WO2016170183A1 - Procédé perfectionné de mesure d'une interaction entre des molécules - Google Patents

Procédé perfectionné de mesure d'une interaction entre des molécules Download PDF

Info

Publication number
WO2016170183A1
WO2016170183A1 PCT/EP2016/059173 EP2016059173W WO2016170183A1 WO 2016170183 A1 WO2016170183 A1 WO 2016170183A1 EP 2016059173 W EP2016059173 W EP 2016059173W WO 2016170183 A1 WO2016170183 A1 WO 2016170183A1
Authority
WO
WIPO (PCT)
Prior art keywords
tdc
target compound
compound
test compound
conjugate
Prior art date
Application number
PCT/EP2016/059173
Other languages
English (en)
Inventor
Wilhelmus Jacobus LAAN
Bieke Van De Broek
Vanessa Adrienne Therese BONNARD
Original Assignee
Pharmadiagnostics Nv
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
Priority claimed from PCT/EP2015/058871 external-priority patent/WO2015162234A1/fr
Application filed by Pharmadiagnostics Nv filed Critical Pharmadiagnostics Nv
Publication of WO2016170183A1 publication Critical patent/WO2016170183A1/fr

Links

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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • 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
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance

Definitions

  • the present invention relates to improved methods and tools for determining an interaction between two molecules, in particular between a target compound and a test compound, based on the monitoring of the localized surface plasmon resonance (LSPR) properties of metallic nanoparticles.
  • LSPR localized surface plasmon resonance
  • Nanoparticles, particularly metal nanoparticles, coated with molecules such as proteins can be used to determine interaction or binding events with a second molecule by monitoring a change in optical properties of the particles, such as by Localized Surface Plasmon Resonance (LSPR) sensing.
  • LSPR Localized Surface Plasmon Resonance
  • the specific binding of an analyte to the molecules coated on said recognition interface is converted into an optical signal, e.g. a change in absorbance which is detected and analyzed.
  • LSPR sensing is based on the sensitivity of the localized plasmon absorbance of (metal) nanoparticles to changes in the dielectric properties of the contacting medium. In practice, however it is observed that such methods where nanoparticles are used in solution often do not attain the required accuracy.
  • Competition assays also known as competitive binding assays, are an important tool for determining the concentration of an analyte and/or identifying interactions between two compounds or molecules such as a protein and an antibody.
  • a substance competes for labeled versus unlabeled ligand, although there is an increasing need for label-free assays.
  • a number of label-free assays are based on the use of surface plasmon resonance (SPR), for example as described in US2012/0157328. These assays typically involve the detection of changes in the refractive index at the surface of an SPR detection system as a result of the immobilization of the compounds on a solid surface.
  • SPR surface plasmon resonance
  • Warsinke et al. (Analytica Chimica Acta 2005, 550, 69-76) have described methods for the quantification of human tissue inhibitor of metalloproteinases-2 (TIMP-2) using SPR and functionalized gold nanoparticles for signal enhancement.
  • the methods involve the immobilization of TIMP-2 or a protein with high affinity to TIMP-2 to a sensor surface.
  • the presence of TIMP-2 in a test solution influences the interaction between the sensor surface and the functionalized nanoparticles, which is detected via an SPR signal.
  • WO 2004/042403 relates to methods for the detection of an analyte in a sample involving the use of functionalized nanoparticles which are immobilized on a surface. The methods involve measuring scattered light emitted by individual nanoparticle structures.
  • the present inventors have surprisingly found that, in an LSPR based assay for determining the interaction between two molecules, in particular for determining the interaction between a target compound and a test compound, when at least the solution comprising the test compound comprises a detergent, unwanted background signals can be suppressed.
  • the present invention relates to a method for suppressing background signals in a method or assay of determining an interaction between a test compound and a target compound, and to improved methods of determining an interaction between a test compound and a target compound, comprising providing said test compound in a solution comprising at least one detergent.
  • said method or assay is an LSPR based assay, particularly an LSPR based competition assay using a nanoparticle suspension.
  • a first aspect of the present invention relates to a method for suppressing background signals in an LSPRD assay comprising a test compound, wherein said test compound is provided as a solution comprising at least one detergent, which has been added to suppress background signals in the LSPR assay.
  • Preferred embodiments relate to a method for suppressing background signals in a method of determining an interaction between a test compound and a target compound, wherein said test compound and, optionally, said target compound is provided as a solution comprising at least one detergent.
  • said method of determining an interaction between a test compound and a target compound is an LSPR based assay using a metal nanoparticles suspension wherein the metal nanoparticles are conjugated to a compound.
  • said LSPR based assay is an LSPR based competition assay comprising a target definition compound as envisaged herein conjugated to a metal nanoparticle, said target compound and said test compound.
  • Particular embodiments of the methods of the present invention relate to a method for suppressing background signals in an LSPR based competition assay, wherein said competition assay comprises the steps of
  • TDC target definition compound
  • test compound modulates binding of said target compound to said TDC, based on the presence or absence of a change in refractive index surrounding the NPs due to the binding of said target compound to said TDC when contacting said suspension comprising said TDC-NP conjugate with said target compound and said test compound.
  • said TDC is a first polypeptide P1 and said target compound is a second polypeptide P2.
  • step (c) comprises a data processing step, as further defined herein, wherein the raw absorbance data (as measured) potentially reflecting a change in refractive index surrounding the NPs are transformed, such as via background subtraction and normalization, in data that are suitable for curve fitting or modelling, and, subsequently, performing the curve fitting or data modelling.
  • a second aspect of the present invention relates to an LSPR based method of determining an interaction between a target compound and a test compound, comprising providing said test compound in a solution comprising at least one detergent.
  • said LSPR based method of determining an interaction between a target compound and a test compound comprises the steps of:
  • TDC target definition compound
  • test compound modulates binding of said target compound to said TDC, based on the presence or absence of a change in refractive index surrounding the NPs due to the binding of said target compound to said TDC when contacting said suspension comprising said TDC-NP conjugate with said target compound and said test compound.
  • the concentration of said detergent in the solution comprising the test compound is above the critical micelle concentration.
  • the concentration of the detergent in the liquid mixture obtained in step (b) is between 5 times and 10 times the CMC of the detergent.
  • the detergent is a nonionic, cationic and/or zwitterionic detergent, preferably a nonionic detergent.
  • said metal nanoparticles are gold nanorods (GNRs).
  • step (b) comprises:
  • step (c) comprises
  • steps (c1 ) and (c2) are repeated at least once.
  • said test compound and, optionally, said target compound are provided in a solution further comprising DMSO thereby obtaining in step (b) a liquid mixture comprising said conjugated nanoparticles, said test compound, said detergent, optionally said target compound, and between 0.5w% and 50w% (DMSO) and wherein step (c) comprises correcting the change in refractive index surrounding the nanoparticles for the presence of DMSO in said liquid mixture.
  • said liquid mixture obtained in step (b) comprises between 0.5 w% and 10w% DMSO.
  • said step (a) comprises:
  • said TDC is a first polypeptide P1 and said target compound is a second polypeptide P2 and step (a) comprises:
  • (a1 ) providing a suspension of metal nanoparticles (NPs), wherein said suspension has a pH between (pl-1 ) and pi, wherein pi is the isoelectric point of P1 ;
  • said linker molecule is coupled to P1 via a maleimide functional group.
  • said method further comprises determining the target compound concentration to be used in step (b) via a concentration titration of said TDC-NPs with said target compound, wherein a suitable amount of the target compound is an amount which results in a detectable change of the LSPR properties of the TDC-NPs but which does not saturate the available TDC binding sites.
  • kits comprising a target compound; optionally, a suspension of a TDC-NP conjugate wherein the TDC is a compound that can bind to the target compound and wherein said TDC is conjugated to a metal nanoparticle, and one or more test compounds, each provided as a solution comprising the test compound and at least a detergent as envisaged herein.
  • the methods and tools described herein are particularly suitable for the accurate high- throughput screening of compounds interacting with biomolecules such as proteins. More particularly, the present methods can allow for compound library screening for determining the binding specificity, kinetics and affinity of a plurality of pre-determined test compounds on a target compound such as a protein of interest, while minimizing/suppressing undesired background signals.
  • the present inventors further found that such competition assays, particularly in the presence of a detergent, are surprisingly effective for screening compounds which can modulate the interaction between peptides and/or proteins.
  • Fig. 1 Titration curve for the titration of biotin-GNR with neutravidin, showing A max in function of the concentration of added neutravidin.
  • Fig. 2 Plot of A max against the amount of biotin and HABA preincubated with a fixed concentration of neutravidin and then added to a biotin-GNR suspension.
  • Fig. 3 Titration curve for the titration of p53-GNR with MDM2, showing A max in function of the concentration of added MDM2.
  • Fig. 4 Plot of A max against the amount of p53 (A) and nutlin-3 (B) preincubated with MDM2 and then added to a p53-GNR suspension.
  • Fig. 5 Plot of the wavelength of maximal absorbance max ) of TDC-conjugated nanorods against the amount of various test compounds (1 -4) in the absence of detergent.
  • Fig. 6 Plot of the wavelength of maximal absorbance ( max ) of TDC-conjugated nanorods against the amount of various test compounds (1 -4) in the presence of detergent (0.1 v% Triton X-100).
  • Fig. 7 Plot of A max against the amount of test compound 1 incubated with a target compound and a TDC-GNR, in the presence or absence of a detergent (0.1 v% Triton X-100).
  • LSPR localized surface plasmon resonance
  • LSPs localized surface plasmons
  • the nanoparticles act as nanoscale antennas, concentrating the electromagnetic field into very small volumes adjacent to the particles. Exceptionally large enhancements in electromagnetic intensity can be obtained this way.
  • the nanoparticles used in the LSPR enable the occurrence of the resonance oscillations.
  • the term "absorbance” refers to the extent to which a sample absorbs light or electromagnetic radiation in the UV, visual or near infrared range of the spectrum.
  • LSPR changes in refractive index may be detected through monitoring changes in the absorbance.
  • changes in the LSPR extinction band of the nanoparticles cause changes in the intensity and/or the wavelength of maximum absorbance.
  • colloids refers to a fluid composition of particles suspended in a liquid medium.
  • the particles therein are between one nanometer and one micrometer in size.
  • azido refers to -N 3 .
  • amino by itself or as part of another substituent, refers to -NH 2 .
  • aqueous as used herein means that more than 50 percent by volume of the solvent is water. Aqueous compositions or dispersions may further comprise organic liquids which are miscible with water.
  • the present invention generally relates to LSPR based methods and tools for determining an interaction between a first compound and a second compound, which are herein also referred to as “target compound” and “test compound”, respectively.
  • target compound and test compound
  • the interaction measured between the test compound and target compound is referred to as "binding”.
  • binding refers to two molecules associating with each other in a non-covalent or covalent relationship.
  • said target compound is conjugated to a nanoparticle and said test compound is a compound potentially interacting with the target compound attached to the nanoparticles.
  • the nature of the target compound and test compound is not critical to the methods and tools envisaged herein, provided that the binding of the test compound to the target compound attached to the nanoparticle results in a measurable change in LSPR parameters, or optical parameters such as absorbance, refractive index, absorption, scattering, fluorescence, luminescence or photoluminescence.
  • said method for determining an interaction between a first compound or "target compound” and a second compound or “test compound” is a competition assay, further comprising a so-called “target definition compound” or TDC, which is conjugated to a nanoparticle and wherein the TDC and the test compound compete for binding to the target compound.
  • the TDC is thus a known interaction partner of the target compound, i.e. it is a compound which is known to (specifically) bind to the target compound. It typically serves as a probe for the binding site of the target compound.
  • the TDC is bound to the NP surface in such a way that it still is able to bind to the target compound.
  • the nature of the TDC and target compound is not critical to the methods and tools described herein, and may include small organic molecules, nucleic acids, peptides, proteins, polysaccharides, lipids, or other molecules.
  • the TDC may be selected from enzyme inhibitors, protein cofactors, drugs, small molecule antigens of antibodies, and targets of aptamers, proteins, peptides, antibodies.
  • the "target definition compound” or TDC and “target compound” are members of a specific known binding pair or couple, such as antigen- antibody, sugar-lectin, receptor-receptor binding agent, nucleic acid strand-antisense strand, and others.
  • the TDC or target compound as envisaged herein include, but are not limited to biomolecules, where the term "biomolecule” refers to any organic or biochemical molecule, group or species of interest, e.g., that can specifically bind to an analyte of interest.
  • biomolecules include, but are not limited to peptides, proteins, amino acids and nucleic acids.
  • the competition assay as envisaged herein the test compound is a small molecule, such as present in compound or small molecule libraries. Small organic and inorganic molecules refer to small compounds having a molecular weight of more than 50 and less than about 2500 dalton.
  • Small organic compounds may include functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • Such compounds may include cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • NPs nanoparticles
  • the methods described herein involve the use of nanoparticles (NPs), and generally comprise the steps of (a) Providing a suspension comprising a first compound conjugated to metal nanoparticles (NPs), (b) Contacting the suspension comprising the NPs conjugated to said first compound with a solution comprising a test compound; and (c) measuring the interaction between the first compound conjugated to the NPs and a second molecule via changes in the optical properties of the nanoparticles, in particular via a change in refractive index surrounding the NPs due to the binding of a second molecule to said first compound.
  • the methods described herein relate to a competition assay involving the use of nanoparticles (NPs), and comprise the steps of (a) Providing a suspension comprising a target definition compound (TDC) conjugated to metal nanoparticles (NPs), wherein the TDC can bind to the target compound.
  • NPs nanoparticles
  • TDC conjugated to the metal nanoparticles is also referred to herein as "TDC-NP conjugate", (b) Contacting the suspension comprising the TDC-NP conjugate with the target compound and the test compound; and (c) Determining whether the test compound inhibits binding of the target compound to said TDC, based on the presence or absence of a change in Localized Surface Plasmon Resonance (LSPR) properties (in particular a change in refractive index surrounding the NPs due to the binding of said target compound to said TDC) of the TDC-NP conjugate when contacting the TDC-NP conjugate with the target compound and the test compound, in particular by measuring the absorbance data of the TDC-NP suspension comprising the target compound and the test compound.
  • LSPR Localized Surface Plasmon Resonance
  • test compounds in the absence and presence of the target compound can cause a change in the measured LSPR signal of the TDC-NP conjugate at high test compound concentrations, while not causing a significant change in the LSPR signal at lower test compound concentrations.
  • the present inventors believe that this is caused by formation of test compound aggregates resulting from a limited solubility of the test compound in the nanoparticle suspension or by some kind of aspecific interaction between the test compound and the TDC. Such aggregation/aspecific interaction can lead to unwanted background signals, which influence the accuracy of the assay.
  • the present inventors have found that when the solution comprising the test compound comprises a detergent, such background signals can be suppressed/minimized.
  • the background signal as envisaged herein thus relates to any changes in the measured absorbance of a mixture comprising nanoparticles conjugated to a first molecule and at least a second molecule, which cannot be attributed to the interaction of a first molecule conjugated to the nanoparticles and a second molecule in solution, or which cannot be attributed to the absorbance by the test compounds (in case of colored compounds). These unwanted background signals thus reduce the accuracy of the LSPR based methods as envisaged herein.
  • detergents for suppressing background signals as described herein are particularly suitable for the specific methods of determining an interaction between a target compound and a test compound as described herein.
  • detergents may also be used for the suppression of background signals in other assay methods.
  • a first aspect of the present invention relates to a method for suppressing background signals in an assay comprising a test compound, in particular a method of determining an interaction between a test compound and a target compound, comprising providing said test compound in a solution comprising at least one detergent.
  • Preferred embodiments relate to a method for suppressing background signals in an LSPR based assay using a metal nanoparticle suspension wherein the metal nanoparticles are conjugated to a compound, more preferably in an LSPR based competition assay comprising a target definition compound as further defined below conjugated to a metal nanoparticle, said target compound and said test compound, wherein said test compound is provided in a solution comprising at least one detergent, added to suppress the background signals in said LSPR based assay.
  • the detergent as envisaged herein is a nonionic, cationic and/or zwitterionic detergent. It will be understood to the skilled person that reference herein to the use of a nonionic, cationic and/or zwitterionic detergent includes the use of combinations of different nonionic, cationic and/or zwitterionic detergents.
  • the detergent is a nonionic detergent, more preferably is an octylphenol ethoxylate or polysorbate.
  • nonionic detergent refers to a detergent which does not have any ionic groups.
  • the nonionic detergent is selected from the group comprising octylphenol ethoxylates, polysorbates, glucamines, lubrol, Brij®, Nonidet®, Pluronic®, Genapol® and Igepal®.
  • the polysorbate is chosen from the group comprising polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80 and polysorbate 85.
  • the nonionic detergent is an octylphenol ethoxylate.
  • the octylphenol ethoxylate is selected from the group comprising TRITON® X-15, TRITON® X-35, TRITON® X-45, TRITON® X-100, TRITON® X-102, TRITON® X-1 14, TRITON X-165 (70%), TRITON® X-305 (70%), TRITON® X-405 (70%) and TRITON® X-705 (70%).
  • the glucamine is selected from the group comprising of N-octanoyl-N-methylglucamine (MEGA-8), N-nonanoyl-N-methylglucamine (MEGA-9) and N-decanoyl-N- methylglucamine (MEGA-10).
  • cationic detergent refers to a detergent with a positive ionic charge.
  • the cationic detergent is selected from hexadecyltrimethyl ammonium bromide (CTAB) or trimethyl(tetradecyl) ammonium bromide (TTAB).
  • zwitterionic detergent refers to a detergent which has ionic groups, but no net charge.
  • the zwitterionic detergent is selected from the group comprising amidosulfobetaines, alkylbetaines and ammonio propanesulfonates.
  • the zwitterionic detergent is selected from the group comprising amidosulfobetaine-14, amidosulfobetaine-16, 3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1 -propanesulfonate (CHAPSO), 3-(4-heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate (C7BzO), EMPIGEN® BB, 3-(N,N-dimethyloctylammonio) propanesulfonate inner salt, 3-(decyldimethylammonio) propanesulfonate inner salt, 3-(dodecyldimethylammonio) propanesulfonate inner salt, 3-(N,N-dimethylmyristylammonio) propanesulfonate inner salt
  • the detergent is used in a concentration equal to or above the critical micelle concentration (CMC).
  • CMC critical micelle concentration
  • the CMC of a detergent is the concentration at which the detergent forms higher aggregates, so-called micelles.
  • the CMC can be determined via titration and by determining the jump in physical properties such as for example the surface tension, the osmotic pressure, the equivalent conductivity, the interfacial tension and/or the density. Each of these parameters can be measured with known methods.
  • the detergent is used in a concentration which is equal to or above the CMC, before and after contacting the solution comprising the test compound and detergent with the solutions comprising the other compounds of the test, particularly the solution comprising the target compound and the suspension comprising the TDC- NP.
  • concentration of the detergent in the solution comprising the test compound is between 1 time and 20 times the CMC, more particularly between 5 times and 10 times the CMC. It is not excluded that in other embodiments, the detergent may be used in a concentration below the CMC, for example between 10% and 95% of the CMC.
  • particular embodiments of the methods of the present invention relate to a method for suppressing background signals in an LSPR based competition assay, wherein said competition assay comprises the steps of
  • TDC target definition compound
  • test compound modulates binding of said target compound to said TDC, based on the presence or absence of a change in refractive index surrounding the NPs due to the binding of said target compound to said TDC when contacting said suspension comprising said TDC-NP conjugate with said target compound and said test compound.
  • said TDC is a first polypeptide P1 and said target compound is a second polypeptide P2.
  • step (c) comprises a data processing step for determining whether said test compound modulates binding of said target compound to said TDC, such as by data processing including curve fitting, as further discussed below.
  • a second aspect of the present invention relates to an LSPR based method of determining an interaction between a target compound and a test compound, preferably an LSPR based competition assay for determining an interaction between a target compound and a test compound, wherein the test compound is provided in a solution comprising a detergent.
  • the LSPR based method of determining an interaction between a target compound and a test compound as envisaged herein comprises the steps of:
  • test compound (b) contacting said suspension of metal nanoparticles conjugated to said first molecule with said test compound; wherein said test compound potentially interacts with said target compound on said nanoparticle and wherein said test compound and, optionally said target compound-NP conjugate, is/are provided in a solution comprising at least one detergent;
  • TDC target definition compound
  • NPs metal nanoparticles
  • LSPR Localized Surface Plasmon Resonance
  • the tools provided herein are specifically adapted to carry out one or more steps of the methods described herein. This will be explained further herein below. Particularly, the different steps of the LSPR based methods for determining an interaction between a test compound and a target compound as envisaged herein are further discussed below, mainly in the context of a competition assay, wherein the TDC is attached to the nanoparticles. However, the skilled person understands this is equally valid for or can easily be adapted to nanoparticles conjugated to the target compound (i.e. in a non-competition assay).
  • the LSPR based methods envisaged herein typically comprise, in a first step (a) providing a suspension of nanoparticles (NPs) to which a first molecule, particularly a TDC or the target compound, is adsorbed, attached, coupled, linked, or bound, generally referred to herein as "conjugated".
  • NPs nanoparticles
  • conjugated to nanoparticles is also referred herein as a "TDC-NP conjugate”.
  • target compound conjugated to nanoparticles is also referred herein as a "target compound NP conjugate”.
  • the methods and tools provided herein make use of a nanoparticle suspension comprising nanoparticles to which a TDC or target compound is conjugated.
  • the nanoparticles can be of any suitable shape and composition and can include but are not limited to nanorods, nanospheres, nanopyramids, nanowires, nanoprisms, nanocubes, nanotetrapods, etc.
  • the nanoparticles are nanorods (NRs). As nanorods exhibit two plasmon resonance peaks at different wavelengths, i.e.
  • NRs can increase the sensitivity of the methods described herein by assessing the changes (absorbance, wavelength) of the longitudinal peak due to refractive index changes upon binding.
  • nanorods particularly gold nanorods, conjugated to the TDC, form a stable nanoparticle suspension both in the presence and absence of the target compound, as formation of TDC-NP/target aggregates may interfere with the optical properties to be measured.
  • the nanorods have an aspect ratio (i.e. length divided by width) ranging between 1 .5 and 10, more particularly between 2 and 5.
  • the nanorods have a width or diameter between 2 and 20 nm, more particularly between 5 and 18 nm, for example about 15 nm. In particular embodiments, the nanorods have a length between 4 and 60 nm, more particularly between 40 and 50 nm, for example about 48 nm.
  • the NPs comprise or are made of one or more metals.
  • the NPs used in the context of the present invention comprise one or more metals selected from Au, Ag, Cu, Ta, Pt, Pd, and Rh.
  • said metal is selected from gold, silver and copper; preferably gold. Particularly good results are obtained if the NPs used are gold nanorods (GNRs).
  • the nanoparticles provided in the methods and tools envisaged herein are typically provided as a colloid, thus as particles suspended in a solvent. Accordingly, the nanoparticles are not immobilized on a solid substrate, which is particularly useful for high-throughput screening.
  • the solvents suitable for suspending the nanoparticles may depend on the nature of the nanoparticle surface.
  • the nanoparticles are provided with a hydrophilic coating, wherein the solvent may comprise one or more solvents selected from water, ethanol, butanol, isopropanol, acetone, etc.
  • the particles are suspended in an aqueous medium.
  • the colloid comprises the nanoparticles in such a concentration that in step (c) of the present method, the colloid has an absorbance at A max between 0.3 and 4, preferably between 0.7 and 1.5 (OD for 20 ⁇ _ in a 384-well plate).
  • a max is the wavelength of maximal absorbance of the nanoparticles between 350 and 1000 nm.
  • the absorbance of the colloid is between 2 and 27, more particularly between 4.8 and 10.2 at A max , for a path length of 1 cm. Colloids having such absorbance are particularly useful for use with well plates, or other recipients which only require low volumes of the colloid.
  • the NPs are at least partially coated with a target definition compound (TDC), wherein the TDC is a known interaction partner of the target compound, i.e. it is a compound which is known to (specifically) bind to the target compound.
  • TDC target definition compound
  • the methods and assays described herein may include the step of conjugating a first molecule or compound, particularly the TDC (in case of a competition assay as envisaged herein) or target compound as envisaged herein, to the NPs.
  • Methods suitable for conjugating a first molecule, such as the TDC, to NPs are known in the art, and typically involve incubating the nanoparticles in a solution comprising said first molecule (such as the TDC) under conditions which allow the attachment of the first molecule (such as the TDC) onto the nanoparticle surface.
  • the first molecule may comprise a metal binding functionality which allows for direct coupling of the TDC to the surface of the metal NPs.
  • a preferred metal binding functionality is a sulfhydryl. Sulfhydryl moieties strongly bind to metal surfaces, particularly to gold surfaces.
  • the first molecule may also be coupled indirectly to the NP surface.
  • the NPs may be coated with ligand molecules, also referred to herein as "Ngands" carrying specific functional groups, such that the surface of the coated NPs exposes these functional groups.
  • the TDC or target compound may then be coupled to the NPs via the (functional groups of the) ligands.
  • the functional groups of the ligands may facilitate the conjugation of the TDC or target compound to the NP surface, but may also improve other characteristics of the nanomaterial such as solubility and/or stability.
  • the stability of the nanoparticle colloids in aqueous media may be improved.
  • the functional groups may be activated prior to reaction with the TDC, target compound or linker (see further).
  • the functional group is a carboxyl
  • the carboxyl may be activated using one or more carboxyl activating groups. Suitable carboxyl activating groups include, but are not limited to, carbodiimide reagents, phosphonium reagents, uranium, and carbonium reagents, as is known by the skilled person.
  • the first molecule may not comprise a metal binding functionality, or a functional group which can react with the functional groups exposed on the NP surface.
  • the TDC or target compound may be coupled to the NP surface or the ligands indirectly, more particularly via a linker molecule, which is also referred to herein as "linker".
  • Linker molecules may also be used in other cases wherein direct coupling of the TDC or target compound to the NP surface or to the ligands is not possible or desired, e.g. to improve the access of the target molecule to the TDC when conjugated to the particles.
  • the linker molecule is coupled to the NPs and the first molecule, particularly TDC or target compound, is not critical.
  • the linker may first be coupled to the NPs or to the ligands provided on the NPs followed by coupling of the TDC or target compound to the linker, or vice versa.
  • the TDC or target compound may first be coupled to the linker molecule, followed by coupling the linker molecule to the NP.
  • step (a) of the present methods may comprise:
  • step (a) of the present methods may comprise:
  • linker molecules are known to those of skill in the art and typically includes bi-functional molecules.
  • linker molecules comprise a spacer group terminated at one end with a first portion capable of coupling to the nanoparticles (e.g. via a metal binding functionality, or via binding to the ligands provided on the nanoparticle surface) and at the other end a second portion which is a functional group capable of forming a covalent bond to the first molecule, particularly TDC or target compound.
  • Spacer groups of interest possibly include aliphatic and unsaturated hydrocarbon chains, spacers containing hetero-atoms such as oxygen (ethers such as polyethylene glycol) or nitrogen (polyamines), peptides, carbohydrates, cyclic or acyclic systems that may possibly contain hetero-atoms.
  • hetero-atoms such as oxygen (ethers such as polyethylene glycol) or nitrogen (polyamines), peptides, carbohydrates, cyclic or acyclic systems that may possibly contain hetero-atoms.
  • short spacer groups are preferred as they typically result in a stronger LSPR signal.
  • a spacer group which is too short may be insufficient to stabilize the nanoparticles in suspension, in particular when the linker directly binds to the NP surface via a metal binding functionality.
  • Preferred spacer groups comprise a hydrocarbon chain with 6 to 18 and preferably 6 to 12 carbon atoms; or a polyethyleneglycol (PEG) chain of 2 to 6 ethylene glycol monomers, preferably 3 or 4 monomers.
  • Potential functional groups capable of covalently binding the TDC or target compound include nucleophilic functional groups (amines, hydroxyls, sulfhydryls, azides, hydrazides), electrophilic functional groups (alkynyles, carboxyls, aldehydes, esters, vinyl ketones, epoxides, isocyanates, maleimides), functional groups capable of cycloaddition reactions, forming disulfide bonds, or binding to metals.
  • nucleophilic functional groups amines, hydroxyls, sulfhydryls, azides, hydrazides
  • electrophilic functional groups alkynyles, carboxyls, aldehydes, esters, vinyl ketones, epoxides, isocyanates, maleimides
  • functional groups capable of cycloaddition reactions capable of cycloaddition reactions, forming disulfide bonds, or binding to metals.
  • the linker is provided with a maleimide functional group.
  • Maleimide functional groups are particularly suitable for conjugating a first molecule, particularly TDC or target compound, to the nanoparticles, wherein the TDC or target compound is a protein or peptide containing a sulfhydryl (from cysteine) which is available for binding.
  • the coupling of a proteinaceous or peptidic TDC or target compound to the nanoparticles via a linker having a maleimide functional group may allow for a uniform and oriented coupling of the protein or peptide to the nanoparticles, which can improve the reliability of the present methods.
  • the linker may be provided with a maleimide functional group (for coupling to the TDC or target compound) and an amine group (for binding to carboxylic acid functional groups provided on the nanoparticle surface).
  • the functional group for coupling the linker to the nanoparticles may be a metal binding functionality.
  • a preferred metal binding functionality is sulfhydryl.
  • the functional group for coupling the linker to the nanoparticles is a functional group which can covalently bind to a functional group provided on the (ligands of the) nanoparticles.
  • the surface of the metal nanoparticles provided in (a1 ) may be provided with one or more functional groups.
  • the one or more functional groups are selected from amino, azido, alkynyl, carboxyl, hydroxyl, maleimide and carbonyl.
  • the nanoparticle surface is provided with carboxyl groups.
  • Carboxyl groups are especially useful for binding proteins, because an activated carboxyl group can react with an amine moiety of a protein, thereby forming an amide bond.
  • the nanoparticles are at least partially coated with a mercaptocarboxylic acid.
  • the sulfhydryl moiety of the mercaptocarboxylic acid can bind to a metal atom of the nanoparticle surface via chemisorption, while the carboxyl moiety can be used to bind to molecules such as proteins.
  • the functional groups provided on the first and/or second portion of the linker may allow a coupling mechanism as used in Click Chemistry.
  • the functional groups may comprise an azide or an alkyne, thereby allowing an azide alkyne Huisgen cycloaddition using a Cu catalyst at room temperature, as known by the person skilled in the art.
  • An azide functional group further provides the possibility of Staudinger ligation, which typically involves reaction between an azide moiety with a phosphine or phosphate moiety.
  • the TDC-NP conjugate or target compound-NP conjugate is further contacted with a blocking reagent which reacts with the remaining unreacted functional groups which may be present on the TDC-NP conjugate or target compound-NP conjugate.
  • a blocking reagent which reacts with the remaining unreacted functional groups which may be present on the TDC-NP conjugate or target compound-NP conjugate.
  • this may be done to prevent nonspecific binding of the target compound to the unreacted functional groups.
  • blocking reagents are known in the art.
  • the blocking reagent is typically chosen such that it does not significantly interact with the substances that shall be tested with the conjugated nanomaterial, in particular the target compound and/or test compound.
  • the blocking reagent is further chosen such that it contributes to a good solubility of the conjugated nanoparticles in one or more solvents, for example by adding charge, hydrophilicity or steric hindrance.
  • the blocking reagent is a carboxyl blocking reagent, for example a reagent comprising an amino functional group.
  • Suitable carboxyl blocking reagents include but are not limited to Bovine Serum Albumin (BSA), Ovalbumin, and an amino functionalized polyethylene glycol.
  • potential blocking reagents are molecules comprising a phosphine or alkyne moiety. If the functional group is an alkyne or phosphine, potential blocking reagents are molecules comprising an azide moiety. Examples of such molecules are modified proteins or peptides which do not significantly interact with the target compound or test compound.
  • the first molecule, particularly TDC or target compound may be conjugated to the NPs in a controlled way, such that the amount of said TDC or said target compound conjugated to said NPs is below 70% of the amount required for full coverage of said NPs with said TDC or target compound, more preferably below 50% of full coverage.
  • full coverage refers to the maximal amount of the first molecule, particularly TDC or target compound, that can be conjugated or attached to a nanoparticle as a monolayer around said nanoparticle. Full coverage may be obtained by exposing the nanoparticles to a large excess of the first molecule, particularly TDC or target compound, in conditions suitable for coating the nanoparticles. The optimal amount of the TDC or target compound may be determined via a titration experiment.
  • this may involve titration of a fixed amount or concentration of NPs with a variable amount or concentration of TDC or target compound.
  • optical properties such as ⁇ max or ARU (see further) against the amount or concentration of added TDC or target compound, the optical properties will change with increasing amount of TDC or target compound until full coverage of the nanoparticles is obtained.
  • the optimal amount may depend on the characteristics of the nanoparticles, such as size and shape, and the TDC and/or target compound.
  • the amount of the first molecule, particularly TDC or target compound, conjugated to the nanoparticles is below 70%, preferably below 50%, of the amount required for full coverage, wherein the nanoparticles are nanorods with a length between 40 and 60 nm and a diameter between 10 and 20 nm.
  • the amount of functional groups provided on the nanoparticle surface determines the maximal amount of the first molecule, particularly TDC or target compound, that can be conjugated to the nanoparticles.
  • the amount of functional groups can be ensured that less than full coverage of the nanoparticles by the TDC or target compound is obtained by limiting the amount of functional groups, for example by coating the NPs with a mixture of ligands of which some do and others do not comprise the required functional group for conjugating the TDC or target compound to the NPs.
  • less than full coverage may also be obtained by only letting a certain fraction of the functional groups provided on the nanoparticle surface react with the first molecule, particularly TDC or target compound.
  • the required amount of the TDC or target compound to reach the desired coverage may be found by a titration experiment.
  • the non-reacted functional groups present on the nanoparticles may be reacted with (an excess of) a blocking reagent, in order to avoid nonspecific binding and/or a reduced stability of the nanoparticle conjugate.
  • a certain fraction of the functional groups provided on the nanoparticles may first be reacted with a blocking reagent, followed by reacting the non-blocked functional groups with (an excess of) the TDC or target compound. Again, a concentration titration may be performed first to determine the optimal amount of blocking reagent required for blocking a specific part of the functional groups provided on the nanoparticles.
  • the methods of determining an interaction between a target compound and a test compound as envisaged herein typically comprise contacting or incubating said suspension of metal nanoparticles conjugated to said first molecule with said test compound; wherein at least said test compound is provided in a solution comprising at least one detergent; thereby obtaining a mixture comprising at least the following components: NPs conjugated to the first molecule, the test compound, and a detergent.
  • the target compound is present in said mixture either conjugated to the metal nanoparticles or, in case of a competition assay as envisaged herein in solution.
  • the present methods, particularly competition assay may be used for high-throughput screening of test compound libraries.
  • the competition assay methods of determining an interaction between a test compound and a target compound envisaged herein typically comprise in a step (b), contacting or incubating the suspension comprising the TDC-NP conjugate with the target compound and the test compound wherein said test compound is provided in a solution comprising a detergent, thereby obtaining a mixture comprising the TDC-NP conjugate, the target compound, the test compound and the detergent.
  • the target compound and test compound are provided in a (relatively) purified form in a fluid composition, for example a (buffered) solution in water and/or DMSO.
  • the present methods may be used for high-throughput screening of test compound libraries.
  • the suspension may be contacted with a plurality of test compounds; thereby obtaining a plurality of mixtures each comprising the TDC-NP conjugate, the target compound, detergent and one of the test compounds.
  • the compound library is provided as solutions of test compounds in a well-plate.
  • test compounds are often dissolved in a solvent which is or comprises dimethylsulfoxide (DMSO).
  • DMSO dimethylsulfoxide
  • the target compound may be dissolved in a solvent comprising DMSO.
  • SPR surface plasmon resonance
  • test compound and/or target compound need not be transferred to another solvent than DMSO prior to contacting with the TDC-NP conjugate or target-compound NP conjugate.
  • the test compound is provided as a solution further comprising at least 50 w% (percent by weight) DMSO, wherein the solution may be mixed with the (suspension comprising the) TDC-NP conjugate and the (solution comprising the) target compound as such.
  • the test compound is provided as a solution comprising a detergent and at least 50 w% DMSO, preferably at least 75 w% DMSO, more preferably at least 90 w% DMSO.
  • the resulting liquid mixture comprising the TDC-NP conjugate, the test compound, the detergent and the target compound may comprise a lower amount of DMSO.
  • this liquid mixture comprises between 0.5 w% and 50w% DMSO, between 1 w% and 50 w% DMSO, or even between 5 w% and 50 w% DMSO.
  • the liquid mixture comprises at most 40 w% DMSO, preferably at most 20 w% DMSO, most preferably at most 10 w% DMSO.
  • the target compound is pre-incubated with the test compound prior to contacting the TDC-NP conjugate with the target compound and test compound.
  • the pre-incubation can significantly shorten the amount of time needed for reaching equilibrium after contacting the TDC-NP conjugate with the test compound and the target compound.
  • the present methods may comprise in a step (b): (b1 ) incubating a solution of the target compound with the test compound; thereby obtaining a pre-incubated target compound solution comprising a detergent and optionally comprising at least 0.5 w% DMSO; and
  • the incubation time i.e. the time between steps (b1 ) and (b2) typically is at least 1 minute, preferably at least 5 minutes, most preferably at least 10 minutes, for example between 15 minutes and 60 minutes.
  • the optimal amount of target compound to be used in step (b) may depend on various factors, in particular the concentration of NPs in the suspension and the average amount of TDC molecules bound to the NPs.
  • a suitable amount for the target compound may be found via a titration experiment, wherein the LSPR properties of the TDC-NPs are monitored with increasing amounts of added target compound. More particularly, the titration may be monitored via the measurement of the absorbance of the nanoparticles. In particular embodiments, the titration may involve determining ARU, i.e.
  • OD(x) refers to the optical density at wavelength x (in nm)
  • max refers to the wavelength of maximal absorbance of the TDC-NP conjugate.
  • a suitable amount of target compound is an amount which results in a detectable change of the LSPR properties of the conjugate, but which does not saturate the available TDC binding sites (i.e. below the plateau in the plot of ARU vs. the amount of added target compound).
  • the titration may involve determining A max , i.e. the change in the max for each amount of target compound added.
  • max refers to the wavelength of maximal absorbance of the TDC-NP conjugate, i.e. the wavelength x for which OD(x) reaches a maximum.
  • a suitable amount of target compound is an amount which results in a detectable change of the LSPR properties of the conjugate, but which does not saturate the available TDC binding sites (i.e. below the plateau in the plot of max vs. the amount of added target compound).
  • the target compound concentration is specifically chosen in the linear part of the titration curve, below the plateau value (or maximal value), with the latter value corresponding to saturation of the available binding sites.
  • this reduces the risk of unwanted aggregation of the TDC-NP/target compound complex.
  • the raw absorbance data are processed prior to determining ARU and/or m ax as described above.
  • Data processing may be based on curve fitting (like polynomials or any other representative curve like Gaussian or Lorentzian curves, preferably in a predefined neighborhood, e.g. around a maximum, or even a model, being representative for the resonance phenomena used) and use of the fitted curve instead of the raw data.
  • Step (c) of the methods, particularly competition assay, of determining an interaction between a test compound and a target compound envisaged herein typically comprise (c1 ) monitoring step (b) by illuminating said nanoparticles with at least one excitation light source and monitoring one or more optical properties of said nanoparticles, such as absorbance, refractive index, absorption, scattering, fluorescence, luminescence or photoluminescence; and (c2) detecting a change of one or more LSPR properties or optical properties of the nanoparticles, more in particular detecting a change in refractive index surrounding said nanoparticles, wherein said change is a result of the presence of an interaction between said first molecule (conjugated to said NPs) and another molecule, such as between the target compound NP conjugate and the test compound, or between the TDC-NP conjugate and the target compound.
  • monitoring step (b) by illuminating said nanoparticles with at least one excitation light source and monitoring one or more optical properties of said nanoparticles
  • Particular embodiments of the competition assay envisaged herein typically comprise in a step (c), the determination whether the test compounds modulate (e.g. inhibit) binding of the target compound to the TDC, based on the presence or absence of a change in Localized Surface Plasmon Resonance (LSPR) properties of the TDC-NP conjugate, in particular based on the presence or absence of a change in refractive index surrounding said nanoparticles, when contacting the (suspension comprising the) TDC-NP conjugate with the target compound and the test compound.
  • LSPR Localized Surface Plasmon Resonance
  • the target compound when the test compound does not interact with the target compound (or when the test compound binds to the target compound but does not compete with TDC because it does not interact with the binding site of the TDC), the target compound will bind to the TDC which is conjugated to the nanoparticles.
  • the proximity of the target compound to the conjugate changes the refractive index surrounding the nanoparticles, which will lead to a detectable change in the LSPR properties of the TDC-NP conjugate.
  • the target compound will not bind to the TDC, or only a reduced amount will bind. Accordingly, no change or a minor change in the LSPR properties of the TDC-NP conjugate will be detected.
  • the change in LSPR properties of the TDC-NP conjugate is detected by measuring one or more optical properties of the conjugate in the presence and absence of the target compound.
  • the present methods typically involve measuring one or more macroscopic optical properties of the suspension comprising the conjugate. Accordingly, the average optical properties of the nanoparticles are measured, rather than measuring the optical properties of single particles.
  • competition assays for determining an interaction between a target compound and a test compound as envisaged herein may comprise in a step (c):
  • step (c1 ) monitoring step (b) by illuminating the TDC-NP conjugate with at least one excitation light source and monitoring one or more optical properties of the conjugate;
  • (c2) detecting a change of one or more optical properties of the TDC-NP conjugate, particularly detecting a change in refractive index surrounding said nanoparticles, wherein said change is a result of the presence of an interaction between the target compound and the TDC.
  • said change in refractive index or absorbance is detected at a wavelength ranging between 600 and 900 nm, such as between 700 and 800 nm.
  • the light source used in (c1 ) typically emits light or radiation at one or more wavelengths between 350 and 1000 nm.
  • an excitation light source is used which emits light or radiation comprising between approximately 1 nanowatt and 100 watts of power.
  • the excitation light source is a (xenon) flash lamp or a laser.
  • step (c1 ) is repeated at least once and said step (c2) is applied to an averaged optical property obtained from said repetition.
  • both step (c1 ) and step (c2) are repeated at least once, wherein the final detection of a change of one or more optical properties is based on an average of the detection obtained from each execution of step (c1 ), followed by (c2).
  • detecting means to ascertain a signal (or a change therein), either qualitatively or quantitatively.
  • the methods described herein comprise the step of detecting a signal, more particularly a change in signal at one or more wavelengths.
  • the terms “monitoring”, “determining”, “measuring”, “assessing”, “detecting” and “evaluating” are used interchangeably to refer to any form of measurement, and includes not detecting any change. Said measurement may include both quantitative and qualitative determinations either relative or absolute and also include determining the amount of something present, as well as determining whether it is present or absent.
  • an optical property of the conjugate which is monitored is the absorbance of the conjugate.
  • the binding of the target compound to the TDC-NP conjugate leads to a difference in refractive index around the nanoparticles and thereby to a red-shift of the m ax that can be detected by reading an absorbance spectrum.
  • the change in absorbance properties is expressed as a change in max (A max ).
  • the change in absorbance properties is expressed as ARU, as defined above.
  • the absorbance of the conjugate is measured at two or more wavelengths between 350 and 1000 nm. Measurement at two or more wavelengths can allow for obtaining more accurate data. In particular embodiments, these wavelengths are discrete wavelengths within that range.
  • the raw absorbance data are processed before use in any of the above embodiments, such as prior to determining ARU and/or A max .
  • processing may be based on curve fitting (like polynomials or any other representative curve like Gaussian or Lorentzian curves, preferably in a predefined neighborhood, e.g. around a maximum, or even a model, being representative for the resonance phenomena used) and use of the fitted curve instead of the raw data.
  • step (b) comprises the following steps to obtain a concentration response curve: (i) for each resulting mixture (comprising the TDC-NP conjugate, said test compound, said target compound, and said detergent - with different mixtures having different concentrations of the test compound), the max of the TDC-NPs is determined from the absorbance spectrum;
  • a reference sample e.g. a TDC-NP solution lacking target and test compound
  • this step is insufficient to correct for all unwanted background signals, such as in case a test compound in the absence or presence of the target compound can cause an unpredictable change in the measured LSPR signal of the TDC-NP conjugate (e.g. only at high test compound concentrations, due to aggregation or aspecific interaction with the TDC-NP conjugate).
  • the test compound is available in small quantities, providing the test compound in a solution comprising detergent surprisingly was able to prevent unwanted background signals;
  • the maximal A max is determined using a sample containing the target, the TDC- NPs and the other components of the mixture (detergent, optionally DMSO) but lacking a test compound (positive control);
  • the data processing may further include the quantitative analysis of the concentration response curve, such as by fitting the data with a sigmoidal dose- response function, which yields an IC50 value.
  • concentration response curve such as by fitting the data with a sigmoidal dose- response function, which yields an IC50 value.
  • Data processing may be performed by a computer program configured to process the data obtained via the absorbance spectrum measurements.
  • Such computer program may be configured to use the processed date (such as based on curve fitting or data modelling and used of the fitted curve or modelled data instead of the raw absorbance data) to determine or quantify the interaction between the test compounds and the target compound.
  • computer programs are provided, which, when running on a computer, determine or quantify the interaction between the test compounds and the target compound.
  • steps (c1 ) and (c2) may be performed more than once, preferably after regular time intervals. This may allow for determining whether the mixture has reached equilibrium. Indeed, the LSPR signal may change as long as the mixture advances towards its equilibrium, only to become stable when equilibrium has been reached. It is preferred that the mixture reaches equilibrium, as this allows for a more precise quantification of the interaction between the test compound and the target compound. It is noted that the methods described herein do not suffer from bleaching of the TDC-NP conjugate, in contrast with other methods such as fluorescence-based assays. Accordingly, there is practically no limit on the amount of iterations of steps (c1 ) and (c2) which can be performed.
  • steps (c1 ) and (c2) are reiterated regularly, with a time interval between successive iterations between 0.5 seconds and 20 minutes. If there are multiple samples, e.g. provided in a multi-well plate, a new iteration preferably starts when the previous iteration has been completed for all samples. In such embodiments, a typical time interval is about 15 minutes. Preferably, the iteration is terminated when the measured LSPR properties are stable or when a predefined time limit has expired, whichever occurs first.
  • step (c) of the methods described herein may comprise correcting the observed change in LSPR properties of the TDC-NP conjugate or target compound conjugate for the presence of DMSO.
  • the correction step may involve correcting the measured change in LSPR properties of the TDC-NP conjugate or target compound conjugate, by subtracting the contribution of DMSO to the change.
  • the correction may involve comparing the optical properties of the sample with the optical properties of a reference (blank) sample comprising the TDC-NP conjugate and target compound, or target compound conjugate, in the same solvent (comprising DMSO) but without the test compound.
  • the methods of the present invention are of particular interest in the context of screening methods.
  • the present invention provides screening methods wherein detection is performed according to the present invention.
  • the methods are high-throughput screening methods, more particularly methods which are at least in part carried out in a high-throughput screening device.
  • the competition assay methods envisaged herein may be used to screen for compounds that modulate the interaction between the target molecule and the TDC.
  • modulating includes both decreasing (e.g. inhibiting) and enhancing the interaction between the two molecules.
  • the competition assay methods envisaged herein are particularly suitable for identifying test compounds that can modulate the interaction between a first (poly)peptide (P1 ) and a second (poly)peptide (P2).
  • P1 first (poly)peptide
  • P2 second (poly)peptide
  • polypeptide includes proteins.
  • the effect of the test compound on the interaction between P1 and P2 can be determined using the methods described herein, wherein P1 can be selected as the target compound and P2 as the TDC, or vice versa.
  • (C) determining whether the test compound modulates the interaction between P1 and P2, based on the presence or absence of a change in LSPR properties of the P1 -NP conjugate when contacting the suspension comprising the P1 -NP conjugate with P2 and the test compound.
  • step (B) may include determining the optimal amount of P2 to be added to the conjugate, via a titration experiment as described above.
  • the method may include the selection of a suitable ionic strength for the suspension comprising the P1 -NP conjugate. In particular embodiments, this may include selecting a maximal value for the ionic strength. It is preferred that an ionic strength below the maximal value is respected prior to and after contacting with P2 and the test compound.
  • the inventors have found that for a large number of proteins an optimal stability of the P1-NP conjugate can be obtained by using a suspension having an ionic strength below 20 mM. Without wishing to be bound by theory, it is believed that an ionic strength below the maximal value avoids shielding of the charges on the nanoparticles and/or on the polypeptides. On the other hand, some P1 -P2 interactions may require a minimal ionic strength. Accordingly, in preferred embodiments, the ionic strength of the suspension may be between 5 mM and 20 mM, for example about 10 mM. The ionic strength of the suspension can be increased through the addition of salts which form ions when dissolved in the solvent of the suspension, as is known by the skilled person.
  • step (A) includes conjugating P1 to the nanoparticles
  • the nanoparticle suspension should preferably be buffered at a pH above (pl-1 ), wherein pi is the isoelectric point of P1 , this is the pH at which P1 or its surface carries no net electrical charge. More preferably, the nanoparticle suspension is buffered at a pH between (pl-1 ) and pi. This is particularly advantageous when the nanoparticles are provided by functional groups which carry negative charges, such as carboxyl groups.
  • step (A) may comprise:
  • (A1 ) providing a suspension of metal nanoparticles (NPs), wherein said suspension has a pH above (pl-1 ), and preferably between (pl-1 ) and pi, wherein pi is the isoelectric point of P1 ;
  • steps (a1 ), (a2), and (a3) as described above apply, mutatis mutandis, to steps (A1 ), (A2), and (A3).
  • the pH of the nanoparticle suspension in steps (B) and (C) may be the same or different than the pH range used in step (A).
  • the purification of peptides and proteins may involve providing the peptides and proteins with a tag such as a Histidine-tag (His-tag), which may bind non-specifically to the metal surface of the nanoparticles.
  • a small amount of imidazole may be added to the P1 -NP suspension. The imidazole will then compete with the His-tag for the metal.
  • imidazole is added to the suspension to a concentration between 5 and 50 mM.
  • the present application further provides tools for carrying out one or more steps of particular embodiments of the methods described herein.
  • kits comprising
  • test compounds as described herein, each provided as a solution comprising the test compound and at least a detergent as envisaged herein;
  • instructions for use of the TDC-NP conjugate in one or more of the methods described herein are optionally, instructions for use of the TDC-NP conjugate in one or more of the methods described herein.
  • the kit may comprise a plurality of test compounds, which may be provided in a multi-well plate, wherein the one or more test compounds each are provided as a solution comprising the test compound and at least one detergent as envisaged herein.
  • the solution comprising said test compound and said at least one detergent further comprises DMSO, such as at least 50wt% DMSO.
  • A) Competition assay Determining of interaction between a small molecule and a protein
  • Biotin is known to bind with high affinity to the protein Neutravidin.
  • HABA ((2-(4- hydroxyazobenzene) benzoic acid)) binds to neutravidin in a similar way as biotin, but with lower affinity.
  • TDC target definition compound
  • A1 Conjugation of biotin to gold nanorods (GNRs)
  • GNRs Gold nanorods
  • MUDA mercaptoundecanoic acid
  • the MUDA-coated GNRs provide an outer layer of carboxyl functional groups on their surface.
  • a biotin derivative (amino-PEG4-biotin) was used containing a polyethyleneglycol linker (4 monomers) having an amino functional group.
  • the carboxyl groups on the GNRs were activated using ethyl(dimethylaminopropyl) carbodiimide (EDO) and Sulfo N-hydroxysuccinimide (Sulfo-NHS).
  • biotin-GNR conjugate was coupled via its amino functional group to the carboxyl groups provided on the GNRs, thereby providing a biotin-GNR conjugate. Potentially remaining unreacted carboxylic acid groups were blocked via reaction with 2-(2-aminoethoxy)ethanol (AEE).
  • AEE 2-(2-aminoethoxy)ethanol
  • the biotin- GNR conjugate was purified from unreacted EDC, Sulfo-NHS, amino-PEG4-biotin and AEE by buffer exchange using a centrifugal ultrafiltration device.
  • the determination of the inhibition of the interaction between HABA and Neutravidin involves contacting the biotin-GNR conjugate with Neutravidin.
  • the optimal amount of Neutravidin to be contacted with the conjugate was determined via titration. More particularly, a fixed amount of biotin- GNR was incubated with various concentrations of Neutravidin, and the absorbance spectra after incubation was recorded. A max was calculated and plotted as a function of the Neutravidin concentration (Fig. 1 ). Suitable Neutravidin concentrations are determined as those concentrations which are sufficiently high to provide a detectable signal (A max ), provided that the signal is not in the plateau of the dose-response curve. Optimal concentrations are those providing a signal in the linear response range.
  • a fixed amount of Neutravidin was pre-incubated with different concentrations of HABA or free biotin, followed by incubation with a fixed amount of biotin-GNR conjugate.
  • HABA or free biotin was provided as a solution in DMSO (and detergent). The final DMSO concentration was 5 w%.
  • the relative amounts of biotin-GNR and Neutravidin used are determined via titration as described above (A2). After incubation of Neutravidin with HABA free biotin and biotin-GNR, the absorbance spectra were recorded. X max was calculated, normalized and plotted as a function of the HABA and free biotin concentration (Fig. 2).
  • the normalized A max was calculated, for each sample as [ max (sample) - max (blank)] / max (positive control) - max (blank)], wherein the positive control corresponds to the sample containing Neutravidin and biotin-GNR, without HABA free biotin, and wherein the blank corresponds to only biotin-GN R, in the same solvent comprising 5 w% DMSO (and detergent).
  • the p53 protein (also known as cellular tumor antigen p53) is known to bind with high affinity to the MDM2 protein (Mouse Double Minute 2 homolog).
  • the compound nutlin- 3 also binds to MDM2, thereby inhibiting further binding of MDM2 to p53.
  • the inhibition of the p53-MDM2 interaction by nutlin-3 was assessed using a method as described herein.
  • GNRs Gold nanorods
  • MUDA mercaptoundecanoic acid
  • the MUDA-coated GNRs provide an outer layer of carboxyl functional groups on their surface.
  • p53 peptide was conjugated to the GNRs, using a N-(2-aminoethyl)maleimide linker. More particularly, the carboxyl groups were activated using EDC and Sulfo-NHS. Then, N-(2-aminoethyl)maleimide is coupled via its amino functional group to the carboxyl groups provided on the GNRs, thereby providing a layer of maleimide groups on the GNR surface. Potentially remaining unreacted carboxylic acid groups were blocked via reaction with AEE. The maleimide- functionalized GNRs were then purified from unreacted EDC, Sulfo-NHS, N-(2- aminoethyl)maleimide and AEE by buffer exchange using a centrifugal ultrafiltration device.
  • the p53 peptide was then coupled via its sulfhydryl group (provided by the N-terminal cysteine of p53) to the maleimide moieties on the GNR, thereby obtaining a p53-GNR conjugate.
  • the pH of the GNR suspension during coupling was buffered at a pH of 7.5, which is above the pi of the p53 peptide (5.66).
  • the p53-GNR conjugate was reacted with sulfhydryl functionalized methoxy polyethylene glycol (mPEG-SH), thereby blocking any remaining unreacted maleimide groups.
  • the p53-GNR conjugate was purified from unreacted p53 peptide and mPEG-SH by buffer exchange using dialysis.
  • the determination of the inhibition of the interaction between MDM2 and p53 according to the methods described herein involves contacting the p53-GNR conjugate with MDM2.
  • the optimal amount of MDM2 to be contacted with the conjugate was determined via a similar titration experiment as described above for Neutravidin (A2).
  • a max was calculated and plotted as a function of the MDM2 concentration (Fig. 3).
  • MDM2 MDM2-incubated with different concentrations of nutlin-3 or p53, followed by incubation with a fixed amount of p53-GNR conjugate.
  • the relative amounts of p53-GNR and MDM2 used are determined via titration as described above (B2). After incubation of MDM2 with nutlin-3 (or p53) and p53-GNR, the absorbance spectra were recorded. A max was calculated and plotted as a function of the added amount of nutlin-3 and p53 (Fig. 4A and 4B).
  • test compounds 1 to 4 Four small molecules (test compounds 1 to 4) were dissolved in DMSO, and subsequently added to a buffer comprising either no Triton X-100 or 0.1 volume % (v%), thereby obtaining liquid mixtures comprising 2 v% DMSO.
  • various solutions were prepared with increasing concentration of the test compound (0-100 ⁇ ).
  • the solutions comprising the test compounds were incubated with a suspension of a TDC conjugated to GNRs (TDC-GNR; 1 v% final DMSO concentration), and absorbance spectra of the resulting suspensions was recorded.
  • TDC-GNR TDC conjugated to GNRs
  • Fig. 5 and 6 show the wavelength of maximal absorbance (Amax) for the suspensions without and with detergent (Triton X-100), respectively.
  • a max of the TDC-NP increases at higher compound concentrations. Presumably, this is indicative of a (non-specific) interaction of the test compounds with the TDC at elevated test compound concentration and generates unwanted background signals.
  • no significant change of A max is observed in the presence of 0.1 v% Triton X-100. Accordingly, these results show that background signals can be suppressed via the addition of a detergent such as Triton X-100.
  • test compound 1 (provided as a solution with or without Triton X-100) with the target compound and the TDC-GNR, the absorbance spectra were recorded. A max was calculated and plotted as a function of the added amount of test compound 1 (Fig. 7). This clearly shows that the detergent suppresses the background signals also in the presence of the target compound, and that test compound 1 is a modulator/inhibitor of the binding between the target compound and the TDC.

Abstract

La présente invention se rapporte à un procédé pour la suppression de signaux de fond dans une méthode ou un dosage comprenant l'utilisation d'un composé d'essai dans une solution comprenant au moins un détergent. La présente invention concerne en outre un dosage amélioré et plus précis, en particulier un dosage par compétition, consistant à déterminer une interaction entre un composé cible et un composé d'essai en présence d'un détergent, sur la base du suivi des propriétés de résonance plasmonique de surface localisée (LSPR) de nanoparticules métalliques.
PCT/EP2016/059173 2015-04-24 2016-04-25 Procédé perfectionné de mesure d'une interaction entre des molécules WO2016170183A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/EP2015/058871 WO2015162234A1 (fr) 2014-04-25 2015-04-24 Dosage concurrentiel
EPPCT/EP2015/058871 2015-04-24

Publications (1)

Publication Number Publication Date
WO2016170183A1 true WO2016170183A1 (fr) 2016-10-27

Family

ID=55963313

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2016/059173 WO2016170183A1 (fr) 2015-04-24 2016-04-25 Procédé perfectionné de mesure d'une interaction entre des molécules

Country Status (1)

Country Link
WO (1) WO2016170183A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108827952A (zh) * 2018-07-02 2018-11-16 北京师范大学 Nor逻辑门及其构建方法
EP3574308A4 (fr) * 2017-01-30 2020-12-30 Abaxis, Inc. Dosages plasmoniques en solution de partenaires de liaison spécifiques, et nanostructures métalliques
US11209430B2 (en) 2014-08-13 2021-12-28 Zoetis Services Llc Signal amplification in plasmonic specific-binding partner assays
US11215614B2 (en) 2015-08-04 2022-01-04 Zoetis Services Llc Signal amplification in solution-based plasmonic specific-binding partner assays
US11255854B2 (en) 2011-11-21 2022-02-22 Zoetis Services Llc Signal amplification in lateral flow and related immunoassays
US11977072B2 (en) 2018-01-30 2024-05-07 Zoetis Services Llc Solution-based plasmonic specific-binding partner assays using metallic nanostructures

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004042403A2 (fr) 2002-11-04 2004-05-21 Ludwig-Maximilian-Uni Versität München Procedes, dispositif et instrument pour la detection d'analytes
WO2006129828A2 (fr) * 2005-05-31 2006-12-07 Canon Kabushiki Kaisha Molecule de capture de substance active
US20070117151A1 (en) * 2001-12-21 2007-05-24 Interuniversitair Microelektronica Centrum (Imec) Method and Apparatus For Detecting An Analyte
WO2010064207A2 (fr) * 2008-12-03 2010-06-10 Koji Kawakami Peptide chimérique anticancéreux sélectif
US20110021748A1 (en) * 2009-02-10 2011-01-27 Wayne State University Non-Natural Peptides as Models for the Development of Antibiotics
WO2012018907A2 (fr) * 2010-08-03 2012-02-09 University Of Washington Through Its Center For Commercialization Polypeptides pour traitement et/ou limitation d'infection par la grippe
US20120157328A1 (en) 2007-04-03 2012-06-21 Dudek Ronald P Label Free Kinase Assays and Reagents

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070117151A1 (en) * 2001-12-21 2007-05-24 Interuniversitair Microelektronica Centrum (Imec) Method and Apparatus For Detecting An Analyte
WO2004042403A2 (fr) 2002-11-04 2004-05-21 Ludwig-Maximilian-Uni Versität München Procedes, dispositif et instrument pour la detection d'analytes
WO2006129828A2 (fr) * 2005-05-31 2006-12-07 Canon Kabushiki Kaisha Molecule de capture de substance active
US20120157328A1 (en) 2007-04-03 2012-06-21 Dudek Ronald P Label Free Kinase Assays and Reagents
WO2010064207A2 (fr) * 2008-12-03 2010-06-10 Koji Kawakami Peptide chimérique anticancéreux sélectif
US20110021748A1 (en) * 2009-02-10 2011-01-27 Wayne State University Non-Natural Peptides as Models for the Development of Antibiotics
WO2012018907A2 (fr) * 2010-08-03 2012-02-09 University Of Washington Through Its Center For Commercialization Polypeptides pour traitement et/ou limitation d'infection par la grippe

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
AYELA ET AL: "Antibody-antigenic peptide interactions monitored by SPR and QCM-D", BIOSENSORS AND BIOELECTRONICS, ELSEVIER BV, NL, vol. 22, no. 12, 16 May 2007 (2007-05-16), pages 3113 - 3119, XP022080451, ISSN: 0956-5663, DOI: 10.1016/J.BIOS.2007.01.020 *
CHARNG-SHENG TSAI ET AL: "Gold nanoparticle-based competitive colorimetric assay for detection of protein-protein interactions", CHEMICAL COMMUNICATIONS, no. 34, 1 September 2005 (2005-09-01), pages 4273 - 4275, XP055007100, ISSN: 1359-7345, DOI: 10.1039/b507237a *
CHARNG-SHENG TSAI ET AL: "Rapid-Throughput Competitive Colorimetric Assay Based on Monosaccharide-Capped Gold Nanoparticles for Detecting Lectin-Protein Interactions", CHEMPLUSCHEM, vol. 77, no. 4, 28 April 2012 (2012-04-28), pages 314 - 322, XP055200858, ISSN: 2192-6506, DOI: 10.1002/cplu.201200014 *
MARIA SOLER ET AL: "Direct Detection of Protein Biomarkers in Human Fluids Using Site-Specific Antibody Immobilization Strategies", SENSORS, vol. 14, no. 2, 29 January 2014 (2014-01-29), pages 2239 - 2258, XP055288315, DOI: 10.3390/s140202239 *
MARK P KREUZER ET AL: "Colloidal-based localized surface plasmon resonance (LSPR) biosensor for the quantitative determination of stanozolol", ANALYTICAL AND BIOANALYTICAL CHEMISTRY, SPRINGER, BERLIN, DE, vol. 391, no. 5, 30 March 2008 (2008-03-30), pages 1813 - 1820, XP019621399, ISSN: 1618-2650 *
WARSINKE ET AL., ANALYTICA CHIMICA ACTA, vol. 550, 2005, pages 69 - 76

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11255854B2 (en) 2011-11-21 2022-02-22 Zoetis Services Llc Signal amplification in lateral flow and related immunoassays
US11209430B2 (en) 2014-08-13 2021-12-28 Zoetis Services Llc Signal amplification in plasmonic specific-binding partner assays
US11215614B2 (en) 2015-08-04 2022-01-04 Zoetis Services Llc Signal amplification in solution-based plasmonic specific-binding partner assays
US11614447B2 (en) 2015-08-04 2023-03-28 Zoetis Services Llc Signal amplification in solution-based plasmonic specific-binding partner assays
EP3574308A4 (fr) * 2017-01-30 2020-12-30 Abaxis, Inc. Dosages plasmoniques en solution de partenaires de liaison spécifiques, et nanostructures métalliques
US11977072B2 (en) 2018-01-30 2024-05-07 Zoetis Services Llc Solution-based plasmonic specific-binding partner assays using metallic nanostructures
CN108827952A (zh) * 2018-07-02 2018-11-16 北京师范大学 Nor逻辑门及其构建方法

Similar Documents

Publication Publication Date Title
Zhang et al. Antibody-gold nanoparticle bioconjugates for biosensors: synthesis, characterization and selected applications
Mahmoudpour et al. Nanomaterials and new biorecognition molecules based surface plasmon resonance biosensors for mycotoxin detection
Mahmoudpour et al. Nanomaterials based surface plasmon resonance signal enhancement for detection of environmental pollutions
WO2016170183A1 (fr) Procédé perfectionné de mesure d'une interaction entre des molécules
Langer et al. Protein analysis by time-resolved measurements with an electro-switchable DNA chip
Mauriz et al. Multi-analyte SPR immunoassays for environmental biosensing of pesticides
Carlucci et al. Several approaches for vitamin D determination by surface plasmon resonance and electrochemical affinity biosensors
Wang et al. Water-soluble ZnO–Au nanocomposite-based probe for enhanced protein detection in a SPR biosensor system
Liu et al. SPR quantitative analysis of direct detection of atrazine traces on Au-nanoparticles: nanoparticles size effect
Filbrun et al. A fluorescence-based method to directly quantify antibodies immobilized on gold nanoparticles
Liu et al. Ultrasensitive detection of deltamethrin by immune magnetic nanoparticles separation coupled with surface plasmon resonance sensor
KR20070085400A (ko) 안정한 금속/도전성 중합체 복합물 콜로이드류, 그의 제조방법 및 그의 용도
DK2628007T3 (en) Method for coating nanoparticles
KR101483725B1 (ko) 표면증강 라만분광을 이용한 시료 내 특정물질의 분석방법및 분석장치
Ribeiro et al. Electrochemistry-assisted surface plasmon resonance biosensor for detection of CA 15–3
US20170248589A1 (en) Method of measuring interaction between molecules
Piletsky et al. A novel assay format as an alternative to ELISA: MINA test for biotin
Liu et al. Sensitivity-enhancement of wavelength-modulation surface plasmon resonance biosensor for human complement factor 4
Banciu et al. Optical biosensing of lysozyme
Kausaite-Minkstimiene et al. An ultra-sensitive SPR immunosensor for quantitative determination of human cartilage oligomeric matrix protein biomarker
Islam et al. Molecular switching fluorescence based high sensitive detection of label-free C-reactive protein on biochip
Kastner et al. The effect of layer thickness and immobilization chemistry on the detection of CRP in LSPR assays
Wang et al. Surface plasmon resonance biosensor based on water-soluble ZnO–Au nanocomposites
Tuckmantel Bido et al. Digital SERS Protocol Using Au Nanoparticle-Based Extrinsic Raman Labels for the Determination of SARS-CoV-2 Spike Protein in Saliva Samples
TW201932300A (zh) 含有二硫化鉬之生物感測晶片以及應用該生物感測晶片之檢測裝置

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16721734

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: COMMUNICATION PURSUANT TO RULE 142 EPC (RESUMPTION OF PROCEEDINGS UNDER RULE 142(2) EPC DATED 28.06.2021)

122 Ep: pct application non-entry in european phase

Ref document number: 16721734

Country of ref document: EP

Kind code of ref document: A1