WO2004077034A1 - Procede bioanalytique base sur la mesure du temps d'extinction de la phosphorescence - Google Patents

Procede bioanalytique base sur la mesure du temps d'extinction de la phosphorescence Download PDF

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Publication number
WO2004077034A1
WO2004077034A1 PCT/EP2004/001972 EP2004001972W WO2004077034A1 WO 2004077034 A1 WO2004077034 A1 WO 2004077034A1 EP 2004001972 W EP2004001972 W EP 2004001972W WO 2004077034 A1 WO2004077034 A1 WO 2004077034A1
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WO
WIPO (PCT)
Prior art keywords
interaction
decay time
luminophore
partners
partner
Prior art date
Application number
PCT/EP2004/001972
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German (de)
English (en)
Inventor
Otto S. Wolfbeis
Axel Dürkop
Original Assignee
Chromeon 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 Chromeon Gmbh filed Critical Chromeon Gmbh
Priority to EP04715275A priority Critical patent/EP1639348A1/fr
Publication of WO2004077034A1 publication Critical patent/WO2004077034A1/fr

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Classifications

    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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

Definitions

  • the present invention relates to a method for the detection of interactions between two or more molecules based on the measurement of the decay time of the luminescence, in particular the phosphorescence of a luminophore attached to at least one of the molecules.
  • Fluorescence and phosphorescence are used in bioanalytics to demonstrate interactions between two reaction partners. Interactions occur, for example, when a) an antigen reacts with an antibody, which is for the purpose of
  • Immunoanalysis and diagnostics can be used; b) an oligonucleotide with its counter strand a double helix. forms what can be used for the purposes of genetic analysis and diagnosis; c) a receptor binds a ligand, which is pharmacological
  • Receptor or an enzyme binds, which can be used for the purpose of quick identification of potential drugs, including in high-throughput screening.
  • the luminescence methods mentioned are often used because they * have a high sensitivity (close to that of radioanalytical methods), * are free from the disadvantages of handling radioanalytical materials, and
  • the procedure is to mark one of the two reaction partners of the molecular interaction with a fluorescent marker (“label”), the fluorescence intensity, decay time, or polarization of which is the result of the molecular interaction measurably changes.
  • label a fluorescent marker
  • both reaction partners are even marked, and so-called fluorescence resonance energy transfer (FRET) occurs, the measurement of which has some advantages, but the implementation of which involves considerable additional effort in terms of the marking.
  • FRET fluorescence resonance energy transfer
  • a method for detecting an interaction between two or more molecules which is characterized in that a) at least one of the interaction partners with a luminophore is marked, which has a luminescence decay time of at least 50 ns, b) the marked interaction partner is brought into contact with its potential interaction partner, c) the system is irradiated with light with wavelengths between 300 and 1000 nm, d) after the end of the light irradiation Delay phase is provided, e) the decay time of the luminophore is determined and f) via a determined change in the decay time as a result of
  • a marker is used whose decay time is sometimes longer than that of the background, but at least 50 ns, that a delay time is provided for the measurement, which can be up to 200% of the natural decay time ⁇ , and that instead of the measurement the intensity, decay time or polarization uses the change in the decay time of the marker as a result of the ligand-receptor interaction as analytical information.
  • interactions between molecules can be encompassed, which allows both qualitative and quantitative detection of an analyte to be determined, as well as statements about the type of binding and kinetics of binding.
  • the interaction between two or more molecules detected according to the invention is, in particular, biomolecular interactions. Both natural and synthetic molecules can be used as interaction partners.
  • the method according to the invention is suitable, for example, in particular for the detection of interactions between receptors and ligands or receptors and potential medicaments or their precursors and particularly preferably for the detection of interactions between antigen and antibody, (oligo) nucleotide and (oligo) nucleotide and Polypeptide and ligand, in particular non-peptide small molecule with a molecular weight of ⁇ 1000 Da.
  • At least one of the interaction partners is labeled with a luminophore.
  • the luminophore is a fluorescent or / and phosphorescent, preferably a phosphorescent marker.
  • the decay time of the luminophore is preferably at least 50 ns, more preferably at least 100 ns, and can preferably be up to 1 second, more preferably up to 1 ⁇ s.
  • Luminescence decay time is understood here to mean the decay time which the luminophore has bound to the one interaction partner without the interaction partner entering into an interaction with another molecule.
  • the marked interaction partner is then brought into contact with its potential interaction partner, in particular into molecular contact. If the potential interaction partner is present in the sample, an interaction can be formed between these two interaction partners.
  • the decay time of the luminophore changes due to the formation of the interaction, which is used in the present invention for analysis purposes.
  • the system is irradiated with light, in particular with light with wavelengths between 300 and 1000 nm.
  • the irradiation can take place monochromatically, for example by means of a laser. It is also possible to radiate light with a broad wavelength spectrum, for example using an incandescent lamp.
  • the irradiation is particularly preferably carried out for a predetermined period of time and even more preferably briefly, for example for ⁇ 1 s, more preferably ⁇ 100 ⁇ s, even more preferably ⁇ 10 ⁇ s, most preferably ⁇ 1 ⁇ s and even more preferably for ⁇ 100 ns.
  • a delay phase is provided according to the invention. During this delay phase, disturbing background fluorescence can subside.
  • the delay phase is particularly preferably at least 10 ns, more preferably at least 30 ns and even more preferably at least 50 ns.
  • the delay phase is further adjusted so that it is preferably up to a maximum of 200% of the luminescence decay time, more preferably up to 50% of the luminescence decay time.
  • the decay time of the luminophore is determined, which can be done according to known methods.
  • the method according to the invention relates to a method for the detection of a biomolecular interaction between natural or synthetic molecules, between receptors and ligands or receptors and potential medicaments or their precursors, which is characterized in that a) at least one of the two the interaction partner or binding partner concerned is marked with a phosphorescent marker which has a luminescence decay time between 50 ns and 1 s, b) the binding partner marked in this way is brought into molecular contact with its potential interaction partner, c) the system is briefly exposed to light of the wavelength between 300 and 1000 nm is irradiated, d) after switching off the light pulse, a delay phase of up to 200% of the natural decay time of the luminophore is provided, during which a disturbing background fluorescence can
  • the luminophore or fluorescent or phosphorescent marker can be bound covalently or non-covalently to one of the binding partners.
  • Suitable phosphorescent markers are, for example, metal-ligand complexes, general protein stains, DNA or RNA intercalators and other compounds which have luminescence.
  • a particularly preferred embodiment of the invention is that (1) one of the two binding partners is marked with a marker which has a luminescence decay time between 50 ns and 10 s,
  • the interaction between a ligand and a receptor can be demonstrated by changing the decay time of the luminescence of a marker.
  • the disadvantage here is that the intrinsic luminescence of the biological test material or the components used, in particular the microtiter plates, makes a not insignificant background contribution to luminescence.
  • marker dyes are used which have a decay time ⁇ of> 50 ns and on the other hand that the
  • the time resolution consists in that the sample to be examined is exposed to a short pulse of light and that the decay time ⁇ is only determined after a short delay time (ti) during which the very short-lived ( ⁇ 10 ns) background luminescence decays, so that after the delay time, the luminescence of the marker can be measured very selectively.
  • the change in the decay time allows above all the qualitative detection of the occurrence of an interaction between any receptor and any ligand (for example between antigen and antibody, between two strands of a polynucleotide, between natural receptors and syn- synthetic ligands and the like). However, it can also be used for the quantitative determination of one of the two interacting partners.
  • FIG. 1 illustrates the invention.
  • the biological system is stimulated to luminescence after the interaction. After switching off the excitation light, wait until time ti, after which the background (shown in gray) has subsided. Then the photodetector is opened and the decay time is measured by any method, for example by the rapid lifetime detection method shown in the figures by determining the areas At and A 2 , which are related to the decay time ⁇ via the given equation , This decay time is a direct measure of whether a biomolecular interaction has occurred or not. For reasons of clarity, the decay curve after the interaction between ligand and receptor has not been entered.
  • FIG. 2 shows a life time assay of HSA marked with 55 DC ruthenium and unlabeled anti-HSA.
  • HSA Human serum albumin
  • RuDCC ruthenium-tris-bipyridyl
  • the decay time of the HSA was determined as a function of the amount of antibody (anti-HSA) added. It turns out that the cooldown is made up of two components. One (approx. 100 ns) is analytically useful, the other (approx. 17 ns) remains constant and is therefore not meaningful.
  • Table 1 Changes in cooldown in the interaction of 55DC ruthenium-labeled HSA with unlabeled anti-HSA (at 20 ° C).

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

La présente invention concerne un procédé pour détecter des interactions entre au moins deux molécules, basé sur la mesure de la variation du temps d'extinction de la luminescence, en particulier de la phosphorescence d'un luminophore appliqué sur au moins une des molécules. Au moins un partenaire d'interaction est marqué avec un luminophore présentant un temps d'extinction d'au moins 50 ns. Après l'arrêt de l'impulsion lumineuse, il est prévu une phase de temporisation au cours de laquelle une fluorescence de fond parasite peut s'éteindre.
PCT/EP2004/001972 2003-02-27 2004-02-27 Procede bioanalytique base sur la mesure du temps d'extinction de la phosphorescence WO2004077034A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP04715275A EP1639348A1 (fr) 2003-02-27 2004-02-27 Procede bioanalytique base sur la mesure du temps d'extinction de la phosphorescence

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2003108814 DE10308814A1 (de) 2003-02-27 2003-02-27 Bioanalytisches Verfahren auf Grundlage der Messung der Abklingzeit der Phosphoreszenz
DE10308814.8 2003-02-27

Publications (1)

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WO2004077034A1 true WO2004077034A1 (fr) 2004-09-10

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DE (1) DE10308814A1 (fr)
WO (1) WO2004077034A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005095982A1 (fr) * 2004-04-02 2005-10-13 Chromeon Gmbh Systemes supports solides destines a la detection homogene d'interactions entre des biomolecules sans etapes de lavage

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR3010521B1 (fr) * 2013-09-12 2015-10-09 Luxor Procede fluorimetrique simplifie pour evaluer l'influence d'une condition sur un echantillon biologique et ses applications

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988007670A2 (fr) * 1987-03-27 1988-10-06 Chimerix Corporation Appareil de fluorescence a resolution dans le temps et immunoanalyse
EP0349520A2 (fr) * 1988-06-28 1990-01-03 Ernst Dr. Koller Immuno-essai basé sur l'extinction de fluorescence
EP0628805A1 (fr) * 1993-06-09 1994-12-14 AVL Medical Instruments AG Indicateur optique luminescent pour déterminer l'activité des ions alcalins dans une solution d'échantillon
WO1998038496A1 (fr) * 1997-02-28 1998-09-03 Lakowicz Joseph R Mesure d'analytes a l'aide de sondes de complexe metal/ligand
DE19702914A1 (de) * 1997-01-28 1998-09-17 Max Planck Gesellschaft Verfahren und Anordnung zum Bestimmen vorgegebener Eigenschaften von Zielpartikeln eines Probenmediums
JPH11164700A (ja) * 1997-12-04 1999-06-22 Hitachi Ltd Ｄｎａ検出方法
DE10133844A1 (de) * 2001-07-18 2003-02-06 Biochip Technologies Gmbh Verfahren und Vorrichtung zur Detektion von Analyten
DE10137530A1 (de) * 2001-08-01 2003-02-13 Presens Prec Sensing Gmbh Anordnung und Verfahren zur Mehrfach-Fluoreszenzmessung

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988007670A2 (fr) * 1987-03-27 1988-10-06 Chimerix Corporation Appareil de fluorescence a resolution dans le temps et immunoanalyse
EP0349520A2 (fr) * 1988-06-28 1990-01-03 Ernst Dr. Koller Immuno-essai basé sur l'extinction de fluorescence
EP0628805A1 (fr) * 1993-06-09 1994-12-14 AVL Medical Instruments AG Indicateur optique luminescent pour déterminer l'activité des ions alcalins dans une solution d'échantillon
DE19702914A1 (de) * 1997-01-28 1998-09-17 Max Planck Gesellschaft Verfahren und Anordnung zum Bestimmen vorgegebener Eigenschaften von Zielpartikeln eines Probenmediums
WO1998038496A1 (fr) * 1997-02-28 1998-09-03 Lakowicz Joseph R Mesure d'analytes a l'aide de sondes de complexe metal/ligand
JPH11164700A (ja) * 1997-12-04 1999-06-22 Hitachi Ltd Ｄｎａ検出方法
DE10133844A1 (de) * 2001-07-18 2003-02-06 Biochip Technologies Gmbh Verfahren und Vorrichtung zur Detektion von Analyten
DE10137530A1 (de) * 2001-08-01 2003-02-13 Presens Prec Sensing Gmbh Anordnung und Verfahren zur Mehrfach-Fluoreszenzmessung

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PATENT ABSTRACTS OF JAPAN vol. 1999, no. 11 30 September 1999 (1999-09-30) *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005095982A1 (fr) * 2004-04-02 2005-10-13 Chromeon Gmbh Systemes supports solides destines a la detection homogene d'interactions entre des biomolecules sans etapes de lavage

Also Published As

Publication number Publication date
EP1639348A1 (fr) 2006-03-29
DE10308814A1 (de) 2004-09-09

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