, The method of the present invention may be applied to a situation in which one of the members is fixed on the surface. When one of the members of the binding pair is immobile, the term " antiligand ", as used herein, refers to a molecule that is usually immobilized on a surface. For example, the anti-ligand may be an antibody, and the ligand may be a molecule such as an antigen that specifically binds to the antibody. For purposes of the present invention, where the antigen is a molecule bound to a surface and an antibody is detected, the antibody may be considered a ligand and an antigen, i.e., an anti-ligand. In addition, once the anti-ligand is bound to the ligand, the resulting anti-ligand / ligand complex can be regarded as an anti-ligand for subsequent binding purposes.

As used herein, the term " molecule " refers to a biological or chemical entity present in the form of a chemical molecule or molecules as opposed to a non-molecular form of a salt or other substance. 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), many molecules contain organic molecules (including carbon atoms linked to other atoms by covalent bonds) Lt; / RTI > The general term "molecule" includes a number of descriptive molecular classifications or groups of molecules, such as proteins, nucleic acids, carbon monoxide, steroids, organic drugs, receptors, antibodies, and lipids. Within the principle that both the generic classification of " molecules " and those denominated as small classes, such as proteins, represent mixtures, where necessary, such as when applying the methods of the invention to small groups of molecules, one or more more specific terms (Such as the term " protein " in the mixture group) will be used herein. When used in its most general sense, " molecule " also includes binding complexes of each molecule as described below. Major covalent molecules may also have ionic bonds (such as a salt of a protein or carboxylic acid in which the amino acid residue is bonded to a residue), and such molecules are still considered molecular structures. Of course, salts (e. G., Sodium chloride) may be present in the sample containing the molecular structure, and the presence of such salts is included in the practice of the present invention. Such salts will participate in the overall genetic response, but even in the presence of their salts, molecular binding events or traits can be detected.

The term " binding partner ", " ligand / antagonist ", or " ligand / antagonist complex ", as used herein, refers to a pair of molecules (or groups of larger ; See below). Typically, such pairs or groups are composed of two or more molecules that interact with each other, usually by the formation of non-covalent bonds (such as dipole-dipole interactions, hydrogen bonds, van der Waals interactions) . As is known in the art, even for molecules with similar binding affinities, the interaction time varies considerably (sometimes referred to as the on-off time). For example, there are antibody-antigen, lectin-hydroxide carbon, nucleic acid-nucleic acid, biotin-avidin pairs. Biological binding partners need not be confined to a single molecule pair. Thus, for example, a single ligand can be bound by the coordination action of two or more anti-ligands, or a first antigen / antibody pair can be bound by a second antibody specific for the first antibody . Binding can occur within a liquid (which may also be referred to as a solution) or a solid (which may be referred to as a surface) phase and includes conjugation involving continuous or simultaneous bonding of three or more individual molecular entities . Possible examples include GPCR-ligand binding followed by GPCR / G-protein binding; Nuclear receptor / cofactor / ligand / DNA binding; Or a small molecule ligand, to the target of the complex chaperone protein. Other examples will be readily apparent to those skilled in the art.

As used herein, the term " ligand " is generally used to refer to a molecule (i.e., an " anti-ligand ") that binds to a ligand by a beneficial change in free energy (i.e., negative value) upon contact between the ligand and the anti- Means any molecule present. There is no limit on the size of the interacting substrate, and thus a broader sense of ligand (or anti-ligand) may constitute each molecule and a larger organelle, such as a cell, cell membrane, cell organelle, Molecular groups may also be constituted. As used herein, the terms " ligand " and " anti-ligand " are both broad and may be used interchangeably. However, it can be seen that there is a general tendency to use the term " ligand " in the biological field to mean two smaller binding partners that interact with each other, and where possible, this practice is followed.

As used herein, " ligand / anti-ligand complex " means a ligand bound to an anti-ligand. The binding may be specific or non-specific, and the interacting ligand / anti-ligand complexes are typically bound to each other through noncovalent bonding such as hydrogen bonding, van der Waals interactions, or other forms of intermolecular interactions.

As used herein, the term "specific binding" refers to a binding reaction selected for a related ligand in a heterogeneous population of potential ligands, when referring to a protein, nucleic acid or other binding partner as described herein. do. Thus, under specified conditions (e. G., Immunoassay conditions in the case of antibodies), a specific anti-ligand binds to a specific " target " and does not bind to any other potential ligand present in the sample. For example, cell surface receptors (e. G., Estrogen receptors) for hormone signals can bind to other steroid hormones, including similar steroids such as estriol, The same hormone). Similarly, nucleic acid sequences that are fully complementary will hybridize to each other under preselected conditions, thereby not even hybridizing to other nucleic acids whose sequence is at a single nucleotide position.

As used herein, the terms " isolated ", " purified ", and " biologically purified " refer to a substance that is substantially or essentially free of components .

As used herein, the term " nucleic acid " refers to a deoxyribonucleotide or ribonucleotide polymer in the form of a single- or double-stranded form, and, unless otherwise constrained, can be hybridized in a manner similar to naturally occurring nucleotides Lt; RTI ID = 0.0 > natural < / RTI > nucleotide analogs.

As used herein, "polypeptide", "peptide", and "protein" are used interchangeably to refer to a polymer of amino acid residues. These terms appear to be not used consistently with respect to the size of the molecule, although they mean the size and complexity with which a given order generally increases. All of these terms refer to amino acid polymers in which one or more amino acid residues or peptide bonds are the artificial chemical analogs of the corresponding naturally occurring amino acid or a link to the naturally occurring amino acid polymer.

As used herein, the term " antibody " means a fragment of an immunoglobulin gene or a protein consisting of one or more polypeptide chains substantially encoded by an immunoglobulin gene. The recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes as well as pre-ad immunoglobulin variable region genes. Light chains are classified as kappa or lambda. The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, and it defines the immunoglobulin class of IgG, IgM, IgA, IgD and IgE, respectively.

&Quot; Antigen-binding site " or " binding portion " means a portion of an immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by the amino acid residues in the N-terminal variable (" V ") region of the middle ("H") and light ("L") chains. Three highly-divergent stretches in the V-region of the middle and light chains are believed to result in more conserved flanking stretches known as " framework regions " or " FRs &Quot; means a " hypervariable region " Thus, the term " FR " refers to an amino acid sequence adjacent to and naturally found between hypervariable regions in an immunoglobulin. Within the antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in the three-dimensional space to form the antigen binding " surface ". These surfaces mediate the binding and recognition of target antigens. The three hypervariable regions of each heavy and light chain are referred to as " complementarity measuring regions " or " CDRs ", and are described, for example, in US Department of Health and Human Services, Bethesda, Human Service), and Kabat, which is described in the fourth edition of the US Department of Health and Human Services.

The terms " immunological binding " and " immunological binding properties ", as used herein, refer to non-covalent interactions of forms that occur between an immunoglobulin and an antigen specific to the immunoglobulin.

The term " enzyme " as used herein refers to a protein that acts as a catalyst and lowers the activation energy of a chemical reaction in which a compound is divided into smaller compounds or between other compounds. A compound that undergoes a reaction under the influence of an enzyme is called a " substrate ". The enzyme is not an initial substance or end product of the reaction and does not change after the reaction is completed.

As used herein, the term " test sample " refers to the material (analyte) to be investigated and the medium / buffer in which the analyte is found. Such media or buffers may comprise solid, liquid or gaseous phase materials and the major component of the most physiological media / buffer is water. Solid phase media may be any organic, such as hydrocarbons, proteins, oligo-nucleotide, SiO _2, GaAs, Au, or alternatively, Nylon ^®, Rayon ^®, Dacryon ^®, polypropylene, Teflon ^®, neoprene, Delrin (delin) And may be composed of naturally occurring or synthetic molecules including polymeric materials. Liquid vehicles include those containing aqueous, organic or other major components, gels, gases and emulsions. Exemplary media include cellulose, dextran derivatives, d-PBS aqueous solutions, Tris, deionized water, blood, cerebrospinal fluid, urine, saliva, water, organic solvents.

&Quot; Biological sample " as used herein refers to a biological tissue or fluid that undergoes a health and / or physiological condition to examine an event of interest or structure. Such biological samples include sputum, aminotic fluid, blood, blood cells (e. G., Red blood cells), tissue or micro needle biopsy samples. However, the present invention is not limited to those listed above. Biological samples also include mammals or other cells that have grown in culture. Although biological samples are mostly taken from the patient, it is not limited thereto. (E. G., Birds such as chickens or turkeys) and plants (e. G., Plants and ornamental plants used as food, such as corn or wheat) as well as other mammals such as dogs, cats, sheep, The same measurement may be used to detect molecular events in the sample obtained from < RTI ID = 0.0 > Optionally, the biological sample may be pretreated by diluting or condensing it with a suitable transport medium solution, still in this case also referred to as a " biological sample ". Among a number of standard aqueous transfer medium solutions, one of various transport media such as phosphates, tris, etc., preferably at physiological pH, can be selected and used. As in the case of biological samples, even if a more general sample obtained from a feed material is pretreated (by dilution, extraction, etc.), the feed material may also be referred to as a sample.

As used herein, " fluid storage container " means that where fluid can be held in a position that can be coupled to a single path, regardless of physical size or shape, It is possible to detect a signal obtained as a result of the test signal in the region interacting with the sample. A " fluid reservoir " can be said to mean a fluid rather than a container itself in which the fluid is located. Thus, in its simplest form, a " fluid storage vessel " can be said to mean a fluid drop or layer formed on a flat surface and maintained by inertia and / or surface tension. Such a configuration is primarily intended for a variety of chip designs commonly used in genetic engineering, such as sample fluid passing over the surface of a chip having a specific molecular probe (usually a DNA fragment of known sequence) attached to a known point on the surface Is used. However, " fluid storage containers " are intended to encompass vertical walls (such as sidewalls of a test tube or microtiter plate) that inhibit diffusion by gravity, fully enclosed walls (such as a sealed vessel) And are stored in physical walls that restrict movement of the fluid, such as partially surrounding walls (such as walls of a tube or channel). The last of those described above is referred to herein primarily as the " fluid channel " and may be used to interact with solutions and / or other samples containing reactants or to allow multiple samples to be transported through a single detection region (Such as a microfluidic chip) at a point on the wafer.

As used herein, the term " single path " refers to a transmission medium that supports the delivery of an intended test signal. One embodiment of a transmission path is a two-conducting structure such as a coaxial cable capable of supporting a transmitted electromagnetic (TEM) signal. Other definitions of microstrip lines, strip lines, suspended substrates, slot lines, other multi-conductive parts such as coplanar waveguides, TEM structures are also included in this definition. Other transmission media such as wires, printed circuit board traces, conductive or dielectric waveguide structures, and multipole (eg, 4-pole, 8-pole) structures are also included in this definition.

As used herein, the term " detection zone " refers to a device that receives an electromagnetic signal radiated from a signal path and that is responsive to the signal in a manner that can be detected by a user equipment (e.g., (All or a portion of a well) of a multi-well plate or a fluid transfer channel in a chip. Thus, some of the signals may interact with the sample at other locations (e.g., adjacent walls of the microtiter plate where they are impacted by stray electromagnetic radiation from the probe head), but such external interaction is used If not detected by the equipment, the adjacent area is not part of the " detection area ". On the other hand, if the signal interacts with a portion of the bulk sample, all volumes of the bulk sample that interact with the signal and produce a modulated signal that can be detected by the instrument will be considered as part of the " detection region ". Typically, the detection area of equipment used for the primary purpose of the present invention will be relatively small. This is particularly the case when the aim is to test potential candidate drugs from a library of test compounds for their ability to interact with target receptors as the amount of each individual compound available for a particular assay is small . Thus, a detection zone volume of 1 ml (1 x 10 ^-6 m ³ ) or less is preferred. (1 x ^10-9 m ³ ), 1 nl (1 x 10 ^-12 m ³ ), or 1 pl (1 x 10 ^-15 m ³ ), and less between the detection volumes Is more preferable. Lesser volumes may be used, but are not preferred. This is because a smaller volume may not contain a statistically sufficient number of molecules of interest corresponding to the temperature, pressure and concentration conditions conventionally used in physiological samples.

The term " coupling ", as used herein, refers to the transfer of electromagnetic energy between two structures, either through direct or indirect physical connections or through any form of signal coupling. &Quot; Coupling " as a general term refers to the coupling of a signal that occurs when a molecular event makes direct physical contact with the conductive portion of the signal path (e.g., when the molecule of interest binds to the surface of the signal path) Includes both signal coupling that occurs when an event is physically separated from any surface of the signal path (e.g., the operation described as using a probe coupled to a sample through a wall of a fluid storage vessel). These two types of couplings are commonly referred to as " direct coupling " and " indirect coupling " when it is necessary to distinguish.

As used herein, the term " test signal " refers to an ac time varying signal. Preferably, in a particular embodiment, the test signal is at 10 MHz, 20 MHz, 45 MHz, 100 MHz, 500 MHz, 1 GHz (1 x 10 ⁹ Hz), 2 GHz, 5 GHz, 10 GHz, (10 × 10 ⁶ Hz) or more such as 10 GHz, 18 GHz, 20 GHz, 25 GHz, 30 GHz, 44 GHz, 60 GHz, 110 GHz, 200 GHz, 500 GHz, 1000 GHz (1 x 10 ¹² Hz) or less. The preferred range is 10 MHz to 40 GHz, particularly preferably 45 MHz to 20 GHz.

Ⅱ. summary

The present invention makes it possible to distinguish a number of molecules and to identify structural features and binding capabilities based on unique dielectric properties in the electromagnetic spectrum region that have not previously been used to detect molecular events. This dielectric property can be observed by initially coupling the test signal to a test sample containing the analyte of interest. The dielectric properties of the analyte modulate the test signal and produce an identifiable signal response. This response can be recovered, stored and used to detect and identify molecules in other test samples. Also, as the test signal is further modulated by interaction with the first molecule of the second molecule, it may also detect interactions (e. G., Molecular binding events) of the first molecule with other molecules. Detection and identification of molecular characterization and binding events can occur within a liquid, gas, or solid phase, but is preferably carried out in an aqueous, physiological environment (e.g., an aqueous environment) to characterize the molecule in relation to its function in a biological environment . ≪ / RTI >

The detection assembly of the present invention provides a measurement probe that can be operated to couple a test signal to a test sample where a molecular event occurs. The test sample is placed in a well of a fluid storage vessel, fluid channel or multiple well plate. A test signal is irradiated to a portion of the fluid storage vessel, which is referred to as the detection region. The dielectric properties of the molecules involved in the molecular event modulate the test signal to provide a reflected signal with a signal response that is different than the signal response that would be equally detected when the same signal is applied to a sample that does not contain a molecular event. The signal response is then recovered and provides information about one or more characteristics of the molecules or molecules contained in the molecular event.

Ⅲ. Detector assembly

FIG. 1A illustrates an embodiment of an integrated detection assembly 100 in accordance with the present invention. The detection assembly 100 includes a fluid delivery system 150 integrated with the measurement probe assembly 230. The fluid delivery system 150 includes a fluid channel 151 having an inlet end 152 and an outlet end 154. The movement of the test sample through the channel 151 is controlled by the fluid control 156, which acts to transport the test sample through the channel under the conditions selected by the user and at the appropriate time. Alternatively, the storage vessel 158 may include a second analyte or test sample that may be mixed with the test sample stored in the storage vessel 157 as it is introduced into the fluid channel 151. [ The ability to mix two test samples adjacent to the detector allows for easy measurement of the kinetics of the coupling event from this type of data. The emulsion control unit 156 may move the test sample in one direction of forward and backward or may stop the test sample for a predetermined time in the detection area, for example, to improve the sensitivity.

The probe assembly 230 includes a probe head 230a and a connection end 230b. The probe head 230a is disposed adjacent to the detection region 155 of the fluid channel 151 and acts to electromagnetically couple an incident test signal to a test sample passing through the detection region 155. [ The test sample modulates the test signal, some of which is reflected back to the probe head 230a. The reflected modulated signal is then recovered by the detector assembly described in more detail below. In one embodiment, the probe head may be an open-ended portion of a coaxial cable that transmits a test signal to a test sample that passes through the detection region 155 and recovers the modulated reflected signal from the sample. Those skilled in the art of microwave engineering will appreciate that other embodiments (shorted or loaded ends) and other circuit configurations (strip lines, microstrips, planar area waveguides, Type substrate or waveguide) may be used.

The connection end 230b is electrically connected (via a direct or intermediate circuit) to the measurement port of the molecular detection system described below. In an exemplary embodiment in which the measurement probe is a coaxial structure, the connection end 230b may be a suitable coaxial connection, such as a coaxial cable extending from a molecular detection system, a SMA-type connection or other connection familiar to those skilled in the art of high frequency measurement have. In another embodiment of the invention in which different types of probe structures (e. G., Microstrips, etc.) are used, the connection port may comprise suitable connections for providing signal connections to the molecular detection system.

Fluid transport system

As shown in FIG. 1A, the fluid delivery system 150 includes a fluid channel 151 through which a test sample passes. Depending on the application, the fluid channel 151 may have various forms. For example, in one embodiment, fluid channel 151 is the detection area (155) Tefron ^® for transporting a test sample in and out (polytetrafluoroethylene; PTFE), or any other rigid plastic or polymer tube (e.g., TEZEL ^TM (ETFE) tube). In another embodiment, the channel 151 comprises at least one etched channel (open or closed) in the microfluidic transport system described in more detail below. For improved detection sensitivity, more than one channel may be used to provide a larger detection region 155. [ In another embodiment, the fluid channel 151 is formed through a known semiconductor processing technique. Those skilled in the art will appreciate that other configurations and configurations of fluid channel 151 may be applied under the present invention.

The transport medium is made up of various solutions, gases, or other media depending on the particular analytical object. For example, if the detection system of the present invention is used to detect the presence and / or binding of biological analytes, Dulbecco's phosphate-transfer vehicle saline (d-PBS) It can be used as a transfer solution to provide an environment similar to the natural environment of a biological molecule. DMSO, NaPO4, MOPS, phosphate, citrate, glycine, tris, autate, borate, and other materials may be used in other embodiments of the present invention, as will be appreciated by those skilled in the art.

The fluid channel 151 includes a detection area 155 where the probe 230 irradiates the sample. The area of the detection region 155 includes the material composition and structure of the fluid channel 151, the concentration of the analyte, the intended detection time, the rate at which the test sample passes through the channel, and other factors that one of ordinary skill in the art would expect And is influenced by several factors. In embodiments where an immobilized detection scheme is employed in the detection zone 155, the binding surface is formed within the detection zone 155, the area of which is determined by the chemical nature of the binding surface, the material and shape of the binding surface, It will be influenced by predictable factors. Exemplary sizes of bonding surfaces are 10 ^-1 m ² , 10 ^-2 m ² , 10 ^-3 m ² , 10 ^-4 m ² , 10 ^-5 m ² , 10 ^-6 m ² , 10 ^-7 m ² , 10 ^{-8 m 2, 10 -9 m 2} , 10 -10 m 2, 10 -11 m 2, 10 -12 m 2, 10 -13 m 2, 10 -14 m 2, 10 -15 m 2 or between the It will be the size. Preferably, most of this range is achieved in small volumes by the use of helical or porous surfaces. A small number of the ranges listed above will be typical of systems and microfluidic devices manufactured using semiconductor processing techniques. Instead, the detection region 155 may be altered so that it can be used in other diagnostic applications, such as chips for proteomics known in the art. The size and shape of the detection area need only be capable of signal transmission and analysis target passing, and other limitations are described below.

In the illustrated embodiment of the detection assembly 100, the fluid control 156 is connected to the storage container 157. [ The fluid control 156 uses the fluid from the reservoir 157 to move the test sample through the channel 151 and this requires less test sample than simply pumping the sample through the channel .

Using the second storage vessel 158, the storage vessel 157 can store a second analyte or test sample for mixing with the test sample. In this embodiment, the fluid control 156 may be configured to quickly mix the two test samples and supply the mixture to the detection area 155. By such a technique (as is known in the fluid motion system field, as known as stopped-flow kinetics), the change in signal response as a coupling event occurs between analytes of two test samples So that the observer can see and record it. This data can also be used to measure the kinetic dynamics of the binding events that occur between the analytes of the two samples. Varian Australia Pty Ltd, which is shipped to Victoria, Australia. Such as the model number Cary 50, distributed by the manufacturer, may be employed within the present invention or within the integrated detection assembly 100. [ For more information on fluid technology systems, please visit www.hi-techsci.co.uk/scientific/index.html .

And other components for controlling the flow of the test sample through the channel 151. Fluid control 156, fluid reservoirs 157 and 158, and other components associated with fluid transfer may be configured as separate components of fluid transfer system 150, or may be partially or fully integrated.

Figure IB shows another embodiment of a fluid delivery system. As shown, the fluid delivery system 170 includes a drive circuit 172, a drive member 174, a syringe assembly 176, and a fluid channel 178. Preferably, the fluid delivery system 170 is assembled and operated outside the measurement probe assembly 230. However, in other embodiments of the present invention, some or all of the components of the fluid delivery system 170 may be integrated into the metrology probe assembly 230.

In operation, the drive circuit 172 receives an instruction from the operator and provides a test sample 175 to the measurement probe assembly 230. The instruction may be an instruction to provide a specific amount of sample and / or to provide the sample at a specific rate. Depending on the response, the drive circuit 172 advances the drive member 174 (which may be a thread in one embodiment), thereby advancing the plunger of the syringe assembly 176. The plunger feeds an intended amount of sample to a fluid channel 178 (which in one embodiment is a PTFE tube), and the sample is transported through the channel to a detection region that is illuminated by a test signal emitted from the measurement probe.

When the present invention is used to detect or identify molecules in a liquid phase, several techniques may be used to separate samples that are different from each other. In one technique, a small amount (e.g., 5 [mu] l) of one or more sample plugs may precede or follow a larger volume (e.g., 15 [mu] l) of the main sample plug. The shorter sample plug allows the main sample plug to be insulated from sample concentration changes. The air plug may be introduced as a spacing material before and / or after the sample plug to further minimize mixing of the fluid or to minimize concentration variations. The air plug may also be used as an indicator to inform the test system (or operator) of the location of the test sample in the fluid channel.

In another technique, a transfer medium (which may be air) may be used as a spacing material to separate test samples that are different from each other, the manner of use of which is described in US Pat. Nos. 6,033,456, 5,858,187, 5,126,022, Technology, and other detection systems. When operated in this manner, the spacing material is introduced into the channel after the first test sample, and additional test samples (or samples) are introduced into the channel after the spacing material, and the test samples are passed through the channel As a result, a series of disparate test samples separated by the spacing material are transported through the channel. Such a transfer allows measurements to be made on the detection area 155 separately for each test sample as each test sample passes (or is temporarily stopped) through the detection area 155, by causing the test samples to migrate without intermixing.

A pumping device of a suitable size may be used in the fluid movement control system of the present invention. Such pumps are described in " Fabrication of a Pump for Integrated Chemical Analyzing Systems " by Shoji et al. In Eletronics and Communications in Japa, Part 2, 70: 52-59 (1998) (MEMS) such as "Normally closed microvalve and pump fabricated on a silicon wafer" described in Esashi et al., Sensors and Actuators, 20: 163-169 (1989); Or Moroney et al., Proc. Electro-Pumps as described in "Ultrasonically Induced Microtransport", published in MEMS, 91: 227-282 (1991). However, in many microfluidic devices, the pump (fluid movement control) does not have a moving part and moves the liquid using electrodes and electrostatic force. At least two types of such electrode-based pumping are described under the name " ElectroMagnetic Fluid Dynamics Pump " (EHD) and " Electrosomisis " (EO). The EHD pumping is described in detail in "Sensors and Actuators," edited by Bart et al., Sensors and Actuators, A21-A23: 193-197 (1990), "Microfabricated Electro- A 29: 159-168 (1991). &Quot; A Micromachined Electrohydraulic Dynamic Pump " EO pump is Dasgupta (Dasgupta) etc. Anal. Chem. 66: 1792-1798 (1994), which is incorporated herein by reference in its entirety. [0004] The term " electronic osmotic pressure: A Reliable Fluid Propulsion System for Flow Injection Analysis " A practical consideration for operating a pump, such as EO and EHD, which moves fluids by electrodes in a liquid supply system is disclosed in PCT Application No. WO95 / 14590. The WO 95/14590 application describes a theoretical consideration which provides an appropriate electrode, a method of forming such an electrode, and additional guidance on how to operate such a pump. Additional guidance on fluid movement in microfluidic devices may refer to a number of patents pending in the field. (0.001 μl or less) when the fluid movement control utilizes the electrophoretic movement of the fluid as described in US Pat. Nos. 6,033,546, 5,858,187 and 5,126,022, can do.

Microscopic system devices (including analytical chips with channels that allow for various types of fluid movement, such as introduction of test samples, mixing with additional test or reactant, and separation of mixed mixture, as well as sampling devices, temperature controls, Detection equipment and related equipment such as analytical electronic equipment) have been developed. Because the present invention is directed to a new detection system that can be applied to current (and modified) fluid delivery systems, the use of mechanical pumps and lafge- scale device and the microfluidic system are not themselves an aspect of the present invention, and the device and the microfluidic system are devices and methods by which the detection technique of the present invention can be easily applied. For the basis of these known fields, reference may be made to the publication publication on fluid handling, such as the aforementioned cited patents (and their background patents and other references cited therein). The present disclosure primarily describes a combination of such known systems with the detector and analysis system described below, and such a combination provides new results as described below and as shown in the following examples. However, in order to explain how the method of the present invention is applied to a previous fluid delivery system, a general description will be given.

For example, when a substantially substantially uncharged sample is used in an electro-migration microfluidic device, the electric field of the microfluidic device is adjusted to facilitate the movement of the test sample as a plug, Intercalating materials with relatively high ionic strength are often used as compared to conventional materials. In some cases, it is useful to use a gap fluid that is not substantially mixed with the test sample. However, when the fluid moves relatively quickly (thus there is no time required for diffusion to produce an effect of mixing with the adjacent test sample so as to adversely affect the intended measurement), it is usually from 0.1 to 500 mu m Mixing is not a problem as the entire contents of the microfluidic channel having a cross-sectional diameter of 5 to 50 mu m migrate as a continuous flow without turbulent mixing. The immiscible spacing material is more common in operations large enough to cause turbulent mixing. The most common spacing in such a system is a gas bubble, where the fluid movement control physically pumps the fluid primarily in the form of a peristalic pump. Such large-scale systems were once common in medical analysis systems (for example, commercial clinical analysis systems known as SMA1260), but a significant portion is being replaced by microfluidic systems. However, as disclosed in USP 5,992, 820 entitled " Flow Control in Microfluidic Device by Controlled Bubble Formation ", microfluidic systems also use bubbles as spacers.

When such a wired system is to analyze a series of multiple test samples (although more than one series of measurements may be made in one physical device), the system may be configured to cross a first channel in a fluid delivery system And typically includes one or more channels. In the system, the movement of the fluid in the second fluid channel comprising a series of test mixtures separated by a test mixture or a spacing material is controlled individually. The test mixture (s) from the second fluid channel is introduced into the test sample in the first fluid channel sufficiently upstream from the detection region 155 so that the test mixture 1 < / RTI > fluid channel. Thereafter, an additional change in the test signal determines whether an interaction has occurred. If the interaction is known to occur between two molecular species (such as when two species of molecules are two elements of a known binding pair), the test signal may be a molecular analysis target or agent in the first channel May be used to detect the presence of a test mixture in two channels. Also, as is common in drug screening, molecules not known as elements of a binding pair may be tested for binding (or other interactions such as enzymatic reactions with substrates).

In a preferred arrangement, the fluid delivery system also includes an automatic sampling device (not shown) that introduces a series of test samples into the detection channel separated by a spacing material, which may be a liquid (e.g., a clean transfer medium) or a gas . ≪ / RTI > The handling of a large number of test samples is disclosed in numerous patents and other publications relating to the aforementioned microfluidic systems and in known automation systems. For example, multiple test sample wells may be provided on a single chip, and an automated pipetting system may supply different test samples to each test sample well. In such a detector assembly, each channel leads from each well to a common location where the detection channel is initiated (or a slightly earlier position if further reactants or mixing with the test mixture is required) To a common position. Thereafter, the fluid control continuously moves the test sample from the common position to the other part of the device. Automated handling of other types of small test samples for use in other types of detection devices is disclosed which may be readily adapted to supply test samples to the device of the present invention; See, for example, USP 4,468,331 entitled " Method and System for Liquid Chromatographic Separation. &Quot;

Measuring probe

The measurement probe is activated to oscillate the test signal toward the test sample occupying the detection area 155. [ The dielectric properties of the test sample modulate the test signal and at least some of the modulated signal is reflected towards the measurement probe 230. The reflected modulated signal is recovered by the measurement probe and delivered to a detection system, described in more detail below. The detection system then compares the incident test signal to the reflected modulated signal and generates a return loss or " S ₁₁ " response, as is commonly referred to in the microwave engineering arts. Since the dielectric properties of most molecular events are different, the return loss response of each analyte can also be distinguished and can serve to detect and identify molecular events in unannounced test samples.

The measurement probe 230 may be configured in a number of different forms suitable for propagating test signals of the desired frequency (s). In certain embodiments, the measurement probe 230 is a coaxial cable, but microstrips, strip lines, suspended substrates, slot lines, coplanar waveguides, waveguides, etc. may optionally be used in the present invention. Some exemplary embodiments of various forms include the Microwave Engineering Fundamentals of RE Collins, published by McGraw-Hill Publishers in 1966; Les Besser and Associates, Inc. Of S. See S. March, 1986 Microwave Transmission Lines and Their Physical Realizations .

The frequency or frequencies at which the measurement probe operates will vary depending on the structure of the probe 230, but will generally be 10 MHz to 110 GHz. In certain embodiments in which the measurement probe is coaxially configured, the operating frequency will typically be 45 MHz to 20 GHz. Those skilled in the art will appreciate that probes constructed in accordance with the present invention may be used in other frequency ranges in other embodiments of the present invention.

FIG. 2A illustrates a first embodiment of a measurement probe 230 implemented in a resonant coaxial form in accordance with an embodiment of the present invention. As shown, the probe 230 has two ports: a probe head 230a and a connection end 230b. In a particular embodiment, the probe head 230a is an open-ended coaxial cross-section and the connection end 230b is a coaxial-type connection, and one embodiment of a coaxial-type connection is an SMA connection. Those skilled in the art will appreciate that other circuit configurations (such as microstrips, striplines, slotlines, waveguides, waveguides, etc.) and other ends (shorted or loaded ends) ≪ / RTI >

The probe 230 includes two coaxial portions 238 and 238 each having a central conductive portion 235, a dielectric isolation portion 236, and an outer conductive portion 237 (typically used to provide a ground potential reference) 232 and 234. The first part consists of a pro-head 230a as described above and a first gap end 232a located opposite the head, (Preferably conductive) 231 is attached at the same height as the outer conductive portion 237 of the probe head 230a (preferably solder, conductive epoxy or other conductive Attached by an attachment material).

The second portion 234 has a configuration similar to the first portion 232 having the dielectric insulating portion 236 located between the central conductive portion 235 and the outer conductive portion 237. The second portion 234 further includes a second gap end 234a and a connection end 230b located opposite thereto. The second gap end is implemented as an open-end cross-section of the coaxial cable. The connection end 230b is instantiated as a connection (SMA-shaped in a specific embodiment) that is operated to be connected to a molecular detection system, which is further described below. In an exemplary embodiment, the first portion and the second portion each comprise an RG401 type semi-rigid coaxial cable, wherein a cable of greater or lesser diameter may be used. The length of the first portion 232 can be calculated to about half the wavelength of the desired resonance frequency, as will be described in more detail below. In this description, the first portion is about 4 inches, which corresponds to about half of the wavelength at the 1 GHz test frequency.

In a particular embodiment of the present invention, the probe 230 includes a diversion member 233 that is adjustably engaged between the first and second gap ends 232a and 234a to provide a variable gap distance therebetween . The gap provides a capacitive effect between the first and second portions 232 and 234 and is coupled with the electrical length of the first portion 232 so that the probe 230 Lt; RTI ID = 0.0 > (not) < / RTI > The switchable member 233 can be rotated to stretch or contract the gap between the first and second portions 232 and 234 thereby changing the resonant frequency of the measurement probe 230 to a desired frequency.

In an exemplary embodiment, the gap distance may vary from 0 inches to 0.050 inches, and may also be used in other embodiments of the invention to have other gap sizes that can match the resonance response to a desired frequency point. The seeking resonance response is when the reflected portion of the test signal is substantially null, i. E., The recovery loss or the magnitude of S ₁₁ is minimized. As described below, the presence of an analyte will significantly modify the coplanar signal response, thereby enabling detection and identification of the binding and / or substructure of the analyte.

Preferably, the switching member 233 is a hollow tube made from a material having a relatively large conductivity to maintain the ground potential between the first and second portions at the operating test frequency. The switching member may also include an inner screw 233a that engages an outer screw 238 disposed in the outer conductive portion of the first and second portions near the first and second gap ends 232a and 234a . In another embodiment of the present invention, the diversion member 233 may be omitted, in which case the first and second ends 232 and 234 may comprise one continuous coaxial transmission line structure.

Those skilled in the art will appreciate that the probe 230 may include other circuit elements to provide different signal responses in other embodiments of the present invention. Other circuits may also be included along the probe 230, such as a lumped element type, a dispersed form, or a combination of both. For example, an impedance matching circuit and / or a transmission medium amplification circuit may be employed at the connection end, along the first and / or second portions 232 and 234, or in the probe head 230a, have. Optionally or additionally, an impedance matching circuit and / or one or more output amplifiers may be included to enhance the output signal.

In one embodiment of the present invention as shown in Figure 1, the probe head 230a is disposed adjacent to the test sample but physically separated by the intermediate material. In this example, through an electromagnetic coupling, an incident signal is delivered to a test sample, and the reflected signal is recovered from the test sample. An intermediate material (s) which physically separates the probe head (230a) from the test sample is PTFE, alumina, glass, sapphire, diamond, Lexan ^®, polyimide, or high-frequency circuits, such as other substances used as dielectric materials in the field of design Solid matter; Or other known materials having a relatively high signal transmission rate at a given operating frequency. Optionally or additionally, the liquid and / or gaseous material having a relatively high degree of test signal transmittance may also be used as the material to be tested, Intermediates can be constituted.

The thickness and dielectric properties of the intermediate may vary depending on the oil-filled system employed and the measurement probe used. For example, in systems with large separation distances, a low loss, high dielectric material is desirable to provide maximum coupling between the test sample and the probe 230. In systems with relatively small separation distances, high loss and low dielectric constant materials are permitted. In a particular embodiment, where the TPFE tube is a channel 151 having a 0.031 inch inner diameter, a 0.063 inch outer diameter, a 0.016 inch thickness, and a dielectric constant of about 2, the separation distance is about 0.016 inch, which is approximately the wall thickness of the tube. In other detection assemblies, the separation distance may be 10 ^-1 m, 10 ^-2 m, 10 ^-3 m, 10 ^-4 m, 10 ^-5 m, 10 ^-6 m, and in some cases A metal signal path member having a thin polymer layer on the side of the test sample acting in the etched channel and on the fourth side of the channel, etc.) may be as small as 10 ^-9 m. By increasing the separation distance or increasing the detection area 155, the sample volume or analyte concentration will serve to improve detection sensitivity. The separation material as described above may be a solid material and may alternatively (or additionally) consist of a liquid or gaseous material or a combination thereof.

FIG. 2B illustrates a lid 240 that is disposed opposite the probe 230 to allow a test sample to be placed between the probe and the probe. In one embodiment, the lid 240 is comprised of a conductive material (brass, copper, aluminum, etc.) that is set to ground potential at the operating frequency to provide a ground plane through which the electromagnetic radiation radiated from the central conductive portion 235 terminates. The lid 240 additionally provides a shielding function from an external source that may affect the measurement.

The central conducting portion 235 of the probe head 230a or the measuring probe 230 extends into the channel 151 such that the central conducting portion 235 is in direct contact with the test sample flowing along the channel 151. In other embodiments, do. In this embodiment, the central conducting portion 235 is formed of a material that can support propagation of the test signal and may not negatively affect the analyte. Such materials may include metals such as gold, indium tin oxide, copper, silver, zinc, tin, antimony, gallium, cadmium, chromium, manganese, cobalt, iridium, platinum, mercury, titanium, aluminum, lead, iron, tungsten, Rhenium, osmium, thallium or the like alloys. However, the present invention is not limited thereto. These same materials may be used to form outer probes with other materials that will be apparent to those skilled in the art.

2C and 2D are a cross-sectional view and a plan view, respectively, of a second embodiment of a probe according to the present invention. 2c, the measurement probe 250 includes a first coaxial portion 251, a bracket 252, an attachment platform 253, a contact ring 255, a tuning gap 256, A portion 257, a conductive grounding tube 258, and a metric system shelf 259.

The first coaxial portion 251 is coupled to a signal detection portion (not shown) and a signal source described below. In one embodiment, the first coaxial portion is an RG401 semi-rigid cable. Those skilled in the art will appreciate that other types of semi-rigid cables and other transmission structures may be used in other embodiments of the present invention.

The first coaxial portion 251 is held stationary in the bracket 252 and extends into the gap region 254 near the bottom of the oil system shelf 259. The contact rings 255a and 255b are selectively attached around the outer surface of the first coaxial portion 251 to provide ground conductivity between the first coaxial portion 251 and the inner surface of the grounding tube 258. [ In another embodiment, the contact rings may be high conductive springs, and other structures may also be used. In another embodiment, the outer surface of the first coaxial portion 251 may be sufficiently in contact with the inner surface of the grounding tube 258 (copper in one embodiment), so that the contact ring 255 is not required.

The first and second coaxial portions 251 and 257 are electrically actuated and separated by a tuning gap 256 which provides the resonance response described above. In the illustrated embodiment, the second coaxial portion 257 is secured within the grounding tube 258 within the oil system shelf 259. The first coaxial portion 251 is inserted into the gap region 254 such that the outer surface of the first coaxial portion 251 is in electrical contact with the inner surface of the grounding tube 258 so that a continuous ground potential is provided therebetween do. The tuning gap 256 formed between the first coaxial portion 251 and the second coaxial portion 257 may be shortened or elongated by moving the bracket 252 upward or downward. It will be appreciated that the position of the second coaxial portion 257 in the conductive grounding tube 258 may be adjusted and, alternatively or additionally, the first coaxial portion 251 may also be adjusted. The attachment platform 253 is attached to the housing system shelf 259 such that the first coaxial portion 251 can be inserted into the bracket or the first coaxial portion 251 can be detached from the bracket, . In one embodiment, the bracket 252 is driven by a motor and is contained within a precision motorized relay stage assembly distributed by Newport Corporation of California Irvine.

Figure 2e illustrates an embodiment of a measurement probe implemented in non-resonant coaxial form in accordance with the present invention. In this embodiment, the probe 280 includes an open-ended coaxial line 281 portion, an interactive fixture base 283, an interactive substrate 285, a fluid interface (interface) portion 287, And one or more fluid conduits 289 extending therefrom. In a particular embodiment, the probe 280 is coupled to a vector network analyzer or similar test equipment capable of measuring incident and reflected signal characteristics.

The fluid tube 289 allows the sample to flow into the fluid interface 287. The interactive substrate 285 may be selectively used to separate the supplied sample from the end of the coaxial portion 281. [ The interactive substrate 285 may be comprised of a variety of materials such as, for example, glass, quartz, polyimide, PTFE or silicon dioxide, gallium arsenide, or other materials used in semiconductor processing. In another embodiment, the interactive substrate 285 is removed, and the sample is in direct contact with the coaxial portion 281. The base fixture 283 is used to align and fix fluid interface 287 (and its substrate 285 if an interactive substrate 285 is used) with the open end of coaxial portion 281. In certain embodiments, the base fixture is aluminum, and other materials may be used in other embodiments of the present invention.

During operation, the volume of the sample (which may be a non-analyte buffer used for baseline response) is introduced into fluid interface 287 through fluid conduit 289. A test signal is then applied to coaxial portion 281 from test set 290. As discussed above, the dielectric properties of the supplied sample will modulate the incident signal. The open-ended structure of coaxial portion 281 reflects at least a portion of the modulated signal towards a test signal from which the modulated signal is recovered. Typically, the modulation represented by a change (or lack) of the amplitude and phase of the incident signal represents the presence (or absence) of a molecular event.

If the above-described metaphoric transport system is provided, one of ordinary skill in the art will readily be able to find biology and chemistry related literature for selection of molecular samples to work with. General guidance on biological systems is available from Greene Publishing Associates, Inc. And John Wiley & A joint venture of the Current Protocol editor, F.M. Current Protocols in Molecular Biology , such as FM Ausubel et al., 1997; The Benjamin / Cummings Publishing CO., Located in Menlo Park, California. Watson et al., (1987) Molecular Biology of the Gene, Fourth Edition ; Molecular Biology of Cells from Alberts et al. (1989) published by Second Edition Garland Publishing, New York; Merck & The Merck Manual of Diagnosis and Therapy . Product information provided by manufacturers of biological reactors and experimental equipment also provides useful information for measuring biological systems. Such manufacturers include, for example, SIGMA chemical company (Missouri, St. Louis), R & D systems (Minnesota Minipolis), Pharmacia LKB Biotechnology (Piscataway, NJ), CLONTECH Laboratories, Inc. , Fluka Chemica-Biochemika Analytika (Switzerland, Fluka Chemie AG), Applied Biosystems (Foster City, Calif.), And others who are skilled in the art, such as Aldrich Chemical Company (Wisconsin Milwaukee, Wisconsin Milwaukee, GIBCO BRL Life Technologies, Inc. Gattusburg, There are many other companies known to you.

Biological samples are obtained from patients using known techniques such as venipuncture, lumbar puncture, fluid samples such as saliva or urine, or tissue biopsy. Blood and tissue samples can easily be obtained from a livestock handling facility if the biological material is obtained from a non-human, such as commercially viable livestock. Similarly, the plant material used in the present invention can be readily obtained from agriculture, horticulture and other sources of nature. Alternatively, the biological sample may be obtained from an in vitro source such as a cell or blood bank, or cell culture, which stores cells and / or blood. Cell culture techniques for use as a source of biological material are well known to those skilled in the art. Freshney's Culture of Animal Cells, a Manual of Basic Technique, Fourth Edition (1994) by Wiley-Liss, New York, provides a general overview of cell culture.

Chemical properties of the detection region

In embodiments where the analyte binding or structure is detected without binding to the surface of the detection region 155, the chemical nature of the detection region 155 generally depends on the wall of the channel (Teflon ^® tube in one of the illustrated embodiments) Is selected so as not to react with the passage of the test sample. In another embodiment where detection is performed by binding to a detection area surface, one or more test samples may be delivered by the fluid delivery system for detection of a potential binding interaction between the anti-analyte and the analyte , The detection area surface becomes active and combines with the anti-analyte. In such a case, the surface of the detection region 155 is made of a material having good molecular binding properties. The ligand can be bound to the wall of the channel directly or indirectly through another molecular structure. The possible types of binding interactions identified include protein / protein interactions, DNA / protein interactions, RNA / protein interactions, nucleic acid hybridization, base pair mismatch analysis, RNA / RNA interactions, tRNA interactions , Enzyme / substrate systems, antigen / antibody interactions, small molecule / protein interactions, drug / receptor interactions, membrane / receptor interactions, and lipid / protein interactions. However, the present invention is not limited thereto. The chemistry of the attachment may include only one kind of molecules attached to the surface, and may be an array of several different molecules attached to the surface, or a plurality of binding events between species directly attached to the surface, Lt; / RTI >

IV. Exemplary molecular detection systems

Figure 3A illustrates one embodiment of a molecular detection system 300 in accordance with the present invention that includes a signal source 302, a signal detection unit 304, and a detection assembly 100 . The detection assembly 100 may be coupled to the signal source 302 and the detection portion 304 via a signal path such as, for example, a cable, a transmission line, or other media 310 that may support the propagation of signals at the intended test frequency. . The source 302 is actuated to deliver an incident test signal 312 towards the detection assembly 100. The incident test signal is modulated by the dielectric properties of the test sample and at least a portion of the modulated test signal is reflected toward the resonant probe 230. The reflected signal is coupled to the probe and compared with the incident signal to produce a signal response. Those skilled in microwave engineering community is one of these measures will recognize a port S ₁₁ reflectometry.

In a particular embodiment, signal source 302 and signal detector 304 will be included in a vector network analyzer test set, for example, model numbers 8510 and 8714 of Agilent Technologies, Inc. of Palo Alto, California . Other high frequency measurement systems, such as a system that provides signal information based on transmitted and reflected signals, or a scalar network analyzer, may also be used in other embodiments of the present invention. Although the exemplary detection system 300 is described as a single-port reflection measurement system, in other embodiments of the present invention, a modulated signal that has been reflected using a further signal source (and / or detector) Signal (referred to in the art as S ₂₁ or " pass " measurement).

In one embodiment of the invention, the resonance probe 230 is used in conjunction with the molecular detection system 300 to detect the presence or absence of molecular events in a test sample. The method first measures the baseline response (using air, buffers, or other non-analyte media) for the resonance probe 230, and then measures the baseline response when molecular events are introduced into the detection zone 155 And measuring the change. This method is illustrated in more detail in FIG. 3B.

First, in step 320, the probe 230 is designed to have a resonance response S ₁₁ in a predetermined frequency or in the vicinity of the frequency. In a particular embodiment, this step is achieved by making the length of the first coaxial portion 332 to be half of the wavelength at the intended resonant frequency, using the following equation, as is known to those skilled in the art:

lambda / 2 = c [2 x f _des x _r ^1/2 ]

At this time:

? / 2 = length of first coaxial portion 332 (in meter)

c = speed of light 3 x 10 ⁸ m / s

f _des = desired resonance frequency (Hz)

? _r = relative dielectric constant of insulating material 336

In the disclosed embodiment, where the intended resonant frequency is 1 GHz and the insulating material (Teflon ^® ) 336 dml relative dielectric constant is about 2.1, the length of the first coaxial portion 332 is chosen to be 4 inches. Those skilled in the art of high frequency circuit design will appreciate that the described design techniques and the resulting resonant signal response are only one of many possibilities. In other embodiments, resonant signal responses may be obtained using other known circuit designs (such as short-circuit 1/4-wavelength lines, etc.).

In a next step 321, the probe head 230a is disposed adjacent to the detection region 155 and allows it to be electromagnetically coupled to that region. Typically, this operation involves positioning the probe head 230a as close to the detection area 155 as possible. As described above, the probe head (230a) and an intermediate separating the detection area 155 is PTFE, alumina, glass, sapphire, diamond, Lexan ^®, polyimide, or any other, which is used as a dielectric material in a high-frequency circuit design field Solid materials such as materials; Or other known materials having a relatively high signal transmission rate at a given operating frequency. In certain embodiments, the intermediate may be an electrically insulating material such as glass, PTFF or a modification thereof, quartz, silicon dioxide, gallium arsenide, or other materials as described above. Optionally or additionally, liquid and / or gaseous materials having a relatively high degree of test signal transmittance may also be used.

Subsequently, at step 322, the fluid delivery system 150 feeds the untransformed transport medium to the detection area 155. [ At the next step 323, a test signal is coupled to the detection region 155 and the resulting baseline response is obtained. In the exemplary embodiment described herein, the baseline response is an S (n) obtained by comparing the amplitude and phase data of the reflected signal 312 and 314 with the incident signal in the case where a buffer without analyzing occupies the detection area 155 ₁₁ responses. In other embodiments of the invention, other signal responses may be obtained using known data comparison techniques. During testing, fluid movement may be continuous (e.g., when a single frequency or a small frequency group of signals is measured, or when the detection sensitivity is high, the measurement time is short), or fluid flow may be interrupted. Optionally, for increased sensitivity, multiple scans may be performed and averaged.

In a next step 324, the tuning element is adjusted until it reaches the magnitude of the baseline response at the low point illustrated by the trajectory 334 in Figure 3c (clockwise or counterclockwise, or as shown in Figure 2d Forward using a motorized drive assembly as described above). The frequency point at which the baseline S ₁₁ response reaches the minimum point is called f _res . This point represents the frequency at which the smallest amount of signal intensity is reflected back to the detection system 300. Typically, due to the dielectric effect of the sample and device located in the open-ended portion of the probe head 230a, the resonant frequency f _res deviates from a predetermined frequency f _des . The frequency (f _des ) point is selected at a frequency or at a frequency in which the molecular event is expected to exhibit a sharp change in the dielectric properties of the sample. Although 1 GHz was selected in the illustrative embodiment to illustrate the detection process, in the present invention, the dielectric properties of many molecular events are 10 MHz, 20 MHz, 45 MHz, 100 MHz, 250 MHz, 500 MHz, 1 GHz, 2.5 At a frequency between 1 GHz, 5 GHz, 7.5 GHz, 10 GHz, 12 GHz, 15 GHz, 20 GHz, 25 GHz, 30 GHz, 40 GHz, 50 GHz, 60 GHz, 80 GHz, 100 GHz, Detection is possible. Those skilled in the art will appreciate that other frequencies and frequency ranges may be used in alternative embodiments of the present invention.

The recovery loss or S ₁₁ response in the vicinity of the resonant frequency (f _res ) point will vary considerably sharply. To obtain a more gradual response in the vicinity of the resonant frequency point, the probe can be tuned (by rotating the tuning element) away from the resonance point ( _fres ). This step (and the tuning element 333 itself) may be omitted in another embodiment (e.g., a manufactured IC chip) that represents a tuned S ₁₁ response.

In the next step 325, the test sample is introduced into the detection region 155 using any of the means described above. At step 326, the test signal is coupled to the detection area 155 and the resulting test sample response is obtained.

In the exemplary embodiment described herein, the baseline response is an S ₁₁ response made in the presence of a fluid channel and an unexplained buffer. Those skilled in the art other measurement - it will be appreciated that the (2 S, such as port ₂₁ measurements) can be used as a baseline response. It is also possible to measure the baseline response using different conditions, for example, where the buffer contains a known analyte, or the response is made in the absence of a buffer and / or fluid channel. It will be appreciated that a large number of baseline responses is possible. As described above, the test sample may remain stationary during measurement or may be moved through the detection area 155. [

FIG. 3C shows a test sample response 335 when a molecular event occurs within the detection region 155. FIG. As shown, the test sample response 335 represents a shallower null. Electrically, the dielectric properties of the analyte in the test sample alter the resonance signal response and reduce the amplitude of the test sample response 335 relative to the baseline response 334. [ In an alternative embodiment, the test sample response 335 may be such that the frequency at which the minimum S ₁₁ response occurs is shifted up or down f _res and, in some embodiments, both the amplitude and the frequency of the baseline response are changed will be. If a binding event occurs within the detection region, the response may be monitored in real time (i.e., as the reaction occurs). Those skilled in the art will appreciate that the illustrated test sample response 335 is but one example of a possible response and that other responses can be observed under the present invention. For example, in some embodiments, a test sample may make a deeper null than a buffer resonance point. This is for example when the initial baseline response 334 is tuned away from the minimum resonance point.

In a following step 327, a determination is made whether the baseline and test sample responses 334 and 335 differ by more than a predetermined amount. This step compares the amplitude, frequency, and / or phase data of the baseline and test sample responses 334 and 335 and indicates that a change has occurred when the difference exceeds a predetermined or pre- . An exemplary amount at _fres at 1 GHz is 0.1 dB, 0.5 dB, 1 dB, 3 dB, 5 dB, 10 dB (or between those values) of amplitude; Phase is 1 degree, 10 degrees, 25 degrees, 45 degrees, 90 degrees, 180 degrees (or between them); Frequency is 1 KHz, 3 KHz, 5 KHz, 10 KHz, 1 MHz, 10 MHz, 100 MHz (or between); Or a combination of two or more of such quantities. The above-mentioned frequencies may be increased or decreased according to a higher or lower f _res frequency. For example, a preprogrammed / predetermined frequency for f _res of 10 MHz would be 10 Hz to 1 MHz.

If the difference between the baseline and the test sample response does not exceed a predetermined amount at step 327, the detection system 300 indicates that no molecular event has been detected within the frequency range of f _start to f _stop 328). Such indication may include, for example, conveying the above-described message to the user, graphically displaying the measured amplitude, frequency and / or phase difference between response 334 and response 335, measuring data or other output means And can be delivered to the user in a number of ways, such as by providing. It should be noted that the frequency range of f _start to f _stop described may include two or more smaller frequencies that can expect detection of a combining event or infrastructure.

If the difference between the baseline response and the test sample response exceeds a predetermined amount at step 327, the detection system 300 indicates that the molecular event has been detected within the detection area 155 (step 329). This indication is communicated to the user in a manner similar to that described above. In a preferred embodiment, the test sample response 325 is stored for later retrieval and comparison with other test sample responses (step 330).

Information on the identification of detected analytes can be obtained in a number of ways. If the detection region 155 is operative and is specifically bound to a particular analyte, identification of the bound analyte can be determined therefrom.

In another embodiment where non-specific binding occurs within the detection region 155, or molecular binding does not occur, the identification of the detected binding events results in a multi-step process. First, a known analyte is added to the buffer and fed to the detection region of the fluid channel. Next, using the measurement probe and methodology described above, the signal response 335 of a known test sample is obtained and stored. The identifier of the analysis object (the name of the analysis object, the symbolic code or array, or the other identifier assigned) is successively associated with the test sample response 335 and stored in the database, retrieved from that database, . If another test sample response is closely related to the stored response 335, identification of the molecular event in another test sample can be made.

A step 331 is followed to determine whether another measurement is to continue. Such steps may be employed, for example, in automated molecular detection systems with high yields. If more than one measurement remains, the method returns to step 324, where another test sample is introduced into the detection area 155 using one of the steps described above. If no other measurements are needed, the measurement process ends at step 332. [

In the extension of the described embodiment, the detection system 300 may include 96 probes coupled to N measurement probes 230 _i , for example, one well in each of the 96 well microtiter plates . Each probe will be coupled to each N detection area 155 _i , for example, at the bottom of each well in the microtiter plate. In this embodiment, the detection system preferably comprises a 1 x N switch matrix for delivering a test signal to one of the N detection regions 155 _i , and a 1 x N switch matrix for transmitting the modulated signal from one of the N possible detection regions to the signal detection unit N x 1 switch matrix. In another embodiment, each probe (230 _i) and detection zone (155 _i) is to be designed to have different resonant frequencies, in which case all the detection areas (155 _i) is to be detected over a continuous frequency spectrum . Other configurations of detection system 300 will be apparent to those skilled in the art.

Figure 3D illustrates a method for detecting molecular binding events occurring in a test sample in accordance with the present invention. This process is initiated in a manner similar to that described above with respect to Figure 3b:

Designing the probe to have a resonance response at a frequency at or near a predetermined frequency, placing the probe head adjacent to the detection region of the channel, supplying a buffer that is not to be analyzed to the detection region, (Steps 340-343). ≪ / RTI >

In the next step 344, a first test sample including the first analysis target is supplied to the detection area. In a preferred embodiment, the analyte is supplied, for example, at a concentration of 1 x 6 mg / ml. A response 353 (shown in FIG. 3E) is obtained and stored (step 345). In the next step 346, a second test sample containing a second analyte, preferably having the same concentration as the first analyte (6 mg / ml in the illustrated embodiment) is supplied to the detection region. Preferably, the detection region is washed with a detergent to remove residues of the first analyte. This process will be performed using the spacing material described above. A response 355 (shown in FIG. 3E) to the second test sample is then obtained and stored (step 347).

In a next step 348, the first and second analytes are mixed into a third sample. Preferably, the first and second analytes are mixed at a concentration of 0.5x (3 mg / ml each in the example described) to maintain the overall concentration of analyte for the first and second samples being measured the same. Thereafter, a third sample is supplied to the detection region, and a response is obtained and stored (step 349). Alternatively, two analytes may be introduced and mixed in the detection region / adjacent to that region (e.g., using the stepped-flow system described above) to monitor changes in signal response in real time.

The detection of a binding event that occurs between the first and second analysis objects may include comparing a buffer baseline response, a first test sample response 353, a second test sample response 355, and a third test sample response 357 or 359 .

FIG. 3E illustrates an example of a baseline buffer response 351, a first test sample response 353, an example of a second test sample response 355, and a third sample signal response 357, ≪ / RTI > 359) corresponding to the binding state between the first analyte and the second analyte. As can be seen from the trajectories 353, 355 and 357, if the two analytes are not combined in the third test sample, then the third test sample response 357 is the first and second test sample responses 353 And 355, respectively. If the first and second analysis objects combine in the third test sample, the response 359 distinguishes the mean and size and frequency (as well as phase) of the two samples.

The test system calculates an average response of the given first and second test sample responses, calculates the difference between the calculated response and the measured test sample response, and determines whether the combination has occurred from the difference And the closer the measured response is, the more often the coupling does not occur). In a particular embodiment, a predetermined amplitude and frequency window is calculated for the calculated average value of the two responses. A measurement response occurring outside of a predetermined amplitude / frequency window represents a combination, and the measurement response occurring within that window implies a combination. The amplitude / frequency window at 1 GHz is defined by the amplitude at frequencies of 0.1, 0.5, 1, 3, 5, and 10 dB (or between these values) , 3 KHz, 5 KHz, 10 KHz, 30 KHz, 50 KHz, 100 KHz, 1 MHz, 3 MHz, 10 MHz, 100 MHz (or between). The above-mentioned frequencies may be increased or decreased according to a higher or lower f _res frequency. For example, a preprogrammed / predetermined frequency for f _res of 10 MHz would be 10 Hz to 1 MHz.

In another embodiment of the process shown in FIG. 3D, measurement step 345 includes readjusting the tuning element 233 such that the probe is tuned to the resonance response in the presence of the first test sample. This response is then stored and subtracted arithmetically from the second test sample measurement 347 and / or the combined test sample measurement 349.

Figure 3f shows a second method for detecting molecular binding events in a sample containing two analytes. This method is very similar to the method of Figure 3d except that in step 367 the first and second analytes are mixed in a 1 × concentration (ie, 6 mg / ml each) in the third sample .

In this embodiment, the absence of coupling can be detected by analyzing the change in resonance frequency. In particular, the third sample if the coupling between the first and second analyte in the did not occur, the signal response of the three samples is substantially f _res to the first and the sum of the total change in the resonant frequency point of the second sample response Will represent the resonance frequency. An example of the first, second and third sample responses is shown in FIG. 3g. If coupling occurs, the resonant frequency response of the third sample will vary significantly from the resonant frequency sum shown in FIG. 3g. The amplitude / frequency window as described above will be calculated from the measured responses of the first and second samples. A signal response occurring within a predetermined window indicates that no coupling has occurred between the two analysis objects.

It has been described above that by monitoring the change in the resonance signal response of the probe, the object to be analyzed is detected and identified using the resonance probe. Those skilled in the art of high frequency circuits and systems will appreciate that non-resonant probes (one embodiment shown in Figure 2e) can be used to detect and identify analytes. The process is very similar to the steps shown in Figures 3b and 3d, except that baseline and test sample responses are typically taken over a wide frequency range of a few hundred MHz or GHz. The wideband response offers the advantage of significantly changing the signal response over a wide frequency range by exploring the dielectric properties of the sample over a wide frequency range. A large change in the measured response can be used to indicate that a molecular event has occurred in the test sample. Also, if the signal response is associated with a molecular event, the response may be used to identify a specific molecular event in testing of the subsequent unknown sample.

FIG. 4A illustrates another embodiment of a molecular detection system including a domain measurement system 400. FIG. The system 400 includes a pulse signal source 410 and a detection unit 412 that is coupled to the detection assembly through a signal path such as a coaxial cable, transmission line or other transmission medium 420. Additional pulse sources and detectors may be used to provide full two-port measurement capability. In certain embodiments, the pulse signal source 410 and detector 412 may be integrated within a time domain reflectometer system, such as model number 11801, manufactured by Tektronix Corporation, located in Beaverton, OR. Other high frequency measurement systems, such as network analyzers having a time domain measurement mode, may also be used.

During time domain measurement, a pulsed incident signal 422 is generated and oscillated along the transmission line 420 towards the detection assembly 100. In one embodiment, the pulses are comprised of square waves, but other pulse shapes may be used in other embodiments of the invention. The dielectric properties of the molecule (s) in solution reflect some of the incident pulses toward the signal detection portion 412. The reflected signal 424 will exhibit a specific shape and / or time delay characteristic of the molecular dielectric property. Thus, the pulse shape and delay of the reflected signal 424 can be used to characterize and identify the molecule (s) in solution. Time domain test system 400 may be used in combination with a scalar or vector network analyzer or separately to identify one or more unknown molecules in solution.

As is known in the art, the dielectric relaxation frequency of a molecule is the rate of change in dielectric properties at the molecular level when an electric field is applied to the molecule. Like the dielectric properties of a molecule, the dielectric relaxation frequency is mainly determined by the structure unique to each molecule and the geometrical shape of the molecule. Thus, once measured, the dielectric relaxation frequency of a molecule can be used to identify the molecule.

The dielectric relaxation frequency can be determined by measuring the rate at which the analyte absorbs energy over the frequency. Figure 4B illustrates one embodiment of a system 450 for this type of measurement. The system 450 is similar to the time domain measurement system 450 shown in FIG. 2A and includes a detector 462 coupled to the detection assembly 100 and a pulse signal source 460. Additional pulse sources and detectors may be used to provide full two-port measurement capability. In a particular embodiment, the measurement system 450 is configured with a time domain reflectometer as described above wherein the input signal includes a pulse train that can adjust the pulse spacing. Those skilled in the art will appreciate that other devices capable of generating similar pulse trains and detecting corresponding resultant signals may be used in other embodiments of the present invention.

The incident signal 480 consists of separate pulse groups 482 and 484, each of which has two or more incident pulses and a different pulse interval. Pulse groups 482 and 484 are oscillated toward a portion of the transmission line along the transmission line, i. If the pulse group 482 has a pulse interval that is substantially equal to the dielectric relaxation period (inverse of the relaxation frequency) of the analyte, the analyte will continue to absorb less energy in the subsequent pulse. The decrease in signal absorption may be measured as the response 490 reflected at the detector 462. [ As another alternative measurement amount, the remaining signal energy can be measured at the detector 462. [

The pulse interval and the signal absorption change rate at which the change occurs can be displayed and can be used to identify and identify a combination event including an unknown analysis target and / or an analysis target. These system features may be used with or independently of the time and / or frequency domain test systems described above.

In all of the above systems, those skilled in the art will be able to scale these systems down to chip level using techniques such as Microwave Monolithic Integrated Circuits (MMICs). Such miniaturization systems can be easily extended to parallel systems capable of simultaneously detecting and measuring hundreds, thousands or even tens of thousands of mixtures. These systems are called " logic gates " that are switched by the coupling event itself by changing the characteristic impedance and hence the transmission and / or reflection coefficient, or by changing the bandpass characteristics of the circuit and using the circuit as an on / .

Ⅴ. Exemplary Uses

Using the systems and methods described above, the present invention can be used for a variety of applications. In one aspect, the invention can be used to identify binding events or infrastructures that include an analyte, such as a protein in a first binding step. In the calibration phase, the response of a large number of known proteins can be measured and stored. After introducing an unknown protein into the detection region, the dielectric properties of the system can be measured and the characteristics of the protein can be identified using the dielectric properties of the signal. Since the fingerprint response of each protein is stored, the unknown response can be compared to the stored response, and the unknown protein can be identified using the pattern recognition method.

In another embodiment, the invention can be used in a parallel measurement format. The device has a number of addressable channels, each of which can be independently searched. The test sample or samples are fed to the device and then measured and charaterized at each site response. For example, using this type of device, the presence of a particular nucleic acid sequence in a test sample can be measured and / or identified by attaching a unique nucleic acid sequence as an anti-ligand to the detection region or a portion thereof. When exposed to a test sample, the complement arrangement will be bonded at the appropriate point. The response at each point will indicate whether the array is joined. Such a measurement will also indicate whether the binding sequence is fully matched with the anti-ligand sequence or whether there is more than one mismatch. This embodiment can also be used to identify the types of proteins and proteins.

In another embodiment, the invention can be used to determine a standard curve or titration curve that can be used to measure the unknown concentration of a particular analyte, or to determine a ligand binding curve. For example, the antibody may be attached to the detection region. The device may be exposed to a variety of different concentrations of the analyte and responses to each measured concentration. Such curves are also known to those skilled in the art as dose-response curves. Unknown test samples may be exposed to the device and the measured response. The response may be compared to a standard curve to determine the concentration of the analyte in the unknown test sample. Similarly, the binding curves of different ligands can be compared to determine which of a number of different ligands have the greatest (small) affinity constant for binding to a particular protein or molecule.

In another embodiment, the present invention can be used for internal self-calibration against loss due to aging and other stability problems. For example, in an antibody-antigen system, by the present invention, the amount of active antibody in a test sample can be determined by measuring the primary response before exposure to an unknown test sample. The response is compared and the amount of active antibody remaining is determined.

Detection assemblies are used to obtain information about various properties of test samples, such as detection and identification of molecular binding events, changes in the dielectric properties of bulk test samples, classification of detected binding events, and the like. The sensing assembly also has self-calibration capability, which is useful in terms of quality control and assurance in use. These methods and capabilities are described in more detail below. Based on the described method and structure, one of ordinary skill in the art will be able to easily make improvements and additions.

Molecular structure detection

The present invention can detect the presence of a molecular binding event or the presence of a molecular structure in the detection region 155 of the detection system. Detectable join events include primary, secondary, and higher order join events. For example, the combination of two ligand / anti-ligand pairs can be detected by mixing two test samples. For example, a solution comprising a molecule of interest or a molecular structure is provided. The test signal propagates along the signal path. Optionally, a test signal is applied during or immediately after the mixing operation so that the signal response occurring as a result of the coupling event can be observed in real time. A test signal is retrieved and the response of the signal indicates the detection of an object, infrastructure or combination event.

The dielectric properties of the test sample contribute to induce any number of signal responses, each representing a molecular bond. For example, the dispersive nature of a test sample can vary widely across frequencies. In this case, the signal response represents a large change in amplitude and / or phase response over frequency when a molecular event occurs within the detection region, thereby detecting a molecular binding event or other time-dependent event after mixing of the test sample Lt; / RTI >

In another embodiment, the dielectric relaxation characteristics of the test sample in the detection region will be varied as a function of the pulse time (period) of the input signal. In this case, the test signal response will indicate a change in the absorbed energy or a change in some other parameter of the test signal, such as phase or amplitude, at or near the specific pulse time. By observing changes in absorbed energy or other parameters, a binding event can be detected. Other quantities such as characteristic impedances, propagation speed, amplitude, phase, dispersion, loss, permeability, susceptibility, frequency, and dielectric constant can also be indicative of molecular presence or binding events. In addition, important information about molecular properties may be determined by measuring the signal while the environmental changes in the molecular structure (such as changes in pH or ionic strength) are detected.

The above methods can be used to detect primary binding of an anti-ligand and ligand. Similarly, the method may also be used to detect secondary binding of the ligand and the anti-ligand. The method is not limited to the detection of primary or secondary binding events that occur along the signal path. Substantially, third and higher order coupling events occurring along the signal path or in suspension in solution can also be detected in this way.

For example, first, as described above, the primary binding event is detected and the signal response is measured. The primary binding event signal response is then stored and used as the baseline response. Next, the second molecular solution is added to the measuring apparatus. The detection steps are repeated to obtain a second signal response. Then, the second signal response and the baseline response are compared. A slight change or no change means that the two signal responses are very close together indicating that the structure and dielectric properties of the test sample have not been altered by the addition of molecules in the new solution. In this case, secondary bonding did not occur to a significant degree. If the result of the comparison is out of a predetermined range, then the structure and / or dielectric properties of the test sample have changed and accordingly exhibit a secondary binding event. The amount that can be used to represent the secondary coupling event is similar to the above-mentioned amounts, such as amplitude, phase, frequency, dispersion, loss, permeability, susceptibility, impedance, propagation rate, dielectric constant, and other factors. A third or higher order combination event can be detected using this method.

Other methods of detecting secondary or higher order binding events do not require prior knowledge of a particular primary binding event. In this embodiment, the measuring device is operated with known parameters at the measurement progress stage so that when a predetermined change in one of the parameters is detected, it is determined that, for example, a combination event or an event has occurred in terms of use . In this embodiment, preliminary measurement of the primary binding event is not necessary since the initial characterization is made at the time of manufacture or at design time.

Secondary binding events can also be obtained by detecting structural changes in the primary molecular structure. When the molecules begin to bond, there is a combination of molecular structures and other changes to the unbonded state. This change not only affects the dielectric properties of the primary binding molecule but also causes a change in the surrounding solution, and the change in such solution can be detected as described above. The amount monitored to indicate a change in the dielectric properties of the primary binding molecule includes the aforementioned quantities, such as amplitude, phase, frequency, dispersion, loss, permeability, susceptibility, impedance, propagation rate, dielectric constant, do.

Detect changes in dielectric properties of test samples

The detection system described herein may also be used to measure the dielectric change of a test sample as a resultant change in temperature, pH, ionic strength, and the like. This method is very similar to the joint event identification method described above, except that it is capable of detecting and quantifying changes in the dielectric properties of the test sample without regard to the binding event.

A process in which the solution has an initial dielectric property is added to the detection assembly. As described above, the signal response is measured and stored. After a predetermined time or operation, a second measurement is made and a second signal response is recorded. Thereafter, the first and second signals are compared with each other to determine whether the two signals are associated within a predetermined range. If it is in that range, the properties of the solution are considered to have no dielectric change.

If the signal response is not correlated within a predetermined range, then at least the dielectric properties of the solution will have changed. Optionally, variations in dielectric properties can be quantified. For example, a second signal is stored and associated with a known signal response. The most relevant response will identify the dielectric properties of the solution and the first signal response is related to the initial value of the dielectric properties, which can be used to determine the amount of strain of the identified dielectric properties.

Molecular structure identification

With the above-described detection assembly, a known analyte can be characterized and then the known analyte can be identified in a solution having an unknown analyte composition. For example, using a measurement system described below, a large number of molecular structures and / or infrastructures are measured and their responses stored. Each stored response will correspond to one structure / substructure in solution or multiple structures / substructures in the same solution. A measurement of the unknown solution is then made. Next, the signal response of the solution is compared to the stored signal response to determine the degree of association therebetween. The unknown molecular structure is identified by selecting the storage response most relevant to the unknown response. Such comparisons may be made using one or more data points to determine the association between one or more stored responses and may include the use of pattern recognition software or similar means for determining associations. The process can be used to identify not only each structure / substructure, but also primary, secondary, and higher molecular bonding molecular structures.

Classification of molecular structure

Known molecular substructures such as structural homology or domains common in sequence homologies or similar protein classifications in nucleic acids can be characterized. In one embodiment, the process proceeds as shown in part D above, except that a number of molecular substructures are measured and the response is stored. Each stored signal response will correspond to one or more substructures. The process continues until a sufficient number of structures are detected and characterized to identify the unknown mixture. When a sufficient number of correlations occur, the unknown molecular structure can be classified. There are other processes that can classify unknown analytes. One process identifies unknown analytes by detecting binding to a structural motif of an unknown compound. First, the detection assembly may comprise a plurality of addressable parallel channels each having an anti-ligand for the specific ligand subunit bound in the detection region. Next, the presence of particular substructures is detected by binding each of the substructures to each anti-ligand and subsequently characterizing. In one embodiment, this step is performed as described above, but other variations may be implemented. Each binding event is then characterized by affinity, kinematic, and quantitative identification such as a spectral response. An association between the notification and the unknown response is made. If each unknown response is associated with a known response, the ligand is identified as a ligand corresponding to a known response. If the substructure represents both associative and non-associative, the associated response is used to establish a more general classification of the unknown ligand. Such a process can be used to identify any molecular structure, such as a protein that occurs within the same class or that has a resuscitative structural homology.

As comparisons to known structures are made, structure and function can be investigated by intensity spectrum analysis of a given unknown mixture, and a certain degree of classification can be estimated.

Specific binding versus non-specific binding

Spectrum fingerprints of binding events can distinguish between specific and non-specific binding. Indeed, any two binding events, such as when one molecule binds to two similar but different molecular partners in different states, is generally distinguished by the spectral fingerprint of the two binding events. For example, a given binding event, such as antibody binding to an antigen, may first be characterized in a rectified solution containing only the ligand of interest and an anti-ligand specific for that ligand. Then, a broad spectrum test is performed to find the time when the strongest response is found in the spectrum. Thereafter, the measurement is repeated for a solution such as whole blood, which is found according to the application, to determine the effect of the non-specific binding on the response. Then, a number of points representing a specific binding are searched, a set of each point representing a non-specific binding is selected, and a subset of these frequency points is selected for actual measurement. The degree of specific binding is determined by comparing the response due to the specific binding with the response due to the non-specific binding.

Characterization of a given subject

It is sometimes desirable to determine the specific amount of a given molecule. For example, determining the classification to which the protein belongs, or determining whether a given gene or nucleic acid sequence is homozygous in any form. This is done in a number of ways. In general, proteins are classified by the number and type of structural homology, or by specific substructures found in the same or similar protein classifications. For example, the G-protein, which is mainly found in cell membranes and constitutes a signaling pathway between the extracellular environment and the intracellular environment, always has a structure that traverses the cell membrane seven times. Such a structure conclusively defines the G-protein. Similarly, different protein classifications have structural homology, and accordingly, a method of distinguishing one protein class from another protein class based on such homology is used in many biological studies. If the dielectric properties of a given molecule are determined by the geometric shape of the charge distribution of the molecule and most proteins have a unique structure or geometry, then each protein can be identified by measuring the dielectric properties of the protein. Thus, a simple genetic signature such as that generated by the present invention allows each of the given proteins to be seeded, and also allows classification of the protein into some known protein classifications. By using an anti-ligand group on a specific detection assembly for a specific substructure of a given protein, more precision can be added to the classification methodology. For example, the presence or absence of the substructure can be determined using antibody groups specific to a particular substructure, such as a domain. Thus, any given protein can be identified by determining the presence and absence of a particular substructure and by determining the dielectric properties of the protein itself. In addition to this classification method, more precision can be added by examining temperature, pH, ionic strength, and other environmental influences on the aforementioned properties.

Nucleic acid may also be specified by following a similar manner as described above. For example, we can see that a given gene has a specific base pair arrangement. Naturally there can be slight variations in the arrangement. For example, in a gene that has information on the chlorine ion transport channel in many cell membranes, there is a common single base-pair mutation or modification. Such a modification causes a disease called cystic fibrosis in humans. Thus, it is very important to characterize a given nucleic acid sequence for a few strains. Such modifications are often referred to as homozygous abnormalities and currently such homozygous abnormalities are detected by forming complementary strands for each known homozygous abnormality. Since any given gene can take the form of one of hundreds or thousands of homozygous, it is very difficult to make complementary fibers for each homogenous disorder. Using the present invention, non-complement binding and hybridization can be detected and identified by measuring many of the same physical properties as described above. The dielectric properties of the hybridization event can be specified and associated with known data, and thus the type of hybridization made completely or incompletely. Thus, using an anti-ligand consisting of a given nucleic acid sequence, hundreds of different homologies (as ligands) can be detected by characterization of the binding event. One skilled in the art will understand that it is possible to apply more stringent conditions to alter the hybridization process, or to give additional precision, such as to determine the melting point and to change the temperature, which serves as another indicator of the nature of the hybridization process .

In a similar manner, drug-receptor interactions can be characterized to determine a given binding event that causes the receptor to turn on or off, or cause some other type of allosteric effect . For example, a given receptor can be used as an anti-ligand, and known antigens can be used as the first ligand. Thereafter, the interaction is specified according to the dielectric response, and the response is stored. The selected mixture is then observed for binding properties with the receptor for drug candidates. Molecules that bind and produce similar genomic responses are judged to have similar effects on receptors as known antigens and thus are more likely to be antigens. This example can be used to eventually characterize any form of target-receptor binding event and is a significant improvement over current detection methods that measure only whether a binding event has occurred. Those skilled in the art will appreciate that the invention is also applicable to a number of different coupling event areas.

Examples of substructures that can be used in the above-described method include: proteins such as alpha helices, beta sheets, triple helix, domains, barrel structures, beta-turns, Structure, and various symmetric groups found in quaternary structures such as C ₂ symmetry, C ₃ symmetry, C ₄ symmetry, D ₂ symmetry, cubic symmetry, and icosahedral symmetry. [Heirarchic Organization of Domains in Globular Proteins of G. Rose, published in 1979, J. Mol. Biol. The structure of the nucleic acid that can be analyzed includes, but is not limited to, sequence homology and sequence homology, A, B and Z DNA, single and double fiber forms, supercoiling forms, anticodon loops, T ψ C loops within different classes of tRNA molecules [W. W. 1984, Springer-Verlag, New York). W.Saenger, Principles of Nucleic Acid Structure ; And the Cold Spring Harbor Research Publisher, Cold Spring Harbor, New York. P. Schimmel, D.Soll, and J.Abelson, Transfer RNA]

Concentration quantification

The detection assembly described herein can also be used to quantitate the concentration of the analyte. In one embodiment of this method in which the device is not pre-calibrated, an anti-ligand with the appropriate binding properties such as binding affinity and kinetic is first selected for the analyte to be measured. These properties are chosen so that the equilibrium constant of the ligand is near the middle of the linear action region. If one concentration of ligand is too broad to use an anti-ligand, several anti-ligands with different affinities and / or linear action regions may be used, thereby achieving a wider range of concentration values.

Next, an anti-ligand is added or attached to the detection assembly or chip, and the device is connected to the measurement system. Thereafter, a determination is made as to whether the response needs to be characterized for maximum specificity. If characterization is required, spectrum analysis, where the frequency or frequencies at which the analyte binding maximally affects the signal is determined, the region where the nonspecific binding maximally affects is determined, and the response due to the analyte binding is determined . If characterization is not required, or if necessary, after completion, the device is calibrated. This step is carried out in one embodiment of feeding a known concentration of ligand to the detection assembly and measuring the resulting response. Optionally, if more data points are required for calibration, different concentrations of test sample may be selected and the response to this concentration may be measured. Then, an extrapolation algorithm is generated by recording the calibration points from the above-mentioned responses. Next, a test sample of unknown analyte concentration is measured. This step supplies an unknown test sample to the detection assembly, associates the response with a titration algorithm, and determines the analyte concentration therefrom.

If a given detection assembly is pre-calibrated or calibrated at the design stage, then only the steps of mixing the coupling pairs and measuring the response are needed. Such a detection assembly can be realized in various ways. For example, some circuit parameters such as a particular frequency or impedance of the resonant circuit may be designed to change in a predetermined manner when a coupling event occurs, and the amount of change in the parameter may be designed to have a dose- have. Thus, when analyzed with an appropriate algorithm, the measurement of the circuit parameters immediately provides a quantitative value for the concentration of a given analyte or ligand.

Detection assembly self-calibration

The detection assembly has self-diagnostic capability and thus provides quality control and assurance in terms of use. For a given specific application, a particular ligand (primary binding species) will act as an anti-ligand for some target ligand (secondary binding species) in solution. The primary binding species can be attached at the time of manufacture and the secondary binding species can be attached at the time of use. Thus, manufacturing variations, particularly the attachment of primary species, will cause deviations in the ability of the device to bind specific ligands. However, the amount of binding ligand will be directly proportional to the amount of bound anti-ligand, and thus two ratiometic measurements are possible.

In one embodiment of the method, the molecular binding surface is formed along the signal path, by binding a number of different concentrations of the appropriate antibody and specifying the resulting response for each concentration, and any value " x " . Next, a similar titration curve for the ligand is obtained by measuring the antibody / ligand binding response for a number of different concentrations of ligand, and the ligand titration curve is pre-defined. Next, by taking the response ratio of the antibody bound to the ligand, a scale factor (A) is obtained. From an operational point of view, the uncorrected measurement is first examined to determine the amount " x " of bound antibody and the size factor " y " obtained therefrom. Thereafter, the ligand is applied to the measurement, the response is measured, and the response and the predetermined titration curve are scaled to the magnitude factor " y " to determine the unknown concentration.

This process can be improved to quantify the amount of binding in solution. In this modified embodiment, the binding surface of the detection assembly comprises an anti-ligand having a predetermined affinity and ligand specificity. The solution is then applied to the apparatus and the response is measured. The signal response will be proportional to the amount of ligand bound. Thus, by selecting an appropriate ligand of linear activity range, titration of any given ligand is achieved. Wherein the range of action is within a range of log unit couplings of an intended range of concentrations at which the equilibrium constant is to be detected. By applying the same proportional analysis as described above and by internal control and calibration necessary to ensure reliability, reliable and accurate quantitative analysis can be obtained.

VI. Software execution

Each of the measurement and detection methods described herein can be implemented in a variety of ways (i.e., software, hardware, or a combination thereof) and various systems. In one embodiment, the above-described method may be implemented as a software program.

5A shows a schematic block diagram of a computer system 510 that is operative to execute a software program designed to execute each of the above-described methods. The computer system 510 includes a monitor 514, a screen 512, a cabinet 518, and a keyboard 534. Other input / output interfaces, such as a mouse (not shown), a light pen, or a virtual reality interface, to provide input / output commands. The cabinet 518 accommodates a CD-ROM drive 516, a hard disk drive (not shown) or other storage data media that can be used to store and read digital data and software programs including methods of the present invention. Although CD-ROM 516 is shown as a removable medium, other removable media including floppy disks, tape, and flash memory may be used. Cabinet 518 also houses other common computer components (not shown) such as a processor, memory, and the like.

FIG. 5B shows an internal configuration of the computer system 510. FIG. The computer system 510 optionally includes a monitor 514 that interacts with the input / output control 524. The computer system 510 may also include other components such as a system memory 526, a central processor 528, a speaker 530, a removable disk 532, a keyboard 534, a fixed disk 536, and a network interface 538 Subsystem. Other computer systems suitable for use in the methods described above may include additional subsystems or may include fewer subsystems. For example, other computer systems may include one or more processors 528 (i. E., A multi-processor system) for digital data processing. Arrows 540 and the like represent the system bus structure of the computer system 510. However, this arrow 540 illustrates any interconnect configuration that connects subsystems. For example, a local bus may be used to connect the central processor 528 to the system memory 526. [ The computer system 510 shown in Figure 6 is merely an example of a computer system suitable for use with the present invention. Those skilled in the art will readily understand other subsystem structures suitable for use with the present invention.

VII. Experiment

Using the system and method of the present invention, several experiments were conducted to detect and identify analytes in the transfer medium. Figure 6a shows the employed molecular detection system and Figures 6b-6f show the measurement results.

6A shows a molecular detection system used in the following experiments, which system includes a vector network analyzer, a computer, and a measurement probe of model number HP 8714 supplied by Agilent Technologies, Inc. (formerly Hewlett Packard) , The probe comprising a grooved lid piece on which the detection area is formed above and a PTFE tube of a predetermined length used as a transport channel and as a fluid channel for transporting the test sample to the detection area of the measurement probe ≪ / RTI > Cole-Parner Instrument Company). The resonance probe 230 shown in Fig. 2A was used in this experiment. The resonance probe was designed at 1 GHz f _des and exhibits a f _res of about 1.163 GHz when a fluid channel (PTFE) containing buffer solution is present and assembled.

The computer runs Labveiw ^® software to control the operation of the network analyzer and display and store data from it. A coaxial-shaped measuring probe as shown and described in Fig. 2A was used. A PTFE tube (0.031 " inner diameter, 0.063 " outer diameter, 0.016 in wall thickness) is placed across the detection area of the measurement probe and secured using a grooved top cover screwed into the shelf of the measurement probe. (Not shown), and the other end is immersed in a fluid test sample to be analyzed. The syringe pump is used to draw a test sample through the tube to the detection area In the following experiments, the spectra of various test samples were measured while moving the test sample over the detection area 155. The syringe pump was used to inhale the fluid at a rate of ~0.0 mL / min Separate the end of the tube from the first test sample, wait for ~ 1 sec to allow an air gap of ~ 1 cm in length to flow, then place one end of the tube in a second test sample The air gap is used to prevent the two test samples in the tube from intermixing. When the fluid is held stationary over the detection area (i.e., when the syringe pump Similar to the measurements obtained when the tube described above was replaced with a tube of smaller internal diameter such as 0.020 " inner diameter, 0.063 " outer diameter, 0.021 " wall thickness PTFE.

The analytes used in the experiments shown in FIGS. 6B-6H are carbonic anhydrase II (CA, bovine), fibrinogen (IS type, bovine), lysozyme (egg white), pepsin (porcine gastric mucosa) (I type, horse chestnut), bovine serum albumin (BSA), water (18 megohm), sodium dodecylsulfate (SDS) and transfer medium phosphate. The salt solution was purchased from Sigma St. Louis, MO). Monobasic sodium phosphate and dibasic sodium phosphate were purchased from EM Science (Gibbstown, NJ). The PBS transfer medium was used as purchased and 25 mM sodium phosphate transfer medium was prepared at pH 7.8 using water (18 megohms) just prior to use. A 1.0% solution (w / w) of CA, fibrinogen, lysozyme, BSA and pepsin is provided in 25 mM sodium phosphate transport medium adjusted to pH 7.8 just prior to use. 1.0% solution (w / w) ferritin and 5% (w / w) SDS.

In a first experiment, the PBS delivery medium is introduced into the tube and transferred to a detection area where a S ₁₁ (recovery loss) measurement is taken. Next, a ferritin test sample (1% w / w in PBS) was transferred to the detection area and a second S ₁₁ measurement was performed. Ferritin measurements were performed 4 times for measurement reliability. The S ₁₁ response in PBS solution and the four ferritin responses are shown in FIG. 6 b (in db) and in FIG. 6 c (in phase angle).

As shown in FIG. 6B, the S ₁₁ response with respect to the PBS solution has a deep null at 1.163 GHz. Also, the difference between PBS and ferritin S ₁₁ can be seen in Figure 6b. In particular, the ferritin S ₁₁ response is less pronounced and frequency converted than the PBS response. All or any of these differences can be used as a parameter to detect the presence of an analyte as described in the above-described process. In addition, the ferritin S < ₁₁ > response (size and / or phase) can be stored and used as an identifying indicia to identify unknown analytes in other test samples, as in the process described above.

Figure 6c further illustrates size differences between PBS and feritin S ₁₁ responses over frequency. At the maximum point, the difference approaches 20 dB, which is a large difference to be able to represent the analyte present in the test sample. In addition, a small deviation between the ferritin measurements can be seen, indicating high measurement repeatability and stability.

Figure 6d shows the phase of the S ₁₁ response of the PBS and ferritin samples, and Figure 6e shows the differences between them. The phase difference between the PBS and the ferritin measurement is obvious, and the repeatability of the ferritin sample measurements is also apparent. Therefore, the phase information can be used as a parameter for detecting an analyte in a solution according to the above-described process.

Next, the above-described molecular detection system was used to specify detection of different analytes. Figures 6f and 6g illustrate the size and phase S ₁₁ measurements taken from six analytes of carbonic anhydrase, fibrinogen, lysozyme, BSA, and pepsin and transfer medium (25 mM sodium phosphate transport medium, pH 7.8). As can be seen in the figure, each analyte provides a signal response that can be distinguished and identifiable, thereby allowing detection and identification of each subject in the test sample as described above.

Figure 6h shows the S ₁₁ response (size) of the five solutions of sodium chloride obtained using the system described above. The solutions were obtained from a 1.0 M stock solution by continuous dilution with deionized water. Fluid samples are introduced into the detection region of a coaxial resonant structure through a tube as described above. First, deionized water is introduced to the top of a resonance probe operating at a f _des of 1.2 GHz. The probe is then tuned to the fres shown to obtain a reflection measurement of -65 dB in magnitude. Each sodium chloride solution is then introduced and the signal response recorded. As shown in Figure 6h, the test system can identify 0.010 mM NaCl solution and deionized water.

Figures 6i and 6j show S ₁₁ signal responses that occur when detecting a specific molecular binding event using the method of Figure 3d (using the same weight concentration of 1/2 x) according to the present invention. Figure 6i shows the signal response for the denatured HSA, SAL, and unbound mixture, and Figure 6j shows the signal response for the native HSA, SAL, and mixture of the native HSA and the native SAL. Each measured response representing amplitude resonance (minimum amplitude point) and frequency resonance (frequency at which minimum amplitude is measured) is described below.

Human serum albumin (HSA) and salbutamol were purchased from Aldrich Chemical Company, Milwaukee, Wis. Phosphate saline (PBS) was purchased from Life Technologies, Inc. of Grand Island, NY Lt; / RTI > The parent solution of SAL was prepared in 1x PBS (pH 7.2) at a concentration of 50 μM. The parent solution of HSA was prepared in 1x PBS (pH 7.2) at a concentration of 50 μM. 50 μM concentration HSA was denatured in 1 × PBS buffer at 60 ° C. for 15 minutes. Freshly prepared 50 [mu] M HSA (native and denatured) were preincubated for 10 min in the same volume of 50 [mu] M SAL. For a solution containing 50 μM SAL in 1 × PBS (pH 7.2) for 50 μM HSA (native and denatured) in 1 × PBS (pH 7.2) and for 1 × PBS pH 7.2), signal responses were obtained for a mixture having a final concentration of 25 [mu] M HSA and 25 [mu] M SAL. Signal responses were obtained at room temperature using a vector network analyzer from Model 8714 from Agilent Technologies, Palo Alto, California. A resonant coaxial probe having a _fres of 1.2 GHz was used as a measurement probe.

Referring to Figure 6i, a buffer is in response approximately 1232.86 MHz, represents the f _res up to amplitude resonances at about -62 dB. The SAL solution exhibits slightly increased resonant amplitude (-60 dB), and the change in resonant frequency is negligible. The modified HSA solution indicates a slightly reduced amplitude to -73 dB, the frequency indicates that the conversion of about +3.5 KHz from the buffer f _res. Mixture at an amplitude of about -67 dB from the buffer f _res represents the frequency resonance of about +1.9 KHz. As shown, the unbonded signal response represents the amplitude and frequency resonance close to the average value between the denatured HSA and SAL responses.

As shown in Figure 6i, the buffer and SAL responses are shown in comparison to Figure 6j. Natural HSA is converted from about +2.3 KHz from the buffer f _res, has a measured amplitude resonance of -55 dB. (Combined), the combined mixture has an amplitude resonance of about +2.3 KHz, and the frequency-converted, -53 dB from the buffer f _res, these amounts are clearly distinct from the average value of the SAL and the original HSA response.

Figures 6k and 61 show S ₁₁ signal responses obtained when detecting a specific molecular binding event using the method of Figure 3f (using 1x concentration of the same volume in the combined mixture) according to the present invention. Figure 6k shows the signal responses for the denatured HSA, SAL, and unbound mixture, and Figure 61 shows the signal response of the combined HSA, SAL, and combination mixture in which the native HSA is combined with the SAL. As described below, each measured response represents amplitude resonance (minimum amplitude point) and frequency resonance (frequency at which minimum amplitude is measured).

Moisture solutions of salbutanol and HSA were prepared in 1 x PBS (pH 7.2) at a concentration of 200 μM. HSA with a final concentration of 200 μM was denatured overnight in 1 × PBS buffer adjusted to pH 2.73. Prior to the binding experiment, the PBS buffer was rapidly converted to pH 7.2 by thin-film filtration.

A freshly prepared 100 μM final concentration of HSA was pre-incubated for 10 minutes in salbutamol at a final concentration of 100 μM. For 100 μM HSA (native or denatured) in 1 × PBS (pH 7.2), for a solution containing 100 μM SAL in 1 × PBS (pH 7.2) and for 100 μM in 1 × PBS (pH 7.2) For solutions with HSA and 100 [mu] M SAL, signal responses were obtained. Signal responses were obtained at room temperature using a vector network analyzer from Model 8714 from Agilent Technologies, Palo Alto, California. A resonant coaxial probe having a _fres of 1.2 GHz was used as a measurement probe.

Referring to FIG. 6k, the buffer response represents f _res , at about 1232.86 MHz, reaching an amplitude resonance at about -62 dB. The SAL solution exhibits slightly increased resonant amplitude (-60 dB), and the change in resonant frequency is negligible. The modified HSA solution represents a reduced amplitude to -87 dB, the frequency indicates that the conversion of about +7.2 KHz from the buffer f _res. Unbound mixture at an amplitude of about -63 dB from the buffer f _res represents the frequency resonance of about 7.2 KHz. As shown, the non-associated signal responses HSA (about 7.2 KHz) and SAL (approximately 0 Hz) ratio to the sum of the frequency transformation due to the coupling component f _res to plus as about the same frequency 0 people (f _res + 7.2 KHz of ).

As shown in Figure 61, buffer and SAL responses are shown in comparison to Figure 6k. Natural HSA is converted from about +6.3 KHz from the buffer f _res, has a measured amplitude resonance of -61 dB. The binding mixture represents a frequency approximately 10.8 KHz converted from the buffer _fres , and this frequency is clearly deviating from the aggregate contribution range of SAL (0 Hz) and HSA (6.3 KHz).

6M shows the dose response curve obtained according to the present invention. HSA was purchased from Aldrich Chemical Company of Milwaukee, Wis., And PBS was purchased from Life Technologies, Inc. of Grand Island, NY And mecaptohexadecanoic acid (C16) was purchased from Gateway Chemical Technology, Inc. of St. Louis, Missouri. Lt; / RTI > The parent solution of C16 was prepared in DMSO at a concentration of 1 mM. The parent solution of HSA was prepared in 1 x PBS (pH 7.2) at a concentration of 200 μM. HSA was prepared to a final concentration of 10 μM and pre-incubated with C16 at a final concentration of 0.1 to 1000 μM for 10 minutes. All final concentrations include 5% DMSO. A baseline response is obtained for a 1x PBS solution containing 5% DMSO (pH 7.2). A measurement response is obtained for a solution containing various concentrations of C16 in 1x PBS containing 5% DMSO (pH 7.2). Signal responses were obtained at room temperature using a vector network analyzer from Model 8714 from Agilent Technologies, Palo Alto, California. A resonant coaxial probe having a _fres of 1.2 GHz was used as a measurement probe.

Of HSA, C16, and (divided), the average is obtained for f _res of the HSA and is measured a number of the amount of resonant frequency change produced by the C16 mixture, PBS / 5% DMSO baseline response of the solution, C16 Coordinates are plotted against the concentration. The response (Figure 6m) clearly shows a saturation curve with a saturation point of about 50 [mu] M for C16.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. For example, other transmission media such as conductive or dielectric waveguides as well as other fluid delivery systems may be selectively used. In addition, all publications and patent documents cited in this application are cited for any purpose, to the extent that each publication and patent document is individually indicated. In particular, the present application relates to the applicant's pending applications as set forth below, all of which are incorporated herein by reference:

09 / 243,193 (Attorney Docket No. 19501-000200US) entitled " Method and Apparatus for Detection of Molecular Binding Events ", filed February 1, 1999;

09 / 243,196 (Attorney Docket No. 19501-000300US) entitled " Method and Apparatus for Detection of Molecular Binding Events ", filed February 1, 1999;

09 / 365,578 (Attorney Docket No. 19501-000210) entitled " Method and Apparatus for Detection of Molecular Binding Events ", filed August 2, 1999;

09 / 365,978 (Attorney Docket No. 19501-000500) entitled " Test System and Sensor for Detection of Tom Binding of Molecular Bond Events " filed on August 2, 1999;

09 / 365,581 (Attorney Docket No. 19501-000600) entitled " Method for the Analysis of Nucleic Acids " filed on August 2, 1999;

09 / 265,580 (Attorney Docket No. 19501-000700) entitled " METHOD FOR ANALYSIS OF PROTEIN BINDING EVENT " filed on August 2, 1999;

09 / 480,846 (Attorney Docket No. 19501-000310) entitled " Method and Apparatus for Detection of Molecular Binding Events ", filed on January 10,2000;

09 / 480,315 (attorney docket number 19501-000320) entitled " Method and Apparatus for Detection of Molecular Binding Events, " filed January 10,2000.

Claims (28)

A molecular detection system for detecting molecular events within a test sample: (1) a fluid storage container comprising a detection area having a volume less than 1.0 mL; (2) a signal source operative to transmit an electromagnetic incident test signal at a frequency of 10 MHz to 1000 GHz; (3) As a measurement probe, (a) a probe including (i) a waveguide coupled to the signal source, or (ii) a transmission line coupled to the signal source, a ground plane, and a dielectric layer disposed between the transmission line and the ground plane. Wherein the probe head is configured to electromagnetically couple an incident test signal to a test sample in a detection area, wherein interaction of the incident test signal with a test sample generates a modulated test signal, A probe head configured to recover a portion of the modulated test signal; (b) a measurement probe comprising a connection end; (4) a signal detection unit coupled to the connection end of the measurement probe and configured to recover a modulated test signal. 3. The method of claim 1, wherein the first aqueous sample, except that a first modulation test signal is obtained while the first aqueous sample comprising 0.3 g or less of fibrinogen is present in the detection region and does not contain fibrinogen, When the second modulated test signal is obtained while the second aqueous sample, which is the same as the second modulated test signal, is present in the detection region, the signal detection section is set to a sensitivity that is high enough to detect the difference between the first modulation test signal and the second modulation test signal The system to be configured. The system of claim 1, wherein the probe head is physically separate and electromagnetically coupled to a sample in the detection region. 4. The system of claim 3, wherein the probe head includes an open-ended cross-section of a coaxial transmission line. The measuring probe according to claim 3, wherein the measuring probe comprises: A first coaxial portion comprising the probe head and a first gap end; A second coaxial portion including a second gap end and the connection end; And a tuning element configured to be adjustably coupled between the first and second gap ends and to provide a variable gap distance between the ends. 6. The system of claim 5, wherein the tuning element comprises a hollow conductive tube surrounding the gap end. 2. The system of claim 1, wherein the signal source and the signal detector are included in a vector network analysis system, a scalar network analysis system, or a time domain reflectometer. 2. The system of claim 1, wherein the fluid storage vessel comprises a well of a microtiter plate. 2. The system of claim 1, wherein the fluid storage vessel comprises a channel configured to transfer fluid from one point to another. 10. The system of claim 9, wherein the fluid channel comprises an internal channel of a PTFE tube. 10. The system of claim 9, wherein the fluid storage vessel comprises one or more channels in a microfluidic transport system. 10. The apparatus of claim 9, further comprising a fluid delivery system configured to transfer fluid through the channel, The fluid delivery system comprising: A fluid storage container configured to store a transfer fluid; And And a fluid control coupled to the fluid storage vessel and to the fluid channel, Wherein the fluid control is configured to control the transfer rate of fluid through the fluid channel from the fluid storage vessel. A method for detecting molecular events in an aqueous test sample comprising: (1) introducing the first sample into a fluid storage vessel having a detection area of 1.0 mL volume; (2) applying a signal to the detection region, (a) as a measurement probe: (A) a probe head including (i) a waveguide coupled to a signal source, or (ii) a transmission line coupled to the signal source, a ground plane, and a dielectric layer disposed between the transmission line and the ground plane. Wherein the probe head is configured to electromagnetically couple an incident test signal to a test sample in a detection region, wherein interaction of the incident test signal with a test sample generates a modulated test signal, A probe head configured to recover a portion of the modulated test signal; (B) a measurement probe comprising a connection end, and (b) a signal detection unit coupled to the connection end of the measurement probe and configured to recover the modulated signal; Applying a test signal of 10 MHz to 1000 GHz to the detection region using the test signal; (3) measuring whether a change in the test signal has occurred as a result of interaction of the test signal with the sample. 14. The method of claim 13, wherein the molecular event is structural or functional similarity of a first molecular substrate to a reference molecular substrate, wherein the similarity is determined by comparing a test signal detected when the sample comprises the first molecular substrate, Lt; RTI ID = 0.0 > a < / RTI > substrate. 14. The method of claim 13, wherein the molecular event is a binding of a first molecular substrate to a reference molecular substrate. A method for detecting molecular events in an aqueous test sample comprising: (a) introducing a first sample into a fluid channel of a fluid delivery system having a sample inlet end, a detection area having a volume less than 1 mL, and a fluid channel having a sample outlet end and a fluid movement control; (b) moving the sample through the channel from the sample inlet end to the sample outlet end under the control of the fluid control; (c) applying a test signal from 10 MHz to 1000 GHz to the detection region of the fluid channel; And (d) detecting a test signal change as a result of the interaction of the test signal with the sample. 17. The method of claim 16, (e) introducing a spacing material into the channel after the first test sample, (f) introducing an additional test sample into the channel after the spacing material, (g) moving the further test sample through the channel under the control of the fluid control, so that a plurality of different samples spaced apart by the spacing material are transported in the channel without being mixed with each other Way. 18. The method of claim 17, wherein the spacing material comprises a solution of ionic strength that is large enough to be transported by an electroosmotic pump, and wherein the fluid movement controller pumps the fluid using an electroosmotic pump. 18. The method of claim 17, wherein the spacing material comprises a fluid that is not substantially mixed with the test sample. 18. The method of claim 17, wherein the spacing material comprises a gas bubble, and wherein the fluid movement control unit pumps the fluid using a physical pump. 17. The method of claim 16, Providing an additional second fluid channel crossing the first fluid channel, the fluid transfer system being adapted to control fluid movement in a second fluid channel comprising a test mixture or a series of test mixtures, Providing a fluid channel, Introducing a test mixture from the second fluid channel into a test sample in a first fluid channel that is sufficiently upstream from the test signal to cause the test mixture to be introduced into the first fluid channel prior to reaching the test signal, Introducing the test mixture so as to have sufficient time to combine with the structure, And detecting the binding by a change in the test signal. CLAIMS What is claimed is: 1. A method for detecting molecular binding in a test sample in a detection region of a fluid storage container comprising: Placing a measurement probe proximate to the detection region that exhibits a resonance signal response at a predetermined frequency of 10 MHz to 1000 GHz and electromagnetically coupling the signal to the detection region; Supplying a reference medium to the detection area; Coupling a test signal to the detection region and recording a baseline signal response; Feeding a test sample that is believed to contain or contain a molecular event to a detection region; Coupling a test signal to the detection region and obtaining a test sample response; If there is a difference between the test sample response and the baseline response, measuring the difference; And And correlating the difference to a molecular event. The method of claim 22, wherein the measurement probe is shown the S ₁₁ 0 people response. 23. The method of claim 22, wherein coupling the test signal to a detection region and obtaining a baseline signal response comprises: Generating an incident signal; Coupling an incident signal to a detection region; Recovering a signal reflected from the detection area; And And comparing the amplitude or phase of the incident signal to the amplitude or phase of the reflected signal. 25. The method of claim 24, Storing a first test sample response; And Further comprising comparing a subsequent test sample response with the stored first test sample response. A measurement probe configured to detect a molecular event in a test sample, comprising: A center conductor portion extending in a longitudinal direction and having a first gap end and a probe head having an open-end coaxial end face portion, a dielectric insulating portion disposed around the longitudinal axis of the central conductive portion, and an outer side portion disposed around the longitudinal axis of the dielectric insulating portion A first coaxial portion including a ground plane; A second gap end having an open-end coaxial cross-section and a connection end having a coaxial connection, wherein the central conduction section extends in the longitudinal direction, the dielectric isolation section is arranged around the longitudinal axis of the central conduction section, A second coaxial portion including an outer ground plane disposed circumferentially; And And a tuning element adjustably coupled between the first and second gap ends and configured to provide a variable gap distance between the ends. 27. The measurement probe of claim 26, wherein the first coaxial portion comprises a shelf that is conductively attached to the outer conductive portion and has the same height as the open end of the probe head. 14. A computer-readable storage medium containing information obtained by the method of claim 13.