WO2004077034A1

WO2004077034A1 - Bioanalytical method based on measurement of the phosphorescence decay period

Info

Publication number: WO2004077034A1
Application number: PCT/EP2004/001972
Authority: WO
Inventors: Otto S. Wolfbeis; Axel Dürkop
Original assignee: Chromeon Gmbh
Priority date: 2003-02-27
Filing date: 2004-02-27
Publication date: 2004-09-10
Also published as: EP1639348A1; DE10308814A1

Abstract

The invention relates to a method for detecting interactions between two or more molecules by measuring the change in the luminescence decay period, especially the phosphorescence decay period of a luminophore fixed to at least one of the molecules. At least one interacting partner is marked with a luminophore having a decay period of at least 50 ns. A delay phase during which interfering background fluorescence is able to decay is provided after switching off the light pulse.

Description

Bioanalytical method based on the measurement of the decay time of phosphorescence

description

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 (generally: luminescence methods) 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

Studies on sites of activity and binding affinities are permitted, and d) in drug screening when a so-called prodrug is applied to one

Receptor or an enzyme binds, which can be used for the purpose of quick identification of potential drugs, including in high-throughput screening.

These and other interactions are often subsumed under the term (bio) molecular affinity.

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

* are comparatively easy to perform instrumentally.

In practically all cases, 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. In another method, 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.

However, the measurement of fluorescence intensity, decay time or polarization suffers from the serious disadvantage that practically any background fluorescence is also recorded. Numerous biomolecules have their own fluorescence, which is often difficult to differentiate from that of the marker. The (often used) microtiter plates made of plastic also fluoresce strongly. Both fluorescence form an underground signal of unknown size. As a result, the measured change in fluorescence intensity, decay time or polarization not only reflects the presence (or concentration) of an antibody, a DNA counter strand, a ligand or a prodrug, but is also falsified by an unknown fluorescence background.

It was therefore an object of the invention to provide a method in which the background signal is largely switched off.

This object is achieved by 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

Interaction between the two or more molecules An interaction between the two or more molecules is detected.

In the method described here, these problems of the prior art are solved by using a time-resolving measuring method. The fluorescence of many markers decays - just like that of the background - in the nanosecond range (typically 1 - 100 ns). The decay time is the time after which the luminescence intensity of a luminophore (F _t ) has dropped to a fraction 1 / e of the initial fluorescence intensity (F ₀ ); in the simplest case, the relationship F _t = F ₀ -e ^", ' ^τ applies. Current methods (for example pulse fluorometry or phase fluorometry) cannot distinguish between background fluorescence and analytical signal. that 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 τ, 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.

According to the invention, therefore, the determination of the decay time of an interaction partner marked with a luminophore and the If the interaction between this marked interaction partner and one or more other interaction partners takes place, the change in the cooldown is determined, and this is then used as an analytical parameter.

With the method according to the invention, 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.

According to the invention, 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 μs. 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.

To excite the luminescence, 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 μs, even more preferably <10 μs, most preferably <1 μs and even more preferably for <100 ns. After the end of the light irradiation, which for the purposes of the present invention is equated with the switching off of the light pulse, 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. After the delay phase, the decay time of the luminophore is determined, which can be done according to known methods. A change in the decay time as a result of an interaction between the two or more molecules can then be used to determine the presence of an interaction partner qualitatively and / or quantitatively or to determine the mode of action and binding affinity of the interaction partners. In a particularly preferred embodiment, 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 subside, e) the decay time of the luminophore s is determined according to a known method, and f) a molecular interaction of the binding partners is detected via a change in the decay time, or the concentration of a reaction partner whose concentration is determined quantitatively on the basis of previously established quantitative relationships between the change in the decay time.

In the method according to the invention, 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,

(2) the binding partner enters into a molecular interaction with its counterpart, (3) irradiates the product of this molecular interaction with a pulse of light (300 to 1000 nm),

(4) then provides for a delay phase of up to 50% of the luminophore's decay time,

(5) then the decay characteristic of the luminophore is determined, and (6) from the change in the decay characteristic determined in this way

Evidence of the occurrence of the interaction, or based on previously determined quantitative relationships between the change in the decay time and the concentration of one of the reaction partners to its presence or the concentration is calculated.

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. To solve this problem, it is proposed on the one hand that marker dyes are used which have a decay time τ of> 50 ns and on the other hand that the

Measurement is made time-resolved. The time resolution consists in that the sample to be examined is exposed to a short pulse of light and that the decay time τ 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.

The invention is further illustrated by the attached figures and the following examples.

Figure 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 τ 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.

example 1

A typical experimental result is shown below. Human serum albumin (HSA) was labeled in a manner known per se with the fluorescent marker ruthenium-tris-bipyridyl (RuDCC) (see the article: Polarization Immunoassays Using Reactive Ruthenium Metal-Ligand Complexes as Luminescent Labels, A. Dürkop, F. Lehmann, OS Wolfbeis, Anal. Bioanal. Chem. 372 (2002) 688-694). 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. The following table shows the measured values: Table 1. Changes in cooldown in the interaction of 55DC ruthenium-labeled HSA with unlabeled anti-HSA (at 20 ° C).

The data are shown graphically in FIG. 2. You can see from this that

(a) the interaction that has occurred between the antigen (USA) and the antibody manifests itself in a change in the cooldown and can therefore be demonstrated by measuring it;

(b) the measured decay time correlates with the concentration of antibody;

(c) the change in the cooldown is approx. 130 ns, which is much easier to measure than e.g. Are changes that are in the sub-nanosecond range.

Claims

Expectations

1. A method for detecting an interaction between two or more molecules, characterized in that a) at least one of the interaction partners is labeled with a luminophore which has a luminescence decay time of at least 50 ns, b) the labeled 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) a delay phase is provided after the end of the light irradiation, e) the decay time of the luminophore is determined and f) via a determined change in the decay time as a result of a Interaction between the two or more molecules An interaction between the two or more molecules is detected.

2. The method according to claim 1, characterized in that the determined change in the decay time determines at least one of the molecules qualitatively and / or quantitatively.

3. The method according to any one of claims 1 or 2, characterized in that in step a) at least one of the interaction partners is marked with a fluorescent and / or phosphorescent luminophore which has a luminescence decay time between 50 ns and 1 s.

4. The method according to any one of the preceding claims, characterized in that in step c) the system is irradiated with light for a period of <1 second, in particular <10 μs.

5. The method according to any one of the preceding claims, characterized in that the delay phase is at least 10 ns, in particular at least 20 ns and up to 200% of the decay time of the luminophore, in particular up to 50% of the decay time of the luminophore.

6. The method according to any one of the preceding claims, characterized in that a quantitative determination of one of the interaction partners on the basis of planned quantitative relationships between the

Change in cooldown and concentration of this interaction partner is performed.

7. The method according to any one of the preceding claims for the detection of a biomolecular interaction between natural or synthetic molecules, between receptors and ligands or receptors and potential medicaments or their precursors, characterized in that a) at least one of the two interaction partners or binding partners concerned with one phosphorescent marker is marked, which has a luminescence decay time between 50 ns and 1 s, b) the binding partner thus marked is brought into molecular contact with its potential interaction partner, c) the system is briefly irradiated with light of wavelength between 300 and 1000 nm, d ) after switching off the light pulse, a delay phase of up to 200% of the natural decay time of the luminophore is provided is, during which a disturbing background fluorescence can subside, e) the decay time of the luminophore is determined according to a known method, and f) a molecular interaction of the binding partners is detected via a change in the decay time that has occurred or on the basis of previously established quantitative relationships between the change in the Cooldown and the concentration of a reactant whose concentration is determined quantitatively.

8. The method according to any one of the preceding claims, characterized in that the marker is covalently bound to a binding partner.

9. The method according to any one of the preceding claims, characterized in that the phosphorescent marker is a metal-ligand complex.

10. The method according to any one of the preceding claims, characterized in that one of the binding partners is a protein, an antibody, an antigen or a proteinaceous receptor.

11. The method according to any one of the preceding claims, characterized in that at least one binding partner is a natural or synthetic nucleic acid oligomer.

12. The method according to any one of the preceding claims, characterized in that at least one binding partner is a natural or synthetic ligand or a prodrug.

13. The method according to any one of the preceding claims, characterized in that one of the binding partners is non-covalently marked with a phosphorescent marker.

14. The method according to any one of the preceding claims, characterized in that the phosphorescent marker is a so-called general protein stain.

15. The method according to any one of the preceding claims, characterized in that the phosphorescent marker is a so-called DNA or RNA intercalator.

16. The method according to any one of the preceding claims, characterized in that the interaction is detected using the method according to the invention using microtiter plates.