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EP1192007B1 - Dispositif matrice a microcircuit integre destine a l'amplification et a la caracterisation d'acides nucleiques - Google Patents

Dispositif matrice a microcircuit integre destine a l'amplification et a la caracterisation d'acides nucleiques Download PDF

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Publication number
EP1192007B1
EP1192007B1 EP00952983A EP00952983A EP1192007B1 EP 1192007 B1 EP1192007 B1 EP 1192007B1 EP 00952983 A EP00952983 A EP 00952983A EP 00952983 A EP00952983 A EP 00952983A EP 1192007 B1 EP1192007 B1 EP 1192007B1
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EP
European Patent Office
Prior art keywords
chip
chamber
nucleic acids
sample
optically transparent
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.)
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EP00952983A
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German (de)
English (en)
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EP1192007A1 (fr
Inventor
Ralf Ehricht
Thomas Ellinger
Jens Tuchscherer
Eugen Ermantraut
Siegfried Poser
Torsten Schulz
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Clondiag Chip Technologies GmbH
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Clondiag Chip Technologies GmbH
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30—Micromixers
    • B01F33/3031—Micromixers using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/142—Preventing evaporation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0418—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic electro-osmotic flow [EOF]

Definitions

  • the invention relates to a device for reproduction and Characterization of nucleic acids.
  • DNA deoxyribonucleic acid
  • PCR polymerase chain reaction
  • This routine two-stage duplication enables an enormously large number of identical molecules to be produced from a few starting nucleic acid molecules, but has the disadvantage that it is very laborious and time-consuming, has a low sample throughput (the number of processed nucleic acids in a unit of time) and therefore very much is expensive.
  • the one-step duplication by PCR is relatively fast, enables a high sample throughput in small batch volumes due to miniaturized processes and is not so labor intensive due to automation. Characterization of nucleic acids by duplication alone is not possible. Rather, after duplication, it is necessary to use analysis methods, such as nucleic acid sequence determinations or electrophoretic analyzes of the PCR products or their enzymatically produced individual fragments, to characterize the PCR products.
  • thermocyclers for carrying out the PCR.
  • 5,856,174 discloses a system with which it is possible to pump sample liquids back and forth between, for example, three miniaturized chambers.
  • the PCR is carried out in one chamber of this system, a workup reaction is carried out in the next and the reaction products are detected in the third, for example with a DNA chip.
  • the PCR chamber is a standard tube, as described in the literature (S. Poser, T. Schulz, U. Dillner, V. Baier, JM Köhler, D. Schimkat, G. Mayer, A. Siebert; Chip elements for fast thermocycling, Sensors and Actuators A, 1997: 62, 672-67).
  • the genetic characterizations e.g. for identification and Taxonomic classification of microorganisms is currently taking place based on DNA-DNA hybridization studies, rRNA gene sequence comparison (e.g. by means of the 16S or 23S rRNA gene segments) after sequencing these sections and using Restriction fragment length polymorphism (RFLP) studies or PCR tests using specific primers gel electrophoretic separation and detection of restriction or PCR products (T. A. Brown, 1996, genetic engineering for beginners, Spectrum Academic Publishing House Heidelberg, Berlin Oxford).
  • RFLP Restriction fragment length polymorphism
  • the known RFLP studies are based on an individual-specific distribution of restriction endonuclease interfaces, which relates to DNA sequence differences in the area of genomic DNA, which has a high degree of homology to a labeled DNA probe used for hybridization (TA Brown, 1996, Genotechnologie für beginnerers, Spectrum Academic Publishing House Heidelberg, Berlin Oxford).
  • the RFLP examination which is used, for example, in HLA diagnostics (human leukocyte antigen) in immunology prior to transplantation or transfusion (cf.
  • Gene probes are single-stranded nucleic acid molecules of a known nucleotide base sequence with an optimal length of 100 to 300 bases, which specifically lead to a double-stranded nucleic acid pairing with single-stranded nucleic acid sections, e.g. a gene, and mostly with a non-radioactive or radioactive reporter element (marker), e.g.
  • a radionucleotide dye which serve the detection of the gene probes.
  • hybridization a distinction is made between the hybridization of probes with isolated single-stranded nucleic acid (DNA or RNA) and the so-called in situ hybridization (on-site hybridization in tissues, cells, cell nuclei and chromosomes), in which the gene probe spreads to one in the cell (Single-stranded) nucleic acid (DNA or RNA) couples (Leitch, AR, Schwarzacher, T., Jackson, D. and Leitch IJ, 1994, in situ hybridization, Spektrum Akademischer Verlag Heidelberg, Berlin Oxford). With this in situ hybridization it is particularly important that the target sequence and the tissue morphology are preserved and that the preserved tissue is permeable to the probe and the detection reagents.
  • in situ hybridization it is particularly important that the target sequence and the tissue morphology are preserved and that the preserved tissue is permeable to the probe and the detection reagents.
  • Essential for the hybridization is the presence of single-stranded nucleic acid target and nucleic acid probe molecules, which usually occurs through heat denaturation, as well as the selected optimal stringency (setting of the parameters: temperature, ionic strength, concentration of helix-destabilizing molecules), which ensures that only probes with almost perfectly complementary ones (corresponding) sequences remain paired with the target sequence (Leitch, AR, Schwarzacher, T., Jackson, D. and Leitch IJ, 1994, in situ hybridization, Spektrum Akademischer Verlag Heidelberg, Berlin Oxford).
  • Classic applications of probe technology that enable the identification of unknown organisms or the detection of certain organisms in a mixture of organisms are, for example, phylogenetic studies or the detection of germs in medical diagnostics.
  • rRNA ribosomal RNA
  • rDNA ribosomal RNA
  • the rDNA contains flanking sequence sections that are highly conserved within the respective organism kingdom. Primer sequences directed against these sections can be used for species-independent amplification of the rDNA (G. Van Camp, S. Chapelle, R. De Wachter; Amplification and Sequencing of Variable Regions in Bacterial 23S Ribosomal RNA Genes with conserved Primer Sequences.
  • McCabe Bacterial species identification after DNA amplification with a universal Pprimer pair. Mol. Genet. Metab, 1999; 66: 205-211) describe a method in which rDNA is amplified from clinical bacterial isolates lysed on filter spots by using universal primers and then identified by hybridization with specific probes. This process is sensitive; however, the number of species to be detected is also relatively narrow.
  • Oyarzabal and co-workers Oyarzabal and co-workers (OA Oyarzabal, IV Wesley, KM Harmon, L. Schroeder-Tucker, JM Barbaree, LH Lauerman, S.
  • the invention has for its object a device for Specify the duplication and characterization of nucleic acids, which has an almost simultaneous duplication and characterization allows a high sample throughput, and thus the disadvantages of Bypasses state of the art.
  • the task is characterized by the distinctive features of the first Claim resolved. Advantageous refinements are due to the subordinate claims recorded.
  • the essence of the invention is that the device spatially combines the PCR and the parallel hybridization against chip-bound nucleic acid in a temperature and flow controllable cell (chamber).
  • the inside of the chamber has a chip that generates a capillary gap between the chamber bottom and the detection surface of the chip, which receives the sample liquid, the sample liquid being mixed by an induced electroosmotic flow.
  • the chamber around the capillary gap and the chip advantageously forms a gas reservoir through which a gas reservoir leads to the capillary gap, which separates an inlet from an outlet, so that the samples can be injected via the inlet, due to the capillary forces from the inlet into the capillary gap arrive and can be removed from it via the outlet.
  • the capillary gap When the capillary gap is filled, an air gap is generated as a ring around the chip stored in the chamber and the capillary gap (which serves as a sample reservoir) due to surface tension effects, so that the chip and the capillary gap are thermally insulated from the chamber body, which leads to the fact that the Samples in the chamber gap can be quickly heated and cooled by heating and cooling elements, which, together with temperature sensors and electrodes, are placed on a chamber support that holds the chamber and is in heat-conducting contact with it over the chamber floor. Because the capillary gap serves as a sample reservoir, the evaporation rate of the sample liquid is greatly reduced even at temperatures near the boiling point of the sample liquid, since the sample can only evaporate over the edge of the capillary gap.
  • the capillary gap (the sample reservoir) is the location of the nucleic acid amplification in the sample liquid by PCR with specific primers and the genetic characterization of the sample.
  • the labeled PCR products are fished out of the sample liquid by the immobilized specific probes, which are bound on the nucleic acid chip.
  • the chamber and the chip are optically transparent and, due to their design, enable the on-line detection of the marking signal of the PCR products bound to the probes.
  • the device according to the invention has the advantage over the previously used methods that a maximum genetic typing using specific probes can be automated in a minimal diagnosis time with minimal sample volumes and is possible with a high sample throughput in a temperature and flow controllable cell, with PCR being used to highlight the diagnosis relevant gene structures against a sequence background and the almost simultaneous, parallel hybridization of the PCR products against the chip-bound nucleic acid results in a specific detection.
  • the device according to the invention finds e.g. for the simultaneous Detection of various microbial pathogens (e.g. based on 16S or 23S rRNA analysis), the screening for Resistance of individual pathogenic microorganisms or a genomic typing of diagnostically relevant allele structures of Eukaryotic cells use, the parallel recognition by the chip with its different, for the different Target sequences specific probes is made possible.
  • various microbial pathogens e.g. based on 16S or 23S rRNA analysis
  • Characterization of nucleic acids consists of a chamber body 1 and a chamber carrier 5.
  • the chamber body 1 is provided with a bearing surface 4, via which it is sealingly connected to the chamber carrier 5, so that a sample chamber 3 is formed.
  • This sample chamber 3 consists of a gas reservoir 6 and a capillary gap 7 and is provided with at least one inlet 81 and at least one outlet 82.
  • the inlet 81 and the outlet 82 lead into the sample chamber 3 and are spaced apart by an intermediate gas reservoir nose 9 of the gas reservoir 6.
  • This chip 2 which carries detection surfaces 12 in the form of spots 13, is held in the chamber body 1 in such a way that the detection surfaces 12 in the form of spots 13 face the surface of the chamber carrier 5 and through the edge 42 of the chamber body 1 from the chamber carrier 5 are positioned evenly spaced so that the chip 2 and the chamber carrier 5, as shown in FIG. 2, generate the capillary gap 7, which serves as a sample reservoir.
  • This capillary gap 7 receives the sample liquid 19.
  • the chamber body 1 consists, for example, of optically transparent plastic or glass, the sample chamber 3, which represents a space for filling the sample liquid 19, by milling, and the inlet 81 and the outlet 82, which are routes for the sample liquid, by drilling into the Chamber body 1 can be introduced.
  • the nucleic acid chip 2 consists of an optically transparent support, the material of which can be, for example, silicon or glass, and of nucleic acid molecules of a specific sequence (for example probes) immobilized on this support.
  • the sample chamber 3 comprises the gas reservoir 6 and the capillary gap 7, gas and air bubbles collecting in the gas reservoir 6 when filling the sample liquid 19 due to surface tension effects, so that the chip 2 and the capillary gap 7 are thermally insulated from the chamber body 1.
  • the capillary gap 7, which forms the sample reservoir (for example with a volume of 1.8 ⁇ l), ensures that the detection surface 12 is completely wetted with the sample liquid 19.
  • the inlet 81 and the outlet 82 serve to direct the sample liquid 19, which enables filling and emptying of the sample chamber 3, and thus also filling and emptying of the capillary gap 7 as a result of the acting capillary forces.
  • the inlet 81 and the outlet 82 which can for example run side by side as shown in FIG. 1, are spatially separated from one another by a gas reservoir 9, so that the sample liquid 19 is prevented from flowing from the inlet 81 to the outlet 82 without entering the Capillary gap 7 to arrive.
  • the chamber support 5, which is optically transparent and has good thermal conductivity, is made, for example, of glass and, as shown in FIGS.
  • the chamber body 1 can be provided with the means for applying temperature 17 and the miniaturized temperature sensors 16 and the electrodes of the quadropole 18.
  • the temperature sensors 16 can be designed, for example, as nickel-chrome thick-film resistance temperature sensors.
  • the length of the temperature sensor 16 is, for example, in the case that the chamber support 1 has an area of 8 x 8 mm and the chip 2 has an area of 3 x 3 mm or less, 10.4 mm and the width of the temperature sensor 16 in this example 50 ⁇ m, so that the resistance at 20 ° C is 4 kOhm and the temperature coefficient TK R at 0 ° C is 1500 ppm.
  • the temperature sensors 16 can also be designed as optically transparent thin layers.
  • the means for applying temperature 17 can, for example, be designed as a nickel-chrome thick-film resistance heater.
  • the means for applying temperature 17 have a length of 2.6 mm and a width of eight individual webs, each 50 ⁇ m wide, so that the resistance at 20 ° C. is 300 ohms.
  • the means for applying temperature 17 can also be designed as optically transparent thin layers.
  • the quadrupole 18 can be designed, for example, as gold-titanium electrodes. In the dimensions of the previous example, these electrodes have a length of 2.2 mm and a width of 0.5 mm.
  • the quadropole serves to induce an electroosmotic flow, which leads to the mixing of the sample liquid 19 in the sample chamber 1.
  • the quadruple 18 can also be designed as an optically transparent thin layer.
  • Fig. 2 shows the chamber body 1, which over the bearing surface 4 with the chamber support 5 is in rigid, non-detachable connection.
  • This Connection can be made, for example, by gluing.
  • the capillary gap 7 which serves as a sample reservoir, which Due to its capillary action it is able to extract sample liquid from the Record sample chamber 3.
  • the inlet 81 and the outlet 82 lead in the gas reservoir 6 of the sample chamber 3, so that through the inlet 81st Sample liquid 19 via the gas reservoir 6 into the capillary gap 7 can be filled and discharged via the outlet 82.
  • the chip 2 is like the chamber support 1 made of optically transparent or transparent Material such as Glass, so a conical opening in the Chamber body 1, the continuous cone forming a viewing cone Recess 11, optical and photometric evaluations, such as. Fluorescence measurements from which the detection surface 12 are possible.
  • Fig. 3 shows the inlet 81 and the outlet 82 and the Recess 11 through which the detection surface 12 with spots 13 of the Chips 2 are optically accessible. This visual accessibility enables the above optical and photometric evaluations of the Signals emanating from the detection surface 12, which are not in the example shown fluorescence signals.
  • the means for applying temperature 17 consist of eight individual microstructured in parallel Resistance heating lines 171 through which the under the chamber body 1st located chamber support 5 and with it the filled Sample liquid 19 in the capillary gap 7 is homogeneously enicable.
  • the Resistance line 171 of the means for applying temperature 17, those with a different, definable temperature can be acted upon have dimensions such that the above optical Accessibility of the detection surfaces 12 of the chip 2 is ensured.
  • the temperature sensor 16 is around the detection surface 12 of the chip 2 is mounted, so that said optical accessibility of the detection surface 12 is ensured.
  • the Temperatucleler 16 are by a not shown in the figure Passivation layer electrically opposite subsequent elements of the Device 20 and opposite the sample liquid 19 electrically isolated.
  • Fig. 7 shows the positioning of the temperature sensor 16 on the Chamber body 1 facing surface side of the chamber support 5, which is also the surface side of the chamber support 5 with the chip 2 held by the chamber body 1 has the capillary gap 7 generated.
  • Fig. 8 shows one on the, not shown in detail Passivation layer of the temperature sensor 16 applied Quadrupole 18 including assigned conductor tracks 1518 and Pads 1418.
  • Quadrupole 18 is electrically conductive Contact with the sample liquid 19 so that the alternating Applying a voltage of +1 V to two electrodes 181 of the Quadrupole 18 an inducible by the electroosmotic flow Swirling in the capillary gap 7 filled with sample liquid 19 can be caused.
  • Another pair of electrodes 181 of quadrupole 181 put under tension, so change Turbulence conditions. By constantly alternating the pairs electrodes 181, which are energized, are made effective mixing of the sample liquid 19.
  • the applied Low voltage of only one volt prevents the Sample liquid 19 in the capillary gap 7 electrochemical Changes are subject to and gas bubbles form, for example.
  • the Quadrupole 18 is designed, as shown in this figure, that the optical accessibility of the detection surface 12 ensures is.
  • the quadrupole 18 can also be an optically transparent one Thin layer.
  • FIG. 9 shows the positioning of the quadrupole 18 on the surface side of the chamber carrier 5 facing the chamber body 1.
  • FIGS. 10a and b schematically show the sample liquid 19 stored in the capillary gap 7 between the chamber body 1 and the chamber carrier 5. Due to the size of the gas reservoir 6, driven by the minimization of the interfacial energy, any air bubbles, not shown in detail, can be discharged from the capillary gap 7 into the gas reservoir 6 of the sample chamber 3. This forms an air ring around the sample liquid 19, which thermally insulates it and the chip 2 from the chamber body 1, so that the sample liquid 19 can be quickly heated and cooled in the capillary gap 7 with low energy consumption.
  • the evaporation rate of the sample liquid 19 is greatly reduced even at temperatures near the boiling point, since the sample liquid 19 can only evaporate over the edge of the capillary gap 7.
  • the need for sample liquid 19 is low (in the .mu.l range) sample reservoir 7, since the capillary gap 7 forms only a small volume, which means that the required sample volumes are very small.
  • the heating and cooling rates customary for microthermal cyclers described by Posner and others can be achieved (S. Poser, T. Schulz, U. Dillner, V. Baier, JM Köhler, D. Schimkat, G. Mayer, A.
  • FIG. 11 shows the installation of the device 20 for the duplication and characterization of nucleic acids in an analysis system 200.
  • the analysis system 200 consists of a temperature controller 21, a mixing control 22, electrical lines 23, 24, 33, 34, a total inlet 25, a waste container 26 , a conditioner 27, valves / pumps 28, storage containers 29, connecting hoses 30, a conditioner control 31, an automatic control 32, a control computer 35, a computer bus 36 and an automatic pipetting device 37.
  • the device 20 is directly connected via the inlet 81 and the outlet 82 , Conditioner 27 and the waste vessel 26 and via the electrical lines 23 and 24 directly to the temperature controller 21 and the mixing control 22 in connection, the temperature controller being coupled to the temperature sensors 16 and the means for applying temperature 17 and the mixing control to the Quatrupol 18.
  • the sample liquid 19 can be pipetted into the total inlet 25 via the automatic pipetting device 37 from microplates not shown in detail.
  • the sample liquid 19 can be conducted through the connecting hoses 30 into the conditioner 27, the conditioner 27 being used for processing the sample liquid 19 (for example pH adjustment and Filter out interfering substances).
  • the buffer liquids and reaction solutions for this workup can be supplied from the storage containers 29, which are in a liquid-conducting connection with the conditioner 29.
  • the automatic pipetting device 37 and the conditioner 29 are connected to the conditioner control 31 and the machine control 32 via the electrical lines 33 and serve to control and regulate them.
  • the sample liquid 19 can be tempered and mixed in the area of the capillary gap 7 by means of the temperature controller 21 and the mixing control 22.
  • the capillary gap 7 is therefore the site of the amplification and characterization of a nucleic acid, in the example the target DNA.
  • Figures 12a to c show an example of an embodiment of the device 20 that the chamber body 1 has a length and width of 8 mm and a height of 3 mm, the gas reservoir length and width of 5.4 mm and a height of 0.5 to 0.8 mm, the chamber support 5 has a thickness of 0.9 mm, the recess 11 on its side facing the chip 2 has a diameter of 2.8 mm and the inlet 81 and the outlet 82 have a diameter of 0.5 mm have, the inlet 81 and the outlet 82, and the recess 11 with respect to the chamber support 5 have an inclination of 70.
  • the device 20 shows the optical beam path through a further embodiment of the device 20, in which the support surface 4 is detachably and sealingly connected to the chamber support 5 via an additional sealing surface 43, for the dark field fluorescence image of the detection surface 12 chips 2.
  • the excitation light is directed by the dark field mirror 38 onto the detection surface 12 along the excitation light beam path 39.
  • the fluorescent light emanating from the detection surface 12 is directed along the detection light beam path 40 onto a microscope objective 41.
  • the distance between the dark field mirror 38 and the detection surface 12 is approximately 4.6 mm and the distance between the detection surface 12 and the microscope objective 41 is approximately 22.0 mm.
  • the optical readout of the interaction signal between the target DNA 50 shown in FIG. 14 and the probe DNA 56, 57, 58, 59 on the surface of the chip 2 can take place online due to the construction of the device 20.
  • the chip 2 is held in the chamber body 1 in such a way that it can be irradiated by light in a wide solid angle, so that the hybridization can be tracked online or in situ by means of the marked probes 56, 57, 58, 59, for example fluorescence measurements.
  • the arrangement and size of the temperature sensor 16 and the quadrupole 19 is designed in such a way that the beam path for the online detection or the subsequent in situ detection is not disturbed and the detection of the interactions on the spots 13 by all forms of optical detection or spectroscopy (for example, photometry, differential photometry, confocal fluorescence measurement, dark field fluorescence measurement, transmitted light fluorescence measurement, incident light fluorescence measurement, etc.) can be evaluated, whereby the label 60 and measurement method must be coordinated.
  • optical detection or spectroscopy For example, photometry, differential photometry, confocal fluorescence measurement, dark field fluorescence measurement, transmitted light fluorescence measurement, incident light fluorescence measurement, etc.
  • Fig. 14 shows the schematic representation of the chip 2, which bears the primers 54 (A) and 53 (B '), these showing the specific sequence region of the target DNA 50, ie the sequences A, X, S1, X, B and B ', X, S1', X, A '.
  • sequences A and B or A 'and B' define the region of the target DNA 50, which is identical for all species, or of the single-stranded AB target DNA 51 and A'B 'target DNS 52
  • the primers 53 and 54 carry, for example, a fluorescent label 60 which can be incorporated into the secondary amplification products 61 and 62 by the amplification process, as a result of which the hybridization to the probes 56 and 57 can be detected during amplification by fluorescence measurement, so that the decision is made possible whether the target DNA 50 has the sequence S1 or S1 'and / or the sequence S2 or S2' between the sequence areas A and B or A 'and B'. Since the probe sequences can be specific for a particular species, for example, this method can be used to provide evidence of the presence of a particular species in a sample.
  • Figure 15 shows the schematic representation of the secondary and tertiary Amplification products 61, 62 and 63, which are generated by the device 20
  • the amount of secondary amplification product 61 and 62 is from the second reaction cycle within the Capillary gap 7 almost doubled with each cycle, so that the Concentration of secondary amplification product after a few Cycles sufficient to attach to probes 56, 57 attached to spots 13 are immobilized to hybridize, extending the Probes 56, 57 complementary to the secondary amplification product 61, 62 takes place.
  • This tertiary amplification product 63 out Probes 56, 57 and secondary amplification product 61, 62 can, for example. via a label 60, which is coupled to the primers 53, 54 used, be detected by means of fluorescence detection.
  • the chip 2 of the device 20 is a DNA chip in this example and serves, during or after the DNA amplification, for the detection of the amplification products and possibly also for the provision of solid phase coupled DNA primers (Figs. 14 and 15).
  • a sequence S1 which is specific for a species for example Escherichia coli
  • the thermal amplification process for example PCR
  • immobilization of the corresponding probes on chip 2 can be used to identify all species, strains and Detect diseases in parallel with only one thermal amplification reaction in the device 20.
  • the range of applications can be expanded by using several pairs of primers 53, 54.
  • the fluorescence detection of the tertiary amplification products 63 is carried out by means of fluorescent labeling 60 of the primers 53, 54.
  • Other types of labeling such as intercalators, radioisotopes, FRET systems, fluorescence-labeled nucleotides, etc., are not thereby excluded.
  • the molecular biological process taking place in the device 20 will be described below with reference to FIGS. 14 and 15.
  • the target DNA 50 originating from a biological sample is added to the sample reservoir (the capillary gap) 7 together with primers 53, 54, which can be labeled 60.
  • the spots 13 of the chip 2 on the detection surface 12 carry, via spacers 55, probe DNA with sequences S1, S1 ', S2, S2' etc., which are characterized in that they can be complementary to those in the target DNA 50 occur.
  • the target DNA contains 50 sequences that are complementary to probes 56 and 57. Each sequence S1, S1 'and S2, S2', etc.
  • the probes (56, 57, 58, 59) was chosen in such a way that it is specific for a specific problem. If, for example, certain pathogens are to be detected by means of the device 20, S1 and S1 'are specific for the Bacillus cereus pathogen , S2 and S2' for the Campylobacter jejuni pathogen etc. If there is only the Bacillus cereus pathogen in a stool sample, so After the sample has been properly processed, there will be a target DNA 50 in the sample liquid which only contains the sequences S1 and S1 '. In order to make them detectably hybridize on the detection surface 12, the number of copies of target DNA 50 must generally be increased significantly.
  • a noise-suppressing, specific DNA amplification method is therefore carried out in the sample reservoir (capillary gap) 7.
  • two primers 53, 54 with sequences A and B ', which are the same for all pathogens, are selected, which frame all possible pathogen-specific probe sequences (S1, S2, S3 ...) (as in Fig. 14) Sequences S1 and S1 'are framed by sequences A and B').
  • the target DNA 50 is denatured at approx. 90 ° C
  • the primers 53, 54 aneal at approx. 65 ° C at B or A 'and it becomes a primer at approx.
  • a second application example describes a parallel detection of bacterial pathogens in stool samples:
  • the chip 2 of the device 20 is a DNA chip and serves for the parallel detection of several bacterial pathogens in human or animal stool samples.
  • the total DNA from each stool sample is isolated using standard techniques (eg using the Qiagen kit provided for this).
  • the DNA is taken up in a volume of a standardized, optionally commercially available buffer system suitable for use in the device 20, in which a PCR amplification can be carried out.
  • this contains at least one thermostable polymerase, an optionally isomolar mixture of the four natural deoxynucleotide triphosphates, a divalent salt, optionally components to increase the effectiveness of the PCR, and building blocks for labeling the PCR products (e.g. fluorescence-biotin or similarly labeled deoxynucleotide triphosphates ).
  • a chip 2 is used, on the surface of which oligonucleotide probes 56, 57, 58, 59 are immobilized, which are complementary to one or more variable regions of the 16S rRNA genes and / or the 23S rRNA genes and / or the internal genes Ranges between 16S and 23S rRNA genes of different organisms to be detected are.
  • the probes 56, 57, 58, 59 are directed, for example, against one or more of the corresponding sequences from Aeromonas spec.
  • the oligonucleotide probes 56, 57, 58, 59 are arranged in spots 13, so that each individual spot 13 contains a multiplicity of oligonucleotide probes (for example the probe 56) of the same sequence.
  • the probes 56, 57, 58, 59 are immobilized either at their 3 'end or at the 5' end or at an internal position, the 3 'end of the probes 56, 57, 58, 59 possibly blocking, for example by amination is so that it can not serve as a substrate for DNA polymerases.
  • each of the probes has a high sequence specificity for the organism to be detected and on the other hand there are motifs in the genomes of the germs at a short distance from the binding site of the specific probes all or for groups of the organisms to be detected have the same sequence.
  • universal primers 53, 54 are directed, which are suitable for PCR amplification of a sequence section, which contains the binding site of the probes immobilized on chip 2, in all organisms to be detected.
  • This Primers 53, 54 are added to the DNA isolated from the stool sample and taken up in the amplification solution (sample liquid 19).
  • the primer 53, 54 which specifies the synthesis of the strand which contains the sequence complementary to the sample immobilized on the chip 2 during the subsequent PCR amplification, can be added as a labeled component.
  • the amplification mixture is filled into the device 20 provided with a chip 2 as described.
  • the solution in the device 20 is subjected to a cyclic temperature regime, so that the target DNA 50 is amplified according to a typical PCR mechanism and, if necessary, simultaneously labeled.
  • there is a hybridization step in which the target sequences amplified with the universal primers 53, 54 hybridize with the specific probes 56, 57, 58, 59 immobilized on the chip 2.
  • a rinsing step follows in which DNA molecules which are not linked to the chip and are bound non-specifically are removed. Subsequently, the marking remaining on the chip 2 is detected. Organisms present in the stool sample are identified by marking the sample spots 13 specific to them on the chip 2.
  • sample liquids 19 for example, from stool samples or tissue a large number of processing steps are required. It cells have to be broken down, proteins, lipids and solids are separated and the DNA is worked up and cleaned. The enzymes, primers and necessary for the use of the device other substances must also be in the sample liquid 19 are fed. These steps can be done by installing the Device 20 for the duplication and characterization of Nucleic acids in the analysis system 200 that i.a. from pumps and Valves 28 that move and control the liquids, from filters and Reaction chambers (conditioner 27) in which the individual Process steps are carried out sequentially and from Storage containers 29, which supply the chemicals required for this, exists (shown in Fig. 11), automatically and continuously.
  • the Device 20 for the duplication and characterization of Nucleic acids in the analysis system 200 that i.a. from pumps and Valves 28 that move and control the liquids, from filters and Reaction chambers (conditioner 27) in which the individual Process steps are carried out sequentially and from Storage containers 29, which supply the chemicals required for this, exists (show
  • the samples are not made from one by a pipetting robot 37 Standard delivery system shown in detail in the Total inlet 25 filled for conditioning.
  • the through that Analysis system 200 processed samples pass through inlet 81 in the device 20, so that a duplication and Automated characterization of sample nucleic acids can be carried out.
  • the whole process is done by one Control computer 35 monitors the over a computer bus 36 with electronic controllers and control devices 21, 22, 31, 32 is connected.

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  • Dispersion Chemistry (AREA)
  • Physics & Mathematics (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Claims (19)

  1. Dispositif de multiplication et de caractérisation d'acides nucléiques dans un espace de réaction,
    caractérisé en ce qu'un corps optiquement transparent de chambre (1) qui contient une microplaquette optiquement translucide (2) comportant des acides nucléiques qui lui sont liés et une surface de détection (12), est disposé sur un porte-chambre optiquement transparent (5) de façon à faire étanchéité, de manière telle qu'une chambre d'essai (3) avec une fente capillaire (7) entre le porte-chambre (5) et la surface de détection (12) de la microplaquette (2) est formée, pilotable en température et en débit.
  2. Dispositif selon la revendication 1,
    caractérisé en ce que les moyens de régulation en température sont reliés avec le porte-chambre (5) et autorisent un chauffage et/ou un refroidissement rapide de la chambre d'essai (3) avec la fente capillaire (7).
  3. Dispositif selon la revendication 2,
    caractérisé en ce que les moyens de régulation en température figurent sur le côté du porte-chambre (5) dirigé vers le corps de chambre (1).
  4. Dispositif selon l'une quelconque des revendications précédentes,
    caractérisé en ce que les moyens de régulation en température (16, 17) sont réalisés sous la forme de couches minces optiquement transparentes et/ou structurées assez finement pour que la transparence de la microplaquette (2) demeure inchangée au moins dans la zone des acides nucléiques liés par points à la surface de détection (12).
  5. Dispositif selon la revendication 4,
    caractérisé en ce que les moyens de régulation en température comprennent des éléments chauffants (17) microstructurés, de préférence des chauffages à résistance en couche épaisse nickel-chrome et/ou des capteurs de température (16) microstructurés, de préférence des capteurs à résistance en couche épaisse nickel-chrome.
  6. Dispositif selon l'une quelconque des revendications précédentes,
    caractérisé en ce que le porte-chambre (5) comprend des systèmes pour mélanger le liquide d'essai réalisés sous la forme de couches minces optiquement transparentes et/ou structurées assez finement pour que la transparence de la microplaquette (2) demeure inchangée au moins dans la zone des acides nucléiques liés par points à la surface de détection (12), s'agissant de préférence d'un système quadripôle pour l'induction d'un écoulement électro-osmotique.
  7. Dispositif selon la revendication 6,
    caractérisé en ce que le système quadripôle est réalisé en tant qu'électrodes or-titane.
  8. Dispositif selon l'une quelconque des revendications précédentes,
    caractérisé en ce que le porte-chambre (5) et le corps de chambre (1) sont constitués de préférence de verre et/ou d'une matière synthétique optiquement translucide, de manière particulièrement préférée en polycarbonate et/ou en acrylate d'éthyle de polyméthane.
  9. Dispositif selon l'une quelconque des revendications précédentes,
    caractérisé en ce que le porte-chambre (5) se constitue de matériau conducteur de la chaleur.
  10. Dispositif selon l'une quelconque des revendications précédentes,
    caractérisé en ce que la microplaquette est en verre et/ou en silicium.
  11. Dispositif selon l'une quelconque des revendications précédentes,
    caractérisé en ce que le corps de chambre (1) présente un creux conique optiquement translucide au moins dans la zone de la microplaquette (2).
  12. Dispositif selon l'une quelconque des revendications précédentes,
    caractérisé en ce que le corps de chambre dispose d'une admission (81) et d'une évacuation (82) séparées spatialement l'une de l'autre pour charger la chambre d'essai (3) et la fente capillaire (7).
  13. Dispositif selon la revendication 12,
    caractérisé en ce que l'admission (81) et l'évacuation (82) sont disposées unilatéralement par rapport à la microplaquette (2) et séparées par un nez de réservoir à gaz (9).
  14. Dispositif selon l'une quelconque des revendications précédentes,
    caractérisé en ce que le corps de chambre (1) est relié au porte-chambre (5) de façon inséparable et en formant étanchéité, par collage et/ou soudage, ou, de façon amovible, au travers d'une surface d'étanchéité (43) supplémentaire.
  15. Dispositif selon l'une quelconque des revendications précédentes,
    caractérisé en ce que la surface de détection (12) est constituée sous forme de points où des sondes (56, 57, 58, 59) sont immobilisées sous forme de molécules d'acide nucléique, les molécules d'acide nucléique étant de préférence des molécules d'ADN ou d'ARN.
  16. Dispositif selon la revendication 15,
    caractérisé en ce que les sondes (56, 57, 58, 59) sont immobilisées au travers de pièces d'écartement (55).
  17. Dispositif selon l'une quelconque des revendications précédentes,
    caractérisé en ce que l'exploitation de la caractérisation d'acides nucléiques basée sur microplaquette peut se réaliser sous la forme de la détection optique et/ou par spectroscopie, de manière particulièrement préférée par mesure de fluorescence de transparence, mesure de fluorescence de champ sombre, mesure de fluorescence confocale, mesure de fluorescence en éclairage direct, photométrie et/ou photométrie différentielle.
  18. Utilisation d'un dispositif selon l'une quelconque des revendications précédentes pour l'amplification et la caractérisation basée sur microplaquette quasi-simultanées d'acides nucléiques.
  19. Utilisation d'un dispositif selon l'une quelconque des revendications précédentes 1 à 17 pour l'amplification PCR et la caractérisation basée sur microplaquette quasi-simultanées d'acides nucléiques.
EP00952983A 1999-07-02 2000-06-30 Dispositif matrice a microcircuit integre destine a l'amplification et a la caracterisation d'acides nucleiques Expired - Lifetime EP1192007B1 (fr)

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DE19932423 1999-07-02
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PCT/EP2000/006103 WO2001002094A1 (fr) 1999-07-02 2000-06-30 Dispositif matrice a microcircuit integre destine a l'amplification et a la caracterisation d'acides nucleiques

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CA (1) CA2379125C (fr)
DE (1) DE50006164D1 (fr)
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ATE264718T1 (de) 2004-05-15
US7888074B2 (en) 2011-02-15
AU6559900A (en) 2001-01-22
EP1192007A1 (fr) 2002-04-03
IL147227A0 (en) 2002-08-14
CA2379125A1 (fr) 2001-01-11
CA2379125C (fr) 2009-04-07
US20020150933A1 (en) 2002-10-17
ES2219374T3 (es) 2004-12-01
WO2001002094A8 (fr) 2001-06-21
DE50006164D1 (de) 2004-05-27
AU768113B2 (en) 2003-12-04

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