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WO2016015701A1 - Dispositif d'analyse biologique, sa fabrication et procédé de détection d'analytes biologiques au moyen du dispositif - Google Patents

Dispositif d'analyse biologique, sa fabrication et procédé de détection d'analytes biologiques au moyen du dispositif 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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WIPO (PCT)
Prior art keywords
layer
functional layer
carrier
cover layer
sensor elements
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PCT/DE2015/000373
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German (de)
English (en)
Inventor
Hans Scheefers
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Schebo Biotech Ag
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Publication of WO2016015701A1 publication Critical patent/WO2016015701A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements

Definitions

  • the invention relates to a novel apparatus for bioanalytics and to methods for detecting analytes (such as bioanalytes, cells, immune complexes, supramolecular structures, cell compartments, etc.) from biological samples by means of the device according to the invention.
  • analytes such as bioanalytes, cells, immune complexes, supramolecular structures, cell compartments, etc.
  • It is a sensor-receiver sandwich assembly for measuring the local electrical, optical, biophysical properties of substances in an immunofunction thin-film, which also serves as a bioanalytical 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.
  • Techniques used include: immunoagglutination, immunoprecipitation, one-dimensional simple immunodiffusion according to Oudin, two-dimensional immunodiffusion according to Ouchterlony (for the generation of immunoprecipitation patterns), immuno and cross-electrophoresis, electrodiffusion, immunofixation, radial diffusion and rocket electrophoresis, preparative immunoprecipitation. zipitation, radioimmunoassay (RIA), nephelometry.
  • RIA radioimmunoassay
  • the visualization of an immune reaction / immune binding is based on a stable bridge between the sought-after reaction partners (antigen or antibody) and the e.g. Amplification system.
  • amplifiers or indicators: fluorescent dyes, luminescent dyes, radioactivity, enzymes, nanoparticles, e.g. colloidal gold.
  • the visualization of the antibody binding is carried out by direct and indirect methods, eg PAP methods of Sternberger, biotin-avidin bridge (ABC technique), direct or indirect Proof of, for example, a labeled protein A acting as an amplifying agent, eg direct protein A gold method, indirect protein A gold method but also proof of a labeled lectin functioning as an amplifying agent.
  • direct and indirect methods eg PAP methods of Sternberger, biotin-avidin bridge (ABC technique)
  • direct or indirect Proof of, for example, a labeled protein A acting as an amplifying agent eg direct protein A gold method, indirect protein A gold method but also proof of a labeled lectin functioning as an amplifying agent.
  • Demonstration of Specificity of Antibodies in Cells and Tissues by Sandwich Method DOT Immunoassay, Western Blotting, Protein Transfer, Immobilization, and Immunodetection.
  • Biacore technology is based on a sensor chip that can generate surface plasmon resonance (SPR-S stands for surface, PR stands for plasmon resonance).
  • SPR-S surface plasmon resonance
  • PR plasmon resonance
  • the invention is based on the technical problem of providing devices for bioanalytics which allow a more objectivatable reading.
  • possibilities for automatic, computer-based, Reading will be provided.
  • the analysis of larger units, such as cell compartments, cell substructures, whole cells or cell superstructures should be made possible.
  • the invention teaches a device for bioanalytics for selected biological analytes in a liquid phase, comprising a) a carrier layer,
  • the functional layer can absorb and transport liquid and has at least one capture molecule for the selected analyte or analytes
  • the carrier layer and / or the cover layer contains transmitter elements, which in turn by printing technology Method on or in the carrier layer and / or on or in the cover layer were introduced, wherein the transmitter elements are tuned to the selected analyte (s) and / or characterized in that the carrier layer and / or the cover layer contains sensor elements, which in turn by printing process on or in the carrier layer and / or on or in the cover layer were introduced, wherein the transmitter elements are tuned to the selected analyte (s).
  • An essential component of the device according to the invention is a functional layer which is capable of absorbing and transporting liquids.
  • This is, for example, a porous, sponge-like material. It may be z. Silica gel, diatomaceous earth, alumina,
  • Such functional layers generally contain pores having an average pore diameter of, for example, 4-1000 nm.
  • the functional layer contains at least one biological scavenger molecule as at least one site as a "biofalle.” In this way, a microcavity is made available in which the Further evidence leading biochemical reaction can be done (agglutination or precipitation according to "Heidelberger curve").
  • the biological capture molecule may, for example, be an antibody for a selected antigen, for example a protein.
  • the layer is arranged flat and is wetted along one edge with a liquid containing the selected analyte.
  • the liquid migrates through the functional layer and strikes the capture molecule at the appropriate location.
  • the course of the liquid through the functional layer can also be effected by using pumps, in particular micropumps (piezo pumps).
  • pumps in particular micropumps (piezo pumps).
  • other mechanical forces may act, such as a centrifugal force, when the functional layer is rotated.
  • the capture molecule will not react nor agglutinate or precipitate
  • the liquid may be any polar or non-polar solvent, such as DMSO, water, acetonitrile
  • a second recognition molecule which recognizes the complex formed from catcher molecule and analyte, for example a second antibody .
  • This second recognition molecule can be, for example, a radioactive label, a luminescent component, an ionic component. te or metal, for example gold particles, e maintained to ensure or improve the detectability of the agglutinate / precipitate formed.
  • the immune function layer contains capture molecules that are bound to both mobile particles and the solid phase.
  • the immune function layer also contains micro-wells (micro-wells) in which an agglutination reaction with the analytes according to Ouchterlony occurs.
  • RNA and DNA sequences for example RNA and DNA sequences, carbohydrates, lipids, hormones (steroid or sugar hormones), enzymes, cell compartments, cell substructures, cell superstructures and / or whole cells, protein complexes, protein-protein complexes, protein-lipid complexes, protein-carbohydrate complexes, protein-nucleic acid complexes and viruses.
  • catcher molecules must be based on the analyte to be captured:
  • antibodies (polyclonal or monoclonal), aptamers, Spiegelmers, lectins or mixtures thereof come into consideration.
  • several different capture molecules may also be contained in the functional layer, for example: different antibodies or antibodies and aptamers or aptamers and lectins, etc. These may in particular also be located at different positions.
  • the device according to the invention and the methods according to the invention carried out therewith are suitable for the quantitative and qualitative analysis of the different analytes. Many embodiments are suitable for quantitative and qualitative analysis of multiple analytes over the same time period.
  • the functional layer used can be, for example, a nitrocellulose or polyacetate layer, as is known from lateral-flow systems, which is treated at one point with an antibody.
  • the functional layer is applied to a carrier layer in the system according to the invention.
  • This is preferably made of plastic, which in turn is also preferably transparent.
  • the carrier layer is characterized in that it contains sensor elements which in turn have been applied to or into the layer by means of printing technology.
  • sensor / sensors and detector / detectors are to be regarded as synchronous.
  • conductive or semiconducting elements can be introduced as sensor elements. Such elements are known for example from the production of touch screens.
  • the carrier slides around a polyester layer coated with indium tin oxide. Indium tin oxide is a translucent semiconductor.
  • the optional cover layer is in principle of the same construction as the carrier layer, ie a plastic layer in which sensor elements have been applied in or on the layer by means of printing technology.
  • the printing technology of the layer can be done for example by ink jet (inkjet) printing using appropriately conductive ink, but can also be done by appropriate application of methods such as gravure, high-pressure, offset, flexographic or screen printing. Such conductive ink techniques are known in the art.
  • each of cover layer and carrier layer serves as electrodes to which a voltage is applied. Between these electrodes, the resistance can be measured, which can depend, for example, on the liquid flow, in particular the analyte contained. For example, ionic molecular fragments, but also gold particles that bind to a given analyte via antibodies, effectively affect a change in resistance that can be measured.
  • two respective opposing electrodes of carrier layer and cover layer can also form a capacitor whose capacitance can be measured. Also in this case causes the presence of an analyte, in particular an analyte containing metal z.
  • Example 1 Various methods for measuring capacitance are known from the prior art, which can be used to perform the capacitance measurement. These include the measurement of the resonant frequency of an LC resonant circuit formed with the capacitance, the application of an ac voltage under measurement of the current profile or charging with constant current and observing the voltage rise speed.
  • the carrier layer and / or the cover layer contains sensors for photon detection.
  • Photosensitive sensor elements for example a photoresistor or a photodiode, are used for this purpose.
  • Incident light, z. For example, from a laser beam, for example from a tunable dye laser, for example, can radiate through the functional layer and be measured on the carrier layer by means of the sensor elements according to the invention. The passage of a sample leads to a weakening of the beam, for example due to absorption, which causes a change in measured value.
  • the term light does not only cover visible light, but covers the entire range of electromagnetic radiation from the infrared range over the visible range to the UV range.
  • X-rays can also be used from an X-ray laser or appropriate synchrotron radiation.
  • a measuring circuit for measuring light by means of a photodiode is reproduced by way of example in FIG. 17.
  • LEDs based on organic materials so-called OLEDs.
  • Corresponding organic transistors are also applicable as receiver elements based on the CMOS technology.
  • Such optical emitters and sensors are available for example from the companies NovaLED and Hamamatsu on the market.
  • integrated components can be used, for example diode arrays or sensor arrays.
  • a light beam e.g. a laser beam perpendicular to the direction of the sample liquid to be sent through the functional layer.
  • a scattering 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 cuvette in this structure.
  • the term "light” is to be understood as meaning that electromagnetic radiation from the infrared range, the visible range, or the UV range is used.
  • a further embodiment of the invention is that extremely light-sensitive carriers and / or cover layers are applied, which contain, for example, elements of a photomultiplier (working, for example, according to the avalanche principle). Such an embodiment requires a bio trap which can be excited to glow, for example on the basis of luminol. The optoelectronic sensor elements from the cover or carrier layer are able to measure the imitated light.
  • a further embodiment of the invention consists in applying light-emitting carrier and / or cover layers which contain, for example, elements of an LED. For this purpose, flat-bed LEDs are produced by printing technology and brought into contact with the functional layer (laminated and / or glued).
  • OFET organic field effect transistors
  • OLED organic light-emitting diodes
  • OVC organic solar cells
  • All of these components can be used according to the invention.
  • the excitation should preferably be done with ultraviolet radiation (transmitter).
  • the sensor should be sensitive to visible or infrared radiation; for the ultraviolet - resp.
  • visible - radiation should be as insensitive as possible.
  • Such infrared sensors are available on the market, such as in the form of the Micro NIR 1700 spectrometer from JDSU. Further embodiments of the invention are based on so-called microbolometers, which recently can also measure uncooled at room temperature.
  • infrared sensors also allows embodiments of the invention in which antibody-provided magnetic nanoparticles are used, which are thermally excited by applying an electromagnetic alternating field of, for example, 100 kHz.
  • the temperature change produced thereby can be measured by the microbolometers mentioned or by thermopiles (see FIG. 16). In these considerations on the choice of electromagnetic
  • a transmitter e.g. an infrared diode
  • a receiver e.g. a photo diode
  • the transmitter-receiver semiconductor components which can be printed on foils (flexible or inflexible), are preferably used.
  • a plurality of light-emitting elements are applied to the support and / or cover layer, e.g. an LED panel.
  • the functional layer can be illuminated simultaneously at several points. For example, corresponding to each individual LED of an LED panel, different antibodies can be introduced into the functional layer.
  • a CCD or a CMOS chip is possible.
  • chips can be used, which are typically installed in digital cameras.
  • a large number of individual sensors are already present, which can be used to measure the incident light (corresponding to the individual pixels of a digital camera).
  • the light emission takes place in the form of an LED panel and the light measurement by means of a corresponding CCD or CMOS element.
  • an array of Photodiodes are used. Such arrays of photodiodes are also known in the art and need no further explanation at this point.
  • the cover and / or carrier layer contains semiconducting elements which act as field-effect transistors (FET).
  • FET field-effect transistors
  • CHEMFETs very sensitive detection systems
  • the metal layer is replaced by a chemically sensitive layer, in this case the functional layer of the device according to the invention with the applied catcher molecule. If there is an antibody-antigen reaction, the potential changes, which can be measured.
  • Such an embodiment of the invention is also particularly suitable for detecting complete cells or cell compartments.
  • IGFET, NIGFET, MISFET, MOSFET, OFET, ISFET, ENFET, EOSFET, CNTFET or floating gate transistors and variants of those mentioned can also be used.
  • entire cells can be detected, for example by light scattering methods, as already mentioned above. Different light scattering methods, such as “forward-scatter” or “side-scatter”, make it possible to differentiate between different cell sizes and shapes. Fluorescence measurements are also possible.
  • the printing elements applied in the cover and / or carrier layer are printed.
  • sensors are used as amperometric probes, in particular for glucose tests, oxygen-sensitive tests and oxidase tests.
  • the sensor elements incorporated in the functional and / or cover layer are used as potentiometric biosensors, for example as pH-sensitive electrodes, as ammonia-sensitive electrodes, as C02-sensitive electrodes.
  • the functional layer has at least one site antibody to the selected analyte, while the liquid contains a second antibody, which leads to the reaction with the analyte.
  • the second antibody is coupled with a dye. Agglutination of the analyte with a complementary coupling to the dye-carrying second antibody thus takes place at the selected location of the functional layer.
  • the reaction with the second antibody may take place before the analyte passes through the corresponding site of the functional layer. Alternatively, the reaction is possible after the first agglutination reaction has already taken place. In any case, care should be taken that the two antibodies do not cross-react.
  • the determination and quantitative measurement of the construct formed is then carried out in the usual way by optical methods.
  • the second antibody in the liquid can be present, for example, in or in the vicinity of the application site of the functional layer in a soluble, unfixed form.
  • Application of the liquid sample causes a solution of the second antibody (or a corresponding colloid formation), which then moves capillary forces through the functional layer and reacts with it at the location of the fixed first antibody and precipitates or agglutinates it. niert.
  • the second antibody can also be added to the sample and then applied to the functional layer as a common solution.
  • the second antibody is preferably bound to nanoparticles, for example silver, gold, iron, lattice or plastic nanoparticles. In this case, in particular, colloids are formed.
  • the embodiments described here are also used for other detection methods (eg.,
  • metal nanoparticles are preferably of a rod-shaped design.
  • particle growth may still be excited prior to detection by the sensor elements, e.g. Growth of silver particles by reaction with silver nitrate in the presence of chloride or bromide ions.
  • the device may be constructed in the manner of an AlphaLIS assay in which an analyte is bound by two different antibodies. Both antibodies are coated so that the irradiation of a laser beam (for example of 680 nm wavelength) leads to an emission of singlet oxygen, which excites the immediately adjacent second antibody to a light emission at a different wavelength (eg 615 nm) , which in turn is detectable.
  • a laser beam for example of 680 nm wavelength
  • the excitation by the laser beam takes place in the visible light range
  • the light emission takes place in the infrared range, preferably in the near infrared range.
  • Suitable dyes are, for example, cyanine dyes (CY7, CY.l or CY7.5).
  • the emission in the area of the near infrared range facilitates the measurement of the light emission.
  • the additional arrangement of a filter layer in front of the sensor elements is recommended, to prevent the disturbing incidence of visible light on the sensor element.
  • the functional layer is formed by carbon nanotubes.
  • Carbon-containing nanosets can also be supplemented and / or replaced by nanotubes and / or other open-porous materials, membranes or flows, such as those manufactured by companies such as Milliporre, Pall, General Electric, Satorius, etc., where the Liquid phase must contain appropriate catcher molecules.
  • glass fibers it is also possible to guide light through glass fibers to the functional layer.
  • light from laser diodes e.g., blue light at 405 nm
  • appropriate glass fibers may be supplied by appropriate glass fibers.
  • Multi-mode readers can also be used to detect the electromagnetic waves.
  • Such hybrid readers can realize one or more types of detection, e.g. B.
  • Raman spectroscopy can also be used in accordance with the invention, for example in the form of Surface Enhanced Raman Spectroscopy (SERS).
  • SERS Surface Enhanced Raman Spectroscopy
  • solar cells which are based on the material copper indium gallium diselenide (CIGS) can also be used as photocells.
  • CGS copper indium gallium diselenide
  • indium aluminum gallium nitride which mimic in the UV range
  • Materials based on indium aluminum gallium nitride which mimic in the UV range can also be used according to the invention.
  • wavelengths between 400 and 305 nm are generated.
  • the functional layer can be arranged in a circle, with the capture molecules sitting on concentric circles (see FIG.
  • Such embodiments can be read 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 production of CD players, so that at this point further explanations are unnecessary.
  • lanthanoid-containing particles or antibodies which are coupled to lanthanide-containing chelate ligands are particularly advantageous.
  • Such compounds can be used, for example, on the basis of europium, terbium, samarium or dysprosium. In these compounds, there is a so-called Stokes shift, d. H. a distance between see absorption and emission maximum which avoids interference in the measurement.
  • the detection of the analyte can in principle also be carried out by means of magnetic methods.
  • principle known magnetic proteins can be coupled to an analyte and by magnetic techniques) z. B. by means of SQUIDS).
  • the magnetically-labeled analyte can also be held in a specific position by appropriately applied (homogeneous) magnetic fields.
  • Corresponding magnets can also be introduced by printing techniques into the carrier layer and / or the cover layer, for example on the basis of neodymium.
  • Magnetoresistive voltage sensors magnetoresistive current sensors
  • the reading of the magnetic particles of the functional layer can also be effected by an arrangement as known from hard disk drives or CD-ROM drives: A correspondingly small sensor is transferred a rotating plate, the plate containing magnetic particles.
  • the rotating plate is designed as a functional layer and the magnetic particles trapped by the catcher molecules are read out by means of the magnetic sensor.
  • the sensor elements incorporated in the cover and / or carrier layer must receive an electrical discharge, so that the corresponding measured values can also be supplied to a measuring device.
  • Derivatives of this kind are known in principle from the production of a wide variety of components, for example from the manufacture of touchscreen displays.
  • the discharges are likewise introduced into the carrier and / or cover layer by printing technology.
  • the measured values can then be fed into corresponding analog and / or digital measuring devices.
  • the evaluation of the measurement signals also takes place on a microchip, which in turn was introduced by printing technology onto or into the layer.
  • the electronic derivation of the measurement signals can also be performed after analog-to-digital conversion in a PC, tablet or other Head Hero Computer or Smartphone. This may require a USB and / or Bluetooth interface.
  • the transmission of the measured values can also be carried out by means of other non-contact techniques (eg RFID technology).
  • the methods of statistics, stochastics using the line of algebra by means of feature reflectors, pattern recognition and corresponding algorithms for evaluating the measured data are known in principle. Color changes of the functional layer can also be detected and evaluated via the digital camera of a smartphone or another mobile device.
  • different catcher molecules can be present at several points of the functional layer, so that a wide variety of analytes can be determined from a sample.
  • the sensor elements from the carrier and possibly the cover layer must be adapted to the respective analytes with regard to their position and the type of sensor elements.
  • the functional layer can also be flowed through successively from different sides with different solvents. Such a solvent course corresponds to the techniques known in principle from 2D chromatography. In this way, it is possible to achieve a planar distribution of different analytes, which can then be detected by the sensor elements contained in the carrier and, if appropriate, top layer.
  • the sample can also be applied in the middle of the functional layer so that the liquid course runs in a radial direction around the central point.
  • the course of the liquid in the radial direction can be influenced by the fact that the functional layer is rotated. The occurring centrifugal forces then drive the liquid to the outside.
  • a round embodiment of the functional layer is preferred for such an embodiment.
  • the corresponding catcher molecules can hereby be incorporated into the functional layer in one or more concentric circles (see figures).
  • the cover layer does not cover the entire functional layer. Rather, the functional layer must be free at least one point, so that the sample can be applied.
  • the cover layer it is possible, for example, for the cover layer to cover only a part of the functional layer and to apply the sample in the remaining free part of the functional layer.
  • the cover layer it is possible for the cover layer to have openings at one or more locations which serve for sample application. Only in extreme cases, it will be possible that the wetting of the functional layer takes place solely through the edges of the device according to the invention, wherein the liquid "pulls" through the entire layer only by capillary forces starting from the edge of the functional layer.
  • the analyte or the analytes can also be moved through the functional layer by application of an electrical voltage, for example according to the model of gel electrophoresis.
  • the captured capture molecule-analyte complex eg an antibody-antigen-agglutinate or precipitate
  • the functional layer can be mechanically removed or eluted by appropriate solvents.
  • the isolated complex can then be subjected to further analysis, such as, for example, mass spectroscopy (see, for example, Himmelsbach et al., sympat al., 63, Feb. 2015, pp.144-146).
  • the device according to the invention offers a platform for optimizing the cost / benefit ratio in medical diagnostics, food analysis, forensics and basic research.
  • the device according to the invention permits the quantitative determination of biomarkers present in plasma, blood or other body fluids (proteins, peptides, nucleic acids, etc.) for the diagnosis of cancer.
  • biomarkers can be determined.
  • several biomarkers can be measured simultaneously, which greatly increases the quality of the analysis. It was found in particular that a panel of the following biomarkers, which can be determined both in the stool and in the blood or plasma by means of the device according to the invention:
  • IGFBP2 Insulin like growth factor binding protein 2
  • PK-M2 Pyruvate kinase ⁇ 2
  • the presence of one or more of these biomarkers in blood, plasma or stool may be an indication of colorectal cancer or its precursor.
  • the device according to the invention also offers a platform for analyzing larger cell units, for example cell compartments or other cell substructures, whole cells or even superstructures. Also, dimers, trimers, tetramers or polymers of peptides, proteins, antibodies, carbohydrates, lipids and other biomolecules can be fed to a simplified analysis. Further supramolecular structures that can be analyzed with the device according to the invention and the method described here include protein-nucleic acid complexes, protein-lipid complexes,
  • a particular advantage of the invention is that denaturations of the analytes by chemicals (such as detergents) are largely avoided.
  • the functional layer may have capture molecules for selected analytes at several sites. For example, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024 or 2048 digits may be occupied by catcher molecules.
  • versions with 384 or 1,536 positions have proven to be useful.
  • the catcher molecules can be designed identically (ie for the same analyte) or differently (eg for different analytes).
  • the various locations may be arranged, for example, like a checkerboard, z. B. 32 measuring points in a 8x8 matrix, so that there are sufficient gaps between the measuring points that the measurement results are not falsified.
  • the immune function thin layer represents a platform for the purpose of bioanalysis.
  • the structure of this arrangement serves to investigate the measurement of the local electrical properties of this functional layer.
  • the two (located on both sides) electrodes represent an electric plate capacitor.
  • the plates of the plate capacitor can take any shape, for example, square, rectangular, hexagonal or round.
  • comb-like shaped plates have proven useful.
  • comb expansions arranged in a comb-like manner can be realized at one or more edges of a rectangle (FIG. 15).
  • Various such electrodes can be confined with one another in order to utilize the surface particularly well and to realize the smallest possible electrode distances (FIG. 15, bottom).
  • such "restricted electrode combs” can be realized in only one layer (eg the cover layer or the carrier layer.)
  • such "entangled electrode combs” can be formed in only two layers (the cover layer and the carrier layer ) be realized opposite each other.
  • the arrangement and these preferred semiconductor components are used for measuring / investigating the optical, biophysical, biochemical properties of the analytes (antigens) which are agglutinated in the immune function layer and thus locally concentrated.
  • the arrangement according to the invention allows the analytes (antigens) selected by selected catcher molecules and concentrated locally in the functional layer to be accessed by various analysis and examination methods.
  • the mobile capture molecules on the nanoparticles serve 1. the selection, the stationary capture molecules serve 2. the local concentration of the analytes (antigens) in the 3D structure of the functional layer and thus closing examination by means of various physical methods accessible.
  • the local arrangement of the capture molecules causes "traps" in which the analytes / antigens agglutinate and are thus concentrated.
  • Electrodes represent an electrical capacitor. This capacitor can be electrically read by means of electroanalytical methods, which are used in analytical chemistry.
  • Voltametry The term voltametry (English: controlled-current potentiometry titration) is an electrochemical analysis method. It must not be confused with voltammetry.
  • Voltammetry is a collective term for various electroanalytical methods for the qualitative and quantitative analysis of a sample, with which one can determine the chemical composition of substance mixtures on the basis of the voltage-dependent current profile, as well as for the elucidation of reaction mecha- nisms.
  • these electrical properties can be measured (eg by special measuring methods, by selected sensors, active principles and their practical implementation, also eg by automated measuring systems).
  • the measurement of the electrical properties should preferably be carried out by so-called application-specific integrated circuits. The measurement can be carried out in certain embodiments cost and mass suitable.
  • a so-called ASIC (English: application specific integrated cireuit), also "custom chip” is an electronic circuit, which was implemented as an integrated circuit.This particularly suitable, but also other microchips are to find application.Only developed ASIC building block on the market For example, the company Elmos is a company among many who are familiar with the so-called amplifier ASIC, and the corresponding evaluation algorithms could, for example, be acquired by Siemens.
  • the e.g. Dispensing the antibody solutions into the XYZ dimension of the functional layer could e.g. carried out by devices of the company M2 -Automation.
  • Nano-Elektronics is the term used to refer to the use of nano technology on electronic components, including transistors so small that inter-atomic, interactions and quantum mechanical properties.
  • Nano-Photonics should be used. That on the nanometer scale.
  • the invention makes use of the special properties of the immune function layer. This is based on nano-mechanics. Nano-mechanics is a nano-science field that investigates the elastic, thermal and kinetic properties of a physical system on a nanometer scale. According to the invention, the use of "nanomaterials", fillers and carbon forms nanoparticles and colloids.
  • the invention is used inter alia. studying on nanomaterials. This means: Field web studies materials with morphological features on the nanoscale, especially those that have special properties stemming from their nanoscale dimensions.
  • Nanoparticles and their colloids are to be used in the invention. Colloidal Gold, Nanocomposites, Nanofiber Nanobots, Nanobeads, Nanomesh, tubequantumdots etc ..
  • the device or the immune function layer is used for the study and measurement of eg DNA proteins, DNA-RNA, DNA-protein-lipid, DNA-protein-sugar. In the particular embodiment of the device, a so-called quantum point contact can come into play.
  • the immune function layer can later be replaced by certain techniques, e.g. Microscopy and special techniques, atomic force microscopy, scanning panelmicroscopy, scanning probe microscopy etc.
  • the device according to the invention is used for analytical instrumentation and as a tool for characterizing biological materials, e.g. by means of broadband with electrical impedance spectroscopy (EIS), e.g. in the frequency range from a few megahertz up to 100 gigahertz.
  • EIS electrical impedance spectroscopy
  • the measurement of bioimpedance can be used as a high-performance tool for the rapid and minimally or non-invasive characterization of biological objects.
  • the selection of the frequency range, the time resolution (measurement speed) and the electrode arrangements determines the interpretability of the measured properties.
  • the competence of the specialist lies in the choice, adaptation and characterization of the technical measuring environment (hardware and software) to the biological-medical measuring task to be solved in the different application areas.
  • macro and microelectrodes with a wide variety of geometries can be used.
  • amperometric and impedimetric biosensors are used. Electrode production can be carried out by means of electroplating on various substrates. According to the state of the art, commercially available impedance measuring systems which are tuned, for example, to the interests of communications engineering or materials science can not be used to measure the electrochemical properties of biomaterials.
  • thin-film transistor TFT thin-film transistor TFT
  • the capacitor electrodes which can be controlled individually.
  • These thin-film transistors TFT can preferably be embedded in the functional layer during the production of the immune function layer, in which the casting process, ie the production process of the functional layer, for example nitrocellulose or polyacetate, is direct running on a thin film in which, for example, these TFT are embedded.
  • each cavity has two active components (FIG. Electrodes) and a fluid moves through this layer by capillary forces, this immune function layer may also be referred to as an active matrix. If you replace the o.g. electrical components TFT by optical components, so the cavities can be optically measured and examined. Due to the fact that with the help of the capacitor
  • Electrodes or with the aid of the two opposing TFTs larger charges over a longer period of time, "stored” and measured, can be read out, the mean charge density of each cavity and thus the contrast during electrical and / or optical "triggering" increases. the functional layer.
  • LEDs or an LED panel should preferably be installed according to the invention.
  • the functional layer contains microcavities in which agglutination occurs.
  • the colored colloidal gold particles provided with fluorescent dyes, for example can be excited by the photodiodes to emit radiation in the "agglutination lump", for example by electromagnetic waves which serve for the excitation by, for example, electronic photosensitive semiconductor components (“receptors”) by, for example, photoresistors, photodiodes, photomultiplier diodes, etc.
  • the electrical radiation effects an "electrical current flow" which is amplified with corresponding measurement technology known from the prior art
  • This emission radiation is detected by the underlying detection layer (sensor layer) in which sensors of different types are located.
  • sensor layer In the detection layer, sensor layer, it is preferable to use field effect transistors with insulated gel IGFET or with non-isolated gel NIGFET paten according to the application.
  • Functional layer according to the invention consists of an open-pored material with pore sizes of 0.1 ⁇ m to 8 ⁇ m and with
  • the functional membrane is used, for example, for particle analysis, ie analysis of the clots which have arisen through the agglutination reaction.
  • the functional layer can be examined and measured electrically or optically as shown.
  • the functional membrane according to the invention may be made from nitrocellulose, polyacetate, cellulose acetate, cellulose nitrate, cellulose triacetate, hydrocellulose, nylon, polycarbonate, polyester, polyethersulfone, polytetrafluoroethylene or regenerated cellulose, e.g. consist, with pore sizes of 0.1, 0.2, 0.4, 0.45, 0.65, 0.8, 1.2, 3 microns, 5 microns, 8 microns with molecular weight cut-offs of 30,000, 50,000 , 100,000, 1,000 daltons, 5,000 daltons, 10,000 daltons, 20,000 daltons, 30,000 daltons.
  • the functional membrane may also consist of glass fiber membranes, preferably partially altered by appropriate silane chemistry, such that covalently proteins, e.g. can be covalently coupled to the 3D structure of the glass fiber membrane by means of succinimide.
  • An alternative embodiment is the use of graphene as a functional layer.
  • glass / microfibers which have been produced with synthetic binders and which serve for the strength of the membrane.
  • the functional membrane is mechanically and chemically stable and either hydrophobic or hydrophilic. Preference is given to hydrophilic membranes.
  • papers are also conceivable as a functional membrane, in particular tissue paper. Blotting papers, chromatographic papers, filter cardboard, glass microfiber filters, germination paper, kieselguhr filter paper, line cleaning paper, surface protection paper, parchment weighing paper, phase separation paper, etc.
  • the functional layer according to the invention preferably consists of microporous membrane material.
  • the Sephadex are polymeric sugars that are three-dimensionally crosslinked. The material has different porosity, between 20 and 30 ⁇ m. Pore size e.g. Sephadex G200, 1000 to 200,000 Molecular Weight. Manufacturers for this are Sartorius, but also GE (General Electric), Health Care Life Sciences with different brands, Emerson, Sephacryl, Sephadex etc.
  • a particular embodiment is the use of a flat form of "Capto", a material for purifying large biomolecules
  • different molecules can be agglutinated in the microcavities of the functional layer by means of, for example, antibodies and / or lectins or other scavengers. structures are brought to "lump".
  • the agglutinated material may e.g. from proteins, nucleic acids, DNA, RNA, siRNA, microRNA, iRNA, sRNA, shRNA, qPCR, cDNA, lipids and carbohydrates and their aggregates in supramolecular architecture, which until now was difficult to access for analytics ,
  • the functional layer may consist of various porous materials used for chromatography, eg, Superdex, Superose (cross-linked with agarose) materials from Whatman, Merck / Millipore, Satorius, Pall, and other companies that are used for lateral flow and flow through and immunoassays, eg, absorbent pads, bloodseparator, glassfibre, blood, components for dipsticks, membranes for immunoassay, conjugate release, glassfiber pads with different absorption properties can be used according to the invention.
  • porous materials used for chromatography eg, Superdex, Superose (cross-linked with agarose) materials from Whatman, Merck / Millipore, Satorius, Pall, and other companies that are used for lateral flow and flow through and immunoassays, eg, absorbent pads, bloodseparator, glassfibre, blood, components for dipsticks, membranes for immunoassay, conjugate release, glassfiber pads with different absorption properties can be used according
  • a particular inventive embodiment is the use of PCR, in particular real-time PCR method.
  • the invention also relates to the use of super-paramagnetic nanoparticles whose surface is functionalized with bio-affine ligands.
  • a particular inventive embodiment is the use of a magnetic detector. This leads to the development of a mobile measuring device that is suitable for fast, highly sensitive evaluation of magnet-> marker-based multiplex assays.
  • GMR Magneto-Resistive
  • AMR Magnetic MR
  • TMR Magnetic MR
  • Maxwell bridges and / or resonance coils could be used according to the invention.
  • a particular embodiment is the provision of an array-i arrangement of the electronic components in the functional layer.
  • the array arrangement of the individual sensors in the transmitter and receiver layer should be arranged according to the arrangement of olfactory cells in the mammalian olfactory epithelium.
  • the sensors of the array layer and their electrical signals are electronically detected as patterns, i.
  • the activities or the signals of the individual sensor cells are compared by means of an algorithm, so that an activity pattern is generated which is typical for the presence of many simultaneously present analytes.
  • a similar arrangement of the sensor cells can also be found in the retina. Also the principle “cabling" of the individual sensor cells, both in the olfactory system and in the optical system of the retina (see bionics).
  • the antibodies in a particular production step, e.g. by nanodispensing directly onto e.g.
  • the "base region" of an organic field-effect transistor is applied before the contact with the open-pored functional layer is established, and the absorptive or covalent bonding of the capture molecules to the specially designed "base” of the transistor is to be optimally pursued.
  • the material used at the base of the transistor may e.g. Graphene (?) Or other material materials.
  • Streptavidin-biotin-protein A can be used for administration.
  • the exitation of the immune function layer takes place by means of electromagnetic waves using LEDs, preferably LEDs printed on foils.
  • the radiation penetrates into the 3D structure of the functional layer and excites there in the microcavities agglutinated particles, nanoparticles, colloids, which are provided with catcher molecules and / or are preferably labeled with dyes, for emission of electric-magnetic radiation.
  • the emitted radiation is converted into electrical signals by a sensor that functions as a transducer.
  • the signals are evaluated by means of state-of-the-art micro- or nanobased measurement technology and apps in a smartphone, tablet, PC, which function as a computer and display, and the corresponding measured values or the "result" are visually displayed on the screen Display shown.
  • the exitation layer (sensor layer) preferably contains flat-bed LEDs, which are preferably applied to adhesive-coated films and are replaced by e.g. Lamination can be connected during production with the functional layer.
  • the detection layer (sensor layer) preferably contains printed on adhesive film z.
  • the flatbed LEDs can also be designed as thin-film panels.
  • the detection layer can also be designed as a thin-film receptor layer panels.
  • the device according to the invention is used for bioanalytics and diagnostics.
  • the functional layer can be used, for example, as a disposable article.
  • An embodiment of the functional layer can identify different running distances. This is inventively achieved by z. Bd. The application of liquid wax. This will created the individual, separate tracks in the immune function layer. These 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 e.g. various liquid samples by means of e.g. a multichannel pipette at the start line (ped) can be applied simultaneously.
  • This multiple functional layer can also be processed and measured "simultaneously" in a laboratory automatic machine according to the invention.
  • thin-layer LEDs printed, preferably thin-layer field-effect transistors, in the exitation layer or detection layer.
  • the present invention may also be coupled to a mass spectrometer by means of a TLC MS interface.
  • a mass spectrometer by means of a TLC MS interface.
  • the supramolecular structures immunoprecipitated in the functional layer can be extracted and eluted by means of liquid and infused directly or after LC into the ion source of a mass spectrometer for further characterization.
  • the use of MALDI-MS or HPLC-MS techniques is likewise possible.
  • Electronic processors are in the smartphone, tablet, PC and their different operating systems.
  • Smartphone, tablet, PC additionally serve as transmitters and provide access to the "cloud", to the Internet.
  • the functional layer according to the invention is produced either by conventional production methods known from thin-layer chromatography ("casting of TLC plates") or else by printing techniques, in which case the "casting" of the functional layer (which according to the invention is also known as Gel can be formed) can also be made directly on Sen- sorimplantation.
  • a functional layer can be applied directly to a commercially available CMOS or CCD chip, as known from digital cameras. correspond The same applies to light-emitting elements, eg LEDs or semiconductor lasers.
  • the functional layer of the device according to the invention can also be produced by 3D printing techniques, which nowadays work just as quickly and reliably as injection molding.
  • the application of antibodies in the functional layer can be done by printing techniques.
  • a particularly preferred production method for the devices according to the invention is the lamination technique, in which one or two layers which already contain the sensor elements are laminated to a functional layer on one or both sides, for example by using moderate pressures and / or temperatures ,
  • a particular application of the invention is in the context of histology.
  • tissue samples are examined histologically.
  • Particularly noteworthy here are the early diagnosis of tumors, the detection of metabolic disorders, parasitic, bacterial, inflammatory diseases and the like.
  • Classical examination methods in the histodiagnostic laboratory include tissue fixation, paraffin ingrowing, paraffin block cutting, and microscopic examination of the layer.
  • tissue fixation By means of the device according to the invention using appropriate antibodies, it is possible to physicochemically bind and visualize the various cells and cell structures.
  • serve a fluorescent dye which is bound at the site of a protein, or a cell or a cell compart- ment and allows its detection.
  • tissue such as connective and supporting tissue, adipose tissue, bone tissue, cartilage tissue, epithelial tissue, muscle tissue and nerve tissue is possible.
  • the functional layer present within the device can be measured by microvolume spectrophotometry, e.g. through the DeNovix system from Zeiss. This system allows measurement in the range of 190nm to 840nm and allows 3D imaging, clinical routine, correlative microscopy, deep-tissue imaging, fluorescence imaging, image handling, and life-cycle analysis. Cell imaging, spectral imaging, ultrastructural studies, X-ray microscopy for biomedical engineering, cell biology, developmental biology, neuroscience, pathology, pharmacology and toxicology, structural biology and / or zoology and plant sciences. In any case, a coordination of the agents involved is essential to the invention, so that no hook effect occurs.
  • the functional layer is applied to a carrier layer and covered with a cover layer. Both the carrier layer and the cover layer contain electrically conductive sensor elements which have been applied by printing to the layers and face each other. Each form two opposing sensor elements
  • a capacitor in analogy to the representation in Figure 1.
  • Between a plate pair are antibodies as catcher molecules.
  • the capture molecules are able to catch gold microparticles.
  • the capacitance of the capacitor changes, which can be measured by means of the arrangement described below.
  • the following diagram shows the comparison measurement of two capacitors constructed in this way (C1 and C2) by means of an Atmega88 processor, which displays a display in the form of a light-emitting diode display.
  • the clock generator on the UART specifies the standard clock frequency between the transmitter (controller) and the receiver (required for external data transmission).
  • a uniform baud rate must be set in order to "synchronize" the systems.
  • the transmitted data stream consists of a start bit, the following data bits and a stop bit with parity.
  • the baud rate can be determined as a function of the processor clock frequency; UBRR_Val ((F_CPU + BAUD * 8) / (BAUD * 16) -1). In the usage variant described here, a variable in the "unsigned-long" area was chosen.
  • the UART interface is ready for transmission. To avoid a collision of data in the send buffer, it must be checked for an assignment. A check of the UCSROA register with reference to UDRE0 reveals whether the buffer is empty. An empty register outputs the status "1." In a variable, the value to be sent is output via the interface.
  • the data to be transferred is written to an array.
  • a string with about 10 elements of the type "char" should suffice for a port, the index logically starts at 0 and ends at 9.
  • the capacity measurement is 3.94, this value is arranged as follows:
  • a variable (here C-time) 0 is initialized.
  • a port (here PB is declared as an output and switched to a LOW level in order to discharge the applied capacity at the beginning of the measuring cycle.
  • the corresponding port is switched from LOW to HIGH and an internal pull-up is activated. From the switchover time, the capacitor is charged until the controller detects (via a loop) a high level. This level depends on the set reference voltage.
  • the reference voltage is currently 3.5 V. However, this voltage may depend very much on the selected controller and the power supply (button battery / AA battery / USB port / power supply). No matter which power supply is selected, it will take some time until a high level is detected. This time must be measured in a do loop and forms a ratio to the capacitance over an approximately constant charging current. If a long charging time is measured, the capacity also becomes very large. In order to convert the measured time into a capacitance, a voltage and current-dependent factor must be determined. This is in the already known example at 0.000622. This changes with a different reference voltage or a strongly deviating capacitance.
  • the system should be able to display the capacitance of the capacitor using simple optical indicators.
  • a reference value (yet to be determined) is stored in the system. This reference value becomes that in the variable (here "c"). If the two values deviate only minimally from each other, a green LED lights up. If the deviation is greater, the yellow or the red LED is energized.
  • one antibody specifically binds to the antigen
  • the second antibody binds to a particular protein, on the nanoparticle on the one hand, and bound to one piece of DNA
  • the agglutinate can be greatly enlarged in space by greatly extending the short pieces of DNA present on the antibodies by means of a DNA polymerase reaction.
  • the resulting 3D DNA agglutination structure is spatially large ( Figure 26), e.g. bring the electrodes (teeth) of the two combs, which act as electrodes (see Figure 15, bottom), into electrical contact. If the two comb-shaped electrodes are bridged by means of the 3D structure, this causes a very great change in the electrical resistance.
  • the change in the electrical resistance can be measured very sensitively by means of an electronic measurement setup.
  • the resistance change serves to quantify e.g. of the desired antigen (analyte).
  • microchannels are formed in a wide variety of materials, such as glass, plastic silicon, and fluids are moved through these channels.
  • the movement can be carried out by means of appropriate micro pumps, but also by application of centrifugal force.
  • Printed field effect transistors are i.a. described by Dimos Poulikakos, ETH Zurich.
  • Various conductive printing inks are commercially available, for. From BASF.
  • gur 1 shows schematically the construction of an embodiment of a device according to the invention in cross-section:
  • the electrode pairs 1/1 ⁇ 2/2 'and 3/3 ⁇ each form (plate) capacitors whose capacitance (inter alia) depends on the dielectric properties of the intermediate space. Change in the dielectric properties of the gap, for example due to trapping of antibody-coated gold particles lead to capacity changes that can be measured (eg in the form of permittivity) and stored time-dependent.
  • FIG. 2 shows schematically the structure of another embodiment of a device according to the invention in cross section:
  • photosensor 2 e.g. CCD CMOS chip
  • photosensor 1 e.g. CCD or CMOS chip 1
  • the LEDs 1, 2 and 3 each form with the opposite photosensors 1, 2 and 3, light barriers.
  • the light transmission through the functional layer depends on the optical properties of the intermediate space. Changes in the light absorption of the interspace, for example due to trapping of antibody-coated gold particles, lead to a location-dependent change in the light absorption that can be measured and stored over time.
  • the evaluation can also be carried out on a wavelength-dependent basis.
  • FIG. 3 shows schematically the construction of an expanded embodiment of a device according to the invention.
  • the device already described in FIG. 2 is supplemented by two filter layers (18 and 19).
  • This embodiment makes it possible to filter out light radiation having specific wavelengths.
  • This embodiment is particularly suitable for capture molecules which, when irradiated with light of a certain wavelength, emit light of a different wavelength, as described above. Thanks to the built-in filter layers, it is thus possible to ensure that on the part of the
  • Transmitter only light with the transmission wavelength is emitted and that only light with the receiving wavelength reaches the receiving sensor on the part of the receiver. In this way, disturbances are largely suppressed, which leads to correspondingly more precise measurement results.
  • Figure 4 shows schematically an embodiment of the invention with five different measuring points (shown here as black bars). Capture molecules for selected analytes can be positioned at each of these measurement sites. The corresponding sensor elements will be contained in the carrier and / or cover layer matching these locations. In this way For example, five different analytes can be analyzed in one measurement run.
  • FIG. 5 shows a checkered arrangement of catcher molecules and sensor elements. In this way, many different analytes can be measured.
  • FIG. 6 shows an embodiment of the invention in which the application point is in the middle and the direction of travel of the applied solution runs concentrically, that is to say uniformly in all directions of the plane.
  • FIG. 7 schematically shows an embodiment of the invention based on FIG. 6, in which several rings with measuring points are positioned concentrically around the application site.
  • FIG. 8 shows schematically the arrangement of ten measuring points within a device according to the invention, wherein the measuring points have spacings to each other in all directions of the plane.
  • Such an arrangement may be required to prevent disturbances of the measurements.
  • Such disorders can z. B. occur in that - in the case of the application of optical analysis methods - light emissions, for example, due to scattering, fall on adjacent measuring sensors len.
  • electrical measuring methods in which, for example, the capacitance measurement of a capacitor unit formed can be disturbed by electrical influences of the neighboring measuring stations.
  • Another source of interference may be the application of the catcher molecules during the production of the functional layer. This can (depending on the type of application) lead to a certain bleeding 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.
  • FIG. 9 again shows the basic functional structure in the side view.
  • the functional layer is located in the middle of the device, with an application site on the left and a "Wiek" on the right, which absorbs incoming liquid and thus ensures that the capillary flow is maintained.
  • the capillary flow direction is shown by the arrow.
  • the carrier layer and the cover layer are laminated to the functional layer by means of a pressure-sensitive adhesive.
  • two capacitors are formed by a respective capacitor plate in the carrier and the cover layer and adjacent Kondensatorabtubplatten.
  • the right-hand condenser unit shown in Figure 9 includes a microcavity with immunoagglutination. At this point are the catcher molecules.
  • the capacitor shown on the left in FIG. 9, which is initially reached by the capillary flow serves only as a reference or for control purposes.
  • the carrier and cover layers are formed as a film.
  • the illustrated illustration of reasons distance between the film and the functional layer is not present in reality.
  • the film is adhered directly to the functional layer by means of the adhesive.
  • FIG. 10 shows a structure corresponding to FIG. 9 at a time 4 minutes after application.
  • the reference capacitor shown in FIG. 9 is omitted for reasons of clarity.
  • the catcher molecules according to the invention are located in the microcavities between the plates of the measuring capacitor. Since at this time the analyte to be measured has not yet passed this place, it is not yet have come to an immunoagglutination.
  • the resonant circuit formed by the capacitor plates makes it possible to measure the capacitor capacitance. You can also see supplementary capacitor shielding plates to reduce interference.
  • the liquid flow flowing from the application site in the capillary flow direction has reached the microcavities already shown in FIG. 10 and led to immunoagglutination.
  • the capture molecules reacted with the analyte accordingly.
  • the agglutinates that accumulate as a result lead to a change in the capacitor capacitance, which is measured by means of the resonant circuit formed.
  • FIG. 12 again shows the structure according to FIG. 11 in the top view.
  • an immunoagglutination which changes the capacitance of the capacitor.
  • FIG. 12 again shows the structure according to FIG. 11 in the top view.
  • an immunoagglutination which changes the capacitance of the capacitor.
  • FIG. 13 shows the associated functional measurement setup for determining the capacitor capacitance. This will be two
  • the capacitor is formed by corresponding plates, which are located above or below the functional layer and are printed, for example, on a foil.
  • the resonant circuit 2 is a trim capacitor. At the beginning of the measurement, if no immunagglutination has taken place yet. has found, the trim capacitor is tuned to the measuring capacitor. If the capacitance of the measuring capacitor changes due to immunoagglutination, the deviation can be determined and measured by resonance measurement.
  • FIG. 14 again shows a device according to the invention with two foils, between which a functional layer is located. Applied to the films are electrically conductive plates which act as a capacitor. Between the capacitor plates is an electric field. By located next to the actual capacitor plates additional plates, a shield is caused.
  • FIG. 15 shows various embodiments of comb-shaped capacitor plates.
  • the bottom figure shows two such comb-like shaped capacitor plates, which are interlocked.
  • FIG. 16 schematically shows an embodiment with measurement of a temperature increase, for example by means of a bolometer, after electromagnetic excitation of nanoparticles by an alternating field by means of IR radiation.
  • FIG. 17 describes the basic functional structure of an embodiment of the present invention, in which various individual pads are embedded in a non-electrically conductive polymer film.
  • FIG. 18 shows the basic functional structure of an optical detection method.
  • the sample is applied at the application site and flows in the illustrated capillary flow direction to a "Wiek" located at the opposite end, which contains liquid. can.
  • the functional layer contains microcavities prepared for immunoagglutination at one site.
  • the coupling of the antibody to a fluorophore is provided.
  • the transmitter is arranged below the functional layer, for example in the form of an LED, a laser or another light emitter. Above the layer, the emitted light is measured by the receiver.
  • the receiver can be designed, for example, as a photoresistor or phototransistor.
  • Optional optical filters may be set up between the functional layer and the transmitter or the receiver (see below).
  • Figure 19 shows a similar arrangement in which the receiver is in the form of an avalanche diode.
  • FIG. 20 shows an embodiment of the invention in which the detection in the microcavities is carried out by thermodetection.
  • An electromagnetic alternating field is applied at the detection site, which thermally excites the nanoparticles bound in the microcavity.
  • a corresponding detector for.
  • a thermal camera a microbolometer or a similar detector indicates the presence of nanoparticles by the resulting increase in temperature.
  • FIG. 21 shows another embodiment with an optical detection in which both transmitter and receiver are arranged on the same side of the functional layer. This is possible because the fluorescence generated in the microcavities is naturally radiated in all spatial directions.
  • FIG. 22 shows an expanded embodiment of the device shown in FIG. Here the fluorescence excitation and measurement takes place at two different points of the functional shift takes place.
  • the emission detection point shown on the left in FIG. 22 serves as the reference measurement range, while the emission or detection arrangement shown on the right in FIG. 22 represents the measurement of the analyte in the microcavity formed.
  • FIG. 23 shows a similar arrangement as in FIG. 23, wherein only the beam directions for excitation or for measurement are arranged at an angle to the functional layer.
  • Figures 18-23 exemplify the use of filters. Because of the Stokes shift and the preferred use of filters e.g. As a result, only the light emitted by the fluorescence (emission light (for example 800-770 nm)) reaches the receiver.
  • the blocking filter is designed so that it is only transparent to the longer-wavelength emission light (about 790-870 nm) of the fluorescence. The still existing, in the example shorter wavy excitation light (about 600-750 nm) is absorbed.
  • FIG. 24 shows schematically a circuit for measuring emitted light by means of a photodiode.
  • FIG. 25 shows an embodiment of the invention based on a "magnetoresistive current sensor" which is very sensitive and can be obtained inexpensively together with the necessary circuitry.
  • FIG. 26 shows the structure formed in accordance with Example 2 by using two antibodies, one antibody specifically bindable to the antigen, the second antibody binding to a particular protein, on the nanoparticle on the one hand and bound to a piece of DNA.
  • 27 shows an example of a device according to the invention in which the functional area ("Active Area") is applied directly to a semiconductor element (above schematically, below as a photograph). Nanoparticles can be detected extremely sensitive to this fact.
  • the new inventive platform utilizes the natural agglutination of nanoparticles present in the colloid.
  • the colloid was created artificially by the addition of nanoparticles.
  • the nanoparticles are provided with catcher molecules.
  • the agglutination takes place in the 3D structure of the functional layer.
  • capture molecules by using, for example, specific microdispensing devices are introduced into the functional layer during production of the platform, locally in the coordinates xyz.
  • the capture molecules are mainly biophysical to e.g. the open pore structure of e.g. Nitrocellulose adorptively bound.
  • a covalent binding of the capture structures in the 3D structure is only an additional variant in the production of the functional layer used.
  • the nanoparticles provided with catcher molecules cause, for example, a "bridge" between
  • the transmitter / receiver / sensor / detector preferably consists either of opto-electronic, magneto-electronic or thermo-electronic components.
  • the nanoparticles present in the colloid, to which catcher molecules are coupled may consist of gold, silver, etc., but magnetic dipoles may also be used with preference.
  • the measurement of locally occurring agglutination can be carried out using various technologies, which are known in principle, but in other contexts of the prior art.
  • the aim is to derive measured values and from them a quantification of the measured analytes.
  • the inventive platform allows the so-called multiplexing of analytes, ie an analyte sample (eg liquid) which has previously been applied to the functional layer with a mixture of different nanoparticles which are provided with different capture molecules and, as stated above, the agglutination takes place at various locations xy (previously provided with different capture molecules bound to the solid phase in the preparation) this leads to a pattern of "agglutination sites", ie also to a "special signature" of the analytical sample. Agglutination at different locations in the functional layer leads to a pattern of supervision
  • the agglutination patterns with the different analytes can, as stated above, advantageously be measured according to the invention, for example by opto-electronic array sensors (with corresponding pixels), for example by using SPAD Single Photon Avalanche Diodes, fabricated in the HV 0.35 ⁇ m CMOS Technology
  • the use of these diodes according to the invention allows the use of
  • each receiver each sensor is provided with the corresponding deflection sensor and leakage measuring sensor.
  • a particular variant of the inventive approach uses e.g. in addition to the variants described, chemoluminescence, e.g. using luminol.
  • chemoluminescence e.g. using luminol.
  • Chemiluminescence for the measurement of analytes belongs to the state of the art. However, the use of luminescence in connection with the functional layer and in connection with the It is self-evident that the reagents to achieve chemoluminescence after completed agglutination have to be applied with a time delay to the sample application site of the functional layer.
  • the radiation emitted in the chemiluminescence radiation is received in accordance with the inventive structure highly sensitive of suitable receivers / sensors / detectors and amplified and evaluated.
  • the device / platform can be realized according to the invention.
  • the device / platform can be realized according to the invention.
  • the device / platform can be realized according to the invention.
  • the device / platform can be realized according to the invention.
  • the device / platform can be realized according to the invention.
  • the device / platform can be realized according to the invention.
  • the device / platform can be realized according to the invention.
  • the device / platform can be realized according to the invention.
  • the devices / platforms produced can have different technical requirement profiles with respect to their use.
  • the transmitter and receiver and the structure of the circuits should be specially designed to implement the required technical solution, eg what sensitivity, separation performance, etc. but also as far as the production costs are concerned.
  • the platform and the structure and the procedure in carrying out the analysis is inventively optimized so that an enrichment, concentration of the analyte to be determined in the sample is localized in the functional layer (the analyte or cages are in microcavities cages as already stated above captured) .
  • the site-specific agglutinated, precipitated analytes e.g. to elute and other techniques of analysis.
  • the user can elute the analytes in a localized manner and supply them to other modern analytical methods, e.g. mass spectrometry and / or modern Raman spectroscopy.
  • the aim is to further analyze the analytes enriched in accordance with the invention in order to obtain further information from the analysis sample which the user can use.
  • inventive platform represents a composition of different technologies
  • NIR radiation near-infrared
  • the preferred use of NIR radiation according to the invention furthermore leads to an optimized signal-to-noise ratio during the measurement.
  • the selectivity is achieved by the choice of bindable catcher molecules suitable for the analyte.
  • Measurement setup The measuring, i. the characterization, quantification and standardization are carried out by the structure according to the invention.
  • the relatively low production costs are obtained according to the invention by the choice and by the production methods described in accordance with the invention (for example, mass-production-suitable).
  • the transmitter in the inventive design can e.g. an LED, a laser diode, etc.
  • the receiver can - as described in the text - e.g. consist of different electronic components or used.
  • the transmitter represents e.g. a coil which, according to Lenz's rule, can generate an alternating electromagnetic field.
  • the receiver may be used in this construction e.g. a CMOS linear sensor, e.g. Visual spectrum from 400 nm to 1000 nm or FAR infrared detector for thermal imaging (use of a microbolometer with readout IC), which is sensitive to long-wave infrared 8 ⁇ m to 14 ⁇ and can provide an IR image by means of an IR camera.

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  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un nouveau dispositif d'analyse biologique pour des analytes biologiques choisis, contenant a) une couche de support, b) une couche fonctionnelle, c) facultativement une couche de recouvrement, la couche fonctionnelle pouvant recevoir et transporter du liquide et présentant une molécule de capture pour les analytes choisis sur au moins un site, le dispositif étant caractérisé par le fait que la couche de support et/ou la couche de revêtement contiennent des éléments de capteur pour les analytes choisis, lesquels sont introduits à leur tour sur ou dans la couche de support et/ou la couche de recouvrement par procédé typographique. L'invention concerne également un procédé de détection d'analytes biologiques au moyen du dispositif selon l'invention.
PCT/DE2015/000373 2014-07-31 2015-07-22 Dispositif d'analyse biologique, sa fabrication et procédé de détection d'analytes biologiques au moyen du dispositif WO2016015701A1 (fr)

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CN108489867A (zh) * 2018-04-19 2018-09-04 中国科学院地球化学研究所 一种测定植物叶片细胞输运能力的方法
DE102017221542A1 (de) * 2017-11-30 2019-06-06 Contitech Schlauch Gmbh Hohlkörper, vorzugsweise zylindrischer Hohlkörper, besonders vorzugsweise Schlauch
CN110142036A (zh) * 2019-05-29 2019-08-20 中国科学院兰州化学物理研究所 一种葡萄糖量子点键合硅胶亲水色谱固定相的制备及应用
CN110187091A (zh) * 2019-04-18 2019-08-30 浙江大学 一种用于抗肿瘤药物筛选的高通量3d细胞阻抗传感器及检测方法
CN110333227A (zh) * 2019-06-20 2019-10-15 广西科技大学鹿山学院 一种萘酚/石墨烯/联吡啶钌电致化学发光传感器的制备方法及其在测定盐酸赛庚啶的应用
CN111122540A (zh) * 2019-12-25 2020-05-08 桂林电子科技大学 基于时间相关单光子探测技术的多功能光纤探针系统
US10650312B2 (en) 2016-11-16 2020-05-12 Catalog Technologies, Inc. Nucleic acid-based data storage
WO2021004564A2 (fr) 2019-07-05 2021-01-14 Schebo Biotech Ag Dispositif pour sélectionner un kit de test pour mettre en évidence la présence de biomarqueurs
US11227219B2 (en) 2018-05-16 2022-01-18 Catalog Technologies, Inc. Compositions and methods for nucleic acid-based data storage
US11286479B2 (en) 2018-03-16 2022-03-29 Catalog Technologies, Inc. Chemical methods for nucleic acid-based data storage
US11306353B2 (en) 2020-05-11 2022-04-19 Catalog Technologies, Inc. Programs and functions in DNA-based data storage
US11535842B2 (en) 2019-10-11 2022-12-27 Catalog Technologies, Inc. Nucleic acid security and authentication
CN115704800A (zh) * 2021-08-13 2023-02-17 北京大学 一种病毒检测器件及制备方法和病毒检测方法
US11610651B2 (en) 2019-05-09 2023-03-21 Catalog Technologies, Inc. Data structures and operations for searching, computing, and indexing in DNA-based data storage
US11763169B2 (en) 2016-11-16 2023-09-19 Catalog Technologies, Inc. Systems for nucleic acid-based data storage

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US11763169B2 (en) 2016-11-16 2023-09-19 Catalog Technologies, Inc. Systems for nucleic acid-based data storage
US12001962B2 (en) 2016-11-16 2024-06-04 Catalog Technologies, Inc. Systems for nucleic acid-based data storage
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DE102017221542A1 (de) * 2017-11-30 2019-06-06 Contitech Schlauch Gmbh Hohlkörper, vorzugsweise zylindrischer Hohlkörper, besonders vorzugsweise Schlauch
US12006497B2 (en) 2018-03-16 2024-06-11 Catalog Technologies, Inc. Chemical methods for nucleic acid-based data storage
US11286479B2 (en) 2018-03-16 2022-03-29 Catalog Technologies, Inc. Chemical methods for nucleic acid-based data storage
CN108489867A (zh) * 2018-04-19 2018-09-04 中国科学院地球化学研究所 一种测定植物叶片细胞输运能力的方法
US11227219B2 (en) 2018-05-16 2022-01-18 Catalog Technologies, Inc. Compositions and methods for nucleic acid-based data storage
CN110187091A (zh) * 2019-04-18 2019-08-30 浙江大学 一种用于抗肿瘤药物筛选的高通量3d细胞阻抗传感器及检测方法
CN110187091B (zh) * 2019-04-18 2020-11-27 浙江大学 一种用于抗肿瘤药物筛选的高通量3d细胞阻抗传感器及检测方法
US12002547B2 (en) 2019-05-09 2024-06-04 Catalog Technologies, Inc. Data structures and operations for searching, computing, and indexing in DNA-based data storage
US11610651B2 (en) 2019-05-09 2023-03-21 Catalog Technologies, Inc. Data structures and operations for searching, computing, and indexing in DNA-based data storage
CN110142036B (zh) * 2019-05-29 2021-11-19 中国科学院兰州化学物理研究所 一种葡萄糖量子点键合硅胶亲水色谱固定相的制备及应用
CN110142036A (zh) * 2019-05-29 2019-08-20 中国科学院兰州化学物理研究所 一种葡萄糖量子点键合硅胶亲水色谱固定相的制备及应用
CN110333227A (zh) * 2019-06-20 2019-10-15 广西科技大学鹿山学院 一种萘酚/石墨烯/联吡啶钌电致化学发光传感器的制备方法及其在测定盐酸赛庚啶的应用
WO2021004564A2 (fr) 2019-07-05 2021-01-14 Schebo Biotech Ag Dispositif pour sélectionner un kit de test pour mettre en évidence la présence de biomarqueurs
US11535842B2 (en) 2019-10-11 2022-12-27 Catalog Technologies, Inc. Nucleic acid security and authentication
CN111122540A (zh) * 2019-12-25 2020-05-08 桂林电子科技大学 基于时间相关单光子探测技术的多功能光纤探针系统
US11306353B2 (en) 2020-05-11 2022-04-19 Catalog Technologies, Inc. Programs and functions in DNA-based data storage
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