Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Designs of labels for detection with a surface-selective nonlinear optical
technique.
BACKGROUND OF THE INVENTION
The prior art shows that second harmonic-active moieties, when coupled to a
protein as a label, can
render the protein detectable at an interface by second harmonic generation.
In this case, the
adsorption of a labeled protein at an air-water interface was monitored to
measure the adsorption
curve and surface density of protein. 'The second harmonic-active label was an
oxazole dye derivative
which could he covalently coupled to surface amines or sulfhydryls on the
protein's surface. Single-
molecule labels may not provide enough scattering cross-section and the target
objects - such as cells,
proteins, viruses or nucleic acids - may have only one or several sites
available for labeling and these
are typically at random orientations to each other, further reducing the net
cross-section. Because the
second harmonic labels used to date may not be sufficient to monitor many
processes of interest,
designs of labels with significantly higher hyperpolarizabilities (eg., second
harmonic cross-sections)
are needed.
Relevant portions of cited references are incorporated by reference herein.
DESCRIPTION OF THE INVENTION
The present invention offers a number of designs of second-harmonic active
labels with high
hyperpolarizabilities. Designs are proposed which involve the use of non-
centrosymmetric metallic
particles, oxazole dyes, linear chains of nonlinear-active dyes and solid
scaffolds on which to build
nonlinear active moieties, nanocrystals or nanoparticles. These high-cross-
section
(hyperpolarizability) labels may then be used to label viruses, cells,
proteins, nucleic acids or other
particles, especially biological particles ("bioparticles").
One means of determining whether a particular molecule or particle is a
candidate for use as a
nonlinear-active label is by studying it using second harmonic generation.at
an air-water interface.
For instance, in the case of particles, if the particles assemble at the air-
water interface in a manner
which gives a net orientation of the particles (on a length scale of the
coherence length) the layer of
particles will generate second harmonic light. Another means of doing this is
by measuring a sample
of a suspension of the particles and detecting the hyper-rayleigh scattering.
Yet another means is by
EFISH (Electric-field induced second harmonic generation). EFISH can be used
to determine if a
candidate molecule or particle is nonlinearly active. Electric field induced
second harmonic (EFISH)
is well known in the field of nonlinear optics. This is a third order
nonlinear optical effect, with the
polarization source written as: P(z~(c~3) = x(z~ (-t~3; cy,c~z) : E~'' E'~z.
The effect can be used to measure
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the hyperpolarizabilty of molecules in solution by using a do field to induce
alignment in the medium,
and allowing SHG to be observed. This type of measurement does not require
that the particle
themselves be ordered at an interface, but does require that the particles be
nonlinear active.
In one important aspect of the invention, the use of linkers which couple the
labels to their targets can
be made long enough so that the orientation of the targets at the interface
does not significantly affect
the orientation of the label. Because the intensity of the nonlinear light
generated will depend on the
net orientation of the labels at the interface - and the orientation of the
targets at an interface can be
difficult to control (i.e., the targets may even be randomly oriented at the
interface) - the use of
linkers can separate the labels sufficiently from the targets so that the
orientation of the targets does
not necessarily determine the orientation of the labels, resulting in a net
orientation of labels. In cases
where this is less important, for example with integral membrane proteins in
supported Iipid bilayers
on glass - where the orientation of the membrane protein presented to the
targets is generally uniform
- this aspect of the linkers can be less important. Nevertheless, in many
cases, linkers are necessary
in order to couple the label to the targets.
Certain terms used herein are intended to have the following general
definitions:
1. Complementary: Refers to the topological and chemical compatibility of
interacting surfaces
between two biological components, such as with a ligand molecule and its
receptor (also referred
to in the prior art as: 'molecular recognition'). Thus, the receptor and its
ligand can be described
as complementary, and, furthermore, the contacts' surface characteristics are
complementary to
each other.
2. Biological (Components): These may include any naturally occurring or
modified particles or
molecules found in biology, or those molecules and particles which are
employed in a biological
study. Examples of these include, but are not limited to, a biological cell,
protein, nucleic acids,
antibodies, receptors, peptides, small molecules, oligonucleotides,
carbohydrates, lipids,
liposomes, polynucleotides and others such as drugs, toxins and genetically
engineered protein or
peptide.
3. Ligand: A ligand is a molecule that is recognized by a particular receptor.
Examples of ligands
that can be studied by this invention include, but are not restricted to,
antagonists or agonists for
cell membrane receptors, toxins and venoms, viral epitopes, hormones, hormone
receptors,
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peptides, enzymes, enzyme substrates, cofactors, drugs (e.g. opiates,
steroides, etc.), lectins,
sugars, oligonucleotides, nucleic acids, oligosaccharides, proteins, and
monoclonal antibodies.
4. Receptor: A molecule that has an affinity for a given ligand. Receptors may
be naturally
occurring or rnan-made molecules. Also, they can be used in an unaltered state
or as aggregates
with other species. Receptors may be attached, covalently or noncovalently, to
a binding partner,
either directly or via a specific binding substance. Examples of receptors
which can be employed
by this invention include, but are not limited to, antibodies, cell membrane
receptors, monoclonal
antibodies and antisera reactive with specific antigenic determinants (such as
on viruses, cells or
other materials), drugs, polynucleotides, nucleic acids, peptides, cofactors,
lectins, sugars,
polysaccharides, cells, cellular membranes and organelles. Receptors are
occasionally referred to
in the art as anti-ligand. As the term receptors is used herein, no difference
in meaning is
intended. A "Ligand Receptor Pair" is formed when two macromolecules have
combined through
molecular recognition to form a complex.
Other examples of receptors which can be investigated by this invention
include but are not restricted
to:
a) Microorganism receptors: Determination of ligands which bind to receptors,
such as specific
transport proteins or enzymes essential to survival of microorganisms,
is useful in developing a new class of antibiotics. Of particular value would
be antibiotics against
opportunistic fungi, protozoa, and those bacteria resistant to the
antibiotics in current use.
b) Enzymes: For instance, one type of receptor is the binding site of enzymes
such as the enzymes
responsible for cleaving neurotransmitters; determination of ligands
which bind to certain receptors to modulate the action of the enzymes which
cleave the different
neurotransmitters is useful in the development of drugs which can be
used in the treatment of disorders of neurotransmission.
c) Antibodies: For instance, the invention may be useful in investigating the
ligand-binding site on the
antibody molecule which combines with the epitope of an
antigen of interest; determining a sequence that mimics an antigenic epitope
may lead to the
development of vaccines of which the immunogen is based on one or
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4
more of such sequences or lead to the development of related diagnostic agents
or compounds useful
in therapeutic treatments such as for autoimmune diseases (e.g., by blocking
the binding of the "self'
antibodies).
d) Nucleic Acids: Sequences of nucleic acids may be synthesized to establish
DNA or RNA binding
sequences.
e) Catalytic Polypeptides: Polymers, preferably polypeptides, which are
capable of promoting a
chemical reaction involving the conversion of one or more reactants
to one or more products. Such polypeptides generally include a binding site
specific for at least one
reactant or reaction intermediate and an active functionality
proximate to the binding site, which functionality is capable of chemically
modifying the bound
reactant. Catalytic polypeptides are described in, for example, U.S.
Pat. No. 5,215,899, which is incorporated herein by reference for all
purposes.
f) Hormone receptors: Examples of hormone receptors include, e.g., the
receptors for insulin and
growth hormone. Determination of the ligands which bind with high
affinity to a receptor is useful in the development of, for example, an oral
replacement of the daily
injections which diabetics must take to relieve the symptoms of
diabetes, and in the other case, a replacement for the scarce human growth
hormone which can only
be obtained from cadavers or by recombinant DNA
technology. Other examples are the vasoconstrictive hormone receptors;
determination of those
ligands which bind to a receptor may lead to the development of
drugs to control blood pressure.
g) Opiate receptors: Determination of ligands which bind to the opiate
receptors in the brain is useful
in the development of less-addictive replacements for morphine
and related drugs.
h) Ion channel proteins or receptors, or cells containing ion channel
receptors.
5. Surface-selective: Refers to a non-linear optical technique such as second
harmonic generation or
sum/difference frequency generation in which, by symmetry, only a non-
centrosymmetric surface
(comprising array, substrate, solution, biological components, etc.), is
capable of generating non-
linear light.
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6. Array: Refers to a substrate or solid support on which is fabricated one
type, or a plurality of
types, of biological components in one or a plurality of known locations. This
includes, but is not
limited to, two-dimensional microarrays and other patterned samples. Other
terms in the prior art
which are often used interchangeably for 'array' include: gene chip, gene
array, biochip, DNA
chip, protein chip and microarray.
7. Label: Refers to a nonlinear-active moiety, particle or molecule which can
be attached
(covalently or non-covalently) to a molecule, particle or phase (e.g., lipid
bilayer) in order to
render the latter more nonlinear optical active. The labels are pre-attached
to the molecules or
particles and unbound or unreacted labels separated from the labeled entities
before a
measurement is made.
8. Linleer: A molecule which serves to chemically link (usually via covalent
bonds) two different
objects together. Herein a linker can be used to couple targets to non-linear
active particles or
moieties, targets to nonlinear-active derivatized particles, surface layers to
targets, surface layers
to nonlinear-active particle or moieties, etc. A linker may be a
homobifunctional or
heterobifunctional cross-linker molecule, a biotin-streptavidin couple wherein
the biotin is
attached to one of the two objects and the streptavidin to the other, etc.
Many linkers are
available commercially, for example from Pierce Chemical Inc., Sigma-Aldrich,
Fluka, etc. In
some prior art, the term 'tether', 'spacer' or 'cross-linker' is also used
with the same meaning.
9. Elements: When used with 'array' or 'microarray', the meaning is a specific
location among the
plurality of locations on the array surface. Each element is a discrete region
of finite area formed
on the surface of a solid support or substrate.
10. Nonlinear: Refers herein to those optical techniques capable of
transforming the frequency of an
incident light beam (called the fundamental). The nonlinear beams are the
higher order frequency
beams which result from such a transformation, e.g. second harmonic, etc. In
second harmonic,
sum frequency or difference frequency generation, the nonlinear beams are
generated coherently.
In second harmonic generation (SHG), two photons of the fundamental beam are
virtually
scattered by the interface to produce one photon of the second harmonic. Also
referred to herein
as nonlinear optical or surface-selective nonlinear (optical) or by various
combinations thereof.
11. Target: Refers herein to a particle or molecule to be labeled with a
nonlinear-active moiety, in
order to render said particle or molecule for study by a nonlinear-active
technique at an interface
of interest. Biological targets, for example, may include the following: a
protein, oligosaccharide,
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peptide, nucleic acid, liposome, small molecule, oligonucleotide, liposome, or
biological cell,
liposome, receptor, antibody, antigen, peptide, receptor, drug, enzyme,
ligand, carbohydrate.
12. Attached: Refers herein to biological components which are either prepared
or engineered in-
vitro to be attached to some surface, via covalent or non-covalent means,
including for example
the use of linker molecules; or are naturally part of the surface such as in
the example of
membrane receptors embedded in cell membranes, liposomes, tissues, organs (in-
vitro or in-vivo)
or supported lipid bilayer membranes..
13. Centrosymmetric: A molecule or material phase is centrosyrnmetric if there
exists a point in
space (the 'center') through which an inversion (x,y,z) -3 (-x,-y,-z) of all
atoms is performed that
leaves the molecule or material unchanged. A non-centrosymmetric molecule or
material
lacks this center of inversion. For example, if the molecule is of uniform
composition and
spherical or cubic in shape, it is centrosymmetric.
14. Nucleic Acid Analog: A non-natural nucleic acid which can function as a
natural nucleic
acid in some way. For example, a Peptide Nucleic Acid (PNA) is a non-natural
nucleic acid
because it has a peptide-like backbone rather than the phosphate background of
natural
nucleic acids. The PNAs can hybridize to natural nucleic acids via base-pair
interactions.
Another example of a Nucleic acid analog can be one in which the base pairs
are non-natural
in some way.
15. Binding Affinity or Affinity: The specific physico-chemical interactions
between binding
partners, such as a probe and target, which lead to a binding complex
(affinity) between them.
The binding reaction is characterized by an equilibrium constant which is a
measure of the
energetic strength of binding between the partners. Specificity in a binding
reaction implies that
probe-target binding only occurs appreciably with specific binding partners -
not any at random.
For example, the protein Immunoglobulin G (IgG) has a specific binding
affinity for protein G
and not for other proteins. In some prior art, the term 'molecular
recognition' is used to describe
the binding affinity between components.
16. Electrically Charged or Electric Charge: Defined herein as net electric
charge on a particle or
molecule, which confers a mobility (velocity) of said particle or molecule in
an electric field. The
net charge could be part of a molecular moiety such as phosphate group on
nucleic acid
backbones, side-chains of amino acid residues in proteins, lipid head groups
in membrane lipids
or cellular membranes, etc. The charge can be positive or negative and would
determine the
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direction of mobility of the particle or molecule if said particle or molecule
is placed in an electric
field of a given orientation (direction of positive to negative electric
potential). The charge can be
non-integer multiples of the fundamental unit of charge (q ~ 1.6 x 10-19 C) or
a fraction of the
fundamental unit of charge - so-called 'partial charges', well known to those
skilled in the art.
17. bipolar: Defined herein as possessing an electric dipole or 'dipole
moment' (no net charge) on a
particle or molecule, which takes the standard definition known to one skilled
in the art: the sum
of all vectors ~, = Q~R where Q is the amount of charge (positive or negative)
at a particular
spatial location (x,y,z in Cartesian coordinates) in the particle or molecule
and R is the vector
which points from an origin of reference (x,y,z) to the net charge Q. If the
sum of these vectors
results in a vector with a non-zero trace (sum of x,y,z components of the
resultant vector), the
particle or molecule possesses a dipole moment and is electrically dipolar.
1 ~. Electrically Neutral: Defined herein as zero net (sum of positive and
negative) electric charge on
a particle or molecule, which would result in no appreciable mobility
(velocity) of said particle or
molecule in an electric field.
19. Hyperpolarizability or Nonlinear Susceptibility: The properties of a
molecule, particle, interface
or phase which allows for generation of the nonlinear light. Typical equations
describing the
nonlinear interaction for second harmonic generation are: atz~(2c~) _
(3:E(~)~E(w) or P~~~(2ee~) _
x~z~:E(co)E(c~) where a and P are, respectively, the induced molecular and
macroscopic dipoles
oscillating at frequency 2w, ~3 and x~2~ are, respectively, the
hyperpolarizability and second-
harmonic (nonlinear) susceptibility tensors, and E(w) is the electric field
component of the
incident radiation oscillating at frequency co. The macroscopic nonlinear
susceptibility x~2~ is
related by an orientational average of the microscopic (3 hyperpolarizability.
For sum or
difference frequency generation, the driving electric fields (fundamentals)
oscillate at different
frequencies (i.e., w, and w2) and the nonlinear radiation oscillates at the
sum or difference
frequency (w1 ~ w2). The terms hyperpolarizability, second-order nonlinear
polarizability and
nonlinear susceptibility are sometimes used interchangeably, although the
latter term generally
refers to the macroscopic nonlinear-activity of a material or chemical phase
or interface. The
terms 'nonlinear active' or 'nonlinearly active' used herein also refer to the
general property of
the ability of molecules, particles, an interface or a phase, to generate
nonlinear optical radiation
when driven by incident radiation beam or beams.
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20. Polarization: The net dipole per unit volume (or area) in a region of
space. The polarization can
be time-dependent or stationary. Polarization is deftned as: f p,(R) dR where
an integration of the
net dipole is made over all volume elements in space dR near an interface.
21. Radiation: Refers herein to electromagnetic radiation or light, including
the fundamental beams
used to generate the nonlinear optical effect, or the nonlinear optical beams
which are generated
by the fundamental.
22. Near-field techniques: Those techniques known in the prior art to be
capable of measuring or
imaging optical radiation on a surface or substrate with a lateral resolution
at or smaller than the
diffraction-limited distance. Examples of near-field techniques (or near-field
imaging) include
NSOM (near-field scanning optical microscopy) whereby optical radiation (from
fluorescence,
second harmonic generation, etc.) is collected at a point very near the
surface.
23. Detecting: Refers herein to methods by which the properties of surface-
selective nonlinear optical
radiation can be used to detect, measure or correlate properties of probe-
target binding reactions
or effects of the binding reactions.
24. Interface: For the purpose of this invention, the interface can be defined
as that region which
generates a nonlinear optical signal.
25. Surface layer: Refers herein to a chemical layer which functionally
derivatizes the suxface of a
solid support. For instance, the surface chemical groups can be changed by the
derivatization
layer according to the particular chemical functionality of the derivatizing
agent. In the case of
solid objects used as 'scaffolds' for creating power nonlinear-active labels
(see below), the solid
surface can be derivatized to produce a different chemical functionality which
can be presented to
nonlinear active moieties or particles, or to targets. For instance, a silica
bead with negatively
charged silanol groups on its surface can be converted to an amine-reactive,
amine-containing,
etc. surface via organosilane reagents.
26. Conjugated: Refers herein to the state in which one particle, moiety or
molecule is chemically
bonded, covalently or non-covalently linked or otherwise attached to a second
particle moiety or
molecule. The second particle, moiety or molecule is often a target, i.e. a
species of interest
which must be labeled for detection by a nonlinear optical technique.
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ORGANIC MOLECULES
It was demonstrated that an oxazole dye 4-[5-methoxyphenyl)-2-
oxazolyl]pyridinium
methanesulfonate (also known as 4PyMP0-MeMs) is strongly second harmonic-
active and
chemically stable at neutral pH (Salafsky and Eisenthal, Chemical Physics
Letters). Furthermore, the
Stokes shift of the fluorescence which results from two-photon absorption is
large so that the second
harmonic beam can readily be separated from the fluorescence. Other dyes in
this family have similar
properties, including but not limited to (J.H. Hall, 1992):
5-(4-methoxphenyl)-2-(4-methoxyphenyl)-2-(4-pyridyl)oxazole
2-(4-methoxyphenyl)-5-(4-pyridyl)oxazole
2-(4-methoxyphenyl)-5-(4-pyridyl)oxadiazole
2-(4-methoxyphenyl)-5-(4-pyridyl)furan
2-(4-pyridyl)-4,5-dihydronapthol[1,2-d]-1,3-oxazole
5-Aryl-2-(4-pyridyl)-4-R-oxazole where R is a hydrogen atom, methyl group,
ethyl group or other
alcyl group.
2-(4-pyridyl)cycloalkano [d] oxazole
2-(4-pyridyl)phenanthreno[9,10-d]-1,3-oxazole
6-Methoxy-4,4-dimethyl-2-(4-pyridyl)indeno[2,1-d]oxazole
4,5-Dihydro-7-methoxy-2-(4-pyridyl)napthol[1,2-d]-1,3-oxazole
These dyes can readily be made into labels, that is, reactive to various
functional groups on targets, by
using synthetic methods known to one skilled in the art. Furthermore, the
following two
commercially available dyes (Molecular Probes, Inc.) can be readily conjugated
- without further
modification- to protein amines [1] or cysteines [2].
[1] 1-(3-(succinimidyloxycarbonyl) benzyl)-4-(5-(4-methoxyphenyl) oxazol-2-
yl)pyridinium
bromide.
[2] 1-(2,3-epoxypropyl)-4-(5-(4- methoxyphenyl)oxazol-2-yl) pyridinium
trifluoromethanesulfonate (PyMPO epoxide).
Other molecules which can, by synthetic means known to one skilled in the art,
be made reactive to
various functional groups on targets include members of the following
families:
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Merocyanines
Stilbenes
W dodicarbocyanines
Hemicyanines
Stilbazims
Azo dyes
Cyanines
Stryryl-based dyes
Methylene blue
Diaminobenzene compounds
Polyenes
Diazostilbenes
Tricyanovinyl aniline
Tricyanovinyl azo
Melamines
Phenothiazine-stilbazole
Polyimide
Sulphonyl-substituted azobenzenes
Indandione-1,3-pyidinium betaine
Fluorescein
Benzooxazole
Perylene
Polymethacrylates
Oxonol
Thiophenes
Bithiophenes
DERIVATIZED PARTICLE LABELS
A solid microparticle or a nanoparticle of size nanometers to microns in scale
including, but not
limited to, a sphere (latex, polystyrene, silica, etc.) or a strip, offers a
surface area which can be
derivatized with a nonlinear-active moiety via chemical or electrostatic means
so that the entire object
has a much higher hyperpolarizability than may be obtained otherwise. For
instance, nonlinear-active
dyes can be assembled on silica bead surfaces via electrostatic interactions
(dye is positively charged,
silica surface is negatively charged) and the entire bead, if derivatized with
target-reactive linkers, can
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then function as a nonlinear active label. If the nonlinear active moieties
are assembled on the solid
surface so that phase interference between moieties is small, the overall
hyperpolarizability will scale
nonlinearly (eg., quadratically) in their number. The solid particle can vary
in shape and its size can
range from nanometers to microns in scale. Linkers which allow attachment to
the target object (e.g.,
cells, viruses, proteins, nucleic acid) can be attached to the particle
surface - at low density if
necessary, for example to prevent multiple attachment points of the label
target to the label - using
commercially available bifunctional linkers. The nonlinear active moieties
will ideally all have the
same orientation, or the same orientation with respect to the solid particle,
for an optimal scattering
cross-section.
Examples of the particles to be used include, but are not limited to,
polystyrene beads and silica
beads, both readily commercially available.
a. Covalent attachment
The solid particles can be surface derivatized using a variety of chemistries
available in the prior art.
Nonlinear-active moieties are covalently coupled either to the solid particles
or to a derivatized layer.
The nonlinear-active moieties themselves can contain linkers for making the
covalent attachment, if
necessary.
For instance, polystyrene beads can be derivatized with dextran, lactose or
amines (the latter case for
example, via chloromethyl groups with ethylenediamine). Silica can be
derivatized using
organofunctional silanes, for example using trichlorosilanes or other
functional silanes (such as
methoxy, amine, or other functional groups), to produce surfaces with a
variety of chemical
functionalities. The surfaces of the derivatized beads can then be reacted
with a nonlinear active
moiety via appropriate chemistry.
b. Electrostatic attachment
Nonlinear active moieties can also be electrostatically bound to a micron- or
nanometer-sized particle
surface. This has been demonstrated in the prior art with charged nonlinear
active moieties using
silica or polystyrene beads and malachite green or oxazole dyes. A charged
nonlinear active moiety,
an organic dye for example, can be oriented at a counter-charged microparticle
surface, thus allowing
for a net hyperpolarizability of the object when using an appropriate
geometry. An example of an
appropriate geometry is a microparticle sphere where the diameter is
approximately the wavelength of
the fundamental light, i.e. from tens of nanometers to microns so that
destructive phase interference
between nonlinear active moieties on opposing faces of the sphere is
minimized. The
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hyperpolarizability of each dye at the spheres's surface, when integrated
across the entire surface of
the sphere of wavelength of light size, is large and positive.
Preferred embodiment:
For instance, a commercially available oxazole dye with functionality for
binding to amines (1-(3-
(succinimidyloxycarbonyl) benzyl)-4-(5-(4-methoxyphenyl) oxazol-2-
yl)pyridinium bromide
(PyMPO-SE) can be coupled to beads with amine or surface groups. Spherical
silica beads 0200 nm
diameter) are derivatized with surface amines using 3-
aminopropyltrimethoxysilane according to
means well known in the prior art. The PyMPO dye is then reacted with the
beads, covalently linking
the dye to the surface of the beads. About 5-10% of the available amines can
be left unreacted with
the dye by varying the reaction conditions. These unreacted amines can then be
covalently coupled to
a heterobifunctional crosslinking agent N(4-Azidosalicylamido)Butyl
3'(2'Pyridyldithio)Propionamide
(available from Pierce Chemical, Inc.). The crosslinker allows covalent
attachment to amines and
sulfhydryls. The sulfhydryl-functional end is available for reaction with
sulfhydryl groups on the
target's surface.
Preferred embodiment:
Silica beads 0200 nm, roughly spherical) are reacted with a low concentration
of 3-
aminopropyltrimethoxysilane or 3-aminooctyltrimethoxysilane so that only ~5-
10% of the surface
silanols become covalently coupled to the silane agent. These amine groups are
then reacted with the
amine-reactive homobifunctional crosslinker Disuccinimidyl glutarate (DSG,
Pierce Chemical) to
create amine-reactive linkers on ~5-10% of the bead surface. The beads are
then incubated with 4-[5-
methoxyphenyl)-2-oxazolyl]pyridinium methanesulfonate (also known as 4PyMP0-
MeMs), a
positively charged dye which binds electrostatically to the charged silanols
on the surface and orients
to some degree. The excess dye is removed from the beads by centrifugation.
The electrostatic
adsorption can be sufficiently high in some cases to immobilize the charged
dye, even in the absence
of a bulk concentration of it. The beads can then be attached covalently to
target objects containing
amines using the amine-reactive tethers. This coupling can produce target
objects containing amines
(cg., proteins, viruses, cells, oligonucleotides, nucleic acids, etc.) coupled
to a strongly nonlinear-
active label.
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Fig. 1 illustrates in (A) two possibilities for an organic nonlinear-active
molecule or moiety. (B)
illustrates some of the various possibilities involving linkers, solid objects
('scaffolds') and non-linear
active components, the components being molecules, particles, proteins or
moieties which are
nonlinear active.
LINEAR CHAINS OF NONLINEAR ACTIVE MOIETIES
Linear chains of nonlinear active moieties which are aligned in a manner to
maximize the overall
scattering cross-section and have functionality for attaching to target
objects would be useful as
nonlinear-active labels. For example, rigid nonlinear active dyes can be
coupled in a head-to-tail
polymeric fashion, with each monomer of dye oriented in the same direction.
For example, dyes such
as the oxazole can be created so that they can covalently connect to each
other in a head-to-tail
fashion, and with a coupling moiety at one end only for attachment to the
target object. For example,
as depicted in Fig. 3, by reacting a nonlinear active molecule containing
functional group (X) with an
excess of a nonlinear-active molecule containing both (X) and (Y) where (Y) is
reactive towards (X),
one could easily construct chains of monomers of the dye under conditions
where (X) and (Y) are
reactive. By synthetic means available to one skilled in the art, one could
easily functionalize
nonlinear-active molecules or moieties with (X) and (Y) groups.
DERIVATIZED NON-CENTROSYMMETRIC, METALLIC NANOCRYSTALS, NANOPARTICLES,
CLUSTERS AND COLLOIDS
Prior art shows that metallic nanoparticles and clusters, ranging from about 1
nm to 25 or more
microns in size, can be derivatized and conjugated to biomolecules for use in
staining for electron
microscopy, x-ray scattering and other applications. Prior art also shows that
non-centrosymmetric
metal nanoparticles can exhibit extremely high hyperpolarizabilities (3,5,9).
Another aspect of the present invention therefore is to use nora-
centr~osynarnetric metal nanocrystals or
nanoparticles as labels for nonlinear optical studies. A variety of shapes and
sizes of metal
nanoparticles are available in the prior art. To use these particles as
labels, one must derivatize them
for conjugation to a target biological component or particle. Either the
labels must be derivatized with
linkers; or the targets must be derivatized with linkers allowing for their
coupling to the labels (the
labels can be derivatized if necessary). A number of embodiments employing
these metal particles
are described herein.
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14
Fig. 2 depicts some of the various combinations of linkers and metallic or
semiconductor particles.
The particles can be both centrosymmetric or non-centrosymmetric. If
centrosymmetric, they must be
joined together in clusters to create a composite particle which is overall
non-centrosymmetric; or
they must be greater than or equal to 10% of the wavelength of the fundamental
light used in the
nonlinear optical technique.
Preferred Embodiment:
Non-centrosymmetric gold particles can be prepared by lithographic means
(according to means
found in references 2 and 5) or by synthetic methods (according to means found
in reference 7).
These can then be derivatized with X-R-SH where -SH is a sulfhydryl moiety, R
is an alkyl chain
and X is a terminal group suitable for conjugation to amines or sulfhydryls.
The gold particles can be
derivatized, for example, with HS-(CHZ)ls-COOH (obtained commercially)
according to means well
known in the prior art. The carboxyl groups on the derivatized particle can
then be made amine-
reactive by reaction with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC,
Molecular Probes,
Inc.) according to protocols supplied by Molecular Probes. The particles are
now amine-reactive and
can be conjugated directly to protein amines, or to protein sulfliydryls via a
variety of
heterobifunctional crosslinkers which are commercially available (Pierce Inc.,
Rockford, IL.)
In an alternate embodiment, the carboxyl group of the functional allrylthiol
listed above can be
coupled to oligonucleotides or nucleic acids via similar synthetic means.
In an alternate embodiment, a tris(aryl) phosphine ligand bearing a single
primary amine is mixed
with tris(p-N-methylcarboxamidophenyl) in a ratio 1:5 to derivatize the gold
particle as described in
U.S. Pat. No. 5,521,289, which reference is incorporated by reference herein.
The gold particles are
then reacted with N methoxycarbonylmaleimide (NMCM) in DMSO, mixed and
incubated at 0
degrees C for 30 minutes. The maleimido-gold particles can be separated from
unreacted NMCM on
a gel filtration column. The maleimido-gold particles can then be reacted with
a variety of biological
moieties including amines, sulflrydryls and carboxylic acids by using cross-
linking agents if
necessary.
In an alternate embodiment, a tris(aryl)phosphine ligand bearing a single
nonlinear active moiety (eg.,
oxazole dye) is reacted with the tris(aryl)phosphine ligand and tris(p-N-
methylcarboxamidophenyl) as
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described in U.S. Pat. No. 5,521,289 to produce particles which contain
nonlinear active molecules as
well as the means to make them reactive with biological molecules.
In an alternate embodiment, silica beads can be used (readily available from a
number of commercial
sources). A bead with ~ 200 nm diameter is reacted with 3-
aminopropyltrimethoxysilane so that the
charged silanols are functionalized to produce an amine-surface. These beads
can then be reacted
with a mixture of 1-(3-(succinimidyloxycarbonyl) benzyl)-4-(5-(4-
methoxyphenyl) oxazol-2-
yl)pyridinium bromide (PYMPO-SE; Molecular Probes Inc.) and (N-[ -
Maleimidobutyryloxy]succinimide ester (GMBS, Pierce Chemical Inc.). This
reaction produces a
bead with a surface that has both a covalently attached oxazole dye and a
tether for reaction with
sulfliydryl-containing targets.
In an alternate embodiment, the noncentrosymmetric metal particles can
themselves be complexed to
a larger object which, in turn, contains linkers for coupling the object to a
molecule or particle. The
Au particles can be chemically derivatized with SH-X-SH where X is an
alkylthiol according to
methods well known in the prior art. If a silica sphere is used, its surface
can be readily derivatized
with amines by using a well known reaction with an arninoalkyltrichlorosilane.
The silica surface can
then be covalently coupled to the Au particles via a number of commercially
available
heterobifunctional crosslinkers (Pierce Chemical, Inc.).
In an alternate embodiment, metallic or semiconductor particles (either
centrosymmetric or non-
centrosymmetric) can be coupled to an SHG-active particle (such as oxazole, a
stryrl dye, or some
other molecule or particle). These resonantly enhancing particles are well
known in the prior art to
strongly increase the intensity of nonlinear light scattered from a nearby
nonlinear active moiety. For
example, gold nanoparticles have been used to strongly enhance the SH-activity
of a styryl dye [14].
Because these resonantly enhancing particles are not themselves generating the
nonlinear light, they
can be centrosymmetric or non-centrosymmetric. They must be close enough to
the SH-active moiety
to create the resonant enhancement effect, which occurs
through a dipole-dipole interaction; the distance between the two species is
typically on the order of
angstroms to nanometers. The general resonance enhancement effect on nonlinear
optical phenomena
is discussed in the context of roughened silver surfaces in references 15 and
16. The resonantly
enhancing particles are available commercially with a variety of surfaqe
chemistries amenable to
coupling to an SH-active molecule such as oxazole (succinimidyl ester,
maleimide, etc. offered by
Molecules Probes, Eugene, OR). Or the particle-nonlinear-active moiety complex
can be constructed
according to a number of schemes available in the prior art.
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In an alternate embodiment, groups or chains of the metallic particles bound
together via linking
molecules can be used as labels with additional functional linkers to the
targets. For example, Au
particles can first be sparsely derivatized with linkers for a variety of
targets according to the method
of the preferred embodiment. The remaining underivatized surface area on the
particles can then be
used to chemically couple the particles together via prior art chemistry
involving dimercapto-alkyl
chains
In an alternate embodiment, the particles can be centrosymmetric (eg., a
sphere or a cube shape) if
their size (i.e., diameter or edge length, respectively) is larger than about
10% of the wavelength of
the fundamental light. For instance, a spherical particle of 80 nm diameter is
expected to produce a
nonlinear response when illuminated with 800 nm wavelength fundamental light.
PROTEINS AS NONLINEAR-ACTIVE LABELS
Some proteins are strongly SH-active and can be used as fusion-protein labels
for in-situ or in-vivo
studies. For example, the gene encoding a protein of interest X can be fused
at the N-terminal or C-
terminal to a gene encoding one of the SH-active proteins. These include, but
are not limited to, green
fluorescence protein (GFP) and bacteriorhodopsin. Detailed procedures in the
prior art exist for
creating the fusion of such proteins to another protein of interest
(references 12 and 13). For example,
GFP (an intrinsically fluorescent protein) can be fused to many other proteins
in order to render those
proteins fluorescent. GFP has been used in this way in prior art to monitor
gene expression and
cellular location of the GFP-X construct via fluorescence detection.
DESCRIPTION OF THE DRAWINGS
Fig. 1:
A. Organic Molecules
In the first drawing, (5) represents an organic, nonlinear-active moiety or
molecule and
(X) denotes the functional group which is reactive towards a target, a linker,
a surface
layer or a solid object.
In the second drawing, (10) represents an organic, nonlinear-active moiety or
molecule
and (Y-Z) denotes a linker molecule where (Y) is the group which bonds the
linleer to
the nonlinear active moiety or molecule and Z is the functional group which is
reactive
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towards a target, a linker, a surface layer or a solid object.
B. Derivatized Particle Labels
In drawing (a), (15) represents a solid object used as a 'scaffold' whose
surface area is
used to attach linkers (Y) and nonlinear-active moieties, molecules or
particles (X). In
this case, the linker Y is directly attached to the surface groups of the
solid object and
the nonlinear active components (20) are non-covalently adsorbed to the
surface of the solid
object. Functional group (Y) on the linker (25) is reactive towards a target,
another linker,
or a solid object.
In drawing (b), (30) represents a solid object used as a 'scaffold' whose
surface area is
used to attach linkers and nonlinear-active moieties, molecules or particles
(40), (35) is a
surface derivatized layer and (45) is a linker group. Functional group (Y) on
the linker is
reactive towards a target, another linker, or another solid object.
In drawing (c), (50) represents a solid object used as a 'scaffold' whose
surface area is
used to attach linlcers and nonlinear-active moieties, molecules or particles,
(55) are the
covalently attached nonlinear-active moieties, molecules or particles and (60)
represents
the linker. In this case, (X) is a functional group on the nonlinear-active
component
which covalently reacts with a surface group on the solid object and (Y) is
the
functional group on the linker which is reactive towards a target, another
linker or
another solid object.
In drawing (d), (65) represents a solid object, (70) a surface layer which
derivatizes the
surface of the solid object and presents a functional group to the nonlinear-
active
components (75). (80) represents a linker which is directly attached to the
surface of the
solid object while (X) denotes the functional group on the nonlinear-active
component
which is reactive towards the functional group of the surface derivatization
layer.
In drawing (e), (85) represents a solid object, (90) represents a nonlinear-
active
component, (X) denotes the functional group of the nonlinear-active component
which
allows the latter to be covalently linked to the surface of the solid object,
and (Y)
denotes the functional group of a linker (95) which is also part of the non-
linear active
component. (Y) is reactive towards a target, another linker or another solid
object.
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Fig. 2: Metallic and Semiconductor Particles
In drawing (A), a non-centrosymmetric metallic or semiconductor particle (100)
is
derivatized with linkers containing functional end-groups (Y). The functional
groups
(Y) is reactive towards a target, another linker or another solid object. The
shape of
the metallic particle (1) is drawn to emphasize its non-centrosymmetric
nature.
In drawing (B), a non-centrosymmetric metallic or semiconductor particle (105)
is
capable of directly attaching to a target without the need for a linker.
In drawing (C) non-centrosymmetric particles (110) are attached together
covalently via
linkers (115) to create a composite particle which is overall non-
centrosymmetric.
Another linker (120) is used to covalently link the composite to a target or
solid object
via an end-functional group (Y).
In drawing (D), non-centrosymmetric particles (122) are adsorbed or aggregated
together
to create a composite particle which is overall non-centrosymmetric. Linkers
(124) containing an end-functional group (Y) are used to link the composite to
a target or
solid object.
In drawing (E), centrosymmetric particles (125) are connected to each other
via linkers
(130) to create a composite particle which is overall non-centrosymmetric. As
drawn, the
centrosymmetric particles are covalently linked to each other via linkers, but
they
can also be adsorbed or aggregated to each other to create the composite. The
spherical shape of the particles is intended to emphasize their
centrosymmetry.
Fig. 3: Linear chains of nonlinear active moieties where X indicates a
chemically reactive group
on a first nonlinear active moiety capable of bonding to Y on a second
nonlinear active
moiety. The moieties can be assembled into a chain of desired length.
