Method and apparatus for detecting molecular binding events

Practically every field of biomedical sciences needs System for the analysis of chemical and biochemical reactions and Determining the presence and quantity of certain analytes. These needs range from basic research in the lab, where biochemical courses shown and correlated their functions with the disease processes be up to clinical diagnosis, in patients routinely the values ​​of clinically relevant analytes are monitored. Other areas include pharmaceutical research, military applications, and applications in veterinary science, in the food sector and in environmental protection. In all these cases have the Presence and amount of a specific analyte or a Of analytes are determined.

For analysis in the fields of chemistry, biochemistry, biotechnology, molecular biology and in numerous other areas, it is often useful to one or more molecular locate structures and the bond between the structures to measure. The interesting molecular structures contain u. a. cells, antibodies, Antigens, metabolites, proteins, drugs (drugs), small molecules, enzymes, nucleic acids and other ligands and analytes. In medicine, it is z. B. very helpful the existence of cellular components such as receptors or cytokines or antibodies and determining antigens as markers for various disease processes serve, of course, exist or has been introduced into the system in physiological fluids are. also are the DNA and RNA analysis very useful in the diagnosis, at genetic testing and research, agriculture and in pharmaceutical development. Through the rapid progress in molecular cell biology and in the knowledge of normal and diseased Systems there is an increasing need for detection method, wherein which no objective evidence such as fluorophores or radioisotopes needed are, the quantitative and qualitative specific interest for the molecule are to implement highly sensitive and relatively simple.

in the Over the years, numerous methods have been developed to to meet the requirements in these areas, z. B. Enzyme-Linked Immunosorbent Assays (ELISA) (enzyme-linked immunoassay), radio-immunoassay (RIA), numerous fluorescence analysis, mass spectroscopy, calorimetric analysis, Gel electrophoresis, and a large number of special analyzes. The Most of these analytical techniques require special preparations, in particular the binding of an indicator or the extreme cleaning and reinforcement the sample to be tested. To a binding event between a ligand to be able to recognize, and an anti ligand, is a detectable Signal is required, the presence or the extension the binding relates. Normally the signal from an indicator is provided that either the ligand of interest or antiligand conjugated. Physical or chemical effects which detectable generate signals and for the appropriate indicators exist, include radioactivity, fluorescence, Chemiluminescence, phosphorescence and enzymatic activity to only to name a few. The indicator can then by spectrometry, Radiometry or optical tracking methods detected. in many cases unfortunately it is difficult or even impossible, any or all of for a mark specific analysis necessary molecules. In addition, the presence of an indicator, the recognition between two molecules of many reasons including steric effects cancel out. Further, none of these Procedures to mark the exact nature of the binding event certainly; so z. B. is the binding of the active page to a receptor indistinguishable from the binder of the non-active side as the allosteric binding, and thus provide the current detection methods no functional information. A method for the detection of Binding events that both the need for an indicator eliminates and provides functional information would therefore the above approaches significantly improve.

Other approaches for studying biochemical systems use different types dielectric measurements to certain classes of biological systems characterize as tissue samples and cellular systems. In the 50-ies of the last century were experiments Measuring the dielectric properties of biological tissues under Application of the then-known standard techniques for measuring dielectric Properties of materials carried out. Since then included different approaches for execution these measurements frequency domain measurements and time domain techniques as the dielectric time-domain spectroscopy. were In these approaches, the experiments generally using different types coaxial transmission lines or other transmission lines and Structures, which are typically in the dielectric characterization be used of materials is performed. This included studies to evaluate the usefulness and relevance of the dielectric properties a wide range of biological systems: extended Interest from full tissue samples, the various organs of mammalian species were removed, to cellular and sub-cellular systems including cell membrane and organelles effects. In the recent past have been attempts <?page 3?>made to miniaturize the above techniques (They eg, US Patent No. 5,653,939;.. 5,627,322 and 5,846,708) to changes detect the dielectric properties of molecular systems better to. Typically, the biological sample - which is to tissue Cellular systems or molecular systems can act - in the electrical circuit topology as shunt or series element used. This configuration has several disadvantages including some significant restrictions the useful in the detection strategy frequencies and a serious restriction of Sensitivity for detecting molecular systems.

in the Generally consist in most analytical systems restrictions in the areas of specificity and sensitivity. Cellular particles and non-specific binding often cause a noisy analysis and make the extraction meaningful information difficult or impossible. are, as mentioned above some systems too complicated to all analytes of interest to be provided with indicators or to a precise optical measurement perform. Further, as mentioned above provide most of these detection technologies no information on the functional nature of the binding event. Consequently, any practical and economical universal method that without indicator the presence of analytes or the extent, the function and the type the binding events that actually take place in a given system, directly monitor real-time can, a significant breakthrough.

More accurate requires said the biomedical industry improved technology on a general platform that has a very wide applicability to various physiological systems based on water or other fluid base has, such as nucleic acid binding, Protein / protein interactions, small molecule binding, and other compounds of interest. Ideally, the analysis no highly specific nucleic acid strands as specific antibodies and exactly complementary require nucleic acid strands; they should in native environments such as whole blood, cytosolic mixtures and other natural occurring systems function; they should by measuring the native properties of the molecules function and no additional Indicators or tracers require to actually monitor the binding event; in some applications, it should be capable of certain desired information on the to provide the type of binding event, eg. as whether a given compound as agonist or antagonist to a particular drug receptor acts or not, and not just act as a marker to indicate whether the binding event has occurred or not. For many Applications should be miniaturized to a great extent and alike be so complex biochemical pathways can be mapped, or extremely small, with numerous amounts of combinatorial compounds can be used in Drogensichtungsprotokollen. In numerous applications it should also be able to monitor a complex series reactions in real time, so that accurate kinetics and affinity information almost immediately can be obtained. Perhaps most importantly for most commercial applications is that they are cheap and are easy to use, a few steps for sample preparation, inexpensive Electronics and disposable components such as surface chips for bio-analysis, by an analysis to be disposed of, contain and to a wide range are well adapted analytical applications.

it is important to note that other industries have similar requirements regarding have detection, identification or additional analysis. If worked well in most applications with biological molecules is, virtually any molecule be detected if a specific binding partner is available or when the molecule itself can adhere, as described below to the surface.

The meets present invention many of the requirements set forth above and other needs.

SUMMARY THE INVENTION

The This invention provides systems and methods for detecting molecular binding events and other environmental effects ready by the unique dielectric properties of bound molecular structure or structures and the local Near advantage of making, and to identify the presence and the concentrations of molecular species, and the physical Characteristics of the local environment in a particular biological System.

On Aspect of the invention thus provides a method of assaying of interaction of an analyte with its environment using a modulated signal willing to investigate the interactions, the method comprising: contacting a first aqueous Surroundings (<figref>157</figref>) That the analyte and optionally additional includes components, with a bio-assay device (<figref>150</figref>), full: &lt;?page 4?&gt;<ul><li>(1) a signal path, said signal path comprising: (A) a waveguide or (B) a transmission line (<figref>120</figref>) a base member (<figref>130</figref>) And a dielectric material (<figref>158</figref>) Interposed between the transmission line and the disposed basic element, the signal path is designed to that electromagnetic signals with one or more frequencies in the range of 10 MHz to be transferred to 1000 GHz, and</li><li>(2) a molecular binding layer (<figref>56</figref>), The electromagnetic is coupled to the signal path, whereby after said contacting Analyte interacts with the molecular binding layer;</li></ul>Transfer a first input signal in the range from 10 MHz to 1000 GHz along the signal path, whereby said input signal is electromagnetically is coupled to the molecular binding layer; detecting of the modulated input signal of the interaction during first input signal according to the molecular binding layer the contact of the molecular binding layer with said first aqueous Environment is obtained.

at one embodiment of the invention is the method of detecting a molecular Binding event and includes the steps of providing a signal path and a molecular Binding layer which is formed along the signal path. On Test signal is transmitted along the signal path and coupled to to the molecular binding layer. In response to the coupling gives the signal a response that both the molecular binding event as well as the molecular bonding layer is itself significant.

at a second embodiment of the invention is the method for determining the classification an unknown ligand. The method comprises the step of Providing a signal path of a first molecular Binding layer with N corresponding antiligand binding to N corresponding ligand substructures coupled. Thereafter a solution, containing a number of unknown ligands on the molecular binding layer applied. In response, a second molecular binding layer formed along the signal path, said second molecular binding layer N ligands containing. N ent speaking test signals are transmitted to the N respective ligand. N known signal responses that a known ligand classification define, can be provided. Most recently, coupled each of Test signals to the N ligand / anti-ligand complexes and outputs as the reaction N corresponding measured responses for the presence of each of the N sub-structures are indicative, so that when a predetermined number of N known signal responses with a predefined correlated area with the N measured responses, it is determined that the ligand is within the known classification.

On second aspect of the invention provides an apparatus for inspecting the interaction between an analyte and its environment that comprising: a signal, wherein the signal (a) a waveguide or (b) a transmission line (<figref>120</figref>), A basic element (<figref>130</figref>) And a dielectric layer (<figref>158</figref>) Connected between the transmission line and is arranged to the base element, wherein the signal path configured to is that electromagnetic signals with one or more frequencies are transmitted in the range of 10 MHz to 1000 GHz; a molecular binding layer (<figref>156</figref>), The electromagnetic is coupled to the signal path; a signal source (<figref>110</figref>) for transferring a first test signal in the range from 10 MHz to 1000 GHz along the Signal, whereby the test signal electromagnetically with the molecular Bonding layer is coupled; and a detector (<figref>160</figref>) for detecting a first modulated signal during the Interaction of the first test signal with said molecular binding layer is obtained.

at one embodiment the invention, the apparatus (claim 25) using a test signal for detecting one or more of the molecular Bonding layer associated Properties such as are used, the presence of a ligand. The device contains a signal path having a first terminal and a second terminal to transfer the test signal and having a continuous conductive zone between. The molecular binding layer may include a ligand, coupled to the signal path. The bio-assay device may further comprise a included with the molecular binding layer coupled solution, the ligands the can transport the molecular binding layer.

in a further embodiment the invention, a system for detecting a molecular binding event presents. The system includes a signal source for issuing a test signal, &lt;?page 5?&gt;to the signal source coupled device according to claim 27 and a second coupled with the device detector. The contains device a signal path and a first molecular binding layer, the ligands a or anti-ligand may contain, and with a solution may be the signal path coupled. The test signal travels the pathway along which continuously throughout the zone of the molecular binding layer is, couples to the molecular binding layer and has as a response signal in response to which the presence of the molecular Binding event occurred.

The Nature and advantages of the present invention are based on the following drawings and detailed description yet more clear.

SUMMARY THE DRAWINGS

<figref idrefs="S108">1A</figref> shows an embodiment of the bio-assay system according to the present Invention.

<figref idrefs="S108">1B</figref> shows a second embodiment of the bio-assay system according to the present Invention.

<figref idrefs="S109">1C</figref> is a sectional view of the in <figref idrefs="S108">1B</figref> shown bio-assay system.

<figref idrefs="S109">1D</figref> shows an embodiment of a molecular Bonding layer according to the present Invention.

<figref idrefs="S110">1E</figref> shows an embodiment of a molecular Bonding layer spatially with several separate anti-ligand according to the present Invention.

<figref idrefs="S110">1F</figref> shows an embodiment of a molecular Binding layer with several classes antiligand according to the present Invention.

<figref idrefs="S111">1G</figref> showing one or more cells having molecular binding layer in accordance with the present Invention.

<figref idrefs="S111">1H</figref> shows a molecular binding layer with cell membranes related and membranes structures according to the present Invention.

<figref idrefs="S112">2A</figref> shows an embodiment of the bio-assay device according to the present Invention.

<figref idrefs="S113">2 B</figref> shows a second embodiment of the bio-assay device according to the present Invention.

<figref idrefs="S114">3</figref> shows an embodiment the surface-binding chemistry, along the conductive layer bioelectrical interface present.

<figref idrefs="S115">4A</figref> shows an embodiment of an equivalent circuit model for the in <figref idrefs="S112">2A</figref> illustrated bioelectrical interface structure.

<figref idrefs="S116">4B</figref> shows an embodiment of a circuit according to the in <figref idrefs="S115">4A</figref> Equivalent circuit model shown.

<figref idrefs="S117">4C</figref> shows an embodiment of an equivalent circuit model for the in <figref idrefs="S113">2 B</figref> illustrated bioelectrical interface structure.

<figref idrefs="S118">4D</figref> shows an embodiment of a circuit according to the in <figref idrefs="S117">4C</figref> Equivalent circuit model shown.

<figref idrefs="S119">5A</figref>-<figref idrefs="S120">5G</figref> show specific embodiments of a two-wire circuit topology according to the present Invention implemented bioelectrical interface.

<figref idrefs="S120">6A</figref> shows an embodiment of a method for detecting molecular binding events according to the present Invention.

&lt;?page 6?&gt;

<figref idrefs="S121">6B</figref> shows an embodiment of a method for detecting secondary molecular binding events and molecular binding events higher Procedure according to the present Invention.

<figref idrefs="S122">6C</figref> shows an embodiment of a method for measuring dielectric changes the molecular binding layer in accordance with the present invention.

<figref idrefs="S122">6D</figref> shows an embodiment of a method for identifying a ligand in an unknown solution in accordance with the present Invention.

<figref idrefs="S123">6E</figref> shows an embodiment of a method to identify the class of a ligand according to the present invention.

<figref idrefs="S124">6F</figref> shows an embodiment of a method for quantifying the ligand concentration of a solution according to the present Invention.

<figref idrefs="S125">6G</figref> shows an embodiment of a method for providing a self-diagnostic function of the bio-assay device according to the present Invention.

<figref idrefs="S126">7A</figref> shows an embodiment of a computer system for execution a software program for carrying out each of the <figref idrefs="S120">6A</figref> to <figref idrefs="S125">6G</figref> illustrated is designed procedure.

<figref idrefs="S126">7B</figref> shows a simplified system block diagram a typical computer system, the one as described for the embodiment Method containing software program is used.

<figref idrefs="S127">8A</figref> shows an embodiment of a frequency measuring system according to the present Invention.

<figref idrefs="S127">8B</figref> shows a first measured frequency response, of detecting or identifying a molecular structure according to the present Invention can be used.

<figref idrefs="S127">8C</figref> shows a second measured frequency response, of detecting or identifying a molecular structure according to the present Invention can be used.

<figref idrefs="S128">9</figref> shows a second embodiment, a frequency measuring system of the present Invention.

<figref idrefs="S128">10</figref> shows an embodiment of a time domain measurement system according to the present Invention.

<figref idrefs="S128">11</figref> shows one embodiment of a measurement system for the The dielectric relaxation according to the present Invention.

<figref idrefs="S129">12A</figref>-<figref idrefs="S129">12B</figref> show the measurements of the return loss and the transmission loss the primary Binding of urease to an ITO surface.

<figref idrefs="S130">12C</figref> and <figref idrefs="S130">12D</figref> show the measurements of the transmission loss the primary Binding effects of collagenase and lysozyme.

<figref idrefs="S131">12E</figref> shows the transmission loss response of bound and unbound dextran.

<figref idrefs="S131">12F</figref> shows the response of non-glucose and of glucose bound ConA.

<figref idrefs="S132">12G</figref> shows the transmission loss biotin / Avidin relative to the Avidinantwort.

<figref idrefs="S132">12H</figref> shows the results of Wettbewerbstitration between dextran and glucose.

<figref idrefs="S133">12I</figref> the return loss ConA depending of glucose concentration at resonance.

<figref idrefs="S133">12J</figref> shows the transmission loss of DNS / Polysin complexes relative to Polysin response.

&lt;?page 7?&gt;

<figref idrefs="S134">12K</figref> shows the change the transmission loss response dependent on the pH for a number Buffers at 100 MHz, 1 GHz and 10 GHz.

<figref idrefs="S134">12L</figref> shows the change the transmission loss response dependent on on the ion concentration for a number buffer at 100 MHz, 1 GHz and 10 GHz.

<figref idrefs="S135">12M</figref> shows the transmission loss response for at 1 GHz tested 10 whole blood samples, including a display of the detection capability delivers in a complex environment.

<figref idrefs="S135">12N</figref> shows the result of avidin binding, the the Quadrapolmomentdetektion indicates.

DESCRIPTION THE SPECIFIC EMBODIMENTS

Table of Contents <ul><li>I. definitions</li><li>II. Introduction A. Bio-Analysis System B. Chemistry of the system</li><li>III. The bio-assay device A. Structure of Apparatus B. Chemistry of the binding surface C. Bioelectrical interface D. Specific embodiments</li><li>IV. Measurement methods A. General Overview B. detecting molecular binding events C. detecting changes of the dielectric properties D. Identify molecular binding events E. Identifying Classes of bound molecular structures F. quantifying concentrations G. Self-calibration of the bio-assay device</li><li>V. Measurement Systems A. Frequency Measurement System B. time domain measurement system C. Measurement system the dielectric relaxation</li><li>VI. Examples</li><li>VII. Applications</li></ul>

I. definitions

As used herein, the terms biological "binding partners" or "ligand / antiligand" or "ligand / Antiligandkomplex" refer to molecules other molecules specifically recognize (z. B. bind) to a binding complex such as Antibody-antigen, lectin-hydrocarbon, Nucleic acid-nucleic acid, biotin-avidin etc. to form. Biological binding partners need not to couples individual molecules limited to be. For example, a single ligand coordinated by the Effect of two or more "anti-ligand" is attached.

As used herein, the terms "ligand" or "analyte" or "marker" refers to any molecule that is detected. It will detected through its interaction with an anti-ligand the Ligands specifically or non-binding, or by the characteristic dielectric properties of the ligand. The ligand is generally as each molecule defined, for the other molecule (Ie, an anti-ligand) is present which, owing to the ligand the detection of a part of the ligand, a specific or nonspecific Alternative binding. The anti-ligand may be, for. Example, be an antibody and the ligand is a molecule as an antigen which specifically binds with the antibody. In the case in which the antibody on the surface bound and the antibody the recognized molecule is, in the context of this document, the antibody of the ligand and the antigen is the antiligand. The ligand may also consist of cells, cell membranes, Organelles and synthetic analogues thereof consist.

Suitable Ligands for the realization of this invention include antibodies (the an antibody / epitope complex form), antigens, nucleic acids (Z. B. natural or synthetic DNA, RNA, Gdns, &lt;?page 8?&gt;cDNA, mRNA, tRNA, etc.), lectins, Sugar (z. B. a lectin / sugar complex form), glycoproteins, Receptors and their cognate ligands (eg. As growth factors and their associated Receptors, cytokines and their associated receptors, signaling Receptors etc.), small molecules as Arneimittelkandidaten (either from natural products or from synthetic analogs developed in combinatorial libraries and are stored), metabolites, drugs that are subject to abuse and their metabolic by-products, such as vitamins and cofactors other naturally and synthetically occurring compounds, oxygen and other Gases in physiological fluids, cells, cellular components, Cell membranes and associated Structures, other natural Products in plant and animal sources, other partially or completely synthetic products and the like.

As used herein, the term "antiligand" refers to a molecule which is another molecule (ie a ligand) specifically or non-binding. The antiligand is also through its interaction with a ligand to which it is a specific Alternative binding, or by its own dielectric properties recognized. the anti-ligand as used herein is normally either alone or as an element of an immobilized binding pair on the surface on the surface immobilized. In some embodiments, the anti-ligand from the molecules may be in the signal path or on the conductive surface exist. Alternatively, after the binding of an antiligand to ligand the resulting Antiligand- / ligand complex for the purpose of subsequent bond be considered as a Antilgand.

As used herein, the term "specifically bind" in the context of a protein, polypeptide, nucleic acid or a receptor or other binding partners described herein refers to a binding reaction which one of the related Determinativum ligand of interest in a heterogeneous population of proteins and / or other biological substances. Under predetermined Conditions is (z. B. immunoassay conditions in the case of an antibody) the predetermined ligand or antibody to its respective "target" (z. B. a go Hormone specifically binds to its receptor a) is a bond and not to a significant extent with other proteins in the Sample or with other proteins, with which the ligand or antibody in an organism or in a derived from an organism sample may come into contact. Similarly way can nucleic acids under preselected Conditions hybridize to each other.

As used herein, the terms "isolated" "purified" or "biologically pure" refers to material that Mostly or substantially free of components, which in its native occurring condition with him.

As used herein, the term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single or double-stranded form, and unless otherwise limited, it comprises known analogues of natural Nucleotides in a similar Way to act as naturally occurring nucleotides.

The Terms "polypeptides", "peptides" and "protein" are used interchangeably herein used and refer to a polymer of amino acid residues. The terms apply to Amino acids polymers in which one or more amino acids residues artificial chemical analogues a corresponding naturally occurring amino acid are, as well as for Naturally occurring amino acids polymers.

As used herein, the term "antibody" refers to a protein consisting of one or more polypeptides consists, essentially by immunoglobulin or fragments of immunoglobulin genes are encoded. recognized Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, Epsilon and mu genes for the constant region, and countless Immunoglobulin fo r the variable region. Light chains are classified as either kappa or Lambda classified. Heavy chains are classified as gamma, mu, alpha, Delta, or epsilon, which in turn immunoglobulin IgG, IgM, IgA, IgD and IgE, respectively are defined.

From a typical immunoglobulin (antibody) -Struktureinheit is known that it comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and a "heavy" chain (50 - 70 kDa) has. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids for primarily the antigen recognition are responsible. The terms' variable light (variable light) (VL) "and" variable heavy refer (variable heavy (VH) Chain itself. In this light and heavy chains

antibodies exist well characterized as intact immunoglobulins or as a number Fragmen&lt;?page 9?&gt;te that produced by digestion with various peptidases will. So concludes z. B. Pepsin antibody among the disulfide bridges in the movable region to produce F (ab) '₂ to create, a dimer of Fab which itself by a disulfide bond with a VH-CH1 associated light chain is. The F (ab) '₂ can under mild Conditions can be reduced to the disulfide bridge in the movable region to solve, thereby converting the F (ab) '₂Dimer is converted to a Fab 'monomer. The Fab 'monomer is essentially a Fab with part of the movable region (with respect to a detailed Description of other antibody fragments see Fundamental Immunology, WE Paul, ed. Raven Press, N. Y. (1993)). While various antibody fragments by the digestion an intact antibody are defined, it is for Those skilled in the art that such Fab 'fragments either chemically or by Application of recombinant DNA methodology synthesized de novo can. The term 'antibody' as used herein includes so antibody fragments one that produced by a modification of whole antibodies, either or synthesized de novo by the application of recombinant DNA methodology will. Preferred antibodies include antibodies of a chain, preferably single-chain Fv (scFv) antibody, in which a variable heavy and a variable light chain together are connected (directly or through a peptide bridge) to form a continuous polypeptide.

A single chain Fv ( "scFv" or "scFv") polypeptide is an equivalent linked VH :: VL heterodimer which consists of a nucleic acid having VH and VL coding sequences either directly connected or connected by a bridge Peptidcodierende are expressed can be. Huston et al. (1998) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. A number structures for converting the naturally aggregated - but chemically separated light and heavy polypeptide chains from an antibody V-region to a scFv molecule folds into a three dimensional structure which substantially is identical to the antigen binding site. See, for. Example, the US patents No. 5,091,513. 5,123,405 and 4,956,778.

An "antigen binding site" or "binding portion" is a part of a Immunoglobulin molecule that is involved in antigen binding. The antigen binding site is by amino acid residues the N-terminal variable ( "V") regions of the heavy ( "H") and light ( "L") chains formed. Three highly divergent Routes within the V regions of the heavy and light chains are referred to as "hypervariable" regions, protected between better flanking routes are known as "framework regions" or "FR" (framework regions) are known. The term "FR" refers So to amino acid sequences, the course occur between and adjacent to hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable Regions of a heavy chain relatively in three-dimensional Space arranged so that they form an antigen binding "surface". This surface mediates the recognition and binding of the target antigen. The three hypervariable Regions of each of the heavy and light chains are referred to as "complementary determining regions" or "CDRs" (complementarity determining regions) denotes and z. B. from Kabat et al. in Sequences of proteins of immunological interest, 4th edition, US Dept. Health and Human Services, Public Health Services, Bethesda, MD (1987) characterized.

As used herein, the terms "immunological binding" and "immunological binding properties" in the non-equivalent Interactions of the type connected between an immunoglobulin molecule and an Antigen occurs, for which the immunoglobulin is specific. As used herein, a biological sample is a sample of biological tissue or Fluid, which in a healthy and / or pathological state to the (S) of the analyte of interest to be analyzed. Such samples include sputum amniotic fluid, blood, blood cells such. as white cells) Tissue samples or cannulas biopsy samples, urine, peritoneal fluid and pleural fluid, or cells thereof. Biological samples also sections of tissues include such frozen for histological Purposes removed sections. Although the sample is typically a human patient is taken, the analysis also to be able Detection of (the) analyte of interest in samples from any mammal such as dogs, cats, sheep, cattle and pigs are used. The sample may be pretreated as necessary by dilution in an appropriate buffer solution or, if desired, be concentrated. One of a number of aqueous standard buffer solutions a buffer of various buffers such as phosphate, Tris or the like. preferably with a physiological pH of each buffer solution can be used will.

As used herein, the term "signal" to a transfer medium along the bioelectric Interface, which is capable of an electromagnetic signal with any useful frequency including to perform a static DC field. A non-exhaustive list includes signal paths conductive and dielectric waveguide structures Mehrtachleiter transmission media such as electromagnetic Transverse (transverse electromagnetic - TEM) transmission lines, transmission lines with three or more conductive Elements which support TE, TM or TEM mode transmission &lt;?page 10?&gt;as Quadrupole or Achtpolleitungen, coupled waveguide, Hohlraumresonatorstrukturen which coupled could be or not, other non-modal Struktren as wires, printed circuits and other distributed circuits and conductive structures with punctiform distributed Impedance, and the like. The structure of the signal path, the signal level, have the basic level or a combination of both structures. Typically, the signal path in one direction is formed, the non-perpendicular to the surface the molecular binding layer (molecular binding layer - MBL) runs. at Embodiments, in which the signal path consists of a conductive layer or region, extends the conductive Region continuously this area. In embodiments with a non-metallic signal ie with a dielectric waveguide is the signal defined as the way of the lowest signal loss or a conductivity of more than 3 mhos / m has.

As used herein, the term "molecular binding layer" or "MBL" in a layer with at least one molecular structure (ie with an analyte, Anti ligand or a ligand / anti-ligand pair, etc.) that match coupled signal path along the bioelectrical interface is. The molecular binding layer may consist of one or more Ligand, antiligand, ligand / Antiligandkomplexen, Coupling groups, matrices of polymers and other materials molecular structures described herein or other exist. The molecular binding layer can also be extremely diverse and a or more components including matrix layers and / or containing insulating layers, one or more linking groups can have. The MBL is the Sig path cuts through either a direct or indirect physical connection or via coupled electromagnetic coupling when the ligand is physically is separated from the signal path. The MBL may consist of a derivatized surface made as of thiol coupling groups biotip metal units contained and the like., all of which the standard practice in the state of the art.

As used herein, the term "binding event" refers to an interaction or an interaction between at least two molecular structures as a ligand or antiligand. The interaction may occur when two molecular structures in direct or indirect physical contact stand or if the two structures physically separate but interrelated are electromagnetically coupled. Examples of interesting binding events in the medical context include ligand / receptor, antigen / antibody, enzyme / substrate, DNA / DNA, DNA / RNA, RNA / RNA, nucleic acid mismatches, complementary nucleic acids, and Nucleic acid / proteins. Alternatively, the term "binding event" is a single molecule or a single molecular structure concern as described herein, such as a Ligand or a ligand / Antiligandkomplex that the signal path is bound. In this case the signal path is the second molecular Structure.

As used herein, the term "ligand / Antiligandkomplex" refers to the bound to the antiligand Ligands. The binding may be specific or non-specific and the bonds are typically covalent bonds, hydrogen bonds, immunological bonds, Van der Waals forces, or other types of binding.

As used herein, the term "coupling" the energy transfer between the two Structures, either a direct or indirect physical connection or through any Form of signal coupling, such as electrostatic or electromagnetic Coupling.

As used herein, the term "test signal" to an be useful with any propagating frequency within the electromagnetic spectrum Signal. The frequency of the test signal lies, for example at or above 1 MHz, z. B. 5 MHz, 10 MHz, 20 MHz, 45 MHz, 100 MHz, 500 MHz, 1 GHz, 5 GHz, 10 GHz, 30 GHz, 50 GHz, 100 GHz, 500 GHz 1000 GHz and intervening Frequencies.

As used herein, the term "enzyme" refers to a protein that as catalyst acts to the activation energy of a chemical reaction in reduce other compounds or "substrates", but is not the final product of the reaction.

As includes herein the term "solution" means a material in which is a ligand. A non-exhaustive list solutions includes materials in solid, liquid or gaseous state. Celebrations solutions can Naturally occurring or synthetic molecules including carbohydrates, proteins, Oligonucleotides, or other, any organic polymeric material such as nylon, rayon, Dacryon, polypropylene, teflon, neoprene, delrin or the like. Liquid solutions include aqueous, organic or other primary Components, gels, gases or emulsions. Exemplary solutions are inter alia, celluloses, dextran, an aqueous solution of d-PBS, Tris buffer, deionized water, blood, phy&lt;?page 11?&gt;siologischer buffer, cerebrospinal fluid, Urine, saliva, water, organic solvents. The solution in the present Context refers to a material in which the ligand and / or antiligand on the binding surface is applied. The solution contains the sample to be analyzed.

As used herein, the term "linkage group" or "coupler" in chemical structures to adhere any two components are used in the bio-assay device. The So coupling groups have a first portion of the se binds a component such as the conductive surface, and a second binding portion, which is located at a different component as the matrix or the antiligand binds.

As used herein, the term "bio-assay device" refers to a structure, in which the molecular binding layer is formed. The bio-assay device may from a surface, a recessed area, or a hermetically sealed housing are made, all of which any size or may form.

As used herein, the term "bio-assay system" on the bio-assay device described above in connection with the necessary components to the bio-assay device to investigate electromagnetic and detect. These components contain, inter alia, the (s) signal (s), (a) substrate (s), electronic devices such as signal generators, oscilloscopes and vector analyzers, the required for inspecting and detecting signals from the bio-assay device are, microchips and microprocessors which electromagnetic signals be able to scan and detect and analyze data and like.

As used herein, the Betriffe relate "resonant" or "resonance" refers generally to a rapidly changing dielectric response function of frequency.

As used herein, the term "bio-electrical interface" refers to an interface structure between a signal for the transfer a test signal and a molecular binding layer.

As used herein, the term "matrix" or "binding matrix" refers to a material layer on the bioanalysis chip, the to as spacers or to increase the surface vertügbaren Napkins or to optimize the alignment of the molecules for better Bond serves, or to enhance any other property of binding to to optimize the bio-assay device. The matrix layer may from carbohydrates such as dextran, polyamino crosslinked and crosslinked proteins and the like. exist.

II. Introduction

A. The Bio-Analysis System

The present invention takes advantage of the observation that a very large number of molecules on the basis of the unique dielectric properties which most of the molecules have, can be distinguished. This distinguishable dielectric properties may be observed by a signal with the bound molecular Structure is coupled. The distinctive dielectric properties modulate the signal, making it an unmistakable signal response receives. The unique signal response can then for detecting and identifying the ligands and other molecules, forming the molecular binding layer may be used.

<figref idrefs="S108">1A</figref> shows an embodiment of a bio-assay system <figref>100</figref> according to the present Invention. The system<figref>100</figref> is in a two-wire, Signalebene-, Basic level circuit topology shown that in a variety Architectures can be realized, including point-like or distributed element circuits in microstrip, stripline systems, coplanar waveguide or coaxial systems. It should also it for the skilled person that the system in a simple manner to a single conductor waveguide system or a three- or multi-conductor system can be modified.

As contains illustrated the system <figref>100</figref> a signal source <figref>110</figref>, Transmission lines <figref>120</figref>. a ground plane <figref>130</figref>, A bio-assay device <figref>150</figref> and a signal detector <figref>160</figref>, The illustrated embodiment shows two transmission lines, although in alternative embodiments, a single transmission line with the bio-assay device can be coupled, or in further alternative embodiments, three or more transmission lines can be coupled to the bio-assay device. The transmission lines<figref>120</figref> consist of a material which makes the transmission a signal over the &lt;?page 12?&gt;desired support operating frequency can. The transmission lines<figref>120</figref> are as a conductive layer z. B. Gold realized, which, on a substrate such as alumina Diamond, sapphire, polyimide, or glass using conventional photolithographic or semiconductor processing techniques is deposited.

The system <figref>100</figref> contains Further, the transmission lines with the <figref>120</figref> coupled Bio-assay device <figref>150</figref>, The bio-assay device<figref>150</figref> includes a receiving substrate <figref>151</figref>. on which a conductive layer <figref>153</figref> upset is. The conductive layer <figref>153</figref> forms an interface for supporting the transmission a test signal. The receiving substrate<figref>151</figref> may be of any insulating material such as glass, alumina, diamond, sapphire, Silicon, gallium arsenide or others in the semiconductor processing used insulating materials.

A molecular binding layer (MBL) <figref>156</figref> with one or several areas of the interface transmission line <figref>153</figref> coupled. For the Electronic engineers, it is obvious that the coupling either a direct connection between the interface transmission line <figref>153</figref> and the MBL <figref>156</figref> as illustrated, or alternatively through signal coupling can be carried out, the later will be described.

The MBL <figref>156</figref> consists mainly of one or more ligands, although other molecules and structures may be present, is as described herein. The MBL<figref>156</figref> may consist of only one bound ligand combination consist, for. example, in the case of the primary binding, or it may in those cases where secondary or binding Acts place order of two, three, four, five or more ligands bound combinations consist. Several ligand combinations on different binding surfaces<figref>155</figref> about the entire interfacial transmission line <figref>153</figref> present.

at of the illustrated embodiment is the dielectric substrate <figref>158</figref> between the solution <figref>157</figref> and the ground plane <figref>159</figref>, In the illustrated embodiment are the dielectric layer <figref>158</figref> and the ground plane <figref>159</figref> within the bio-assay device <figref>150</figref>Although one or both in alternative embodiments, may also be arranged externally. also the arrangement of the MBL <figref>156</figref> and the solution <figref>157</figref> alternative and vice versa in the direction of the base plane or in the close Interfacial transmission line <figref>153</figref> shifted will.

The system <figref>100</figref> contains a signal source <figref>110</figref>That the test signal on the transmission line <figref>120</figref> and in Direction of the bio-assay device <figref>150</figref> settles. A signal detector<figref>160</figref> is positioned along the transmission path, to the resulting signal (either reflected or transmitted or both) to capture. When the signal along the transmission line interfaces<figref>153</figref> of the Bio-assay device <figref>150</figref> travels, modulating the dielectric properties of the MBL <figref>156</figref> the Test signal. The modulated signal can then be regenerated and Detection and identification of within the bio-assay device the ongoing molecular binding events are used, how later will be described.

at an alternative embodiment, of the invention are the detection and identification of a ligand, described Antiligand- / ligand complex or another herein molecular structure possible when this physically from the interfacial transmission line <figref>153</figref> separated are. In this embodiment, is the ligand of the interface transmission line <figref>153</figref> separated, but electrically or electromagnetically coupled thereto. The Coupling between the interface transmission line <figref>153</figref> and the in suspension ligand alters the response of along the interfacial transmission line <figref>153</figref> migratory Test signal, thereby providing a means for its detection and / or identification provided. The maximum separation between the interface transmission line<figref>153</figref> and the suspended ligand will be on such factors as the effective permittivity of the medium between the interface transmission line <figref>153</figref> and the ligand, the entire coupling region, the sensitivity the signal detector, the concentration of the ligands in solution and the desired Detection time determined. The separation distances are typically, in the order of 10^-1 m, 10^-2 m, 10^-3 m, 10^-4 m, 10^-5 m, 10^-6 m, 10^-7 m, 10^-8th m, 10^-9 m, 10^-10 m or in between.

at some embodiments as with cell-based analysis, the MBL solution electromagnetically are coupled to the signal path. Thus, cells and in particular Cell membranes and membrane-based structures to the signal coupled.

<figref idrefs="S108">1B</figref> shows a second embodiment of the bio-assay system, an arrangement of microstrip resonant circuits <figref>170</figref> having. Each resonant circuit <figref>170</figref> consists of a transmission line <figref>172</figref>, the in an open stub <figref>176</figref> ends. To those skilled in the field of circuit design, it is understood that other resonant structures in punctiform Element, distributed element or in a combi&lt;?page 13?&gt;nation of two can be used circuit topologies.

<figref idrefs="S109">1C</figref> shows a sectional view of a resonant circuit <figref>170</figref>, The open stub <figref>176</figref> forms the boundary surface of the bioelectrical the resonant circuit <figref>170</figref> and is in the <figref idrefs="S108">1A</figref> illustrated bioelectrical interface very similar. In particular, there is the open stub <figref>176</figref> from a Interfacial transmission line <figref>176a</figref>. a dielectric layer on the <figref>176b</figref> above the ground plane <figref>176c</figref> is applied.

at this embodiment the MBL <figref>176d</figref> about a direct connection to the transmission line <figref>176a</figref> coupled. The MBL <figref>176d</figref> can along the interfacial transmission line bind to specific or non-specific manner. As above, the concerned molecular structure at the interface transmission line <figref>176a</figref> hanging, but be electrically coupled or electromagnetically therewith to Detection and identification information with respect to a Binding event ready rust ellen.

The Dimensions of the interfacial transmission line <figref>176a</figref> will aspects such as the desired Measurement time (a larger area resulting in faster detection time), the desired Resonance frequency f_res, Certain impedance matching conditions, to a higher to achieve performance or to cause interruptions highlight the binding events, and the process by which the entire assembly is prepared affected. For example, conventional applied microwave photolithography, the binding surface can Using a relatively thick dielectric layer such as aluminum oxide, Diamond, sapphire, Duriod or other conventional substrate material between 10^-1 m² and 10^-6 m² be. Becomes alternatively applied semiconductor processing, the bonding surface may be a relatively thin use dielectric layer of silicon or gallium arsenide between 10^-6 m² and 10^-12 m² be.

Among using conventional Microwave design techniques or CAD tools like Microwave Spice^TM, EEsof Touchstone^TM and Libra^TM the length and impedance of the transmission line <figref>172</figref>, the Dimensions of the interfacial transmission line <figref>176a</figref> as the thickness and the dielectric constant the dielectric layer <figref>176b</figref> be chosen so that the resonant structure a resonance signal response at the desired resonant frequency point f_res supplies. The desired resonant frequency point f_res is typically in the frequency range over which the molecules of interest a dramatic change have their dielectric properties, their measurement their Detection allows. Alternatively, the resonance point f_res as Center the desired Test frequency range are defined to the area farthest the signal detection enable. In the illustrated embodiment contains the resonance frequency f_res 10 MHz, 20 MHz, 45 MHz, 100 MHz, 500 MHz, 1 GHz, 5 GHz, 10 GHz, 30 GHz, 50 GHz, 100 GHz, 500 GHz or 1000 GHz or intermediate frequencies lying.

During the Measurement is the solution <figref>176e</figref> over a or more of the open stubs <figref>176</figref> applied. An MBL <figref>176d</figref> is formed when one or more molecules in the solution the interfacial transmission line <figref>176a</figref> tie. In this case, the MBL behave <figref>176d</figref> and the solution electrically a parasitic Kreis, who later is described by the point of the resonance frequency f_res over or under its original Resonance frequency point is shifted. This resonance shift can be recognized and serves as an indicator for the occurrence of a molecular Binding event. The signal response may also have a wide spectrum be interrogated to find the identity of the bound molecular to confirm structure, is as described below. Each resonant circuit<figref>170</figref> can are prepared so that he different molecular structures binds and each resonant circuit <figref>170</figref> can be made addressable, whereby simultaneously a large Number of molecular structures detected within the same solution and can be identified. In an alternative embodiment can each resonant circuit <figref>170</figref> are designed to it has a particular resonant frequency, in which case all resonant circuits <figref>170</figref> over a continuous frequency spectrum can be queried, to determine a molecular bond.

B. Chemistry of the system

The chemical processes the system generally run within the bio-assay device and in particular along the conductive Layer (interfacial transmission line in the <figref idrefs="S108">1A</figref> to <figref idrefs="S109">1C</figref>) from. The conductive Layer is made of materials that have a conductive morphology to the transfer be able to support the high-frequency test signal. The conductive surface is from materials with appropriate conductivity over the desired test frequency range and with good molecular binding properties as described above. Such materials include gold, indium tin oxide (ITO), copper, Silver, zinc, tin, antimony, gallium, cadmium, chromium, manganese, cobalt, Iridium, platinum, mercury, titanium, &lt;?page 14?&gt;Aluminum, lead, iron, tungsten, Nickel, tantalum, rhenium, osmium, thallium or alloys thereof. The conductive Layer may also consist of semiconductor materials having either crystalline or amorphous structure exist, including chemically doped or pure carbon, silicon, germanium, gallium arsenide, indium gallium arsenide or the like. The conductive Material can also consist of polymers, in particular those the conductive are as polyacetylene, polythiophene and the like. The conductive layer can as required applying thick or more molecular layers deep. The conductive Layer may be composed of an evaporated thin metal layer or a epitaxial layer of gallium arsenide or other semiconductor materials, which are made conductive by semiconductor processing techniques. In addition, the conductive layer are derivatized by a well-known process; see z. B. Kumar et al. "Patterned Self-Assembled Monolayer and Mesoscale Phenomena, "Accounts of Chemical Research, 28: 219-226 (1995).

The conductive Layer is also made of materials with such a morphology which is conductive, to facilitate molecular binding. Ligands can directly, indirectly through other molecular structures or by both configurations Bonds to the conductive received layer. The range of molecules that adhere to the conductive layer can bind includes inter alia proteins, nucleic acids, small molecules, Saccharides, lipids, and other molecules of interest. To the chemical processes , only a single molecular species adhering to the surface, a number of different species or adhering to the surface multiple binding events between species directly abutting on the surface Species and ligand of interest to be involved in the solution.

The involved in the adhesion of a ligand to the conductive layer typical chemistry depends generally on the type of the ligand and any antiligand, the binds of this, and of their functions in the analysis from. A list of possible includes types of interactions that take place on the surface, is but not limited on interactions protein / protein interactions DNA / protein interactions RNA / protein hybridization of nucleic acids including a Analysis of base pair mismatch on, interactions RNA / RNA, tRNA interactions, enzyme / substrate systems, interactions Antigen / antibody, Interaction small molecule / protein Interactions drug / receptor interactions membrane / receptor, conformational in solid phase ligand interactions protein / saccharide and Interactions lipid / protein.

The actual surface Chemistry may at any one of Embodiments as the primary and secondary Binding are described. additional Layers with molecular binding may also occur. The primary Binding relates to the adherence of an antiligand to the conductive surface, can be done by means of a coupling molecule. The secondary binding relates to the binding of a ligand to an antiligand, the a another molecule be in the MBL, can, or directly to the conductive surface itself. Typically in binding a liquid phase ligand binding involved in an immobilized solid phase ligand. A primary bond could For example, the adhesion of a specific antibody to the conductive Layer of the bio-assay device may be, and a secondary bond would the Binding of a specific antigen in a sample solution to the antibody mean. Alternatively, the secondary binding the direct adhesion a protein to the conductive surface be z. B. when the amino-terminus a protein adhered directly to a conductive layer of gold.

The abovementioned bond results in the formation of a molecular Binding layer (MBL) <figref>180</figref> along one or more areas the conductive Layer, one embodiment of which in <figref idrefs="S109">1D</figref> is shown. In this embodiment, is the MBL <figref>180</figref> optionally from a first coupling group <figref>181</figref>, one insulator <figref>182</figref>, A second coupling group <figref>183</figref>. a matrix <figref>184</figref>, A third linking group <figref>185</figref>, a Antiligandschicht <figref>186</figref> and a Ligandschicht <figref>187</figref>,

The first coupling group <figref>181</figref> ensures the adhesion of the insulating layer <figref>182</figref> at the conductive Layer (not shown). The first coupling group<figref>181</figref> consists of molecules such as thiols, amines, amides, or metals such as chromium or titanium. The insulating layer <figref>182</figref> provides a barrier between the conductive Layer and the MBL <figref>180</figref> and a solution (not shown) ready. The insulating layer <figref>182</figref> can a hermetic barrier deploy to a structural deterioration of the conductive layer To prevent the influence of MBL and / or the solution. alternative or additionally, the insulating layer may <figref>182</figref> made of an electrically non-conductive material made to the flow of direct current or low frequency energy from the conductive Layer to MBL and / or solution prevent, whereby the measurement would be disturbed. The insulating layer may Polyimide, alumina, diamond, sapphire, non-conductive polymers, insulating semiconductor material such as silicon dioxide or gallium arsenide or contain other materials that a hermetic seal provide and / or electrically insulating properties. The Insulating layer may also consist of air or another gaseous substance exist, in which case the coupling group <figref>181</figref>&lt;?page 15?&gt;waived can be.

The second coupling group <figref>183</figref> ensures the adhesion of the insulating layer <figref>182</figref> at the matrix <figref>184</figref> and consists of the same or similar molecules as the first coupling group <figref>181</figref>, The matrix layer<figref>184</figref> can consist of a polymer layer, but also optionally a carbohydrate, Protein, Polyaminosäureschicht or the like.. The third coupling group<figref>185</figref> consists of molecules which for adhering the matrix layer on the anti-ligand <figref>186</figref> are, and may consist of the same or similar molecules as the first and / or second coupling group <figref>181</figref> or. <figref>183</figref> consist.

Of the antiligand <figref>186</figref> used for specific or unspecific Ligand binding <figref>187</figref> in the solution and / or for measuring the physical properties of the solution, some of them eg. B. the temperature, the pH, the ionic strength are and the like. The antiligand consists of a molecule or a molecular structure which specifically or non to the ligand <figref>187</figref> binds. In the case, for. Example, in which the Ligand is an antigen, the antiligand is <figref>186</figref> out an antibody. The ligand <figref>187</figref> consists of a molecule or a structure specifically or non-specifically to the anti-ligand <figref>186</figref> binds.

in the Generally sufficient for the MBL a measurable interaction as described herein with an electromagnetic Test signal to the associated Signal route. Thus, essentially any MBL composition, having the varying dielectric properties, analyzed will. With most forms of execution the thickness of MBL in the range between about 1-5 Å to 1 cm. For simple molecular binding events is the area generally between about 10 Å and 10,000 Å, typically between 100 Å and 5000 Å or between 500 Å and 1,000 Å. For larger interactions (Eg cellular), the MBL between 1 .mu.m and 100 .mu.m, preferably between 5 .mu.m and 50 .mu.m. By Insulators, matrices and the like. Rises this size considerably.

The embodiment from <figref idrefs="S109">1D</figref> is not possible MBL configurations exhaustive represent. For the expert, it is understood that a wide variety of combinations can be designed to form the MBL, as special from the Applications required. Thus, for. Example, the first, second and third coupling group <figref>181</figref>. <figref>183</figref>. <figref>185</figref>. the insulating layer <figref>182</figref> and the matrix layer <figref>186</figref> not implemented, and the MBL consists of the anti-ligand <figref>168</figref> and the ligand <figref>187</figref>, As a further alternative to the first coupling group <figref>182</figref> and the insulating layer <figref>182</figref> waived will. Other alternative embodiments which accounts for one or more of the described layers or additional added layers be, are for the skilled artisan.

The MBL may also from heterogeneous molecules exist, the spatially grouped depending on the matrix format in question or may be layered or distributed as desired. So shows z. B.<figref idrefs="S110">1E</figref> a top view of an MBL <figref>180</figref> With Four different anti-ligands <figref>190</figref>. <figref>191</figref>. <figref>192</figref> and <figref>193</figref>. spatially are separated. <figref idrefs="S110">1F</figref> shows an MBL <figref>180</figref>. in four different anti-ligands <figref>190</figref>. <figref>191</figref>. <figref>192</figref> and <figref>193</figref> about the entire MBL are arbitrarily distributed. <figref idrefs="S111">1G</figref> one Alternatively, shows a sectional view in which the MBL <figref>180</figref> cell <figref>194</figref> in a solution <figref>157</figref> contains the to the signal path <figref>153</figref> coupled. In another embodiment There is a cell membrane <figref>195</figref> with membrane-bound Structures (not shown) in the signal path with the <figref>153</figref> coupled solution <figref>157</figref>, The layers may z. B. coupling groups, matrices, antiligand, ligand and a or more insulating layers included. In some embodiments, , one or more membranes are used, such as, for. example, those the ion transport, the size or La formation selectively control or the adhesion of the anti-ligand or a other molecular structure support.

in electrical terms, the MBL distinctive dielectric Properties which in part to the structural and conformational properties and their amendments the bound molecules both isolated and in the presence of environmental changes how binding events, changes pH, ionic strength are and the like due.. The dielectric properties of the bound molecular structures together with the local structures of the dissolving medium (the solution) can also on changes the intramolecular and intermolecular bonds due to primary bond or binding higher Order and the displacement of the dissolving medium near the conductive layer be due.

As soon as a conductive Layer has been provided, those skilled in the prior art is Literature for Biology and chemistry have recourse, to select a system, with which he can work. A general introduction to biological systems can be found in: Current Protocols in Molecular Biology, FM Ausubel et al. Eds., Current Protocols, a joint venture between Greene Publishing Associa&lt;?page 16?&gt;ted, Inc. and John Wiley &amp; Sons, Inc. (adds up 1997) (Ausubel); Watson et al. (1987) Molecular Biology of the Gene, 4th edition, The Benjamin / Cummings Publishing CO, Menlo Park, CA. Alberts et al. (1989) Molecular Biology of the Cell, 2nd Edition, Garland Publishing, NY; The Merck Manual of Diagnosis and Therapy, Merck &amp; Co., Rathway, NJ. Product information from manufacturers of biological reagents and experimental equipment also provide useful information for the analysis of biological systems. Such manufacturers include,. B. Sigma Chemical Company (St. Louis, MO), R &amp; D Systems (Minneapolis, MN), Pharmacia LKB Biotechnology (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Aldrich Chemical Company (Wilwaukee, WI), GIBCO BRL Life Technologies, Inc. (Gaithersberg, MD), Fluka Chemica-biochemicals Analytica (Fluka Chemie AG, Buchs, Switzerland, Applied Biosystems (Foster City, CA) as well as numerous other commercial sources that the skilled person are well known.

Biological samples patients using sufficiently known techniques such as Venipuncture, lumbar puncture and from fluid samples such as saliva or urine, or tissue biopsy and the like. be removed. If the biological material of non-human beings as commercially taken significant cattle, to leave the blood and tissue samples simplest of livestock processing plants to win. Similarly Example, used in the invention plant material well from agricultural or horticultural sources and other sources of natural Products are obtained. Alternatively, a biological sample coming from a cell or blood bank where tissue and / or blood stored be, or from an in vitro source, such as a cell culture. techniques the need for cell culture for use as a source of biological Materials are known to the skilled worker. Freshney, Culture of Animal Cells, a Manual of Basic Technique, 3rd edition, Wiley-Liss, NY (1994) provides a general introduction to the cell culture.

The present invention may by a series embodiments be realized. Some are described in detail below, Other embodiments and applications can be found at the section "Applications".

at one embodiment The invention is to detect the binding of a molecular structure of a signal applied. In this embodiment, a signal transmitted along the signal path. While its transmission coupled it to the bound structure and is modulated. The Analysis of the modulated response indicates binding.

at Alternatively, the invention can be for identifying secondary bonds are applied. A primary Binding can for example to adhesion of an antibody be the conductive surface. secondary binding could measuring the binding between the immobilized antibody and its antigen in the solution imposing keep. After the primary Binding has been detected as described in the previous paragraph, is the antibody containing solution added to the bio-assay device and the response measured again. The answer is compared with the response of the primary binding. A change would it point out that a binding event has occurred.

at one aspect, the present invention for identifying applied antiligand z. B. proteins in the primary binding phase will. In the calibration phase, the responses of a large number can known proteins are determined and stored. After adhering an unknown protein to the analysis interface could the dielectric properties measured of the system and the dielectric properties of the signal be used to identify the protein on the surface. because the fingerprint response of each protein is stored can, compared the unknown response to the stored answers and pattern recognition to identify the unknown protein used will.

at Alternatively, the invention can be applied in a matrix format. The Device has a plurality of addressable locations, wherein a at each specific anti-ligand is bound. After the addition of solution Device be binding answers each point measured and characterized. A device of this type may for measuring and / or Identifying the presence of specific nucleic acid sequences be used in a sample. At each of the addressable locations adheres a unique nucleic acid sequence as the antiligand. After exposure to the sample will bind to complementary sequences the appropriate spaces. The answer at any point indicates whether a sequence is a bond has been received. Such a measurement also indicates whether the bound sequence is a perfect match with the antiligand sequence, or whether one or more mismatches present. This embodiment can also be used to identify proteins and classes of proteins.

&lt;?page 17?&gt;

at Alternatively, the invention can be to generate a standard or titration are used, then the to determine the unknown concentration of a particular analyte or ligand would be used. an antibody example could adhere to the device. The apparatus could of several different exposed to concentrations of the ligand and the response for each concentration be measured. Such a curve is to the skilled man also known as dose-response curve known. An unknown sample can be exposed to the device, and the response can be measured. Your answer can with the standard curve are compared to the concentration of the ligand in the unknown to determine sample.

at Alternatively, the invention may be for internal self-calibration to losses caused be applied from aging and other stability issues. For example, in antibody / antigen systems the invention allows the measurement of the amount of active antibody the surface by measuring the primary Response before the unknown sample the device is exposed. The value of the primary Response is used to adjust the secondary response, the antigen binding by a constant, the amount of active on the device remaining antibodies equivalent.

III. The bio-assay device

A. Structure of Apparatus

Regarding containing structure the bio-assay device includes a signal path and a bio-electrical interface. Of the Signal may consist of a single input / output terminal, an input signal connection path and an output-connection path, or multiple input and / or Output connection paths consist. The (the) signaling pathways) can (can) as a number of different Architectures can be realized, such. As to a transmission line or a waveguide structure that the transmission of the test signal over desired Frequency range supported. With respect to possible Embodiments see RE Collins Foundations for Microwave Engineering, McGraw-Hill Publishing Co., 1966; and S. March, Microwave Transmission Lines and Their Physical Realizations, Les Besser and Associates, Inc., 1986. In addition, , the bio-assay device in diverse various configurations can be realized. Among the possible configurations include among other things, large to miniaturized structures using conventional techniques, conventional etching and Photolithography or semiconductor processing techniques.

<figref idrefs="S112">2A</figref> shows an embodiment of the bio-assay device on average. The bio-assay device<figref>230</figref> consists of a cover plate <figref>231</figref>, Contact terminals <figref>237</figref> and a baseplate <figref>239</figref>, The cover plate<figref>231</figref> contains a lower surface with an applied thereto interfacial transmission line <figref>233</figref>, The dielectric substrate <figref>240</figref> and the ground plane <figref>250</figref> are outside the bio-assay device. The cover plate <figref>231</figref> and / or the dielectric substrate <figref>240</figref> consist of an insulating material such as glass, which is preferably of conventional Photolithography or gold sputtering, etching or processing by suitable - chemical deposition from the vapor phase (CVD chemical vapor deposition) is. Other materials such as alumina, silicon, gallium arsenide or other insulating materials can optionally be used.

As in <figref idrefs="S112">2A</figref> illustrated is the lower surface of Interfacial transmission line <figref>233</figref> in contact with the molecular binding layer (MBL) <figref>234</figref>, As shown can the MBL of bonded molecular structures different Layers, types, or from molecular structures exist, in the solution available. In alternative embodiments, the MBL <figref>234</figref> about small or large Portions of the interfacial transmission line <figref>233</figref> extend and consists of various bound molecular structures as shown. The MBL can only Antiligand- / ligand structures or from a variety of Intermediate structures of coupling group, Matrix and insulating consist, as in <figref idrefs="S109">1D</figref> is shown. provided provided, the insulating layer <figref>182</figref> (<figref idrefs="S109">1D</figref>) Of air, polyimide, alumina, diamond, Sapphire or insulating semiconductor material such as silicon dioxide or Gallium arsenide or additionally to other conventional Insulating materials made of a non conductive material. The Thickness and dielectric constant the insulating layer can be selected so that the MBL <figref>234</figref> and the interfacial transmission line <figref>233</figref> during the signal transmission are tightly coupled. The thickness of the insulating layer<figref>182</figref> may 10^-1 m, 10^-2 m, 10^-3 m, 10^-4 m, 10^-5 m, 10^-6 m, 10^-7 m, 10^-8th m, 10^-9 m, 10^-10 m or less or have a value between what the required degree of Coupling, the dielectric constant the insulating layer and the total coupling surface depends. The coupling can be carried a number of different configurations including cross and offset coupled configurations in multi-layer, coplanar or waveguide circuit topologies can be realized. The implementation an insulating layer can for her&lt;?page 18?&gt;metic sealing of the interfacial transmission line opposite to the dissolution medium be advantageous and / or to prevent or DC low-frequency current in the solution flows, which is held molecular binding events may could be interrupted.

The Interfacial transmission line <figref>233</figref> consists of a material capable of supporting the signal transmission and that is capable of the MBL <figref>234</figref> to bind. The material varies depending on the structure of the MBL, but may include gold, indium tin oxide (ITO), copper, silver, zinc, tin, antimony, gallium, cadmium, chromium, manganese, Cobalt, iridium, platinum, mercury, titanium, aluminum, lead, iron, Tungsten, nickel, tantalum, rhenium, osmium, thallium or alloys thereof be. Optionally, the interfacial transmission line<figref>233</figref> a or more molecular structures (antiligand) (the part the MBL <figref>234</figref> form included) to bonds with one or several target molecules form (ligands). The interfacial transmission line containing material can also be chosen such that it sticking promoting of coupling groups and the signal transmission. Other Materials for forming the interfacial transmission line <figref>233</figref> used can be, are well known in the art.

The Ligands may by means of a solution <figref>260</figref> as z. B. the phosphate buffered saline solution (d-PBS) Dulbecco to MBL <figref>234</figref> be transported. The protein, the nucleic acid or other interest ligand may by a variety Techniques such as wicking, pipetting, dipping, trickling direct Contact or applied by capillary action.

at a specific embodiment, is the interfacial transmission line <figref>233</figref> so designed such that it has a low signal loss and with regard the impedance of the external transmission lines <figref>270</figref> closely matches. A low signal loss is achieved by the interface transmission line <figref>233</figref> out a conductive Material such as gold, copper, aluminum, indium tin oxide prepared (ITO) or other conductive materials described above becomes. A close impedance match is achieved by the width of the interfacial transmission line <figref>233</figref> at about the width of the external transmission lines <figref>270</figref> in dependence of the relative dielectric properties of the substrate, the solution and MBL is determined. The signal passage between the interfacial transmission line<figref>233</figref> and the external transmission lines <figref>270</figref> is via contacts <figref>237</figref> provided. As explained above, the MBL <figref>234</figref> and the dissolution medium <figref>260</figref> in nearby the ground plane <figref>250</figref> be arranged or alternatively close the interfacial transmission line <figref>233</figref>,

additional analog and / or digital circuits in punctiform element form, distributed Form or in a combination of the two can at the input and / or output terminals of the Bio-assay device may be provided. At the input terminal can For example, impedance matching circuits and / or buffer amplifier circuits be provided. Alternatively or in addition, impedance matching circuitry and one or more output amplifiers are implemented, the output signal to increase further. For the electronics skilled clear that in alternative embodiments other types of processing circuits can be used.

<figref idrefs="S113">2 B</figref> shows a second embodiment of the bio-assay device. In this embodiment, takes the solution a space above the interface transmission line <figref>233</figref> on, on the upper surface the bottom plate <figref>239</figref> is trained. The top of the Interfacial transmission line <figref>233</figref> forms the binding surface, where the MBL <figref>234</figref> liable. The dielectric layer<figref>240</figref> is between the boundary surfaces transmission line <figref>233</figref> and the ground plane <figref>250</figref> positioned. contact terminals<figref>237</figref> put a signal to the external transmission lines <figref>270</figref> ready. The interface transmission line, the top plate, the bottom plate, the contact terminals and the dielectric layer can prepared from the materials and the processes as above will. The MBL can above for<figref idrefs="S109">1D</figref> described configure or variations thereof. Further, the MBL <figref>234</figref> and the dissolution medium <figref>260</figref> in nearby the ground plane <figref>250</figref> be arranged or alternatively close the interfacial transmission line <figref>233</figref>,

More structural embodiments contain bio-assay devices with multi-element transmission lines, Waveguides, and resonant cavities, in which the MBL at a or more of the conduits or cavity elements in such a Way can stick that detection specificity and sensitivity improves will. examples for such structures include parallel arranged signal combiners, Cavity resonators or waveguides, which along the bound MBL to a member, the signal transmission properties compared to another parallel element without the bound structural changes and so the fashion characteristics of the combined signal changed, resulting in legible output characteristics. This latter Effects make themselves well-known techniques &lt;?page 19?&gt;measurement of Frequency, the frequency stability and very small frequency changes advantage of ultra-high precision.

B. Chemistry of the binding surface

<figref idrefs="S114">3</figref> shows an embodiment the chemical processes the binding surface, along the conductive drain layer bioelectrical interface. The bio electric interface contains a substrate <figref>320</figref>, A conductive layer <figref>220</figref>. MBL <figref>340</figref> and a solution <figref>350</figref>, substrate <figref>320</figref> As described in each of the herein dielectric layer or substrate materials, including alumina, Diamond, sapphire, glass, and the like., And can be used for the conductive layer <figref>230</figref> a bearing Bearing offer. In an alternative embodiment deleted the substrate <figref>320</figref> and the bearing Bearing is an insulating layer <figref>342</figref> provided.

The conductive layer <figref>330</figref> consists of a material with a morphology the signal transmission via the desired Frequencies and the binding of MBL <figref>340</figref> promotes such described above. When running a two-wire circuit topology the conductive layer <figref>330</figref> have the signal level and the ground plane. In any case, however, a second conductive layer (either the Signal level or the ground plane; not shown) either below the substrate <figref>230</figref> (Arrangement by <figref idrefs="S113">2 B</figref>) disposed, or at least one substrate layer removed from the solution <figref>350</figref> away (Reverse arrangement of <figref idrefs="S112">2A</figref>). Alternatively, conductive Layers on both of these levels are arranged.

The solution <figref>350</figref> is with the MBL <figref>340</figref> coupled to the flow of ligands for MBL <figref>340</figref> permit. The ligands flow of the solution<figref>350</figref> to MBL <figref>340</figref> can be addressed or not addressed. The solution is of any transport medium such as gases, materials in liquid or Solid phase, of which z. B. some aqueous d-PBS, Tris buffer, phosphate buffer and like is..

The MBL is along the bio-electrical interface between at least one Part of the solution and the signal path is positioned such that the MBL along this part closer the signal as is to the solution. In the embodiment from <figref idrefs="S114">3</figref> the MBL <figref>340</figref> between the solution <figref>350</figref> and the conductive layer <figref>330</figref> closer to the latter positioned. In one embodiment, (in <figref idrefs="S112">2A</figref> shown) located the solution between the signal and the ground plane. ment of In a second Ausfüh (in <figref idrefs="S113">2 B</figref> shown) located the solution outside the region of the signal-ground plane.

The typical chemistry involved in binding the MBL to the conductive surface depends in general on the type and content of the MBL its function in the analysis as well as from. The MBL may consist of a ligand, a ligand / Antiligandkomplex molecular structures described herein or other exist. The ligand is typically functionally intact, is so close as possible on the surface and the surface density the antiligand is sufficiently high, the largest dielectric effect to offer, but not so high that it interferes with the binding function, such as by steric hindrance or by the physical blocking the active binding site of the immobilized anti ligand through neighboring molecules.

ligands can specifically or non-specifically either directly to the conductive layer<figref>320</figref> or via intermediate structures bind as in <figref idrefs="S114">3</figref> shown. are specific bound ligand desired, is an optional linking group used for binding to simplify, z. B. all of the proteins to bind so that the conductive layer <figref>320</figref> of the solution is exposed. To a densely packed layer binding ensure can Thiol groups, Fab or proteins are used as protein A, for binding of antibodies or other anti-ligand along the conductive layer <figref>320</figref> to simplify. These and similar substances can in several ways to the conductive layer <figref>320</figref> be applied, inter alia, by photolithography, Semiconductor processing, or any other conventional coating technique.

In addition, some Ligands and anti-ligands to bonds in several ways in the To be able to do. These ligands have a statistically prevailing Binding mode or can be manipulated so that it ties in site-specific manner received. Some anti-ligands bind the surface in a site-specific optional Wise. An oligonucleotide can, for example, to a terminus be bound. In general, the anti-ligand is liable to such Way that the function of the anti-ligand is not adversely affected, z. B. preferably at concentrations Oberflächendenaturierung the minimize.

The varied concentration of the antiligand on the binding surface dependent on the respective analyte. Typical concentrations for proteins are z. B. 10⁷/cm². 10⁸/cm², 10⁹/cm², 10¹⁰/cm², 10¹¹/cm². &lt;?page 20?&gt;10¹²/cm², 10¹³/cm², 10¹⁴/cm², 10¹⁵/cm² or concentrations between. Typical concentrations for nucleic acids are 10⁷/cm², 10⁸/cm², 10⁹/cm², 10¹⁰/cm², 10¹¹/cm², 10¹²/cm², 10¹³/cm², 10¹⁴/cm², 10¹⁵/cm², 10¹⁶/cm², 10¹⁷/cm², 10¹⁸/cm², 10¹⁹/cm², 10²⁰/cm² or concentrations between. Typical concentrations for Analytes in whole blood are 55 M, 25 M, 10 M, 1 M, 0.5 M, 10^-1 M, 10^-2 M, 10^-3 M, 10^-4 M, 10^-5 M, 10^-6 M, 10^-7 M, 10^-8th M, 10^-9 M, 10^-10 M, 10^-11 M, 10^-12 M, 10^-13 M, 10^-14 M, 10^-15 M, 10^-16 M, 10^-17 M, 10^-18 M or Concentrations between.

At the MBL should adhere a sufficient amount of ligand to the transfer a signal over the bioelectrical interface to change. The amount of adhering to the bonding surface ligands can 1, 10, 10², 10³. 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ or more be ligands or any amount in between, depending on the surface conductive Layer. The ligands do not need in predefined regions along the conductive to be applied layer since the signal responses from the inherent dielectric Features of the MBL over determines the arrangement of the bio-assay device or chip will. The MBL will generally have a surface density for smaller molecules in the range of 10¹⁰ cm² at 10²⁴ cm², typically of 10¹⁵ cm² at 10²⁰ cm², The fat the ligand may be only one layer, but 2, 3, 4, 5, 10 or more layers are selectively used.

As soon as a ligand to the conductive Layer is bonded, the chemical and / or structural biology comes of the system to bear. The dielectric properties of the ligands yield a signal response which is characteristic of the bound (s) Structures), whereby the detection of binding events as well as from other properties of interest is made possible in the structure. The provided by the binding event unmistakable answer depends by immobilized anti ligand, its target ligand, and the rearrangement Nearby solution molecules (such as Water and free ions) from. The range of molecules which to the surface can bind, contains inter alia, proteins, nucleic acids, small molecules, Saccharides, lipids, and other molecules of interest.

The molecules the MBL are typically in a solution, which is to be a aqueous solution of Water, d-PBS, Tris, blood, physiological buffer, cerebrospinal fluid, Urine, sweat, Saliva, other bodily secretions, organic solvents can act, and the like.. Other solutions can gases, Emulsions, gels, and organic and inorganic compounds contain.

The secondary Binding reaction occurs at the MBL of the bio-assay device. A ligand in a solution is over the bio-assay device transports that he with the anti-ligands the bond layer comes in contact. The concentration of the ligand in the solution varies and may be 10^-1 M, 10^-2 M, 10^-3 M, 10^-4 M, 10^-5 M, 10^-6 M, 10^-7 M, 10^-8th M, 10^-9 M, 10^-10 M, 10^-11 M, 10^-12 M, 10^-13 M, 10^-14 M, 10^-15 M, 10^-16 M, 10^-17 M, 10^-18 M, 10^-19 M, 10^-20 M be. If an interaction such as a bond between the Ligand and the antiligand is taking place, the ligand is then optional part of the binding layer, by the chemical equilibrium characteristics the binding event is predetermined.

The MBL containing the bound ligand and may also include solution molecules. In the bound Ligands may be any molecules, including proteins, Carbohydrates, lipids, nucleic acids and any other act herein discussed molecules. The MBL may further contain a linking group to the binding of the anti-ligand to the binding surface layer to support.

Also changes the Interaction of the antiligand with the ligand, the characteristic dielectric response of the binding layer, when only the antiligand is adhered. If z. B. the antiligand A one antiligand, the bonding layer forms, are the dielectric response of a transmission line along the transmitted Test signal, the characteristic features of the structure of the Antiligand A again. When the ligand B with the anti-ligand A an alternative binding site, change the structure and / or the dielectric properties of the Binding layer due to the binding of A to B. The structure of A can change if B binds to A, whereby another signal response provided becomes. The signal change because of the binding interaction is characteristic for the binding of A to B. The existence of a binding interaction can therefore from a signal change be determined.

Further can information about the type of bond or structural and / or conformational are obtained in the binding, by determining which Part of the answer signal has changed due to the interaction. The ligand B is optional in the binding to the anti-ligand A by the signal change detected and identified. The binding of ligand B to antiligand A induces a conformational change or other changes in the molecular structure or the surrounding solution at Antiligand A and its surroundings. These changes alter the dielectric own sheep&lt;?page 21?&gt;th of MBL, whereby the signal response of the data transmitted via the signal path changed test signal becomes. The change the test signal can be used to the binding event of the ligand B to recognize, and the specifics of the amendment can for identifying the Ligands B are used. In this respect, the relationship between Structure and function of the molecule is known, for. example, in the case of enzymes, antibodies, receptors and the like., the function of the bound ligand from its spectral Identification is derived.

at one embodiment is a Antiligandtyp applied to the bonding surface to an MBL form and a ligand is the MBL applied to detect a binding event between the two molecules. In another embodiment, can the antiligand be a mixture and the layer on the bond applied Ligand is a known analyte or antibodies. By detecting specific changes the signal response, the ligand in question, with said anti-ligand interacting, due to conformational and other changes, induced in the ligand or anti-ligand and the resulting resulting spectral sensitivity can be determined. A such an embodiment does not require the spatial Separating each of the specific anti-ligand, but passes the desired level of specificity rather from the spectral sensitivity, so that a given binding interaction rather by observing the electromagnetic response than by Determining which part of the analysis of the binding event occurred will have determined.

at Alternatively, can the antiligand be a known molecule on the binding layer and the ligand is a mixture of unknowns z. B. a whole blood sample on the Bio-assay device applied. In this case, the presence of of a particular ligand such as an antibody in the blood by the presence of or absence of a particular point or a specific signal detected in the spectrum from the passage of the signal through the Bio-assay device results. Alternatively, it may be due to changes in the spectrum of the anti-ligands or ligands are detected upon binding of the ligand. such embodiment elevated specificity detection over of the binding chemistry alone, since the signal information on the includes type of binding event. Thus, a specific binding of non-specific binding be distinguished, and the overall specificity of detection may be compared to the specificity just chemistry are significantly improved.

The Detection system formed by the use of the bio-assay device provides a detection system having a high throughput prepared as the Detection optionally occurs in real time and numerous samples quickly can be analyzed. The response period is optional monitored on a nanosecond time scale. Once the molecules are bonded to each other, the detection takes place. to measure low concentrations or binding events between molecules binding affinity low Bin is optionally a longer Period of time required. The actual Time is optional limited by diffusion rates. apart of these potential limitations can thousands of compounds optionally processed very quickly by the system be such. as in an hour. Among application of chip fab technologies of developing for example, a 10,000-channel device (using up Microfluidics technologies) possible and with small volumes and thus short diffusion times and with kinetic measurements, which measure only the beginning of the reaction, optionally 10 million samples per hour measured. At acquaintances Concentrations, the binding affinity optional alone from the kinetics calculated and thus the device can with a very fast sampled time scale and the affinity of the slope of the kinetic Curve calculated and / or estimated will. Information on kinetics and affinities can be found in any standard work on biochemistry or chemistry, for. example, in Mathews and van Holde, Biochemistry, Benjamin Cummings, New York., 1990

C. bioelectrical interface

The bioelectrical interface is a structure along which the MBL and the signal path are formed. As described above, the signal path of a conductive or dielectric waveguide structure, a two conductor structure such as a conventional Signal / ground plane structure or three or more conductor structures exist, as are known in the art. In general is the thickness of the conductive Region of the signal path so designed that a minimum signal loss given is. So is z. B. the typical thickness of a transmission line of gold on the order from 0.1 to 1000 microns, preferably between about 1 - 10 microns.

Of the Signal path is formed along a direction that is not perpendicular runs to MBL. In one embodiment, the test signal in parallel to a tangent to the surface on which is formed the MBL, transferred. In other embodiments, can the test signal at an angle of ± 1 °, ± 2 °, ± 3 °, ± 4 °, ± 5 °, ± 10 °, ± 15 °, ± 20 °, ± 30 °, ± 40 °, ± 45 ° , ± 50 °, ± 60 °, ± 70 °, ± 80 ° or ± 85 ° relative to the MBL-binding top&lt;?page 22?&gt;area or transmitted under any values ​​in between who the. At a first embodiment is the signal from a transmission line in two-wire structure and the direction of the signal path is through the Poynting vector defines what in the field of electromagnetics is well known. In a second embodiment, the transmission line of a conductive Region or layer made that continuously along the bioelectrical interface region extends. In a third embodiment, the signal path be defined as the way along the lowest signal loss bioelectrical interface via the desired Operating frequency range has. In a fourth embodiment , the signal path than to an AC conductivity of more than 3 mhos / m are defined, that is having a conductivity greater than that of a physiological Saline, typically about 5 mhos / m, but ideally in the range of 100 to 1000 mhos / m and about that is.

The Operation of the bioelectrical interface is based on the development an equivalent circuit model for the interface explained in more detail. The presented Equivalent circuit models are shown in two-wire circuit topology, although it the Fachmall in the field of circuit design is obvious, that each model in single-core waveguide topologies, resonant circuit topologies implemented and in circuit topologies with three or more conductors can be.

<figref idrefs="S115">4A</figref> shows an embodiment of an equivalent circuit model <figref>420</figref> for in <figref idrefs="S112">2A</figref> illustrated bioelectrical interface structure. For the Those skilled in the art of circuit design, it is obvious, that the circuit model shown is not exclusively and that other equivalent circuit models of bioelectric interface according to <figref idrefs="S112">2A</figref> can be derived.

The Equivalent circuit model shown includes series blocks <figref>422a</figref>. <figref>424a</figref> and <figref>426a</figref>. the series electrical effects of the interface transmission line <figref>232</figref>. the MBL <figref>234</figref> or the solution <figref>260</figref>. all of which in <figref idrefs="S112">2A</figref> are presented, model. The interface transmission line, the BML and the solution circuit blocks <figref>422a</figref>. <figref>424a</figref> and <figref>426a</figref> are coupled in parallel, since the interfacial transmission line, the MBL and solution each a potential Signal path in the longitudinal direction providing along the interface. In an alternative embodiment, in which the MBL and solution close the ground plane is, the arrangement of the interfacial transmission line and the basic levels of the equivalent circuit model <figref>420</figref> vice versa. In the embodiments, in which both the MBL and the solution near both the interface transmission line and the ground plane are assumed <figref idrefs="S112">2A</figref> the upper half the equivalent circuit is, the lower half (basic level) identical is when the same solution and MBL are used.

The Equivalent circuit model <figref>420</figref> further includes shunt circuit blocks <figref>422b</figref>. <figref>424b</figref> and <figref>426b</figref>. each having the electrical shunt effects of in <figref idrefs="S112">2A</figref> illustrated dielectric layer <figref>240</figref>, of the MBL <figref>234</figref> and the solution <figref>260</figref> model. The series orientation of shunt blocks <figref>422b</figref>. <figref>424b</figref> and <figref>426b</figref> results from the physical arrangement of each of these elements in series of the interface transmission line through the MBL, the solution and the dielectric layer provided to the base plane and in <figref idrefs="S112">2A</figref> is shown.

<figref idrefs="S116">4B</figref> shows an embodiment of a circuit <figref>430</figref>. which the equivalent circuit model of <figref idrefs="S115">4A</figref> equivalent. Those skilled in the art of circuit design will readily recognize, that other circuit configurations are possible. The series circuit blocks<figref>422a</figref>. <figref>424a</figref> and <figref>426a</figref> consist each composed of a resistor and a series inductor. The shunt circuit blocks <figref>422b</figref>. <figref>424b</figref> and <figref>426b</figref> consist each composed of a resistor and a capacitor connected in parallel. Series resistors R_T, R_M, R_S model respectively the resistivity of the interfacial transmission line, the MBL and the solution. Shunts R_M', R_S', R_d modeling each specific Resistance of the MBL, the solution and the dielectric layer. Reihenindukturen L_T. L_M, L_S model each of the inductance of the interface transmission line, the MBL and the solution. Shunt capacitors C_M, C_S. C_d model respectively the capacity of MBL <figref>234</figref>, The solution <figref>260</figref> and the dielectric layer <figref>240</figref>, Defining their entirety the aforementioned resistors, Inductors and capacitors, the circuit <figref>430</figref>, the the Input signal V_i in the output signal V_O converts.

The dielectric properties of the MBL largely determine the values of the circuit elements corresponding to each of these layers. at the in <figref idrefs="S116">4B</figref> embodiment illustrated defined z. B. in particular the sensitivity of MBL the value of Shunt capacitor C_M, Further determine the dispersive properties of the MBL largely the value of the shunt resistor R_M'. The values ​​of C_M and R_M'Define the signal response bioelectrical interface to a significant degree. The signal response bioelectrical interface is so characteristically highly for the dielectric properties &lt;?page 23?&gt;the MBL and can be used, to detect molecular binding events and to identify how is described below.

at Embodiments, in which the solution <figref>260</figref> a aqueous solution is, the dielectric properties associated disadvantageous the signal transmission along the interfacial transmission line. Especially have water and other aqueous solutions pronounced as whole blood a relatively high resistance R_S and one relatively low resistance R_S and adsorptive properties with regard electromagnetic radiation in certain areas of the spectrum on. The size of these Parameter results in a very high signal loss along the Interfacial transmission line. The location of the MBL between the interfacial transmission line and the solution according to the present Invention is used for the isolation from the signal and the solution or the modulation of the coupling with the signal and the solution, whereby the signal loss is modulated and other parameters of the signal transmission changed will.

<figref idrefs="S117">4C</figref> shows an embodiment of an equivalent circuit model <figref>450</figref> for in <figref idrefs="S113">2 B</figref> illustrated bioelectrical interface structure. For the Those skilled in the art of circuit design, it is obvious, that the circuit shown is not only model and that other equivalent circuit models of bioelectric interface according to <figref idrefs="S113">2 B</figref> can be derived.

The Equivalent circuit model shown <figref>450</figref> contains row and shunt circuit blocks <figref>452a</figref> and <figref>452b</figref>. the interfacial transmission line electric model. The equivalent circuit model<figref>450</figref> also contains an MBL circuit block <figref>454</figref>. in series with a solution circuit block <figref>456</figref> coupled is of the MBL and solution electrically modeled. As explained above defines the orientation of the Series and shunt circuit blocks <figref>452a</figref> and <figref>452b</figref> a conventional Interfacial transmission line structure. also results in the series alignment of the MBL and solution circuit blocks <figref>454</figref> and <figref>456</figref> out of the interfacial transmission line by MBL and in the solution extending signal field lines, their arrangement in <figref idrefs="S113">2 B</figref> is shown. Alternative circuit models can as described above for Bio-assay devices having an MBL and a solution close the ground plane as an alternative or in addition to its location near the Interfacial transmission line be derived.

<figref idrefs="S118">4D</figref> shows an embodiment of a circuit <figref>470</figref>. which the equivalent circuit model of <figref idrefs="S117">4C</figref> equivalent. Those skilled in the art of circuit design will readily recognize, that other circuit configurations are possible. The row and Shunt circuit blocks <figref>452a</figref> and <figref>452b</figref> represent together the conventional model for the interfacial transmission line. The MBL circuit block <figref>454</figref> is between the boundary surfaces transmission line and the solution circuit block coupled and is in one embodiment of each one series-connected capacitor C_M, resistance R_M and inductor L_M, Together, the aforementioned resistors, inductors define and capacitors, the circuit <figref>470</figref>That the input signal V_i in the output signal V_O converts.

As explained above affect the dielectric properties of the MBL and solution of the Values ​​of each of the electrical elements. In particular, the sensitivity determine and other dielectric properties of the MBL significantly the value of C_M; the dielectric constant, other dielectric Properties and the surface morphology the MBL significantly determine the value of L_M; and the dispersive properties and conductivity and other dielectric properties significantly determine the value of R_M, The signal response bioelectrical interface is thus highly characteristic of the dielectric properties of the MBL and can be used for detecting used and identify molecular binding events are what later will be described.

D. Specific Embodiments

The <figref idrefs="S119">5A</figref> to <figref idrefs="S120">5G</figref> show specific embodiments bioelectrical interface, which is implemented in a two-wire circuit topology. To those skilled in the field of circuit design, it is understood that each them in a single conductor waveguide topology and in three or More wire circuit topologies may be implemented.

Each the embodiments consists of a signal level <figref>520</figref>, A dielectric layer <figref>530</figref> and a ground plane <figref>550</figref>, An MBL<figref>515</figref> and a solution <figref>510</figref> are either to the signal level <figref>520</figref>, With the ground plane <figref>550</figref> or coupled with both. In each of the embodiments, the MBL<figref>520</figref> either in direct contact with the interface transmission line <figref>530</figref> to stand or be coupled with this. If the signal level<figref>520</figref> the MBL <figref>515</figref> directly affected, it consists of a material which is capable of both the signal transmission as well as the adhesion of ligands such as proteins, nucleic acids, carbohydrates, En&lt;?page 24?&gt;enzymes and the like to support.. Such materials include gold, ITO, copper, silver, zinc, tin, Antimony, gallium, cadmium, chromium, manganese, cobalt, iridium, platinum, Mercury, titanium, aluminum, lead, iron, tungsten, nickel, tantalum, Rhenium, osmium, thallium or alloys thereof. To those skilled it should be understood that other materials can be used can.

The dielectric layer <figref>530</figref> can consist of air, polyimide, teflon, Web materials such Duriod^TM, Alumina, Diamond, sapphire or insulating semiconductor material such as silicon dioxide or gallium arsenide or other insulating materials exist. The Thickness and dielectric constant the dielectric layer <figref>530</figref> are selected such that the desired Impedance of the transmission line is provided, as is known in the art. The solution<figref>510</figref> can of any transport medium such as phosphate physiological saline (D-PBS) Dulbecco exist, which provides the corresponding molecular structure. The protein, which nucleic acid or other interest ligand may by a variety Techniques such as wicking, pipetting, dipping, trickling direct Contact or added by capillary bioelectric interface.

The <figref idrefs="S119">5A</figref> and <figref idrefs="S119">5B</figref> show Sectional views of the interface, the microstrip circuit technology is realized, and in which the solution <figref>510</figref> and the MBL <figref>515</figref> above or below the interface transmission line <figref>530</figref> positioned are. The<figref idrefs="S119">5C</figref> and <figref idrefs="S119">5D</figref> show Sectional views of the interface, the solution <figref>510</figref> and MBL <figref>515</figref> above or below the base plane <figref>550</figref> positioned are.

<figref idrefs="S119">5E</figref> shows a sectional view of the in koplananer Optic topology realized interface. In this embodiment, the solution <figref>510</figref> and MBL <figref>515</figref> above the interface transmission line <figref>530</figref> positioned. Alternatively, the solution <figref>510</figref> and MBL <figref>515</figref> below the interface transmission line <figref>530</figref> or above or below one or both coplanar ground planes <figref>550</figref> be positioned. <figref idrefs="S119">5F</figref> shows a sectional view of the stripline circuit topology realized interface. at this configuration, the solution <figref>510</figref> and MBL <figref>515</figref> above the interface transmission line <figref>530</figref> positioned. In other embodiments, can these layers alternatively or additionally below the interface transmission line <figref>530</figref> or above or below one or both ground planes <figref>550</figref> arranged will.

<figref idrefs="S120">5G</figref> shows an embodiment of the bioelectrical Interface, which is realized in a coaxial circuit topology. A first insulator <figref>530a</figref> with a cavity <figref>570</figref> partially encloses an interfacial center conductor <figref>530</figref>, The MBL <figref>515</figref> is in the vicinity of the uncovered portion the interfacial center conductor <figref>530</figref> positioned. A second insulator <figref>530b</figref> is connected between the outer conductor <figref>550</figref> and the first insulator <figref>530a</figref> provided and encloses the outer conductor <figref>550</figref>. whereby the cavity <figref>570</figref> is formed, in which the solution <figref>510</figref> is. The radii and dielectric constants the first and second insulator <figref>530a</figref> and <figref>530b</figref> can equal have or different values, and each is selected such that the desired Line impedance and the required measurement sensitivity over the Frequency range of the test signal are provided. At a alternative embodiment is the MBL <figref>515</figref> in the vicinity of the outer conductor <figref>550</figref>, In this embodiment, contains the second insulator <figref>530b</figref> a cavity, so that the MBL near of the outer conductor may form, and the first insulator surrounds the center conductor <figref>320</figref> completely. Further can the MBL <figref>515</figref> and the solution optionally outside of the outer conductor <figref>550</figref> . are

The bioelectrical interface can depending on the application in a variety of Forms are prepared for. B. as squares, ellipsoids, rectangles, Triangles, circles, or parts thereof, or with irregular geometric Forms such as those in the bore of a hypodermic needle fit. The size of the bioelectrical interface varies depending on the application and is of the order of 10 m², 1 m², 10^-1 m², 10^-2 m², 10^-3 m², 10^-4 m², 10^-5 m², 10^-6 m², 10^-7 m², 10^-8th m², 10^-9 m², 10^-10 m², 10^-11 m², 10^-11 m², 10^-12 m² or in between. The bioelectrical interface can be prepared so that even in the small bore you a needle fits. The interface may alternatively be modified to other diagnostic applications take as proteomics chips. Size and shape of bioelectrical interface have to only fulfill the condition, that the signal transmission and the molecular bonding can take place along it.

IV. Measurement methods

A. General Overview

The Measurement methods of the present invention takes advantage of the observation advantage that a large number molecules based on their unique dielectric properties, including dispersion effects, resonance effects and effects on the these molecules surrounding solution . count If vorlie in&lt;?page 25?&gt;lowing invention a test signal with the MBL coupled found an interaction of MBL with the energy the test signal instead, resulting in a distinctive signal response results. The unique signal response can for recognition and identification of the MBL-forming molecules are used.

Of the Expert knows that most of the molecules changes of the dielectric properties over different frequencies exhibit. A molecule For example, a dramatic change in its dielectric Properties depending the frequency in one or more regions of the electromagnetic have spectrum. The frequency band over which the molecule a dramatic dielectric change which is often as dispersion regime of the molecule designated. about change this regime the dielectric constant, the permittivity, the dipole and / or multipole moments and the sensitivity of the molecule dependent on dramatically on the frequency. These variables are often complex with real and imaginary Proportions to both the size and the phase changes to take into account in the signal response. The dispersion regime extending over different frequencies, including RF, microwave, millimeter wave, far infrared and Infrared frequencies.

The dielectric properties of the molecule can be prepared by coupling a test signal with the molecule and observing the resulting signal can be determined. If the test signal, the molecule stimulates at a frequency within the dispersion regime of the molecule, especially at a resonant frequency, is a strong interaction the molecule instead of the signal and the resulting signal has dramatic changes its measured amplitude and phase, thereby a distinctive Signal response is generated. This response can to detect and Identify the bound molecular structure are used. In addition, since most of the molecules different dispersion properties on the same or different frequency bands comprise, each generates a unique signal a response that used to detect and identify the molecular structure can be.

detection and identify molecular binding events can Detecting and measuring the dielectric properties at the molecular Level occur. The dielectric properties at the molecular levels can are defined by the multipole moments of the molecule whose Potential energy are represented as infinite series as is known in the prior art: <img img-content="mf" img-format="tif" he="10" wi="61" file="00550001.tif" />

The infinite series consists of multiple terms, each of which in varying degrees dielectric properties in the presence of an electric, magnetic or electromagnetic field describes. The first Term is called monopole moment and represents the scalar quantity of the electrostatic Potential energy of the total charge of the molecule. The second term or "dipole moment" is a vector quantity and is three degrees of freedom. The third term or the "Quadropolment" is a rank-2 tensor and describes the response of the molecule through nine degrees of freedom. in the Generally, the term N. a tensor with rank N-1 3^N-1 Degrees of freedom, although the total number of degrees of freedom can be reduced by symmetries. It is likely to are obvious that higher moments Order more details about provide the dielectric properties of the molecule and thus more about the unmistakable signature of the molecule dielectric divulge. The gradient of the potential in the electric field results in <st32:df xmlns:st32="http://lighthouseip.com/">E = -∇Φ (x)</st32:df> The field intensity the moments of higher falls order dependent on rapidly on the distance, so that their contribution is difficult to is measured. For example, falls the field due to the dipole moment as r^-3 and the field due to the Quadropolmoments as r^-4 from. This approach thus requires close proximity between the binding molecules and the signal path and a low signal loss between. As it often found that the detection of molecular binding events in strong signalabsorbierenden solutions like Whole blood samples or ionic solutions takes place, the signal loss between the binding events and the signal quite high and the detection of higher-order moment is very difficult.

It should also each Multipolterm with the electric field in a different way coupled. This leaves initially using the energy of a given electrostatic field show: <st32:df xmlns:st32="http://lighthouseip.com/">W = ∫P (x) Φ (x) d<st32:sup>3</st32:sup>x</st32:df>

&lt;?page 26?&gt;

The Expansion of the electrostatic potential in a Taylor series results

<img img-content="mf" img-format="tif" he="43" wi="79" file="00560001.tif" />

Further applies to the external field ∇ · E = 0, thus obtained that <img img-content="mf" img-format="tif" he="10" wi="87" file="00560002.tif" />

By Reinsertion into the above equation for the energy results

<img img-content="mf" img-format="tif" he="11" wi="64" file="00570001.tif" />

This shows the type of interaction each Multipolterms with the interrogating Field: The total charge q with the potential of the dipole p with electric field, the quadrupole Q_ij With the gradient of the electric field, etc. This is the second Difficulty in the detection of higher-order multipole moments: In a bulk sample, it is difficult sufficient field gradients for coupling with higher moments to achieve order.

The overcomes present invention the aforementioned obstacles by the bioelectrical described interface implemented. The interface contains a MBL which is coupled along the signal path. The MBL is a very thin and (under dielectric viewpoints) extremely inhomogeneous layer so that they have the necessary proximity Electromagnetic scanning structure and sufficient field gradients provides for coupling to higher order multipole moments. These properties allow the detection of moments of higher Order that a much better view of the dielectric Properties of the molecule offer. The positioning of the MBL in the vicinity of the signal and / or ground planes serves the transmitted thereto to separate signal so that it is not absorbed in the solution, whereby the signal loss is reduced and higher test frequencies are possible for binding events to recognize and identify exactly. In this way enables the present invention a higher measure of Regeneration of the signal response, including the shares from the dipole and other multipole higher Rules of the molecule.

Among Use of the bio-assay device of the present described Invention, numerous belonging to MBL Properties are recognized. <figref idrefs="S120">6A</figref> shows an embodiment this method. First in step <figref>602</figref> formed a MBL and along a coupled portion of a signal path. As described, the MBL consist of a ligand, a Antiligand- / ligand complex etc. and may directly or indirectly physical contact with the Signal are or electromagnetically coupled to this. The signal can, in a two-conductor transmission topology from the Signal or ground plane exist.

then in step <figref>604</figref> transmit a test signal via the signal path. The test signal, each time varying signal at each its frequency between 10 MHz and 1000 GHz, for. example, at a signal frequency of Or 10 MHz with a frequency range of 45 MHz to 20 GHz. After that to couple the test signal in step <figref>606</figref> at the MBL and in response to the coupling, it generates a signal response. The Signal response is then regenerated and provides information on a or more properties of the molecular binding layer.

The Bio-assay device may be used to provide information on numerous Properties of the MBL be used, eg. B. for the detection and Identify molecular binding events, ligand&lt;?page 27?&gt;concentrations, changes the dielectric properties of the MBL, classification of detected Binding events and the like. In addition, contains the bio-assay device, a self-calibration function for quality control and quality assurance useful on site is. Each of these methods and each of these functions will be referred to described in detail. Based on the methods described and Structures for the expert modifications and additional applications arise.

The ability molecular dipole, quadrupole and higher order multipole moments in dissolved to detect and measure state, provides for a number of reasons a have significant step forward in the art. First, many molecules, which are in biomedicine of interest such as proteins, very different Structures and therefore different multipole moments. By identifying the multipole moments of a given molecule are so distinctive Properties of this molecule recognizable, which in turn switched the identification of this molecule gestat. Second, the structure and function in many molecules of biomedical relevance as proteins closely related. The ability, Characteristics to recognize a given molecule that directly with the function of the molecule related, therefore, means that the functionality for all monitors activity areas can be. Third, plays the local physiological environment often an important role in the structure and function of a given molecule, so that the possibility the physical properties can be seen above, means that molecules can be used as monitors and probes to changes in a given to measure system. With the ability to complex and informative properties of molecular and cellular definition system in a detectable electronic data format, resulting in the discussed herein areas completely New opportunities.

B. detecting molecular binding events

The herein described bio-assay device allows the detection of molecular Binding events that take place along the signal path. detectable Binding events include primary, secondary binding events and such higher Order. In a bioelectrical two-wire interface without previously existing MBL z. B. the molecules of the conductive layer the anti-ligand to bind the ligand form, where the ligands MBL form. In another embodiment, both anti-ligand and ligand included in the MBL. In this embodiment, MBL adheres to the signal path via Coupling groups, matrix molecules, Insulating layers or combinations thereof as described in <figref idrefs="S109">1D</figref> shown.

<figref idrefs="S120">6A</figref> shows an embodiment of this process. First, in step <figref>602</figref> a signal of a material formed, the transmission, the a signal over the desired support operating frequency can. The signal may consist of a path to a terminal, a Way with two connections or a way to me several terminals within a herein described bio-assay devices exist. also can the signal as a transmission line, Cavity or waveguide structure can be realized.

After that in step <figref>604</figref> a solution provided that the molecule in question or contains the relevant molecular structure. A step<figref>606</figref> shall one consisting of the ligand MBL formed from the solution and between coupled to at least a portion of the signal path and the solution. Then, in step <figref>608</figref> transmit a test signal along the signal path. Alternatively, the test signal during the application of the solution are issued to the in real time as a result of the binding events occurring signal response observed. In step<figref>610</figref> immigrated the signal via the MBL, and this couples to develop a signal representing the indicating presence of the ligand. In steps<figref>612</figref> and <figref>614</figref> shall then regenerates the test signal, the response the detection of the Ligands indicates.

The dielectric properties of the MBL may help any to induce number of signal responses, each of which for molecular binding representative can be. So can For example, the dispersive properties of the MBL on Change frequency dramatically. In this case, the test signal response has large changes in the amplitude and / or the phase response over the frequency when molecular binding events along the binding surface take place, thereby providing a means for detecting molecular binding events along the binding surface provided.

at Alternatively change the dielectric relaxation properties of the MBL function from the pulse period of the input signal. In this case, shows the test signal response a change absorbed power on or a change of another parameter of the test signal like phase or amplitude, at or near a specific Pulse period. By observing a change in the power absorbed or other parameters can Binding events along the Bindungsoberflä&lt;?page 28?&gt;che be detected. Other the likes characteristic impedances, propagation velocity, amplitude, Phase, dispersion, loss, permittivity, Sensitivity, frequency and dielectric constant are also potential indicators molecular binding events.

The The method described above may for detecting a primary binding an anti-ligand or ligand directly or indirectly along the binding surface be applied. Similarly Example, the process of <figref idrefs="S120">6A</figref> also for detecting a secondary Binding of a ligand to be applied to an anti-ligand. The process of <figref idrefs="S120">6A</figref> is not limited to the detection primary or secondary limited binding events that take place along the signal path. In fact, can tertiary Binding events or binding events of higher order, either along the signal or in solution held suspended, also detected by this method will.

<figref idrefs="S121">6B</figref> showing a second process for detecting secondary Binding events and binding events of higher order, which along the signal take place. First be in step <figref>620</figref> detects the primary binding event and the signal response is measured, of which an embodiment of the steps <figref>602</figref> to <figref>612</figref> illustrated is. subsequently in step <figref>622</figref> the signal in response to the primary binding event stored and used as the measurement based response. Thereafter, in step <figref>624</figref> a second molecular solution of the bio-assay device added so that they the binding surface can circulate. In step<figref>626</figref> then steps <figref>608</figref> to <figref>612</figref> according to <figref idrefs="S120">6A</figref> repeated to generate a second signal response to receive. Then, in step<figref>628</figref> the second signal response and the measurement base response compared. A little or no change Recalls that the two signal responses are very close together are, which means that the structural and dielectric Properties of MBL not by the addition of molecules in the new solution changed have been. In this case, does not have a secondary binding appreciable occurred extent (Step <figref>630</figref>). Once the comparison a change outside of a predetermined Area results, the structure and / or the dielectric properties changed the MBL been what to secondary Binding events indicating (step <figref>632</figref>). Sizes that to indicate secondary Binding events can be used are the same as the above factors, eg. , Amplitude, phase, frequency, dispersion, loss, absolute The permittivity, Sensitivity, impedance, propagation speed, dielectric constant and other factors. Tertiary Binding events or binding events of higher order can under Application of this procedure can be detected.

On alternative method for detecting secondary binding events or Binding Acts Procedure does not require prior knowledge of the specific primary binding event. In this embodiment, is the bio-assay device designed in the analysis phase of development so that they known Parameters operates such that whenever a predefined change one of these parameters is detected, z. B. on site, certainty is that (the) binding events) has occurred (have). In this embodiment, is the previous measurement is not a primary binding event required since the initial characterization already either been made at the manufacturing time or the interpretation is.

Secondary binding events can by detection of changes the structure of the primary bound molecule be achieved. If a molecule is bound, it is subject over its unbound state conformational and other changes its molecular structure. These changes affect the dielectric properties of the primary binding of the molecule and induce changes in the surrounding solution, the means of the steps <figref>620</figref> to <figref>628</figref> according to the above described <figref idrefs="S121">6B</figref> can be detected. Sizes that to indicate a change monitors the dielectric properties of the molecule with primary bond can be, contain the above factors, eg. , Amplitude, phase, frequency, dispersion, loss, permittivity, Sensitivity, impedance, propagation speed, dielectric constant and other factors.

C. detecting changes the dielectric properties of the molecular binding layer

The herein described bio-assay device may also measure the dielectric changes due to changes of the MBL the temperature, pH, ionic strength and dg. be used.

<figref idrefs="S122">6C</figref> shows an exemplary embodiment the process. The process is the disclosed methods for identifying very similar binding events with the exception that the method comprises the detection and quantification of changes allows the dielectric properties of the MBL.

&lt;?page 29?&gt;

Of the Process begins with step <figref>641</figref>In which a solution a dielectric top property for bio-assay device added, is measured the signal response and recorded. at one embodiment this step is in accordance with steps <figref>602</figref> to <figref>612</figref> executed. After a predetermined time or period of operation, a second measurement and the second signal response is recorded (step <figref>642</figref>), again in one embodiment, according to the steps <figref>602</figref> to <figref>612</figref>, In step <figref>643</figref> then the first and second signal compared to determine whether the two signals within a predefined range correlate. If this is the case, assumed that the properties of the solution dielectric no changes have experienced (step <figref>644</figref>).

If the signal can not reply within a predefined range correlate, it is assumed that one or more dielectric Features of the solution a change have experienced (step <figref>645</figref>). Optionally, the change quantified in the dielectric properties in the following manner will. In step<figref>646</figref> the second signal is stored and correlated with a known signal response. The best correlating response identifies the dielectric property the solution and the first signal response can with the initial value of the dielectric Property are correlated, wherein the difference therefrom for determining of the amount by which the identified dielectric property changed has, can be used (step <figref>647</figref>).

D. Identify bound molecular structures

With the bio-assay devices described it is possible to use a to characterize known ligands and it then made into a solution unknown ligand identified. <figref idrefs="S122">6D</figref> shows an embodiment this process. First be in step <figref>652</figref> a large number of molecular structures measured and stored their responses, wherein one or more the measuring systems described below are used. at one embodiment this step is in accordance with steps <figref>602</figref> to <figref>612</figref>, Each canned response can present a single in the solution Ligands or more present in the same solution ligands correspond. subsequently in step <figref>654</figref> made the measurement of an unknown solution. In one embodiment, this step is in accordance with steps <figref>602</figref> to <figref>612</figref>, Thereafter, in step <figref>656</figref> the signal response of the solution with the stored signal responses compared to the degree of correlation using this to determine. In step<figref>658</figref> is the unknown molecular structure identified by the stored response chosen is that best correlates with the unknown response. Of the Comparison can be performed using one or more data points to determine the correlation between one or more stored performed answers and may be the application of pattern recognition software or similar include means for determining the correlation. The process can for identification of bound primary, secondary molecular structures or higher molecular structures Rules are applied.

E. Identifying Classes bound molecular structures

it is possible, too, known molecular substructures as regions or other structural characterize homologies that similar classes proteins or sequence homologies are common in nucleic acids. At a embodiment is the process as described in <figref idrefs="S122">6D</figref> illustrated continued, with the exception that in step <figref>652</figref> N molecular measured substructures and their responses are stored. Each stored signal response may be one or more sub-structures correspond. The process is in accordance with steps<figref>654</figref>. <figref>656</figref> and <figref>658</figref> continued until identified and characterized a sufficient number or structures are to identify the unknown compound. As soon as a sufficient number of correlations is present, it is possible that unknown molecular substructure classify.

<figref idrefs="S123">6E</figref> illustrates another process by which unknown Ligands can be classified. Of the Process identifies the unknown ligand by detecting a bond to structural patterns of the unknown. First in step <figref>660</figref> a bio-assay device is provided, the plurality of addressable matrices, has each of which a Antiligand for having a specific ligand substructure. Thereafter, in step<figref>662</figref> the Presence of certain substructures based on the binding of a each detected at their respective anti-ligand, followed by Characterization. In one embodiment, this step is according to the steps <figref>602</figref> to <figref>612</figref>, subsequently in step <figref>664</figref> each of the binding events by identifying characteristics such as affinity, kinetics and spectral sensitivity characterizes. In step<figref>666</figref> done Then, the correlation between the known and the unknown Reply. If any of the unknown with the known answers Responses correlated, the ligand is identified as the one ligand, of the known response corresponds. If the substructures correlated both exhibit and uncorrelated responses to the correlated responses be used, &lt;?page 30?&gt;a general classification of the unknown create ligands. This process can to identify each molecular structure, for. example, proteins are used, in the same Class present or have recurring homologies.

it is possible, too, that an intensive spectral analysis of a given unknown Connection insights Structure and function returns, as comparisons with known structures can be made and an extrapolation to a certain classification results.

A. Specific against nonspecific Binding:

The specific ligand binding differs from the non-specific Binding by the spectral fingerprint of the binding event. A given binding event z. B. an antibody binding to an antigen can be first in a purified solution containing only the interest Ligands and for the containing ligand specific anti-ligand on the MBL, characterized will. Then, a comprehensive Spektraluntersuchung is performed to to determine when the strongest Replies in the spectrum occur. The analysis is then repeated in the solutions typically present in specific applications such. As whole blood, to determine what effects non-specific binding has on the response. Then various points are determined, the determining factor for a specific bond, and its own set of points is determined, the determining factor for non-specific binding, and a subset of these frequency points is then used for the actual Analysis application selected. By comparing the responses due to a specific binding with which, due to non-specific binding, the extent of specific Binding can be determined.

B. Characterization of a given ligand:

it is often desirable, to determine certain properties of a given molecule. Examples include determining the class to which belongs a protein, or what type polymorphism a given gene or other nucleic acid sequence is. This can be done in several ways. proteins often by the number and types of structural homologies or certain substructures, viewed in the same or similar Find protein classes, classified. To have such. B. G-proteins, usually present in cell membranes and the signal conversion paths between the außerzellularen bring around and in the intracellular environment, always a structure which traverses the cell membrane seven times. such Structure is virtually definitive of a G-protein. Other classes of proteins have similar structural homologies and therefore any method that a different protein class from another based on these homologies may, in the fields of biomedical research of enormous Use. Assuming that the dielectric properties a given molecule completely determined by the geometry of the charge distribution of this molecule be, and further provided that most proteins have a have unique structure or geometry, any protein, by measuring its dielectric properties are uniquely determined. Thus can be a simple dielectric signature, such as those present from the Invention is produced, serve a given protein clearly to identify and further the classification of the protein in to allow a previously known protein class. The methodology of classification can be further refined by a group antiligand on the bio-assay device is used for certain Substructures of a given protein are specific. So can For example, a group of antibodies, specifically for certain substructures such spaces, for determining the presence or the absence of these substructures are used. any given Protein can thus by determining both the presence or the Absence of certain sub-structures as well as the dielectric properties the protein itself be characterized. Further refinements this classification strategy may the detection of the temperature, pH, ionic strength and other environmental influences included in the above-mentioned properties.

Nucleic acids can also by following a similar are paradigm characterized. It may be known for. Example, that a given gene has a specific base pair sequence. To find to z. B. in the gene for a chloride ion transport channel in many cell membranes coded, common Einbasenpaarmutationen or changes. Such changes to lead to a disease which denotes in humans as cystic fibrosis becomes. The characterization of a given nucleic acid sequence regarding minor amendments So of enormous importance. Such deviations are often referred to as Polymorphisms designated and such polymorphisms are currently complementary by forming Strands for each known polymorphism detected. Since any given gene, a take the form of hundreds or even thousands polymorphisms can, it is often an arduous task of generated complementary strands for each polymorphism. Using the invention described herein can &lt;?page 31?&gt;not complementary be recognized and differentiated binding or hybridization by many of the same physical properties as described in previous paragraph have been described to be measured: Dielectric Features of the hybridization event can be characterized and with known data are correlated, whereby the - either fully or incomplete - took place is determined hybridization type. With an anti-ligand from a given nucleic acid sequence can So hundreds of different polymorphisms (as ligands) by the characterization of the binding event can be detected. To those skilled it will be understood that further refinements are possible, eg. as a modification the stringency conditions to alter the hybridization process, or a temperature change for determining the melting point, as a further indicator of the type the hybridization process is used.

Similarly way can Interactions between drug and receptor are characterized, to determine if a given binding event results in that the receptor is activated or deactivated, or other taking place form an allosteric effect. For example, a given receptor can be used as anti-ligand and a well-known Agonist as the first ligand. The interaction is then according to the dielectric characterized response, and this response is saved. Subsequently Compounds which are tested for drug candidates in terms of their binding properties with the receptor observed. Of a Molecule, which binds and a similar dielectric response generated is then known that there is a similar effect on the receptor as the known agonist has, and is therefore at a much higher Probability is an agonist. This paradigm can be used to characterize applied practically every type of target receptor-binding event of interest be and represents a significant improvement over current Detection strategies is that only determine whether a binding event has taken place or not. For the expert it is obvious that there is a lot more classes Binding events are, where applied, the present invention can be.

Examples for substructures which can be used in the above method include the following: Secondary and tertiary protein structures such as alpha helices, beta sheets, helices Dreifch, areas barrel structures, beta-windings and various symmetry groups in quaternary structures as C₂Symmetry, C₃-Symmetry, C₄-Symmetry, D₂Symmetry, cubic symmetry and Ikosaedral symmetry available. [G. Rose (1979), Hierarchic Organization of Domains in Globular protein, J. Mol Biol. 134:. 447-470] substructures nucleic acids, which can be analyzed, include: sequence homologies and sequence polymorphisms, A-, B- and Z forms of DNA, Check and double-stranded forms, super rotating shapes nodoc loops, D-loops and TψC loops in tRNA and in different classes tRNA molecules. [W. Saenger (1984) Principles of Nucleic Acid Structure. Springer-Verlag, New York; and P. Schimmel, D. target and J. Abelson (Ed.) (1979) Transfer RNA. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.]

F. quantifying concentrations

The herein described bio-assay device may also for quantifying use of ligand concentrations. <figref idrefs="S124">6F</figref> shows an embodiment of this process. For the Event that the device is not pre-calibrated (step <figref>679</figref>) are first in step <figref>670</figref> Antiligand with suitable binding properties as binding affinity or kinetics for the measured analyte selected. These properties are selected such that the equilibrium constant antiligand near the middle of its linear operating region. In applications where the concentration range for the use of a single antiligand is too far, several anti-ligands with different affinities be used and / or linear operating ranges, thereby forming a Value of the concentration via a much wider range is achieved.

After that at steps <figref>672</figref> and <figref>674</figref> the antiligand on the bio-assay device or the chip is applied, and the device with the measuring system connected. In step<figref>674</figref> it is decided whether the answer requires a characterization as to the maximum specificity. If so, a spectral analysis is carried out, wherein which the frequencies where analyte binding has maximal binding (step <figref>675a</figref>) the regions where the non-specific binding the maximum effect (step <figref>675b</figref>) Can be determined and the unmistakable Response is determined due to the analyte (step <figref>675c</figref>). The Device will be calibrated when no characterization required is, or if they do, after their completion. This step is in one embodiment, conducted by ligands of known concentration to the bio-assay device added be and the resulting response is measured (step <figref>676a</figref>). Are more data points for the calibration step (Step <figref>676b</figref>) Is required, then can optionally select a sample with a different concentration (step <figref>676c</figref>) and the response to this concentration mea&lt;?page 32?&gt;sen (step <figref>676a</figref>). In one embodiment, the measurement according to the steps <figref>602</figref> to <figref>612</figref> done. subsequently in step <figref>677</figref> generates an extrapolation, by the calibration points recorded in the previous answer will. Thereafter, in step<figref>678</figref> a sample of unknown Ligand concentration measured. This step is, in one embodiment conducted by where the unknown sample to the bio-assay device, correlates the response with the titration algorithm and it the ligand concentration is determined.

in the case in which a given bio-assay device is either pre-calibrated or calibrated design conditionally, consists of only required step in applying the ligand or analyte on the surface and measuring the response. Such a bio-assay device may be realized in many different ways. For example, some circuit parameters such as impedance or characteristic frequency a resonant circuit are designed so that they relate to a Change predetermined manner, when the binding event occurs, and the amount by which the parameter changes, can also be designed with a dose-response. The measurement the circuit parameter so by analyzing a suitable algorithm directly a quantitative value for the concentration a given analyte or ligand deliver.

G. Self-Calibration the bio-assay device

The described bio-assay devices possess a self-diagnostic capability and thus the possibility quality control and quality assurance in the field. acts Given a specific application a particular antiligand (primary Binding species) as a antiligand for a ligand (secondary binding species) of interest in the solution. The primary Binding species, in the preparation and the secondary binding species be mounted on site. Variations in the preparation of - especially When attaching the primary Species - cause Therefore variations of ability the device to bind their specific ligands. The amount the bound ligand, however, is directly proportional to the amount the bound anti-ligand so that a ratio of the two measurement possible is.

<figref idrefs="S125">6G</figref> shows an embodiment of the process. First in step <figref>680</figref> molecular bonding surface along the signal path by binding the appropriate antibody at formed various concentrations and the resulting response for every these concentrations characterizes what a value "x" for each concentration results. Thereafter, in step<figref>682</figref> a similar titration curve for produces the ligand by the antibody / Ligandbindungskonzentrationsantwort for several various concentrations of the ligand is measured, and Ligand titration curve is predetermined. Thereafter, in step<figref>684</figref> on A scaling factor based on the ratio of the responses of the Antibody Binding produced for ligand binding. On site, the uncalibrated Analysis then examined first (step <figref>686</figref>), The amount to determine the bound antibody "x" and the resulting scaling factor "y". The ligand is then measured introduced into the analysis and the response (step <figref>689</figref>) and the response and predetermined titration curve will be with the scaling factor "y" scaled (step <figref>690</figref>) to determine the unknown concentration.

Of the process of <figref idrefs="S124">6F</figref> may also be modified, to quantify the amount of ligand in the solution to allow. contains in the modification the binding surface the bio-assay device at a predetermined antiligand affinity and ligand specificity. The solution then applied to the device and measuring the response. The signal response behave proportional to the amount of bound ligand. Thus, a titration of any given ligand can be performed by an antiligand with a suitable linear operating range - the range in which the equilibrium constant within some logarithmic units of the desired to be detected concentration range is - is chosen. The same analysis as described above can quotient for obtaining a robust and quantitative analysis with internal control and calibration needed to ensure the reliability are required to be applied.

Each the methods described may be in many different ways (Ie, software or hardware or in a combination of both) and in a variety systems be realized. In one embodiment, the described The method may be implemented as a software program.

<figref idrefs="S126">7A</figref> shows an example of a computer system <figref>710</figref> to Processing of a software program, the for execution any of the methods described is designed. The computer system<figref>710</figref> includes a monitor <figref>714</figref>. a screen <figref>712</figref>A housing <figref>718</figref> and a keyboard <figref>734</figref>, A mouse (not shown), a light pen, or other I / O interfaces as virtual reality interface can also be included, &lt;?page 33?&gt;to provide I / O commands. The housing<figref>718</figref> contains a CD-ROM drive <figref>716</figref>. a hard disk drive (not shown) or other storage media, which to store and retrieve digital data and software programs, containing the present invention can be used, and like. Although the CD-ROM<figref>716</figref> as a removable Medium is illustrated, other physically removable media including floppy disks, magnetic tape and flash memory may be used. The housing<figref>718</figref> contains also known Computer components (not shown) such as a processor, Storage and the like.

<figref idrefs="S126">7B</figref> is a simplified system block diagram of a typical computer system <figref>710</figref>Which to finalize a Software program with the desired Method is used. As in<figref idrefs="S126">7A</figref> illustrated contains the computer system <figref>710</figref> the monitor <figref>714</figref>Which selectively interactive with the I / O control <figref>724</figref> is working. The computer system<figref>710</figref> contains further Subsystems such as a system memory <figref>726</figref>, A central processor <figref>728</figref>. a speaker <figref>730</figref>, A removable hard drive <figref>732</figref>. the keyboard <figref>734</figref>, A stationary disk <figref>736</figref> and a network interface <figref>738</figref>, Other for use with the described method appropriate computer systems can more or less subsystems included. so for example, could another Computer system more than one processor <figref>728</figref> (Ie be multi-processor system) included for processing the digital data. arrows as <figref>740</figref> represent The system bus architecture of computer system <figref>710</figref>, These arrows <figref>740</figref> show way of example only, each connection structure, which serves for coupling the subsystems. So could z. B. a local bus to connect the central processor <figref>728</figref> with the system memory <figref>726</figref> used will. This in<figref idrefs="S126">7B</figref> Computer system shown <figref>710</figref> is only one example of the invention for use with the present suitable computer system. Other configurations of the sub-systems, suitable for use with the present invention provide readily to the average skilled person.

V. Measurement Systems

to implementation the methods described above, various measuring systems be used. The<figref>8</figref> to <figref idrefs="S128">10</figref> show three examples of possible Measurement systems: a frequency domain test system, a time domain test system and a measurement system for the dielectric relaxation.

A. Frequency Measurement System

<figref idrefs="S127">8A</figref> shows an embodiment of a frequency measuring system according to the present Invention. The system<figref>800</figref> contains one to the input <figref>852</figref> of the Bio-assay device coupled signal source <figref>810</figref> and to the output <figref>858</figref> of the Bio-assay device coupled signal detector. Optionally, a additional Signal source to the output <figref>858</figref> the bio-assay device and an additional Signal detector connected to the input <figref>852</figref> coupled to the test circuit are to provide a full provide two-terminal measurement function. The system can be a one-port test system are modified, in which a signal detector is coupled to the signal path to a reflected signal to receive. In a specific embodiment, the above- said frequency measurement system consists of a network analyzer, such. as model No. 8510C from the Hewlett-Packard Company. Optionally, other RF measurement systems such as scalar network analyzers, signal information transmitted based will deliver and reflected signals used.

The Measurements are in accordance with the above mentioned methods performed. First is an entry signal <figref>860</figref> deposed to test circuit and transferred and / or reflected signals <figref>870</figref> or. <figref>890</figref> will then regenerated. The resulting signal responses take Form unique frequency responses or "spectral fingerprints" on, of which the <figref idrefs="S127">8B</figref> and <figref idrefs="S127">8C</figref> two Examples show. <figref idrefs="S127">8B</figref> shows a type frequency response, wherein the resonance at the frequency f_res occurs. Here is subject to the answer <figref>870</figref> a steep drop and Increase, which indicates that at this frequency little or no signal energy reaches the output terminal. The response is caused by the dielectric property and impedance of the MBL, wherein the frequency of f_start to f_Stop changes. Different ligands go at different frequency points resonate. also can Some ligands multiple resonant frequency points over the measured band f_start to f_Stop exhibit. Once a ligand has been characterized in that it one or more unique resonance points has, this can Data identifying the presence of the ligand in a unknown solution be used. This characterization can using empirical Data or by means of theoretical calculations of Mehrpolmomenten confirmed and resonant frequencies will. Further, when the presence of secondary binding events detected can these data through a change one or more unique resonance points indicate when to an analyte bound to a ligand.

&lt;?page 34?&gt;

<figref idrefs="S127">8C</figref> shows another type frequency response to used detecting or identifying a molecular structure can be. In this case, the frequency response shows a generally monotonically increasing or decreasing trend with a certain degree of amplitude variation. The slope of the frequency response and / or the amplitude fluctuation can be used to detect and / or uniquely characterize the bound molecule be used. The resonant frequency points, slope, trend and change the phase of the test signal can Thus in the described manner to uniquely identify the molecular binding event can be used. The frequency response can the input terminal <figref>852</figref>, At the output terminal <figref>858</figref> or at both terminals are measured to the bound molecular structure clearly to identify.

<figref idrefs="S128">9</figref> shows a second exemplary embodiment a frequency measuring system of the present Invention. The to be tested bio-assay device<figref>920</figref> consists a coaxial topology (in <figref idrefs="S120">5G</figref> shown) with a central conductor <figref>921</figref>, A first insulator <figref>922</figref> With a cavity <figref>922a</figref>, A second insulator <figref>923</figref> and an outer conductor <figref>924</figref>, The solution <figref>926</figref> is in the cavity <figref>922a</figref>, Of course, devices can other circuit topologies are tested.

As soon as the solution <figref>926</figref> in the cavity <figref>922a</figref> has been introduced, which form the molecules in the solution <figref>926</figref> a MBL <figref>921a</figref> close of the center conductor <figref>921</figref>, During the measurement requires a source <figref>910</figref> an input test signal <figref>912</figref> to the central conductor <figref>921</figref> from. The MBL<figref>921a</figref> modulates the input test signal <figref>912</figref> and the reflected test signal <figref>932</figref> provides an unmistakable signal response ready to identify the the ligand may be used. The coaxial configuration a connection may, for example as a subcutaneous needle structure be realized.

B. time domain measurement system

<figref idrefs="S128">10</figref> shows an embodiment of a time domain measurement system <figref>1000</figref> according to the present Invention. The system includes a pulse source <figref>1002</figref> and a detector <figref>1004</figref>. coupled to the input <figref>1022</figref> coupled to a test circuit are. In an alternative embodiment, one additional Pulse source and an additional Detector to the output terminal <figref>1028</figref> are coupled, to a full two-port measurement capability provide. Furthermore, the system can be a one-port test system have, in which a signal detector coupled to the signal path is to receive a reflected signal. In a specific embodiment is the time domain measurement system from a time domain reflectometer as model <figref>11801</figref> by the Tektronix Corporation. Optionally, other RF measurement systems such as network analyzers with a time domain measurement mode, the signal information transmitted based will deliver and reflected signals used.

at the time domain measurement system consists the input test signal <figref>1060</figref> out three time domain pulses whose shares reflected on the Time can be displayed. is in the present embodiment an input pulse <figref>1060</figref> in the direction of the section of the transmission line discontinued, which is closely linked with the analysis surface. by virtue of the dielectric property of the MBL, a portion of the input pulse <figref>1060</figref> to the detector <figref>1004</figref> reflected. The reflected pulse<figref>1070</figref> has a unique shape and / or time delay, which for the dielectric Properties of the MBL and is characteristic, as in turn highly by the dielectric properties of the ligand, the antiligand and the surrounding solution To be defined. Thus, the pulse shape and delay the reflected pulse <figref>1070</figref> for characterization and Identification of ligands are used. The time domain measurement system can separately and together with the high-frequency measurement system to identify one or more unknown ligands can be used.

C. measuring system for the dielectric relaxation

As is known in the prior art, the dielectric relaxation frequency is a ligand, the rate at which the dielectric properties change at the molecular level, when an electric field is applied to the molecule. As in the case of the dielectric properties of the ligand, the dielectric relaxation frequency is defined primarily by the structure and binding geometries, the for each molecule unmistakably are. If the dielectric relaxation frequency ligand so Once measured, it can be used for its identification.

The dielectric relaxation frequency can be quantified by the rate at which the ligand absorbs power over frequency, is measured. <figref idrefs="S128">11</figref> shows an embodiment of a system <figref>1100</figref> for this measurement implementation. The measuring system<figref>1100</figref> similar to in <figref idrefs="S128">10</figref> Zeitbe shown&lt;?page 35?&gt;rich measurement system <figref>1000</figref> and contains a pulse source <figref>1102</figref> and a detector <figref>1104</figref>. coupled to the input <figref>1122</figref> the test arrangement are coupled. An additional Pulse source and an additional Detector can the output terminal <figref>1128</figref> are coupled to a full two-port measurement capability provide. In a specific embodiment, the time domain measurement system from a time domain reflectometer as model <figref>11801</figref> by the Tektronix Corporation. Optional can other high-frequency measurement systems such as network analyzers with a time domain measurement mode, the signal information transmitted based will deliver and reflected signals used.

The Input test signal <figref>1160</figref> consists of multiple groups of pulses, each of which two or more input pulses, and various Pulse intervals has. The pulse groups<figref>1162</figref> and <figref>1164</figref> will sold to the transmission line section, closely with the binding surface coupled. If a pulse group<figref>1162</figref> an interval has the substantially the dielectric relaxation period (the reciprocal of the relaxation frequency) corresponds, absorbed MBL successively less energy in the pulses. The Decrease in signal absorption can in the reflected response <figref>1170</figref> at the input terminal <figref>1122</figref> or at the output terminal <figref>1128</figref> measured will. As an alternative measure may the residual signal power either at the input terminal <figref>1122</figref> or at the output terminal <figref>1128</figref> be measured.

The rate of change of signal absorption and the pulse interval, in which the change takes place, can then coated and for characterizing and identifying the (S) unknown molecule (Molecules) be used. This system characterization may independently or together with the time and / or frequency domain test systems described above be applied.

the Skilled worker is well aware that all of the above systems by the Application of technologies such as Microwave Monolithic Integrated Circuits (MMIC - Monolithic microwave integrated circuits) and the like. on chip size down can be scaled. Such miniaturized systems can be extremely parallel in a simple manner to enhance operating systems that are capable of hundreds, to detect thousands, or tens of thousands of connections simultaneously and to eat. These systems can are configured so that they produce "logic gate", which by the binding event themselves connected, eg., by changing a characteristic Impedance and thus the transmission and / or reflection coefficients, or by changing the band pass properties such a circuit and use this as the on / off gate.

VI. Examples

A. Example 1: Detection a ligand binding to the surface

The primary Binding of urease to an ITO surface was detected in the bio-assay device as in <figref idrefs="S112">2A</figref> shown. The bonding surface of the Bio analysis device had a ITO with chemical deposition from the vapor phase (Chemical vapor deposition - CVD) treated on glass cover. The ITO transmission line was carefully examined to ensure that they did not contain micro-cracks or fractures. The transmission line was measured with a Tektronix <figref>11801</figref> Signal analyzer with a TDR module measured with a broadband reference impedance of 32 Ω determined has been. The cable length was about 2.6 ns, for the binding surface has an impedance of 34 Ω and a length determined by about 200 ps. The distance between the upper and lower Plate was 10 mils and the chamber had a length of 1/2 inch. There were no side walls used; instead kept the capillary action of the upper and lower plate the solution in their position.

After that the bio-assay device was filled with a d-PBS solution. at filled Bio-assay device using S-parameter measurements of measuring basic transmission loss (S₂₁) And the return loss (S₁₁) A Test frequency range from 45 MHz to 1 GHz performed. The Measurements were made and by means of a network analyzer model no. HP 8510B from the Hewlett-Packard Company stored. Then urease was in a volume excess of 10: 1 was added. The S-parameter measurements of the transmission and return loss were repeated and compared with the measuring base measurements.

The Table 1 below shows these values ​​for 100 MHz and 1 GHz and the repatriation and transmission loss measuring responses are in the <figref idrefs="S129">12A</figref> and <figref idrefs="S129">12B</figref> shown. The data indicate that the bio-assay test device a change the return loss (S₁₁) Of -0.5 dB and -0.42 dB at 100 MHz and 1 GHz between the d-PBS filled chip and the d-PBS filled + Protein &lt;?page 36?&gt;device exhibited. The device had a change of transmission loss (S₂₁) Of +0.325 +0.450 dB and 100 dB at MHz and 1 GHz.

Around to determine whether the signal responses to a mass effect (proteins in solution) or on the binding of proteins are due to the binding surface, each response was recorded and the protein solution d-PBS in a volume excess 25: 1 rinsed (2ml d-PBS to 0.075 ml chamber size). The bio-assay device was then measured from 45 MHz to 1 GHz as described above again.

As the comparison of the last two columns of Table 1, had rinsing the protein from the bio-assay device comprises a minimal effect on the measurements of the repatriation and transmission loss. This shows that the effect measured in fact at the binding surface binding Unease in the bio-assay device is due. In general it was found that the replacement of the solution with the ligand to an identical solution without the ligand a very little or no change the response caused.

table 1 effect the primary Binding of urease <img img-content="tb" img-format="tif" he="39" wi="133" file="00800001.tif" />

B. Example 2: Identification collagenase and lysozyme by primary binding

Among similarly using a bio-assay device of the above example 1, in a similar manner had been prepared and characterized, we have a number Experiments carried out to the different responses of different proteins on the to investigate the frequency range of 1 to 10 GHz. The same device was for each experiment used (to minor manufacturing differences between to prevent the respective devices), but prior to the application each of the proteins thoroughly with SDS. The<figref idrefs="S130">12C</figref> and <figref idrefs="S130">12D</figref> show the transmission loss measurements the primary Binding effects of collagenase or Lysozymproben about Test frequency range from 1 GHz to 10 GHz. In both cases dismissed the signal response to a pattern of peaks and valleys, which for unambiguous Detection and identification of ligands can be used. In particular, the frequency response of the Kollagenaseprobe showed strong positive peak near of 5 GHz. The answer of Lysozymprobe showed a relatively flat Frequency response near of 5 GHz and a strong positive peak near 8 GHz. In each of the numerous other proteins studied was the answer every Protein clearly, so that an unknown protein in the group was easily identifiable. Naturally can be more spectral points also compared and analyzed, to distinguish these and other molecular substances. The answers can stored and later be retrieved to identify unknown samples. In addition, the less pronounced Tips are examined collectively to determine patterns for certain ligands.

C. Example 3: Detection secondary Bonds: Concanavalin A to Dextran

These Application provides an example of the detection of a secondary Binding prepared similarly wherein a bio-assay device of the from the above Example 1, similar to the was had been prepared and characterized manner used. Concanavalin A (ConA) is a glucose binding protein in Jack beans (Canavalia ensiformis) occurs, and as Primärbindungsligand has been used. The ConA used here was from Sigma Chemical Company procured. Dextran, a glucose polysaccharide, was then used as a ligand for binding of ConA with glucose as the displacer served to reverse the Dextranbindung to demonstrate specificity. (Dextran and glucose were also obtained from Sigma Chemical Company procured).

&lt;?page 37?&gt;

The transmission line was the same as in Example 1 discussed with a nominal 32 Ω reference impedance and an ITO cover glass with a DC resistance of 80 Ω and a nominal TDR impedance of 34 Ω. A concentration of about 15 uM ConA solution was introduced directly into the bio-assay device and the wait for reaching the equilibrium state.

Evaporation losses led not to dry out the chamber, by a visual inspection was found. After the system has been flushed and stabilized was, dextran was added to bind the ConA. After Detecting a signal change the chamber containing 10 mg / ml d-PBS and the signal response was rinsed a measured again. This effect is in<figref idrefs="S131">12E</figref> at 1 GHz shown. The unbound response was as a measurement based response used. As can be seen, the bound response appears to 0.25 dB less lossy than the unbound response. The binding specificity was confirmed, by the bound dextran was replaced with glucose, followed by a d-PBS rinse, to release glucose. This last step returned the signal to return to the measurement base, which had been obtained prior to adding the dextran, so that the specificity of the binding event has been detected.

D. Example 4: Detection the binding of small molecules

Among similarly using a bio-assay device of the above example 1, in a similar manner had been prepared and characterized, the bio-assay test assembly were and power analyzer used to prove that small is at large molecules binding molecules can be also detected with the present invention. to Investigation of the bio-assay device at higher frequencies, has been the Device repeatability and carefully placed in a Faraday cage, to protect them against external influences. Thereby the apparatus could at frequencies up to 20 GHz are examined. First ConA was added to the bio-assay device, which at the bioelectrical interface tape. A measurement of transmission loss has been made, the saved and the measurement base Answer <figref>1252</figref> used , as shown in <figref idrefs="S131">12F</figref> shown.

After that Glucose was added at a concentration of 10 mg / ml for the bio-assay device, to bind to the Con-antiligand. A measurement of transmission loss was performed and relative to the measuring base Answer <figref>1252</figref> applied, the change the signal response to determine due to the binding of small molecules.

As out <figref idrefs="S131">12F</figref> It can be seen the binding response <figref>1254</figref>Which corresponds to the binding of glucose to Con A, of the measuring base measurement <figref>1252</figref> distinguishable. especially has the binding response <figref>1254</figref> two large peaks between 16 and 20 GHz, which in the measurement base Answer <figref>1252</figref> not determine are. The difference in the measured signal responses<figref>1252</figref> and <figref>1254</figref> provides a basis for the identification ready when to glucose to the Con-antiligand has bound. subsequently was flushed with a d-PBS buffer and the response was reversed, when the bound glucose from ConA solved. A separate experiment in which the effect of glucose on the bare chip (ie, no ConA as antiligand) was investigated showed that glucose alone if at all only a small Impact on the response to the electromagnetic interrogation in above said frequency spectrum has been thus demonstrated that the result due solely to the effect of the binding of glucose to Con A is.

The Experiment was for the binding of biotin to avidin repeated. Avidin was for bio-assay device added as an antiligand, and a measurement of the transmission loss was made, stored and used as measurement based response <figref>1262</figref> used. Thereafter biotin was added at a concentration of 1 uM and a measurement of the transmission loss relatively made to the base measurement. The results are in<figref idrefs="S132">12G</figref> shown.

The binding response <figref>1264</figref> according to the bound to avidin Biotin is 14-16 GHz a complete zero value and a great tip close of 20 GHz. The difference between the measured base-answer (for unbound Avidin) and the binding response (for bound avidin) is dramatic and can be used for detection of the bound avidin molecule will.

E. Example 5: Quantifizierungstitrationen

These Experiments show that the size of the signal change upon binding of a ligand to an antiligand on the number of occupied sites depends. The test system in which similarly a bio-assay device of the of Example 1 above, in a similar been prepared and characterized ways &lt;?page 38?&gt;was was used was employed with at ConA binding dextran, wherein glucose as displacement inhibitor served. A serial dilutions was generated around the binding constant of ConA. Dextran as antiligand tied to to a 100% binding to ConA. A series displacement glucose concentrations was to displace the dextran used so that the concentration of dextran on the molecular binding surface was reduced accordingly.

The Standard configuration of the transmission line as explained above was used. ConA went with the molecular binding layer a Binding one and the system was stabilized. The bio-assay device was then flushed with d-PBS and data collected at 1 GHz. The results of these Verdrängungstitration are in <figref idrefs="S132">12H</figref> shown. The results show how the signal changes, when the concentration of glucose is increased from 0 to 15 mg / ml. the signal changes of the ConA when the dextran is released and the glucose bound (Which actually the avidity is measured of dextran). Specificity was also by glucose reversal the Dextranbindungseffekts detected.

table 2 shows the amount of change the transmission loss dependent on of glucose concentration for some selected Concentrations.

table 2 <img img-content="tb" img-format="tif" he="39" wi="93" file="00840001.tif" />

<img img-content="tb" img-format="tif" he="16" wi="93" file="00850001.tif" />

A simple Glukosetitration was also at the resonance point in the spectrum performed by ConA. <figref idrefs="S133">12I</figref> shows the change the return loss dependent on of the glucose concentration in this resonance point, whereby two Effects are demonstrated: First, the glucose has as a ligand a dose-response effect, which is based on the effect they on the anti-ligand has (which is in this case ConA). Second, there It regions in the spectrum, a much more sensitive response the ligand / anti-ligand binding event than other Regions.

successive dilutions the dextran, wherein the concentration to below one picomolar (10^-15 Molar) was reduced, showed even at these low concentrations a significant signal response which indicates that a bond has taken place. The for the signal acquisition time required ranged from a few minutes up to ten minutes, but the response was characteristic of the detection dextran at higher Concentrations.

F. Example 6: Detection of nucleic acids

To the Demonstrate the ability nucleic acids to identify a bio-assay device with polylysine as was on a gold surface Adhesive antiligand prepared. similarly using a bio-assay device of the of Example 1 above except for the gold surface, which in a similar had been prepared and characterized way, was a highly concentrated solution (20 μ) from calf thymus DNA prepared in a d-PBS buffer. The polylysine was applied to the bio-assay device applied and the transmission loss response measured. The answer has been tested and stored for their stability over time. The chamber was then flushed with the buffer, the response again to changes by purging and stability checked and the response is stored as a measurement based response.

After that A solution which contained DNA added to the bio-assay device and the variation of the response measured, by the resulting response of the measurement base response subtracted and up &lt;?page 39?&gt;stability checked has been. The bio-assay device was rinsed with the buffer, to the DNS to remove from the set, whereby only the DNA / polylysine complexes on the surface the bio-assay device remained. The resulting change is in <figref idrefs="S133">12J</figref> shown.

G. Example 7: The influences of pH and salinity

The Effects of pH and salinity in the signal were in two various experiments measured. To investigate the influence of pH, a number of buffer with pH values ​​from 3.94 to 9.80 was measured. conductivity at 60 Hz for each buffer was measured by the change in free ions correct. subsequently were transmission loss answers measured at 100 MHz, 1 GHz and 10 GHz. The results are in<figref idrefs="S134">12K</figref> shown.

A similar Experiment was to determine the effects of the change of the ion concentration in a solution performed. There have been several solutions recognized, starting with a simple d-PBS solution, the various amounts Sodium chloride were added. Then, the conductivity was measured at 60 Hz and recorded, and the samples were the Sequentially added to the bio-assay device; the transmission response was measured at 100 MHz, 1 GHz and 10 GHz. These results are in<figref idrefs="S134">12L</figref> applied.

As show both graphs, result certain environmental changes in changes of the measured parameters.

H. Example 8: Detection in whole blood

The Detection of troponin I (TN-I) was carried out in untreated whole blood, the detection capability in disturbed check environments. The untreated human blood was treated with sodium citrate to prevent coagulation. An anti-TN-I antibody corresponding to the epitope of TN-I, was used to rierungszwecken Calib. The interfacial transmission line the bio-assay device was coated with anti-TN-I Ab (antiligand). A blood sample was spiked to a 10ng / ml concentration of TN-I and a second identical sample of blood was ungespikt as a control sample.

The Experiment consisted of the following steps: antiligand anti-TN-I From the device adhere; then first the unspiked sample across the Chamber result; repeated washings the sample chamber to determine the exchange of noise; then spiked Sample which was replaced several times, for a noise floor at determine. In each case, the change of the transmission loss was measured. The experiment was then repeated to check it, to determine if any other properties of the two Blood samples (as identical accepted except the TN-I-Spike) responsible for the change were. The following table shows the result of this experiment for a Test signal at 1 GHz.

<img img-content="tb" img-format="tif" he="23" wi="98" file="00870001.tif" />

in a second series of tests from ten different blood samples a clinical laboratory obtained, with the exception of the anticoagulant were heparin untreated. One of the samples was divided into two parts, one of which with the antiligand TN-I was spiked in the previous Paragraph. The bio-assay device was then coated with the antibody anti-TN-I on the surface prepared. The samples were then sequentially through the bio-assay device sent, the spiked sample was stored last. The responses of each of these samples at 1 GHz as in the previous experiment were measured and are in <figref idrefs="S135">12M</figref> shown. The spiked sample was significantly compared to the rest of the (unspiked) Samples to distinguish.

I. Example 9: Detection the Vierpolmoments a molecule

Of the Effect of binding avidin to a gold surface was investigated to the to determine detectability of Vierpolmoments a molecule. avidin a tetramer with a very small dipole moment in unbound State, by the symmetry of the molecule in the unbound state is caused. <figref idrefs="S135">12N</figref> shows the result of Avidin with the characteristic shown in the graph Spit&lt;?page 40?&gt;Zen. It should be noted that these peaks considerably smaller than the peaks that are due to the biotin binding, as described in <figref idrefs="S132">12G</figref> are shown.

VII. Applications

The Methods and systems of the present invention may be in different applications are used, the herein described will.

The present invention could to quantify the extent of binding between a ligand and an antiligand and thus for the determination of the effect of other molecules on the activity of are enzyme used. So could For example, other molecules in the solution the extent of binding increase or decrease and could in this way the identity be determined by enzyme inhibitors or -inducern.

The presence of infectious Pathogens (viruses, bacteria, fungi or the like.), Or Karzinotumore can be tested using the binding of an antiligand to the pathogen, the tumor cell or a component of the pathogen or tumor such as a protein, a cell membrane, a cell extract, tumor markers as CEA or PSA, other antigenic epitopes or the like. monitors becomes. The invention is for example able to the pathogen or to detect the tumor by the binding of pathogenic or tumor markers in the patient's blood with an antibody on the bio-assay device is detected. also is the binding of an antibody from a patient's blood to a viral protein such as an HIV protein a common Test to monitor the patient in the presence of virus. Another common practice Example is the quantification of Prostate Specific Anti gen (prostate specific Antigen - PSA) in the patient's blood as a marker for the progression of prostate cancer.

In addition, interactions between drug and receptor, including both membrane - and Non-membrane receptors and receptor conformational changes as Result of drug binding with the present invention be determined. In another aspect, the invention for Supplying information on Lipid interactions as Lipo-protein binding to lipids and liposomal Interactions are used with lipids.

at other embodiments can be used to the technology of the invention, gene chips for the Investigation of nucleic acid samples and proteomic chips the cataloging and description of proteins provide. Such chips can the unique ability make the invention advantage, by the same affinity, kinetics and the distinctive dielectric signatures each binding event measured and these measurements at a plurality of addressable measuring points run on the chip. The exact nature of the addressing will depend on the applications, but the general strategy is as follows: Define a vector space by the variable K_ec, k_A and ω = (ω1, ω2, ω3, ...), wherein these variables is the equilibrium constant, the kinetic Constant and a basic set of N frequencies represent, in which the dielectric properties are investigated. On Room with N + 2 dimensions is defined in the each binding event can be mapped. Then a group of reference molecules is selected, the a spectrum of interest binding events represents, z. B. a group oligonucleotides having different nucleic acid sequences or a selection antibody specifically for Protein regions, or other sub-structures of proteins, and attached to the addressable points of the chip. A particular species molecules or group species is introduced into the chip, and each address then the value of each of the points defined in the above vector space (Or a suitable subset thereof) tested. Each Species may be represented by an address in the vector space will. The complexity the system depends the size of the vector space and the total number of different immobilized ligands on the surface from.

As example for the foregoing, consider a simple system comprised of two different nucleic acid strands is, different at four frequencies are analyzed; furthermore each of these frequencies in analyzed ten different amplitudes will. Such a system would 100 million potential Addresses (10⁴ for the first polymorphism and 10⁴ for the second polymorphism). An introduced into the system Unknown is, by a unique address of the form [[1, 5, 3 7) (4, 8, 6, 7)] represents the first four numbers, the spectral response of the first strand in the four selected frequencies and the second four figures, the spectral response of the second strand selected in the four Frequencies represent. Thus, with only two strands and four frequencies are produced 100 million unique addresses.

&lt;?page 41?&gt;

"Smart Needle" IV-analyzes a miniature bio-assay device in the bore of a needle deploy, can the technology of the present invention is also provided. These embodiment can to provide cost effective and safe medical diagnostic equipment for use in emergency rooms and other points of care, and the like are used.. application examples include: diagnosing acute conditions such as heart attacks, infectious diseases perinatal as bacterial meningitis, Group B Step infections in neonatal / Environment, coagulopathy, fetal and neonatal oxygenation in intensive care units, diagnosing chronic conditions in supply points such as medical services and field offices.

A Bio-assay device with a plurality of biological binding parameters allows the simultaneous analysis of a plurality of analytes in Sample. also provides measurement of the binding of a single analyte to a number different species of biological binding partner a check for a non-specific binding. A comparison of the binding of different proportions Analytes a test sample allows the evaluation of the relative Increase or decrease of different analytes.

The Bio-assay device of this invention can be used to detect virtually each analyte can be used in vivo or ex vivo. While a preferred embodiment, The analyte may be a biological molecule, it need not in this respect limited to be, unless a specific binding partner is available described or other characteristic of the analyte in a herein embodiments can be measured. Suitable analytes include virtually every in biological materials or from materials used occurring analyte. Virtually any analyte, preferably in a aqueous solution suspended or dissolved can be, can be detected with the method of the present invention will. examples for Analytes of interest include: 1) antibodies such as antibodies to HIV; 2) Helicobacter pylori, hepatitis (eg., Hepatitis A, B and C), Measles, mumps and rubella; Drugs of abuse and their metabolic byproducts, such as cotinine, Cocaine, benzoylecgonine, benzodiazepine, tetrahydrocannabinol, nicotine, ethanol; 3) therapeutic drugs including. Theophylline, phenytoin, Acetaminophen, lithium, diazepam, Nortryptylin, secobarbital, phenobarbitol , etc .; 4) hormones and growth factors such as Testeron, estradiol, 17-hydroxyprogesterone, Progesterone, thyroxine, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, transforming Growth factor alpha, epidermal growth factor, insulin-like Growth factor I and II, Release Inhibiting growth factor and Sex hormones that bind globulin; and 5) other analytes including glucose, Cholesterol, caffeine, Corticosterid binding globulin, DHEA binding Glycoprotein and the like.

As indicated above suitable analytes include proteins, glycoproteins, Antigens, antibodies, nucleic acids, Sugars, carbohydrates, lectins and the like. However, it can also larger multi-molecular Units such as cells, cell membranes and other cellular components detected with the methods of this invention and / or quantified will. Thus, z. B. microorganisms (eg., bacteria, fungi, algae, etc.) with characteristic Surface markers (Z. B. receptors, lectins, etc.) detected and / or quantified (such. B, in a biological sample from an animal or a Plant). something similar applies to Cell types (eg. B. cells for are a particular tissue characteristic) with the characteristic Markers (eg., Tumor cells expressing IL-13 receptor highlight (see z., US Patent 5,614,191)). Thus, cells to certain Pathologies, certain states of differentiation (or lack thereof) or particular tissue types will point, detected and / or quantified.

conjugation of biological binding partner (ligand or antiligand) -Effektormoleküls with the "smart" user interface In one embodiment is the biological binding partner (ligand or antiligand) chemically with the underlying surface conjugate (for. example, with the bioelectrical interface). Means for chemical conjugation of molecules to the skilled worker known (see, for example, Chapter 4 in Monoclonal Antibodies:. Principles and Applications, Birch and Lennox, Ed. John Wiley &amp; Sons, Inc., N. Y. (1995), in which the conjugation of antibodies to anticancer drugs, Indicators incl. Radio indicators, enzymes and the like. Described becomes).

The Procedure, with a binding partner (eg., Protein, antibody, glycoprotein, Nucleic acid, Lectin, sugar, carbohydrate, etc.) bound to a surface is, is according to the chemical structure of the binding partner different. Polypeptides typically contain various functional Groups such. As carboxylic acid (COOH) or free amino (NH₂)-Groups, capable of reacting with a suitable functional group on the surfaces or coupling groups to which they are attached, are present. Similarly included other biological molecules z. B. nucleic acids, Sugars, carbohydrates all diverse functional groups (eg. as OH, NH₂, COOOH, -S Etc.), the suitable coupling points.

&lt;?page 42?&gt;

alternative the target molecule can and / or the effector be derivatized to additional liberate reactive functional groups or binding. The Deriva tomation may include the adherence of any molecule of a series coupling molecules, as they are from the Pierce Chemical Company, Rockford, Illinois.

in the Scope of the present invention is a "coupler" is a molecule, which for connection of the biological Binding partner (eg. As a ligand or anti-ligand) with the underlying lying (z. B. Device or device) surface can be used. The coupler is in a position equivalent Bonds with both the biological binding partners as well as with the underlying surface to build. Suitable couplers are well known to those skilled and comprise, inter alia Kohlenstoffkoppler having straight or branched Chains, heterocyclic or Kohlenstoffkoppler Peptidekoppler.

On bifunctional coupler having a functional group with a Group on the surface responds, and another group with the binding partner responding, can be used to form the desired conjugation will. Alternatively, the derivatization of a chemical treatment of the binding partner and / or substrate include. For example can be silanized silica or a glass substrate to produce a functional group. Similarly, a protein or glycoprotein can be derivatized, eg., by the glycol a sugar moiety, the protein sticks to the antibody with periodate to to generate free aldehyde groups. The free aldehyde groups on the antibody or Protein or glycoprotein may reacted with free amino or hydrazine groups on the surface in order to bind the binding partner in order (see U.S. patent No. 4,671,958). A method to generate free sulfhydryl of polypeptide such as antibodies or antibody fragments are also known (see U.S. Pat. No. 4,659,839).

the Skilled are numerous methods and coupling molecules to adhere various biological molecules various metal, glass, plastic substrates, etc. sufficiently known. See z. B. European Patent Application No. 188,256. U.S. Patent No. 4,671,958.; 4,659,839; 4,414,148; 4,699,784; 4,680,338; 4,569,789 and 4,589,071 and Borlinghaus et al. (1987) Cancer Res 47: 4071-4075. Methods for conjugating antibodies Proteins and glycoproteins can be found in large numbers in the immunotoxin literature such. as in "Monoclonal Antibody Toxin Conjugates: Aiming the Magic Bullet ", Thorpe et al. Monoclonal Antibodies in Clinical Medicine, Academic Press, p 168 - 190 results (1982) Waldmann, Science, 252: 1657 (1991), U.S. Patents No. 4,545,985 and. 4,894,443.

Use of nucleic acid-binding partners

If the binding partner is a nucleic acid (eg DNA;. RNA, peptide, nucleic acid etc.) specific binding is preferably achieved under "stringent" conditions wherein the hybridization is more specific, the more stringent the Conditions.

The Choice of stringent conditions for any strand / target combination is for the average person skilled a routine task. also can be determined empirically, the stringency by the stringency the conditions gradually reinforced is (z. B. Increase Until the desired level reached the salt concentration, temperature, etc.) of specificity has been.

"Starting points" for stringent Conditions are well known. So hybridize z. B. the desired nucleic acids to complementary Nucleic acid strands under the hybridization and wash conditions of 50% formamide at 42 ° C. Other stringent hybridization conditions can also be selected. In general, stringent conditions are selected such that be about 5 ° C below the thermal melting point (T_m) For the specific Sequence at a defined ionic strength and a defined pH l ying. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence to a perfect paired strand hybridize. Stringent conditions are typically those in which the salt concentration is at least 0.02 M at pH 7 and the temperature is at least 60 ° C. Since the other factors Strin can significantly affect gence hybridization to in particular, the base composition and size of the complementary strands, the Presence of organic solvents and the extent of Base mismatch counts is the combination of parameters important than the absolute measure of a individual. A detailed Guide to the hybridization of nucleic acids is found in Ausubel et al. Current Protocols in Molecular Biology, Current Protocols, a joint venture between Greene Publishing Associated, Inc. and John Wiley &amp; Sons, Inc. (supplemented to 1998).

&lt;?page 43?&gt;

oligonucleotides for use as a binding partner can be chemically synthesized z. B. to the phosphoramidite triester method of the solid Phase, first described by Beaucage, SL and Carruthers, MH, 1981, Tetrahedron Lett, 22 (20):. 1859-1862 was described, using an automatic synthesizer uses was as described by Needham-VanDevanter, DR et al., 1984, Nucleic Acid Res., 12: 6159-6168 will be described. Purification of oligonucleotides by either native acrylamide gel electrophoresis or by Anion exchange HPLC as described in Pearson, JD and Regnier, FE (1983) J. Chrom. 255: 137-149, described. The sequence of the synthetic Oligonucleotide can by means of chemical Degradationsverfahrens Maxam, AM and Gilbert, W. (1980) in Methods Enzymol. 65: 499-560 be varied.

The Bio-assay device has a wide range of applications including, for example, the investigation of a large number of molecules biological activity or the identification of biological samples to the existing or lack of concentration of a particular component or certain Components. In the investigation on biological activity such. B. is the bond layer exposed to one or more receptors such as antibodies or whole cells. By detecting an interaction between antiligand and ligand binding layer can Presence and concentration can be determined. A special Advantage of this technique is that no indicators required are to detect this interaction. The inherent properties serve the individual molecules for the detection of their presence and their quantity, their lack or the interaction with other molecules.

Other potential applications for the bio-assay device or chip relating to the diagnosis, wherein different antibodies for certain Receptors would be used, to form the binding layer, and blood z. B. on immune diseases would be examined. The bio-assay device is selectively prepared so that it fits into the bore of a hypodermic needle. It would be only a Tiny blood sample required to applied a binding to a previously Anti-ligands to detect on the binding layer. A bio-assay device can be used for measurement of a wide range of clinically relevant analytes, of pathogens such as viruses or bacteria to metabolic activities such as Glucose concentration or lipid levels up to the usual tests for liver enzymes, Electrolytes, clotting factors, specific antibodies like (Used in rheumatological disorders) ANA and allergic Responses to antibody prepared oxygenation of arterial blood, drugs of abuse and the like. will.

The used in this function bio-assay device would consist of inexpensive Since these produce disposable chips easily and not to the semiconductor processing limited are. The chips are, for example, optional on cost Materials such as plastic or glass substrates manufactured. The Chips are then either used in a device as described below described, and a signal is transmitted by the bio-assay device, to the binding interactions due to the ligand in the blood detect. In fact, could many different shapes and sizes of bio-assay devices with various binding layers for the countless biological and chemical applications are prepared for the detection without indicator useful would.

unknown and can not characterized proteins classified and / or be identified by the binding of the structural pattern of unknown protein is detected. For example, proteins in the same or a similar class structural homologies; ie substructures such as domains that are repeat within a given class of proteins. By producing a chip with multiple addressable arrays, of which one each a antiligand for a specific substructure has, could be an unknown molecular classified species as follows and / of be identified: The Presence of certain substructures is a by binding each recognized at their respective anti-ligand. Each of these sub-structure binding events then by properties such as affinity, kinetics and spectral sensitivity characterized. Then, the correlation is carried out between the unknown molecular species and the resultant of known proteins data. In the case where no exact match is found, can many of the structural details of the unknown compound are assembled in a similar way as in the Nuclear magnetic resonance (NMR) spectroscopy for organic molecules takes place.

at Alternatively, this technique can be used to develop gene chips for detection of nucleic acids be applied. Gene chips are arrays of nucleic acids for detecting complementary nucleic acids be used in a sample. The presence of the complementary DNA, such as by binding to a particular DNA molecule on the Gene chip measured, is the desired result. If this complementary Binding does not occur, partial hybridization can be detected and are characterized by physical quantities such as Affinity, measured melting point or other stringency and direct spectral sensitivity of the signal and the correlation with previously measured data are determined. In this way, a single Antiligand in the form of a Nukleinsäurese&lt;?page 44?&gt;sequence an entire area detect polymorphisms without a separate sequence for each Polymorphism is required. So could z. B. with a chip with a few hundred different nucleic acid sequences thousands of different polymorphisms are detected.

gene chips can to identify drug targets for bacterial identification, used typing of genes and other diagnostic tasks will.

The Technique requires the attachment of the requisite nucleic acids, typically as strands, to the substrate and a method for measuring the binding of complementary nucleic acids to this substrate. Normally, the nucleic acids need of Sample an indicator of what most is provided by a fluorescent strand. With this technology, is the necessity of an indicator of the sample DNA superfluous and contiguous the order Problems are eliminated. Gene chips can for special needs in drug-target identification, molecular diagnostics and developed the detection and identification of biological agents will. Other types of devices that could be fabricated immunoassay devices, Drug discovery devices and Toxiditäts test devices, analytical devices, and the like.

The invention described herein can also be applied to various aspects be used in the development of new drugs, from initial Investigation process to hinzur patient typing and therapeutic monitoring. In the initial stages of drug discovery, the invention can to simplify the target identification, validation and testing with high throughput (high throughput screening) are applied. Of the Target receptor may be the antiligand on the bio-assay device be and by characterizing the actions of known agonists, Antagonists or allosteric effectors could first scan targets with high throughput are quickly identified and validated. In HTS process hundreds of thousands of compounds tested to determine which can bind to the target. The invention described herein can be miniaturized so that extremely similar test platforms can be realized, which are capable of hundreds or thousands of connections simultaneously to consider and at the same time the effect of binding (eg. as agonist or antagonist), Affinity, Kinetics etc. to determine. Also need such Miniature systems very low connection levels, thereby significantly Costs of obtaining such compounds from combinatorial Libraries can be saved. The through use of the bio-assay device Detection system formed constitutes a detection system with high Throughput ready because the detection is done either in real time and many samples can be analyzed quickly. The response period is Optional monitored on a nanosecond time scale. Once the molecules together are attached, the detection takes place. For the measurement of low concentrations or binding events between molecules with low binding affinity is any more time is required. The actual Time is limited by the diffusion rate. Apart from these through potential restrictions thousands of compounds quickly the system, z. B. within a Hour. At use of chip manufacturing technology, for example, a device with 10,000 channels possible (when using the new Mikrofluidiktechnologien) and in small volumes and thus short diffusion times and with kinetic Measurements, measure only the beginning of the reaction, are optional 10 million samples per hour measured. At known concentrations is the binding affinity optional calculated solely from the kinetics and thus the device sampled at a very fast time scale and the affinity of the inclination the kinetic curve is calculated and / or estimated. Information on kinetics and Affnitäten can be found in any standard work on biochemistry or chemistry, z. B. Mathews and van Holde, Biochemistry, Benjamin Cummings, New York., 1990

The invention can be extended in a simple manner in cell-based analyzes, since the Detection may not require purification and amplification of the sample. These can use classes cellular system on various changes either by the Detecting external expressions or by lysing the cell to release cytosolic components and detecting the presence of one or more of interest monitors analytes will.

The Invention can also be performed for "lab-on-a-chip" applications. Due to the simple miniaturization, very small chips thousands or tens of thousands contained therein addressable bio-assay devices be realized. The detector may be realized as a "logic gate" in which the presence of a particular ligand or analysis has the effect that the gate according to the application either on or is turned off. Such a gate may in any number of ways be realized that the binding event into an electrical Signal convert that one of two possible states according to can be assigned to Off or On, 1 or 0, and the like.. The two conditions could different frequencies of a cavity or Wel&lt;?page 45?&gt;lenleiters bound accordingly and unbound or amplitude changes a transmission line or a waveguide corresponding bound or unbound, or changes be or the like of the bandpass of a particular circuit.

Above was indeed a complete description of possible embodiments given the invention, it can However, various alternatives, modifications and equivalents be used. For the Skilled in the art it is, for example, on the hand, that the signal path of the above bio-assay device is not in a transmission line limited is. Other transmission media as conductive or dielectric waveguides may alternatively be used.