EP1354190A1 - Method for examining a test sample by means of fluorescence spectroscopy, especially fluorescence correlation spectroscopy, and device for carrying out said method - Google Patents

Method for examining a test sample by means of fluorescence spectroscopy, especially fluorescence correlation spectroscopy, and device for carrying out said method

Info

Publication number
EP1354190A1
EP1354190A1 EP02702316A EP02702316A EP1354190A1 EP 1354190 A1 EP1354190 A1 EP 1354190A1 EP 02702316 A EP02702316 A EP 02702316A EP 02702316 A EP02702316 A EP 02702316A EP 1354190 A1 EP1354190 A1 EP 1354190A1
Authority
EP
European Patent Office
Prior art keywords
sample
analytes
measurement
sample receiving
volume
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Withdrawn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
EP02702316A
Other languages
German (de)
French (fr)
Inventor
Lars Edman
Rudolf Rigler
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.)
Gnothis Holding SA
Original Assignee
Gnothis Holding SA
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 Gnothis Holding SA filed Critical Gnothis Holding SA
Publication of EP1354190A1 publication Critical patent/EP1354190A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating 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
    • G01N21/645—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
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N2001/4038—Concentrating samples electric methods, e.g. electromigration, electrophoresis, ionisation
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions

Definitions

  • the invention is concerned with the fluorescence spectroscopic, in particular fluorescence correlation spectroscopic examination of a measurement sample.
  • a measurement sample e.g. a biological sample, e.g. a body fluid, such as blood, serum, plasma, urine, saliva, etc.
  • a molecular biological reaction approach e.g. a sequencing approach.
  • the analytes can be low-molecular substances, such as drugs, hormones, nucleotides, metabolics, etc., or high-molecular substances, such as proteins, sugars, nucleic acids, etc., viruses or cells, such as bacterial cells, and other substances.
  • fluorophore-carrying reagents which emit fluorescence signals upon light, in particular laser irradiation, which signals are detected and evaluated.
  • fluorescence correlation spectroscopy auto or / and cross correlations of the detected fluorescence signals are evaluated. More information on fluorescence spectroscopy and in particular on fluorescence correlation spectroscopy can be found, for example, in EP 0 679 251 B1.
  • the focus of the microscope used for the examination is usually only a small part of the sample - the measuring volume. So that the fluorescent molecules do not fade due to too intense and long light radiation and falsify the measurements, efforts are being made to make this measurement volume ultra-small, for example in the femtoliter Area.
  • the analytes to be identified are only present in the measurement sample in a very low concentration, it may take a comparatively long time for such small measurement volumes and even unacceptably long for large-scale series examinations until one of the analytes sought diffuses into the measurement volume and becomes observable.
  • Such molecular traps are particularly suitable for individual examinations simply because of the space required for the electrodes required to generate the electrical fields, but also because of the frequently required high expenditure for the electrical control of the electrodes.
  • they prove to be unsuitable if, in the context of serial examinations, a large number of measurement samples, for example several thousand to a few hundred thousand, are arranged in closely adjacent depressions of a common sample carrier (for example a multi known from EP 0 679 251 B1) -Well film), should be examined with reasonable effort.
  • the object of the invention is therefore to provide a simpler way of electrically concentrating analytes.
  • the invention is based on a method for the fluorescence spectroscopic, in particular fluorescence correlation spectroscopic examination of a measurement sample, in which method the measurement sample is introduced into a sample receiving chamber recessed in a sample holder, and then electrically charged analytes contained in the measurement sample be electrically concentrated in a measurement volume lying within the sample volume and the measurement volume is examined.
  • this method provides that the boundary walls of the sample receiving chamber are brought to an electrostatic potential in the same direction as the charge of the analytes in order to concentrate the analytes in the measurement volume.
  • the electrical charge applied to the boundary walls of the sample receiving chamber repulsive forces are exerted on the analytes charged in the same direction in the measurement sample.
  • this repulsion leads to the analytes moving specifically to the measurement volume and collecting there.
  • the walls of the sample receiving chamber itself serve as an electrode.
  • a single charge application can be sufficient; at least for the measuring time, the chamber walls will usually be able to hold the load without any special measures.
  • the chamber walls are constantly connected to a potential source during the entire measurement time. There is also no need to control the potential of the chamber walls.
  • the method according to the invention makes it possible to reliably trap the analytes in the measurement volume without having to dynamically change the potential of the chamber walls. It is not even necessary to have a constant wall potential over time. It only has to be so high that sufficiently strong repulsive forces are generated to achieve the desired concentration of the analytes. Any discharge of charge from the chamber walls can therefore be tolerable as long as the basic level of the wall potential is sufficiently high.
  • the invention in another aspect, relates to a device for performing the above method.
  • This device comprises a sample carrier with at least one sample receiving chamber formed in it and means for essentially all-over electrostatic charging of the boundary walls of the sample receiving chamber.
  • the sample receiving chamber can be undercut. This can help to get a point of the Coulomb equilibrium of forces within the volume of the measurement sample. However, it is not fundamentally necessary to have such a point of equilibrium within of the volume of the measurement sample. It is conceivable that a point of the force equilibrium is only above the filling level up to which the measurement sample fills the sample receiving chamber. It is even conceivable that the sample receiving chamber is designed in such a way that no point of the Coulo b equilibrium of forces exists at all. Since the repulsive forces drive the analytes towards lower electrostatic potentials, it should only be ensured that the measurement volume lies in a range of the lowest electrostatic potential within the total volume of the measurement sample. For this reason, an undercut-free sample receiving chamber can also be used.
  • the sample carrier can have a plurality of sample receiving chambers, preferably arranged in a matrix, the boundary walls of which can be electrostatically charged together. It goes without saying, however, that a sample carrier with a large number of sample receiving chambers can also be used, the chamber walls of which can be at least partially individually electrostatically charged.
  • Fig. 1 shows a schematic plan view of a sample carrier
  • FIG. 2 schematically shows a measurement arrangement for examining a measurement sample, which is located in a sample receiving chamber of the sample carrier.
  • the sample receiving chambers 12 are formed by depressions or hollows in the sample carrier 10.
  • the size of the sample receiving chambers 12 will be selected depending on the intended application of the sample carrier 10 in question.
  • the volume of each sample receiving chamber 12 can be between 100 fl and 100 ⁇ l.
  • the area of the sample receiving chambers 12 to be accommodated per unit area of the sample carrier 10 is correspondingly large, and between 100 and 100,000 sample receiving chambers 12 can easily be accommodated per cm 2 of the sample carrier 10.
  • the sample carrier 10 can, for example, be made from a film material. But it can also be designed as a plate part.
  • one column of the matrix of sample receiving chambers 12 can be reserved for each patient.
  • the measurement samples filled into the sample receiving chambers 12 are then mixed line by line with different test solutions.
  • Each test solution contains different analyte-specific reagents that are marked with • fluorescent dyes. Different analytes can be detected from line to line.
  • the measurement arrangement shown in FIG. 2 is used for the fluorescence spectroscopic examination of the measurement samples.
  • This comprises a microscope arrangement, generally designated 1, with a laser source 16, an optics 18, a dichroic mirror 20, a photon detector 22 and an evaluation unit 24.
  • the laser beam provided by the laser source 16 is fed into the optics 18 via the mirror 20 and by the latter focuses on a small volume element indicated at 26 within the volume of the measurement sample filled in the relevant sample receiving chamber 12.
  • This volume element 26 is confocally imaged on the detector 22 via the optics 18 and, if appropriate, a perforated diaphragm, not shown.
  • the laser beam excites the fluorophores located in the volume element 26 (free or bound to the analytes sought) to fluorescence.
  • the light pulses generated in this way are registered by the detector 22 and evaluated in the evaluation unit 24. Detection of the analytes sought is preferably done via auto- and / or cross-correlations of the fluorescence signals
  • the inner wall of the relevant sample receiving chamber 12, designated 28 is charged over the entire surface to an electrostatic potential which is in the same direction (positive or negative) to the electrical charge of the analytes sought , DNA strands, for example, are usually negatively charged; the inner chamber wall 28 is therefore negatively charged in this case.
  • the electrical charge carriers brought to the inside wall 28 of the chamber as a result of the charge exert Coulomb's repulsive forces on all molecules and other particles in the measurement sample charged in the same direction and thus also on the analytes sought. These repulsive forces drive the analytes along the potential gradient prevailing in the sample receiving chamber 12 to regions of lower electrostatic potential.
  • the analytes eventually accumulate in the area that has the lowest electrostatic potential within the measurement sample volume.
  • a particularly steep potential gradient and thus a particularly effective and rapid concentration process, is obtained when the vectorially added repulsive forces exerted by the charge carriers distributed over the inner chamber wall 28 are also present at the location whose electrostatic potential is the lowest within the measurement sample volume , cancel each other out, so there is a balance of Coulomb's force at this place.
  • the sample receiving chambers 1 2 can be designed with an undercut.
  • the sample receiving chambers 1 2 are partially overlapped or overlapped by the edge regions of their openings, designated 30, as can be clearly seen in FIG. 2.
  • edge areas 30 when the chamber inner wall 28 is electrostatically charged, repulsive forces go out with a component directed towards the bottom of the sample receiving chamber 1 2, which counteracts the repulsive forces directed out of the sample receiving chamber 1 2 and thus enables a force balance to be established within the sample receiving chamber 1 2.
  • Force components directed toward the chamber bottom can also be generated, for example, by electrostatically charging a cover element 32, preferably a cover film, by means of which the openings of the sample receiving chambers 12 can be covered. If there is no point of force equilibrium within the sample volume (either because such a point only exists outside the sample volume or because it does not exist at all), the repulsive forces will ensure that the analytes to be detected are concentrated in an area near the surface of the sample. For this reason, it is entirely possible to use undercut-free sample receiving chambers 12a, as is shown by way of example in FIG. 3. The same elements as in Fig. 2 are provided with the same reference numerals, but supplemented by a lower case letter.
  • the chamber walls 28 of the sample receiving chambers 12 can be electrostatically charged, for example, by means of an electrode 36 connected to a direct voltage source 34, which is brought into contact with the sample carrier 10. If the sample carrier 10 is made of an electrically conductive material, the electrode 36 can be contacted at any point with the sample carrier 10. In this case, all of the sample receiving chambers 12 could be loaded together at the same time. However, it is also conceivable that the sample carrier 10 consists of a non-conductive base material, but the sample receiving chambers 12 are coated on the inside with a conductive material. In this way, the sample receiving chambers 12 could be individually chargeable.
  • the electrical potential relating to earth potential for charging the chamber inner walls 28 depends on the dimensions of the sample receiving chambers 12 and on the height of the desired concentration gradient of the analytes in question in the sample receiving chambers 12.
  • the range of potential differences used can be between 10 V and 10,000 V.

Landscapes

  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention relates to a method for examining a test sample by means of fluorescence spectroscopy, especially fluorescence correlation spectroscopy. Said test sample is introduced into a sample receiver chamber (12) which is sunk in a sample carrier (10). The electrically charged analytes contained in the test sample are then concentrated in a measuring volume (26) inside the sample volume. The measuring volume (26) is then examined. According to the invention, the defining walls (28) of the sample receiver chamber (12) are brought to an equidirectional electrostatic potential for charging the analytes, essentially over the entire surface, in order to concentrate the analytes in the measuring volume (26). An electrical molecular trap formed in such a way is very easy to carry out and is especially suitable for examining a series of test samples which are arranged, in a large number, in cavities (12) of the sample carrier (10) in a closely adjacent manner.

Description

Verfahren zur fluoreszenzspektroskopischen, insbesondere fluoreszenzkor- relationsspektroskopischen Untersuchung einer Messprobe sowie Einrichtung zur Durchführung desselbenProcess for fluorescence spectroscopic, in particular fluorescence correlation spectroscopic examination of a measurement sample and device for carrying out the same
Die Erfindung befasst sich mit der fluoreszenzspektroskopischen, insbesondere fluoreszenzkorrelationsspektroskopischen Untersuchung einer Messprobe.The invention is concerned with the fluorescence spectroscopic, in particular fluorescence correlation spectroscopic examination of a measurement sample.
Mittels Fluoreszenzspektroskopie kann das Vorhandensein bestimmter Analyten in einer Messprobe, beispielsweise einer biologischen Probe, z.B. einer Körperflüssigkeit, wie Blut, Serum, Plasma, Urin, Speichel etc., oder einem molekularbiologischen Reaktionsansatz, z.B. einem Sequenzierungsansatz, nachgewiesen werden. Bei den Analyten kann es sich um niedermolekulare Substanzen, wie Arzneimittel, Hormone, Nukleotide, Metaboliken etc., oder hochmolekulare Substanzen, wie Proteine, Zucker, Nukleinsäuren etc., Viren oder Zellen, wie Bakterienzellen, und sonstige Substanzen handeln. Um die gesuchten Analyten identifizieren zu können, werden sie mit fluorophor-tragenden Reagenzien markiert, die bei Licht-, insbesondere Laserbestrahlung Fluoreszenzsignale aussenden, welche detektiert und ausgewertet werden. In der Fluoreszenzkorrelationsspektroskopie werden hierbei Auto- oder/und Kreuzkorrelationen der detektierten Fluoreszenzsignale ausgewertet. Nähere Informationen zur Fluoreszenzspektroskopie und insbesondere zur Fluoreszenzkorrelationsspektroskopie können beispielsweise der EP 0 679 251 B1 entnommen werden.Using fluorescence spectroscopy, the presence of certain analytes in a measurement sample, e.g. a biological sample, e.g. a body fluid, such as blood, serum, plasma, urine, saliva, etc., or a molecular biological reaction approach, e.g. a sequencing approach. The analytes can be low-molecular substances, such as drugs, hormones, nucleotides, metabolics, etc., or high-molecular substances, such as proteins, sugars, nucleic acids, etc., viruses or cells, such as bacterial cells, and other substances. In order to be able to identify the analytes sought, they are labeled with fluorophore-carrying reagents which emit fluorescence signals upon light, in particular laser irradiation, which signals are detected and evaluated. In fluorescence correlation spectroscopy, auto or / and cross correlations of the detected fluorescence signals are evaluated. More information on fluorescence spectroscopy and in particular on fluorescence correlation spectroscopy can be found, for example, in EP 0 679 251 B1.
Im Fokus des zur Untersuchung verwendeten Mikroskops liegt regelmäßig nur ein kleines Teilvolumen der Probe - das Messvolumen. Damit die fluoreszierenden Moleküle nicht durch zu intensive und lange Lichtbestrahlung ausbleichen und die Messungen verfälschen, ist man bestrebt, dieses Messvolumen ultraklein zu machen, beispielsweise im Femtoliter- bereich. Wenn die zu identifizierenden Analyten jedoch nur in sehr geringer Konzentration in der Messprobe vorhanden sind, kann es bei derart kleinen Messvolumina vergleichsweise lange und für Reihenuntersuchungen im Großmaßstab sogar unakzeptabel lange dauern, bis einer der gesuchten Analyten in das Messvolumen diffundiert und dadurch beobachtbar wird.The focus of the microscope used for the examination is usually only a small part of the sample - the measuring volume. So that the fluorescent molecules do not fade due to too intense and long light radiation and falsify the measurements, efforts are being made to make this measurement volume ultra-small, for example in the femtoliter Area. However, if the analytes to be identified are only present in the measurement sample in a very low concentration, it may take a comparatively long time for such small measurement volumes and even unacceptably long for large-scale series examinations until one of the analytes sought diffuses into the measurement volume and becomes observable.
Zur Beschleunigung der Messung wurden deshalb in der einschlägigen Fachliteratur und unter anderem auch in der oben erwähnten europäi- sehen Druckschrift EP 0 679 251 B1 elektrische Molekülfallen vorgeschlagen, mittels der die nachzuweisenden Analyten, sofern sie elektrisch geladen sind, unter dem Einfluss elektrischer Felder in das Messvolumen getrieben und dort konzentriert werden können. Aus der EP 0 679 251 B1 ist es beispielsweise bekannt, die gesuchten Analyten mittels eines rotierenden elektrischen Wechselfelds in dem Messvolumen zu halten. Quer zu diesem Wechselfeld werden sie durch zwei elektrisch gleichsinnig geladene Pole am Verlassen des Messvolumens gehindert. In der DE 1 95 08 366 C2 wird vorgeschlagen, zwischen einer Ringelektrode und einer mit ihrer Spitze im Zentrum der Ringelektrode angeordneten Kapil- lare ein elektrisches Feld zu erzeugen, das die Konzentrierung der gesuchten Analyten um die Kapillarspitze herum bewirkt.To accelerate the measurement, electrical molecule traps were therefore proposed in the relevant specialist literature and, inter alia, in the European publication EP 0 679 251 B1 mentioned above, by means of which the analytes to be detected, if they are electrically charged, under the influence of electric fields into the Measurement volume driven and can be concentrated there. From EP 0 679 251 B1 it is known, for example, to keep the analytes sought in the measurement volume by means of a rotating alternating electric field. At right angles to this alternating field, they are prevented from leaving the measuring volume by two electrically charged poles. DE 1 95 08 366 C2 proposes to generate an electric field between a ring electrode and a capillary with its tip in the center of the ring electrode, which causes the analytes sought to be concentrated around the capillary tip.
Solche Molekülfallen eignen sich allein schon aufgrund des Platzbedarfs der zur Erzeugung der elektrischen Felder benötigten Elektroden, aber auch aufgrund des häufig erforderlichen hohen Aufwands für die elektrische Steuerung der Elektroden vorwiegend für Einzeluntersuchungen. Sie erweisen sich aber als wenig geeignet, wenn im Rahmen von Reihenuntersuchungen eine große Anzahl von Messproben, beispielsweise mehrere Tausend bis hin zu einigen Hunderttausend, die in eng benachbarten Vertiefungen eines gemeinsamen Probenträgers angeordnet sind (etwa einer aus der EP 0 679 251 B1 bekannten Multi-Well-Folie), mit vertretbarem Aufwand untersucht werden sollen. Aufgabe der Erfindung ist es deshalb, einen einfacheren Weg der elektrischen Konzentrierung von Analyten anzugeben.Such molecular traps are particularly suitable for individual examinations simply because of the space required for the electrodes required to generate the electrical fields, but also because of the frequently required high expenditure for the electrical control of the electrodes. However, they prove to be unsuitable if, in the context of serial examinations, a large number of measurement samples, for example several thousand to a few hundred thousand, are arranged in closely adjacent depressions of a common sample carrier (for example a multi known from EP 0 679 251 B1) -Well film), should be examined with reasonable effort. The object of the invention is therefore to provide a simpler way of electrically concentrating analytes.
Bei der Lösung dieser Aufgabe geht die Erfindung aus von einem Ver- fahren zur fluoreszenzspektroskopischen, insbesondere fluoreszenzkorre- lationsspektroskopischen Untersuchung einer Messprobe, bei welchem Verfahren die Messprobe in eine in einem Probenträger vertieft ausgebildete Probenaufnahmekammer eingebracht wird, sodann in der Messprobe enthaltene, elektrisch geladene Analyten elektrisch in einem innerhalb des Probenvolumens liegenden Messvolumen konzentriert werden und das Messvolumen untersucht wird.In solving this problem, the invention is based on a method for the fluorescence spectroscopic, in particular fluorescence correlation spectroscopic examination of a measurement sample, in which method the measurement sample is introduced into a sample receiving chamber recessed in a sample holder, and then electrically charged analytes contained in the measurement sample be electrically concentrated in a measurement volume lying within the sample volume and the measurement volume is examined.
Erfindungsgemäß ist bei diesem Verfahren vorgesehen, dass zur Konzentrierung der Analyten in dem Messvolumen die Begrenzungswände der Probenaufnahmekammer im Wesentlichen ganzflächig auf ein zur Ladung der Analyten gleichsinniges elektrostatisches Potential gebracht werden. Infolge der auf die Begrenzungswände der Probenaufnahmekammer aufgebrachten elektrischen Ladung werden Abstoßungskräfte auf die gleichsinnig geladenen Analyten in der Messprobe ausgeübt. Bei geeigneter Gestaltung der Probenaufnahmekammer führt diese Abstoßung dazu, dass sich die Analyten gezielt zu dem Messvolumen hinbewegen und dort ansammeln. Es hat sich gezeigt, dass auf diese Weise Analyten, deren Konzentration in der Messprobe unterhalb des nM-Bereichs bis hinunter zum aM-Bereich liegt, im Messvolumen ohne weiteres auf Werte im nM-Bereich angereichert werden können, so dass das Vorhandensein dieser Analyten innerhalb vertretbarer Messzeiten (beispielsweise in weniger als einer Sekunde) nachgewiesen werden kann.According to the invention, this method provides that the boundary walls of the sample receiving chamber are brought to an electrostatic potential in the same direction as the charge of the analytes in order to concentrate the analytes in the measurement volume. As a result of the electrical charge applied to the boundary walls of the sample receiving chamber, repulsive forces are exerted on the analytes charged in the same direction in the measurement sample. With a suitable design of the sample receiving chamber, this repulsion leads to the analytes moving specifically to the measurement volume and collecting there. It has been shown that in this way analytes whose concentration in the measurement sample is below the nM range down to the aM range can be easily enriched in the measurement volume to values in the nM range, so that the presence of these analytes within reasonable measuring times (for example in less than a second) can be demonstrated.
Wenn hier von einem Ladungssinn der Analyten die Rede ist, zu dem das Wandpotential der Probenaufnahmekammer gleichsinnig sein soll, so versteht es sich, dass hierunter der Ladungssinn der fluorophor-markierten Analyten verstanden wird, da es diese sind, die im Messvolumen konzentriert werden sollen.If we are talking here about a sense of charge of the analytes to which the wall potential of the sample receiving chamber should be in the same direction, it is understood that this includes the sense of charge of the fluorophore-labeled Analyte is understood, since it is these that are to be concentrated in the measurement volume.
Bei der erfindungsgemäßen Lösung ist keine komplizierte und platzrau- bende Elektrodenanordnung erforderlich. Vielmehr dienen die Wände der Probenaufnahmekammer selbst als Elektrode. Um diese Wände elektrostatisch zu laden, kann eine einmalige Ladungsaufbringung genügen; zumindest für die Messzeit werden die Kammerwände die Ladung im Regelfall ohne besondere Zusatzmaßnahmen halten können. Es ist aber auch denkbar, dass die Kammerwände während der gesamten Messzeit ständig mit einer Potentialquelle verbunden sind. Es ist auch keine Steuerung des Potentials der Kammerwände erforderlich. Das erfindungsgemäße Verfahren erlaubt es, die Analyten zuverlässig im Messvolumen einzufangen, ohne dabei rückgekoppelt das Potential der Kammerwände dynamisch verändern zu müssen. Es ist noch nicht einmal ein zeitlich konstantes Wandpotential erforderlich. Dieses muss lediglich so hoch sein, dass hinreichend starke Abstoßungskräfte erzeugt werden, um die gewünschte Konzentrierung der Analyten zu erreichen. Ein eventueller Ladungsabfluß von den Kammerwänden kann deshalb tolerierbar sein, solange das Grundniveau des Wandpotentials ausreichend hoch ist.No complicated and space-consuming electrode arrangement is required in the solution according to the invention. Rather, the walls of the sample receiving chamber itself serve as an electrode. In order to charge these walls electrostatically, a single charge application can be sufficient; at least for the measuring time, the chamber walls will usually be able to hold the load without any special measures. However, it is also conceivable that the chamber walls are constantly connected to a potential source during the entire measurement time. There is also no need to control the potential of the chamber walls. The method according to the invention makes it possible to reliably trap the analytes in the measurement volume without having to dynamically change the potential of the chamber walls. It is not even necessary to have a constant wall potential over time. It only has to be so high that sufficiently strong repulsive forces are generated to achieve the desired concentration of the analytes. Any discharge of charge from the chamber walls can therefore be tolerable as long as the basic level of the wall potential is sufficiently high.
Nach einem weiteren Gesichtspunkt betrifft die Erfindung eine Einrichtung zur Durchführung des vorstehenden Verfahrens. Diese Einrichtung umfasst einen Probenträger mit mindestens einer vertieft in diesem aus- gebildeten Probenaufnahmekammer sowie Mittel zur im Wesentlichen ganzflächigen elektrostatischen Aufladung der Begrenzungswände der Probenaufnahmekammer.In another aspect, the invention relates to a device for performing the above method. This device comprises a sample carrier with at least one sample receiving chamber formed in it and means for essentially all-over electrostatic charging of the boundary walls of the sample receiving chamber.
Die Probenaufnahmekammer kann hinterschnitten ausgebildet sein. Dies kann helfen, um innerhalb des Volumens der Messprobe einen Punkt des Coulomb'schen Kräftegleichgewichts zu erhalten. Es ist jedoch nicht grundsätzlich notwendig, einen solchen Gleichgewichtspunkt innerhalb des Volumens der Messprobe zu haben. Es ist denkbar, dass ein Punkt des Kraftgleichgewichts erst oberhalb des Füllniveaus liegt, bis zu dem die Messprobe die Probenaufnahmekammer ausfüllt. Es ist sogar vorstellbar, dass die Probenaufnahmekammer so gestaltet ist, dass überhaupt kein Punkt des Coulo b'schen Kräftegleichgewichts existiert. Da die Abstoßungskräfte die Analyten in Richtung zu niedrigeren elektrostatischen Potentialen treiben, sollte lediglich sichergestellt sein, dass das Messvolumen in einem Bereich niedrigsten elektrostatischen Potentials innerhalb des Gesamtvolumens der Messprobe liegt. Aus diesem Grund kann auch eine hinterschneidungsfreie Probenaufnahmekammer verwendet werden.The sample receiving chamber can be undercut. This can help to get a point of the Coulomb equilibrium of forces within the volume of the measurement sample. However, it is not fundamentally necessary to have such a point of equilibrium within of the volume of the measurement sample. It is conceivable that a point of the force equilibrium is only above the filling level up to which the measurement sample fills the sample receiving chamber. It is even conceivable that the sample receiving chamber is designed in such a way that no point of the Coulo b equilibrium of forces exists at all. Since the repulsive forces drive the analytes towards lower electrostatic potentials, it should only be ensured that the measurement volume lies in a range of the lowest electrostatic potential within the total volume of the measurement sample. For this reason, an undercut-free sample receiving chamber can also be used.
Um bei Reihenuntersuchungen die Prozedur zur elektrostatischen Aufladung der Kammerwände möglichst ökonomisch zu gestalten, kann der Probenträger eine Vielzahl vorzugsweise matrixförmig angeordneter Probenaufnahmekammern aufweisen, deren Begrenzungswände gemeinsam elektrostatisch aufladbar sind. Es versteht sich jedoch, dass auch ein Probenträger mit einer Vielzahl von Probenaufnahmekammern verwendet werden kann, deren Kammerwände wenigstens zum Teil individuell elek- trostatisch aufladbar sind.In order to make the procedure for electrostatic charging of the chamber walls as economical as possible during serial examinations, the sample carrier can have a plurality of sample receiving chambers, preferably arranged in a matrix, the boundary walls of which can be electrostatically charged together. It goes without saying, however, that a sample carrier with a large number of sample receiving chambers can also be used, the chamber walls of which can be at least partially individually electrostatically charged.
Ein Ausführungsbeispiel der Erfindung wird nachfolgend anhand der beigefügten Zeichnungen näher erläutert. Es stellen dar:An embodiment of the invention is explained below with reference to the accompanying drawings. They represent:
Fig. 1 schematisch eine Draufsicht auf einen Probenträger undFig. 1 shows a schematic plan view of a sample carrier and
Fig. 2 schematisch eine Messanordnung zur Untersuchung einer Messprobe, die sich in einer Probenaufnahmekammer des Probenträgers befindet.2 schematically shows a measurement arrangement for examining a measurement sample, which is located in a sample receiving chamber of the sample carrier.
Der in Fig. 1 gezeigte und dort mit 10 bezeichnete flächige Probenträger weist in einer Matrixanordnung eine Vielzahl durch schwarze Punkte angedeuteter Probenaufnahmekammern 12 auf, in die Blutproben oder Proben anderer Lösungen eingefüllt werden können, welche auf das Vorhandensein bestimmter Viren, DNA-Fragmente oder anderer Analyten untersucht werden sollen. Die Probenaufnahmekammern 12 sind von Vertiefungen oder Aushöhlungen in dem Probenträger 10 gebildet.The flat sample support shown in FIG. 1 and labeled 10 there has a large number of black dots in a matrix arrangement indicated sample receiving chambers 12, into which blood samples or samples of other solutions can be filled, which are to be examined for the presence of certain viruses, DNA fragments or other analytes. The sample receiving chambers 12 are formed by depressions or hollows in the sample carrier 10.
Die Größe der Probenaufnahmekammern 12 wird man abhängig von der beabsichtigten Anwendung des betreffenden Probenträgers 10 wählen. So kann das Volumen jeder Probenaufnahmekammer 12 beispielsweise zwischen 100 fl und 100 μl liegen. Entsprechend groß ist der Bereich der pro Flächeneinheit des Probenträgers 10 unterzubringenden Probenaufnahmekammern 12. Pro cm2 des Probenträgers 10 können dabei ohne weiteres zwischen 100 und 100 000 Probenaufnahmekammern 12 untergebracht sein. Der Probenträger 10 kann beispielsweise aus einem Folien- material hergestellt sein. Er kann aber auch als Plattenteil ausgebildet sein.The size of the sample receiving chambers 12 will be selected depending on the intended application of the sample carrier 10 in question. For example, the volume of each sample receiving chamber 12 can be between 100 fl and 100 μl. The area of the sample receiving chambers 12 to be accommodated per unit area of the sample carrier 10 is correspondingly large, and between 100 and 100,000 sample receiving chambers 12 can easily be accommodated per cm 2 of the sample carrier 10. The sample carrier 10 can, for example, be made from a film material. But it can also be designed as a plate part.
Im Rahmen einer Reihenuntersuchung von Messproben verschiedener Patienten kann beispielsweise für jeden Patienten je eine Spalte der Ma- trix von Probenaufnahmekammern 12 reserviert werden. Die in die Probenaufnahmekammern 12 eingefüllten Messproben werden dann zeilenweise mit unterschiedlichen Testlösungen gemischt. Jede Testlösung enthält dabei unterschiedliche analytspezifische Reagenzien, die mit fluo- • reszierenden Farbstoffen markiert sind. Von Zeile zu Zeile können so unterschiedliche Analyten nachgewiesen werden.In the course of a series examination of measurement samples from different patients, for example, one column of the matrix of sample receiving chambers 12 can be reserved for each patient. The measurement samples filled into the sample receiving chambers 12 are then mixed line by line with different test solutions. Each test solution contains different analyte-specific reagents that are marked with • fluorescent dyes. Different analytes can be detected from line to line.
Zur fluoreszenzspektroskopischen Untersuchung der Messproben dient die in Fig. 2 gezeigte Messanordnung. Diese umfasst eine allgemein mit 1 bezeichnete Mikroskopanordnung mit einer Laserquelle 16, einer Optik 18, einem dichroitischen Spiegel 20, einem Photonendetektor 22 und einer Auswerteeinheit 24. Der von der Laserquelle 16 bereitgestellte Laserstrahl wird über den Spiegel 20 in die Optik 18 eingespeist und von dieser auf ein bei 26 angedeutetes kleines Volumenelement innerhalb des Volumens der in die betreffende Probenaufnahmekammer 12 eingefüllten Messprobe fokussiert. Über die Optik 18 und ggf. eine nicht näher dargestellte Lochblende wird dieses Volumenelement 26 konfokal auf den Detektor 22 abgebildet. Der Laserstrahl regt die in dem Volumenelement 26 befindlichen (freien oder an die gesuchten Analyten gebundenen) Fluorophore zu Fluoreszenz an. Die dabei erzeugten Lichtimpulse werden vom Detektor 22 registriert und in der Auswerteeinheit 24 ausgewertet. Der Nachweis der gesuchten Analyten geschieht dabei bevorzugt über Auto- oder/und Kreuzkorrelationen der vom Detektor 22 gelieferten Fluoreszenzsignale.The measurement arrangement shown in FIG. 2 is used for the fluorescence spectroscopic examination of the measurement samples. This comprises a microscope arrangement, generally designated 1, with a laser source 16, an optics 18, a dichroic mirror 20, a photon detector 22 and an evaluation unit 24. The laser beam provided by the laser source 16 is fed into the optics 18 via the mirror 20 and by the latter focuses on a small volume element indicated at 26 within the volume of the measurement sample filled in the relevant sample receiving chamber 12. This volume element 26 is confocally imaged on the detector 22 via the optics 18 and, if appropriate, a perforated diaphragm, not shown. The laser beam excites the fluorophores located in the volume element 26 (free or bound to the analytes sought) to fluorescence. The light pulses generated in this way are registered by the detector 22 and evaluated in the evaluation unit 24. Detection of the analytes sought is preferably done via auto- and / or cross-correlations of the fluorescence signals supplied by the detector 22.
Konfokale Mikroskopanordnungen dieser Art sind an sich bekannt. Beispielhaft wird auf die EP 0 679 251 B1 verwiesen, der Einzelheiten eines solchen Mikroskops entnommen werden können. Es versteht sich, dass die Mikroskopanordnung 14 zur Erfassung verschiedener Fluoreszenzwellenlängen auch zwei oder mehr Detektoren 22 aufweisen kann. Sie kann auch als Doppelmikroskop mit zwei beidseits des Probenträgers 10 angeordneten Optiken 18 ausgebildet sein, sofern der Probenträger 10 aus einem lichtdurchlässigen Material besteht. Ein solches Doppelmikroskop kann ebenfalls der EP 0 679 251 B1 entnommen werden.Confocal microscope arrangements of this type are known per se. Reference is made, for example, to EP 0 679 251 B1, from which details of such a microscope can be found. It goes without saying that the microscope arrangement 14 can also have two or more detectors 22 for detecting different fluorescence wavelengths. It can also be designed as a double microscope with two optics 18 arranged on both sides of the sample carrier 10, provided that the sample carrier 10 consists of a translucent material. Such a double microscope can also be found in EP 0 679 251 B1.
Um die Konzentration der nachzuweisenden Analyten in dem Volumenelement 26 zu erhöhen und dadurch die Messzeit zu verkürzen, wird die mit 28 bezeichnete Innenwand der betreffenden Probenaufnahmekammer 12 ganzflächig auf ein elektrostatisches Potential aufgeladen, das gleichsinnig (positiv oder negativ) zur elektrischen Ladung der gesuchten Analyten ist. DNA-Stränge beispielsweise sind im Regelfall negativ geladen; die Kammerinnenwand 28 wird in diesem Fall daher negativ geladen. Die infolge der Aufladung an die Kammerinnenwand 28 gebrachten elektrischen Ladungsträger üben Coulomb'sche Abstoßungskräfte auf alle gleichsinnig geladenen Moleküle und sonstigen Teilchen in der Messprobe und somit auch auf die gesuchten Analyten aus. Diese Abstoßungskräfte treiben die Analyten längs des in der Probenaufnahmekammer 1 2 herrschenden Potentialgradienten zu Bereichen niedrigeren elektrostatischen Potentials hin. In dem Bereich, der innerhalb des Messprobenvolumens das geringste elektrostatische Potential aufweist, sammeln sich die Analyten schließlich an. Einen besonders steilen Potentialgradienten und damit einen besonders effektiven und raschen Konzentrierungsvorgang erhält man, wenn sich an dem Ort, dessen elektrostatisches Potential innerhalb des Messprobenvolumens am geringsten ist, zugleich die vekto- riell addierten Abstoßungskräfte, die von den über die Kammerinnenwand 28 verteilten Ladungsträgern ausgeübt werden, gegenseitig aufheben, an diesem Ort also ein Gleichgewicht der Coulomb'schen Kraft existiert.In order to increase the concentration of the analytes to be detected in the volume element 26 and thereby shorten the measurement time, the inner wall of the relevant sample receiving chamber 12, designated 28, is charged over the entire surface to an electrostatic potential which is in the same direction (positive or negative) to the electrical charge of the analytes sought , DNA strands, for example, are usually negatively charged; the inner chamber wall 28 is therefore negatively charged in this case. The electrical charge carriers brought to the inside wall 28 of the chamber as a result of the charge exert Coulomb's repulsive forces on all molecules and other particles in the measurement sample charged in the same direction and thus also on the analytes sought. These repulsive forces drive the analytes along the potential gradient prevailing in the sample receiving chamber 12 to regions of lower electrostatic potential. The analytes eventually accumulate in the area that has the lowest electrostatic potential within the measurement sample volume. A particularly steep potential gradient, and thus a particularly effective and rapid concentration process, is obtained when the vectorially added repulsive forces exerted by the charge carriers distributed over the inner chamber wall 28 are also present at the location whose electrostatic potential is the lowest within the measurement sample volume , cancel each other out, so there is a balance of Coulomb's force at this place.
Um zu erreichen, dass ein Punkt solchen Kraftgleichgewichts innerhalb der Probenaufnahmekammer 1 2 und vorzugsweise sogar innerhalb des Messprobenvolumens existiert, können die Probenaufnahmekammern 1 2 mit Hinterschnitt ausgeführt sein. Bei dieser Ausbildung werden die Probenaufnahmekammern 1 2 von den mit 30 bezeichneten Randbereichen ihrer Öffnungen teilweise überragt oder überlappt, wie in Fig. 2 gut er- kennbar ist. In diesen Randbereichen 30 gehen bei elektrostatischer Aufladung der Kammerinnenwand 28 Abstoßungskräfte mit einer zum Boden der Probenaufnahmekammer 1 2 gerichteten Komponente aus, die den aus der Probenaufnahmekammer 1 2 heraus gerichteten Abstoßungskräften entgegenwirkt und so die Etablierung eines Kraftgleichgewichts inner- halb der Probenaufnahmekammer 1 2 ermöglicht.In order to ensure that a point of such force equilibrium exists within the sample receiving chamber 1 2 and preferably even within the measuring sample volume, the sample receiving chambers 1 2 can be designed with an undercut. In this embodiment, the sample receiving chambers 1 2 are partially overlapped or overlapped by the edge regions of their openings, designated 30, as can be clearly seen in FIG. 2. In these edge areas 30, when the chamber inner wall 28 is electrostatically charged, repulsive forces go out with a component directed towards the bottom of the sample receiving chamber 1 2, which counteracts the repulsive forces directed out of the sample receiving chamber 1 2 and thus enables a force balance to be established within the sample receiving chamber 1 2.
Zum Kammerboden gerichtete Kraftkomponenten können beispielsweise auch durch elektrostatische Aufladung eines Abdeckelements 32, vorzugsweise einer Abdeckfolie, erzeugt werden, mittels welchem die Öff- nungen der Probenaufnahmekammern 12 abgedeckt werden können. Falls innerhalb des Messprobenvolumens kein Punkt des Kraftgleichgewichts existiert (entweder weil ein solcher Punkt nur außerhalb des Messprobenvolumens existiert oder weil er überhaupt nicht existiert), so werden die Abstoßungskräfte für eine Konzentration der nachzuweisenden Analyten in einem Bereich nahe der Oberfläche der Messprobe sorgen. Aus diesem Grund ist es durchaus möglich, hinterschneidungsfreie Probenaufnahmekammern 12a zu verwenden, wie beispielshaft in Fig. 3 gezeigt ist. Gleiche Elemente wie in Fig. 2 sind dort mit gleichen Bezugszeichen versehen, jedoch ergänzt um einen Kleinbuchstaben.Force components directed toward the chamber bottom can also be generated, for example, by electrostatically charging a cover element 32, preferably a cover film, by means of which the openings of the sample receiving chambers 12 can be covered. If there is no point of force equilibrium within the sample volume (either because such a point only exists outside the sample volume or because it does not exist at all), the repulsive forces will ensure that the analytes to be detected are concentrated in an area near the surface of the sample. For this reason, it is entirely possible to use undercut-free sample receiving chambers 12a, as is shown by way of example in FIG. 3. The same elements as in Fig. 2 are provided with the same reference numerals, but supplemented by a lower case letter.
Die elektrostatische Aufladung der Kammerinnenwände 28 der Probenaufnahmekammern 12 kann beispielsweise mittels einer an eine Gleichspannungsquelle 34 angeschlossenen Elektrode 36 erfolgen, die in Kontakt mit dem Probenträger 10 gebracht wird. Falls der Probenträger 10 aus einem elektrisch leitfähigen Material gefertigt ist, kann die Elektrode 36 an irgendeiner Stelle mit dem Probenträger 10 kontaktiert werden. In diesem Fall könnten alle Probenaufnahmekammern 12 gleichzeitig gemeinsam geladen werden. Vorstellbar ist aber auch, dass der Probenträger 10 aus einem nichtleitenden Grundmaterial besteht, die Probenauf- nahmekammern 12 jedoch innenseitig mit einem leitenden Material beschichtet sind. Auf diese Weise könnten die Probenaufnahmekammern 12 einzeln aufladbar sein.The chamber walls 28 of the sample receiving chambers 12 can be electrostatically charged, for example, by means of an electrode 36 connected to a direct voltage source 34, which is brought into contact with the sample carrier 10. If the sample carrier 10 is made of an electrically conductive material, the electrode 36 can be contacted at any point with the sample carrier 10. In this case, all of the sample receiving chambers 12 could be loaded together at the same time. However, it is also conceivable that the sample carrier 10 consists of a non-conductive base material, but the sample receiving chambers 12 are coated on the inside with a conductive material. In this way, the sample receiving chambers 12 could be individually chargeable.
Das auf Erdpotential bezogene elektrische Potential zur Aufladung der Kammerinnenwände 28 hängt von den Abmessungen der Probenaufnahmekammern 12 und von der Höhe des gewünschten Konzentrationsgradienten der betreffenden Analyten in den Probeaufnahmekammern 12 ab. Der Bereich der zur Anwendung kommenden Potentialdifferenzen kann durchaus zwischen 10 V und 10 000 V liegen. The electrical potential relating to earth potential for charging the chamber inner walls 28 depends on the dimensions of the sample receiving chambers 12 and on the height of the desired concentration gradient of the analytes in question in the sample receiving chambers 12. The range of potential differences used can be between 10 V and 10,000 V.

Claims

Ansprüche Expectations
1 . Verfahren zur fluoreszenzspektroskopischen, insbesondere fluo- reszenzkorrelationsspektroskopischen Untersuchung einer Mess- probe, bei welchem Verfahren die Messprobe in eine in einem1 . Method for the fluorescence spectroscopic, in particular fluorescence correlation spectroscopic examination of a measurement sample, in which method the measurement sample is integrated into one
Probenträger (10) vertieft ausgebildete Probenaufnahmekammer ( 1 2) eingebracht wird, in der Messprobe enthaltene, elektrisch geladene Analyten elektrisch in einem innerhalb des Probenvolumens liegenden Messvolumen (26) konzentriert werden und das Messvolumen (26) untersucht wird, dadurch gekennzeichnet, dass zur Konzentrierung der Analyten in dem Messvolumen (26) die Begrenzungswände (28) der Probenaufnahmekammer (1 2) im Wesentlichen ganzflächig auf ein zur Ladung der Analyten gleichsinniges elektrostatisches Potential gebracht werden.Sample holder (10) is formed in a deeply formed sample receiving chamber (1 2), electrically charged analytes contained in the measurement sample are electrically concentrated in a measurement volume (26) lying within the sample volume and the measurement volume (26) is examined, characterized in that for concentration of the analytes in the measurement volume (26), the boundary walls (28) of the sample receiving chamber (1 2) are brought over their entire surface to an electrostatic potential which is in the same direction as the charge of the analytes.
2. Einrichtung zur Durchführung des Verfahrens nach Anspruch 1 , gekennzeichnet durch einen Probenträger (10) mit mindestens einer vertieft in diesem ausgebildeten Probenaufnahmekammer (1 2) sowie Mittel (34, 36) zur im Wesentlichen ganzflächigen elektrostatischen Aufladung der Begrenzungswände (28) der Probenaufnahmekammer ( 1 2).2. Device for performing the method according to claim 1, characterized by a sample carrier (10) with at least one recessed in this formed sample receiving chamber (1 2) and means (34, 36) for substantially full-surface electrostatic charging of the boundary walls (28) of the sample receiving chamber (1 2).
3. Einrichtung nach Anspruch 2, dadurch gekennzeichnet, dass die Probenaufnahmekammer ( 1 2) hinterschnitten ist.3. Device according to claim 2, characterized in that the sample receiving chamber (1 2) is undercut.
4. Einrichtung nach Anspruch 2 oder 3, dadurch gekennzeichnet, dass die Probenaufnahmekammer (1 2a) hinterschneidungsfrei ist.4. Device according to claim 2 or 3, characterized in that the sample receiving chamber (1 2a) is free of undercuts.
5. Einrichtung nach einem der Ansprüche 2 bis 4, dadurch gekennzeichnet, dass der Probenträger (1 0) eine Vielzahl vorzugsweise matrixförmig angeordneter Probenaufnahmekammern (12) auf- weist, deren Begrenzungswände (28) gemeinsam elektrostatisch aufladbar sind. 5. Device according to one of claims 2 to 4, characterized in that the sample carrier (1 0) a plurality of preferably matrix-shaped sample receiving chambers (12) on has, the boundary walls (28) are electrostatically charged together.
EP02702316A 2001-01-25 2002-01-25 Method for examining a test sample by means of fluorescence spectroscopy, especially fluorescence correlation spectroscopy, and device for carrying out said method Withdrawn EP1354190A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10103304A DE10103304A1 (en) 2001-01-25 2001-01-25 Process for fluorescence spectroscopic, in particular fluorescence correlation spectroscopic examination of a measurement sample and device for carrying it out
DE10103304 2001-01-25
PCT/EP2002/000797 WO2002059582A1 (en) 2001-01-25 2002-01-25 Method for examining a test sample by means of fluorescence spectroscopy, especially fluorescence correlation spectroscopy, and device for carrying out said method

Publications (1)

Publication Number Publication Date
EP1354190A1 true EP1354190A1 (en) 2003-10-22

Family

ID=7671705

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02702316A Withdrawn EP1354190A1 (en) 2001-01-25 2002-01-25 Method for examining a test sample by means of fluorescence spectroscopy, especially fluorescence correlation spectroscopy, and device for carrying out said method

Country Status (4)

Country Link
US (1) US7227631B2 (en)
EP (1) EP1354190A1 (en)
DE (1) DE10103304A1 (en)
WO (1) WO2002059582A1 (en)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100526932C (en) * 2004-02-09 2009-08-12 皇家飞利浦电子股份有限公司 Fluorescence microscope arrangement
WO2009021732A2 (en) * 2007-08-14 2009-02-19 Rudolf Rigler Method for determining the flow of a fluid close to a surface of an object immersed in the fluid
DE102008049877A1 (en) * 2008-09-30 2010-04-01 Carl Zeiss Microimaging Gmbh Method for evaluating correlation spectroscopy measurement data
WO2013116508A1 (en) 2012-01-31 2013-08-08 University Of Utah Research Foundation Measurement of depth and energy of buried trap states in dielectric films by single electron tunneling force spectroscopy
CN108865880A (en) * 2018-06-25 2018-11-23 中南林业科技大学 A kind of screening has the device and method of the electric heavy metal resistance microorganism for moving ability
US11758256B2 (en) 2019-06-20 2023-09-12 Cilag Gmbh International Fluorescence imaging in a light deficient environment
US11937784B2 (en) 2019-06-20 2024-03-26 Cilag Gmbh International Fluorescence imaging in a light deficient environment
US11012599B2 (en) 2019-06-20 2021-05-18 Ethicon Llc Hyperspectral imaging in a light deficient environment
US11533417B2 (en) 2019-06-20 2022-12-20 Cilag Gmbh International Laser scanning and tool tracking imaging in a light deficient environment

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3517241B2 (en) 1993-01-18 2004-04-12 エボテック バイオシステムズ アクチェン ゲゼルシャフト Method and apparatus for evaluating the fitness of biopolymers
DE19508366C2 (en) 1995-03-10 1998-01-29 Evotec Biosystems Gmbh Methods for the direct detection of fewer strands of nucleic acid
US7244349B2 (en) * 1997-12-17 2007-07-17 Molecular Devices Corporation Multiaperture sample positioning and analysis system
US20030015425A1 (en) * 2001-06-20 2003-01-23 Coventor Inc. Microfluidic system including a virtual wall fluid interface port for interfacing fluids with the microfluidic system

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO02059582A1 *

Also Published As

Publication number Publication date
US20040144929A1 (en) 2004-07-29
US7227631B2 (en) 2007-06-05
DE10103304A1 (en) 2002-08-01
WO2002059582A1 (en) 2002-08-01

Similar Documents

Publication Publication Date Title
DE69736633T2 (en) DETECTION OF SUBSTANCES MOVING INTO A MICRO-CHANNEL USING FOURIER ANALYSIS
DE69722800T2 (en) SAMPLE ANALYSIS METHOD BY DETERMINING A FUNCTION OF SPECIFIC BRIGHTNESS
DE112015005465B4 (en) Device and method for measuring biomolecules
DE69405233T2 (en) BIOCHEMICAL MULTIPLE CAPILLARY ANALYZER
EP1311655B1 (en) Device and method for making electrical contact with biological cells suspended in a fluid
DE69232592T2 (en) AUTOMATIC CAPILLARY ELECTROFORESE UNIT
DE102005003911B4 (en) Method for measuring the concentration or concentration change of a redox-active substance and associated device
DE69428000T2 (en) ANALYSIS AND SEPARATION METHOD
DE60219040T2 (en) DIELECTROPHORETIC PROCESS AND ARRANGEMENT FOR HIGH-THROUGHPUT SCREENING
DE102006053540B3 (en) Optospectroscopic analysis of biological sample in fluid, comprises: fixing the biological sample in a sample holding unit, categorizing particles in biological sample based on an analysis of the image data and analyzing biological sample
WO1998009154A1 (en) System for distinguishing fluorescent molecule groups by time resolved fluorescence measurement
WO2003056330A2 (en) Cell sorting system for the size-based sorting or separation of cells suspended in a flowing fluid
DE60110137T2 (en) biochip
WO2002059582A1 (en) Method for examining a test sample by means of fluorescence spectroscopy, especially fluorescence correlation spectroscopy, and device for carrying out said method
DE10023423B4 (en) Direct detection of single molecules
DE3856583T2 (en) Automatic capillary electrophoresis device
DE10224567B4 (en) Sensor arrangement and method for operating a sensor arrangement
DE60117180T2 (en) METHOD FOR MEASURING THE ACTIVATION CONDITION OF SIGNALING PATHS IN CELLS
DE10218325A1 (en) Chip arrangement used in biotechnology and gene technology for detecting biological molecules, such as DNA, RNA, proteins and polypeptides comprises a channel containing electrodes, a control switching circuit, and sensor elements
DE10214719A1 (en) Sensor for the qualitative and quantitative determination of (bio) organic oligomers and polymers, analysis methods for this as well as methods for producing the sensor
DE10117723A1 (en) Carrier for biological or synthetic samples has a sample holding plate with reservoirs and a dosing plate with projections, fitted with membranes, of an optically transparent material with trouble-free light beam transparency
DE19950823A1 (en) Quantitative determination of a target nucleic acid in a biological sample comprises amplification using a known number of labeled primer molecules
WO2002099417A2 (en) Method for detecting a substance and microtiter plate
DE3009901A1 (en) DEVICE FOR DETECTING CHANGES IN THE MINERALION CONCENTRATION IN ORGANIC TISSUES AND THE USE THEREOF
DE10221885B4 (en) Sensor unit, sensor arrangement and method for operating a sensor unit

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20030724

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

17Q First examination report despatched

Effective date: 20070102

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20100803