EP1658496A1 - Procede a cinetique de relaxation photochromique - Google Patents

Procede a cinetique de relaxation photochromique

Info

Publication number
EP1658496A1
EP1658496A1 EP04763779A EP04763779A EP1658496A1 EP 1658496 A1 EP1658496 A1 EP 1658496A1 EP 04763779 A EP04763779 A EP 04763779A EP 04763779 A EP04763779 A EP 04763779A EP 1658496 A1 EP1658496 A1 EP 1658496A1
Authority
EP
European Patent Office
Prior art keywords
fret
photochromic
state
light
fret acceptor
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.)
Ceased
Application number
EP04763779A
Other languages
German (de)
English (en)
Inventor
Thomas Jovin
Elizabeth A. Jares-Erijman
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.)
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Original Assignee
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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 Max Planck Gesellschaft zur Foerderung der Wissenschaften eV filed Critical Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Publication of EP1658496A1 publication Critical patent/EP1658496A1/fr
Ceased legal-status Critical Current

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

  • the invention relates to a method for determining a characteristic kinetic size of a chemical reaction between a plurality of chemical species in a sample, wherein at least one species contains at least one fluorophore, comprising the following steps: generating an imbalance state of the chemical reaction by exposing the sample to light and time-resolved observation of at least a portion of the relaxation of the concentrations of the species involved using a fluorescence signal of at least one fluorophore.
  • Such methods represent special forms of the methods generally known as relaxation spectroscopic methods with fluorescence detection.
  • the classic relaxation kinetic methods as described, for example, in Eigen, M .; DeMaeyer, L .: “Theoretical basis of relaxation kinetics” in “Investigations of rotes and mechanisms of reactions", Part III, 3, 3rd edition (G. Hammes, ed.), Techniques ofChem., Vol. 6, p.
  • Time-resolved fluorescence spectroscopy has proven to be a suitable method of observation. If at least one species involved in the reaction to be examined has a flurophor (be it that the species itself is fluorescent or that it is labeled with a suitable fluorophore), the fluorescence of which varies depending on the binding state of the species, the relaxation process can suitable excitation can be observed very precisely based on the fluorescence.
  • a disadvantage of the known method is that an intensive thermodynamic variable must be changed in each case, which on the one hand involves a comparatively great technical outlay and on the other hand represents a possible load on the chemical species. Sensitive biological material in particular can easily be damaged. Repetitive methods are known in which small changes in the relevant intensive thermodynamic variable are repeated many times in order to build up a low-noise signal. Although the strain on the species involved can be reduced compared to a one-time strong deflection, these reduced loads can already be too great for particularly sensitive material, in particular when examining living cells, etc. In addition, the repetitive methods usually require the balance to be restored before each deflection.
  • FRET fluorescence resonance energy transfer
  • FRET experiments are due to The strong dependence on distance, for example, is used in the investigation of the attachment of certain substances to biological structures, where certain areas of the structures to be examined and the attached substance each comprise a partner species of a FRET pair, and the FRET donor is then excited with a light of a suitable wavelength , its excitation energy can at least partially transfer to the FRET acceptor without radiation.
  • Such a chromophore can be used as a switchable FRET acceptor, namely if there is a suitable FRET donor whose emission spectrum overlaps in a very different way with the excitation spectra of the different conformations of the chromophore,
  • the FRET efficiency can be varied by irradiation with the conformation-changing light
  • irradiation of the sample with light of different wavelengths, in particular UV light and visible light can be carried out as desired between two photochromic Switch states back and forth.
  • At least one product of the chemical reaction to be investigated comprises a compound of two species, each of which contains a partner of a FRET pair consisting of FRET donor and FRET acceptor, wherein the FRET acceptor is a photochrome, the absorption spectrum of which can be changed by exposure to light of a suitable wavelength, the FRET donor is a fluorophore, the Emission spectrum with the absorption spectrum of the FRET acceptor has an overlap area, the size of which depends on the photochromic state of the FRET acceptor and the light used to generate the imbalance state of the chemical reaction has a wavelength switching the photochromic state of the FRET acceptor.
  • An imbalance state arises because, as will be explained in more detail below with the aid of an example, an FRET-mediated de-excitation channel is available in the bound state, which is not given for the free ligand. At the end of the switching process, therefore, the proportion of the bound state with the FRET acceptor in the changed photochromic state is too low compared to the free ligand.
  • the system can return to its equilibrium state in different ways by time-resolved fluorescence measurement, since the photochromic states in the bound state can be distinguished from one another on the basis of the different FRET efficiency.
  • the term “free ligand” encompasses all states in which the distance between the FRET partners is too large for a significant FRET to take place; in contrast, “bound state” or “complex” means any state in which the FRET partners are arranged sufficiently close to one another, in particular these terms should not be understood as restricting them to certain chemical bond forms.
  • a first species includes a FRET donor and is generally referred to as D.
  • a second species comprises a photochromic FRET acceptor and is generally referred to as A.
  • Short-term irradiation with an intense UV light pulse triggers a change in the conformation of the photochromic acceptor, the photochromic acceptor is "switched on”.
  • the spectra of D and A are matched to one another in such a way that D with A in the "switched on” state (A + ) forms an efficient FRET pair, whereas in the "switched off” state (A.) only a minimal FRET is possible between D and A.
  • the chemical reaction of interest involves the complex formation of species D and A to complex DA, where DA + and DA denote in each case the switched-on or switched-off state of the FRET acceptor in the bound state
  • DA + and DA denote in each case the switched-on or switched-off state of the FRET acceptor in the bound state
  • the reaction scheme is illustrated below, which illustrates the entire system when it is exposed to a wavelength that switches the photochromic state of A (eg in the UV range).
  • k f and k r are the rate constants of the forward and reverse reactions of the complex formation. For the sake of simplicity, they are assumed to be the same for the on and off state of A, although this is not essential for the basic idea of the present invention.
  • the method according to the invention has the advantage over the conventional relaxation methods that it is technically particularly simple to implement, since only a controllable light source of a suitable wavelength has to be available to set the imbalance state. Due to the simplicity of its construction, the method is also suitable for use in portable devices for quick on-site Measurement, for example when detecting special chemical substances that influence the kinetics of a reaction.
  • the faster applicability of a radiation flash compared to a change in temperature or pressure enables a better temporal resolution of the e-electronics to be measured.
  • the method is suitable for use on very small volumes and can therefore also e.g. to be used for imaging kinetic measurements in a microscope. living cells are also possible.
  • the fluorescence of the FRET donor can be measured to observe the relaxation.
  • the product to be examined comprises a further flurophor, which is a further FRET acceptor to the FRET donor.
  • This additional FRET acceptor can be present, for example, on the same molecule that also comprises the first, photochromic FRET acceptor.
  • the further FRET acceptor it is also possible for the further FRET acceptor to be encompassed by a third party molecule involved in the reaction.
  • the further FRET acceptor need not necessarily be a chromophore. Based on this experimental constellation, the FRET donor can be isolated in order to observe the relaxation and the fluorescence of the further FRET acceptor can be measured.
  • the Another FRET acceptor competes with the first photochromic acceptor in terms of energy transfer from the FRET donor.
  • fluorescence therefore depends on the one hand on the spatial constellation to the FRET donor and on the other hand on the photochromic state of the first FRET acceptor.
  • the required kinetic information can thus also be obtained from the fluorescence of the further FRET acceptor.
  • the first, photochromic FRET acceptor itself is not fluorescent and if the reaction to be investigated, for example DNA hybridization, it attaches itself much closer to the FRET donor than the other, fluorescent FRET acceptor, the and switching off the photochromic FRET acceptor of the energy transfer to the further, fluorescent FRET acceptor and thus switching its fluorescence on or off.
  • Monitoring only the fluorescence of the further, fluorescent FRET acceptor is therefore a particularly sensitive measurement method, since only complexes are detected in the switched-off state, the relaxation of which can therefore be observed in isolation.
  • the photochromic FRET acceptor it is possible to change its photochromic state in a first direction by irradiating the sample with light of a first wavelength, while changing its photochromic state in a second direction by irradiating it with light of a second wavelength.
  • This behavior is particularly favorable, since the switching direction, ie. H. from “On” to "Off” or from "Off” to "On".
  • the photochromic molecule can assume more than two photochromic states that can be switched with different wavelengths.
  • the change in the photochromic state of the FRET acceptor to take place in at least one direction by irradiation with ultraviolet light.
  • This light can also be used to excite the FRET donor, since most fluorophores can be excited in the ultraviolet spectral range.
  • the opposite circuit can, like also the case with diheteroarylethenes, for example by excitation with visible light.
  • the FRET donor can advantageously also be excited in the visible spectral range. This enables, for example, the simultaneous excitation of the donor together with the photochromic switching of the FRET acceptor regardless of the switching direction. This also enables targeted excitation of the FRET donor without simultaneously triggering the UV-mediated switching of the photochromic acceptor.
  • the latter is possible in particular if, as provided for in a preferred embodiment, the irradiation intensity for changing the photochromic state of the FRET acceptor is substantially stronger than the irradiation intensity for generating the fluorescence to be observed.
  • Such an experimental constellation is generally possible, since with conventional flurophores for the excitation of fluorescence, significantly lower irradiation intensities are required than for the switching of common photochromes.
  • the sample is irradiated in a time-modulated manner to change the photochromic state of the FRET acceptor. This means that radiation intensities change over time.
  • the circuit is then preferably according to. a repetitive "forcing function".
  • the detected signal will then be a convolution of the forcing function with the actual relaxation signal.
  • a special case of modulated radiation can be used if the photochromic FRET acceptor is switched on and off by means of light of two different wavelengths, namely it can be provided that to change the photochromic state of the FRET acceptor, the sample alternating with light of the first and the second wavelength is irradiated.
  • any switching pattern can be implemented. The most favorable choice of a switching pattern has to be made in coordination with the other experimental conditions and the experimental goal.
  • the specific choice of the radiation pattern also depends on the Properties of the photochrome used, which can show, for example, a different stability of its photochromic states, so that a return to one of the photochromic states based on thermal de-excitation can take place even without active switching back.
  • a device for carrying out the method explained above it is basically only necessary to take a sample, at least one controllable light source for the temporally and spectrally controlled irradiation of the sample in the sample, at least one light detector suitable for time-resolved measurement for detecting fluorescent light, which from the sample is emitted as a result of the radiation, and to provide a control unit which, as a rule, in terms of programming, is set up to control the at least one light source and the at least one light detector according to the inventive method.
  • a correspondingly programmed evaluation unit can preferably also be provided for the automated evaluation.
  • the simplifications that result from the inventive method compared to. conventional relaxation processes can, in a particularly preferred embodiment, e.g. All device components for mobile use can be integrated in a portable housing.
  • Figure 1 a circuit diagram of a photochromic FRET acceptor as a free ligand.
  • Figure 2 a circuit diagram of a photochromic FRET acceptor in the bound state with a FRET donor.
  • Figure 3 three simulated concentration curves as a result of the inventive method.
  • Figure 4 a schematic representation of a complex formation reaction with two FRET acceptors.
  • FIG. 1 shows the circuit diagram of a photochromic FRET acceptor A as a free ligand in the broad sense explained above.
  • large biological molecules are also included, which are labeled with the photochromic acceptor A and which, for example, attach themselves to other biological structures in the course of the reaction to be investigated.
  • A. denotes the FRET acceptor when it is switched off.
  • A. * represents the photochromic FRET acceptor when switched off, energetically excited.
  • a + denotes the FRET acceptor in the switched-on ground state
  • a + * denotes the FRET acceptor in the switched-on, photonically excited state.
  • Molecules in state A + can also be energetically excited by the incident light. There is therefore an excitation in parallel to state A + , namely with the velocity constant ke X ⁇ + . Similar to the case described above, some of the molecules in state A + return to the basic state A4- with the rate constant k d A + , while other parts use the absorbed excitation energy to switch to the switched-off state A.
  • the circuit diagram can be represented as a simple reaction with monoexponential kinetics and the speed constants k-4- 2 and k + - 2 for the switch-on and switch-off process.
  • FIG. 2 shows the same circuit diagram as FIG. 1, but for the case in which the photochromic FRET acceptor is present in the form bound to the FRET donor.
  • the term “bound” in the sense explained above is to be understood broadly here.
  • reaction partners of a DNA hypothesis can be labeled with the corresponding flurophores.
  • the “bound” state is generally referred to as complex DA.
  • the inner circle of the circuit diagram corresponds to that in Figure 1.
  • the radiation stimulates the FRET donor, so that a state D A results. This arises with the velocity constant ke X D. A large number of the molecules excited in this way return to the ground state DA. with the speed constant kd D. This includes the fluorescence emission.
  • FRET leads to a transition to state DA.
  • the FRET acceptor is switched off (A.), the efficiency of this transition is very low.
  • molecules in the DA + state are also brought into the energetically excited state DA + by the irradiation with the rate constant ke X D.
  • a partial return to the basic state DA with the speed constant k d D Another part of the molecules in the state D A + undergoes an energy transition via FRET to the state DA +. Since the FRET acceptor (A +) is switched on with a high FRET efficiency, there is an asymmetry of the entire system, which leads to an under-occupation of the state DA + of the bound FRET pair compared to the occupation of the state A + of the free FRET acceptor.
  • FIG. 3 shows three simulated concentration curves as a result of the method according to the invention.
  • Figure 3a shows the total concentration of the bound FRET pair in arbitrary units.
  • the steep rise 10 in the left area of the diagram represents the behavior during a UV radiation pulse.
  • the area 12 following on the right which is shown enlarged in FIG. 3b, shows the relaxation into the equilibrium state. Due to the asymmetry explained above, the state DA + is understaffed. There is therefore a relaxation in favor of this state.
  • FIG. 3c shows the concentration of the donor and the FRET pair in the switched-off state. It is exactly the opposite of the curve previously explained.
  • the excitation irradiation of the sample is carried out with considerably less intensity.
  • the wavelength of this detection radiation is generally selected so that the FRET donor is energetically excited, but without this being associated with any noteworthy switching of the photochromic FRET acceptor.
  • the fluorescence of the FRET donor itself can be measured. This will decrease with increasing concentration of bound FRET pairs in the switched-on state, since the overall FRET efficiency increases and thus a growing competitive channel for donor fluorescence arises.
  • the fluorescence of the FRET acceptor can also be measured, provided that it is a fluorescent photochrome. This is exactly the opposite of donor fluorescence. It is also possible to measure the fluorescence of a third fluorophore, which is also present in the reaction product that contains the bound FRET Contains pair, and which acts as a further, non-photochromic, but fluorescent FRET acceptor to the specially chosen FRET donor.
  • This additional FRET acceptor represents an excitation channel that competes with the donor fluorescence and the fluorescence of the first FRET acceptor.
  • a diagram of a corresponding exemplary complex formation reaction for example a DNA hybridization with two FRET acceptors, is shown in FIG. "D” here denotes the FRET donor, "pc" the first photochromic FRET acceptor and "A” the further, non-photochromic, fluorescent FRET acceptor.
  • D and pc are arranged close to each other.
  • A is at a greater distance arranged to D.
  • the examples described here and shown in the drawings represent only particularly advantageous embodiments of the method according to the invention, which can be varied in many ways within the scope of the teaching disclosed.
  • the special choice of the species used, the wavelengths and intensities of the light used to switch the photochrome and / or to excite the FRET donor can be modified to a large extent.
  • the method according to the invention allows, since essentially only reversible processes are involved, repetitive method variants to improve the signal / noise ratio. Spatially meandering suggestions by moving the sample (e.g. in a microscope) and / or the stimulating light beam can also be used depending on the specific experimental goal.

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  • Health & Medical Sciences (AREA)
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  • Engineering & Computer Science (AREA)
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Abstract

La présente invention concerne un procédé pour déterminer une grandeur cinétique caractéristique d'une réaction chimique entre plusieurs espèces chimiques dans un échantillon, au moins une espèce contenant au moins un fluorophore. Ce procédé consiste à induire un état de déséquilibre de la réaction chimique en exposant l'échantillon à de la lumière et en observant avec une résolution temporelle au moins une partie de la relaxation des concentrations des espèces impliquées, à l'aide d'un signal de fluorescence d'au moins un fluorophore. Au moins un produit à analyser de la réaction chimique comprend un composé de deux espèces, qui contiennent respectivement un partenaire d'une paire de fluorescence de transfert de l'énergie de résonance constituée d'un donneur de fluorescence de transfert de l'énergie de résonance et d'un accepteur de fluorescence de transfert de l'énergie de résonance. Cette invention est caractérisée en ce que l'accepteur de fluorescence de transfert de l'énergie de résonance est un photochrome dont le spectre d'absorption peut être modifié par exposition à de la lumière d'une longueur d'onde adaptée, en ce que le donneur de fluorescence de transfert de l'énergie de résonance est un fluorophore dont le spectre d'émission présente une zone de chevauchement avec le spectre d'absorption de l'accepteur de fluorescence de transfert de l'énergie de résonance, dont la taille dépend de l'état photochromique de l'accepteur de fluorescence de transfert de l'énergie de résonance et en ce que la lumière utilisée pour induire un état de déséquilibre de la réaction chimique présente une longueur d'onde qui change l'état photochromique de l'accepteur de fluorescence de transfert de l'énergie de résonance.
EP04763779A 2003-08-11 2004-08-04 Procede a cinetique de relaxation photochromique Ceased EP1658496A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10337108A DE10337108B3 (de) 2003-08-11 2003-08-11 Photochromes relaxationskinetisches Verfahren
PCT/EP2004/008729 WO2005017527A1 (fr) 2003-08-11 2004-08-04 Procede a cinetique de relaxation photochromique

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EP1658496A1 true EP1658496A1 (fr) 2006-05-24

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EP04763779A Ceased EP1658496A1 (fr) 2003-08-11 2004-08-04 Procede a cinetique de relaxation photochromique

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US (1) US20070184445A1 (fr)
EP (1) EP1658496A1 (fr)
JP (1) JP2007501936A (fr)
DE (1) DE10337108B3 (fr)
IL (1) IL173643A0 (fr)
WO (1) WO2005017527A1 (fr)

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Publication number Priority date Publication date Assignee Title
DE102008024567A1 (de) * 2008-05-21 2009-12-03 Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts Universitätsmedizin Verfahren zur Messung einer Konzentration einer Messsubstanz in einer biologischen Probe
US8547533B2 (en) * 2009-12-28 2013-10-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Composite probes and use thereof in super resolution methods
DE102010012857B4 (de) * 2010-03-25 2015-02-19 Tim Liedl Vorrichtung zum Erfassen einer Weggröße
FR2983740B1 (fr) * 2011-12-07 2014-02-14 Centre Nat Rech Scient Procede de determination du mecanisme reactionnel d'une reaction et dispositif associe

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Publication number Priority date Publication date Assignee Title
DE2408646A1 (de) * 1974-02-22 1975-08-28 Max Planck Gesellschaft Reaktionskinetisches messgeraet
DE3938598A1 (de) * 1989-11-21 1991-05-23 Bayer Ag Optischer biosensor
JP3448090B2 (ja) * 1994-02-16 2003-09-16 浜松ホトニクス株式会社 エネルギー移動検出法およびその装置
EP1271133B1 (fr) * 2000-02-28 2008-05-28 Daiichi Pure Chemicals Co., Ltd. Méthode de mesure basée sur la fluorescence par transfert d'énergie utilisant un donneur fluorescent à longue durée de vie

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Title
See references of WO2005017527A1 *

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Publication number Publication date
DE10337108B3 (de) 2005-05-04
IL173643A0 (en) 2006-07-05
WO2005017527A1 (fr) 2005-02-24
JP2007501936A (ja) 2007-02-01
US20070184445A1 (en) 2007-08-09

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