U.S. patent application number 15/594427 was filed with the patent office on 2017-08-31 for method of measuring interaction between molecules.
The applicant listed for this patent is PHARMADIAGNOSTICS NV. Invention is credited to Vanessa Bonnard, Sylviane Boucharens, Meike Roskamp.
Application Number | 20170248589 15/594427 |
Document ID | / |
Family ID | 45939693 |
Filed Date | 2017-08-31 |
United States Patent
Application |
20170248589 |
Kind Code |
A1 |
Roskamp; Meike ; et
al. |
August 31, 2017 |
METHOD OF MEASURING INTERACTION BETWEEN MOLECULES
Abstract
The present invention relates to methods of measuring
interaction between a first and a second molecule, for example a
protein and an antibody, by conjugation of one of these molecules
with nanoparticles, and measuring the interaction between the first
and second molecule via changes in the optical properties of the
nanoparticles. The present invention further relates to methods of
coating nanoparticles.
Inventors: |
Roskamp; Meike; (Papenburg,
DE) ; Bonnard; Vanessa; (Uccle, BE) ;
Boucharens; Sylviane; (Lanarkshire, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PHARMADIAGNOSTICS NV |
Zellik |
|
BE |
|
|
Family ID: |
45939693 |
Appl. No.: |
15/594427 |
Filed: |
May 12, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14376503 |
Aug 4, 2014 |
9678066 |
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PCT/EP2013/053103 |
Feb 15, 2013 |
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15594427 |
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61599455 |
Feb 16, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B82Y 30/00 20130101;
G01N 33/54313 20130101; G01N 33/54353 20130101; G01N 33/587
20130101; G01N 33/5306 20130101; B82Y 40/00 20130101; G01N 33/54346
20130101; G01N 33/54306 20130101; B82Y 15/00 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 16, 2012 |
GB |
1202631.6 |
Claims
1. A method of coating a metal nanoparticle with a compound,
comprising: i) providing a liquid composition comprising metal
nanoparticles at least partially coated with cetyl trimethyl
ammonium bromide (CTAB); ii) adding a thiol-polyethylene glycol to
said composition of step i), thereby obtaining a liquid composition
comprising metal nanoparticles coated with thiol-polyethylene
glycol; iii) purification of said composition obtained in step ii)
by separating said nanoparticles from free thiol-polyethylene
glycol and free CTAB; iv) adding a mercaptocarboxylic acid to said
composition obtained in step iii), thereby obtaining a liquid
composition comprising metal nanoparticles coated with
mercaptocarboxylic acid, and v) contacting said metal particles
obtained in step iv) with said compound.
2. The method according to claim 1, wherein step iv) further
comprises providing said metal nanoparticles coated with
mercaptocarboxylic acid with one or more functional groups.
3. The method according to claim 2, wherein said functional groups
are selected from the group consisting of carboxyl, amino, azido,
alkynyl, carbonyl, hydroxyl and maleimides.
4. The method according to claim 1, wherein said liquid composition
in steps i), ii), iii) and iv), is an aqueous composition.
5. The method according to claim 4, wherein said liquid composition
in step v) is an aqueous composition.
6. The method according to claim 1, wherein the mercaptocarboxylic
acid added in step iv) is a compound of formula (I) ##STR00005##
wherein n is an integer from 6 to 16.
7. The method according to claim 3 wherein the mercaptocarboxylic
acid is 11-mercaptoundecanoic acid.
8. The method according to claim 1, wherein the thiol-polyethylene
glycol added in step ii) is a compound of formula (IV) ##STR00006##
wherein nc is such that the molecular weight of compound (IV) is
between 100 Da and 10 kDa; and R.sup.1 is C.sub.1-4alkoxy, wherein
said C.sub.1-4alkoxy is optionally substituted by one or more
groups such as hydroxyl.
9. The method according to claim 8 wherein the thiol-polyethylene
glycol is methoxy-PEG-thiol or ethoxy-PEG thiol.
10. The method according to claim 1, wherein said metal
nanoparticles comprise a metal selected from the group consisting
of Au, Ag, Cu, Ta, Pt, Pd, and Rh.
11. The method according to claim 1, wherein said metal
nanoparticles are nanorods
12. The method according to claim 11, wherein said nanorods have an
aspect ratio between 1.5 and 5.
13. The method according to claim 1 wherein said compound is a
biomolecule.
14. The method according to claim 13 wherein said biomolecule is a
peptide, protein, amino acid or a nucleic acid.
15. The method according to claim 1 wherein the amount of said
compound coated onto the metal nanoparticles is less than 70% of
the minimum amount required for full coverage of said metal
nanoparticles with said compound.
16. The method according to claim 15, wherein the amount of said
compound coated onto the metal nanoparticles is more than 30% of
the minimum amount required for full coverage of said metal
nanoparticles with said compound.
17. The method according to claim 1, wherein said compound is
provided with at least one functional group, prior to the step v)
of contacting said metal particles obtained in step iv) with said
compound, to thereby conjugate said compound to the metal
nanoparticles.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 14/376,503, filed Aug. 4, 2014 which is the U.S. National Phase
under 35 U.S.C. .sctn.371 of International Application
PCT/EP2013/053103, filed Feb. 15, 2013, which claims priority to GB
1202631.6, filed Feb. 16, 2012 and U.S. Provisional Application No.
61/599,455, filed Feb. 16, 2012.
FIELD OF THE INVENTION
[0002] The present invention relates to methods of measuring
interaction between a first and a second molecule, for example a
protein and an antibody, by conjugation of one of these molecules
with nanoparticles, and measuring the interaction between the first
and second molecule via changes in the optical properties of the
nanoparticles. The present invention further relates to methods of
coating nanoparticles.
BACKGROUND OF THE INVENTION
[0003] Metal nanoparticles coated with molecules such as proteins
can be used to determine binding events by monitoring a change in
optical properties of the particles, such as by Localized Surface
Plasmon Resonance (LSPR) sensing. In the common scheme a
recognition interface is constructed on the metal nanostructure.
The specific binding of an analyte to said recognition interface is
converted into an optical signal, e.g. a change in absorbance
(wavelength, intensity) which is detected and analyzed.
[0004] 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.
[0005] In principle LSPR can be used in the detection of
antibody-ligand interactions, receptor-ligand interactions,
enzyme-ligand binding and antibody-antigen association-dissociation
kinetics.
[0006] In practice, however it is observed that such methods where
nanoparticles are used in solution often do not attain the required
accuracy.
[0007] Thus, there remains a need in the art to provide methods
which allow the accurate determination of interactions between
molecules using nanoparticles.
SUMMARY OF THE INVENTION
[0008] The present invention relates to methods of measuring
interaction between a first and a second molecule by conjugation of
one of these molecules with nanoparticles, and measuring the
interaction between the first and second molecule via changes in
the optical properties of the nanoparticles. The present invention
allows using nanoparticles in quantities which are sufficient for
reliable detection, while avoiding or at least reducing ligand
depletion.
[0009] In a first aspect, the present invention provides a method
of determining an interaction between a first and a second molecule
comprising: [0010] a) providing nanoparticles comprising one or
more metals; [0011] b) providing said nanoparticles with one or
more functional groups, or coupling the first molecule to a
molecule comprising a metal binding functionality; [0012] c)
conjugating said first molecule to said nanoparticles, whereby the
amount of said first molecule attached to said nanoparticles is
less than 70%, and preferably between 10% and 70%, of the amount
required for full coverage of said nanoparticles with said first
molecule; [0013] d) incubating said nanoparticles with said second
molecule; [0014] e) monitoring step d) by illuminating said
nanoparticles with at least one excitation light source and
monitoring one or more optical properties of said nanoparticles;
and [0015] f) detecting a change of one or more optical properties
of said nanoparticles wherein said change is a result of the
presence of an interaction between said first molecule and said
second molecule.
[0016] In particular embodiments, the nanoparticles comprise gold,
silver or copper. In certain embodiments, the nanoparticles are
nanorods.
[0017] In particular embodiments, step f) comprises determining an
association-dissociation equilibrium between said first and second
molecule. In certain embodiments, step f) comprises measuring said
one or more optical properties at two or more wavelengths ranging
between 350 and 1000 nm.
[0018] In particular embodiments, step b)) comprises providing
nanoparticles having attached to their surface: [0019] one or more
molecules comprising a metal binding functionality, and a
functional group selected from carboxyl, amino, azido, alkynyl,
carbonyl and hydroxyl; and [0020] one or more molecules comprising
said metal binding functionality and not comprising said functional
group selected from carboxyl, amino, azido, alkynyl, carbonyl and
hydroxyl.
[0021] In certain embodiments, step c) comprises: [0022] c1)
optionally, selecting a suitable pH and ionic strength for
conjugation of said first molecule with said nanoparticles via a
buffer test; [0023] c2) determining the amount of said first
molecule needed for conjugation of said first molecule to said
nanoparticles; [0024] c3) conjugation of said first molecule to
said nanoparticles, based on the information obtained in step c2)
and optionally c1).
[0025] In further embodiments, step c2) comprises a concentration
titration of said nanoparticles with said first molecule,
optionally at the pH and ionic strength selected in step c1). In
particular embodiments, step b) comprises coupling said first
molecule to a linker molecule having a metal binding functionality
and step c) comprises conjugation of said first molecule to said
nanoparticle via said linker molecule. In further embodiments, the
linker molecule is a mercaptocarboxylic acid.
[0026] In certain embodiments, the first molecule is a protein and
the surface of said nanoparticles is provided with carboxyl groups.
In further embodiments, the present method further comprises
reacting free carboxyl groups on the surface of said nanoparticles
with a carboxyl blocking compound.
[0027] In a further aspect, the present invention provides a method
of coating a metal nanoparticle with a compound, comprising: [0028]
i) providing a liquid composition comprising metal nanoparticles at
least partially coated with cetyl trimethyl ammonium bromide
(CTAB); [0029] ii) adding a thiol-polyethylene glycol to said
composition, thereby obtaining a liquid composition comprising
metal nanoparticles coated with thiol-polyethylene glycol; [0030]
iii) purification of said composition obtained in step ii) by
separating said nanoparticles from free thiol-polyethylene glycol
and free CTAB; [0031] iv) adding a mercaptocarboxylic acid to said
composition obtained in step iii), thereby obtaining a liquid
composition comprising metal nanoparticles coated with
mercaptocarboxylic acid, and [0032] v) contacting said metal
particles obtained in step iv) with said compound.
[0033] In certain embodiments, the liquid composition in steps i),
ii), iii) and iv) is an aqueous composition.
[0034] In a further aspect, the present invention provides a kit
comprising: [0035] a medium comprising a plurality of metal
nanoparticles; [0036] instructions for use of said nanoparticles in
the method of determining an interaction between a first and a
second molecule according to the present invention; [0037]
optionally, said first molecule; and [0038] optionally, said second
molecule.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] The invention will now be described, inter alia with
reference to the accompanying Figures, which are provided by way of
example only and should not be considered to limit the scope of the
present invention.
[0040] FIG. 1A demonstrates a protein titration curve for human
Serum Albumin (HSA) and represents a plot of .DELTA.RU (i.e.
.DELTA.OD(.lamda..sub.max+80)/OD(.lamda..sub.max)) against the
amount of HSA per mL nanoparticle suspension. FIG. 1B shows the
absorbance spectrum of identical nanorods, conjugated with
different amounts of a protein.
[0041] FIG. 2 represents a plot of .DELTA.RU against the amount of
antibody per mL nanoparticle-HSA conjugate suspension.
[0042] FIG. 3 represents a plot of .DELTA.RU against the amount of
BSA_MUDA added to a suspension of mPEG-SH coated gold nanorods.
[0043] FIG. 4 shows a conjugation between a first molecule (1) and
a nanoparticle (2) according to a particular embodiment of the
present invention.
[0044] FIG. 5 shows a conjugation between a first molecule (1) and
a nanoparticle (2) according to a particular embodiment of the
present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0045] The present invention will be described with respect to
particular embodiments but the invention is not limited thereto but
only by the claims. Any reference signs in the claims shall not be
construed as limiting the scope thereof.
[0046] As used herein, the singular forms "a", "an", and "the"
include both singular and plural referents unless the context
clearly dictates otherwise.
[0047] The terms "comprising", "comprises" and "comprised of" as
used herein are synonymous with "including", "includes" or
"containing", "contains", and are inclusive or open-ended and do
not exclude additional, non-recited members, elements or method
steps. The terms "comprising", "comprises" and "comprised of" when
referring to recited components, elements or method steps also
include embodiments which "consist of" said recited components,
elements or method steps.
[0048] Furthermore, the terms first, second, third and the like in
the description and in the claims, are used for distinguishing
between similar elements and not necessarily for describing a
sequential or chronological order, unless specified. It is to be
understood that the terms so used are interchangeable under
appropriate circumstances and that the embodiments of the invention
described herein are capable of operation in other sequences than
described or illustrated herein.
[0049] The term "about" as used herein when referring to a
measurable value such as a parameter, an amount, a temporal
duration, and the like, is meant to encompass variations of +/-10%
or less, preferably +/-5% or less, more preferably +/-1% or less,
and still more preferably +/-0.1% or less of and from the specified
value, insofar such variations are appropriate to perform in the
disclosed invention. It is to be understood that the value to which
the modifier "about" refers is itself also specifically, and
preferably, disclosed.
[0050] The recitation of numerical ranges by endpoints includes all
numbers and fractions subsumed within the respective ranges, as
well as the recited endpoints.
[0051] All documents cited in the present specification are hereby
incorporated by reference in their entirety.
[0052] Unless otherwise defined, all terms used in disclosing the
invention, including technical and scientific terms, have the
meaning as commonly understood by one of ordinary skill in the art
to which this invention belongs. By means of further guidance,
definitions for the terms used in the description are included to
better appreciate the teaching of the present invention. The terms
or definitions used herein are provided solely to aid in the
understanding of the invention.
[0053] As used herein, the term "localized surface plasmon
resonance" or "LSPR" relates to methods which detect changes at or
near the surface of metal nanoparticles. Typically, these changes
are detected by detecting changes in one or more optical properties
of the particles. When the metal surfaces of the nanoparticles are
excited by electromagnetic radiation, they exhibit collective
oscillations of their conduction electrons, known as localized
surface plasmons (LSPs). When excited in this fashion, 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.
[0054] As used herein, 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. In LSPR changes
in refractive index may be detected through monitoring changes in
the absorbance. Upon illumination of a sample, changes in the LSPR
extinction band cause changes in the intensity and/or the
wavelength of maximum absorbance.
[0055] The term "colloid" refers to a fluid composition of
particles suspended in a liquid medium. In representative colloids,
the particles therein are between one nanometer and one micrometer
in size.
[0056] The term "sample" as used herein refers to a fluid
composition, where in certain embodiments the fluid composition is
an aqueous composition. While a skilled person would understand
that any type of sample may be used in the context of the present
invention, non-limiting examples include biological samples,
including patient samples and environmental samples, plasma,
hybridoma supernatants etc.
[0057] The term "C.sub.4-16alkyl", as a group or part of a group,
refers to a hydrocarbyl radical of Formula C.sub.nH.sub.2n+1
wherein n is a number ranging from 4 to 16. Alkyl groups may be
linear, or branched and may be substituted as indicated herein.
When a subscript is used herein following a carbon atom, the
subscript refers to the number of carbon atoms that the named group
may contain. Thus, for example, C.sub.6-12alkyl means an alkyl of 6
to 12 carbon atoms. Examples of alkyl groups are octyl, decyl,
undecyl and its chain isomers.
[0058] The term "C.sub.4-16alkylthiol" refers to HS--R.sup.w,
wherein R.sup.w is C.sub.4-16alkyl. Non-limiting examples of
suitable C.sub.4-16alkylthiol include undecane-1-thiol,
decane-1-thiol or octane-1-thiol.
[0059] The term "azido" refers to --N.sub.3. The term "amino" by
itself or as part of another substituent, refers to --NH.sub.2.
[0060] The term "alkynyl" refers to a branched or unbranched and
cyclic or acyclic unsaturated hydrocarbon group comprising at least
one triple bond. Non-limiting examples of alkyl groups include
ethynyl, propynyl, 1-butynyl, 2-butynyl and the like.
[0061] The term "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.
[0062] Reference throughout this specification to "one embodiment"
or "an embodiment" means that a particular feature, structure or
characteristic described in connection with the embodiment is
included in at least one embodiment of the present invention. Thus,
appearances of the phrases "in one embodiment" or "in an
embodiment" in various places throughout this specification are not
necessarily all referring to the same embodiment, but may.
Furthermore, the particular features, structures or characteristics
may be combined in any suitable manner, as would be apparent to a
person skilled in the art from this disclosure, in one or more
embodiments. Furthermore, while some embodiments described herein
include some but not other features included in other embodiments,
combinations of features of different embodiments are meant to be
within the scope of the invention, and form different embodiments,
as would be understood by those in the art. For example, in the
appended claims, any of the features of the claimed embodiments can
be used in any combination.
[0063] The present invention relates to the use of nanoparticles
for determining the interaction between a first and a second
molecule, more particularly the provision of methods and tools to
increase the accuracy of detection of such interactions. Indeed,
the present inventors have found that the accuracy of detection is
very much dependent on the coating of the nanoparticles.
[0064] In general, the binding of a ligand to a specific receptor
or binding site may be characterized by the equilibrium
dissociation constant (K.sub.d). According to the law of mass
action the K.sub.d of receptor or binding site ligand binding is
dependent on both the association (k.sub.on) and dissociation
(k.sub.off) rates and determined as the ratio of k.sub.off to
k.sub.on.
K.sub.d=k.sub.off/k.sub.on
[0065] Standard methods of analyzing K.sub.d parameter (saturation
binding) typically assume that the concentration of free ligand is
constant during the experiment and the free ligand concentration
does not significantly differ from the total added ligand
concentration. In order to meet this constraint, the receptor or
binding site concentration must be rather low, compared with the
K.sub.d value. However, in some experimental situations, where the
receptors or binding site are present in high concentration and
have a high affinity for the ligand, that assumption is not true
and the free ligand concentration is depleted by binding to the
receptors or binding sites. This is known as "ligand depletion".
Ligand depletion significantly impedes data analysis, and may lead
to inappropriate values being derived for the binding parameters
k.sub.off, k.sub.on and k.sub.d.
[0066] Therefore, when using bioconjugated metal nanoparticle
suspensions for the determination of ligand-receptor interactions,
and more particularly a K.sub.d and/or k.sub.on value, ligand
depletion should be avoided. In practice, this means that less than
10% of the ligand in solution should bind to the receptors or
binding sites. Although ligand depletion may already be reduced by
reducing the concentration of nanoparticles comprising the
receptor, a minimum amount of nanoparticles is required for
reliable detection, the ratio of surface area to volume of
nanomaterials is very high compared to bulk materials (for example
SPR sensors or wells in a microplate) and enables the presentation
of a large number of binding sites per nanoparticle. Therefore,
reducing the nanoparticle concentration is often not sufficient to
adequately reduce ligand depletion as the signal to noise ratio is
decreasing too. The inventors have found a method for determining
interactions between molecules without reducing the nanoparticle
concentration, which maintains an acceptable signal to noise ratio
value and decreases the effects of ligand depletion, thus enabling
the determination of more accurate binding parameters.
[0067] Thus, in a first aspect, the present invention provides a
method for studying potential interactions between a first molecule
and a second molecule with improved accuracy, particularly in terms
of derivation of correct binding parameters. The methods of the
invention comprise the use of nanoparticles which are conjugated
with the first molecule and then contacted with the second
molecule, whereby the interaction between the first and the second
molecule is determined by monitoring a change in optical
properties. Most particularly, the methods comprise the use of
nanoparticles, whereby the amount of the first molecule attached to
the nanoparticles is less than 70% of the amount required for full
coverage of said nanoparticles with the first molecule. Indeed, in
the present methods, the amount of the first molecule attached to
the nanoparticles can be controlled to be less than 70% of the
amount required for full coverage. In particular embodiments, the
methods according to the present invention comprise the steps of:
[0068] a) providing nanoparticles comprising one or more metals;
[0069] b) providing said nanoparticles with one or more functional
groups, or coupling the first molecule to a linker molecule
comprising a metal binding functionality; [0070] c) conjugating
said first molecule to said nanoparticles, wherein the amount of
said first molecule attached to the nanoparticles is less than 70%
of the amount required for full coverage of said nanoparticles with
said first molecule; [0071] d) incubating the nanoparticles with
the second molecule; [0072] e) monitoring step d) by illuminating
said nanoparticles with at least one excitation light source and
monitoring one or more optical properties of said nanoparticles;
and [0073] f) detecting a change of one or more optical properties
of the nanoparticles wherein this change is a result of the
presence of an interaction between the first molecule and the
second molecule.
[0074] In particular embodiments, the first and second molecule are
a member of a specific known or envisaged binding pair or couple.
Thus, the second molecule (or potential cognate ligand) may refer
to a molecule which potentially interacts with the first molecule.
Typically the first molecule and second molecule are both sensing
moieties which are members of a binding couple such as
antigen-antibody, receptor-ligand, enzyme-ligand, sugar-lectin,
receptor-receptor binding agent, and others. In these embodiments,
the methods according to the present invention may serve for
sensing the interaction between the two members of the binding
pair. Sensing moieties of interest 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. Exemplary biomolecules
include, but are not limited to peptides, proteins, amino acids and
nucleic acids, small organic and inorganic molecules, ligands, etc.
In particular embodiments, the first molecule is a protein.
[0075] In particular embodiments, the interaction measured between
the first and second molecule is referred to as "binding". The term
"binding" refers to two molecules associating with each other in a
non-covalent or covalent relationship.
[0076] 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. One of skill in the art will appreciate that
other nanoparticles may also be useful in the present
invention.
[0077] In particular embodiments, the nanoparticles are nanorods.
In further embodiments, the nanorods have an aspect ratio (i.e.
length divided by width) ranging between 1.1 and 10, more
particularly between 1.5 and 5. In certain embodiments, 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.
[0078] The nanoparticles comprise or are made of one or more
metals. In certain embodiments, the nanoparticles used in the
context of the present invention comprise one or more metals
selected from Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Y, Zr, Nb, Mo,
Tc, Ru, Rh, Pd, Ag, Cd, La, Hf, Ta, W, Re, Os, Ir, Pt, Au and/or
Ac. In an embodiment, the nanoparticles comprise a metal selected
from the group comprising Au, Ag, Cu, Ta, Pt, Pd, and Rh. In
certain embodiments, said metal is selected from gold, silver and
copper.
[0079] The nanoparticles provided in step a) of the methods
according to the invention are typically provided as a colloid.
[0080] The solvents suitable for suspending the nanoparticles may
depend on the nature of the nanoparticle surface. For example, the
nanoparticles may be coated with a hydrophobic or hydrophilic
coating. If the nanoparticles are provided with a hydrophobic
coating, the solvent may comprise one or more solvents selected
from toluene, hexane, heptane, pentane, cyclohexane, cyclopentane,
chloroform, etc. If the nanoparticles are provided with a
hydrophilic coating, the solvent may comprise one or more solvents
selected from water, ethanol, butanol, isopropanol, acetone, etc.
In certain embodiments, the particles are suspended in an aqueous
medium.
[0081] In particular embodiments, the colloid comprises the
nanoparticles in such a concentration that the colloid has an
absorbance at .lamda..sub.max between 0.3 and 4, more particularly
between 0.7 and 1.2, wherein .lamda..sub.max is the maximal
absorbance of the nanoparticles between 350 and 1000 nm.
[0082] In the methods of the present invention, the metal
nanoparticles are optionally provided with one or more functional
groups (referred to as step (b) above), preferably selected from
amino, azido, alkynyl, carboxyl, hydroxyl and carbonyl. The
functional groups may be used for conjugating the first molecule to
the nanoparticles (as provided in step c) above) of the present
method, and will be discussed further below.
[0083] The nanoparticles used in the methods of the present
invention are conjugated to a first molecule. It has been found
that the conjugation density is critical to ensure the required
accuracy for optical detection of the interaction between the first
and the second molecule, more particularly in the determination of
association/dissociation kinetics. The methods of the present
invention thus involve controlling the binding density of the first
molecule to the nanoparticles, by conjugating the first molecule to
the nanoparticles such that the nanoparticles are not fully covered
with the first molecule. Thus, in a next step (corresponding to
step c) described above) of the methods of the present invention,
the first molecule is conjugated to the nanoparticles (provided in
step b)), whereby the amount of the first molecule conjugated to
the nanoparticles is less than 70% of the amount required for full
coverage of said nanoparticles with said first molecule. In
particular embodiments, the amount of the first molecule conjugated
to the nanoparticles is more than 10% of the amount required for
full coverage. This allows using nanoparticles in quantities which
are sufficient for reliable detection of interactions between the
first and second molecule, while avoiding or at least reducing
ligand depletion. In certain embodiments, the amount of the first
molecule conjugated to the nanoparticles is more than 15% of the
amount required for full coverage. In certain embodiments, the
amount of the first molecule conjugated to the nanoparticles is
more than 20% of the amount required for full coverage. The term
"full coverage" as used herein refers to the maximal amount of the
first molecule 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 in conditions suitable for coating the
nanoparticles. In certain embodiments, the amount of the first
molecule conjugated to the nanoparticles is less than 85, 80, 75,
70, 65, 60, 55, 50, 45, 40, 35, 30 or 25% of the amount required
for full coverage. In particular embodiments, the amount of the
first molecule conjugated to the nanoparticles is between 10% and
70%, preferably between 20% and 70%, more preferably between 20%
and 60%, and even more preferably between 30 and 50% of the amount
required for full coverage. The optimal amount of the first
molecule may be determined via titration experiments and may depend
on the characteristics of the nanoparticles, such as size and
shape, and the first and/or second molecule. In particular
embodiments, the amount of the first molecule conjugated to the
nanoparticles is between 30 and 50%, preferably between 40 and 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.
[0084] The first molecule is typically adsorbed on/attached
to/coupled to/linked to/bound to the surface of the nanoparticle,
generally referred to herein as "conjugated to", by incubating the
nanoparticles in a solution comprising the first molecule under
conditions which allow the attachment of the first molecule onto
the surface of the nanoparticles. Specifically, the first molecule
may be conjugated to the nanoparticles by any one of a variety of
methods, for example: [0085] I) incubating nanoparticles which are
provided with one or more functional groups with said first
molecule, wherein said one or more functional groups are suitable
to covalently bind the first molecule; [0086] II) coupling the
first molecule to a linker molecule which has a metal binding
functionality, followed by conjugation of the first molecule to the
nanoparticles via the linker molecule. Each of these methods can be
used in a strategy to reduce the number of binding sites that are
presented per nanoparticle.
[0087] Thus, in particular embodiments, the step of functionalizing
the nanoparticles (corresponding to step b) described above) of the
present method comprises providing the surface of nanoparticles
(provided in step a)) with one or more functional groups suitable
to covalently bind the first molecule. In particular embodiments,
said functional groups are selected from carboxyl, amino, azido,
alkynyl, carbonyl or hydroxyl.
[0088] Methods for functionalization of nanoparticles are well
known to the skilled person, and may for example involve attachment
of a linker molecule to the nanoparticle surface, wherein said
linker molecule comprises a first portion linked to the
nanoparticle (e.g. via a metal binding functionality) and a second
portion which is a functional group capable of forming a covalent
bond to the first molecule.
[0089] A variety of linker molecules is known to those of skill in
the art and typically includes bi-functional molecules. Generally,
such linker molecules will comprise a spacer group terminated at
one end with a metal binding functionality and at the other end a
functional group capable of covalently binding the first
molecule.
[0090] 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. The spacer group is
preferably as short as possible, because it has been observed that
the optical detection of the interaction of a biomolecule with a
second molecule improves with reduced distance of the interaction
to the nanoparticle surface. However, a spacer group which is too
short may be insufficient to stabilize the nanoparticles in
suspension. For optimal results, the spacer group preferably
comprises a hydrocarbon chain with 6 to 18 and preferably 6 to 16
carbon atoms, for example 11 carbon atoms.
[0091] Potential functional groups capable of covalently binding
the first molecule include nucleophilic functional groups (amines,
alcohols, thiols, azides, hydrazides), electrophilic functional
groups (alkynes, carboxyl, aldehydes, esters, vinyl ketones,
epoxides, isocyanates, maleimides), functional groups capable of
cycloaddition reactions, forming disulfide bonds, or binding to
metals. Specific examples include primary and secondary amines,
hydroxamic acids, N-hydroxysuccinimidyl esters,
N-hydroxysuccinimidyl carbonates, oxycarbonylimidazoles,
nitrophenylesters, trifluoroethyl esters, glycidyl ethers,
vinylsulfones, and maleimides.
[0092] Specific linker molecules that may find use in the subject
bifunctional molecules include compounds such as mercaptocarboxylic
acids such as 11-mercaptoundecanoic acid,
11-[2-(2-azido-ethoxy)-ethoxy]undecane-1-thiol, azidobenzoyl
hydrazide,
N-[4-(p-azidosalicylamino)butyl]-3-[2'-pyridyldithio]propionamid),
bis-sulfosuccinimidyl suberate, dimethyladipimidate,
disuccinimidyltartrate, INI-maleimidobutyryloxysuccinimide ester,
N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N-succinimidyl
[4-azidophenyl]-1,3'-dithiopropionate, N-succinimidyl
[4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl
4-[N-maleimidomethyl]cyclohexane-1-carboxylate,
3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester
(SPDP), 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid
N-hydroxysuccinimide ester (SMCC), and the like.
[0093] In particular embodiments, the one or more functional groups
provided on the nanoparticle surface comprise 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. Accordingly, in particular
embodiments, the nanoparticles are coated with one or more (linker)
molecules comprising a carboxyl group and a metal binding
functionality. In certain embodiments, the metal binding
functionality is a sulfhydryl. Sulfhydryl moieties strongly bind to
metal surface, particularly to gold surfaces. In further
embodiments, the nanoparticles are at least partially coated with a
mercaptocarboxylic acid. The sulfhydryl moiety of the
mercaptocarboxylic acid can form a (coordination) bond with a metal
atom of the nanoparticle surface, while the carboxyl moiety can be
used to bind to molecules such as proteins. In particular
embodiments, the mercaptocarboxylic acid is a molecule of formula
(I):
##STR00001##
wherein n is an integer from 6 to 16. In particular embodiments the
mercaptocarboxylic acid is 11-mercaptoundecanoic acid.
[0094] In particular embodiments, one or more functional groups
provided on the nanoparticles allow a coupling mechanism as used in
Click Chemistry. For example, 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. Accordingly, in particular
embodiments, the nanoparticles are at least partially coated with a
(linker) molecule of formula (IIa), (IIb), (IIIa) and/or
(IIIb):
##STR00002##
wherein na, nb, ng and ni are independently an integer from 6 to 16
and nh and nj are independently an integer from 1 to 5.
[0095] An azide functional group further provides the possibility
of Staudinger ligation. Staudinger ligation typically involves
reaction between an azide moiety with a phosphine or phosphate
moiety. Accordingly, in certain embodiments, the nanoparticles are
at least partially coated with a molecule of formula (IIa) or (IIb)
as described above, wherein na and ng are independently an integer
from 6 to 16.
[0096] In particular embodiments, the first molecule may as such
not comprise a functional group suitable for covalently binding the
functional groups provided on the nanoparticles. The methods
according to the present invention may therefore comprise a further
step of providing at least one functional group to the first
molecule, prior to conjugation of the first molecule to the
nanoparticles.
[0097] If the first molecule is conjugated to the nanoparticles via
the functional groups as described above, the amount of functional
groups provided on the nanoparticle surface determines the maximal
amount of the first molecule that can be conjugated to the
nanoparticles. Thus, by limiting the amount of functional groups,
it can be ensured that less than full coverage of the nanoparticles
by the first molecule is obtained, as required in the methods of
the present invention. In particular embodiments this is ensured by
contacting the nanoparticle with different linker molecules, one
carrying the reactive functional group, the other not carrying the
reactive functional group, such that the nanoparticle is coated
with a mixture of reactive and non-reactive linkers. Thus, in
particular embodiments, the nanoparticles used in the methods of
the present invention are generated such that they comprise [0098]
one or more linker molecules comprising a functional group and a
metal binding functionality; and [0099] one or more molecules
comprising said metal binding functionality and not comprising said
functional group. These one or more molecules are herein also
referred to as "non-linker molecules".
[0100] The metal binding functionality ensures that the linker and
non-linker molecules can be attached to the metal nanoparticle
surface, while the functional group of the linker ensure that the
first molecule can be covalently bound to the nanoparticles. As the
non-linker molecules do not comprise the functional group,
attachment of a sufficient amount of these molecules to the
nanoparticle surface ensures that the amount of proteins conjugated
to the nanoparticles is less than 70% of the amount required for
full coverage of said nanoparticles with that protein.
[0101] Such a coating may be obtained by exposing the nanoparticles
with a mixture of the linker and non-linker molecules.
Alternatively, the coating may be obtained by fully or almost fully
coating the nanoparticles with linker molecules, and partially
exchanging the coating with non-linker molecules, or vice versa.
Thus, the selection of the linker comprising non-functional groups
is ideally selected such that a) it can be partially exchanged with
a linker with a reactive functional group and b) it does not
interfere with later reactions.
[0102] Examples of suitable linker molecules are described above.
The one or more non-linker molecules are preferably structurally
similar to the linker molecule. In certain embodiments, the
non-linker molecules are identical to the linker molecule, except
in that they lack the functional group. This typically results in a
similar affinity and exchange characteristics of the linker and
non-linker molecules to the nanoparticle surface. In certain
embodiments, the non-linker molecules have a reduced length
compared to the linker molecule. This reduces steric hindrance by
the non-linker molecules upon reaction of the functional group of
the linker molecules with the first molecule. However, in
particular embodiments, the non-linker molecule(s) has/have a
length similar to or greater than the linker molecule. This can
further by the stability of the coating.
[0103] In particular embodiments, the non-linker molecule(s)
comprise one or more functional groups which improve other
characteristics of the nanomaterial such as solubility and/or
stability. In particular embodiments, the presence of the
non-linker molecules improves the stability of the nanoparticle
suspensions. For example, if the non-linker molecules comprise
sulfate, hydroxyl or polyethyleneglycol (PEG) moieties, the
stability of the nanoparticle colloids in aqueous media can be
improved.
[0104] In certain embodiments, the functional group of the linker
molecule(s) is selected from carboxyl, azido, alkynyl, amino,
carbonyl and hydroxyl. In certain embodiments, the functional group
is selected from carboxyl, azido and alkynyl. In particular
embodiments, the functional group is a carboxyl. In certain
embodiments, said metal binding functionality is a sulfhydryl. In
certain embodiments, the one or more linker molecules comprise a
mercaptocarboxylic acid of formula (I) above such as
11-mercaptoundecanoic acid, and the one or more non-linker molecule
is a compound of formula (IV) and/or (V):
##STR00003##
wherein R.sup.1 and R.sup.2 are independently selected from
sulfate, hydroxyl, hydrogen or methoxy; wherein nc is such that the
molecular weight of compound (IV) is between 100 Da and 10 kDa,
more particularly between 100 Da and 1 kDa; and nd is an integer
from 6 to 16.
[0105] In specific embodiments, nc is an integer from 1 to 10.
Typically, for the mixed monolayers the linker molecule is chosen
to be relatively shorter, but provided the linker is sufficiently
flexible (e.g. PEG), longer linkers can be used.
[0106] 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. The required
amount of the first molecule to reach the desired coverage may be
found by a titration experiment (see further). However, the
unreacted functional groups still present on the nanoparticle
surface area may cause nonspecific binding and a reduced stability
of the nanoparticle conjugate, for example during purification and
towards buffers. Accordingly, in particular embodiments, the
non-reacted functional groups present on the nanoparticles are
reacted with (an excess of) a blocking reagent after conjugation of
the nanoparticles with the first molecule. The blocking reagent
reacts with the remaining unreacted functional groups present on
the nanoparticles, thereby preventing nonspecific binding.
[0107] Alternatively, less than full coverage may also be obtained
by blocking a certain fraction of the functional groups provided on
the nanoparticles with a blocking reagent, followed by reacting the
non-blocked functional groups with (an excess of) the first
molecule. To determine the optimal amount of blocking reagent
required for blocking a certain part of the functional groups
provided on the nanoparticles, a concentration titration may be
performed.
[0108] 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 embodiments, 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 sterical
hindrance. Solubility of the conjugated nanoparticles in polar
solvents may be increased by providing blocking reagents comprising
one or more ethylene glycol moieties, and/or providing blocking
reagents comprising one or more polar functional groups such as a
sulfate, hydroxyl or methoxy.
[0109] If the functional group is a carboxyl, then the blocking
reagent is a carboxyl blocking reagent. The carboxyl blocking
reagent may comprise an amine group so as to react with the
remaining activated carboxyl groups. In particular embodiments, the
(carboxyl) blocking reagent is a compound of formula (VI) or
(VII)
##STR00004##
wherein R.sup.3 and R.sup.4 are independently selected from
sulfate, hydroxyl, hydrogen and methoxy; ne is such that the
molecular weight of compound (VI) is between 89 Da and 10 kDa,
preferably between 1 kDa and 10 kDa, for example 1 kDa; and nf is
such that the molecular weight of compound (IV) is between 30 Da
and 500 Da. In particular embodiments, nf is an integer from 1 to
16, more particularly from 1 to 8.
[0110] As described above, the choice of blocking reagent may
depend on various factors such as the substances that shall be
tested with the protein conjugated nanomaterial. In particular
embodiments, the carboxyl blocking reagent is selected from Bovine
Serum Albumin (BSA), Ovalbumin, and an amino polyethylene glycol of
formula (VI) as described above. These blocking reagents are
particularly useful if the first molecule is Human Serum Albumin
(HSA). In certain embodiments, any functional groups provided on
the nanoparticles may be activated prior to reaction with the first
molecule. If the functional group is a carboxyl, the carboxyl may
be activated using one or more carboxyl activating groups. Useful
carboxyl activating groups include, but are not limited to,
carbodiimide reagents. In particular embodiments, activation of the
carboxyl groups comprises addition of a N-hydroxysuccinimide (NHS)
such as sulfo-NHS together with a coupling reagent such as
ethyl(dimethylaminopropyl) car carbodiimide (EDC) or
dicyclohexylcarbodiimide (DCC), preferably EDC. In certain
embodiments, activation of the carboxyl groups comprises addition
of sulfo-NHS together with EDC. This is herein also referred to as
"sulfo-NHS/EDC coupling".
[0111] Alternative carboxyl activating groups include phosphonium
reagents such as benzotriazolyloxy-tris-(dimethylamino) phosphonium
hexafluorophosphate (BOP) and the like, uronium or carbonium
reagents such as O-(benzotriazol-1-yl)-N,N,N',
N'-tetramethyluronium hexafluorophosphate (HBTU),
N-hydroxy-succinimide (NHS),
benzotriazol-1-yl-oxy-tripyrrolidinophosphonium hexafluorophosphate
(PyBOP) and the like; kethoxycarbonyl-2-ethoxy-1,2-dihydroqunoline
(EEDQ); I-methyl-2-chloropyridinium iodide (Muikaiyama's reagent)
and the like.
[0112] If the functional group is an azide, 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 which do not significantly interact
with the second molecule.
[0113] An alternative method for coupling the first molecule to the
nanoparticles is by coupling the first molecule to a linker
molecule which has a metal binding functionality in step b),
followed by conjugation of the first molecule to the nanoparticles
via the linker molecule in step c)(method II referred to above).
The use of a linker with a metal binding functionality eliminates
the need to provide the nanoparticles with functional groups for
binding the first molecule. The first molecule is then conjugated
to the nanoparticles via the linker molecule, more particularly via
the metal binding functionality of the linker molecule. This is
obtained by incubating the nanoparticles with (an excess of) the
first molecule with the linker molecule attached thereto. In
particular embodiments, the linker molecule comprises a carboxyl
group and a metal binding functionality. The carboxyl group allows
binding of the linker molecule to amine functions present on
proteins. In certain embodiments, the linker molecule is a
mercaptocarboxylic acid, for example mercaptoundecanoic acid.
[0114] In certain embodiments, the metal nanoparticles, e.g. gold
nanoparticles, are coated with a thiol-PEG of formula (IV) as
described above, prior to incubation with (an excess of) the first
molecule with the linker molecule attached thereto. The thiol-PEG
molecules are then at least partially exchanged for the first
molecule, via the linker molecule. In particular embodiments, the
use of nanoparticles coated with neutral thiol-PEG molecules such
as methoxyl-PEG-thiol is particularly useful to prevent
nanoparticle agglutination during conjugation with the first
molecule. Indeed, for some proteins which are highly positively
charged at pH 6-8, the use of the EDC/Sulfo-NHS coupling method on
carboxyl-functionalized nanomaterials which are negatively charged
can be challenging due to agglutination of the nanoparticles.
[0115] In particular embodiments, the step of conjugating said
first molecule to said nanoparticles, includes the step of
determining the amount of the first molecule required for obtaining
the desired coverage of the nanoparticles. In further embodiments,
step c) of the present method comprises: [0116] c1) optionally,
selecting a suitable pH and ionic strength for conjugation of said
first molecule with said nanoparticles via a buffer test; [0117]
c2) determining the amount of said first molecule needed for
conjugation of said first molecule to said nanoparticles; [0118]
c3) conjugation of said first molecule to said nanoparticles, based
on the information obtained in step c2) and optionally c1).
[0119] Step c2) is typically performed by a concentration
titration, wherein the nanoparticles are mixed with different
amounts of protein and analyzed. The titration may be performed at
the pH and ionic strength selected in step c1). The titration may
be monitored via the measurement of the absorbance of the
nanoparticles, preferably at two or more wavelengths.
[0120] In particular embodiments, step c2) comprises addition of
different amounts of the first molecule to a fixed amount of
nanoparticles and recording of absorbance spectra. Then, the ratio
OD(.lamda.max+80)/OD(.lamda.max) (.DELTA.RU) may be plotted versus
the amount of first molecule added. The conjugation of the first
molecule to the nanoparticles leads to a difference in refractive
index around the nanoparticles and thereby to a redshift of the
Amax that can be detected by reading an absorbance spectrum. With
increasing amount of the first molecule conjugated to the
nanoparticles, the redshift also increases. After reaching the
maximum amount of first molecule that can be conjugated to the
nanoparticles the spectrum doesn't change anymore resulting in a
plateau in the plot of OD(.lamda.max+80)/OD(.lamda.max) (.DELTA.RU)
versus the amount of first molecule. The lowest amount of protein
on the plateau is the minimum amount of first molecule needed to
fully cover the nanoparticle surface with the first molecule (or at
least the LSPR sensitive part of the nanomaterial). In order to
obtain a lower number of binding sites than what corresponds to
full coverage of the nanoparticles with the first molecule, a lower
amount of first molecule than the maximal amount can be used for
conjugation. The optimal amount of first molecule to use for
conjugation is a compromise between two tendencies, to ensure
optimal sensitivity. Usually the ideal amount of protein to use is
the amount that corresponds to 30-60% of the plateau.
[0121] If the nanoparticles are provided as a colloid, the colloid
is preferably purified after conjugation of the first molecule to
the nanoparticles, particularly when the conjugation of the first
molecule to the nanoparticles occurs by incubating the
nanoparticles in a solution comprising an excess of the first
molecule. The excess of first molecules which are not bound onto
the nanoparticles are preferably removed after conjugation. This
may be obtained by one or more cycles of precipitation and
resuspension of the nanoparticles as known by the skilled
person.
[0122] After having obtained the nanoparticles suitably coated with
the first molecule, in a next step (step d) of the present methods,
the nanoparticles are incubated with the second molecule.
Typically, the second molecule is provided in a (relatively)
purified form in a fluid composition, which may be an aqueous
composition and/or a buffer. In particular embodiments, the second
molecule may be present in a biological sample.
[0123] Incubation of the nanoparticles with the second molecule
allows interaction of the second molecule with the first molecule,
which is conjugated to the nanoparticles. The proximity of the
second molecule to the nanoparticles changes the dielectric medium
surrounding the nanoparticles, which typically leads to changes of
certain optical properties of the nanoparticle, such as the
absorbance. Measuring one or more of these properties can therefore
provide information of the interactions between the first and
second molecule.
[0124] Accordingly, a further step (step (e) referred to above) of
the presently claimed methods comprises monitoring the interaction
between the first and second molecule as they are contacted (in
step d) by illuminating the nanoparticles with at least one
excitation light source and monitoring one or more optical
properties of said nanoparticles. The light source typically emits
light or radiation at one or more wavelengths between 220 and 1000
nm. In particular embodiments an excitation light source is used
which emits light or radiation comprising between approximately 1
nanowatt and 100 watts of power. In more particular embodiments the
excitation light source is a (xenon) flash lamp or a laser.
[0125] The monitoring of the optical parameters is used to detect a
change therein, e.g., a change in absorption at a given wavelength.
Any convenient optical parameter may be assessed or monitored in
this step, where representative parameters include, but are not
limited to: absorbance, refractive index, absorption, scattering,
fluorescence, luminescence and the like. The optical parameter may
be monitored using any convenient device and protocol, where
suitable protocols are well known to those in the art. The presence
or absence of a change in the optical parameter is then used to
make a determination of whether or not the first molecule interacts
with a second molecule, which in particular embodiments is used to
provide an indication of the presence of an analyte of interest
(second molecule) in a sample.
[0126] Accordingly, in a further step (step (f) referred to above)
of the methods of the present invention, a change of one or more
optical properties of said nanoparticles is detected. This change
is a result of the presence of an interaction between the first
molecule and the second molecule. Indeed, where the first and the
second molecule interact, this will be detected as the optical
properties of the nanoparticle will change. Where there is no
interaction between the first and the second molecule, no change in
optical properties will be detected.
[0127] 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.
[0128] Thus, as used herein, the term "detecting" means to
ascertain a signal (or a change therein), either qualitatively or
quantitatively.
[0129] In particular embodiments, the one or more optical
properties of the nanoparticles are measured at two or more
wavelengths between 220 and 1000 nm, preferably between 350 and
1000 nm. Measurement at two or more wavelengths allows obtaining
more accurate data. In particular embodiments, these wavelengths
are discrete wavelengths within that range.
[0130] The methods of the present invention typically further
comprise the step of analyzing the detected signal or change in
signal and determining a particular property of the first or second
molecule or of their interaction, based thereon.
[0131] In particular embodiments, the step of determining a change
of one or more optical properties of said nanoparticles comprises
determining the dissociation constant (K.sub.d) or binding or
association constant (K.sub.a) for the association-dissociation
equilibrium between the first and second molecule. The
association-dissociation equilibrium can be represented by equation
1:
M1+M2.revreaction.M1M2 (equation 1)
wherein M1M2 represents an association or complex of M1 and M2.
[0132] The reaction is characterized by the on-rate constant
k.sub.on and the off-rate constant k.sub.off. In equilibrium, the
forward binding transition M1+M2.fwdarw.M1M2 are balanced by the
backward unbinding transition M1M2.fwdarw.M1+M2. Assuming a
first-order reaction, that is,
k.sub.on[M1][M2]=k.sub.off[M1M2] (equation 2)
where [M1], [M2] and [RL] represent the concentration of unbound
free M1, the concentration of unbound free M2 and the concentration
of M1M2 complexes, respectively. The association constant K.sub.a
and dissociation constant K.sub.d are defined by:
K.sub.a=1/K.sub.d=k.sub.on/k.sub.off=[M1M2]/([M1][M2]) (equation
3)
[0133] Accordingly, if the concentrations of M1, M2 and M1M2 at
equilibrium are known, K.sub.a and K.sub.d can be calculated.
Alternatively, K.sub.a and K.sub.d may be calculated from a binding
isotherm (see further).
[0134] From the association or dissociation constant, more
information such as the association energy may be obtained.
[0135] In a further aspect, the present invention provides the use
of the present method of determining an interaction between a first
and a second molecule for preparing binding isotherms for binding
of said second molecule to said first molecule, thereby assessing
the binding affinity, binding constant K.sub.a or dissociation
constant K.sub.d of said second molecule for said first molecule
from said binding isotherm.
[0136] A binding isotherm may be constructed from the detected
changes in the optical properties such as the refractive index or
absorbance and the amount (or concentration) of second molecule
added to the nanoparticles.
[0137] The methods of the present invention are also of interest in
the context of screening methods. Thus in particular embodiments
the present invention provides screening methods wherein detection
is performed according to the present invention. In further
embodiments, the methods are high-throughput screening methods,
more particularly methods which are at least in part carried out in
a high-throughput screening device.
[0138] The term "screening" refers to determining the presence of
something of interest, e.g., an analyte, an occurrence, etc. As
such the methods according to the present invention may be used to
screen a sample for the presence or absence of one or more target
analytes in the sample. As such, the invention provides methods of
detecting the presence of one or more target analytes in a sample.
In addition, the subject methods may also be used to screen for
compounds that modulate the interaction of a given specific binding
member pair. The term modulating includes both decreasing (e.g.
inhibiting) and enhancing the interaction between the two
molecules. For example, where the colloid displays a first member
of a binding pair and the colloid is contacted with the second
member in the presence of a candidate agent, the effect of the
candidate agent on the interaction of the binding member pairs can
be evaluated or assessed.
[0139] In yet a further aspect the present invention relates to
tools for carrying out the methods of the present invention. More
particularly, the tools include nanoparticles to which a first
molecule has been conjugated. More particularly, the nanoparticles
are conjugated such that there is not full coverage of the
nanoparticles with the first molecule. In particular embodiments,
the coverage is less than 70%, less than 60%, particularly between
30 and 50%.
[0140] The nanoparticles of the present invention can be obtained
as described above by conjugating them with less than 70% of the
amount required for full coverage of the nanoparticles with the
first molecule.
[0141] The invention further provides kits for carrying out the
methods of the present invention. More particularly the kits for
carrying out the methods of the present invention comprise [0142] a
medium comprising a plurality of metal nanoparticles; [0143]
instructions for use of said nanoparticles in the methods according
to the present invention; [0144] optionally, said first molecule;
and [0145] optionally, said second molecule.
[0146] In particular embodiments, said nanoparticles are provided
with functional groups, preferably selected from carboxyl, amino,
azido, carbonyl and hydroxyl.
[0147] The kits of the present invention optionally also include
solvents, buffers and/or stabilizers. The kits optionally also
include one or more linker and/or non-linker molecules as described
herein.
[0148] In particular embodiments, the nanoparticles are gold
nanorods. Many methods for manufacturing gold nanorods are known to
the skilled person. Typically, these methods involve the use of
gold nanoparticle seeds and cetyl trimethyl ammonium bromide (CTAB)
as a coordinating molecule, and result in nanoparticles which are
fully coated with CTAB. However, in the methods of the present
invention, particles fully coated with CTAB may not be suitable for
conjugation to the first molecule. Therefore, an exchange of the
CTAB coating to a coating of different molecules, such as
mercaptocarboxylic acids, may be required. However, such an
exchange typically requires long reaction times and/or various
phase transfers between polar and non-polar phase. The inventors
have found optimized coating methods which are particularly
suitable for obtaining nanoparticles conjugated with a first
molecule. These methods are of general interest for coating
nanoparticles but can also be used in the methods of the present
invention. Thus, in a further aspect, the present invention
provides methods of coating a metal nanoparticle, comprising using
metal nanoparticles with a CTAB coating to obtain nanoparticles
with a mercaptocarboxylic acid coating. In particular embodiments,
the methods of the invention comprise the steps of: [0149] i)
providing a liquid composition comprising metal nanoparticles at
least partially coated with CTAB. Typically, the metal
nanoparticles provided in this step are fully coated with one or
more molecules, wherein the coating comprises CTAB. The coating may
further comprise other molecules, such as
benzyldimethylammoniumchloride. [0150] ii) Adding a
thiol-polyethylene glycol to said composition, thereby obtaining a
liquid composition comprising metal nanoparticles coated with a
thiol-polyethylene glycol. [0151] iii) Purification of said
composition obtained in step ii) by separating said nanoparticles
from free thiol-polyethylene glycol and free CTAB. [0152] iv)
Adding a mercaptocarboxylic acid to said composition obtained in
step iii, thereby obtaining a liquid composition comprising metal
nanoparticles coated with a mercaptocarboxylic acid.
[0153] In contrast with known methods which require hydrophobic
solvents in some steps, all steps of the method of coating a metal
nanoparticle according to the present invention may be performed in
aqueous phase. This significantly reduces potential health and
safety issues and facilitates scaling up the nanoparticle
production. Furthermore, this also results in reduced costs related
to chemicals and waste management. Accordingly, in particular
embodiments, the liquid composition in steps i), ii), iii) and iv)
is an aqueous composition. In particular embodiments, the
mercaptocarboxylic added in step iv) is a compound of formula (I)
described above.
[0154] In particular embodiments, the thiol-polyethylene glycol
added in step ii) is a compound of formula (IV) as described above,
wherein nc is such that the molecular weight of compound (IV) is
between 100 Da and 10 kDa, preferably between 1 kDa and 10 kDa, for
example 5 kDa; and R.sup.1 is C.sub.1-4alkoxy, preferably methoxy
or ethoxy, wherein said C.sub.1-4alkyl is optionally substituted by
one or more groups such as hydroxyl.
[0155] The mercaptocarboxyl acid-coated nanoparticles obtained in
step iv) as described above can be used to conjugate a molecule
thereto. Accordingly, the present invention further provides a
method for conjugating a molecule to a metal nanoparticle,
comprising: [0156] i) providing a liquid composition comprising
metal nanoparticles at least partially coated with CTAB; [0157] ii)
adding a thiol-polyethylene glycol to said composition, thereby
obtaining a liquid composition comprising metal nanoparticles
coated with a thiol-polyethylene glycol; [0158] iii) purification
of said composition obtained in step ii) by separating said
nanoparticles from free thiol-polyethylene glycol and free CTAB;
[0159] iv) adding a mercaptocarboxylic acid to said composition
obtained in step iii), thereby obtaining a liquid composition
comprising metal nanoparticles coated with a mercaptocarboxylic
acid; and [0160] v) contacting said metal particles obtained in
step iv) with said molecule.
[0161] In a further aspect, the present invention provides a method
for conjugating a molecule to a nanoparticle, comprising: [0162] a)
providing nanoparticles comprising one or more metals; [0163] b)
providing said nanoparticles with one or more functional groups, or
coupling the first molecule to a molecule comprising a metal
binding functionality; [0164] c) conjugating said first molecule to
said nanoparticles, whereby the amount of said first molecule
attached to said nanoparticles is less than 70% of the amount
required for full coverage of said nanoparticles with said first
molecule;
[0165] The following examples are provided for the purpose of
illustrating the present invention and by no means are meant and in
no way should be interpreted to limit the scope of the present
invention.
EXAMPLES
A) Preparation of Nanorods Coated by 11-Mercaptoundecanoic Acid
a1) Preparation of CTAB-Coated Nanorods
[0166] The method used for preparing the gold nanorods is similar
to the method as described by Yu and Irudayaraj (Anal. Chem. 2007,
79, 572-579). In short, a seed solution was prepared by adding 0.5
mM hydrogen tetrachloroaurate (HAuCl.sub.4) to 0.1M CTAB, after
which 0.01M NaBH.sub.4 solution is added.
[0167] Then, a growth solution was prepared by adding 42 mM
AgNO.sub.3 and then 1 mM HAuCl.sub.4 to 0.1M CTAB solution.
Immediately after preparing the growth solution, ascorbic acid
solution is added to the growth solution. After change in color,
seed solution is added, thereby generating CTAB-coated gold
nanorods.
a2) Removal of Excess CTAB
[0168] After one week, excess CTAB was removed from the nanorod
suspension by centrifuging the suspension at 10000 g for 30
minutes. Then, the supernatant (which contains most of the excess
CTAB) was removed and the precipitated nanorods in the pellet were
resuspended in (double-distilled) water.
a3) Exchange of CTAB to mPEG-SH
[0169] After removal of excess CTAB, the CTAB coating of the
nanorods was exchanged to a sulfhydryl functionalized methoxy
polyethylene glycol (mPEG-SH, available from Nanocs as "PEG3-0021")
coating. First, a 10 g/L solution of mPEG-SH in (double-distilled)
water was prepared. To this solution the same volume of purified
nanorod suspension was added under stirring. Then, the mixture was
sonicated for 5 minutes, followed by stirring for 12 hours at room
temperature.
a4) Removal of Excess mPEG-SH
[0170] The suspension obtained in step a3) was centrifuged at 8000
g for 30 minutes at 25.degree. C. After centrifugation, the
supernatant was removed and the precipitated nanorods were
resuspended in a (1.times. concentrate) tris/borate/EDTA (TBE)
buffer. The centrifugation and resuspension cycle was repeated
three times. After the last centrifugation step, the nanorods were
resuspended in (1.times. concentrate) TBE buffer.
a5) Exchange of mPEG-SH Coating to MUDA
[0171] For the exchange of the mPEG-SH coating of the nanorods to a
mercaptoundecanoic acid (MUDA) coating, a 60 g/L solution of MUDA
in ethanol was prepared. This solution was added to the nanorod
suspension obtained in step a4) resulting in a ca. 1.2 g/I MUDA
concentration. Then, the suspension was sonicated for 5 minutes,
followed by stirring for two hours. The suspension was then
centrifuged at 5000 g for 30 minutes at 25.degree. C., followed by
removal of the supernatant and resuspension of the precipitated
nanorods in 25 mL of TBE buffer. Then, 60 g/L MUDA solution in
ethanol was added to the nanorod suspension (resulting in a final
MUDA concentration of 1.2 g/I), followed by stirring for 12
hours.
a6) Removal of Excess MUDA
[0172] The nanorod suspension obtained in step a5) was centrifuged
at 5000 g for 30 minutes at 25.degree. C., followed by removal of
the supernatant and resuspension of the precipitated nanorods in
TBE buffer. The cycle of centrifuging and resuspension was repeated
twice. After the last centrifugation step, the pellet was
resuspended in 10 mM MES buffer comprising 0.002% (by volume) Tween
(polysorbate). The suspension was then dialyzed against 10 mM MES
buffer comprising 0.002% (by volume) Tween for one hour (3 L of
buffer per 25 mL of nanorod suspension).
[0173] To obtain nanorods which are coated with a mixture of MUDA
and another molecule, the method as described above can be used,
with the difference that in step a5) a solution containing MUDA and
that other molecule is used.
B) Conjugation of Nanorods with HAS
[0174] The amount of HSA necessary to obtain a full coverage of
nanoparticles was determined by a concentration titration. A
suspension comprising carboxylated (i.e. MUDA-coated) gold nanorods
as described in example A) was provided. The carboxyl groups were
activated using EDC and sulfo-NHS, and the nanorod suspension was
mixed with increasing amounts of HSA in a range of 0 to 200 .mu.g
of protein per mL nanorod suspension.
[0175] Absorbance spectra of these samples were recorded and
.DELTA.RU, which is the change in the ratio
(OD(.lamda..sub.max+80))/(OD.lamda..sub.max), i.e. the ratio of the
optical density at .lamda..sub.max+80 nm (OD(.lamda..sub.max+80)))
and the optical density at the peak value (OD.lamda..sub.max) was
plotted against the amount of HSA per mL of nanoparticle
suspension. The ratio of these optical densities provides
information about the amount of conjugated HSA. A plot of .DELTA.RU
vs. the HSA amount is shown in FIG. 1A, which shows that increased
amounts of added HSA result in an increased change of .DELTA.RU,
which can be understood as an increased amount of conjugated HSA
surrounding the particles. Indeed, the attachment of protein to the
nanomaterial leads to a difference in refractive index around the
nanomaterial and thereby to a redshift of the .lamda..sub.max that
can be detected by reading an absorbance spectrum. The effect of
protein conjugation on the optical density (OD) of a gold nanorod
suspension is shown in FIG. 1B. Higher amounts of conjugated
protein lead to increasing redshifts of the spectrum i.e. shifts to
higher wavelengths.
[0176] FIG. 1A shows that after reaching the maximum amount (100%)
of protein that can be attached to the nanomaterial, the spectrum
does not change anymore resulting in a plateau in the plot of
.DELTA.RU versus the amount of protein.
[0177] The lowest amount of protein on the plateau is the minimum
amount of protein needed to fully cover the nanomaterial with the
protein of interest.
[0178] In a particular embodiment of the present invention, the
optimal amount of protein to use is the amount that corresponds to
30-60% of the plateau. In the present example this corresponds to
9-25 .mu.g/mL protein, as shown in FIG. 1.
[0179] In this range, a sufficiently low amount of protein is
attached to the rods to avoid ligand depletion, and a sufficiently
high amount of protein is attached for providing a significant
redshift of .lamda..sub.max upon binding of a binding partner.
C) Effect of Binding Site Density on K.sub.d Determination
[0180] To study the effect of the amount of binding sites provided
on the nanoparticles on the measured binding properties such as the
dissociation constant (K.sub.d), a carboxylated nanorod suspension
as used in example B) was divided in two groups. The two groups
were conjugated with HSA by activation of the carboxyl groups with
EDC/sulfo-NHS and mixing the suspension with 600 .mu.g/mL HSA
(sample 1) and 18.75 .mu.g/mL HSA (sample 2), respectively. The
unreacted carboxyl groups present on the nanorod surface were
blocked by addition of BSA. In accordance with the results of
Example A), full coverage of the nanorods with HSA was obtained in
sample 1, while only a partial coverage was obtained in sample
2.
[0181] The two solutions were contacted with Anti-Human Serum
Albumin monoclonal antibody (ab18081, available from Abcam.RTM.) in
identical conditions. After an incubation time of 30 minutes,
absorption spectra of the suspensions were recorded.
[0182] FIG. 2 shows the effect of addition of various amounts of
the antibody to the optical properties of the two suspensions at
equilibrium. In all cases, increased antibody addition results in
an increased change of in the ratio of
OD(.lamda..sub.max+80))/OD.lamda..sub.max, indicative of increased
amounts of HSA molecules binding with the antibody.
[0183] From the absorbance data, dose response curves could be
constructed, from which the dissociation constant K.sub.d for the
HSA-antibody association-dissociation equilibrium was calculated.
The results are given in Table 1. With full coverage (sample 1),
the K.sub.d calculated from the experiments strongly deviate from
the correct value as determined for sample 2. This deviation can be
attributed to ligand depletion.
TABLE-US-00001 TABLE 1 Results of K.sub.d determination with
different binding site density Sample [HSA] (.mu.g/mL) K.sub.d (nM)
1 600 99.39 2 18.75 6.413
[0184] Similar results are obtained when using other blocking
agents. Table 2 shows Kd values obtained using methoxy-PEG-amine
(mPEG-NH.sub.2), BSA and ovalbumine. The incubation time was 60
minutes for each blocking agent. The results shown in Table 2
indicate that the obtained values for K.sub.d are equal within the
margin of error.
TABLE-US-00002 TABLE 2 Results of K.sub.d determination with
different blocking agents Amount of blocking Blocking agent agent
used (.mu.g/mL) K.sub.d (nM) mPEG-NH.sub.2 16700 2.5 .+-. 1.6 BSA
200 8.44 .+-. 5.7 Ovalbumine 200 5.22 .+-. 3.3
[0185] A similar batch of gold nanorods was divided in three
groups. The groups were conjugated with HSA by mixing the
suspension with 600 .mu.g/mL HSA (sample 1), 37.5 .mu.g/mL HSA
(sample 2) and 18.75 .mu.g/mL HSA (sample 3), respectively. The
unreacted carboxyl groups present on the nanorod surface were
blocked by addition of BSA. In accordance with the results of
Example B), full coverage of the nanorods was obtained in sample 1,
while only a partial coverage was obtained in sample 2 and 3.
[0186] The three solutions were contacted with Anti-Human Serum
Albumin monoclonal antibody (ab18081, available from Abcam.RTM.) in
identical conditions. After an incubation time of 60 minutes,
absorption spectra of the suspensions were recorded, from which the
dissociation constant K.sub.d for the HSA-antibody
association-dissociation equilibrium was calculated. The results
are given in Table 3. With full coverage (sample 1), the K.sub.d
calculated from the experiments again strongly deviate from the
correct value as determined for sample 2 and 3. Again, this
deviation can be attributed to ligand depletion.
TABLE-US-00003 TABLE 3 Results of K.sub.d determination with
different binding site density Sample [HSA] (.mu.g/mL) K.sub.d (nM)
1 600 252.4 .+-. 47.1 2 37.5 5.5 .+-. 1.25 3 18.75 3.8 .+-. 1
D) MUDA-Functionalization of a Protein and mPEG-SH Exchange
[0187] The first molecule of interest in this example is BSA. BSA
was covalently modified on its primary amine with a thiol-linker,
more particularly MUDA. For this reaction, the carboxyl moiety of
MUDA was activated with EDC and sulfo-NHS.
[0188] An excess of the modified BSA (BSA_MUDA) was then added to a
suspension of purified mPEG-SH coated nanorods, as obtained in step
a4) of example A) described above. This results in a (partial)
exchange of mPEG-SH by BSA_MUDA at the nanorod surface. The
exchange is slow and takes a few hours to several days.
[0189] FIG. 3 shows a concentration titration of mPEG-SH coated
nanorods with BSA_MUDA. Addition of larger amounts of BSA_MUDA
typically leads to an increased exchange of mPEG-SH by BSA_MUDA,
indicated by an increased .DELTA.RU. From measurement of the same
samples after 120 minutes and 3 days, it is clear that longer
exchange times lead to increased exchange.
E) Conjugation Via Click Chemistry or Staudinger Ligation
e1) Click Chemistry
[0190] FIG. 4 shows a conjugation between a first molecule (1) and
a nanoparticle (2) according to a particular embodiment of the
present invention. The first molecule (1) is a protein and the
nanoparticle (2) is a gold nanorod provided with azide functional
groups. In a first step (A), the protein (1) is provided with an
alkyne moiety by reacting an amino moiety of the protein with the
succinimidyl moiety of compound (3). In a second step (B), the
functionalized protein (4) is conjugated to the nanoparticle (2)
via an azide alkyne Huisgen cycloaddition reaction, thereby
providing a nanoparticle conjugate (5).
e2) Staudinger Ligation
[0191] FIG. 5 shows a conjugation between a first molecule (1) and
a nanoparticle (2) according to a particular embodiment of the
present invention. The first molecule (1) is a protein and the
nanoparticle (2) is a gold nanorod provided with azide functional
groups. In a first step (A), the protein (1) is provided with a
triarylphosphine moiety (comprising an ester group situated ortho
to the phosphorus) by reacting an amino moiety of the protein with
the succinimidyl moiety of compound (6). In a second step (B), the
functionalized protein (7) is conjugated to the nanoparticle (2)
via a Staudinger reaction, thereby providing a nanoparticle
conjugate (8).
* * * * *