WO2016015701A1 - Bioanalysis device, the production thereof and method for detecting bioanalytes by means of the device - Google Patents

Bioanalysis device, the production thereof and method for detecting bioanalytes by means of the device Download PDF

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Publication number
WO2016015701A1
WO2016015701A1 PCT/DE2015/000373 DE2015000373W WO2016015701A1 WO 2016015701 A1 WO2016015701 A1 WO 2016015701A1 DE 2015000373 W DE2015000373 W DE 2015000373W WO 2016015701 A1 WO2016015701 A1 WO 2016015701A1
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layer
characterized
device according
functional layer
example
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PCT/DE2015/000373
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German (de)
French (fr)
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Hans Scheefers
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Schebo Biotech Ag
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366Apparatus specially adapted for solid-phase testing
    • G01N33/54386Analytical elements

Abstract

The invention relates to a novel bioanalysis device for selected biological analytes, containing a) a carrier layer, b) a functional layer and c) optionally a capping layer, wherein the functional layer can hold and transport liquid and has a capture molecule for the selected analyte at at least one point, characterized in that the carrier layer and/or the capping layer contain sensor elements for the selected analyte, which sensor elements in turn were introduced by printing methods onto or into the carrier layer and/or the capping layer, and to a method for detecting bioanalytes by means of the device according to the invention.

Description

Device for bioanalytics, their preparation and method for the detection of bioanalytes means of the device

Field of the Invention

The invention relates to a novel apparatus for bioanalytics and method for the detection of analytes (such as bioanalytes, cells, immune complexes, supramolecular structures, Zellkom- parti elements, etc.) from biological samples by means of the inventive device. This is a transmitter of the receiver sandwich arrangement for measuring the local electrical, optical, biophysical properties of materials in an immune-function thin film which also serves as a bio-analytical platform. The invention further describes the use of nanoparticles, nanodots, sensors: z. As magnetic sensors, sensor circuits, combined sensors, measurement techniques for this purpose, etc.

Prior art and background of the invention

From the prior art a series of devices for the analysis of biological analytes verschiedenster are known, such as detection assay, which operate on the principle of a Immunoas- says means verschiedenster antibody. In most of these tests, this test system is achieved a color change at a certain point, which is then read visually off. Therefore, the reading is partly subjective. In addition, many of these tests only semiquantitative nature. The analysis of larger biological input units, such as cellular compartments, Zellsubstruk- structures, whole cells or cell superstructures has so far been very limited with very specific and complex measurement methods possible.

From the prior art are still a number of advantages directions and known methods for the analysis of biological analytes verschiedenster and cells.

To this end, the techniques used are, for example: immune agglutination, import munpräzipitation, simple one-dimensional immunodiffusion according Oudin, two-dimensional immunodiffusion procedure according to Ouchterlony (for generating Immunpräzipitationsmustern), immune and without intersections zelektrophorese, electrodiffusion, immunofixation, radial immunodiffusion and Raketenelektrophorese, preparative Immunprä- zipitation, radioimmunoassay (RIA), nephelometry.

All of these techniques use the bio-physical phenomenon of immune binding. After optimal fixing one of the reaction partners by adsorption or covalent coupling to a UN-soluble support (nitrocellulose, glass fiber, nylon, or other carrier).

The visualization of an immune response / immune binding is based on a stable bridge between the reaction partners are (antigen or antibody) and the example amplificate cation system.

As Amplifikatoren (or indicators) are used: fluorescent dyes, luminescent dyes zyme, radioactivity, ene, nanoparticles such as colloidal gold.

The visualization of the antibody binding takes place through direct and indirect methods, for example PAP methods of Sternberger, biotin-avidin bridge (ABC technique), direct or indirect detection, for example via an acting Amplifikator marking tes Protein A, including direct protein a gold method, indirect protein a-gold method but also evidence of a er than amplificate, functioning labeled lectin. Detecting the specificity of antibodies in cells and tissues by means of the sandwich method, the DOT immunoassays, Western blotting, Proteintrans- fer, immobilization and immunodetection.

Biacore technology / method

This method is easy on the proteins, it makes do with very small amounts of protein without labeling. In addition, in accordance with availability of pure antigens or of specific antibodies from complex mixtures, such as affinity Chromatrographisch the corresponding binding can be cleaned, partner isolated in minutes. The technique can also focus from highly diluted solutions binding partner. The Biacore technology is a sensor chip based on the sur- faces plasmon resonance (SPR, the S stands for Surface, PR represents plasmon resonance) can generate. The sensor chip which extends in a continuous flow-through befin- det microcell can, various ligands and buffer are exposed and register the kinetics and the binding and separation profile for JE stays awhile binding pair of macromolecules. The change of the SPR in quantitative measurements is trans- lated.

TECHNICAL PROBLEM OF THE INVENTION The invention is the technical problem underlying Vorrich- obligations for bioanalysis to make available that allow a more objectified reading. In addition, opportunities for automatic, computer-based reading should be made available. In addition to the analytical tik larger units, such as cellular compartments, Zellsubstrukturen, whole cells or cell superstructures ER values ​​made possible.

Principles of the invention and preferred embodiments

To solve this technical problem, the invention teaches an apparatus for bioanalysis for selected biological analytes in a liquid phase containing a) a support layer,

b) A functional layer

c animals) optionally a top layer which absorb the functional layer liquid and transported can and at least one place a capture molecule for or having the selected analyte, characterized in that the carrier layer and / or the cover layer transmitter elements con- tains, which in turn by typographical method on or in the carrier layer and / or were on or incorporated into the top layer, wherein the transmitter elements are coordinated with the or the selected analyte and / or characterized in that the carrier layer and / or the top layer sensor elements con- tains, which in turn by printing processes were introduced onto or into the carrier layer and / or on or in the cover layer, wherein the transmitter elements are coordinated with the or the selected analyte.

Advantageous embodiments are subject of the subclaims.

Essential part of the apparatus of the invention is a functional layer, which is capable of increasing up liquids and to transport. These are examples of playing a porous, sponge-like material. This can be for. B. is silica gel, diatomaceous earth, alumina,

Cellulose, nitrocellulose or other fine-grained Mate rial act. Such functional coatings typically contain pores having an average pore diameter of examples of play. 4 - 1000 nm, the functional layer contains at least one location at least one biological capture molecule as "Biofalle" In this way, there is provided a microcavity, in which the. can be carried out further evidence leading biochemical reaction (agglutination or precipitation according to the "Heidelberger curve"). In the biological Fängermole- kül may be, an antibody to a selected antigen such as a protein, for example. In a preferred embodiment according to the inventions dung the layer is arranged flat and is moistened along one edge with a liquid containing LYTEN the selected analog. By capillary forces the liquid migrates through the functional layer and impinges on the corresponding location on the capture molecule. The fluid path through the functional layer therethrough can also be done using pumps, in particular micro-pumps (piezo pumps). Alternatively, other mechanical forces can act, such as a centrifugal force when the functional layer is set in rotation. If the desired analyte is in the required concentration in the liquid, a reaction with the scavenger molecule will occur, so that it zipitation agglutination or pre- or can co-precipitation according to the "Heidelberger curve" arrive. If the liquid contains the selected analyte is not in the required amount, the capture molecule will not react and no agglutination or Präzipitati- on occur. the liquid may be an arbitrary saturated polar or nonpolar solvent, such as examples game, DMSO, water, acetonitrile Optional or the like. a second recognition molecule can also be added, WEL ches knows the formed of capture molecule and the analyte complex ER, such as a second antibody. This second recognition molecule may, for example tion a radioactive marking, a luminescent component, an ionic compo- te or metal, for example gold particles, e nthalten to ensure the detectability of the formed agglutinate / precipitate or improve.

In other words: The immune function layer con- tains capture molecules that are bound to both moving particles as well as to the solid phase. The immune function layer further contains micro-cavities (micro-wells), in which an agglutination reaction with the analyte according to Ouchterlony ER- follows.

As an analyte can be virtually any conceivable biological entity or molecule occur: In addition to peptides and proteins, for example, RNA and DNA sequences, carbohydrates, lipids, hormones come (steroid or sugar hormones), enzymes, cell compartments elements, Zellsubstrukturen, cell superstructures and / or te entire cells, protein complexes, protein-protein complexes, protein-lipid complexes, protein-carbohydrate complexes, protein-nucleic acid complexes and viruses into account. It goes without saying that the corresponding capture molecules must be parked on the analyte to be caught: Here, for example, are antibodies (polyclonal or monoclonal), Ap Tamere, Spiegelmers®, lectins, or mixtures thereof. In further embodiments, the invention also re several different capture molecules in the functional layer may be contained th, including: different antibodies or antibodies and aptamers or aptamers and lectins, usw..Diese may be located in particular also at different positions.

The apparatus and the method carried out therewith according to the invention are suitable for the quantitative and qualitative analysis of the different analytes. Many exemplary shapes are suitable for the quantitative and qualitative analysis of several analytes in the same period.

As a functional layer such as a nitrocellulose or Polyacetatschicht can be used, as it is known from lateral flow systems, which is offset body at a location with an anti.

The functional layer is applied in the system according to the invention onto a support layer. This consists preferably of plastic, which in turn is also preferably transparent. The backing layer is characterized in that it includes sensor elements, which for their part were introduced by printing processes on or in the layer. In the present application, the terms sensor / detector sensors and / Detektioren are to be regarded as a system-nonym. As sensor elements tend or semi-conductive elements can be introduced in particular managerial. Such elements are known for example from the production of touch screens. For example, it may be a push polyester layer coated with oxide Indiumzinno- in the carrier. Indium tin oxide is a transparent semiconductor. The optional cover layer is in principle the same construction as the backing layer, ie a layer of plastic, in which method the sensor elements have been applied into or onto the layer by printing technology. The pressure-scale production of the layer may be effected for example by ink (inkjet) printing using conductive ink according to, but may be printed, flexographic printing or screen printing effected by appropriate application of methods such as gravure printing, letterpress printing, offset. Such techniques with conductive ink are known from the prior art.

In the simplest case, is a respective sensor element of cover layer and carrier layer as an electrode, adapted to the sets, a voltage is. Between these electrodes, the resistance can be measured overall, which may depend, for example, from the liquid flow, especially the analyte contained. Thus affect influence, for example, ionic molecular fragments, as well as gold particles that bind LYTEN via antibodies to a specific analog, effectively a change of resistance which can be charged measure. Two mutually opposite elec- clear from the carrier layer and covering layer but can also form a capacitor whose capacitance can be measured. Also in this case, the presence of an analyte, especially an analyte containing causes metal z. B. Goldpar- Tikel a change in the dielectric properties, serving as the dielectric functional layer, which - beispiels- example in the form of the permittivity is-measurable. This is an embodiment of the so-called "capacitivesen- sing", namely a so-called "proximitysensing". The gold particles by causing change in the electric field is measured by the capacitor and is compared ones shown,. An exemplary embodiment is illustrated in Example. 1 various methods for measuring capacitance are known from the prior art that can be used to carry out the capacitance measurement. These include the measurement of the resonance frequency of a formed with the capacity overall LC resonant circuit, the application of an alternating voltage while measuring the current profile or charging with con- stantem current and observing the Spannungsanstiegsgeschwindig- ness.

In a further embodiment of the invention the carrier layer and / or the covering layer contains sensors detecting the photon. This purpose is served, for example, light-sensitive transmitter sorelemente, such as a photo resistor or a photodiode. Incident light, such. Example, of a laser beam, for example, a tunable dye laser can emit and for example, the functional layer can be measured through layer by means of the inventive sensor elements on the carrier. The passage of a sample leads to a weakening of the beam, for example due to sorption waste, which causes a change in measured value. The term light includes not only visible light, but covers the entire range of electromagnetic radiation from the infrared range over the visible region to the ultraviolet region from. Furthermore, X-rays can also consist of an X-ray laser or corresponding synchrotron uses advertising to. A measuring circuit for measuring light by means of a photo-diode is exemplary shown in FIG 17th According to the invention can also be used LEDs are based on or- ganic materials, so-called OLEDs. Appropriate organic cal transistors as receiver elements based applicable to CMOS technology. Such optical emitters and sensors are available, for example from the companies NovaLED and Hamamatsu in the market. Here, integrated devices can be used, for example, diode arrays or sen- rarrays.

In another embodiment of the invention, a light can ray, a laser beam is transmitted perpendicular to the direction of the sample liquid through the functional layer, for example. In the case of a particle-containing sample is a dispersion of the laser beam is achieved, which can be measured by means of the cover layer or the carrier layer and the optoelectronic sensor elements contained therein. The functional layer has the function of a cell in this configuration. Also in this embodiment is to be understood "light" as the term that electromagnetic radiation from the infrared range, the visible range or the ultraviolet range may be used.

A further embodiment of the invention is that extremely light-sensitive carrier and / or cover layers are listed wear, keep the example, elements of a Fotomultipli- ers (working example, by the avalanche principle) corresponds. Such an embodiment requires a Biofalle VO out, which can be excited to emit light, such as on the basis of luminol. The opto-electronic sensor elements from the top or support layer are to measure the impersonated light in the situation. Another embodiment of the invention is that light carriers and / or outer layers are applied, which contain, for example, elements of a LED. to be here- flatbed LEDs fabricated on typographical manner and brought into contact with the functional layer (laminated and / or bonded). From the prior art, the manufacture is development of organic field effect transistors (OFET, Incjet pressure), organic light emitting diodes (OLED, screen printing), organic solar cells (OPVC, screen printing), as well as various other optical sensors, such as photoresistors known. All these components mentioned can be used according to the invention the advertising.

In embodiments based on optical methods, the excitation ( "exitation") should preferably be carried out with ultraviolet radiative (transmitter).

The sensor (receiver) should be sensitive to visible or infrared radiation; for the ultraviolet - or

visible - radiation be as insensitive as possible.

Such infrared sensors are available in the market, such as in the form of micro-NIR spectrometer from 1700 JDSU. Further embodiments of the invention are based on so-called microbolometer that can measure at room temperature, more recently, uncooled.

The use of infrared sensors also allows execution of the invention be used in the provided with antibodies magnetically diagram nanoparticles, which are thermally excited by applying an alternating electromagnetic field, for example, 100kHz. The Temperaturän- generated thereby modification may be measured by said microbolometer or thermopile (see FIG 16). With these considerations for selecting the electromagnetic

Radiation between the transmitter and receiver should the absorption onsverhalten of the material of the functional layer are considered.

Alternatives: As an infrared transmitter Diodeais recom- a photo diode may for example be installed catcher example. Basically, according to the invention to the transmitter-receiver semiconductor devices, the films can be printed on are preferably used (unflexib- le flexible or).

In a further embodiment of the invention, a plurality of light emitting elements are applied in the carrier and / or outer layer, such as an LED panel. In this way, the functional layer can be in several places at the same time fluoroscopy tet. For example, corresponding to be introduced into the functional layer to each individual LED of an LED panel, various antibodies.

As an inventive light receiver, a CCD or a CMOS chip, for example. Here, for example chips used which builds typically einge- in digital cameras. In this manner, a plurality of individual sensors already exist, which can be used for measuring the incident light (corresponding to the individual pixels of a digital camera). In a preferred embodiment, the light emission in the form of an LED panel and the light measurement takes place by means of a corresponding thereto CCD or CMOS element. Alternatively, an array of photodiodes can be used in this embodiment, of course. Also, such arrays are odes of Fotodi- known in the art and need at this point no further explanation. In a further embodiment of the invention, the cover and / or backing layer contains semi-conductive elements which act as field effect transistors (FET). These are very sensitive detection systems (so-called CHEMFETs) for biochemical reactions, such as antibody-antigen reactions. In contrast to a conventional field effect transistor, the metal layer is replaced by a chemically sensitive film, in this case, the functional layer of the ER inventive device with the applied Fängermole- kül. If there is an antibody-antigen reaction is the potential that can be measured will change. Such an embodiment of the invention is also particularly suitable for detecting complete cells or cell compartments. Correspondingly also IGFET, NIGFET, MISFET, MOSFET, OFET, IS- FET, ENFET, EOSFET, CNTFET or floating gate transistors can be used and variations of the above.

In particular, for the detection of whole cells or assortments to Zellkompar- processes have proved on the basis of Durchflusszyto- geometry. Within the scope of the inventive device whole cells may for example by light scattering methods, as already mentioned above, proved. different cell size parameters can SEN and distinguished by different shapes of light scattering techniques, such as "forward- scatter" or "side-scatter". Also Fluoreszenzmessun- gen are possible.

In other embodiments of the invention, the printing process applied to the cover and / or support layer sensors are transmitter as amperometric probes, especially to glucose tests, oxygen sensitive tests and oxidase tests verwen- det. In a further embodiment of the invention incorporated in the functional and / or outer layer sensor elements are used as potentiometric biosensors, for example a pH-sensitive electrode as ammonia sensitive electrode as C02-sensitive electrodes.

In another embodiment of the invention, the optical functional layer at least one point an antibody for the selected analyte, while the liquid containing a second antibody, which leads to the reaction with the analyte. The second antibody is coupled to a dye. therefore agglutination of the analyte with egg ner complementary coupling transmitted to the dye second antibody is carried out at the selected location of the functional layer. The reaction with the second antibody may occur before the analyte passes the corresponding site of the functional layer. but the reaction is also possible, alternatively, after the first agglutination reaction has already occurred. In any case, it must be ensured that the two antibodies do not form a cross-reaction. The loading determination and quantitative measurement of the construct formed is then carried out in a conventional manner via optical methods.

The liquid present in the second antibody may be present as examples play in a soluble, non-fixed form at or near the Auftragestelle the functional layer. A plot of the liquid sample causes a solution of the second antibody (or a corresponding Kolloidbil- formation), then the capillary moves in obedience by the function layer and reacts at the site of the first antibody fixed thereto and precipitated or agglutination defined. Alternatively, the second antibody can also be added to the sample and then applied as a layer common solution to the functional. Preferably, the second Antikör- is bound to nanoparticles by, for example, silver, gold iron, laser tex or plastic nanoparticles. In this case, colloids are formed in particular. will shape the execution described here for other detection methods (eg

electrical, electrochemical, magnetic, applied) analog. For better detection are preferably formed stäb- chenförmig metal nanoparticles. In particular embodiments, the compound is a particle growth, prior to the detection by the sensor elements still be excited, for example a growth of silver particles by reaction with silver nitrate in the opposite waiting of chloride or bromide ions.

In a further embodiment of the optical device according to the invention, the type of AlphaLIS assay may be constructed in which an analyte is bound by two different antibodies. Both antibodies are coated to the insolation of a laser beam (for example, of 680 nm wavelength) leads to an emission of singlet oxygen, the mission the immediately adjacent second antibody to a Lichte- at another wavelength (eg 615 nm) stimulates which is detectable. In a preferred further development of this embodiment, the excitation is carried out by the laser beam in the range of visible light, while the light emission in the infrared range, preferably takes place in the near-infrared region. For this purpose, suitable dyes are cya ninfarbstoffe (CY7, CY.l or CY7.5). The emission in the range of the near-infrared range facilitates the measurement of the mission Lichte-. In this embodiment, the additional arrangement of a filter layer is recommended before the sensor elements to prevent the disturbing incident visible light to the management-in sensor.

In a further embodiment of the invention, the radio is formed by carbon -Nanonetze tion layer.

Carbonaceous Nano networks can also be supplemented and / or re- placed by nanotubes and / or other open porous tool materials, membranes or tile, such as those of company-as Milliporre, Pall, General Electric, Sartorius, etc. prepared, the liquid phase must include appropriate capture molecules.

In specific embodiments of the invention it is also possible heranzu- lead Lich light through optical fibers to the functional layer. For example, light from laser diodes (for example, blue light at 405 nm) supplied by corresponding optical fibers to advertising.

For the detection of electromagnetic waves also multi- ti-Mode Readers can be used. Such hybrid readers can implement one or more types of detection such. B.

Fluorescence, UV-VIS absorption, luminescence, Fluoreszenzpola- risation and the like.

For detection of electromagnetic signals and FT-NIR analysis is advantageous. Also, Raman spectroscopy can be applied erfinde- driven, for example in the form of Surface en- hanced Raman Spectroscopy (SERS).

According to the invention, solar cells can be verwen- det as photocells, which are based on the material of copper indium gallium diselenide (CIGS).

Also materials based on aluminum indium gallium nitride (InAlGaN), can mimic in the UV range erfindungsge- Permitted be used. Here, wavelengths from 400 to 305 nm can be generated, for example. In a specific embodiment of the invention, the function layer can be arranged circular, whereby the scavenger molecules sit on concentric circles (see FIG. 6). DER-like embodiments can be read out in the manner of a CD player, in which the functional layer is rotated, and the surface of the functional layer is read by a laser and a correspondingly arranged sensor. Appropriate technologies are known from the manufacture of CD players, so that at this point further explanations are dispensable.

In embodiments, the solution based on the principle of Fluoreszenzmes- are lanthanoid particles or antibodies coupled to Lanthanoids chelating sawn Sonders advantageous. Such compounds can be used as beispiels- prosium on the basis of europium, terbium, samarium or dys-. In these compounds, there is a so-called Stokes -shift, ie, a distance see be- absorption and emission maximum which avoids interference during the measurement.

The detection of the analyte can in principle also be carried out by means of magnetically genetic methods. To express known magnetic proteins can be coupled to an analyte, for example by magnetic and prinzipi- techniques) z. B. are shown by means of SQUIDS) downstream. Alternatively, the magnetically labeled analyte can also be maintained by correspondingly applied (homogeneous) magnetic fields in a certain position. Mag- corresponding items can also be introduced by printing techniques in the carrier and / or the cover layer, for example on the Ba sis of neodymium. According to the invention magnetoresistive voltage sensors can be (magneto resistive current sensor) is used.

In embodiments in which the functional layer is arranged circular, the reading of the magnetic particles of the functional layer can also be effected by an arrangement as known from hard disks or CD-ROM drives: a correspondingly small sensor is a rotating plate passes overall, the panel contains magnetic particles. In DIE sem case, the rotating plate is excluded forms as the functional layer and the captured by the capture molecules magnetic particles are read out by the magnetic sensor.

For this purpose are particularly suitable particles as described by Graham (magnetoresistive-based biosensores and bi- ochips (Trends in Biotechnology vol. 22, no. 9, September 2004, Page 455 et seq.)).

In each case, the incorporated in the cover and / or support layer sensor elements must ER- keep electrical conduction, so that the corresponding measured values ​​can be supplied to a measurement device. Such derivatives are known in principle, loading from the production verschiedenster components, for example from the manufacture of touch-screen displays. Preferably, the derivatives are likewise brought to printing in the single carrier and / or cover layer. The measured values ​​can then be fed into corresponding analog and / or digital instrumentation. In the apparatus of a preferred further development of ER according to the invention and the evaluation of the measurement signals follows on a microchip, which in turn by printing technology or has been introduced into the layer.

The electronic derivation of the measuring signals can even after analog-digital conversion in a PC, a tablet or On the other reindeer carried Head held computer or a smartphone. For this purpose, if necessary, need a USB and / or Bluetooth interface. The transmission of the measured values ​​can also be done using other touch-free techniques (eg. As RFID technology). The necessary for evaluating the measurement data statistical methods, stochastics using the line of algebra by feature editors, pattern recognition and corresponding algorithms for this purpose are known in principle. Color changes the functional layer can be captured using the digital camera of a smartphone or other mobile devices and evaluated.

In more advanced embodiments of the invention the functional layer different capture molecules may be present for example, at several points, so that can be determined from a sample most varied analytes. The sensor elements of the carrier and optionally the covering layer must be adapted to the respective analyte with respect to their position and the type of sensor elements. In a further advanced embodiment of the invention, the function layer can also be sequentially th of different sides are flowed through at various solvents. Such solvents course meets the techniques as they are known in principle from the 2D chromatography. This allows a two-dimensional distribution of various analytes are obtained, which can then be detected by the carrier and if necessary in the outer layer comprising sensor elements.

For this, the sample can be applied also in the middle of the functional layer, so that the liquid runs in the course radialis ler direction around the central point. As already explained, one passage of the liquid in the course can be influenced radialis ler direction characterized in that the functional layer is set in rotation. The centrifugal occurring galkräfte then push the liquid to the outside. It will be appreciated by those skilled saying that such an embodiment a circular embodiment of the functional layer is preferred. The corresponding capture molecules may be introduced tion layer in the radio (see figures) at here- in one or more concentric circles.

In any case, make sure that the top layer does not cover the entire functional layer. Rather, the functional layer must be free at least one location, so that the sample can be applied. For this purpose, it is beispielswei- se possible, that the cover layer covers only a part of the functional layer and the sample is applied in the remaining free portion of the functional layer. Alternatively, it is possible that the cover layer has at one or more locations openings which serve to sample application. Only in extreme cases, it will be possible that the wetting of the functional layer is effected solely by the edges of the device according to the invention, wherein the liquid "therethrough pulls" only by capillary action from the edge of the functional layer through the entire layer.

In a further advanced embodiment of the inventions dung the analyte or the analyte may also be moved by applying an electric voltage through the functional layer, such as rese, for example, along the lines of Gelelektropho-. In a specific form of the analysis method using the inventive apparatus of the formed Fängermole- kül-analyte complex can be removed mechanically or physico-chemically (eg., An antibody-antigen or agglutinate -Präzipitat). For this, the functional layer may, for example, mechanically removed or ated by appropriate elution solvent. The isolated complex is then to further analysis, such as mass spectroscopy be supplied (see, for example Himmelsbach et al., Nachrichten aus der Chemie, 63, Febr. 2015, p.144-146).

Alternatively, the model of the Western blots an over-transmission of the analyte or the complex formed by the analyte on a second layer, z. B. a gel possible. Of course, this embodiment of the invention assumes that the functional layer does not contain any top layer, or that the overcoat layer was removed by mechanical means in a preceding step. The device provides a platform in order to optimize the cost / benefit ratio in medical diagnostics, food analysis, forensics and basic research. The device allows, among other quantitative determination of in plasma, blood or other body perflüssigkeiten existing biomarkers (proteins, peptides, nucleic acids, etc.) for the diagnosis of cancer. proven particularly useful in the study of pre-cancerous lesions, as examples play as occur in the case of colon cancer has. For this fecal biomarkers can be determined. By means of the device erfindungsgemä- SEN multiple biomarkers are measured simultaneously, which greatly increases the quality analysis. It was particularly found that a panel of the following biomarkers that can be determined in the stool as well as in the blood or plasma by means of the inventive device so-good:

• 14-3-3 protein • Serotonin

• Insulin like growth factor binding protein 2 (IGFBP2)

• stubborn -3 (DKK3)

• pyruvate kinase Μ2 (PK-M2)

· Gamma enolase

The presence of one or more of these biomarkers in blood, plasma or stool may be a sign of colon cancer or its precursor.

The inventive apparatus also offers a platform for the analysis of larger cell units, for example, cell compartments or other substructures of the cell from whole cells or even superstructures. Also includes dimers, trimers, tetramers or polymers of peptides, proteins, additions tikörpern, carbohydrates, lipids and other biomolecules can be supplied to a simplified analysis. Further with the apparatus according to the invention and the beschriebe- here NEN method analyzable supramolecular structures comprehensive sen protein Nükleinsäure complexes, protein-lipid complexes,

Protein Glycocylphosphatidyl-inositoi complexes Sialyglycocon- jugate, ubiquitin-proteasome system, CRL1-CSN supercomplex, etc., only for expenditures were extremely dig detectable by the prior art. A particular advantage of the invention that denaturation of the analytes by chemicals (such as detergents) are largely avoided.

In particular embodiments of the invention, the funct can onsschicht at several locations capture molecules for selected analytes have. So may be occupied with Fängermolekü len for example, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1,024 or 2,048 digits. Execution have been proven beyond approximately form 384 or 1,536 points. Depending on the application purpose can dung equal to the capture molecules (ie, for the same analyte) or different (for. Example, for different analytes) to be configured. The different points can be arranged, for example, a checkerboard pattern, eg. B. 32 measuring points in a 8x8 matrix so that sufficient spaces between the measurement points are present, that the measuring results are not falsified. Analog also an embodiment on a round function layer is conceivable, wherein the measuring points are arranged concentrically around the center point. An alternative embodiment con- sists in the fact the capture molecules in strip form to arrange ° angle to the running direction in the 90th Respective embodiments are shown in Figures 4 - illustrated. 8 Other aspects of the invention,

Basic structure:

2 plates (films or glass) between them there is a thin functional layer. On the inside of the plates befin- to the electrodes. This feature film is According to the invention designed as an immune function layer. The Immunfunk- ti-thin film provides a platform for the purpose of Bioana- is lytik.

The structure of this arrangement is used to examine the measurement of the local electrical properties of these functional layer. The two (located on both sides) electrodes constitute an electric plate capacitor. The plates of the plate capacitor can in this case assume any desired shape, for example square, rectangular, hexagonal or round. In some embodiments of the inventions dung is comb-shaped plates have been found. Here kön- nen at one or more edges of a rectangle like a comb disposed surface enhancements realized be (Figure 15). A variety of such electrodes can be locked to each other limit to exploit the surface particularly well and to possible to realize lichst small electrode spacings (Figure 15, bottom). It goes without saying that such "crossed electrode combs" can be implemented in only one layer (for example, the cover or the support layer. Alterna- tively to such "crossed electrode combs" in only two layers (the top and the carrier layer ) be realized opposite each other.

These two plates can be preferred by semiconductor terelemente (photo resistors, photo diodes, transistors, field effect transistors, etc.), replace. The arrangement and these preferred semiconductor devices are used for measuring / investi- gation of the optical, biophysical, biochemical properties of the egg analyte agglutinated in immune function layer and thus locally concentrated (antigens). The inventive arrangement allows, the selected selected by capture molecules and locally concentrated in the layer functional analytes (antigens) access through various analyzes and tests methods. The located on the nanoparticles capture molecules mobile 1 serve the selection, the stationary catcher molecules serve 2. the local concentration of the analytes (antigens) in the 3D structure of the functional layer and thus a subsequent forming investigation by various physical methods are accessible.

Passes through a fluid / suspension, the immune function according to the invention thin-film, thus causing the local arrangement of the 'yields germoleküle, "traps" in which the analyte / antigens is glutinieren Ag and are thus concentrated.

By the inventive arrangement of the capture molecules on the anchored "fluid" phase on the one hand and the local catcher molecules in the solid phase of the functional layer.

The opposite in immune function layer

Electrodes constitute an electrical capacitor. This capacitor can be by means of electromagnetic analytical methods which are used in analytical chemistry, electrically read out.

Methods: eg voltammetry, voltammetry, chronoamperometry, potentiometry, Coulometry, polarography, amperometry, potentiometric titration, cyclic voltammetry, Differenti- al pulse voltammetry.

Voltammetry: The term voltammetry (English: Controlled- current potentiometry titration) is an electrochemical method of analysis. It must not be confused with voltammetry.

Voltammetry is a collective term for various electro-analytical methods for the qualitative and quantitative analysis of a sample, with which one can run determine the chemical composition of material mixtures based on the voltage-dependent electrical supply source, as well as for the elucidation of reaction mechanisms. According to the state of measurement technology, these electrical properties can measure (eg by special measuring methods by selected sensors, active principles and their practical implementation see, also, for example by automated measurement systems). The measurement of the electrical properties should preferably be carried out by so-called application specific integrated circuits. The measurement can be made in certain embodiments, inexpensive and suitable for mass production. A so-called ASIC (English: application specific integrated cireuit).. Even "custom chip" is an electronic circuit that has been implemented as integrated switching circle this purpose particularly suitable, but other microchips are to be applied once developed ASIC chip can on the market as application-specific standard product apply (ASSP). For example, the company Elmos a company among many, which is familiar with so-called intensifying ASIC. the corresponding evaluation algorithms could for example be taken over by Siemens.

For example, dispensing of antibody solutions in the XYZ dimensions of the functional layer could be done for example by equipment from M2 automation package.

In the case of sensors lot of different sensor circuits of the prior art are known. For example, CU converter ASIC etc.

Service provider for ASIC design for development / production measurement technology for sensors and customizable individual complete solution providers are known For example, the company GEMAC - Corporation for Microelectronics application Chemnitz mbH. When using microsensors all optimized micro-sensor technologies and so-called MEMS sensor Generations of the 1st, 2nd, 3rd and 4th generation available in the prior art are to be used for the application.

It should be used according to the state of the art Nano-Electronics'. Under Nano-Electronics' refers to use of nano tech- nology on Electronic Components, Including transistors so small did interatomic, interactions and quantum mechanical properties need can be of studied.

Furthermore, nano-photonics is to be applied. Ie, the behavior of light on the nanometer scale. The invention utilizes the specific properties of the immune function layer. This is based on nano-Mechanics. Under Nano-Mechanics is defined as a field of nano-science, which examines the elastic, thermal or kinetic properties of a physical system on the nanometer scale. According to the invention is the use of "Nanomaterials", fullerene renes and Carbon Nanoparticle forms and colloid.

The invention is inter alia to the study of nanomaterials. This means that web Field studies materials with morphological features on the nanoscale, love especially thosethat have special properties stemming from Their nanoscale dimensions.

In the invention, nanoparticles and their colloids should be used. Colloidal gold nano-composites, nano fiber nanobugs, nanobeads, Nanomesh, tubequantumdots etc .. The device or immune function layer serves the study and measurement of, for example, DNA-protein, DNA-RNA, DNA, protein-lipid, DNA-protein sugar. In the particular embodiment of the devices a so-called quantum point contact can come to fruition.

The immune function layer can be examined later by certain techniques such as microscopy and special techniques, atomic force microscopy, scanning panel microscopy, scanning probe microscopy, etc..

The device according to the invention is used for analytical measurement technology and as a tool for characterization of biological materials, for example by means of High electric Impedanzspektrosko- pie (EIS), such as in the frequency range of several megahertz to 100 gigahertz. The measurement of the bioimpedance is usable with an exact knowledge of the sensor and measuring properties of a performance-powerful tool for the rapid and minimally or non-invasive characterization of biological objects. The selection of the frequency range, time resolution (measuring speed) and the electrode assemblies is determinative of the interpretability of the measured properties. The competence of the skilled person lies in the choice, customization and characterization of the technical measurement environment (hardware and software) to the problem to be solved biomedical measurement tasks be in the different fields of application.

are to be examined biological objects

- tissue extract

- cell extract

- extracts from single cells

- Adherent cells and suspended - cell population

- biomolecules, etc.

Many of the studies, structural analysis, Biomorphose, inter- action of biological objects, molecular biological objects with different physicochemical structures.

To apply macro- and micro-electrodes can be used with the most varied geometries.

According to the prior art electrodes> and up to 100 microns can be produced today.

Using established thiol functionalization and the Real estate - tion of appropriate receptors and amperometrisehe impedimetric biosensors are used. The Elektrodenherstel- development can be done by plating on different substrates. According to the prior art can be commercially available impedance measurement systems, for example, are tuned to the requirements of the communications engineering or materials science, do not use for measuring the electrochemical properties of biomaterials.

For electrical readout of immune function layer is preferably in place of the capacitor electrodes so-called thin-film transistors (thin-film-transistor TFT) can be used to advertising, which can be controlled individually. This Dünnschichttran- sistoren TFT can be preferred in the preparation of the import munfunktionsschicht in the functional layer, "embed" in which one of the casting process, ie denHerstellprozess the functional layer, such as nitrocellulose or polyacetate, executes directly on a thin film in which, for example, this TFT are embedded.

Since the electronic component TFT on the one hand and the TFT on the other side of the functional layer befin- det, this arrangement provides a cavity in the functional layer (condenser). Since in this arrangement, each cavity two active components ( electrode), and a fluid to move by capillary action through this layer, this immune function layer may also be referred to as an active matrix. By replacing the above-mentioned electrical components TFT by optical components, the cavities can be optically measured and examined. Due to the fact that with the help of the capacitor

can be (electrode or with the aid of the two opposing TFT) larger loads for an extended period of time "stored" and measured, read the average laser rises dung density of each cavity and thus the contrast in electrical and / or optical, "triggering" the functional layer.

The arrangement and the electrical discharge that is, "conductive paths" of the sensors is preferably designed in instructions to the model in nature see retina receptor arrangement of the olfactory mucosa.

This so-called lateral inhibition produced by the inter- connection of PR to the bipolar cell and serves contrast enhancement in the receptive field center. In the Exzitationsschicht LEDs or an LED panels are to be installed according to the invention is preferred. The functional layer contains microcavities in which agglutination occurs. The provided with, for example, fluorescent dyes, colored piston loidalen gold particles can be in the "agglutination clumps" nen for example, by electromagnetic waves DIE the Exitation stimulate from the photodiodes to emit radiation. The electromagnetic radiation at the emission inventions should dung according for example, by electronic light-sensitive semi-conductor devices ( "receptors") by, for example photoresistors, photodiodes, Fotomultiplierdioden etc. In these electric devices, the electric radiation causes an "electrical current flow," the comparable strengthens with corresponding prior art known metrology and can be measured. This emission radiation from the underlying Detek- tion layer (sensor layer), in which sensors are of different type detected. layer in the detection, sensor layer are preferably field effect transistors with insulated gel IGFET or uninsulated gel NIGFET paten come tgemäß used.

The following field effect transistors with insulated gate gel should be covered in the present invention: the functional layer according to the invention consists of an open-pored gene material with pore sizes of 0.1 micron to 8 μη and

Molecular Weight Cut Offs 300000-1000 Dalton. The function of membrane serves as the particle analysis, that the analysis of the clot, which were by the agglutination reaction are corresponds. The functional layer can be electrically or optically inspect and measure as illustrated.

A whole range of spectroscopic and microscopic techniques are possible, eg epifluorescence microscopy.

The functional membrane according to the invention can acetate from nitrocellulose, polyacetate, cellulose acetate, cellulose nitrate, Cellulosetri-, lyethersulfon Hydro cellulose, nylon, polycarbonate, polyester, polyvinyl, for example, consist of polytetrafluoroethylene or of regenerated cellulose, with pore sizes of 0.1, 0.2, 0, 4, 0.45, 0.65, 0.8, 1.2, 3 microns, 5 microns, with 8 micron molecular weight cut-offs of 30,000, 50,000, 100,000, 1000 daltons, 5000 daltons, 10,000 daltons, 20,000 Dalton, 30,000 Dalton. Proteins, for example, can be coupled by means of succinimide covalently to the 3D structure of the glass fiber membrane, the membrane can function hen also from glass fiber membranes consist, preferably partially changed by appropriate silane chemistry so that covalent. An alternative embodiment is the use of graphene as a functional layer.

According to the invention can also glass / microfiber, made with synthetic binders and tables serve the strength of the membrane can be used. The functional membrane is mechanically and chemically stable and either hydrophobic or hy- rophil. hydrophilic membranes are preferred. According to the invention include paper as a function of membrane are also conceivable, in particular tissue paper. Blotting, phiepapiere chroma-, filter cardboard, glass microfiber filters, Keimtestpa- pier, Kieselgurfilterpapier, line cleaning paper, surface chenschutzpapier, parchment weighing paper, phase separation paper, etc.

The function layer is preferably made of microporous membrane material. When Sephadex is polymeric sugars, which are three-dimensionally cross-linked. The Mate rial has different porosity, between 20 and 30 microns. Pore ​​size eg Sephadex G200, 1000 to 200,000 Molecular- Weight. Manufacturers of these are Sartorius, as well as GE (Gen- eral Electric) Healthcare Life Sciences with different brands, Emerson, Sephacryl, Sephadex, etc.

A particular embodiment is the use of an area-speaking form of "Capto", a material for the purification of large biomolecules. In the functional layer different molecules can with- by agglutination in the microcavities of the functional layer, for example, means of antibodies and / or lectins, or other scavenger be brought "lump-off" strukturenzum. The agglutinated material may, for example from proteins, nucleic acids, DNA, RNA, siRNA, micro-RNA, iRNA, sRNA, shRNA, qPCR, cDNA, lipids and carbohydrates and their aggregates in supramolecular architecture which previously nichtodr the analysis only was difficult to access exist.

Also, the agglutination of different cell types, organelles, microorganisms and subcellular structures possible. The functional layer may rial consist of different porous Mate, which is used for the chromatography, for example, Superdex, Superose (gecrosslinked with agarose) materials of the Whatman, Merck / Millipore, Sartorius, Pall and other Companies, which for lateral flow and flow through and immunoassays are used, such as absorbent pads, bloodseparator, glass fiber, blood, components for dip-sticks, membranes for immunoassay, conjugaterelease, glass fiber pads with different absorption propertieskonnen inventiveness come according to the application.

A special inventive embodiment is the appropriation of PCR, in particular Real -Time PCR method.

According to the invention is also the use of super-paramagnet- regard nanoparticles whose surface having bio-affinity ligand which is functionalized.

A particular inventive embodiment is the appropriation of a magnetic detector. This leads to the development of a mobile measuring device suitable> marker-based multiplex assays for rapid, highly sensitive evaluation processing of magnetic-.

Various detection methods such as Magneto-Resistive (GMR, AMR, TMR) and permitive sensors can be adapted to the test format and to the environment condition. Also could so-called Maxwell "bridges and / or resonance -Coils erfindungsge- Mäss apply.

A particular embodiment is to provide an array of electronic components iAnordnung onsschicht in the functional. The array arrangement of the individual sensors in the transmitter and receiver layer to len be arranged in the olfactory epithelium of mammals according to the arrangement of Riechzel-.

The sensors of the array layer and the electrical signals are recorded electronically as a pattern, that is, the activities or the signals of the individual sensor cells are compared by egg nes algorithm so that an activity pattern is corresponds, which for the presence of many analytes simultaneously existing is typical. A similar arrangement of the sensor cells are also found in the retina. The principle "wiring" of the individual sensor cells, both in the olfactory system of the retina as well as in the optical system (see bionics).

In order to optimize the spatial vicinity of the capture structure with the sensors, the antibodies in a separate production step, for example, be applied by Nanodispensierung directly to as "base portion" of an organic field effect transistor in a particular embodiment, for example, before the contactless is made with the open-pored functional layer . Absorptive or covalent binding of capture molecules to the specially designed "base" of the transistor in this case are optimally desirable. which is used at the base of the transistor, the material may, for example, graphs (?) or other material substances.

Biotin - - also known, for example, streptavidin, protein A are used for application.

Also, the application of graphene, carbon, proteins, for example protein-iA, biotin, can be carried out in the gas phase according to the prior art and is desirable. Principle of the transducer array in the 3D structure or functional layer

General construction and claims: The Exitation immune function layer is effected by means of electromagnetic waves waves using LEDs, preferably printed on films LEDs.

The radiation penetrates into the 3D structure of the functional layer and excites in the microcavities agglutinated particle kel, nanoparticles, colloids, which are provided with capture molecules and / or are preferably labeled with dyes for the emission of electro-magnetic radiation to. The emitt- ierte radiation is converted by a sensor, which functions as a transducer to electrical signals.

The signals are let on micro- or nanoba- lized measuring technology and by means of apps in a smartphone, Tab means of modern, PC, soft as a computer and serve as display excluded, and evaluates the corresponding measured values ​​or the "result" visually on the display shown.

The Exitationsschicht (sensor layer) preferably contains flatbed LEDs that are preferably applied to adhesive-of foils, and are joined by lamination for example, in the preparation of the functional layer.

contains the detection layer (sensor layer) Favor z printed on adhesive film. B. flatbed photoresistors or flatbed type field effect transistors (organic FETs).

The flatbed LEDs can be also provided as thin film Panels excluded. The detection layer can also be designed as thin-film receptor layer panels.

The device of the invention serves bioanalytics and di- agnostik.

The functional layer can for example be found -Article use as disposable.

One embodiment of the functional layer can identify different running routes. This is inventively achieved by z. Bd., The application of liquid wax. Thereby advertising the individual, separate running trails in the Immunfunktions- produced layer. This running tracks are electrically, mechanically and optically separated from each other.

A claim of the functional layer is:

This multiple functional layer should be designed such that, for example, various liquid samples simultaneously at the start line, for example by means of a multichannel pipette can be (päd) listed wear.

This multiple functional layer can be inventively executed "simultaneously" in a laboratory machine and measured.

As electronic components should preferably thin-film LEDs preferably (printed) thin film field effect transistors in the Exitationsschicht or Detekti- onsschicht be used.

Others

The present invention may be coupled to a mass spectrometer by means of a TLC-MS interface. Thus, the immunoprecipitated in the functional layer supramolecular structures by means of liquid can be extracted and elute and infundie- ren for further characterization directly or after LC in the ion source of a mass spectrometer. The use of MALDI-MS or HPLC-MS techniques is also possible.

Another device according to the invention and with the described here method analyzable supramolecular structures include protein-nucleic acid - complexes, protein-lipid

Complexes, protein complexes Glycocylphosphatidylinositol, Sia lyglycoconjugate, ubiquitin-proteasome system CRL1-CSN super complex, etc., which processing means of the inventive Vorrich- can be investigated particularly gentle.

SCHEME 1:

Figure imgf000040_0001

Electromagnetic Wave (Exitation) generated Electromagnetic Wave Transducer = electronic components in the detection layer Electronic Processors tablet PC and its different operating systems are in the smartphone.

Various apps for the various operating systems provide algorithms ready for data processing.

Smartphone, tablet, PC also serve as transmitters and provide access to the "cloud" to the Internet.

production method

For manufacturing the device according to the invention in principle known techniques from other technologies herange- be covered, such as the aforementioned production of touch screens. The preparation of the functional layer according to the invention is carried out either by classical Herstellungsverfah- ren, which are known ( "casting of TLC plates"), or by printing techniques of thin layer chromatography. Here, the "molding" of the functional layer (which according to the invention as ) may be formed directly on gel also sorelemente transmitter can take place. To a commercially available CMOS o- the CCD chip, as is known from digital cameras may un- a functional layer to be applied indirectly. The same applies for light emitting Elemete such as LEDs or semiconductor lasers. The functional layer of erfindungsgemä- SEN device can also be produced by 3D printing techniques which nowadays works just as fast and reliable as injection molding. In particular, the application of purchase tikörpern in the functional layer can be done by printing techniques. A particularly preferred production method for the inventive devices is the lamination technique, in which a functional layer on one or both sides th one or two layers, which already contain the sensor elements may be laminated, for example by the application of moderate pressures and / or temperatures ,

The printing techniques used in the invention for producing African drucktech- the sensor elements are known from the prior art and need at this point no further explanation.

Other applications of the invention

One particular application of the invention is in conjunction with histology. Here tissue samples are examined histological. Mention should be made here in particular the early diagnosis of tumors, the detection of metabolic diseases, parasitic, bacterial, inflammatory diseases and similar royal. To the classic methods of investigation in histodiag- nostischen laboratory includes the fixation of tissue "Einblo- CKEN" in paraffin, slicing the paraffin block and microscopic examination of the layer. By means of the inventive apparatus body using appropriate anti-, it is possible that different cells and foam structures physicochemically to bind and to make them visible. For this purpose can be used, which timentes is bound at the site of a protein, or a cell or a Zellkompar- and the detection of which allows for example a fluorescent dye. The diagnostic nostik verschiedenster types of tissue, such as connective and supporting tissue, adipose tissue, bone tissue, cartilage tissue, epithelial tissue, muscle tissue and nerve tissue is possible.

The presence in the frame of the device functional layer can be measured by Mikrovolumenspektrophotometrie, such as by the DeNovix system from Zeiss. This system enables light measurement in the range of 190nm to 840nm and allows measurement of the 3D imaging, clinical routine, correlative microscopy, the deep-tissue imaging, fluorescence - imaging, image handling and analysis, Life cell-Imaging, Spekt- ro imaging, ultrastructural studies Röntgenmikrosko- pie, for example, biomedical engineering, biology the cell, ology developmental biology, neuroscience, pathology, pharmacology and toxicology, the Strukturbi- and / or zoology and plant sciences. The invention is characterized in any case a vote of all involved agents, so no hook effect occurs.

Example 1:

An example of apparatus according to the invention, the principle of the capacitance measurement is used, is described successor neighborhood. The functional layer is applied to a carrier layer thereby Trä- and covered with a cover layer. Both the carrier layer, as well as the cover layer include electrically conductive sensor elements which have been applied by printing on the layers and opposed to each other. In each case two opposing sensor elements thus forming a capacitor in analogy to the representation in the fi gure 1 are located between a pair of plates antibody as capture molecules. The capture molecules are captured microparticles able gold. By enriching the appropriate Gold microparticles between the capacitor plates, the capacitance of the capacitor which can be measured by the below-described arrangement changes. The following diagram shows the comparative measurement of two such aufgebau- ter capacitors (Cl and C2) by means of a processor of the type ATMega88 which show shows a display in the form of a Leuchtdiodenan-.

Figure imgf000043_0001

Block diagram of the controller:

Figure imgf000044_0001

Figure imgf000045_0001

The clock generator at the UART are the uniform clock frequency between the transmitter (controller) and the recipient before (necessary for the external data transfer). In the source code must be a uniform baud rate can be set to the SYS "synchronize" systems. The transmitted data stream is composed of a start bit, the stop bit following the data bits and a parity together with.

The determination of the baud rate can be made depending on the product zessortaktfrequenz; UBRR_Val ((+ F_CPU BAUD * 8) / (BAUD * 16) -1). In the presently described use Description wur- de a variable in the "unsigned long" selected.

Figure imgf000046_0001

For the use of the standard baud setting with 8 data bits, the Baudregister UBRROH is first with "left shift" described Ben. The "Casten" done with "unsignchar". Through the initiative taping with UBRR_VAL (simple cast) are automatically the upper eight bits cut off.

Figure imgf000047_0001

To enable the required ports with UCSROB RXENO and TXENO as a receiver and transmitter, the respective registers are set to "1". The protocol of the UART is set as follows :;

• Bit O TXB8n: Transmit Data Bit 8

• Bit 1 RXB8n: Receive data bit 8

• Bit 2 UCSZn2: size character

• Bit 3 TxENn: transmitter enable

• Bit 4 RXENn: Receiver enable

• Bit 5 UDRIEn: USART data register emty interrupt enable

• Bit 6 TXCIEn: TX complete interrupt enable

• Bit 7 RXCIEn: RX Complete interrupt enable

Figure imgf000047_0002
Figure imgf000048_0001
Figure imgf000049_0001

Bilder5-10 excerpts from the manufacturer's data sheet for the gege- enclosed application

With just explained setting the UART interfaces ler is ready to send. To avoid collision of data in the transmit buffer it must be tested for occupancy. An examination of the UCSROA register with regard to UDRE0 offers some insight whether the buffer is empty. An empty tab gives the state "1". In a variable to send the value is output via the interface.

The items to be writing data into an array. . In the given case, a string should be "char" sufficient with about 10 elements of the type for a port index starts logically fashioned at 0 and ends at 9. Is the capacity measurement, for example 3.94 of this value is arranged as follows:

[3] [.] [9] [4] [SPC] [u] [F] [CR] [LF]

At the time of measurement start, a variable (C- time here) = initialized 0th A port (here PB = declared as an output and a LOW level connected to the anliegen- de capacity at the beginning of the measurement cycle to discharge.

After the discharge time (about 7 min), the corresponding port is switched to HIGH from LOW and an internal pull-up activated. From the switch, the capacitor is charged so long until the controller detects a high level (a loop). This level Conference voltage according to the current reform. The reference voltage is currently at 3.5 V. However, this voltage can strongly troller of the selected Con and the power supply depend (Knopfbatte- rie / AA battery / USB port / power supply). No matter what chip is selected voltage supply, it is up to the recognition of a high level for a certain time lapse. This time must be measured in a do-loop and forms a approxi- hernd a constant charging current to the capacitance ratio. If a long charging time is measured, the capacitance is very large. In order to convert the measured time in a capacity must be a live and current-dependent factor determines the advertising. This is in the already well-known example 0.000622. This changes with another reference voltage or a highly different capacity.

The system is to display the capacitance of the capacitor to be able to use simple visual indicators. For this one is inserted (yet to be determined) reference value one behind in the system. This reference value is then compared to that in the variable (in this case "c"). If the two values ​​only mi- nimal from each other, a green LED lights. Higher deviations, the yellow and the red LED is energized.

Example 2:

By using two antibodies, one antibody capable of binding specifically to the antigen, the second antibody capable of binding for a particular protein, on the one hand and overall nanoparticles linked to a piece of DNA

After the agglutination reaction in the functional layer, the agglutinate can be spatially very greatly increase by greatly extends the short retained on the antibodies pieces of DNA by a DNA polymerase reaction. The obtained DNA agglutination 3D structure is spatially large (Figure 26), such that the electrodes (teeth) of the two combs, which act as electrodes (see Figure 15, bottom), brought into electrical contact. If the two comb-like electrodes with- means of the 3D structure bridges, causes this a very strong AEN alteration of electrical resistance. The change in electrical resistance can be measured very sensitive means of an electronic measurement setup. The change in resistance is used for the quantification for example, of the desired antigen (analyte).

Further prior art

Another analytical method based on electrochemical processes described by Nemiroski et al. , PNAS, 111 (2014) 11984-11989. Further prior art is known from the documents DE 2020130007536 Ul and PCT / US 2004/021187. A system for the determination of malaria was described by Qmed (www. Qmed. Com).

A portable spectrophotometer for different product teinbestimmungen is described at www. colorimetrix. com.

Another smartphone-based analysis tool for Colo- rimetrische tests described by Yetişen et al. , Sensors and Actuators B: chemical, 196 (2014) 158-160.

Further prior art is formed by various systems in the field of microfluidics. Here, micro channels can be in a wide variety of materials, such as for example, formed of glass, plastic and silicon moving liquids through channels DIE se. The movement can be pumped through appropriate micro, but powerful by application of centrifugal.

Other devices of the prior art, the term "pocket-lab" or "lab-on-a-chip" known. Here, using microfluidic SYS temen an analysis of very small quantities of material on a chip performed. The other systems are offered under the name Micrux (www.micruxfluidic. Com).

A review on devices that operate in the field of microfluidics is found in Fiorini, biotechnical ques 38 (2005) 429-446. In the context of the BMBF Proj ect "BeadPlus" different bioanalytical methods have been studied, for example, based on coated magnetic nanoparticles and GMR sensors. This should be detected up to 32 different marked magnetically overall biomolecules. Medical applications were not realized this.

Printed circuit traces, sensors and actuators of Nanomaterials are known from the project PRINTS Munich Technical University

(Www.lme.ei.tum.de) Heinz Nixdorf Chair of Medical Electronics, Professor Dr. Bernhard Wolf. See also

DE.Wikipedia.org/wiki/gedruckte_elektronik. Printed Feldef- be fekttransistoren among others described by Dimos Poulikakos, ETH Zurich. Various conductive inks are com- mercially available, for example. B. from BASF.

Further prior art is formed by the document WO 2012/007537 Al and WO 2010/040851 A2. Furthermore, by WO

2009/040782 A2 and WO 2009/021964 A2.

Furthermore, by WO 2011/053147 Al and WO 2010/090514 Al, WO 2009/040782, WO 2009/021964, WO 2009/090267,

WO 2010/040851 and WO 2002/084276 as well as WO 2014/122094, US20130052748A1, GB 2474888A, WO 2002/084276, EP 2352590 Bl, T2 DE69937631, DE 102008/025680 Alsowie EP 1614725 Al,

WO2013 / 130995Al, US8354270B2, EP1992951B1, EP1133694B1,

US5401667A, CN101509924B, WO2014027225A1, WO2013 / 083686A1, WO2014 / 056987A1, WO2008 / 000461A1, DE3121523C2, EP1336089B1, US2012 / 0267240A1.

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Friedrich Lottspeich, Joachim W. Engels, Bioanalysis, 3. up would be in 2012, Springer-Verlag Berlin Heidelberg

JS Levine, H.-U. Klör, G. Oehler, making processes Gastroenterological decision, in 1988, Schattauer AM Gressner, T. Arndt, Encyclopedia of Medical Laboratori- umsdiagnostik, Volume 1, first edition 2007, Springer Medizin Verlag Heidelberg

Lela Buckingham, Molecular Diagnostics, Fundamentals, Methods and Clinical Applications, Second Edition, 2012, FA Davis Company Ekbert Hering, Rolf Martin, Martin Stohrer, Physics for engineers, 9th edition 2004, Springer-Verlag Berlin Heidelberg

W. Funk, V. Dammann, G. Donnevert, Quality Assurance in Analytical Chemistry, 1992, VCH Verlagsgesellschaft mbH, Weinheim

Gerald Karp, Cell and Molecular Biology, Third Edition 2002, John Wiley & Sons, Inc. Donald J. Voet, Judith G. Voet, Charlotte W. Pratt, Principles of Biochemistry, Third Edition 2008, John Wiley & Sons, Inc.

Georg Löffler, Petro E. Petrides, Biochemistry and Pathobioche- mie, 7th edition 2003, Springer-Verlag Berlin Heidelberg

Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter, Molecular Biology of the Cell, 5th edition 2011, WILEY-VCH Verlag GmbH & Co. KGaA Weinheim

Albert L. Lehninger, David L. Nelson, Michael M. Cox, Principles of Biochemistry, Second Edition, 1993, Worth Publishers New York David L. Nelson, Michael M. Cox, Principles of Biochemistry, Fourth Edition 2005, WH Freeman and Company New York

Lothar Thomas, laboratory and diagnostics, 8th edition (Volume 1) 2012 TH Books Publisher mbH Frankfurt

None of the cited references nor any combination of publications adopting the present invention anticipated ode: puts them even close.

Explanation of the Figures gur 1 schematically shows the structure of an embodiment ner device according to the invention in cross-section:

1: Auftragestelle for the sample

(If necessary, for filtering and chromatographic

Pre-separation prepared)

2: Derivation of the electrode 1

3: electrode 1

4: outer layer with the printing process applied

Electrodes and derivatives

5: Derivation of the electrode 2

6: electrode 2

7: Derivation of the electrode 3

8: electrode 3

9: Absorbent material (Wiek)

10 electrode 3 λ

11: derivation of the electrode 3 λ

12 electrode 2 Λ

13: derivation of the electrode 2 y

14: support layer with printing technology applied

Electrodes and derivatives

15 electrode 1 Λ

16: derivation of electrode 1 λ

17: Function layer containing microcavities,

this, in turn, at least one location containing capture molecules

The electrode pairs 1/1 \ 2/2 'and 3/3 λ each form (plate-TEN) of capacitors, whose capacitance (and others) will depend on the DIE lektrischen properties of the intermediate space. Change in the dielectric properties of the space eg as a result of trapping of antibody-coated gold particles lead to the capacitance change (eg in the form of Permitti- tivity) is measured and may be stored time-dependent.

Figure 2 shows schematically the construction of another exemplary form of an inventive apparatus in cross section:

1: Auftragestelle for the sample

(If necessary, for filtering and chromatographic

Pre-separation prepared)

2: electrical connection of LED 1 (schematic)

3: LED 1

4: outer layer with the printing process applied

LEDs and derivatives

5: electric connection of LED 2 (schematic)

6: LED 2

7: electrical lead to LED 3 (schematically)

8: LED 3

9: An absorbent article (Wiek)

10 Photo sensor 3, such as CCD or CMOS chip

11: electric leakage of photosensor 3

(Schematically)

12 photo sensor 2, for example CCD CMOS chip

13: electric leakage of photosensor 2

(Schematically)

1: support layer with the printing process applied electrodes and derivatives

15 photo sensor 1, for example CCD or CMOS chip 1

16: electric leakage of photosensor 1

(Schematically)

17: Function layer containing microcavities,

this in turn at least one location containing capture molecules LEDs 1, 2 and 3 each form with the opposed photosensors 1, 2 and 3, light barriers. The light slot through the functional layer depends on the optical properties of the space depends. Change in light absorption of the gap, for example due to the catching of antikör- perbeschichteten gold particles result in the location-dependent change in light absorption, the measured time-dependent and can be gespei- chert. The evaluation can be performed wavelength-dependent.

Figure 3 shows schematically the structure of an expanded exemplary form of an inventive apparatus. The device already described in Figure 2 is two layers to filter (18 and 19) added. This embodiment enables light to filter out light having certain wavelengths. This embodiment is particularly suitable for 'yields germoleküle that with light of a particular wavelength to emit light of a different wavelength of radiation, as they are described above. terschichten by the built-FIL can be so sure that the part of the

Transmitter only light is emitted at the transmission wavelength and that passes part of the receiver only light having the Empfangswellen- length to the receiving sensor. In this way, faults are largely suppressed, resulting in correspondingly präzise- ren measurement results.

Figure 4 shows schematically an embodiment of the invention with five different measuring points (here as black bars shown). At each of these measuring points Fängermolekü- le can be positioned for selected analytes. Pairing WAIVED points, the respective sensor elements will be included in the support and / or top layer. In this way, five different analytes can be analyzed in one measurement cycle, for example.

Figure 5 illustrates a checkerboard arrangement of Fängermole- cules and sensor elements. In this way, a lot of different analytes can be measured.

Figure 6 shows an embodiment of the present invention, in which the Auftragestelle is in the middle and the running direction of the applied solution concentrically, i.e. equally in all directions in the plane, extends.

Figure 7 shows schematically a rating based on the Figure 6 exemplary form of the invention in which multiple rings with measuring points are positioned concentrically around the Auftragestelle around.

Figure 8 shows schematically the arrangement of ten measurement points within an inventive device, the measuring points facing in all directions of the plane distances from one another up. Such an arrangement may be necessary in order to prevent interference of the measurements. Such disorders such can. B. occur characterized in that - in the case of application of optical analysis - light emissions, for example, caused by scattering len, on adjacent measurement sensors wrong. The same applies for electrical measurement methods, for example, where the capacitance measurement of a capacitor unit formed may be disturbed measuring stations by electrical influences of neighboring. Another source of interference may be onsschicht in the production of functional, the application of capture molecules. This can lead (depending on the application) to a certain spreading of the applied solution. In this case, immediately adjacent plots could even lead to mixing effects in the border areas, which is effectively prevented by the arrangement shown in Figure 8.

Figure 9 again shows the basic functional structure in the side view. The functional layer is located in the center of the device, wherein a left and right Auftragestelle is gebil- det a "Wiek", which receives incoming fluid, and in this way ensures that the capillary flow is maintained. The capillary flow direction is shown by the arrow. The carrier layer and the deck are laminated in layers by means of a pressure sensitive adhesive on the functional layer. In this configuration, two capacitors are each formed by a capacitor plate in the carrier and the cover layer and adjacent Kondensatorabschirmplatten. The shown in Figure 9 right capacitor unit includes a microcavity with an immune agglutination. are the capture molecules at this point. The presented in Figure 9 left ones shown, capacitor that is initially extends from the ER capillary flow, is only for reference and for control purposes.

As illustrated, the carrier and cover layer are formed as a film. The distance shown to Veranschaulichkeitsgründen between the film and the functional layer does not exist in reality. The film adhesive is adhered by means of adhesive directly on the functional layer.

10 shows a structure corresponding to the figure 9 egg nem time 4 minutes after application. The reference capacitor shown in Figure 9 is left for reasons of clarity gone. Between the plates of Messkon- densators capture molecules of the invention in the microcavities. Since has not yet passed this point at this time of the analyte to be measured, it has not yet come to an immune agglutination. The resonant circuit formed by the capacitor plates allows the measurement of the con- densatorkapazität. One can also see additional capacitor shield to reduce interference.

Figure 11 shows the functional already shown in Figure 10 onsaufbau at a later time (t = 8 minutes). The current flowing from the Auftragestelle in capillary flow direction liquid-sigkeitsstrom has reached the Mikrokavi- already shown in Figure 10 activities and led to the immune agglutination. The scavenger molecules have responded accordingly with the analyte. The accumulating characterized agglutinates lead to an alteration in the capacitor capacity, which is measured by means of the formed resonance circuit.

12 shows the structure according to FIG 11 again in the supervisory. At the location where the capture molecules sit, there is an immune agglutination by which the capacitance of the capacitor is changed. Listed are here schematically the incorporated into the films of the carrier or covering layer, or applied measurement cables and their connections. One can also see additional Kondensatorabschirmplatten.

Figure 13 shows the corresponding functional test setup for determining the capacitor capacitance. For this purpose, two

Resonant circuits formed which are coupled to each other. In the oscillating circuit 1, the capacitor is formed by respective plate-th, which are located above or below the functional layer, and for example, are printed on a sheet.

In the oscillation circuit 2 is a trimmer capacitor. At the start of measurement when no immune agglutination has taken place, the trimmer capacitor is matched to the measuring capacitor. changes the capacitance of the measuring capacitor by an immune agglutination occurred, the deviation is CAPS LOCK by resonance measurement and measurable.

Figure 14 again shows a device according to the invention with two films, between which a functional layer befin- det. applied to the films are electrically conductive plates which act as a capacitor. Between the capacitor plates is an electric field. By left-hand side the actual capacitor plates auxiliary plates, a shield is caused.

Figure 15 shows various embodiments of comb-shaped capacitor plates. The lowermost figure shows two such comb-shaped capacitor plates which are entangled with each other.

Figure 16 shows schematically an embodiment with measurement of a temperature increase, for example by means of a Bolome- ters by electromagnetic excitation of nanoparticles by an alternating field by means of IR radiation.

Figure 17 describes the basic functional structure of an embodiment of the present invention are embedded at which various indipendent pads in a non-electrically conductive polyvinyl lymerfolie.

Figure 18 shows the basic functional structure of an optical see detection method. Within the functional layer the sample is applied to the application site, and flows in the shown capillary flow direction of a at the opposite end befindliches "Wiek" which gene aufsau- liquid can. The functional layer contains at one point micro- cavities prepared for immune agglutination shown. in this embodiment, the coupling of the antibody to a fluorophore is provided. the transmitter is layer arranged underneath the function, for example in the form of an LED, a laser or other light emitter. Above the layer, the emitted light is measured by the receiver. the receiver can be configured transistor, for example as a photo resistor or photo. between the functional layer and the transmitter or the receiver optional optical filters may be configured (s. below).

Figure 19 shows a similar arrangement in which the receiver is designed in the form of an avalanche diode.

Figure 20 shows an embodiment of the invention, wherein the detection is carried out in the microcavities by thermal detection. an electromagnetic alternating field is applied at the detection site, which stimulates the bound in the microcavity Na nopartikel thermally. A corresponding detector such. B. a thermal camera, a microbolometer, or a similar detector indicates the presence of the nanoparticles by the decision-related increase in temperature. Figure 21 shows another embodiment with an optical detection, in which both the transmitter as well as receiver on the same side of the functional layer are arranged. This is possible because the fluorescence generated in the microcavities is naturally radiated in all directions.

Figure 22 shows an expanded embodiment of the apparatus shown in FIG 21st Here, the fluorescence excitation and measurement takes place in two different locations of the functional layer instead. The illustrated in the figure 22 left-emission detection site serves as a reference range, while the emission shown in the figure 22 on the right - or detection device is the measurement of the analyte in the history formed microcavity.

Figure 23 shows a similar arrangement to Figure 23 wherein the le- beam directions for excitation and for measurement in an angle to the functional layer are arranged diglich.

Figures 18-23 show an example of the use of filters. Because the Stokes shift and the preferred use of filters, for example, thereby only the oreszenz emitted by the light passes Flu- (emission light (eg, 800-770 nm)) to the receiver. The notch filter is designed so that it oreszenz only for the longer wavelength emission light (approximately 790-870 nm) of the Flu- is transparent. The remaining, shorter in the example wavelength excitation light (about 600-750 nm) is biert absorbers.

Figure 24 schematically shows a circuit for measuring oriented emit light by means of a photodiode.

Figure 25 shows an embodiment of Eefindung based on a "Magnetoresistive Current Sensor" which acquire highly sensitive and thereby cost, along with the necessary circuitry.

Figure 26 shows the structure formed according to Example 2 by using two antibodies, one antibody capable of binding specifically to the antigen, the second antibody capable of binding for a particular protein, on the one hand, and on the nanoparticles attached to a piece of DNA. Figure 27 shows an example of a Vorrich- invention processing in which the functional layer ( "active area") is applied directly to a semiconductor element (above schematically un- th as a photograph). Glutinat The Ag formed in the functional layer containing, for example silver nanoparticles can be proved extremely sensitive to these Wise.

Conclusion

The new innovative platform uses the natural agglutination nation existing in colloidal nanoparticles. The colloid was produced artificially by the addition of nanoparticles. The nanoparticles are provided with capture molecules.

Agglutination occurs in the 3D structure of the functional layer.

In the 3D structure of the functional layer (hard phase) are introduced to-einge- additionally in the preparation of the platform, stationary relative to the xyz coordinate capture molecules (using, for example, speziel- 1er microdosing devices) in the functional layer. The capture molecules are mainly biophysical on eg the open-pore structure of eg nitrocellulose ad- sorption bound. Covalent attachment of the capture structures in the 3D structure is just an additional variant in the production of the functional layer used.

The nanoparticles provided with capture molecules cause examples game, a "bridge" between

1. the bound antigen in the liquid phase and the catcher molecules on the other hand on the solid phase and

2. between the bound antigen in the liquid phase and the capture molecules on the other hand on the solid phase of the nanoparticles contained in the liquid phase. There is a Ag glutination (using a biophysical process in the piston loid of the test sample). The locally occurring agglutination can be measured and quantified thereby. As a technical solution to this look is used, an inventive transmitter / receiver configuration.

The transmitter / receiver / sensor / detector is preferably either from the opto-electronic photography, magneto-electronic- or thermo-electronic components.

Present in the colloid nanoparticles are coupled to the capture molecules may be selected from gold, silver, etc., however, there also preferred magnetic dipoles can be used.

The measurement of the agglutination locally occurring can be performed using different technologies, but which are known in other contexts from the prior art in principle. The aim is to derive measurements and from a quantification of the measured analyte to obtain.

In a particular variant, the inventive platform allows form the so-called multiplexing of analytes, that is, analog lysenprobe (eg liquid) previously equilibrated with a mixture of different nano-particles, which are provided with different cules Fängermole-, may be applied to the functional layer and since, as stated above, xy is carried out, the agglutination at various locations, (which has been previously provided with different bound to the solid phase receptor molecules in their manufacture), this leads to a pattern of "Agglu- Tina tion places", that also a "special signature" of the test sample. Agglutination at various locations in the functional layer leads to a resulting in a plan pattern of

"Agglutinationsflecken / Agglutinationsbereichen". The agglutination pattern with the different analytes can, as stated above, advantageously according to the invention for example by opto-electronic array sensors are measured (with corresponding pixels). For example, by using SPAD Single Photon Avalan- che Diodes, fabricated in the HV 12:35 μιη CMOS Technology. the use of these diodes according to the invention allows the use of

- Optical time-domain reflectometry

- Fluorescence lifetime spectroscopy

Or using CMOS linear sensor for Spectroscopy Ap plications or

FAR infrared detectors for thermal imaging

(All detectors were developed by Fraunhhofer Institute for Microelectronic Circuits and Systems IMS.)

It is to be understood that each receiver, each sensor with the corresponding Ableitsensorik and arrester is provided for surveying sensors.

A particular variant of the inventive approach uses eg in addition to the described variants of the Chemolumi- neszenz eg using luminol. The use of

Chemiluminescence for measuring analytes is part of the prior art. However, the use of luminescence in connection with the functional layer and in connection with the ER find innovative structure represents a novelty. It goes without saying that the reagents to achieve the Chemolumi- neszenz after completion of agglutination delayed the Probenauftragestelle the functional layer applied to advertising need.

The emitted when chemiluminescence radiation is received according to the inventive construction of suitable highly sensitive receivers / sensors / detectors and amplified and evaluated.

This was a particular variant of the inventive up superstructure, as can be dispensed with in this particular structure to the transmitter.

Different platforms can be realized according to the invention. The device / platform can

First as a disposable article

2. as an analysis platform

3. as a research platform

4. as a screening platform (eg for screening of hybridoma supernatants)

be prepared and used.

In the inventive structure preferably so-called printed circuits and printed circuit traces are to be used.

It goes without saying that the Vorrich- manufactured obligations / platforms to their use different technical requirement profiles can own. Ie transmitter and receiver and circuit design should be specific, etc. but also as regards the production costs to implement the required technical solution, for example, what sensitivity, separation power, be different.

The platform and the structure and the procedure for performing the analysis is inventive optimized so that an enrichment, concentration of the analyte in the sample localized occurs in the functional layer (referred to the or the analyte as already cavities in micro- cages captured) .

It allows the user of the platform according to the invention, the location-related agglutinated, precipitated analyte, for example, to elute and to supply other techniques of analysis. For example, the user location based elute the analytes and perform On the other reindeer modern analytical methods, eg spectrometry of mass and / or the modern Raman spectroscopy.

The aim is to analyze the so inventively enriched analytes on to get more information from the analytical senprobe that can benefit the user.

For example, one could analyze the tumor tissue and / or tissue having pathological through physiologic inflammatory processes in these complex manner and compare the different sample results with each other. This superiors hens as it is possible, for example, as opposed to the prior art, better than before, "tumor-action" of "inflammatory action" to distinguish pathogenic in the patient. The innovative platform provides a composition of various technologies

1. Ouchterlony Technology

2. Solid State technology 3. Modern Electronics

4. Modern printing techniques

5. Modern manufacturing techniques

represents.

To prepare the platform can inventively different dene, known in the art, modern manufacturing procedures are used. In addition to the various methods described above for locking the performed in the functional layer agglutination measure to the measurement setup preferably, the near infrared (NIR) radiation are used, since this radiation penetrates deep into the 3D structure of the functional layer, and hence the agglutination, by absorption the NIR radiation on the 3D structure optimally utilized. Furthermore, obviates the NIR radiation largely the intrinsic fluorescence of the analyte to be measured as the proteome of inen. The preferred use of NIR-radiation according to the invention leads to a further optimized signal to noise ratio in the measurement.

Conclusion :

In the application several technical solutions are ben described:

The selectivity is done by choosing the suitable for analytes overall bondable capture molecules.

For Biophysical events in the functional layer: 1. Sample Preparation

2. Location-based Analytenkonzentrierung

3. Location-based agglutination of the analytes, the sensitivity is done by choice and by the

Measurement setup. Surveying, ie the characterization, quantification and standardization is made by the erfindungsge- MAESSEN construction. The relatively low cost of production will be according to the invention (suitable for mass production, for example) by the choice and the invention described manufacturing method conservation th. In general, the transmitter in the inventive construction as an LED, a laser diode etc.

The receiver can - as described in the text - for example, consist of different electronic components or used.

In the particular variant of the use of magnetic noteilchen Na (dipoles), the transmitter is for example a coil see rule may generate an alternating electromagnetic field according to the Lenz's. The receiver can in this structure, for example, a CMOS linear sensor, for example, Visual Spectrum of 400 nm to 1000 nm or FAR infrared detector for thermal imaging (using a microbolometer with Readout IC) μτη, which is sensitive to long wavelength infrared 8 .mu.m to 14 and an IR image can provide by means of an IR camera.

Claims

claims:
1. A device for bioanalysis for selected biological
Analyte in a liquid phase containing
a) a support layer,
b) A functional layer
c) a cover layer wherein the functional layer can absorb liquid and portable use optional and at least one place a Fängermo- lekül for or having the selected analyte, characterized in that the carrier layer and / or the top layer comprises transmitter elements, which in turn by typographical methods have been introduced onto or into the carrier layer and / or on or in the cover layer, wherein the transmitter elements are coordinated with the or the selected analyte and / or characterized in that the carrier layer and / or the top layer comprises sensing elements, which in turn by printing technology methods were placed on or in the carrier layer and / or on or in the cover layer, wherein the transmitter elements are coordinated with the or the selected analyte.
2. Device according to claim 1, characterized in that the functional layer is a porous, sponge-like material, for example. B. oxide porous glass, nylon, silica gel, kieselguhr, aluminum containing cellulose, nitrocellulose or polyacetate.
3. A device according to claim 2, characterized in that the functional layer is open-pored with a pore size of the functional layer of 4-1000 nm.
4. The device according to claim 1, characterized by a se- parate application surface for the sample application is carried out, which is in fluid contact with the functional layer and causes a pre-filtration or chromatographic pre-separation of the sample.
5. The device according to claim 1, characterized by Any artwork least a second antibody Soft in fluid contact with the functional layer is and which binds to the analyte of the sample, wherein the second antibody, in turn, optical onal (to nanoparticles such as silver, gold, iron , latex or plastic nanoparticles) is bound.
6. The device according to claim 1, characterized in that as a capture molecule at least one antibody (monoclonal or polyclonal), an aptamer, a spiegelmer, a lectin or a mixture thereof is present.
7. Device. contains according to claim 1, characterized in that the functional layer dye molecules, such as fluorescent dyes (markers), in particular NIR fluorescent dyes.
8. The device according to claim 6 or 7, characterized gekennzeich- net that the capture molecule and / or dye molecule was applied by an ink-j et method.
9. The device according to claim 1, characterized in that the functional layer is by a 3D printing process has been manufactured.
10. The device according to claim 1, characterized in that the capture molecule and / or the dye molecule is covalently attached to the functional layer.
11. The device according to claim 1, characterized in that the capture molecule and / or the dye molecule is covalently attached by coupling with N-hydroxysuccinimide to the functional layer.
12. The device according to claim 1, characterized in that the carrier layer and / or the optional top layer of transparent plastics material and that it contains conducting or semiconducting sensor elements which were in turn introduced by printing processes on or in the layer.
13. The apparatus according to claim 12, characterized in that the carrier layer and / or the optional top layer to laser light of certain wavelengths, particularly in the NIR region is transparent.
14. The apparatus according to claim 13, characterized in that the carrier layer and / or the optional top layer WEI terhin includes an optical filter layer for filtering of other wavelengths.
15. The device according to claim 12, characterized in that the are adapted in the carrier layer and / or the cover layer enthalte- NEN sensor elements to carry out the voltammetry, in particular the cyclic voltammetry, the "differential pulse voltametry" or Squarewavevolta- spectrometry, amperometry, danzmessung particular Chronoamperomet- rie, potentiometry, conductometry or impedance-.
16. The device according to claim 1, characterized in comprising that the carrier layer and / or the top layer, one or more light-sensitive sensor elements, for example a photoresistor, a photodiode (for example, Si PIN), a single photon emitting diode or an avalanche photo multiplier (CMOS), wherein the sensor elements may also be present in an array arrangement.
mineszenzdetektion 17. The apparatus according to claim 16, characterized in that the enthalte- nen in the carrier layer and / or the top layer sensor elements are arranged to perform fluorometry (FRET assay), spectrophotometry, the LU or the Elektrochemilumineszenzdetekti- on.
18. The apparatus according to claim 16, characterized in that the carrier layer or the cover layer as-in sensor element includes a solid state photomultiplier, a CMOS chip, a CCD chip, a microbolometer on a CMOS chip or an array of photodiodes ,
19. The apparatus according to claim 16, characterized in that the carrier layer or the cover layer includes light emitting units as the transmitter elements.
20. The apparatus according to claim 19, characterized in that
the light-emitting units are formed by LEDs or laser diodes, in particular flat bed LEDs or flat bed laser diodes.
21. The device according to claim 1, characterized in that the one or in the cover and / or backing up accommodated transmitter or sensor elements semiconducting elemene te and are, for example, as field effect transistors achievements were.
22. The device according to claim 1, characterized in that the surfaces, the sensor elements contained in the carrier and / or the cover layer ermögli the detection of magnetic particles.
23. The device according to claim 22, characterized in that the switch in the cover and / or backing layer or listed mounted sensor elements produced by printing technology SQUIDs illustrate.
24. The device according to claim 1, characterized in that the functional layer at least one position protein. having and / or protein G.
25. The device according to claim 1, characterized in that the functional layer graphene, carbon -Nanonetze, nano tubes or other open porous materials containing.
26. The device according to claim 1, characterized in that the functional layer has at several locations different capture molecules.
27. The device according to claim 26, characterized in that the functional layer has at least 24 points at least 24 different capture molecules.
28. The device according to claim 1, characterized in that latex or metal nanoparticles (eg silver or gold nanoparticles) or quantum Dotsmit capture molecules or ene are zymengelabelt.
29. The device according to claim 1, characterized in that magnetic metal nanoparticles (eg iron particles) are labeled with capture molecules.
30. The device according to claim 29, characterized in that the magnetic metal nanoparticles (eg iron particles) are thermally excited by an electromagnetic alternating field, and that the heating by means of IR sensors is determined.
31. The device according to claim 1, characterized in that the printing process switched into the carrier and / or the optional top layer or coated sensors are connected to electrical leads, which lead to contact points which allow the contact to evaluation.
32. The device according to claim 28, characterized in that the evaluation unit consists of a special measuring device, a computer or a tablet computer or a smart phone, on top of each run, the corresponding evaluation software.
33. The device according to claim 1, characterized in that the sensor elements and / or the derivatives thereof setdruck using ink jet printing, gravure printing, letterpress printing, off-takes place flexographic printing or screen printing using electrically conductive ink. 34. Use of the device according to at least one of arrival lekularpathologie claims 1-33 as an examination platform, platform or screening analysis platform (disposable) for histology, for histopathology, for cytopathology, for Mo, genesis for the pathophysiology for the Patho- , 35. Use according to claim 34 for the detection of peptides, proteins, RNA and DNA sequences, carbohydrates, lipids, hormones (steroid or sugar hormones), enzymes, Zellkom- parti elements, Zellsubstrukturen, cell superstructures
and / or whole cells, protein complexes, protein-protein complexes, protein-lipid complexes, protein carbohydrate complexes, protein-nucleic acid complexes and viruses.
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