EP1872127A1 - Systeme de detection micro-optique et procede pour determiner des parametres d'analytes variables avec la temperature - Google Patents

Systeme de detection micro-optique et procede pour determiner des parametres d'analytes variables avec la temperature

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Publication number
EP1872127A1
EP1872127A1 EP06724318A EP06724318A EP1872127A1 EP 1872127 A1 EP1872127 A1 EP 1872127A1 EP 06724318 A EP06724318 A EP 06724318A EP 06724318 A EP06724318 A EP 06724318A EP 1872127 A1 EP1872127 A1 EP 1872127A1
Authority
EP
European Patent Office
Prior art keywords
detection system
support structure
microoptical
micro
optical
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06724318A
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German (de)
English (en)
Inventor
Holger Klapproth
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
TDK Micronas GmbH
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TDK Micronas GmbH
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Filing date
Publication date
Application filed by TDK Micronas GmbH filed Critical TDK Micronas GmbH
Publication of EP1872127A1 publication Critical patent/EP1872127A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • 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
    • G01N33/557—Immunoassay; Biospecific binding assay; Materials therefor using kinetic measurement, i.e. time rate of progress of an antigen-antibody interaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N2035/00099—Characterised by type of test elements
    • G01N2035/00158—Elements containing microarrays, i.e. "biochip"

Definitions

  • the invention relates to a micro-optical detection system and to a method for detecting analytes by means of time-resolved luminescence. This serves to determine temperature-dependent parameters of analytes, in particular the determination of point mutations of nucleic acids (DNA), for which a time-resolved detection is required.
  • DNA nucleic acids
  • biosensors For qualitative and / or quantitative detection of certain substances, such as biomolecules in a sample to be analyzed, the use of essentially planar systems is known, which are referred to in the art as biosensors or biochips.
  • biochips form a support structure, on the surface of which, as a rule, a large number of detection areas arranged mostly in the form of a grid are formed, the individual areas being formed or range groups by their specificity to a particular analyte to be detected differ from each other.
  • nucleic acid probes such as oligonucleotides or cDNA in mostly single-stranded form, whose respective specificity towards the nucleic acid to be detected is essentially located by the sequence of sequences, are located within the individual regions of the carrier surface, directly or indirectly immobilized is predetermined.
  • the chip surface functionalized in this way is brought into contact with the sample possibly containing the DNA analytes which may be present under conditions which, in the case of the presence of the previously detectably labeled target nucleic acid (s), hybridize with the immobilized probe molecules guarantee.
  • the qualitative and optionally quantitative detection of one or more specifically formed hybridization complexes is then usually carried out by opto-physical luminescence measurement and assignment of the data obtained to the respective detection areas, whereby the determination of the presence of the DNA analyte (s) in the Sample and possibly its quantification is made possible.
  • EP 1 248 948 B1 discloses a method for the parallel determination of temperature-dependent parameters, such as, for example, the association / dissociation constants or equilibrium constants of complexes, which is based on total internal reflection fluorescence (TIRF).
  • TIRF total internal reflection fluorescence
  • this technology which makes use of the evanescent field, is very expensive in terms of apparatus, which leads to a cost factor which is not justifiable for routine analysis.
  • Another disadvantage which excludes such systems in the field of routine analysis is the high expenditure of time associated with these systems. Proceeding from this, it was an object of the present invention to provide a detection system for analytes, which on the one hand pursues the known for biochips miniaturization and at the same time allows a time-resolved measurement.
  • the associated apparatus and time complexity of such a system should be kept as low as possible.
  • micro-optical detection system having the features of claim 1
  • diagnostic device having the features of claim 30
  • method for determining temperature-dependent parameters having the features of claim 32.
  • inventive method uses of the inventive method may be mentioned.
  • the other dependent claims show advantageous developments.
  • a micro-optical detection system for determining temperature-dependent parameters of analytes. This is based on the following elements:
  • a support structure having at least one surface on which receptors for the analytes are immobilized, wherein the receptors form a plurality of measurement points,
  • the detection system according to the invention is based on the essential feature that at least the detector is monolithically integrated into the carrier structure. This makes it possible that the micro-optical detection system can be designed in the form of a biochip or DNA chip. To date, such miniaturized systems are not known from the state of the art, which make it possible to develop systems within the scope of chip technology which permit the determination of temperature-dependent parameters.
  • the detection system according to the invention can be used for the detection of nucleic acids as well as for detectably labeled analytes, in particular proteinose substances, for example peptides, proteins, antibodies and functional fragments thereof.
  • the present invention encompasses any detection of a complex formed from a detectably labeled analyte, ie a component from the sample to be analyzed, and a receptor, ie an immobilized carrier component, according to the invention also including those systems in which the analyte already characterized, for example, by a detectable autofluorescence and therefore requires no further markings.
  • amino acid thyroxine which is a Has intrinsic fluorescence, even without additional labeling of thyroxine residues containing proteinaceous substance.
  • proteinose substances for example antibodies or fragments thereof, can be detected as analytes, even without these having been previously labeled with a luminescent substance which is suitable according to the invention.
  • the detection system according to the invention has a device for continuous contacting, which may preferably be designed as a flow cell, as a cuvette or as a sample container.
  • the mentioned device for contacting is channel-shaped.
  • a plurality of channel-shaped devices are integrated parallel to each other in the array-like support structure.
  • the device for contacting is formed as a recess in the support structure, which is provided on the side facing away from the surface of the support structure with a cover layer.
  • a recess can be etched into the support structure, for example.
  • This cover layer preferably has at least two point-shaped recesses which allow the inflow and outflow of the fluid.
  • the at least one contacting device consists of a photocured polymer and is applied to the support structure by photopolymerization.
  • a further preferred variant provides that a device for contacting of any material is applied to the support structure, wherein this is effected by means of an adhesive, by means of bonding and / or a pressing operation.
  • the flow cell is coupled to at least one pump for transporting fluids.
  • the flow cell is coupled to at least one pump for transporting fluids.
  • tempering element Another essential point of the detection system according to the invention is the use of a tempering element.
  • the tempering allows this as desired, a temperature increase or a decrease in temperature of the fluid or the surface of the support structure.
  • the tempering element must be thermally connected to the fluid or at least to a surface in contact with the fluid.
  • this represents the only limitation with respect to the arrangement of the tempering element.
  • the tempering element is monolithically integrated into the support structure.
  • thermosensor or temperature sensor which may be e.g. forms a control loop in combination with a controller and the tempering element. In this way, a targeted control of the temperature of the fluid in the device for incontact is made possible.
  • the support structure of the biochip is preferably made of metal or semimetal oxides, e.g. Silicon wafer, alumina, quartz glass, glass or a polymer.
  • the support structure of the detection system according to the invention preferably consists of a semiconductor material with an integrated, preferably a plurality of detectors comprising optical detector layer, wherein preferably photodiodes are incorporated as detectors.
  • the signal processing takes place at least partially within the biosensor.
  • the receptor can now be attached to this support structure both directly and via a spacer.
  • a spacer ie a bifunctional molecule
  • compounds are preferably used which have a halosilane or alkoxysilane group for coupling to the surface of the support structure.
  • Particular preference ' is here under a chlorosilane.
  • the carrier structure can be coated with glycidyltriethoxysilane, which can be achieved, for example, by immersion in a solution of 1%.
  • Silane in toluene slow extraction and immobilization by drying at 120 0 C can be done.
  • a coating created in this way generally has a thickness of a few ⁇ .
  • spacer and receptor takes place via a suitable further functional group, for example an amino or an alkoxy group.
  • suitable bifunctional spacers for coupling a variety of different receptor molecules to a variety of support structure surfaces are well known to those skilled in the art (GT Hermanson, "Bioconjugate Techniques", Academic Press, 1996).
  • biomolecules to be detected are nucleic acids
  • suitable DNA probes can then be applied and immobilized by means of common pressure devices.
  • Biosensors made in this way can now be hybridized with, e.g. biotinylated DNA. This can e.g. be generated by PCR and the incorporation of biotin-dUTP. During hybridization, the biotinylated DNA now binds to the opposite strand on the sensor (if present). Positive hybridization events can now be achieved by the addition of dye conjugates, e.g. Streptavidin / avidin conjugates. Suitable dye conformers according to the invention are particularly suitable: europium, terbium and samarium chelates, Microspheres
  • Beads which are loaded, for example via avidin / streptavidin with Eu, Sm, Tb chelates, said chelates are characterized by their property, with appropriate excitation luminescent light with a half life of the excited state of about 5 ns
  • luminescent ornamental Microspheres such as FluoSpheres Europium (Molecular Probes F-20883), since they are able to immobilize a large number of fluorochromes with one binding event.
  • nanocrystals as described, for example, by Quantum Dot Corp. be offered under the name "Quantum Dots ® ".
  • the binding is measured by means of a suitable excitation and the measurement of the time-resolved fluorescence with the excitation light source switched off.
  • the term “luminescence” encompasses all light emissions (in a broader sense also the emission of ultraviolet and infrared radiation) caused by an excitation source of gaseous, liquid and solid substances which are not caused by high temperatures but by preceding ones
  • fluorescence and fluorophores
  • luminescence can be caused by irradiation from an excitation source with light, ie preferably shorter-wave light and X-rays, photoluminescence, with electrons, eg cathodoluminescence, ions, eg ionoluminescence, sound waves, eg sonoluminescence, with radioactive substances, eg radioluminescence, by electric fields, eg electroluminescence, by chemical reactions, eg chemoluminescence or mechanical processes, eg triboluminescence.
  • the thermoluminescence is luminescence initiated or enhanced by thermal influence.
  • the chemiluminescence is performed by coupling the analytes with an enzyme label capable of catalyzing the chemical reaction of a substrate to luminescent radiation.
  • an enzyme label capable of catalyzing the chemical reaction of a substrate to luminescent radiation.
  • all enzymes are suitable which can catalyze the corresponding excitation of the substrate, e.g. Alkaline phosphatase (AP), horseradish peroxidase and other peroxidases, in particular thermostable peroxidases, glucose-6-phosphatase dehydrogenase or xanthine oxidase.
  • Substrates are all chemiluminescent molecules in question, in particular luminol, isoluminol, lucigenin, Peroxioxalate, acridine esters, thioesters, sulfonamides and Phenantridiniumester.
  • a system consisting of Horseradish peroxidase as an enzyme label conjugated to a receptor for a hapten, and luminol together with hydrogen peroxide as a substrate.
  • Avidin or streptavidin for example, which can then be coupled with an analyte biotinylated with biotin or its derivatives, are suitable as the receptor.
  • Another particularly preferred variant provides a system of alkaline phosphatase with adamantyl - 1, 2 - dioxethanphenylphosphat as a substrate. Again, there is again a conjugation with, for example, avidin, streptavidin or anti-dioxygenin as a receptor. These can then be probed with analytes linked to the corresponding partners, e.g. Biotin or its derivatives and dioxygenin, modified, are coupled. It is preferred that the said enzymes are temperature-stable enzymes. The use of temperature-stable enzymes makes it possible to determine the temperature-dependent parameters of the analytes.
  • Another variant relates to the photoluminescence.
  • a time-resolved fluorescence can be evaluated directly on the chip with analog circuits by recording a value after the excitation source has been switched off, for example every nano-second, which then also has a reference value of a previously performed measurement. solution, which was also stored on the chip, is compared.
  • nonspecific interference signals such as, for example, intrinsic fluorescence from possibly existing system components. Assuming that it is now possible to dissolve in the GHZ range ( ⁇ 1 ns), the autofluorescence can be differentiated from the artificial fluorescence.
  • the detection of the field of view signal values is preferably carried out sequentially, e.g. entire rows or columns of the sensor surface or parts thereof are detected sequentially (multiplex application).
  • the electronic output signals of the detectors can be supplied to an external evaluation device by means of suitable switching devices after analog-to-digital conversion.
  • Suitable optical detectors or sensors according to the invention are, in addition to the photodiode (pn, p-i-n, avalanche) CCD sensors, photoconductors or a camera into consideration, which are preferably incorporated monolithically in the form of a row or array arrangement in the semiconductor substrate of the device.
  • Photodiodes can be advantageously used in the context of a time-resolved luminescence measurement, since they have a low detection surface compared to photomultipliers. Particularly preferred here is the use of CMOS photodiodes or CMOS cameras.
  • the choice of detection or the material depends on the emission wavelength of the dye to be detected.
  • the detector has different wavelength sensitivities due to the so-called “semiconductor bandgap", depending on the choice of material (eg silicon or germanium)
  • a sensitivity range is therefore created which ranges from infrared to into the ultraviolet wave spectrum, with the sensitivity being greatest between these regions (B. Streetman, Pricice-Hall, Inc., “Solid State Electronic Devices", 1995, ISBN 0-13-436379-5, p 201-227).
  • the optionally exposed surface of each photodiode consists of SiO 2 or Si 3 N 4 .
  • certain process parameters of the receptor / analyte binding and the detection can be positively influenced by the choice of the surface material for the sensor chip. For example, in some places Si 3 N 4 , on others SiO 2 or Al 2 O 3 or a noble metal may be applied, whereby on the sensor chip or even in the detection field for biomolecules or spacers preferred areas with eg more hydrophobic or rather hydrophilic properties can be provided in order to promote or prevent the application of, for example, DNA receptors in a location-directed manner.
  • controllable noble metal electrodes it is possible according to the invention to provide preferred devices in which, for example, hybridization events can be accelerated by applying, if necessary, detection points or fields of different voltages, or fluorescence can be triggered from electrically excitable dyes.
  • the detectors can additionally be arranged in groups, whereby individual detection fields are created whose input signals ensure a reliable result than would be the case for a single occupancy per detection range.
  • the individual photodiodes can advantageously be combined into defined detection groups or measurement fields, thereby significantly increasing the sensitivity of the subsequent luminescence measurement as well as the reproducibility and reliability of the measurement data obtained thereby ,
  • the excitation source is an integral part of the detection system and is characterized by the
  • a pn diode made of direct semiconductor material allows the following:
  • the activation means the application of a voltage, whereby a light signal (pn diode is used as an LED) is emitted, which depending on the nature and condition of the pn diode in a certain emission wavelength band and causes the excitation of a ligand bound in the region of this pn diode.
  • pn-diode is used as a photodiode
  • it is then activated again to perform the desired measurement (s).
  • the excitation radiation in the previously described embodiment is coupled in via the same component with which the luminescence radiation is also collected makes it possible to selectively irradiate a very small area of the sensor surface or of the detector field and luminescence radiation emanating from this area is evaluated. By doing so, the inspected detector array is to be imaged very accurately, and a disturbance of the measurement by the luminescence from outside the inspected area can be prevented.
  • CMOS complementary metal-oxide semiconductor
  • Manufacturing processes which are likewise suitable according to the invention are, for example, NMOS processes or bipolar processes (Wolf, Silicon Processing for the VLSI ERA, Vol. 1, Lattice Press, Sunset Beach (1986)).
  • NMOS processes or bipolar processes are, for example, NMOS processes or bipolar processes (Wolf, Silicon Processing for the VLSI ERA, Vol. 1, Lattice Press, Sunset Beach (1986)).
  • organic semiconductor conductors EP-A-085 319
  • the individual detection points or fields are separated from each other in such a way that substantially no light emission of one point or field can be received by the detector or detectors of another point or field.
  • the individual types of detection can be arranged in respective depressions, as known, for example, from conventional microtiter plates.
  • trough-like depressions and those whose lateral walls are arranged substantially perpendicular to the surface of the sensor chip are preferred.
  • the respective dimensions of such a depression can be freely selected by those skilled in the art, as long as the luminophore (s) of the expected ligand / receptor complex are within the depression and essentially no emission light can penetrate into adjacent depressions.
  • a particularly preferred depression is sunk into the surface of the device according to the invention by at least 100 nm.
  • the same effect can alternatively be achieved by arranging on the essentially planar detector surface vertically upwardly directed release agents, the dimensions of which can be easily selected by a person skilled in the art with regard to the desired field of application and the spatial dimension of an anticipated receptor / ligand complex.
  • the attachment according to suitable release agent can be done for example by anodic bonding or by so-called.
  • Flip-chip method Such a system according to the invention allows a sensor-based electro-optical image recording method.
  • channels are applied to the detector chip so that several different analytes are measured in parallel on a chip can.
  • the channels may provide rows of sensor elements to which the arrays of receptors are bound.
  • calibration measurements could be carried out.
  • a parallel measurement of n identical arrays is performed so as to drastically reduce the cost per analysis.
  • the chip is divided by microchannels into, for example, 8 identical compartments.
  • a monolithically integrated semiconductor material is used as a carrier device for the receptors and the formation of the sensors, a monolithically integrated circuit can also be produced on the same substrate, whereby a preprocessing of the electronic sensor output signals in the immediate vicinity of the examination subject (receptor / analyte complex) can be done.
  • this preferred embodiment of the present invention is an "intelligent" sensor device that performs significantly more than purely passive sensors, for example, the outputs of the electro-optical sensors can be conditioned by integrated circuits to provide output circuits and outputs Further, the preprocessing may consist of digitizing the analog sensor signals and converting them to a suitable data stream In accordance with the invention, further processing steps are possible with which, for example, the amount of data can be reduced or that of the external one Processing and presentation via a personal computer (PC) can be done. Furthermore, the device according to the invention can be designed so that the preferably compressed or processed data can be transmitted via infrared or radio link to appropriately equipped receiving stations.
  • PC personal computer
  • control of the associated devices on the substrate can be carried out via control signals from a control device, which may preferably also be wholly or partially formed on the substrate or is connected externally.
  • Data analysis by the use of the device according to the invention is subject to no restrictions compared to data generated using conventional external imaging optics.
  • the direct detection of the luminescences on the device according to the invention is realized in that the receptor molecules required for a specific detection - directly or e.g. over a conventional spacer, i. Spacer, or a coupling matrix - located on the surface of an optical detector, which is designed as an integral part of the device according to the invention.
  • the excitation source as in the form of a or several white light lamps LED's, (semiconductor) laser, UV tubes, and by piezoelectric elements (ultrasound) or by light energy emitting gases and / or liquids (chemical excitation) can be provided should be sufficiently powerful and preferably repetitive with high frequency. The latter property is given when the light source can both be activated and deleted for a short time. If an optical excitation source is used, it should be able to be switched off so that after switching off, essentially no further photons, such as afterglow, strike the detector. If necessary, this can be ensured, for example, by the use of mechanical shutters (“shutter”), as well as by selecting LEDs or lasers as an optical excitation source.
  • shutter mechanical shutters
  • the excitation source with the device is preferably optically and mechanically coupled to the optical detector units in such a way that a radiation field is generated in the direction of the optical sensors, the spatial distance of the excitation source to the detection plane being possibly small. However, the distance must be sufficient so that the reactions between ligand and receptor required for the intended use are not impaired.
  • wavelength-specific photoelements or conventional photodiodes are selected which are applied with applied, applied, upright photodiodes. steamed or integrated wavelength filters. It is known, for example, that silicon nitride, in contrast to silicon oxide, does not penetrate UV light, and that polysilicon absorbs UV radiation (VP Iordanov et al., Integrated high rejection filter for NADH fluorescence measurements, Sensors 2001 Proceedings, Vol. 1, 8-10 May, pp. 106-111, AMA-Service (2001)).
  • nitride or polysilicon can be deposited on the gate oxide layer in the context of the customary CMOS process, whereby corresponding filters are created on the photodiode.
  • NADH nicotinamide adenine dinucleotide
  • the sensitivity can be increased.
  • this effect can be used to enable differential detection in parallel use of, for example, two different luminophores, of which, for example, only one light emits in the UV range, since the detectors provided for this purpose are designed to be UV-sensitive or not.
  • this effect offers the possibility of removing possibly interfering autofluorescences of materials present at a known emission wavelength by providing appropriate filters from the measuring method.
  • An example of this is the parallel use of europium chelates (emission at ca. 620 nm) and copper-doped zinc sulfide (emission at ca. 525 nm), which allow two-color detection by sufficiently different emission wavelength ranges, eg within a range of Detector point or field, for example, by half the sensors of a detector point or field with a low-pass filter and the other half of the sensors of the same point or field with a high-pass filter Is provided.
  • different luminophores can be used in parallel, provided that their physical or optical properties differ sufficiently from one another.
  • the different excitation wavelengths of two luminophores A and B to be used and / or their different half-lives are used. This can e.g. by providing two differently doped nanocrystals.
  • the receptor / analyte-specific detection with this layer of optical sensors can be coated with a substance capable of coupling.
  • the sensor chip surfaces of metal or Halbmetalloxiden such as alumina, quartz glass, glass, in a solution of bifunctional molecules, so-called.
  • Linker for example, a halosilane, eg chlorosilane, or alkoxysilane for coupling to the carrier structure, so that a self-assembling monolayer (SAM) is formed, by which the covalent bond between sensor surface and receptor is produced
  • SAM self-assembling monolayer
  • a coating created in this way generally has a thickness of a few angstroms.
  • a diagnostic device which contains a microoptical detection system as described above.
  • diagnostic devices are meant here all measuring arrangements for which the use of micro-optical detection systems, e.g. in the form of biochips, technically useful and practicable.
  • handheld devices which are used in the field, i. e.g. in a hospital or in a medical practice, can be used in portable use.
  • the invention also provides a method for determining temperature-dependent parameters of analytes. This is based on the following process steps:
  • receptors for the analytes are bound to at least one surface of a support structure, wherein the receptors form a plurality of measurement points.
  • C) The receptor-analyte complexes are stimulated with at least one excitation source to cause a detectable optical change of the complex, the receptor or the analyte.
  • D) The optical change caused is then registered with at least one detector monolithically integrated in the carrier structure and directed onto the surface of the carrier structure and subsequently evaluated.
  • a particular feature of the method according to the invention is that the excitation and the detection takes place at at least two different temperatures in order to register and evaluate the temperature-dependent parameters at the at least two temperatures.
  • the method steps described above are carried out at different temperatures under otherwise identical conditions.
  • a temperature program so that, for example, in a temperature window of 20 to 80 0 C in 10 0 C steps, the temperature is increased and for these respective temperature levels, the detection method for the analyte is performed.
  • This then allows the determination of a melting curve of the analytes, which reflects the temperature dependence of the dissociation of the receptor-analyte complexes.
  • the detection takes place in the form of a
  • the association constant, the dissociation constant and / or the equilibrium constant can be determined as temperature-dependent parameters.
  • the method according to the invention is used in all areas in which the determination of temperature-dependent parameters of analytes is of importance. Concrete examples of such applications are pathogen detection in hospitals, proof of paternity, evidence of perpetrators or a P450 isoenzyme analysis. In this regard, especially the malignant hyperthermia, which is based on a mutation of the ryanodine receptor, to call.
  • the detection system according to the invention can enable early detection of hyperthermia here.
  • Another important field of application is the monitoring of the blood coagulation cascade in order to be able to recognize and treat the risk of thrombosis in patients with an increased tendency to coagulate in a timely manner.
  • FIG. 1 shows a schematic representation of the structure of a variant of the optical detection system according to the invention.
  • FIG. 2 shows a schematic representation of the detection of proteins using the microoptical detection system according to the invention.
  • FIG. 3 shows a schematic representation of the detection of nucleic acids with a microoptical detection system according to the invention.
  • Fig. 1 is a micro-optical according to the invention Detection system 1 shown.
  • a support structure 2 which consists of the known from the prior art materials for the production of chips. These include, in particular, a carrier structure made of polychlorinated biphenyl (PCB).
  • PCB polychlorinated biphenyl
  • a detector 3 is arranged, in the present case in the form of a single sensor chip.
  • This variant concerns a chemoluminscence measurement.
  • an excitation source can additionally be contained in the sensor chip 3.
  • the sensor chip 3 can also be integrated directly into the substrate.
  • the sensor chip is attached to the substrate via two adhesion sites 8 and 8 ', which can be made of an adhesive or a solder, for example.
  • a flow cell 4 is arranged, which serves for the continuous contacting of a fluid with the surface of the sensor chip.
  • the flow cell in this case has an inflow 6 and an outflow 7, via which the fluid can be transported into or out of the flow cell.
  • a temperature control element 5 is integrated in the flow cell 4, which allows a temperature increase or a decrease in the temperature of the fluid.
  • the tempering element 5 can also be used for tempering the surface of the support structure 2 or the sensor chip.
  • the arrangement of the tempering 5 is therefore arbitrary, as long as the arrangement allows a corresponding temperature.
  • FIG. 2 shows the measuring principle for the determination of proteins.
  • a detector 3 in the form of a photodiode is integrated here.
  • a primary antibody 9 is immobilized, which acts as a receptor. The receptor immobilized in this way is then brought into contact with the sample containing the analyte 10, whereby binding between receptor 9 and analyte 10 takes place.
  • the surface of the chip is then brought into contact with a detector molecule which can bind to the analyte 10.
  • This detector molecule consists of a receptor 11, in the present case a secondary antibody, and a thermally stable enzyme 12 coupled thereto, which can catalyze an optical detection reaction.
  • horseradish peroxidase is used as the thermally stable enzyme.
  • the substrate consisting of hydrogen peroxide and luminol is added. The associated luminous reaction can then be registered and evaluated by means of the photodiode 3.
  • a support structure 2 is shown, in which a photodiode is integrated as a detector 3. in the surface of the support structure, a DNA receptor 14 is immobilized here.
  • the sample is then brought into contact with the analyte 15 in the form of a DNA molecule with the biochip.
  • the DNA molecule can be labeled by biotin-dUTP.
  • the substrate consisting of luminol and hydrogen peroxide is then added.
  • the detection principle also corresponds here to that described under FIG. example 1
  • Microorganisms are processed in a suitable extraction system (Buchholz et al., 2002).
  • PCR all known and unknown bacteria can be detected by PCR using suitable primers (consensus primer) and their 16s rRNA amplified.
  • the PCR is preferably carried out asymmetrically, ie one of the two PCR primers is deficient in the reaction, so that single-stranded DNA is formed in addition to double-stranded DNA.
  • biotin-dUTP the DNA molecules are labeled. These labeled molecules are then hybridized on the chip. The temperature is below the expected melting point (eg 20 0 C lower than the melting point).
  • the flow cell of the chip is rinsed with wash buffer and streptavidin-HRP is added. After about 10 minutes, the unbound HPR is removed by washing with wash buffer and ECL substrate is added (luminol plus hydrogen peroxide). After the onset of the light reaction, the temperature is gradually increased from 20 0 C to 80 0 C and the signal is measured at each Tempe- ratur intimid. Thereby, a slow perfusion of the flow cell takes place so that separated analyte no longer disturbs the measurement.

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Abstract

L'invention concerne un système de détection micro-optique et un procédé pour mettre en évidence des analytes par luminescence à résolution temporelle, afin de déterminer des paramètres d'analytes variables avec la température, notamment des mutations ponctuelles d'acides nucléiques (ADN), pour lesquelles une détection à résolution temporelle est nécessaire.
EP06724318A 2005-04-20 2006-04-13 Systeme de detection micro-optique et procede pour determiner des parametres d'analytes variables avec la temperature Withdrawn EP1872127A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102005018337A DE102005018337A1 (de) 2005-04-20 2005-04-20 Mikrooptisches Detektionssystem und Verfahren zur Bestimmung temperaturabhängiger Parameter von Analyten
PCT/EP2006/003427 WO2006111325A1 (fr) 2005-04-20 2006-04-13 Systeme de detection micro-optique et procede pour determiner des parametres d'analytes variables avec la temperature

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EP1872127A1 true EP1872127A1 (fr) 2008-01-02

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US (1) US20080160548A1 (fr)
EP (1) EP1872127A1 (fr)
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WO (1) WO2006111325A1 (fr)

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CN102192902B (zh) * 2010-03-19 2013-12-11 瑞鼎科技股份有限公司 生化检测单元及其生化仪器
CA2751947C (fr) * 2010-09-29 2018-10-16 Econous Systems Inc. Revetement anticorps a surface orientee pour la reduction d'une restenose apres la pose d'une endoprothese
US9140684B2 (en) 2011-10-27 2015-09-22 University Of Washington Through Its Center For Commercialization Device to expose cells to fluid shear forces and associated systems and methods
SG11201608897SA (en) * 2013-06-26 2016-12-29 Univ Washington Ct Commerciali Fluidics device for individualized coagulation measurements
CN114381363B (zh) * 2021-12-28 2024-04-30 深圳市思坦科技有限公司 Pcr快速检测系统制备方法及pcr快速检测系统

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US20080160548A1 (en) 2008-07-03
DE102005018337A1 (de) 2006-11-02

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