Classifications

• • 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
  • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
  • B01L3/50851—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates specially adapted for heating or cooling samples
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
  • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
  • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N2021/6463—Optics
  • G01N2021/6478—Special lenses
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
  • G01N21/6458—Fluorescence microscopy

Definitions

• the invention relates to a method for determining an analyte in a sample by fluorescence correlation spectroscopy, the distance between the measurement volume in the sample and the optical excitation / detection direction being> 1 mm and the sample liquid being thermally insulated from the optical device.
• the method is particularly suitable for measuring temperature-variable processes, e.g. the determination of nucleic acid hybridization melting curves or to carry out nucleic acid amplification reactions.
• a device suitable for performing the method according to the invention is disclosed.
• FCS fluorescence correlation spectroscopy
• DE-A-36 42 798 discloses a microscope with a tripod which is displaceably mounted on an object table, the drive device for vertically displacing the object table forming an independent support of the object table spaced apart from the guide device.
• the temperature of the microscope can be regulated by supplying cooling air and by inserting heat protection filters. There is no reference to the use in fluorescence correlation spectroscopy.
• GB-A-2 351 556 describes a method for measuring radiation, in which a plurality of confocal single-channel optical systems and photoelectric detectors are arranged in parallel in order to form a plurality of reading heads which are arranged next to one another in order to be able to simultaneously evaluate radiation from corresponding areas.
• 10 x microscope objectives with a numerical aperture of 0.4, a focal length of about 8 mm and an aperture diameter of 5 mm can be used as focusing optics. There is no indication that the focusing optics can be thermally isolated from the sample.
• DE-C-42 1 8 729 discloses a capillary electrophoresis device with an optical, spatially resolving detection system.
• the temperature inside the capillary can be thermostatted. There is no reference to confocal fluorescence correlation spectroscopy.
• DE-A-1 99 1 9 092 describes an arrangement for the optical evaluation of an object array, which are used for fluorescence analysis can. Again, there is no evidence of thermal insulation of the focusing optics and samples.
• the object on which the present application is based was to provide methods and devices for carrying out fluorescence correlation spectroscopy which at least partially avoid these disadvantages of the prior art.
• the invention thus relates to a method for determining an analyte in a sample by fluorescence correlation spectroscopy, comprising the steps:
• Measuring volume characterized in that the distance between the measuring volume in the sample liquid and the focusing optics of the light source is> 1 mm and that the sample liquid is thermally insulated from the light source and in particular from the focusing optics.
• thermoelectric device independent of the optical excitation or detection device and in particular its focusing optics, e.g. one or more micro lenses, can be set and changed.
• the method can basically be carried out according to the method described in EP-B-0 679 251. This is preferably done the measurement of one or a few analyte molecules in a measurement volume, the concentration of the analyte molecules to be determined preferably being ⁇ 1 0 "14 l.
• Substance-specific parameters are determined which are determined by luminescence measurement on the analyte molecules. These parameters can be translation diffusions - Coefficients, rotational diffusion coefficients or / and the excitation wavelength, the emission wavelength or / and the lifetime of an excited state of a luminescent molecule or the combination of one or more of these measured variables EP 0 679 251.
• the distance between the measurement volume in the sample liquid and the focusing optics of the light source is 1 mm, preferably 1.5 to 10 mm and particularly preferably 2 to 5 mm. It is further preferred that a gas phase region is arranged between the carrier containing the sample liquid and the optical focusing device, which can contain air, protective gas or vacuum.
• the carrier used to carry out the method according to the invention is preferably a microstructure which contains several, preferably separate containers for holding samples.
• the volume of these containers is preferably in the range of 10 6 I and particularly preferably 10 8 I.
• the carrier can comprise a microwave structure with several recesses for receiving sample liquid, which for example have a diameter between 10 and 1000 ⁇ m. Suitable microstructures are described, for example, in DE 100 23 421 .6 and DE 100 65 632.3
• the carrier preferably comprises at least one temperature control element, for example a Peltier element, which enables temperature control of the carrier and / or individual sample containers therein.
• the carrier used for the method is expediently designed such that it enables optical detection of the sample.
• a carrier which is optically transparent at least in the region of the sample containers is therefore preferably used.
• the carrier can either be completely optically transparent or contain an optically transparent base and an optically opaque cover layer with cutouts in the sample containers.
• Suitable materials for supports are, for example, composite supports made of metal (for example silicon for the cover layer) and glass (for the base). Such supports can be produced, for example, by applying a metal layer to the glass with predetermined cutouts for the sample containers.
• plastic carriers made, for example, of polystyrene or polymers based on acrylate or methacrylate can be used. It is further preferred that the carrier has a cover for the sample containers in order to provide a closed system which is essentially isolated from the surroundings during the measurement.
• a carrier which contains a lens element which is arranged in the beam path between the measurement volume and the light source or detector of the optical device.
• the lens element can be attached to the bottom of a microwave structure.
• a lens element can be produced, for example, by heating and shaping a photoresist using a master mold, for example made of metal such as silicon, and then applied to the carrier.
• the lens elements can be integrated into the carrier, for example when using carriers made of fully plastic structure, for example by injection molding during production.
• the numerical aperture of the optical measuring arrangement can be enlarged by using a lens element, preferably a convex lens element. This numerical aperture is preferably in the range from 0.5 to 1.2.
• the support is also preferably coated with a transparent anti-reflection coating in order to generate a higher refractive index.
• a transparent anti-reflection coating for example, transparent oxides or nitrides can be used as antireflection coatings.
• Anti-reflective coatings are preferably also used on the optics.
• electric fields can be generated in the carrier, in particular in the area of the sample containers, in order to achieve a concentration of the analytes to be determined in the measurement volume.
• Examples of electrodes which are suitable for generating such electric fields are e.g. described in DE 1 01 03 304.4.
• the method according to the invention allows a determination to be carried out at a temperature different from the environment and, in particular, a change in the temperature during the measurement.
• the spatial decoupling of carrier and optics allows a simplified scanning of carriers, in particular microwave structures with several separate sample containers.
• the method according to the invention is basically suitable for the detection of any analytes.
• One or more analyte-binding substances are preferably added to the sample, which carry a labeling group that can be detected by luminescence measurement, in particular a fluorescence labeling group.
• the method according to the invention preferably comprises determining the binding of the labeling substance to the analyte to be detected. This detection can take place, for example, by changing the mobility of the labeling group due to the binding to the analyte or by changing the luminescence of the labeling group (intensity or / and decay time) due to the binding to the analyte or by using several labeling groups by means of the so-called cross-correlation.
• the cross-correlation determination uses at least two different markings, in particular fluorescence markings, the correlated signal of which is determined within the measurement volume.
• This cross-correlation determination is, for example, by Schwüle et al. (Biophys. J. 72 (1997), 1 878-1886) and Rigler et al. (J. Biotechnol. 63 (1 998), 97-109).
• the method according to the invention is particularly suitable for the detection of biomolecules, e.g. Nucleic acids, proteins or other analyte molecules occurring in living organisms, especially in mammals such as humans.
• biomolecules e.g. Nucleic acids, proteins or other analyte molecules occurring in living organisms, especially in mammals such as humans.
• analytes that have been generated in vitro from biological samples can also be detected, e.g. cDNA molecules which have been produced from mRNA by reverse transcription or proteins which have been produced from mRNA or from DNA by in vitro translation.
• the method is also suitable for the detection of analytes which are present from elements of a library and have predetermined characteristics, e.g. Binding to the detection reagent. Examples of such libraries are phage libraries or ribosomal libraries.
• the determination comprises nucleic acid hybridization, one or more luminescence-labeled probes binding to a target nucleic acid as analytes.
• Such hybridization methods can be used, for example, to analyze gene expression, for example to determine a gene expression profile, to analyze mutations, for example single nucleotide polymorphisms (SNP).
• SNP single nucleotide polymorphisms
• the method according to the invention is also suitable for determining enzymatic reactions and / or for determining nucleic acid amplifications, in particular in one or more thermocycling processes.
• Preferred methods for determining nucleic acid polymorphisms are in DE 100 56 226.4 and DE 100 65 631 .5.
• a two-color or multi-color cross-correlation determination is particularly preferably carried out.
• the determination comprises the detection of a protein-protein or protein-ligand interaction, the protein ligands being e.g. low molecular weight active substances, peptides, nucleic acids etc. can be used.
• the protein ligands being e.g. low molecular weight active substances, peptides, nucleic acids etc. can be used.
• a two- or multi-color correlation measurement is preferably carried out.
• so-called “molecular beacon” probes or primers can be used, which - if they are in free form - give a different measurement signal in terms of luminescence intensity or / and decay time than in the bound state.
• the determination can comprise the measurement of an energy transfer which is caused by at least one luminescence marker as an energy donor and at least one luminescence marker as an acceptor.
• the donor and acceptor are present in a complex containing the analyte and one or more detection reagents, preferably at least 2 detection reagents.
• Another object of the invention is a device for determining an analyte by means of fluorescence correlation spectroscopy (FCS), in particular for carrying out the method
• a carrier with at least one container for holding a sample liquid which contains the analyte to be determined,
• an optical excitation device comprising luminescence in a measurement volume that is part of the sample liquid and
• an optical detection device for detecting luminescence from the measurement volume, characterized in that the distance between the focusing optics and the measurement volume is> 1 mm and that the carrier is thermally insulated from the excitation device.
• the carrier is preferably a microstructure with a plurality of preferably at least 10 2 containers for holding a sample liquid, the sample liquid in the separate containers being able to come from one or more sources.
• the sample liquid can be introduced into the containers of the carrier, for example by means of a piezoelectric liquid dispensing device.
• the containers of the carrier are designed in such a way that they enable the detection reagent to be bound with the analyte in solution.
• the containers are preferably depressions in the carrier surface, whereby these depressions can in principle have any shape, for example circular, square, diamond-shaped, etc.
• the carrier can also comprise 10 3 or more separate containers.
• the optical excitation device comprises a strongly focused light source, preferably a laser beam, which is focused on the measurement volume in the sample liquid by means of corresponding optical devices.
• the light source can also contain two or more laser beams, which are then focused on the measurement volume by different optics before entering the sample liquid.
• the detection device can contain, for example, a fiber-coupled avalanche photodiode detector or an electronic detector.
• excitation and / or detection Matrices consisting of a dot matrix of laser dots generated by diffractive optics or a quantum well laser and a detector matrix generated by an avalanche photodiode matrix or an electronic detector matrix, for example a CCD camera, can be used.
• the carrier can be provided in a prefabricated form, with luminescence-labeled n a c h w e i s r e a g e n zi e n, prior to sw ei s e l u m i n e sz m a r ki e rte hybridization probes or primers being filled into several separate containers of the carrier.
• the carrier containing the detection reagents is then advantageously dried.
• a prefabricated carrier which comprises a plurality of separate, e.g. Contains 100 containers in which different detection reagents, e.g. Reagents for detection of nucleic acid hybridization, such as primers and / or probes, are present.
• This carrier can then be mixed with an organism to be examined, e.g. a human patient, sample can be filled so that different analytes are determined from a single sample in the respective containers.
• Such carriers can be used, for example, to create a gene expression profile, e.g. for the diagnosis of diseases or for the determination of nucleic acid polymorphisms, e.g. for the detection of an above-defined genetic predisposition.
• Another object of the invention is a method for the determination of nucleic acid polymorphisms. This method is preferably used in combination with the aforementioned fluorescence correlation spectroscopy method, but it can also be applied to other types of single-molecule detection or to conventional detection methods.
• This procedure includes the steps: (a) Provision of a nucleic acid template to be examined and two probes that bind to the template under hybridization conditions, the probes being selected such that
• nucleotides at the 3'-end of the first probe and / or at the 5'-end of the second probe are complementary to the respective positions on the template if this has a first predetermined variant of the polymorphism and not complementary to the respective one
• the nucleic acid polymorphism is a single nucleotide polymorphism (SNP).
• SNP single nucleotide polymorphism
• the polymorphism can also relate to two or more nucleotides.
• DNA of any origin for example from natural sources, but also recombinantly produced DNA or synthetic DNA can be used as the nucleic acid template.
• the DNA preferably originates from an organism in which nucleic acid polymorphisms occur which are to be determined by the method.
• the DNA is preferably used in single-stranded form, for example as a single-stranded cDNA. However, it is also possible to use double-stranded DNA, which is separated into single-stranded DNA by heating and then used for hybridization with the probes.
• the hybridization probes preferably consist at least partially of single-stranded DNA.
• the 3 'end of the first probe must be ligatable to the 5' end of the second probe, preferably using a DNA ligase.
• the probes can also be made partially from nucleic acid analogs, e.g. Peptide nucleic acids.
• the first probe contains a free 3'-OH group at the 3 'end and the second probe contains a 5'-phosphate group at the 5' end.
• the first and the second probe preferably each carry a different marking group, the joint occurrence of which can be proven by a cross-correlation determination.
• ligation product The presence of both different labeling groups in a single molecule (ligation product) can be detected separately from the presence in two separate molecules (first and second probe).
• first and second probe The size differences between the ligation product and individual probes result in very different hybridization melting points and thus in a high sensitivity of the method. For example, if you carry out a melting point curve measurement of the hybrids, the cross-correlation disappears above the lowest melting point of an individual probe during the individual probes, while the
• Ligation product up to much higher temperatures gives a cross-correlated signal.
• Fluorescent labels such as fluorescein, rhodamine, phycoerythrin, CY3, CY5 or derivatives thereof, are preferably used as labeling groups.
• the fluorescent labeling groups can be differentiated using the emission wavelength, the lifetime of the excited states or a combination thereof.
• the detection can preferably be carried out by means of single molecule determination, e.g. done with location and / or time-resolved fluorescence spectroscopy, which is able to detect fluorescence signals down to single photon counting in a very small volume element, such as in a microchannel or in a microwave.
• the detection can be carried out by means of confocal single-molecule detection, such as by fluorescence correlation spectroscopy.
• the detection can also be carried out by a time-resolved decay measurement, a so-called time gating, the fluorescence molecules being excited within a measurement volume and then preferably at a time interval of> 100 ps, the detection interval being opened at the photodetector.
• This measurement method is described, for example, by Rigler et al. in: "Ultrafast Phenomena" D.H. Auston, ed. Springer 1984.
• Figure 1A the schematic representation of a carrier (2) suitable for carrying out the method according to the invention with a large number of containers (4) formed in the form of depressions on the carrier for receiving sample liquid.
• a carrier with an area of 1-2 cm 2 can contain, for example, up to 10 4 wells.
• an excitation and detection device preferably arranged under the carrier base can be used.
• This device can be a light source (6), e.g. contain a laser with which light can be irradiated into a measurement volume (10) within the sample liquid via an optical focusing device (8).
• the luminescence beam em emitted by the measurement volume is directed via a optical focusing device (8; 8a) to a detector (1 2).
• the measuring volume (10) is arranged at a working distance (14) of> 1 mm and preferably> 1 mm from the focusing device (8).
• the carrier (2) is thermally isolated from the optical excitation and detection device, e.g. in that a gas phase region is arranged between the optical focusing device (8) and the carrier (2).
• FIG. 1B the schematic representation of a particularly preferred embodiment for a carrier (20) suitable for carrying out the method according to the invention.
• the carrier (20) also has a large number of separate containers (22) for holding a sample liquid.
• a preferably convex lens element (24) is arranged in the beam path between the optical excitation and detection device (not shown) and the measurement volume (not shown) contained in the sample liquid.
• This lens element advantageously has an anti-reflective coating (26).
• Figure 2 the schematic representation of a particularly preferred, suitable for carrying out the method according to the invention (30) with a plurality of separate containers (32) for receiving Sample liquids.
• the carrier further contains a temperature control element (34), for example a Peltier element.
• the temperature control element is preferably arranged at least partially around the carrier circumference.
• the containers (32) are provided with a cover (36) from the surroundings, which essentially isolates the sample liquid from the surroundings. Seals (not shown) can be used for this purpose, for example.
• Figure 3 a plan view of the carrier (30) shown in Figure 2 with the temperature control element (34). This arrangement can optionally be attached to a frame holder (36) using additional heating or cooling elements (not shown).
• Figure 4 the filling of the sample in a carrier (40) with separate containers (42a; 42b) via entrances (44a, 44b).
• the containers (42a) and (42b) are separated by a barrier (46).
• the barrier can be used as a permanent barrier or as a valve, e.g. a hydrophobic membrane valve permeable by pressurization.
• the sample liquid can be poured into several containers at the same time or - after filling the first container - into the second container via a valve (46).
• Figure 5 a particularly preferred embodiment for filling sample liquid into the carrier.
• the sample liquid is fed from a reservoir (50), optionally integrated on the carrier itself, into feed containers (52) in parallel into sample containers (54). If necessary, the sample liquid can be passed from the container (54) via a valve (56) into a further container (58), i.e. parallel filling can be combined with serial filling.

Applications Claiming Priority (5)

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• 2002
  • 2002-03-08 JP JP2002572386A patent/JP2004527742A/ja active Pending
  • 2002-03-08 CA CA002440183A patent/CA2440183A1/en not_active Abandoned
  • 2002-03-08 WO PCT/EP2002/002582 patent/WO2002073172A2/de active Application Filing
  • 2002-03-08 AU AU2002256658A patent/AU2002256658A1/en not_active Abandoned
  • 2002-03-08 EP EP02726152A patent/EP1373871A2/de not_active Withdrawn

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