EP1129336A1 - Procede et dispositif d'evaluation rapide de reactions de liaison dans des plaques de microtitration interferometriques - Google Patents

Procede et dispositif d'evaluation rapide de reactions de liaison dans des plaques de microtitration interferometriques

Info

Publication number
EP1129336A1
EP1129336A1 EP98966390A EP98966390A EP1129336A1 EP 1129336 A1 EP1129336 A1 EP 1129336A1 EP 98966390 A EP98966390 A EP 98966390A EP 98966390 A EP98966390 A EP 98966390A EP 1129336 A1 EP1129336 A1 EP 1129336A1
Authority
EP
European Patent Office
Prior art keywords
layer
substrate
thickness
change
interference
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Withdrawn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
EP98966390A
Other languages
German (de)
English (en)
Inventor
Gunnar Brink
Joachim RÄDLER
Erich Sackmann
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.)
Jandratek GmbH
Original Assignee
Jandratek 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 Jandratek GmbH filed Critical Jandratek GmbH
Publication of EP1129336A1 publication Critical patent/EP1129336A1/fr
Withdrawn legal-status Critical Current

Links

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
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a device and a method for rapidly evaluating binding reactions in structured interferometric microtiter plates, in particular a device and a method for determining the change in the optical thickness of a layer by means of light interference.
  • microtiter plates are plastic trays made of transparent plastics (polystyrene, polypropylene etc.) with a number (typically 96) of small reaction pots. Proteins, such as antibodies, can be bound in the hydrophobic containers by physisorption. The addition of the solutions to be tested and the subsequent washing cycles are often carried out by automated pipetting machines.
  • the reading of the plates can also be automated.
  • the color reaction is measured based on the optical absorption of the solution, in radiological tests the amount of radioactive substances.
  • radiological tests the amount of radioactive substances.
  • firstly fluorescence or radioactive labels are necessary and secondly, in particular ELISA tests are not always quantitative, ie only detect the presence but not the exact amount of a substance.
  • Biosensors In contrast to this are biosensors, which deliver exact values, for example the optical layer thickness of an adsorbate or the mass coverage of a surface.
  • Biosensors are reversible and continuously working sensors for the detection of proteins, nucleic acids or poly sugars. Such highly sensitive transducers play a major role if, in addition to the presence of a ligand, its concentration is also to be determined.
  • Quantitative sensors can be used in particular to determine the binding constant of a ligand. In addition to the development of suitable substrates and chemical coupling processes, the automation of the quantitative determination of the receptor-ligand binding must still be optimized.
  • optical layer thicknesses include surface plasmon resonance (Liedberg, et al. 1983; Surface Plasmon Resonance for Gas Detection and Biosensing, Sensors and Actuators, 4: 299-304), which reflecto Electric interference spectroscopy (Brecht et al. 1993; Interferometric immunoasay in a FIA system: a sensitive and rapid approach in label-free immunosensing. Biosensors & Bioelectronics. 8: 387-392), as well as quartz scales.
  • the first two techniques measure optical layer thicknesses with a resolution in the Angstrom range, while the Quartz scale measures mass loadings with a detection limit in the ⁇ g range.
  • an interference layer of constant thickness D in this case 48 nm
  • D in this case 48 nm
  • a planar interference layer only provides a signal which is subject to numerous interferences.
  • the object of the invention is to provide a device, a method and a method for producing the device which allow (s) to determine the change in the optical thickness of a layer, preferably an adsorbate layer in microtiter plates. This object is achieved with the features of the claims.
  • the invention is based on the basic idea that due to variations in the spacing between the top and bottom of a layer, for example an adsorbate layer, there are varying interference patterns when irradiated with light if the optical thickness of the layer changes.
  • a biofunctionalized microtiter plate microstructured interference layers introduced or applied.
  • the optical reflection interference of the plate is imaged and evaluated with the help of image processing.
  • the mean square difference of the nominal intensities of the interference patterns before and after adsorption is used to determine the change in the optical layer thickness of the adsorbate.
  • the device / method according to the invention is based on optical interference, the thickness of the interference-capable coating varying. This is resolved by optical imaging and evaluated by a simple image processing method.
  • the ligands to be detected do not have to be pretreated or labeled.
  • the determination of the change in the optical layer thickness of the adsorbate from the mean square difference of the nominated intensities has a high sensitivity with a layer thickness resolution of one angstrom (1 ⁇ ).
  • the method is robust against external fluctuations in the lighting intensity, as well as against irregularities in the exact microstructuring of the plate.
  • microtitre test is based on an imaging process, obvious malfunctions such as the Precipitation of reagents or the accumulation of a speck of dust can easily be noticed. 6.
  • the imaging process can be easily combined with fluorescence techniques.
  • the method should therefore be used as an interferometric immunoassay, the advantages being particularly evident in automated systems with a high throughput of microtiter plates. It is therefore also explained below how structured microtiter plates can be produced inexpensively on a plastic basis.
  • La shows a representation of an integrated interference layer with a laterally varying layer thickness D, the adsorbate layer ⁇ carrying receptors and an optical layer thickness change ⁇ being measured;
  • FIG. 1c shows the mean square difference of the normalized intensities S as a function of the optical layer thickness change ⁇ ;
  • 2a shows a representation of a microtiter plate with 12 reaction droplets
  • 2b shows an interfering recess integrated in each potty (and filled with the plastic 2); 2c shows a representation of an interference pattern. Of a depression, as can be seen in the imaging reflection interference microscopy;
  • FIG. 3 shows a structure for the optical imaging of the reflectivity of the microtiter plate by means of reflection-interference-contrast microscopy
  • FIG. 4 shows a structure for the optical imaging of the reflectivity of the microtiter plate by means of laser scanning.
  • the basis of the method according to the invention is to apply a biofunctionalized layer directly to an interference-capable substrate.
  • This first layer is provided with a biocompatible adsorbate layer 3 with calculation index n3, preferably a polymer layer, which contains antibodies or receptors.
  • Layer thickness ⁇ of this biocompatible layer 3 increases when ligands 4 are bound from an aqueous solution 17 with a calculation index /.
  • the substrate is illuminated from below, ie from the side facing away from the biofunctionalized layer 3, with preferably monochromatic light, and the resulting interference pattern is imaged on a camera.
  • the intensity of the reflected light is determined by the superposition of all reflected partial beams (see: Azza, R. et al. 1975; Ellipsometry and polarized light, Amsterdam, North Holland).
  • For the exact determination of a change in layer thickness ⁇ use is now made of the fact that the thickness D of the interference layer 2 varies laterally, so that a large number of interference maxima and minima are produced.
  • Fig. La shows a first embodiment. This shows, for example, a triangular depression and thus a linear change in the thickness D corresponding to the oblique course of the triangular surfaces from the location.
  • the interference images are now processed in such a way that the change in layer thickness ⁇ is determined only from the mean square phase shift. This corresponds to the shift in the reflectivity curves shown in FIG. 1b. This processing makes the determination of the change in thickness of the adsorbate layer 3 independent both of fluctuations in the incident light and of the precise shape and quality of the microstructured interference layer.
  • a number of maxima and the same number of minima are preferably used for the evaluation.
  • only the first layer 2 is applied to the carrier 1, so that an attachment to the layer 2 takes place.
  • the first layer 2 or second layer 3 does not have the receptors to which ligands bind, but conversely the ligands to which the receptors bind.
  • the mutual binding effect of receptors and ligands is used here.
  • a flat or smooth carrier is used, on which the layer 2 is applied in such a way that the distance between the top and bottom varies. In this way it is achieved in this embodiment that the thickness D of the layer 2 varies.
  • Layer 2 is preferably applied in the form of drops or droplets (for example sprayed on or applied with a pipette). These are allowed to dry or polymerize.
  • a flat layer 2 is applied, to which a structure is then printed.
  • a swellable material is preferably used for the second layer 3, which in the dry state has a thickness of approximately 5 to 200 ⁇ , preferably approximately 10 to 20 ⁇ , while in the swollen state it has thicknesses between 10 and 100,000 ⁇ , preferably 10,000 ⁇ . In this swollen state, this layer preferably has a refractive index n3 of 1.31.
  • the change in the optical thickness of the layer to be determined can take place, for example, in the following ways.
  • the physical thickness and thus also the optical thickness of the layer change due to deposits on the other hand, a change in the optical thickness can be based on a change in the refractive index, which results from deposits in the layer.
  • An interference image is recorded before and after (or during) the addition of the ligand and the following operations are carried out over the surface of the interference pattern. (In the case of a multiple test, such as the microtiter plate shown below, these operations are of course carried out separately for each binding surface.)
  • the intensities are standardized:
  • the ⁇ > means the averaging over all NxM pixels. Then the difference between the normalized intensities I ⁇ , N of the images before and after the binding is formed and divided into squares:
  • averaging is carried out over all NxM pixels of an image section which is preferably the same size or smaller than the interference pattern at the bottom of a microtiter potty. Averaging is preferably carried out over a square area.
  • S is referred to as the "mean square difference of the normalized intensities" of two interference patterns (I v , I j j) before and after the adsorption. The following applies to the value S obtained in this way:
  • Equation (3) is used in many practical cases where NEN the adsorbate layer is small compared to ⁇ / 4 is sufficient to directly determine the optical layer thickness increase ⁇ from the measured value for S.
  • the respective accuracy of equation (3) can be determined by comparison with the numerical values for given refractive indices and layer thicknesses ⁇ .
  • S is proportional to the change in layer thickness ⁇ .
  • the exact relationship can be determined numerically and stored in a table. For the borderline case that the adsorbate layer is exactly zero before adsorption, the above relationship (3) can be derived by neglecting multiple reflection.
  • Fig. Lc shows the size S of a simulated sample, which was calculated according to equations (1) and (2) as a function of
  • Optically high-quality, laterally structured interference layers can be produced by standard vapor deposition of dielectric layers on glass.
  • the structuring would take the form of masks which are either shifted or exchanged between the individual vapor deposition cycles.
  • plastic plate or the carrier substance is structured.
  • An optically low or non-birefringent polymer (polycarbonate, cyclic olefir-e) can, for example, be injection molded onto a microtiter plate mold (FIG. 2a).
  • This consists of an arrangement of small plastic pots with a flat bottom.
  • a depression 5 of a few micrometers depth is pressed in each potty with the aid of a stamp (FIG. 2b).
  • the recess is between 5 and 2000 nanometers deep.
  • a second polymer (layer 2) with a different refractive index is applied from the solution to the depression 5 in the casting process, but can - as shown in Fig. La - extend beyond the recess 5. Since the diameter of the stamp is constant, the layer thickness D can be produced very reproducibly by applying a defined amount of solvent.
  • the second plastic 2 should be soluble in a solvent that does not attack the plastic 1. By drying the layer 2, a meniscus can form on the surface.
  • the reflectivity of the microtiter plate can be imaged with the aid of reflection microscopy under reflected light (see FIG. 3). If monochromatic light is generated with the aid of a narrow-band interference filter, the interference becomes visible in the laterally structured depressions 5 described above. In practice, further the ratio of interference signal to background brightness verbes ⁇ lets fibers by the anti-Flex technology for Pluta (Pluta, M; Advanced Light Microscopy, Elsevier, Amsterdam 1989) uses be ⁇ . 3 shows the beam path of such a reflection-interference-contrast microscope according to Pluta. If the sample chamber is placed directly on the immersion oil ⁇ / 4 plate 7 applied, so the first reflective interface is the glass-magnesium fluoride 2 interface (Fig. La).
  • the carrier 1 with the introduced interference layer 2 is arranged over a ⁇ / 4 plate 7 (optional). In between is a medium 6 with the same refractive index as the support
  • This medium is preferably an immersion oil or silicone.
  • the light emitted by a mercury vapor lamp 14 is directed onto the layer to be measured via a bandpass filter 13, which generates monochromatic light, a polarizer 12 (optional), a semi-transparent mirror 9 and an objective 8.
  • the reflected light penetrates the semitransparent mirror 9 and reaches an camera 11 via an analyzer 10 (optional). This can be a CCD camera.
  • a second possible readout method consists of scanning the microtiter plate with a laser or a laser diode. The reflected light is recorded with a one-dimensional detector. If the plate is scanned, an interference signal again arises, as shown in FIG. 1b, and the evaluation is carried out analogously to the imaging reflection interference.
  • Fig. 4 shows an embodiment with laser scanning. Scanning the microtiter plate with a laser 19 is advantageous in applications in which the speed of the reading process is critical.
  • imaging optics are dispensed with and the microtiter plate is scanned by a laser beam, the intensity of the reflected beam being measured.
  • the laser beam is deflected by a rotatable mirror 20 so that it sweeps over the sample and is reflected on a one-dimensional detector 21 (preferably CCD).
  • the depressions of the microtiter plate in this embodiment of the scanning are preferably roof-shaped or wedge-shaped depressions, that is to say only varied in a direction corresponding to the scanning direction.
  • the averages according to formulas (1) and (2) correspond to the averaging along a line.
  • Reference numeral 18 denotes a glass block with an anti-reflective coating.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Plasma & Fusion (AREA)
  • Clinical Laboratory Science (AREA)
  • Length Measuring Devices By Optical Means (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

L'invention concerne un procédé interférométrique permettant de déterminer la modification de l'épaisseur optique d'une couche d'adsorbat dans des plaques de microtitration. Ce procédé est fondé sur l'intégration de couches d'interférence structurées latéralement dans le fond d'une plaque de microtitration biofonctionnalisée. L'interférence de réflexion de la plaque est représentée et est évaluée par traitement d'images. A cet effet, la différence quadratique moyenne des intensités normalisées est utilisée pour déterminer la modification de l'épaisseur optique de l'adsorbat. Ce procédé s'avère très sensible, quantitatif, économique et robuste par rapport aux perturbations extérieures. Un des principaux avantages qu'il présente réside dans le fait que les ligands n'ont pas à être marqués.
EP98966390A 1997-12-23 1998-12-21 Procede et dispositif d'evaluation rapide de reactions de liaison dans des plaques de microtitration interferometriques Withdrawn EP1129336A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19757706A DE19757706C2 (de) 1997-12-23 1997-12-23 Substrat, Vorrichtung und Verfahren zur schnellen Auswertung von Bindungsreaktionen durch interferometrische Schichtdickenmessung
DE19757706 1997-12-23
PCT/EP1998/008390 WO1999034196A1 (fr) 1997-12-23 1998-12-21 Procede et dispositif d'evaluation rapide de reactions de liaison dans des plaques de microtitration interferometriques

Publications (1)

Publication Number Publication Date
EP1129336A1 true EP1129336A1 (fr) 2001-09-05

Family

ID=7853276

Family Applications (1)

Application Number Title Priority Date Filing Date
EP98966390A Withdrawn EP1129336A1 (fr) 1997-12-23 1998-12-21 Procede et dispositif d'evaluation rapide de reactions de liaison dans des plaques de microtitration interferometriques

Country Status (4)

Country Link
EP (1) EP1129336A1 (fr)
AU (1) AU2275299A (fr)
DE (1) DE19757706C2 (fr)
WO (1) WO1999034196A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111389473B (zh) * 2020-03-25 2021-05-04 武汉大学 一种垂直沟道可调谐高通量声流控分选芯片及其制备方法

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4293224A (en) * 1978-12-04 1981-10-06 International Business Machines Corporation Optical system and technique for unambiguous film thickness monitoring
US4355903A (en) * 1980-02-08 1982-10-26 Rca Corporation Thin film thickness monitor
DE3509512A1 (de) * 1985-03-16 1986-09-18 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V., 8000 München Vorrichtung zur interferometrischen pruefung der optischen homogenitaet von transparenten plattenfoermigen werkstuecken
US4824230A (en) * 1985-12-13 1989-04-25 E. I. Du Pont De Nemours And Company Visualization device
US5116765A (en) * 1989-04-25 1992-05-26 Olympus Optical Co., Ltd. Method for automatic chemical analyzing
DE3915920A1 (de) * 1989-05-16 1990-11-22 Messerschmitt Boelkow Blohm Mikromechanische struktur
US5129724A (en) * 1991-01-29 1992-07-14 Wyko Corporation Apparatus and method for simultaneous measurement of film thickness and surface height variation for film-substrate sample
DE4115414C2 (de) * 1991-05-10 1995-07-06 Meinhard Prof Dr Knoll Verfahren zur Herstellung von miniaturisierten Chemo- und Biosensorelementen mit ionenselektiver Membran sowie von Trägern für diese Elemente
DE4200088C2 (de) * 1992-01-04 1997-06-19 Nahm Werner Verfahren und Vorrichtung zum optischen Nachweis einer An- oder Einlagerung mindestens einer stofflichen Spezies in oder an mindestens einer dünnen Schicht
FR2694809A1 (fr) * 1992-08-11 1994-02-18 Mikralgen Dispositif réactionnel pour identifier un produit, et son procédé d'obtention.
IL110466A (en) * 1994-07-26 1998-07-15 C I Systems Israel Ltd Film thickness mapping using interferometric spectral imaging
US5555471A (en) * 1995-05-24 1996-09-10 Wyko Corporation Method for measuring thin-film thickness and step height on the surface of thin-film/substrate test samples by phase-shifting interferometry

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9934196A1 *

Also Published As

Publication number Publication date
AU2275299A (en) 1999-07-19
DE19757706C2 (de) 2002-01-24
DE19757706A1 (de) 1999-07-01
WO1999034196A1 (fr) 1999-07-08

Similar Documents

Publication Publication Date Title
EP1259796B1 (fr) Systeme de detection par resonance plasmonique de surface
DE4024476C1 (fr)
EP0226604B1 (fr) Senseur optique pour etablir selectivement la presence de substances ainsi que la variation d'indice de refraction dans les substances objet de mesures
EP0617273B1 (fr) Procédé et dispositif optique pour l'analyse de substances sur des surfaces de capteurs
DE69929075T2 (de) Biochip mit einer mehrzahl von bereichen zur molekularen erkennung, und dazu geeignete auslesevorrichtung
US8283156B2 (en) Method and apparatus for assay based on light diffraction
EP2137514B1 (fr) Capteur optique intégré
DE19955556B4 (de) Meßanordnung zum parallelen Auslesen von SPR-Sensoren
US20080280374A1 (en) Methods and systems for detecting biological and chemical materials on a submicron structured substrate
EP1190236A1 (fr) Plate-forme de capteurs et procede permettant de determiner plusieurs substances a analyser
WO1998057151A1 (fr) Dispositif de detection de substances chimiques et biochimiques par excitation de lumiere fluorescente et son procede de production
WO2003056308A1 (fr) Dispositif et procede d'examen de couches minces
DE4200088C2 (de) Verfahren und Vorrichtung zum optischen Nachweis einer An- oder Einlagerung mindestens einer stofflichen Spezies in oder an mindestens einer dünnen Schicht
DE3719524C2 (fr)
WO1993001487A1 (fr) Procede optique de detection specifique de substances specifiques dans des echantillons chimiques, biochimiques et biologiques de mesure
DE69817329T2 (de) Vorrichtung und Verfahren zur Identifikation einer Substanz durch Oberflächenwechselwirkungen
DE19757706C2 (de) Substrat, Vorrichtung und Verfahren zur schnellen Auswertung von Bindungsreaktionen durch interferometrische Schichtdickenmessung
DE4315211A1 (de) Vorrichtung und Verfahren zum Nachweis der Adsorption von Molekülen mittels Nahinfrarot-Oberflächenplasmonenresonanz
DE10052165A1 (de) SPR-Sensorsystem
DE19837437A1 (de) Vorrichtung und Verfahren zum Bestimmen von Abständen und ein zugehöriges Nanodosiersystem
DE102010038718B4 (de) Messvorrichtung und Verfahren zum Bestimmen eines eine elektrische Eigenschaft einer Dünnschicht auf einem Träger beschreibenden ersten Parameters
AT5152U1 (de) Spr-sensorsystem
EP3899494A2 (fr) Arrangement et procédé pour l'acquisition de propriétés optiques d'un échantillon, notamment pour la détection sélective de molécules biologiques et pour la lecture de l'occupation d'une molécule
DE102009019717A1 (de) Verfahren und Verwendung eines optischen Gitters zum Nachweis des Vorhandenseins von Molekülen

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: 20010522

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT CH DE FR GB LI NL SE

17Q First examination report despatched

Effective date: 20040322

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: 20040803