EP2140260A1 - Procédé de détection de liaison et de réactions chimiques dans un échantillon aqueux - Google Patents

Procédé de détection de liaison et de réactions chimiques dans un échantillon aqueux

Info

Publication number
EP2140260A1
EP2140260A1 EP08741833A EP08741833A EP2140260A1 EP 2140260 A1 EP2140260 A1 EP 2140260A1 EP 08741833 A EP08741833 A EP 08741833A EP 08741833 A EP08741833 A EP 08741833A EP 2140260 A1 EP2140260 A1 EP 2140260A1
Authority
EP
European Patent Office
Prior art keywords
water
substance
binding
properties
substances
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Withdrawn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
EP08741833A
Other languages
German (de)
English (en)
Other versions
EP2140260A4 (fr
Inventor
Andreas Barth
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.)
Individual
Original Assignee
Individual
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Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP2140260A1 publication Critical patent/EP2140260A1/fr
Publication of EP2140260A4 publication Critical patent/EP2140260A4/fr
Withdrawn legal-status Critical Current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
    • 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
    • G01N21/78—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 producing a change of colour
    • 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
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7783—Transmission, loss
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G01N21/359—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using near infrared light
    • 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
    • G01N33/18—Water

Definitions

  • the present invention generally relates to a method to detect binding between substances or chemical reactions in an aque- ous sample, by observing or measuring a change in the spectroscopic properties of the water. It is based on the fact that water's vibrational properties, like vibrational frequencies, absorption and scattering cross sections, are different when it is bound to molecules or ions than when sur- rounded by other water molecules, i.e. as in "bulk water”.
  • vibrational spectroscopy examples include infrared spectroscopy in the near-infrared, mid-infrared and far-infrared spectral range, terahertz spectroscopy, Raman spectroscopy, photoacoustic spectroscopy, vibrational circular dichroism, and non-linear techniques like sum frequency generation spectroscopy.
  • a non-exhaustive list includes the binding of a small molecule to a macromole- cule, for instance in ligand-receptor interactions, the binding of two macromolecules, the interaction of ions, molecules or macromolecules with membranes, conformational changes in a macromolecule such as an enzyme or other protein, measurement of chemical reactivity, for instance enzymatic activity etc.
  • the ability to accurately monitor such processes is of key importance to all Life Sciences.
  • Vibrational spectroscopy including e.g. infrared spectroscopy (in the near-infrared, mid-infrared and far-infrared spectral range) , terahertz spectroscopy, Raman spectroscopy, photoacoustic spectroscopy, vibrational circular dichroism, and non-linear techniques like sum frequency generation spectroscopy, is one of the classical methods for structure de- termination of small molecules. This standing is due to its sensitivity to the chemical composition and architecture of molecules . The high information content in a vibrational spectrum carries over also to biological systems.
  • infrared spectroscopy in the near-infrared, mid-infrared and far-infrared spectral range
  • Raman spectroscopy Raman spectroscopy
  • photoacoustic spectroscopy photoacoustic spectroscopy
  • vibrational circular dichroism vibrational circular dichroism
  • non-linear techniques like sum frequency generation spectroscopy is one of
  • vibrational spectroscopy a valuable tool for the investigation of protein and DNA structure, of the molecular mechanism of protein reactions and of protein folding, unfolding and misfolding.
  • the wealth of information in the vibrational spectrum can be exploited even for complex systems.
  • a strik- ing example is the possibility to identify bacterial strains from the infrared spectrum and to differentiate and classify microorganisms (Naumann in: Infrared and Raman Spectroscopy of Biological Materials, eds. Gremlich &Yan, Marcel Dekker Inc., New York, 2001, 323-377).
  • vibrational spectroscopy is a large application range from small soluble molecules to large mac- romolecules like DNA or proteins, a high time resolution, short measuring times, the low amount of sample required (typically 10 - 100 ⁇ g) and the relatively low costs of instrumentation.
  • the molecules of interest are observed directly. Contrary to fluorescence spectroscopy, no labelling that might be difficult to achieve or disturb the biological system under investigation is required.
  • the molecules can be studied in aqueous solution under near physiological conditions in contrast to X-ray crystallography which requires time-consuming crystallization, and to surface plasmon resonance which can detect interactions between molecules only when one of them is attached to a solid substrate.
  • a drawback of using vibrational spectroscopy for structural analysis in an aqueous medium is the strong absorption of water, which interferes with the vibrational data for the molecules of interest.
  • infrared spectroscopy for example, the absorption near 1645 cm "1 overlaps with the important amide I band of proteins and some amino acid side chain bands. Similar problems are encountered throughout the infrared spectral region.
  • the obstacles imposed by the strong water absorption have so far prevented the wide-spread use of infrared spectroscopy to study the binding of substances to macromolecules directly, although specialised techniques have been applied (Barth & Zscherp FEBS Lett. 477 (2000) 151-156). Both, small path- length and high concentrations make the use of standard mixing devices in the mid-infrared range difficult (though not impossible) and the high water absorption decreases the sig- nal to noise ratio in regions of water absorption.
  • the present invention generally relates ' to a method to detect binding between substances or chemical reactivity in an aqueous sample, by observing or measuring a change in the spectroscopic properties of water.
  • the applicant has discovered that the strong absorption of water, previously considered a drawback of using vibrational spectros- copy, can be used in itself to detect molecular interactions in aqueous media indirectly.
  • the present invention therefore relates to a method to detect binding of at least one substance A to at least one other substance B in an aqueous sample, wherein the binding changes the spectroscopic properties of water, characterized in that the binding is detected by observing or measuring any change in the spectroscopic properties of water.
  • the present invention relates to a method to detect a chemical reaction or measure its reaction rate, wherein the reaction changes the spectroscopic properties of water, characterized in that the reaction is detected by ob- serving or measuring any change in the spectroscopic properties of water.
  • the change in the properties of water is measured using infrared, terahertz, Raman, sum frequency generation, or photoacoustic spectroscopy, or vibrational circular dichroism.
  • substances A and B are biomolecules such as proteins, nucleic acids or parts of a biomembrane. More specifically, substances A and B may be therapeutic targets, candidate drugs, herbicide targets or herbicide candidates.
  • the method may also comprise a mixing device for mixing substances A and B, and/or high throughput screening, for instance on a microtiter plate.
  • a catalyst like an enzyme, catalyzes a reaction from at least one reactant to at least one product.
  • Water is an essential participant in many molecular interactions, in particular in living organisms. Its vibrational properties depend on the hydrogen bonding pattern. Therefore they are different for water around hydrophobic solutes, around ions and in bulk water (Worley & Klotz 1966, J. Chem. Phys. 2868-71, Sharp et al. 2001, J. Chem. Phys . 114, 1791- 6) .
  • the water shell gives rise to a particular vibrational signature, since for example vibrational frequencies depend on the degree of hydrogen bonding.
  • This hydrogen bonding is different as compared to bulk water because the macromolecular surface is generally composed of hydrophobic and ionic groups not found in bulk water. Binding of a substance to a macromolecule partially alters this water shell and transfers some of the bound water from the macromolecular surface to bulk water where these water molecules have different vibrational properties. This change in water's vibrational properties can be detected and thus gives evidence for the binding process.
  • the water shells around the reactants of a given chemical reaction and the products of this reaction are different, in particular when the number of charged species changes. Therefore the vibrational properties of the hydration shells of reactants are different from those of the products which can be detected and gives evidence for the chemical reaction and enables measurement of the reaction velocity.
  • the present invention proposes to detect the binding and/or reactivity of molecules in aqueous media by monitoring bound and/or bulk water using vibrational spectroscopy.
  • the invention therefore generally relates to a method to detect binding or reaction of at least one substance A to at least one other substance B in an aqueous sample, wherein the binding or the reaction changes the spectroscopic properties of water, characterized in that the binding or the reaction is detected by observing or measuring any change in the spectro- scopic properties of water.
  • the method according to the invention comprises a wide variety of prac- tical approaches.
  • the at least one substance A and/or B may be a principally pure substance in aqueous solution, or a mixture of substances.
  • substance A may be a mixture of i binding candidates Al, A2... Ai, suspected of binding to either of j binding targets Bl, B2... Bj.
  • a change in the spectroscopic properties of the water would indicate binding between one or several of substances A and B, and additional trials may be necessary to pinpoint the binding pair(s) .
  • the method according to the invention is not dependent on any particular detection means for observing or measuring the change in spectroscopic properties.
  • Any known means in the art is suitable, for instance infrared, Raman, sum frequency generation, photoacoustic , or terahertz spectroscopy, or vibrational circular dichroism.
  • the inventive method may be practically incorporated into any conceivable device or array of devices, and may for instance comprise a mixing device for mixing substances A and B, a mi- crotiter plate for high throughput scanning of several substances, a thermal light source, a laser, or a light-emitting diode, a multi-array detector etc.
  • the method is performed wherein: a) substance B is supplied as an aqueous solution to a mixing device; b) substance A is supplied as a separate aqueous solution to the mixing device; c) the two aqueous solutions are mixed to form a mixed solution; and d) the spectroscopic properties of water in the mixed solution in c) are compared to the properties of water in a) and/or b) , to detect any changes in said properties.
  • the method is performed wherein: a) substance A is supplied as an aqueous solution to a mixing device; b) a catalyst or enzyme is supplied as a separate aqueous solution to the mixing device; c) the two aqueous solutions are mixed to form a mixed solution; and d) the spectroscopic properties of water in the mixed solu- tion in c) is compared to the properties of water in a) and/or b) , to detect any changes in said properties.
  • substance A is the substrate of a catalyst or of an enzyme and substance B its product.
  • the term "bulk water” generally refers to water molecules in an aqueous medium that are substantially surrounded by other water molecules or that are not associated with the substances of interest, such as macromolecules and ligands of which binding is studied or reactants and products of a reaction. It will be appreciated that bulk water can still contain non-water molecules, for' example comprising an aqueous buffer or ion solution. Specifically, “bulk water” has spectroscopic properties that are distinct from “bound water”, as defined below. Substances, like ions, polar or hydrophobic molecules, may be added to "bulk water” in order to enhance the difference between the spectroscopic properties of "bulk water” and "bound water”.
  • bound water generally refers to water molecules in the hydration layer (s) around the substances of interest, such as macromolecules and ligands of which binding is studied or reactants and products of a reac- tion. Generally, “bound water” is in rapid exchange with “bulk water”. It will be appreciated that “bound water molecules” may hydrate a non-water molecule in several layers, and that each bound water molecule need not be associated di- rectly with the hydrated molecule. Specifically, "bound water” has spectroscopic properties that are distinct from those of "bulk water”.
  • water generally refers to water consisting of any oxygen or hydrogen isotope or of a mixture of isotopes.
  • spectroscopic property such as in the phrase “spectroscopic properties of water” means any property that can be measured using any known spectroscopic technique, for instance absorption, scattering, vibrational circular dichroism, photoacoustic signal, the non-linear interaction of photons etc.
  • vibrational spectroscopy comprises methods that detect properties of vibrations or properties that are modulated by vibrations, like absorption and scattering cross section.
  • vibrational spectroscopy comprises infrared, Raman, terahertz, sum frequency generation and photoacoustic spectroscopy as well as vibrational circular dichroism'.
  • the term “near infrared spectral range” refers to the spectral range of 700 to 2500 nanometer; the term “mid infrared spectral range” refers to the spectral range of 2.5 to 50 micrometer and the term “far infrared spectral range” refers to the spectral range of 50 to 100 micrometer.
  • the present invention may generally be used to monitor molecular interactions and reactions in aqueous media.
  • the invention provides an easy means to determine whether one molecule or substance binds to another or not, and whether at least one molecule or substance reacts or not, as such a binding or reaction would register in the vibrational signature of the bound water.
  • Quantitative measure- ments are also possible, as the degree of change in vibrational signature would correlate to strength of binding or amount of bound molecules, i.e. allowing several binding candidates to be compared, or to amount of reacted molecules for example.
  • the invention has particular application in the fields of pharmaceutical development, or in the development of herbicides.
  • the present invention will be especially useful in the field of ' proteomics, i.e. in monitoring protein- protein interactions.
  • Nucleic acid interactions for instance DNA-RNA interactions or DNA-protein interactions, and the measurement of enzymatic activity are also applicable fields for the invention.
  • inhibitor binding to a protein may for instance be observed by a change in water absorption.
  • the ab- sorption spectra of water in the presence of protein, in the presence of inhibitor and in the presence of the inhibitor- protein complex can be recorded separately - either one by one in conventional cuvettes or with more sophisticated approaches .
  • Binding can be observed in a time-resolved way with mixing devices if the time for binding is slower than the mixing time.
  • Mixing devices can be used that allow the recording of spectra in the presence of inhibitor and protein separately before binding and of the complex after binding.
  • the device can be constructed such that the flow of inhibitor and the flow of protein combine and mix by diffusion. Water absorption can then be detected at different distances from the point of combining the two flows. Close to that point, inhibitor and protein will still be largely separated, allowing the detection of the combined absorption of inhibitor and protein before complex formation. Distant from the point of combining the flows, diffusion has mixed inhibitor and protein and the complex has formed.
  • a measurement here measures the water absorption in the presence of the complex, (iii)
  • absorption of the separated inhibitor and the separated protein can be measured in the two channels leading to the mixing chamber, and the absorption of the complex in the channel that leads away from the mixing chamber.
  • a variation of this method is the separation of inhibitor and protein by an optically transparent wall or membrane in the cuvette, which is removed to initiate binding.
  • the inhibitor can be added to the protein via a semipermeable membrane.
  • Binding can be blocked initially by chemical modification of inhibitor or protein which is then relieved to initiate binding.
  • An example is the use of caged compounds or caged proteins (Dynamic Studies in Biology. Phototriggers, Pho- toswitches and Caged Biomolecules, eds . Goeldner & Givens, Wiley-VCH, Weinheim, 2005) . In this case the blockage is removed by photocleavage.
  • complex formation can be revealed in a titration without recording the water spectra in the presence of the two separated interaction partners.
  • a reference signal in the presence of one of the interaction partners (A) is meas- ured first.
  • the other interaction partner (B) is added.
  • the water property will change because of complex formation.
  • the binding constant can be determined.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plasma & Fusion (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

L'invention concerne un procédé de détection de liaison et de réactions de matières dans un échantillon aqueux, la liaison ou la réaction modifiant les propriétés spectroscopiques de l'eau et étant détectée par l'observation ou la mesure d'une modification des propriétés spectroscopiques de l'eau.
EP08741833A 2007-03-21 2008-03-25 Procédé de détection de liaison et de réactions chimiques dans un échantillon aqueux Withdrawn EP2140260A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE0700700 2007-03-21
PCT/SE2008/000221 WO2008115120A1 (fr) 2007-03-21 2008-03-25 Procédé de détection de liaison et de réactions chimiques dans un échantillon aqueux

Publications (2)

Publication Number Publication Date
EP2140260A1 true EP2140260A1 (fr) 2010-01-06
EP2140260A4 EP2140260A4 (fr) 2012-05-02

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP08741833A Withdrawn EP2140260A4 (fr) 2007-03-21 2008-03-25 Procédé de détection de liaison et de réactions chimiques dans un échantillon aqueux

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EP (1) EP2140260A4 (fr)
WO (1) WO2008115120A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2642276A1 (fr) * 2012-03-22 2013-09-25 Inoviem Scientific Procédé de spectroscopie dynamique dans des conditions physiologiques
WO2018052746A1 (fr) * 2016-09-19 2018-03-22 Genovus Biotechnologies Inc. Système et méthode de distribution de fréquences thérapeutiques répliquées par champ électromagnétique pulsé
CN110333202B (zh) * 2019-04-08 2021-11-23 上海理工大学 一种水体除草剂浓度的判定方法

Family Cites Families (1)

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Publication number Priority date Publication date Assignee Title
US7709247B2 (en) * 2004-08-04 2010-05-04 Intel Corporation Methods and systems for detecting biomolecular binding using terahertz radiation

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
A. P. ZHUKOVSKY ET AL: "Comparative study of the structure of microlayering water-dioxane and water-dimethylsulfoxide solutions", JOURNAL OF STRUCTURAL CHEMISTRY, vol. 34, no. 4, 1 January 1994 (1994-01-01), pages 562-566, XP55022357, ISSN: 0022-4766, DOI: 10.1007/BF00753526 *
CZARNIK-MATUSEWICZ B ET AL: "Study of the temperature-dependent near-infrared spectra of water by two-dimensional correlation spectroscopy and principal components analysis", VIBRATIONAL SPECTROSCOPY, ELSEVIER SCIENCE, AMSTERDAM, NL, vol. 40, no. 2, 17 March 2006 (2006-03-17) , pages 235-245, XP025081226, ISSN: 0924-2031, DOI: 10.1016/J.VIBSPEC.2005.10.002 [retrieved on 2006-03-17] *
KHALOIMOV A I ET AL: "Spectroscopic investigation of conformational changes in certain proteins in aqueous solution and in mixed solvents", BIOPHYSICS, ELSEVIER SCIENCE, GB, vol. 21, no. 6, 1 January 1976 (1976-01-01), pages 991-995, XP008149930, ISSN: 0006-3509 *
MAX J -J ET AL: "IR spectroscopy of aqueous alkali halide solutions: Pure salt-solvated water spectra and hydration numbers", JOURNAL OF CHEMICAL PHYSICS AIP USA, vol. 115, no. 6, 8 August 2001 (2001-08-08), pages 2664-2675, XP8149997, ISSN: 0021-9606 *
NOZAKI D ET AL: "Water structural changes in the activation process of the LOV2 domain of Adiantum phytochrome3", JOURNAL OF MOLECULAR STRUCTURE, ELSEVIER, AMSTERDAM, NL, vol. 735-736, 14 February 2005 (2005-02-14), pages 259-265, XP004714147, ISSN: 0022-2860, DOI: 10.1016/J.MOLSTRUC.2004.10.111 *
See also references of WO2008115120A1 *
SUZUKI M ET AL: "Myosin-induced volume increase of the hyper-mobile water surrounding actin filaments", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 322, no. 1, 10 September 2004 (2004-09-10), pages 340-346, XP004526739, ISSN: 0006-291X, DOI: 10.1016/J.BBRC.2004.07.111 *
U. HEUGEN: "Solute-induced retardation of water dynamics probed directly by terahertz spectroscopy", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 103, no. 33, 15 August 2006 (2006-08-15), pages 12301-12306, XP55022367, ISSN: 0027-8424, DOI: 10.1073/pnas.0604897103 *

Also Published As

Publication number Publication date
WO2008115120A1 (fr) 2008-09-25
EP2140260A4 (fr) 2012-05-02

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