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EP1510254A2 - dispositif et procédé de détection d'un analyte dans un fluide - Google Patents

dispositif et procédé de détection d'un analyte dans un fluide Download PDF

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Publication number
EP1510254A2
EP1510254A2 EP20040020359 EP04020359A EP1510254A2 EP 1510254 A2 EP1510254 A2 EP 1510254A2 EP 20040020359 EP20040020359 EP 20040020359 EP 04020359 A EP04020359 A EP 04020359A EP 1510254 A2 EP1510254 A2 EP 1510254A2
Authority
EP
European Patent Office
Prior art keywords
analyte
liquid
detection
transport plane
substrate
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Withdrawn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
EP20040020359
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German (de)
English (en)
Other versions
EP1510254A3 (fr
Inventor
Hans-Peter Wahl
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.)
F Hoffmann La Roche AG
Roche Diagnostics GmbH
Original Assignee
F Hoffmann La Roche AG
Roche Diagnostics GmbH
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by F Hoffmann La Roche AG, Roche Diagnostics GmbH filed Critical F Hoffmann La Roche AG
Priority to EP20040020359 priority Critical patent/EP1510254A3/fr
Publication of EP1510254A2 publication Critical patent/EP1510254A2/fr
Publication of EP1510254A3 publication Critical patent/EP1510254A3/fr
Withdrawn legal-status Critical Current

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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
    • B01L3/502769—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 characterised by multiphase flow arrangements
    • B01L3/502784—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 characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • B01L3/502792—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 characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics for moving individual droplets on a plate, e.g. by locally altering surface tension
    • 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
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • 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
    • B01L2300/089—Virtual walls for guiding liquids
    • 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
    • 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
    • B01L2400/0427—Electrowetting
    • 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
    • B01L2400/0433—Moving fluids with specific forces or mechanical means specific forces vibrational forces
    • B01L2400/0436—Moving fluids with specific forces or mechanical means specific forces vibrational forces acoustic forces, e.g. surface acoustic waves [SAW]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones

Definitions

  • the application relates to a method for the determination of an analyte in a liquid and a device for determining an analyte in a liquid accordingly the inventive method.
  • SAW Surface Acustic Waves
  • the determination of analytes in microanalytical systems is usually carried out by means of Sensors integrated into the channels of the chips.
  • the measuring methods of the used Sensors are based in particular on the previously used microanalytical methods on spectroscopic methods such as fluorescence or Absorption measurements, electrochemical methods, conductivity measurements, luminescence or electrochemiluminescence method and detection method by means of Optic sensors.
  • biosensors have ion-selective electrodes and other sensors which are widely used in routine macroscopic diagnostics are previously proven in microanalytical systems not routinetauglich.
  • microstructured Sensors and electrodes are in particular protein arrays and arrays for the determination of nucleic acids.
  • sensor modules which in clinical and / or chemical analyzers are installed. These are above all Modules for the determination of electrolytes and other analytes such as Glucose or lactate.
  • these methods established in laboratories usually work with significantly larger sample volumes.
  • microfluidic channels are a few microns wide and deep, but usually very long, so the volume of these channels relative to whose cross-section is large. This can be a significant proportion in these systems the sample volume is not used to determine the analyte in the sensory areas used by the system, but represents not usable dead volume. So are a further reduction of the required sample quantity in these channel systems principle limits set. Furthermore, such channels have the great disadvantage that the surface in direct contact with the sample in relation to the Volume is very large.
  • Ion-selective electrodes are currently used mainly in macroscopic analysis systems, especially in clinical and chemical electrolyte analysis modules Analysis systems, used. Such macroscopic detection systems have considerable Disadvantages. So require such modules and systems in addition to the clear larger sample volumes numerous hoses, valves and pumps that flow control the fluids within these systems. For example, have air segments be introduced into the liquid flow to the hoses and the sensors between the individual measurements and calibrations. For the controller the liquid streams are other sensors, in particular light barriers or Conductivity sensors, needed to ensure that the air segments are correctly or be discharged.
  • microarrays are understood as analytical systems which on a carrier many have sensory elements, usually arranged at regular intervals, so that these used for many simultaneously or temporally offset expiring provisions can be.
  • microarrays are used to analyze proteins and nucleic acids used. Such arrays are difficult to regenerate, so that Such systems, for the reasons described above also not for a multiple Use are suitable.
  • microarrays for protein determination work with planar surfaces. These Arrays, however, require relatively large volumes. So must in such systems For example, incubate with about 50 microliters of sample liquid to bind allow the analyte with the detection molecules. To a depletion of To prevent analytes, the sample must be mixed, which is a large represents technical problem.
  • German patent application DE 10117771 A1 describes methods and devices for the manipulation of small quantities of liquid by means of surface acoustic waves.
  • the object of this patent application described in the published patent application is to locate liquids on a solid state chip and, where appropriate to mix.
  • devices and methods are described, which liquids by means of surface acoustic waves on a flat substrate to move to so-called functionalized areas. In such functionalized For example, a chemical or biological reaction may take place in areas.
  • DE 10117771 A1 describes for this purpose devices in which at certain Make functionalized directly in / on the surface of the solid-state chip Areas are used which, among other things, used as sensors in analytical procedures can be.
  • the functionalized areas for analyzing the liquid are integrated directly into the substrate of the solid-state chip, on which the transport the liquid takes place, i. the relevant devices for liquid transport and the devices relevant to the determination of the analyte in the fluid are combined in a single plane, the transport plane.
  • the sensors integrated into the surface of the carrier chip introduce inhomogeneities the surface of the carrier substrate, for example by different surface wetting properties or spatial elevations or depressions.
  • the object of the present invention is to overcome the disadvantages of the prior art remove.
  • it is an object of the present invention to provide microanalytical To provide methods and devices that meet the needs of a cost-effective and user-friendly routine analytics and which become one multiple reuse.
  • the inventive solution of the disadvantages described in the prior art microanalytical methods and devices and subject of the invention are methods and devices for the determination of analytes in liquids, the characterized in that the liquid to be examined on a substrate is applied and the liquid volume to be examined on the flat surface of the substrate, the so-called transport plane, at the location of the investigation is moved, the liquid during transport exclusively with the Substrate of the transport plane is in contact.
  • the movement can be done in particular by Procedures such as surface acoustic waves or Elektrowetting be effected.
  • the methods and devices according to the invention are characterized in that that they have at least one sensory element and optionally further analytical Have units which are separated from the substrate of the transport plane in one second plane opposite the substrate, the so-called detection level, are located.
  • This detection level is designed such that the liquid volumes during their movement to the place of investigation or away from there with the Be in contact or disturbed by it in their movement.
  • the evidence level indicates where the location of the Investigation reveals special formations or devices which only on This defined location of the investigation, a contact with the examined Make liquid and thus allow determination of analytes in the liquid.
  • This contact surface may in particular be formed such that it has a Permanent reduction of the distance of the sensory element or the Has evidence of the transport level at the locations of the investigation, or that the sensory element or the detection plane is designed to be movable is, so that the sensory element then temporarily with the liquid volume to be examined can be contacted if this is at the site of the investigation located.
  • the transport level and the proof level can become a device be connected, which can be placed in an external device.
  • the external device can in particular to control the movements of the liquid volumes to be examined serve, electrical and / or fluidic contacts to the invention Produce device and is possibly able to part of the analytical Unit.
  • a preferred embodiment consists of a closed device, which the Substrate of the transport plane includes as a bottom surface and a lid, which preferably Having side walls, so that a closed device are constructed can.
  • the lid may further have openings for liquid application, which with a cover, in particular a pierceable septum, are closed.
  • a cover in particular a pierceable septum
  • the lid corresponds thus in most cases the detection level of the device.
  • the sensory Element can hereby directly on the site of the investigation in / on the transport plane facing Surface of the lid integrated / applied.
  • the movably formed sensory element on Place of examination for the determination of the analyte for a short time from a transport position moved to a measuring position.
  • the transport position corresponds to a spatial position of sensory element in which it is not in contact with the liquid volume, so that the transport of the liquid volume over the transport plane not from the sensory Element is affected.
  • the measuring position corresponds to a spatial position of the sensory element, in which the distance to the transport plane compared to the transport position is reduced and the sensory element in contact with the fluid volume so that the determination of the analyte by means of the sensory element can be carried out.
  • the substrate of the transport plane may further devices to generate a movement of the liquid volumes to be examined, in particular interdigital electrodes for generating surface acoustic waves or Electrodes as used in electrowetting processes.
  • the Devices which generate the forces required for fluid movement in the case of transportation by surface acoustic waves, they do not necessarily have to be mounted directly on the substrate of the transport plane, but it is also possible the devices for generating these forces are located outside the substrate, for example, as part of an external drive device. In this case will be the externally generated forces then coupled into the transport plane, for example by means of electric fields or mechanical vibrations. This is particularly advantageous if disposable devices are to be used, because then on expensive devices to dispense with generating a movement on the transport plane itself can, which causes a significant production cost reduction.
  • the subdivision of the devices according to the invention in a plane, which of the movement the liquid volume to be examined (transport plane), and a plane, which serves for the analytical examination of the liquid volumes (detection level), allows on the one hand an undisturbed movement of the liquid volumes on the substrate of the Transport level and, on the other hand, the production of these two levels into two distinct Processes. These two manufacturing processes do not have to be compatible. So for example, the transport level can be created independently of the verification level become. Only in the final assembly, these two are critical in each case in the production Elements brought together, preferably in the form of a closed device.
  • liquid to be examined Through openings in the housing, different volumes of liquid to be examined, but also calibration solutions, reference solutions, rinsing or cleaning solutions, Solutions with standardized analyte concentrations, so-called standards, or reagents, be applied to the transport plane, for example by means of pipetting or Injection.
  • the liquid volumes can also be coupled by means of fluidic Systems, such as capillaries and dispensers, applied to the transport plane become.
  • the device according to the invention additionally has in a preferred embodiment a waste container, which is connected via a panel with the device and thus belongs to the closed area of the device.
  • the fluids passing through The panel can be transported in the waste container, by this of Transportund Detection level can be separated, allowing a return to the sensory area and thus an impairment of subsequent measurements is avoided.
  • advantageous embodiments of the invention are the sensory Areas or the entire device against such external interference shielded, preferably by means of a Faraday cage, to remove the signals from interfering electrical influences to keep free or by means of a radiation-reducing envelope, to keep away direct light or stray light from optical detectors.
  • Analytes containing a method according to the invention or the corresponding Devices can be determined are all within the meaning of the present application Particles of interest in analytics, especially in clinical diagnostics.
  • the term "analytes” includes atoms, ions, molecules and macromolecules, in particular biological macromolecules such as nucleic acids, peptides and proteins, lipids, Metabolites, cells and cell fragments.
  • the analytes can be both free and on Particles, in particular artificially produced particles such as so-called "beads", bound.
  • liquids can be pure liquids and homogeneous or heterogeneous mixtures such as dispersions, emulsions or Be suspensions.
  • atoms, ions, molecules and Macromolecules in particular biological macromolecules such as nucleic acids, peptides and Proteins, lipids, metabolites or biological cells or cell fragments may be included.
  • biological fluids of interest are blood, plasma, serum, Urine, cerebrospinal fluid, tear fluid, cell suspensions, cell supernatants, Cell extracts, tissue digestions or the like.
  • Liquids can also be calibration solutions, Reference solutions, rinsing or cleaning solutions, reagent solutions or Solutions with standardized analyte concentrations, so-called standards.
  • liquid volumes For the purposes of the present application, in principle, any form and However, they are preferably in the form of round or flattened drops Volumes in the range of 100 nl to 10 ul before. In particular, liquid volumes are also in elongated form possible, the multiple juxtaposed sensory elements can cover.
  • Sensory elements in the sense of the present application are all systems for determination of analytes containing analyte-specific chemical, biochemical, biological or physical Determine sizes and their changes.
  • the term sensory Element is not in the context of the present application to a purely technical definition of a sensor, but is fully applicable to all systems, which can enable the detection of an analyte in a direct or indirect manner.
  • binding partners of the analyte in particular labeled Binding partner, which allows the detection of the analyte through a specific interaction enable with it (e.g., antibodies, nucleic acids with complementary Sequences, complexing agents) or specific reaction partners of the analyte, which with this one specific reaction and so by means of evidence of the corresponding Reaction products or -ucts indirectly to determine the analyte (e.g. Substrates, enzymes), as sensory elements.
  • These sensory elements are located According to the invention, at the site of the examination, preferably in immobilized form and allow the specific detection of the analyte there.
  • the reagents required for the determination of the analyte in dry chemical Form bound present.
  • the physical location of the evidence by means of a physical or chemical sensor not necessarily with the sensory Elements at the site of the investigation, especially indirect ones Detection methods.
  • the detection of peptidic analytes by fluorescence-labeled Antibody the detection of the resulting fluorescence radiation by an optical sensor, which in a suitable embodiment of the invention also outside may lie the actual device according to the invention, while the proof of Analytes by the antibodies as sensory elements only at the site of the investigation takes place.
  • sensor elements can also be conventional sensors, in particular electrochemical sensors, biosensors, optical sensors such as absorption or Fluorescence detectors and immunological sensors such as optodes, waveguide sensors and evanescent field laser spectrometry sensors.
  • sensors are included, which can determine physical quantities, such as sensors, which determine the viscosity, the density or the mass of a liquid. This is included Reactions that exhibit these properties in the course of an analyte-specific reaction the fluid changes, especially of interest. Examples are coagulation reactions or methods which allow the attachment of analyte molecules by means of them detected, called mass change.
  • Sensors can be present in all possible geometric embodiments, in particular as pointed sensors, as area sensors or as thick-film sensors. Especially preferred are pointed sensors, as in the case of moving away the sensor only one minimal residual volume of the liquid to be examined adheres to this and thus Carryover artifacts can be largely avoided.
  • Such methods are characterized in particular by the fact that the determination of the Analytes indirectly by the detection of a specific interaction with a binding partner, in particular a labeled binding partner in the form of immunoassays or detection methods using polymerase chain reactions, or a specific Reaction of the analyte with detection reagents, in particular in the form of chemical or enzymatic reactions, or a specific change in a physical or chemical size, in particular the viscosity occurs.
  • a binding partner in particular a labeled binding partner in the form of immunoassays or detection methods using polymerase chain reactions, or a specific Reaction of the analyte with detection reagents, in particular in the form of chemical or enzymatic reactions, or a specific change in a physical or chemical size, in particular the viscosity occurs.
  • the analyte For the determination of the analyte For example, you bring a volume of the liquid to be examined and a volume the reagent solution in contact by these volumes of liquid, for example by suitably controlled acoustic surface acoustic waves, to move towards each other, so that they eventually merge into a common fluid volume. Especially It is advantageous if the combined liquid volumes are subsequently mixed To ensure a rapid and complete reaction of the analyte with the reagents and thus to allow the most accurate determination of the analyte. to Blending can in particular suitably controlled surface acoustic waves be used. The contacting and mixing of the liquid volumes can be done directly at the site of the investigation or previously in another area of the Device, so that in the latter case, possibly after a certain reaction time, this mixture is moved to the site of the investigation.
  • Analyte concentrations may be introduced through special openings in the device especially through an already existing sample application septum or through a specially designed septum.
  • the above-described application of the solutions does not necessarily have to be done by pipetting or injection through a septum, but rather In other embodiments of the invention, liquids may be contained in containers be kept ready inside or outside the housing, which at given times for example, with the help of a microdispenser or piezodispenser in the Housing brought or released there.
  • the liquid volumes are preferably in the form of round or flattened drops before, but there are also volumes in elongated form possible, the several side by side lying sensory elements can cover.
  • This embodiment can then be used particularly well if several sensory elements simultaneously with the need to be in contact with the liquid volume to be examined, as for example in the electrochemical measurement of electrolytes is necessary.
  • the general one or more measuring electrodes and a reference electrode with a reference electrolyte Moving a volume of liquid to be examined and a Volume of a reference electrolyte solution toward each other and brings them into contact, it takes place the mixing of the two volumes of liquid without additional mixing Forces initially purely diffusive and thus very slow.
  • the two Partial volumes are located in an electrically conductive connection without interfering effectively mix thoroughly.
  • the liquid volume is the one to be examined Liquid in contact with one or more measuring electrodes and the reference electrolyte containing liquid volume in contact with the reference electrode, so that after the transient oscillation of the measurement signals can be an exact determination of the analyte.
  • the device according to the invention is preferably installed in a closed housing, which is suitable for multiple use.
  • a closed Design becomes an evaporation of liquids and thus a falsification of the concentration prevents the analyte.
  • a Waste container into which already examined liquids as well as reagent solutions, Calibration solutions, reference solutions, rinsing or cleaning solutions and solutions with standardized analyte concentrations are transported after their use.
  • Waste containers may in particular be designed so that these already used liquids can no longer get to the places of investigation and / or other analyte determinations can falsify. This can be done for example by a mechanical Aperture can be achieved.
  • the already used samples can be adsorbed, for example by means of a fleece or sponge. This ensures that the Humidity kept constant high in the device according to the invention and so the Evaporation of small volumes of liquid is prevented without subsequent Determinations of analytes are influenced by previous provisions.
  • the devices or parts according to the invention are the devices are designed in such a way that the devices or parts of the devices, In particular, sensory elements are provided for a single use. This embodiment is particularly suitable for the determination of analytes in which Carryover problems can occur.
  • the present invention includes embodiments which are modular Modular system for simple as well as multiple and multiple determinations of Analytes in liquids are suitable. Particularly advantageous for these embodiments it is that the transport level and the evidence level with different materials and methods can be produced and only to carry out the determination of Analytes must be merged. Furthermore, transport level and Detection level not be directly connected to each other, for example by spacers or a common housing, but can initially independently present and only for the actual determination of the analyte in common contact be brought to the liquid volume to be examined.
  • embodiments are advantageous in which the substrate of the transport plane as a multipurpose usable Substrate is used, which is the transport of many liquid volumes to the corresponding locations of the investigation causes, and the evidence level for a one-time Use is provided, especially in sensory elements, which are not on are based on reversible reactions and thus can only be used once.
  • Examples of this are optical detection based glucose sensors or sensors which based on immunological methods or DNA hybridization.
  • the proof level be designed so that it contains only one sensory element and to each Determination is exchanged while the transport plane to move many volumes of liquid can be used for many analyte determinations.
  • the evidence level but can also be designed so that it contains several sensory elements, which each can be used only once and which are different from each other separate locations of the determination, so that for each analyte determination another sensory element is used.
  • This embodiment has the advantage that with it several analyte determinations with only once usable sensory elements can be performed sequentially in the same device.
  • the device according to the invention is configured in such a way, in that methods are used for the determination of analytes in liquids, which include one or more dry chemical steps.
  • An example of this is the reflectometric glucose determination on test strips.
  • Dry chemical processes are methods which contain at least one reagent in dry form. This must be ensured that the lowest possible humidity prevails in the device. This can be achieved in particular by the fact that in the device or in it in Connecting components such as a waste container, a moisture and / or liquid-absorbing agent such as silica gel, wherein the moisture and / or liquid-absorbing agent through a membrane or a Fleece can be wrapped.
  • Such in dry Form present reagents can be used in particular in the form of a spot spot Provision or, in the case of indirect participation in the determination of the Analytes may be fixed to other locations of the device. Should such devices, which work with dry chemical reagents, used for several determinations For example, multiple spots may be at different locations in the device be attached, which independently with different to be examined Liquid volumes can be brought into contact. That is how you are in the Able to accommodate dry chemical reagents even in reusable devices, without the dry chemical reagents for further determinations damaged by the liquid volumes used in the preceding determinations become.
  • various embodiments are included.
  • a job site be provided on which several to be examined liquid samples be applied, and several spatially separated locations of the Investigation, which all apply the same detection method, so that at the different Places of investigation Analyte determinations carried out in an identical manner become.
  • multiple job locations and multiple locations of investigation which all use the same detection method should be included. This is especially true advantageous if on a device several identical analyte determinations simultaneously to be carried out.
  • a job site and several locations of the investigation, demonstrating the different Analytes allow to be included. This is especially advantageous when from a Sample several different analytes should be determined. This is the sample preferably first divided into several liquid segments, which then subsequently can be transported to the different locations of the investigation.
  • multiple order locations and multiple locations may also be used Investigation, which allow the detection of different analytes included be.
  • the contact of the sensory element or the detection level to investigating fluid volume is carried out according to the invention only at the site of the investigation.
  • the evidence level may be shaped in this area to be a permanent one Reducing the distance of the sensory element from the transport plane at Has the place of examination, or that the sensory element or the Detection level is designed to be movable, so that the sensory element only with the liquid volume to be examined is brought into contact, if this on Place of investigation is located.
  • the device may be formed such that the distance between the detection plane and transport level at the site of the investigation is permanently reduced, preferably in that the sensory elements from the actual detection plane in the direction protrude the transport plane.
  • the distance between the verification level and transport plane outside the sensory area greater than the vertical extent the volumes of liquid, within the sensory area, i. at the place of Examination, smaller or equal to the vertical extent of the liquid volumes.
  • This is a movement of the liquid volumes outside the sensory areas possible, which is not influenced by the sensory elements or the detection level becomes.
  • An interaction of the liquid volumes with the sensory elements occurs on the other hand, only at the constrictions in the places of investigation.
  • the actual determination of the analytes takes place, whereby the respective liquid volume preferably does not change its position during the determination.
  • the device may further be formed such that the distance between the sensory element or the detection level and the transport level locally the examination can be temporarily reduced.
  • the detection level and the transport level temporarily be moved toward each other, for example by means of an electric drive.
  • not the whole level of evidence is at the transport level moved, but only the sensory areas, for example, by movably mounted Sensor electrodes for determining the analyte by external drives be brought into contact with the liquid volume to be examined. This matches with the previously described measuring position.
  • the Detection level or the sensory element again from the liquid volume moved into the transport position and forces are applied, which the liquid volume Move away from the places of investigation, for example, in a waste container or to another place of investigation, with the movement after leaving the investigation, in turn, takes place exclusively in contact with the transport level.
  • the methods and devices of the invention may further be used for determination of analytes by measuring physical and physico-chemical parameters be used. For example, they can be used to determine global coagulation parameters used as the prothrombin time or the activated partial thromboplastin time become. This measurement can on the one hand via electrochemical reactions with the corresponding electrochemical sensors, as described for example in US Pat US 6,352,630 are described. On the other hand, the determination can also be made via a viscosity measurement respectively. This can, in addition to the known methods, such as optical Method or method with magnetic particles, in particular with sensors which are based on surface acoustic waves.
  • the device can be used several times, if after reaching the necessary measurement signal, the device is regenerated, preferably with the aid of reagents known to those skilled in the art, which prevent the formation of complete coagulation.
  • reagents can in liquid form also with the inventive methods and devices at the transport level, in particular by means of surface acoustic waves, to the reaction mixture be transported and transported after mixing with this in a waste container become.
  • the methods and devices according to the invention can furthermore be carried out homogeneous or heterogeneous immunoassays can be used.
  • the reaction in particular on the measurement of turbidity or the optical density by means of optical sensors.
  • the sensor as Waveguide be formed, in particular in the case of a measurement of the reaction means Evanescent field laser spectrometric method.
  • heterogeneous immunoassays can In particular, specific antibodies are used which, for example, to magnetic Particles are bound. The assays are then in a manner known to those skilled in the art Manner performed.
  • the device can furthermore be designed in such a way that analyte-specific detection reactions carried out with one or more separation steps or washing steps can be.
  • one or more substances to be separated with a specific label or probe for example by binding to magnetic particles or labeled antibodies.
  • Bound-free separation takes place in the case of a magnetic marking in that at a certain location of the device from outside a magnetic field is applied, which the magnetic particles with the substances bound thereto holds and thus allows a washing of the particles or a media change.
  • These Reagents and media may also be in liquid form at the transport level, in particular by means of surface acoustic waves.
  • the measurement can there with the known sensors (fluorescence sensors, luminescence sensors or others). This allows in particular the Perform a complete immunoassay in a single closed device.
  • the device can be used several times if, after reaching the necessary Measuring signal cleaned the device analogously to the above method or regenerated.
  • the spent reagents can be placed in a waste container be transferred.
  • the methods and devices according to the invention can furthermore be used in methods for Determination of analytes which are based on biological or chemical Reproduction reactions are based.
  • methods for genetic engineering or DNA comparisons Of living things are often only traces of cellular material available, which is why For determining these molecules methods are needed which nucleic acids in vitro reproduce in sufficient quantities.
  • Polymerase chain reaction Polymerase chain reaction, PCR
  • One cycle of a PCR consists of three discrete temperature steps: 1. Denaturation: When heated to about 95 ° C, the DNA to be amplified melts and gives single strands. 2.
  • Annealing By rapid cooling to about 55 ° C, the reassociation of the single strands prevented and the primer (2 different oligonucleotides with gegensinniger Orientation) attach to corresponding complementary strand sections of the DNA.
  • Third Extension The DNA polymerase prolongs the strand at about 72 ° C starting from primer and thus completes the single strand to the double strand by the incorporation of nucleotides. These new molecules will serve as a template in the next cycle. It comes to an exponential multiplication of the starting DNA and in several, usually 20 to 50, Cycles, the material is often copied identically.
  • the devices according to the invention can be designed in such a way that they are suitable for Performing such a PCR test are suitable.
  • the implementation of the Temperature control can be implemented in the device in various ways.
  • the device is particularly suitable because of its microscopic size, volumes in the micro and nanoliter range to adjust to the desired temperature in the shortest possible time, which shortens the cycle times of the duplication steps. With such small volumes of liquid
  • Various measures are suitable for this purpose.
  • the aqueous phase (the actual PCR reaction mixture) with a with this aqueous phase immiscible medium, which has a higher boiling point than water has, for example mineral oil, are overcoated.
  • the Reaction is carried out at very high humidity or vapor saturation.
  • the cyclical Controlling and adjusting the temperature of the reaction mixture can be different Species take place.
  • the entire device or certain parts of the device, which contain the reaction mixture are heated or cooled from the outside.
  • the devices according to the invention make very rapid temperature changes achieved, since preferably the volume of the liquid to be tempered very can be small and the device can be made of materials which are very high Have thermal conductivity.
  • thermoelectric generation and regulation can be achieved by methods known in the art.
  • Heating or cooling elements may be installed in the detection level. These will externally driven such that the required for the PCR amplification various Temperatures at different locations of the device permanently or temporarily are set. These set to a certain temperature ranges or the heating elements apply in the sense of the invention as places of investigation or sensory elements, because only in these places by the tempering elements the specific amplification reaction may take place to detect the analyte.
  • the heating elements or the detection level in all before be executed described embodiments.
  • the different temperature zones by reductions the distance between the transport plane and the lid are defined, wherein the Heating elements located at these locations at a reduced distance and the reaction mixture at these points in direct contact with the lid or to be there located heating elements is located.
  • the transport plane can now be carried out DNA amplification, the reaction mixture to the respective preheated Areas are moved in the device.
  • the heat dissipation is preferably carried out on the lid side by direct liquid contact with additional mixing of the reaction mixture for example, by surface acoustic waves or by electrowetting, but also embodiments are conceivable without additional mixing the reaction mixture or with non-direct heat transfer or with lateral or bottom-side heat supply work.
  • Detection of analytes using PCR methods can be done in different ways. In particular, this can be performed by so-called "real time PCR” method in a person skilled in the known manner with the measurement of fluorescence used in these methods Choice of a material that is as transparent as possible, either from the side of the transport plane or the side of the evidence level.
  • a so-called End-point PCR can be carried out in a manner known to the skilled worker, in the end the reaction, the product is moved in a detection area, where the corresponding Nucleic acid sequences are present there as specific template probes have been immobilized. This area can advantageously also be tempered, to ensure a specific hybridization. The detection then takes place with known in the art, wherein in general any known post-PCR detection method suitable.

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  • Chemical & Material Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
EP20040020359 2003-08-30 2004-08-27 dispositif et procédé de détection d'un analyte dans un fluide Withdrawn EP1510254A3 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006124458A2 (fr) 2005-05-11 2006-11-23 Nanolytics, Inc. Procede ou dispositif pour conduire des reactions chimiques ou biochimiques a des temperatures multiples
DE102007036060A1 (de) * 2006-08-03 2008-02-07 Yokogawa Electric Corp., Musashino Prüfvorrichtung
WO2009153189A1 (fr) * 2008-06-17 2009-12-23 Biosensor Gmbh Cartouche avec capteur à ondes acoustiques de surface (saw) intégré
US9513253B2 (en) 2011-07-11 2016-12-06 Advanced Liquid Logic, Inc. Droplet actuators and techniques for droplet-based enzymatic assays

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US4517160A (en) * 1979-04-14 1985-05-14 Olympus Optical Company Limited Automatic analyzing apparatus
WO2000051720A2 (fr) * 1999-03-03 2000-09-08 Symyx Technologies, Inc. Microsystemes de traitement chimique, microreacteurs a diffusion mixte et procedes de preparation et d'utilisation associes
WO2002018785A1 (fr) * 2000-08-31 2002-03-07 Advanced Sensor Technologies Systeme microfluidique
US20020187564A1 (en) * 2001-06-08 2002-12-12 Caliper Technologies Corp. Microfluidic library analysis
WO2004101152A1 (fr) * 2003-05-09 2004-11-25 Caliper Life Sciences, Inc. Analyse automatisee d'echantillons

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4517160A (en) * 1979-04-14 1985-05-14 Olympus Optical Company Limited Automatic analyzing apparatus
WO2000051720A2 (fr) * 1999-03-03 2000-09-08 Symyx Technologies, Inc. Microsystemes de traitement chimique, microreacteurs a diffusion mixte et procedes de preparation et d'utilisation associes
WO2002018785A1 (fr) * 2000-08-31 2002-03-07 Advanced Sensor Technologies Systeme microfluidique
US20020187564A1 (en) * 2001-06-08 2002-12-12 Caliper Technologies Corp. Microfluidic library analysis
WO2004101152A1 (fr) * 2003-05-09 2004-11-25 Caliper Life Sciences, Inc. Analyse automatisee d'echantillons

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006124458A2 (fr) 2005-05-11 2006-11-23 Nanolytics, Inc. Procede ou dispositif pour conduire des reactions chimiques ou biochimiques a des temperatures multiples
EP1885885A2 (fr) * 2005-05-11 2008-02-13 Nanolytics, Inc. Procédé ou dispositif pour conduire des réactions chimiques ou biochimiques à des températures multiples
EP1885885A4 (fr) * 2005-05-11 2008-08-27 Nanolytics Inc Procédé ou dispositif pour conduire des réactions chimiques ou biochimiques à des températures multiples
AU2006247752B2 (en) * 2005-05-11 2012-04-12 Advanced Liquid Logic, Inc. Method and device for conducting biochemical or chemical reactions at multiple temperatures
US9452433B2 (en) 2005-05-11 2016-09-27 Advanced Liquid Logic, Inc. Method and device for conducting biochemical or chemical reactions at multiple temperatures
US9517469B2 (en) 2005-05-11 2016-12-13 Advanced Liquid Logic, Inc. Method and device for conducting biochemical or chemical reactions at multiple temperatures
DE102007036060A1 (de) * 2006-08-03 2008-02-07 Yokogawa Electric Corp., Musashino Prüfvorrichtung
DE102007036060B4 (de) * 2006-08-03 2009-01-08 Yokogawa Electric Corp., Musashino Prüfvorrichtung
WO2009153189A1 (fr) * 2008-06-17 2009-12-23 Biosensor Gmbh Cartouche avec capteur à ondes acoustiques de surface (saw) intégré
US9513253B2 (en) 2011-07-11 2016-12-06 Advanced Liquid Logic, Inc. Droplet actuators and techniques for droplet-based enzymatic assays

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