Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Method and Apparatus Using a Surface-Selective Nonlinear Optical Technique
for Detection of Probe-Target Interactions.
FIELD OF THE INVENTION
The present invention relates to a method and apparatus for detecting
interactions between
biological components using a surface-selective nonlinear optical technique.
In particular, the present
invention relates to detection of the binding between biological probes and
nonlinear-active labeled
targets.
BACKGROUND OF THE INVENTION
Detecting and quantifying interactions such as binding between biomolecules is
of central interest
in modern molecular biology and medicine. Genomics and proteomics research is
increasingly directed
toward this problem, which demands high-throughput analysis of a variety of
biological interactions.
Many schemes for doing this rely on immobilization of molecules, often
oligonucleotides or proteins, to
solid surfaces. In particular, a microarray format of samples can be used to
obtain information in a highly
parallel process. For example, Fodor et al. (1991, relevant portions of which
are incorporated by
reference herein) disclose high density arrays formed by light-directed
synthesis - in this case, the
surface-attached probes are oligonucleotides and are tested for binding
(hybridization) against targets.
The targets, freely diffusing in solution, are fluorescently-labeled
oligonucleotides and at places where the
nucleotide sequence of the probe matches the sequence of the target, binding
occurs. When non-bound
targets are removed by washing, the sequence of the remaining targets can be
determined by scanning the
surface for fluorescence since the probe sequence is known, by design, at each
location on the surface,
and targets and probes must have matching, complementary sequences to
hybridize. A number of
variations on this method have been introduced including: studying SNPs
(single nucleotide
polymorphisms), where the binding strength, and hence the fluorescence
intensity, between sequences
differing by one base-pair; detection of protein-protein interactions, where
one protein (the probe) is
immobilized to the surface and tested for binding against a variety of
targets; protein-drug interactions
where protein-protein interactions are modulated by the presence of a drug;
and others.
In all these cases, the xead-out step involves fluorescence-based detection.
However, detection
with fluorescence has several drawbacks: the samples are generally dry (to
remove background
fluorescence; i.e., non-bound targets in the bulk) and therefore no
equilibrium (free energy, dissociation
constant, etc.) measurement is typically possible due to fluorescence
background from the bulk. The non-
bound targets must first be removed from the sample via a wash step and this
obviates equilibrium or
kinetics measurements and, furthermore, can be time-consuming when many scans
must be made on a
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given sample or many samples must be examined. The excitation source for
fluorescence may also
contribute a background signal since it can be scattered by the substrate into
the detection optics, and may
be difficult to completely filter from the fluorescence. Furthermore, there
may be background
autofluorescence or bottlenecks in the "read-out" or detection step because
the scan can require pixel-by-
pixel acquisition with, for example, confocal-based detection schemes. Auto-
focusing routines at each
step in the scan can also lead to significant slow-down in image acquisition.
Another method for quantifying biomolecular binding interactions - with an
ability to measure
both equilibrium and kinetics properties of the interaction is surface plasmon
resonance (SPR). SPR
requires a conductive or semiconductive layer (typically gold) between the
substrate (typically glass) and
the liquid solution it is immersed in. Incident light is coupled into the
conductive layer by means of a
prism or grating and, at a specific wavelength or angle of incidence, a
resonance occurs, resulting in a
sharp minimum or decrease in reflectivity. Generally, a bio-compatible layer
or layers are built on top of
the conductive layer. In one example, proteins are immobilized to the
biocompatible layer (often dextran-
based) and target proteins are brought into contact with the layer. The
resonance wavelength or angle
depends on the refractive index of the solution near the substrate and this,
in turn, depends on the amount
and mass of adsorbed biomolecules within an evanescent wavelength from the
conductive layer. When
target protein binds to the immobilized protein, a change in the resonance
wavelength or angle occurs.
However, the SPR technique is not convenient for detecting samples in an array
format because of the
difficulty in coupling the excitation into each array element separately.
Furthermore, the detection
sensitivity may be low, the technique cannot distinguish between specific and
non-specific binding, and
SPR typically requires an extra, biologically compatible layer to prevent
destructive interactions which
can occur if the biomolecules make contact with the conductive layer. This
biocompatible layer may not
always be stable or prevent destructive interactions with the gold surface and
the immobilized proteins
must often be truncated in order to render them suitable for coupling to the
bio-compatible layer, thus
risking the possibility that their properties may change. A particularly acute
problem occurs with
membrane proteins. Membrane proteins are best studied in a native-like
environment such as a laterally
fluid phospholipid membrane which can be prepared on glass surfaces. However,
it is not possible to
prepare these membranes on gold surfaces due to destructive interactions
between the gold and the lipids.
Surface-selective nonlinear optical (SSNLQ) techniques such as second harmonic
generation
(SHG) allow one to detect interfacial molecules or particles (the interface
must be non-centrosymmetric)
in the presence of the bulk species. An intense laser beam (the fundamental)
is directed on to the
interface of some sample; if the interface is non-centrosymmetric, the sample
is capable of generating
nonlinear light, i.e. the harmonics of the fundamental. 'The fundamental or
the second harmonic beams
can easily be separated from each other, unlike the typical case in
fluorescence techniques with excitation
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and emission light, which are separated more narrowly by the Stokes shift.
Individual molecules or
particles can be detected if they 1) are nonlinearly active (possess a
hyperpolarizability) and 2) are near
to the surface and through its influence (via chemical or electric forces)
become non-randomly oriented.
This net orientation and the intrinsic SHG-activity of the species are
responsible for an SHG-allowed
effect at the interface. For example, the adsorption processes of dye
molecules to~planar solid surfaces
(glass and silica), liposomes and solid beads (silica and polystyrene) at air-
water interface have been
measured. The technique has also been used to follow such processes as
electron-transfer or solvation
dynamics at an interface.
Nonlinear SSNLO techniques, such as SHG, have previously been confined mainly
to physics
and chemistry since relatively few biological samples are intrinsically non-
linearly active. Examples
include the use of an optically nonlinear active dye that is used to image
biological cells (Campagnola et
al., Peleg, 1999). In this technique, nonlinear active stains are immobilized
in membranes and these
stains are used to image the cell surfaces. However, the stains intercalate
into the membranes in either an
'up' or 'down' direction, thus reducing the total nonlinear signal due to
destructive interference.
Nonlinear optically active dyes have also been used to measure the kinetics of
those dyes crossing lipid
bilayers in liposomes (Srivastava and Eisenthal). Recently, too, the concept
and technique of second
harmonic active labels ("SHG labels") was introduced, allowing any non-linear
active molecule or
particle to be rendered non-linear active. The first example of this was
demonstrated by labeling the
protein cytochrome c with an oxazole dye and detecting the protein conjugate
at an air-water interface
with second harmonic generation.
DESCRIPTION OF THE INVENTION
The present invention is based on the use of nonlinear-active labels, the
surface-selectivity of
second harmonic (or sum/difference frequency) generation and the fact that the
nonlinear beam is
scattered from an interface in a predictable, well-defined direction (in
contrast to fluorescence detection in
which fluorescence is emitted somewhat at random). The surface-selective
nonlinear optical techniques
are coherent techniques, meaning that the fundamental and nonlinear beams have
well-defined phase
relationships, and the wavefronts of a nonlinear beam in a macroscopic sample
(within the coherence
length) are in phase. These properties offer a number of advantages useful for
surface or high-throughput
studies in which either a single surface or a microarray surface is studied.
An apparatus using nonlinear
optical suface-selective-based detection, such as with second harmonic
generation, requires minimal
collection optics since generation of the nonlinear light only occurs at the
interface and thus, in principle,
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allows extremely high depth discrimination and fast scanning. The probe-target
interactions can be
correlated with the present invention to the following measurable information,
for example:
i) the intensity of the nonlinear or fundamental light.
ii) the wavelength or spectrum of the nonlinear or fundamental light.
iii) position of incidence of the fundamental light on the surface or
substrate (e.g., for
imaging).
iv) the time-course of i), ii) or iii).
v) one or more combinations of i), ii) and iii).
For example, probe-target binding can be measured by detecting the intensity
of nonlinear optical light
(e.g., second harmonic light) at some position on a substrate with surface-
attached probes; the intensity of
the second harmonic light changes as labelled targets (targets labelled with
second-harmonic-active labels
possessing a hyperpolarizability) bind to the probes at the surface and become
partially oriented because
of the binding, thus satisfying the non-centrosymmetric condition for
generation of second harmonic light
at the interface. Modeling of the intensity of light with concentration of
probe-target binding complexes
at the interface can be accomplished using a variety of methods, for instance
by calibrating the technique
for a given probe-target interaction using radiolabels or fluorescence tags.
The advantages of the present invention are enumerated as follows:
i) Detection of interfacial species in the presence of bulk species in real
time. This property can be
especially useful when the presence of bulk species are necessary to detect a
binding process
(eg., if equilibrium or real-time kinetics data is required via, for example,
changing target
concentrations) or the wash-away step to remove non-bound material is time-
consuming,
incomplete or gives artifactual results.
ii) Higher signal to noise (lower background) than fluorescence-based
detection since SSNLO is
generated only at non-centrosymmetric surfaces. SSNLO techniques thus have a
very narrow
'depth of field'. Sources of fluorescence in fluorescence-based detection
schemes include that
from materials in the field of view but not in the focal plane,
autofluorescence, and
contamination of the emitted fluorescence with stray excitation light; these
are not sources of
background nonlinear optical radiation.
iii) The technique is useful when the presence of a liquid solution is
required for the measurement,
i.e. where the binding process can be obviated or disturbed by a wash-away
step. This aspect of
the invention can be useful for equilibrium measurements (free energy, binding
constants, etc.),
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which require the presence of bulk species or kinetics measurements with
measurements made
over a period of time.
iv) The scattering process responsible for the nonlinear effect in SSNLO
techniques does not lead to irreversible bleaching of the label as quickly as
with
fluorescent labels - the two-photon absorption cross-section is much lower
than the one-
photon cross-section in a molecule arid the NLO technique involves scattering,
not absorption.
iv) A minimum of collection optics is needed and higher signal to noise is
expected since
the fundamental and nonlinear beams (i.e., second harmonic) have well-defined
incoming and
outgoing directions with respect to the interface. This is advantageous
compared to
fluorescence-based detection in which the fluorescence is emitted
isotropically and there may be
a large auto-fluorescence background out of the plane of interest (e.g., the
interface containing
the probes).
v) Ease of use with beads, cells or other particles whose surface makes an
interface with the
supporting medium, solution, etc.
Examples of the use of the invention
Although the present invention may be used in many scientific areas of
analysis and in particular,
in the chemical and biological arts, the present invention can be especially
useful in genomics or
proteomics, where speed and ease of very high-throughput detection are
critical. It may be advantageous
to detect the surface species in the presence of bulk species - for instance
in DNA hybridization or
protein-protein detection, the wash-away step for unbound molecules would not
be necessary, useful in
cases where this step may contribute artifacts to the desired signal.
Moreover, in many techniques, such
as fluorescence-based detection, a large portion of the sample not at or near
the interface (i.e., in the bulk)
may contribute undesirably or interfere with measurements. It would be
advantageous therefore to use a
surface-selective technique such as second harmonic generation or sum
frequency generation which is
sensitive only to the interface.
SSNLO techniques, when used to study proteins, cells or other molecules in an
array format on
some surface (two-dimensional ordering of the samples on a solid surface),
have other important
advantages over the art. Because the technique relies on a scattering
(reflection-like) process, the
nonlinear beam has a well-defined direction. With fluorescence-based detection
the collection optics may
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be complicated and extensive because the emission is isotropic and only
emission from a narrow depth of
field is desired. When using nonlinear optical techniques, however, the
technique is intrinsically surface-
selective - the 'depth of field' is confined by the nature of the technique to
an extremely thin layer near
the interface. Moreoever, the scattered light from the surface possesses a
well-defined direction, so that
its position at a detector can be mapped directly to a location on the array
surface.
Art scanning of microarrays includes confocal-based schemes and non-confocal
based schemes.
U.S. Pat. No. 5,834,75 S (Trulson et al. - relevant portions of which are
incorporated by reference herein)
describes a non-confocal based scheme for imaging a microarray using
fluorescence detection. However,
the sample must lie very flat in order to image only within a single focal
plane for good out-of plane
discrimination. Therefore, a very finely adjustable translation stage
requiring specialized components
must be used for this purpose adding to the cost of the instrument and
possibly the lifetime as well. The
image quality of this type of apparatus can be sensitive to mechanical
vibrations. Furthermore,
discrimination of the out-of plane (non-surface bound) fluorophores places a
limit on the sensitivity of the
technique. U.S. Pat. No. 6,134,002 (Stimson et al. - relevant portions of
which are incorporated by
reference herein) is an example of a confocal scanning microscope device for
imaging a sample plane, i.e.
a microarray. Although the confocal-based techniques have good depth
discrimination, the scan rate may
be low due to descanning requirements and the light throughput can be low,
reducing the overall signal to
noise ratio and the sensitivity of the technique.
For use with nucleic acid hybridization (oligonucleotide, polynucleotide, RNA,
etc.), target
oligonucleotides can be reacted with the entire surface; at the probe
oligonucleotide sequences in the
array (corresponding to known locations) where sequence-complementary
hybridization occurs, the
fundamental light would give rise to a nonlinear optical signal, or a change
in the background of such a
signal. This can be detected and correlated with the spatial location of the
array element and hence the
oligonucleotide sequence.
For example, two major applications of nucleic acid microarrays are: 1)
Identification of
sequence (gene or gene mutation) - monitoring of DNA variations, for example
and 2) Determination of
expression level (abundance) of genes. There are many formats for preparing
the arrays. For example, in
one case probe cDNA (5005000 base pairs long) can be immobilized to a solid
surface such as glass
using robot spotting and exposed to a set of targets either separately or in a
mixture (ref. Ekins and Chu).
Another format involves synthesizing oligonucleotides (2025 mer oligos) or
peptide nucleic acids probes
in-situ (on the solid substrate, Fodor et al.) or by conventional synthesis
followed by on-chip
immobilization. The array is then exposed to target DNA, hybridized, and the
identity or abundance of
complementary sequences are determined. Protein arrays can be prepared (see
for example, MacBeath
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and Schreiber, 2000) to determine whether a given target protein binds to the
immobilized probe protein
on the surface. These arrays were also used to study small molecule binding to
the probe proteins. Many
reviews of microarray technology and applications are available in the art.
For instance, those of:
Ramsay (1998- relevant portions of which are incorporated by reference
herein), Marshall (1998-
relevant portions of which are incorporated by reference herein), Fodor (1997-
relevant portions of which
are incorporated by reference herein), Duggan et al. (1999-relevant (1998),
Marshall (1998), Fodor
(1997), Duggan et al. (1999), Schena et al. (1995), Brown et al.
(1999),portions of which are incorporated
by reference herein), Schena et aI. (1995- relevant portions of which are
incorporated by reference
herein), Brown et al. (1999- relevant portions of which are incorporated by
reference herein), McAllister
et al. (1997) and Blanchard et al. (1996-relevant portions of which are
incorporated by reference herein).
The invention can be used for studying binding processes between other
biological components:
cells with viruses; protein-protein interactions; protein-ligand; cell-ligand;
protein-drugs, nucleic acid-
drugs, cell-small molecule; cell-nucleic acid; peptide-cell, oligo or
polynucleotides, virus-cell, protein-
small molecule, etc. Biomimetic membranes such as phospholipid supported
bilayers (eg., egg
phosphatidylcholine) can also be used and are particularly useful when studies
involve membrane
proteins as probes.
The invention can be used for drug screening or high-throughput screening
where a candidate
drug is tested for its effect on probe-target binding, i.e., to reduce or
enhance probe-target binding. In
other cases, for example, a drug can be tested for efficacy by its ability to
bind to a receptor or other
molecule on the surface of a biological cell.
Other examples of the technique's use with arrays include cellular arrays,
supported lipid bilayer
arrays with or without membrane or attached proteins, etc. Many methods exist
in the art for coupling
biomolecules (eg., nucleic acid, protein and cells) to solid supports in array
format. A wide degree of
flexibility may be used in providing the means by which the arrays are
created. They can involve, for
example, covalent or non-covalent coupling to the substrate directly, to a
chemically derivatized substrate,
to an intermediate layer of some kind (e.g., self assembled monolayer, a
hydrogel or other bio-compatible
layer lrnown in the art). °The identity of the probes (e.g., protein
structure or oligonucleotide sequence)
can vary from site to site across the solid surface, or the same probe can
uniformly cover the surface.
Targets can be of a single identity or a combination of targets with different
identities. The arrays can be
prepared in a variety of ways including, but not limited to, ink jet printing,
photolithography, micro-
contact printing, or any other manner known to one skilled in the art of
fabricating them.
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Because the binding process can be measured in real time and in the presence
of bulk biological
components due to the surface-selectivity of the nonlinear optical technique,
equilibrium binding curves
and kinetics can be measured, the bulk concentration of the components can be
varied, and a "wash-
away" step to remove unbound components, as is used with fluorescence-based
detection, may be
unnecessary.
In another aspect of the invention, SHG labels - for example, as second-
harmonic active
molecules or particles - can be used for imaging studies of cells, membranes,
tissues involving techniques
such as second harmonic (or sum/difference frequency) microscopy or confocal
microscopy by labeling
specific probes, cell membranes, surfaces, etc. in-vitro or in-vivo. For in-
vivo applications, the labels can
be delivered to the sample of interest by well known techniques that use
fluorescent dyes for imaging or
tracing and, for example, endoscopes.
A wide degree of flexibility is expected in the design of the apparatus
including, but not limited
to, the source of the fundamental Light, the optical train necessary to
control, focus or direct the
fundamental and nonlinear light beams, the design of the array, the detection
system, and the use of a
grating or filters and collection optics. The mode of generation (irradiation)
or collection can be varied
including, for example, the use of evanescent wave (total internal
reflection), planar wave guide,
reflection, or transmission geometries, fiber-optic, near-field illumination,
confocal techniques or the use
of a microcavity or integrating detection system. A number of methods for
scanning a microarray on a
solid surface are described. Examples include U.S. Pat. No.'s Trulson et al.
(1998), Trulson et al. (2000),
Stern et aI. (1997) and Sampas (2000)- relevant portions of which are
incorporated by reference herein.
Because the second harmonic light beam makes a definite angle to the surface
plane, one can
read-out the properties of the nonlinear optical radiation (for instance, as a
function of fundamental
incidence position in a two-dimensional array format) without needing to
mechanically translate the
detector or sample and without extensive collection optics. In the 'beam
scanning' embodiment, no
mechanical translation of sample surface or detector is required - only a
change in a direction and/or
angle of the fundamental incidence on the sample (for a fixed sample and
detector) - the apparatus offers
much faster scanning capability, improved ease of manufacturing and a longer
lifetime.
The interface can comprise a silica, glass, silicon, polystyrene, nylon,
plastic, a metal,
semiconductor or insulator surface, or any surface to which biological
components can adsorb or be
attached. The interface can also include biological cell and liposome
surfaces. The attachment or
immobilization can occur through a variety of techniques well known in the
art. For example,
oligonucleotides can be prepared via techniques described in "Microarray
Biochip Technology", M.
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Schena (Ed.), Eaton Publishing, 1998- relevant portions of which are
incorporated by reference herein.
And, for example with proteins, the surface can be derivatized with aldehyde
silanes for coupling to
amines on surfaces of biomolecules (MacBeath and Schreiber, 2000- relevant
portions of which are
incorporated by reference herein). BSA-NHS (BSA-N-hydroxysuccinimide) surfaces
can also be used by
first attaching a molecular layer of BSA to the surface and then activating it
with N,N'-disuccinimidyl
carbonate. The activated lysine, aspartate or glutamate residues on the BSA
react with surface amines on
the proteins.
Supported phospholipid bilayers can also be used, with or without membrane
proteins or other
membrane-associated components as, for example, in Salafsky et al.,
Biochemistry, 1996- relevant
portions of which are incorporated by reference herein, "Biomembranes",
Gennis, Springer-Verlag, Kalb
et al., 1992 and Brian et al., 1984, relevant portions of which are
incorporated herein. Supported
phospholipid bilayers are well known in the art and there are numerous
techniques available for their
fabrication, with or without associated membrane proteins. These supported
bilayers typically must be
submerged in aqueous solution to prevent their destruction when they become
exposed to air.
If a solid surface is used (e.g., planar substrate, beads, etc.) it can also
be derivatized via various
chemical reactions to either reduce or enhance its net surface charge density
to optimize the detection of
probe-target interactions (e.g., a hybridization process).
The binding process can be performed in the presence of small molecules,
drugs, blocking agents,
or other components which modulate the binding process.
The surface arrays can be constructed according a plurality of methods found
in the art. For DNA
microarrays, most are prepared with one of three non-standard approaches (Case-
Green, 1998):
Affymetrix, Inc. probe arrays are prepared using patterned, light-directed
combinatorial chemical
synthesis (Fodor, 1997); spotted arrays can be made according to Duggan
(1999), Schena (1995), Brown
and Botstein (1999) and McAllister (1997) ; ink jet techniques can also be
used to synthesize
oligonucleotides base by base through sequential solution-based reactions on
an appropriate substrate
(Blanchard, 1996)- relevant portions of all of which references are
incorporated by reference herein..
For example, nucleic acid, oligo- or nucleotide arrays can be constructed
according to Pat. No.
6,110,426, Pat. No. 5,143,8546,110,426- relevant portions of which are
incorporated by reference herein,
Pat. No. 5,143,854-relevant portions of which are incorporated by reference
herein or Fodor (1991).
Soluble protein arrays can be constructed according to Ekins (1999) relevant
portions of which are
incorporated by reference herein. Membrane proteins arrays can be constucted
by micropatterning of
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fluid lipid membranes according, for example, to the method of Groves et al.
(1997- relevant portions of
which are incorporated by reference herein). The array substrate can be
composed of glass, silicon,
indium tin oxide, or any other substrate known in the art. The surface array
under study can contain
physical bOrriers between elements so that the elements (and their
biomolecules) can remain in isolation
from each other during a chemical reaction step. The array locations can
consist of different probes, the
same probes everywhere, or some combination thereof. The array can also be
constructed on the
underside of a prism allowing for total internal reflection of the beam and
evanescent generation of the
nonlinear light. Or an array substrate can be brought into contact with a
prism with the same result.
An electrophoretic system can also be used in conjunction with the surface
array, for example to
provide reagents or biological components to one or a plurality of locations
using flow channels or
microcapillaries. For instance, the sample can include an array of
microcapillary channels, each distinct
from the other and each allowing a target-probe reaction to occur; the imaging
technique would then
consist of array elements, each one a microcapillary channel or reaction
chamber into which the channel
feeds and drains.
The polarization of the fundamental and nonlinear beams can be selected with
polarizing optics
elements. By analyzing the intensity of the nonlinear beam as a function of
fundamental and nonlinear
polarization, more information (e.g., higher signal to noise) about the probe-
target complexes can be
obtained. Furthermore, by selecting and analyzing the polarization of the
fundamental or nonlinear
optical radiation, background radiation can be reduced.
Detection can be accomplished with the use of multiple internal reflection
plates (N.J. Harrick-
relevant portions of which are incorporated by reference herein) allowing the
fundamental beam to make
multiple contacts with the array surface, thus increasing the intensity of the
generated nonlinear light.
Another alternative is to construct an optical cavity with the array surface
on one side and a lossy coupler
at one end to permit the output coupling of the nonlinear light, creating an
optical microcavity which
would allow the buildup of very high intensities under resonance and thus
increase the amount of
nonlinear light generated.
There are many linlting moieties and methodologies for attaching molecules
which can be nonlinear-
active labels to the 5' or 3' termini of oligonucleotides, as exemplified by
the following references:
Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL
Press, Oxford, 1991);
Zuckerman etal., Nucleic Acids Research, 15: 5305-5321 (197) (3' thiol group
on oligonucleotide);
Sharma et al., Nucleic Acids Research, 19: 3019 (1991) (3' sulfliydryl);
Giusti et al., PCR Methods and
Applications, 2: 223-227 (1993) and Fung et al., U.S. Pat. No. 4, 757,141 (5'
phosphoamino group via
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11
Aminolink.TM. II available from Applied Biosystems, Foster City, Cafil.)
Stabinsky, U.S. Pat. No.
4.739,044 (3' aminoalkylphosphoryl group); Agrawal et al., Tetrahedron
Letters, 31: 1543-1546 (1990)
(attachment via phosphoramidate linkages); Sproat et al., Nucleic Acids
Research, 15: 4837 (1987) (5'
mercapto group); Nelson et al., Nucleic Acids Research, 17: 7187-7194 (1989)
(3' amino group); and the
like, relevant portions of which are incorporated by reference herein.
Preferably, commercially available linking moieties are employed that can be
attached to an
oligonucleotide during synthesis, e.g., available from Clontech Laboratories
(Palo Alto, Cafil.).
Rhodamine and fluorescein dyes are also conveniently attached to the 5'
hydroxyl of an oligonucleotide at
the conclusion of solid phase synthesis by way of dyes derivatized with a
phosphoramidite moiety, e.g.,
Woo et al., U.S. Pat. No. 5,231, 191; and Hobbs, Jr., U.S. Pat. No. 4,997,928,
relevant portions of which
are incorporated by reference herein.
Protein arrays can be used to determine whether a given target protein binds
to the immobilized
probe protein on the surface; these arrays were also used to study small
molecule binding to the probe
proteins. Protein arrays can be prepared by the method of MacBeath and
Schreiber (2000), for example,
to determine Whether a given target protein binds to the immobilized probe
protein on the surface.
The support on which the sequences are formed may be composed from a wide
range of material, either
biological, nonbiological, organic, inorganic, or a combination of any of
these, existing as particles,
strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries,
pads, slices, films, plates, slides,
etc. The substrate may have any convenient shape, such as a disc, square,
sphere, circle, etc. The substrate
is preferably flat but may take on a variety of alternative surface
configurations. For example, the
substrate may contain raised or depressed regions on which a sample is
located. The substrate and its
surface preferably form a rigid support on which the sample can be formed. The
substrate and its surface
axe also chosen to provide appropriate light-absorbing characteristics. For
instance, the substrate may be a
polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP,
SiO<sub>2</sub>, SiN<sub>4</sub>,
modified silicon, or any one of a wide variety of gels or polymers such as
(poly)tetrafluoroethylene,
(poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinations
thereof. Other substrate materials
will be readily apparent to those of skill in the art upon review of this
disclosure. In a preferred
embodiment the substrate is flat glass or silica.
According to some embodiments, the surface of the substrate is etched using
well known techniques to
provide for desired surface features. For example, by way of the formation of
trenches, v-grooves, mesa
structures, or the like, the synthesis regions may be more closely placed
within the focus point of
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12
impinging light. The surface may also be provided with reflective "mirror"
structures for maximization of
emission collected therefrom.
The identity of the probes (e.g., protein structure or oligonucleotide
sequence) can vary from site
to site across the solid surface, or the same probe can uniformly cover the
surface. Targets can be of a
single identity or a combination of targets with different identities.
In another aspect of the invention, labels can be attached to the surface of a
cell or liposome
containing ion channel proteins. The nonlinear properties (e.g.,
hyperpolarizability) of the labels is
sensitive to the surface electric potential of the cells or liposomes. When
the ion channels open or close
or their properties are otherwise changed, the surface electric potential of
the cells or liposomes can
change in turn, thus changing the nonlinear properties of the labels and in
turn the detected nonlinear
radiation. Thus, this aspect of the invention can be used, fox example, to
detect ligand binding to ion
channel receptors (or to other receptors which will trigger an ion channel
behavior). The binding can be
monitored in the presence of drugs, agonists, antagonists, etc., and in real-
time if desired.
Proteins can be immobilized to a solid surface. For example, they can be
attached using the
methods of MacBeath and Schreiber (Science, 2000). For example, protein G
molecules can be
immobilized to a derivatized surface via the method of MacBeath and Schreiber.
These are the probes.
Immunoglobulin G (IgG) proteins are used as the targets and a solution of IgG
is brought into contact
with the protein G surface. Protein G molecules should possess a net
orientation at the interface if
possible. Decorators are anti-IgG proteins which have been previously labeled
with "SHG labels",
rendering them detectable via second harmonic generation. The labels can be
oxazole dye (Salafsky et
al., 2000) or a non-centrosymmetric Au particle with long linkers. As IgG
targets bind to protein G
probes, the amount of IgG at the interface increases and so does the SHG
signal intensity since the
number of SHG-labels at the interface increases. The SHG-labels on the anti-
IgG can be attached via
linkers to maximize their orientation when bound to targets, Even if the
targets are randomly oriented
within the population of target-probe complexes on the surface, the labels can
be non-randomly oriented
using linkers or spacer molecules, and this will ensure their detection via
second harmonic generation.
Decorators not associated with the interfacial targets will be isotropically
oriented and will not produce a
second harmonic signal. This signal can be quantitatively modeled using a
Langmuir adsorption curve to
determine the concentration of probe-target complexes using software and a PC
as described for
cytochrome c adsorption to silica in Salafsky, 2000. Relevant portions of the
aforementioned references
are incorporated by references herein.
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13
Applications of the labels include studies of protein-protein binding at an
interface, protein or
virus binding to a cell surface, oligonucleotide hybridization at a solid
interface - such as with
microarrays - two-photon absorption studies, two-photon microscopy, nonlinear
optical microscopy (eg.,
SHG microscopy), cell sorting using a nonlinear optical technique, drug-
receptor interaction, etc.
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
involves the use of EFISH
(Electric-held 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~(w3) = x~z~ (-~3; w,,~z) : Ew' E'~z. The effect can be used to
measure 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 and thus
non-centrosymmetric.
Examples of samples in which the labels can be of use include, but are not
limited to, solid
surfaces with immobilized protein, oligonucleotides or cells and supported
phospholipid bilayers. The
surface geometry can be varied, indeed spherical beads and other non-planar
geometries are generally
accessible with the nonlinear optical techniques.
In one important aspect of the invention, the use of the linkers which couple
the labels to their
targets can be made long enough so that the orientation of the targets at the
interface (i.e., when bound to
the probes) does not significantly effect 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 latter does not necessarily determine the orientation of the former. In
cases where this is less
important, for example, with integral membrane proteins in supported lipid
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 most cases, linkers may still be
necessary in order to couple the
label to the targets.
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Cells bound to a substrate can also be used to determine protein-cell binding,
virus-cell binding,
etc. where the cell is the probe component and proteins, viruses, etc. are the
target components. The next
section discusses the well known art for coupling cells to solid substrates.
Various art not involving the use of a surface-selective nonlinear optical
technique contains relevant
portions for the present invention and the following exemplary list and their
references therein is
referenced herein: King et al.., U.S. Patent 5,633,724 for the scanning and
analysis of the scans; Fork et
al., U.S. Patent 6,121,983 for the multiplexing of a laser to produce a laser
array suitable for scanning;
Foster, U.S. Patent 5,485,277; Fodor et al., U.S. Patent 5,324,633 and Fodor
et al., U.S. Patent 6,124,102
for a substrate containing an array of attached probes and for the analysis of
scans to determine kinetic
and equilibrium properties of a binding reaction between probes and targets;
Kain et al., U.S. Patent
5,847,400 for laser scanning of a substrate; King et al., U.S. Patent
5,432,610 for an optical resonance
cavity for power build-up; Walt et al., U.S. Patent 5,320,814, Walt et al.,
U.S. Patent 5,250,264, Walt et
al., U.S. Patent 5,298,741, Walt et aL, U.S. Patent 5,252,494, Walt et aL,
U.S. Patent 6,023,540, Walt et
al., U.S. Patent 5,814,524, Walt et al., U.S. Patent 5,244,813 for fiber-optic-
based apparatus; Fiekowsky
et al., U.S. Patent 6,095,555 for imaging and software-based analysis of
images; Stern et al., U.S. Patent
5,631,734 for data acquisition; Stimson et al., U.S. Patent 6,134,002 for
confocal imaging techniques;
Sampas, U.S. Patent 6,084,991 for CCD-based imaging techniques; Stern et al.,
U.S. Patent 5,631,734 for
photolithographical preparation of probes attached to surfaces; Shalon et al.,
U.S. Patent 6,110,426 for
methods and apparatus for creating attached probes on a surface; Slettnes,
U.S. Patent 6,040,586 for
position-based scanning techniques; Trulson et a1, U.S. Patent 6,025,601 for
methods of imaging probe-
target binding on a surface.
Microarrays of Cells
This section outlines some of the methods concerned with fabricating arrays of
biological cells on
surfaces, one type of array amenable to study using the present invention.
Many methods have been
described for making uniform micro-patterned arrays of cells for other
applications, using for example
photochemical resist-photolithograpy. (Mrksich and Whitesides, Ann. Rev.
Biophys. Biomol. Struct.
25:55-78, 1996). According to this photoresist method, a glass plate is
uniformly coated with a
photoresist and a photo mask is placed over the photoresist coating to define
the "array" or pattern
desired. Upon exposure to light, the photoresist in the unmasked areas is
removed. The entire
photolithographically defined surface is uniformly coated with a hydrophobic
substance such as an
organosilane that binds both to the areas of
exposed glass and the areas covered with the photoresist. The photoresist is
then stripped from the glass
surface, exposing an array of spots of exposed glass. The glass plate then is
washed with an organosilane
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having terminal hydrophilic groups or chemically reactable groups such as
amino groups. The
hydrophobic organosilane binds to the spots of exposed glass with the
resulting glass plate having an
array of hydrophilic or reactable spots (located in the areas of the original
photoresist) across a hydrophobic surface. The array of spots of hydrophilic
groups provides a substrate
for non-specific and non-covalent binding of certain cells, including those of
neuronal origin (Klienfeld et
al., J. Neurosci. 8:4098-4120, 1988). Reactive ion etching has been similarly
used on the surface of
silicon wafers to produce surfaces patterned with two different types of
texture (Craighead et al., Appl.
Phys. Lett. 37:653, 1980; Craighead et al., J. Vac. Sci. Technol. 20:316,
1982; Suh et al. Proc. SPIE
382:199, 1983).
In another method based on specific yet non-covalent interactions, photoresist
stamping is used to
produce a gold surface coated with protein adsorptive alkanethiol. (Singhvi et
al., Science 264:696-698,
1994). The bare gold surface is then coated with polyethylene-terminated
alkanethiols that resist protein
adsorption. After exposure of the entire surface to laminin, a cell-binding
protein found in the
extracellular matrix, living hepatocytes attach uniformly to, and grow upon,
the laminin coated islands
(Singhvi et al. 1994). An elaboration involving strong, but non-covalent,
metal chelation has been used to
coat gold surfaces with patterns of specific proteins (Sigal et al., Anal.
Chem. 68:490-497, 1996). In this
case, the gold surface is patterned with alkanethiols terminated with
nitriloacetic acid. Bare regions of
gold are coated with tri(ethyleneglycol) to reduce protein adsorption. After
adding Niz+, the specific
adsorption of five histidine-tagged proteins is found to be kinetically
stable.
More specific uniform cell-binding can be achieved by chemically crosslinking
specific
molecules, such as proteins, to reactable sites on the patterned substrate.
(Aplin and Hughes, Analyt.
Biochem. 113:144-148, 1981). Another elaboration of substrate patterning
optically creates an array of
reactable spots. A glass plate is washed with an organosilane that chemisorbs
to the glass to coat the glass.
The organosilane coating is irradiated by deep UV light through an optical
mask that defines a pattern of
an array. 'The irradiation cleaves the Si--C bond to form a reactive Si
radical. Reaction with water causes
the Si radicals to form polar silanol groups. The polar silanol groups
constitute spots on the array and are
further modified to couple other reactable molecules to the spots, as
disclosed in U.S. Pat. No. 5,324,591,
incorporated by reference herein. For example, a silane containing a
biologically functional group such as
a free amino moiety can be reacted with the silanol groups. The free amino
groups can then be used as
sites of covalent attachment for biomolecules such as proteins, nucleic acids,
carbohydrates, and lipids.
Other methods for patterning the adhesion of mammalian cells to surfaces using
self assembled
monolayers on a surface include Lopez et al. 1993 and Stenger et al., 1992.
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The non-patterned covalent attachment of a lectin, known to interact with the
surface of cells, to a
glass substrate through reactive amino groups has been demonstrated (Aplin &
Hughes, 1981). The
optical method of forming a uniform array of cells on a support requires fewer
steps and is faster than the
photoresist method, (i.e., only two steps), but it requires the use of high
intensity ultraviolet light from an
expensive light source.
In all of these methods the resulting array of cells is uniform, since the
biochemically specific molecules
are bound to the micro-patterned chemical array uniformly. In the photoresist
method, cells bind to the
array of hydrophilic spots and/or specific molecules attached to the spots
which, in turn, bind cells. Thus
cells bind to all spots in the array in the same manner. In the optical
method, cells bind to the array of
spots of free amino groups by adhesion. Methods .for attaching a variety of
cell types to the same
substrate for simultaneously binding against these cell types also exist.
Peptide-nucleic acids
In an alternative embodiment, peptide nucleic acids or oligomers, which are
analogs of nucleic
acids in which, for example, the peptide-like backbone is replaced with an
uncharged backbone, can be
used with the present invention. PNAs are well known in the art. References
below give extensive
reviews of the use of these nucleic acid analogs in a wide range of
applications, including surface and
array-based hybridization wherein PNAs are attached to surfaces and allowed to
bind with sequence-
complementary DNAs or RNAs.
For instance, oligomers of PNA can be used as the surface-attached probe
components instead of
DNA oligomers. A key advantage to using PNAs is that the hybridization
reaction with DNAs or RNAs,
for example, (containing charged phosphate groups) is only weakly dependent
(eg., the melting
temperature) on ionic strength because there is much less charge repulsion as
found with conventional
DNA-DNA, etc. hybridization. Thus, one can use the surface-selective nonlinear
optical technique to
follow a probe-target hybridization at any desired ionic strength.
The PNAs are commercially available (for instance via Applied Biosystems,
Foster City, CA) or
other analogs of DNA can be synthesized and used.
The following references are broad reviews of the use of PNAs.
Nielsen, et al. "Peptide nucleic acids (PNA): Oligonucleotide analogues with a
polyamide backbone"
Antisense Research and Applications (1992) 363-372
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Nielsen, et al. "Peptide nucleic acids (PNAs): Potential Antisense and Anti-
gene Agents." Anti-Cancer
Drug Design 8 (1993) 53-63
Buchardt, et al. "Peptide nucleic acids and their potential applications in
biotechnology" TIBTECH 11
(1993) 384-386
Nielsen, P.E., Egholm, M. and Buchardt, O. "Peptide Nucleic Acid (PNA). A DNA
mimic with a peptide
backbone" Bioconjugate Chemistry 5 (1994) 3-7
Nielsen "Peptide nucleic acid (PNA): A lead for gene therapeutic drugs"
Antisense Therapeutics 4 (1996)
76-84
Nielsen, P.E. "DNA analogues with nonphosphodiester backbones"
Annu.Rev.Biophys.Biomol.Struct. 24
(1995) 167-183
Hyrup, B. and Nielsen, P.E. "Peptide Nucleic Acids (PNA): Synthesis ,
Properties and Potential
Applications" Bioorg. Med. . 4 (1996) 5-23
Mesmaeker, A.D., Altman, K.-H., Waldner, A. and Wendeborn, S. "Backbone
modifications in
oligonucleotides and peptide nucleic acid systems" Curr. Opin. Struct. Biol. 5
(1995) 343-355
Noble, et al. "Impact on Biophysical Parameters on the Biological Assessment
of Peptide Nucleic Acids,
Antisense Inhibitors of Gene Expression" Drug.Develop.Res. 34 (1995) 184-195
Dueholm, K.L. and Nielsen, P.E. "Chemistry, properties, and applications of
PNA (Peptide Nucleie
Acid)" New J. Chem. 21 (1997) 19-31
Knudsen and Nielsen "Application of Peptide Nucleic Acid in Cancer Therapy"
Anti-Cancer Drug 8
(1997) 113-118
Nielsen, P.E. "Design of Sequence-Specific DNA-Binding Ligands" Chem. Eur. J.
3 (1997) 505-508
Corey "Peptide nucleic acids: expanding the scope of nucleic acid recognition"
TIBTECH 15 (1997) 224-
229
Nielsen, P.E. and Q~rum, H. "Peptide nucleic acid (PNA), a new molecular
tool." In Molecular Biology:
Current Innovations and Future Trends, Part2. Horizon Scientific Press, (1995)
73-89
Nielsen, P.E. and Haaima, G. "Peptide nucleic acid (PNA). A DNA mimic with a
pseudopeptide
backbone" Chem. Soc. Rev. (1997) 73-78
Q3rum, H., Kessler, C. and Koch, T. "Peptide Nucleic Acid" Nucleic Acid
Amplification Technologies:
Application to Disease Diagnostics (1997) 29-48
Wittung, P., Nielsen, P. and Norden, B. "Recognition of double-stranded DNA by
peptide nucleic acid"
Nucleosid. Nucleotid. 16 (1997) 599-602
Weisz, K. "Polyamides as artificial regulators of gene expression" Angew.
Chem. Int. Ed. Eng 36 (1997)
2592-2594
Nielsen, P.E. "Structural and Biological Properties of Peptide Nucleic Acid
(Pna)" Pure & Applied
Chemistry 70 (1998) 105-110
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Nielsen, P.E. "Sequence-specific recognition of double-stranded DNA by peptide
nucleic acids"
Advances in DNA Sequence-Specific Agents 3 (1998) 267-278
Nielsen "Antisense Properties of Peptide Nucleic Acid" Handbook of
Experimental Pharmacology 131
(1998) 545-560
Nielsen "Peptide Nucleic Acids" Science and Medicine (1998) 48-55
LThImann, E. "Peptide nucleic acids (PNA) and PNA-DNA chimeras: from high
binding affinity towards
biological function"Biol Chem 379 (1998) 1045-52
Wang "DNA biosensors based on peptide nucleic acid (PNA) recognition layers. A
review" Biosens
Bioelectron 13 (1998) 757-62
Uhlmann, E., Peyman, A., Breipohl, G. and Will, D.W. "PNA: Synthetic polyamide
nucleic acids with
unusual binding properties" Angewandte Chemie-International Edition 37 (1998)
2797-2823
Nielsen, P.E. "Applications of peptide nucleic acids" Curr Opin Biotechnol 10
(1999) 71-75
Bakhtiar, R. "Peptide nucleic acids: deoxyribonucleic acid mimics with a
peptide backbone" Biochem.
Educ. 26 (1998) 277-280
Lazurlcin, Y.S. "Stability and~specificity of triplexes formed by peptide
nucleic acid with DNA" Mol.
Biol. 33 (1999) 79-83
Nielsen and Egholm "Peptide Nucleic Acids: Protocols and Applications" (1999)
266 pp.
Eldrup and Nielsen "Peptide nucleic acids: potential as antisense and antigene
drugs" Adv. Amino Acid
Mimetics Peptidomimetics 2 (1999) 221-245
Bentin, T. and Nielsen, P.E. "Triplexes involving PNA" Triple Helix Form.
Oligonucleotides (1999) 245-
255
Falkiewicz, B. "Peptide nucleic acids and their structural modifications" Acta
Biochim. Pol. 46 (1999)
509-529.
The following references are descriptions of the use of PNAs in array-based
detection, including means
for attaching the PNA probes to the solid surface.
Hoffmann, R., et al. "Low scale multiple array synthesis and DNA hybridization
of peptide nucleic acids"
Pept. Proc. Am. Pept. Symp., 15th (1999) 233-234
Matysiak, S., Hauser, N.C., Wurtz, S. and Hoheisel, J.D. "Improved solid
supports and spacer/linker
systems for the synthesis of spatially addressable PNA-libraries" Nucleosides
Nucleotides 18 (1999)
1289-1291.
Decorators
In another aspect of the present invention, a "decorator" molecule or particle
is used to detect
probe-target binding reactions. A decorator molecule or particle will possess
a hyperpolarizability and
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can be used to reveal probe-target binding interactions via a surface-
selective nonlinear optical technique
(e.g., second harmonic generation) through the specific binding affinity it
will have for the targets, the
probes or the target-probe complex, or other species which recognize the
targets, the probes or the target-
probe-complex. The technique is useful when probe or targets are not
appreciably nonlinear optical
active (e.g., do not possess a hyperpolarizability). Decorators can
intrinsically possess a
hyperpolarizability or be themselves labeled with a moiety which is nonlinear-
optically active (e.g.,
second harmonic active). Decorators can be present during the probe-target
binding process, or added
afterwards to reveal the sites where binding has occurred. The decorator
molecule or particle can be
dissolved or suspended in the solution or aqueous phase containing the target
components - and it should
not appreciably alter or participate in the target-probe reaction.
An example of the invention is the case of proteins immobilized to a solid
substrate, either in a
microarray or patterned form, or uniformly across a surface - and with protein
composition either varying
or the same from site to site on the surface. At a given site, site A, protein
P (the probe) will be
immobilized. Protein K (the target) binds to protein P to form their complex,
KP. Also, a decorator
protein - Q- with an "SHG" label attached to it, has a specific binding
affinity for protein K. One can
introduce the substrate with immobilized proteins P to a solution containing
the targets (K); without K
bound to the surface, there is a small background SHG signal present. As K
binds to P, the amount of the
decorator Q (and the SHG Iabel) at the solid surface (and partially oriented
by it) will increase, leading to
an increase in the SHG optical signal intensity. The same type of measurement
can also be made in the
presence of drugs, antagonists, agonists, or any other compounds which
modulate the K-P binding
reaction (for example, the equilibrium constant). The measurements can be made
in real-time if
necessary. Furthermore, the decorator Q can be added to the solution some time
after K has been
introduced to the surface containing the probes P.
Another important use of the invention is in detection of DNA or other nucleic
acid or analog
binding. A single stranded probe is immobilized to a surface, a microsphere
bead at the distal end of a
fiber optic, for example. One is interested in probing a pool of unknown or
known strands for the amount
of sequence-complementary targets for a given probe sequence. The probe and
target strands are single
stranded, while their bound complex is double stranded. An nonlinear-active
(e.g., second-harmonic
active) decorator can be used in this case which intercalates within the DNA,
electrostatically binds to the
backbone phosphates, or both. For example, an SH-active intercalator which can
discriminate in its
intercalation binding between single and double-stranded DNA will produce the
desired affect: when a
complementary target binds to a probe, the amount of SH-active intercalator at
the sold surface will
increase, leading to an increase in the optical SH signal. In another example,
an SH-active decorator will
bind electrostatically to either single or double stranded DNA - the number of
decorators at the surface
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for the bound complex will be greater than the number for the single stranded
probe, since there will be
approximately twice as many phosphate groups available for interaction with
the decorator with the
double stranded probe-target complex. The decorator can be comprised of a
single moiety which
possesses both nonlinear optical activity such as being second harmonic active
and can interact
specifically (has an affinity) with the nucleic acids, for example through
intercalation, electrostatic
interaction, etc. Or, the decorator can be comprised of two or more moieties
in which one part is SH-
active and the other part possesses an affinity for the nucleic acids.
For example, well known molecules which can intercalate or electrostatically
bind to DNA, or
both, are as follows:
Psoralen
Ethidium bromide
Methanphosphonate
Phosophoramidites
Propidium iodide
Acridine
Acridine orange
9-amino acridine
Succinimidyl acridine-9-carboxylate
Cloroquine
Pyrine
Echinomycin
4',6-diamidino-2-phenylindole, dihydrochloride (DAPI)
Single-strand binding protein (SSB)
Tripyrrole peptides
Flavopiridol
Pyronin Y
Hoechst 33258
Bisbenzimide
This list is illustrative and is not intended to be limiting in scope. SH-
active moieties can be
linked, covalently bound or otherwise bonded to, by well-known means available
to one skilled in the art
of synthetic organic chemistry, to any of the above listed compounds to
produce a decorator compound
which has both specificity for nucleic acids and a nonlinear optical activity.
It is also desirable to use a
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decorator which is not intrinsically fluorescent, either due to the SH-active
moiety or the nucleic-acid
affinity moiety.
DEFINITIONS
The following terms used throughout the present specification 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
sometimes in the 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): 'This term includes any naturally occurring or
modified particles or
molecules found in biology, or those molecules and particles which are
employed in a biological
study, including probes and targets. 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 used with the present invention include, but are not restricted to,
antagonists or agonists for
cell membrane receptors, toxins and venoms, viral epitopes, hormones, hormone
receptors, 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 a chemical affinity for a given ligand.
Receptors can be naturally
occurring or man-made molecules. Also, they can be used in an unaltered state
or as aggregates with
other biological components. Receptors can 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
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"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 can 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 can lead to the development of vaccines of which
the immunogen is based on
one or 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 can 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
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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 can
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: This term refers to a non-linear optical technique such
as second harnionic
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.
6. Array or Microarray: 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 restricted to, two-dimensional microarrays and other patterned
samples. Other terms in the
art which are often used interchangeably for 'array' include: gene chip, gene
array, biochip, DNA
chip, protein chip and microarray, the latter being an array with elements of
the array (patterned areas
with attached probes) whose dimensions are on the order of microns.
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. EFISH (Electric-field induced second harmonic generation) or Hyper-
Rayleigh scattering 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~2~(w3) =
x~z~ (-~3; W ,w2) : Ew' Ewa.
The effect can be used to measure the hyperpolarizabilty of molecules in
solution by using a do field
to induce alignment in the medium, and allowing SHG to be observed. This is
sometimes called the
reorientational mechanism.
8. Linker: 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,
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targets to nonlinear-active derivatized particles, surface layers to targets,
surface layers to nonlinear-
active particle or moieties, etc. A linker can, for example, 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 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. Probe: Refers herein to biological components (eg., cells, proteins,
virus, ligand, small molecule,
drugs; oligonucleotides, DNA, RNA, cDNA, etc.) which are attached to a surface
(e.g., solid
substrate, cell surface, liposome surface, etc.), or are cells, lipsomes,
particles, beads or other
components which comprise a surface e.g. freely suspended in some medium in a
sample cell. (In
some literature in the art, this term refers to the free components which are
tested for binding against
the probes).
12. Target: Refers herein to biological components which are unbound to the
probes' surface or surfaces
comprising attached probes, and which may bind to probes.
13. Attached (Attach): 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 to, for example, a solid substrate, a cell surface, a
liposome surface, a gel
substrate, etc.; or the probes are found naturally 'attached' to a surface
such as in the example of
native membrane receptors embedded in cell membranes, tissues, organs (in-
vitro or in-vivo). In
some instances herein, the word 'attached' or 'attach' refers also to the
chemical or physical
attachment of a label to a target or decorator. Also referred to herein as
'surface-attached'.
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14. Centrosymmetric: A molecule or material phase is centrosymmetric if there
exists a point in
space (the 'center' or 'inversion center') through which an inversion (x,y,z) -
~ (-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.
Centrosymmetric molecules or
materials have no nonlinear susceptibility or hyperpolarizability, necessary
for second harmonic, sum
frequency and difference frequency generation.
15. 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.
16. Decorator: Refers to a nonlinear active molecule or particle (possesses a
hyperpolarizability)
which can be bound to targets, probes or target-probe complexes in order to
allow the detection and
discrimination between them. A decorator should not appreciably alter or
participate in the target-
probe reaction itself. The decorator can be dissolved or suspended in the
solution or aqueous phase
containing the target component. A decorator is distinguished from an SH-
active label (J.S. Salafsky,
co-pending application 'SH-labels...') for its specific binding affinity for
targets, probes, or the target-
probe complex. In the art (J.S. Salafsky K.B. Eisenthal, co-pending
application 'SHG labels...'), an
SHG-label is attached to a biological component - via specific chemical bonds
or non-specific (e.g.,
electrostatic) means - and then used to follow that component to an interface.
A decorator can be
used to detect probe-target complexes by its specific binding affinity (in
other art, 'molecular
recognition' to the targets, probes or the target-probe complexes.
17. 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 less
or none for other
proteins. In some art, the term 'molecular recognition' is used to describe
the binding affinity
between components.
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18. 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
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.
19. bipolar: Defined herein as possessing an electric dipole or 'dipole
moment' on a particle or molecule,
which takes the standard definition known to one skilled in the art: the sum
of all vectors w = 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.
20. 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.
21. Hyperpolarizability or Nonlinear Susceptibility: The properties of a
molecule, particle, interface or
phase which allow for generation of the nonlinear light. Typical equations
describing the nonlinear
interaction for second harmonic generation are: a~2~(2~) _ (3:E(e~)~E(w) or
P~2~(2e~) = x~2~:E(~)E(w)
where a and P are, respectively, the induced molecular and macroscopic dipoles
oscillating at
frequency 2~, (3 and x~2~ are, respectively, the hyperpolarizability and
second-harmonic (nonlinear)
susceptibility tensors, and E(co) is the electric field component of the
incident radiation oscillating at
frequency w. 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., w1
and w2) and the nonlinear
radiation oscillates at the sum or difference frequency (w1 ~ e~z). 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
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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.
22. Polarization: The net dipole per unit volume (or area) in a region of
space. The polarization can be
time-dependent or stationary. Polarization is defined as: f p,(R) dR where an
integration of the net
dipole is made over all volume elements in space dR near an interface.
23. 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. Also referred to herein as 'waves', 'signal' or 'nonlinear
signal', 'beams', 'light'.
24. Near-field techniques: Those techniques known in the 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.
25. Detecting, Detection: When referring herein to nonlinear optical methods,
refers to those techniques
by which the properties of surface-selective nonlinear optical radiation can
be used to detect, measure
or correlate properties of probe-target interactions, or effects of the
interactions, with properties of the
nonlinear optical light (e.g., intensity, wavelength, polarization or other
property common to
electromagnetic radiation).
26. Interface: For the purpose of this invention, the interface can be defined
as a region which generates
a nonlinear optical signal or the region near a surface in which there are
nonlinear-active labeled
targets possessing a net orientation. Art interface can also be composed of
two surfaces, a surface in
contact with a different medium (e.g., a glass surface in contact with an
aqueous solution, a cell
surface in contact with a buffer), the region near the contact between two
media of different physical
or chemical properties, etc.
27. Conjugated, Coupled: Refers herein to the state in which one particle,
moiety or molecule is
chemically bonded, covalently or non-covalently linked or by some means
attached to a second
particle moiety, molecule, surface or substrate. These means of attachment can
be via electrostatic
forces, covalent bonds, non-covalent bonds, physisorption, chemisorption,
hydrogen bonds, van der
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Waal's forces or any other force which holds the probes with a binding energy
to the substrate (a
corallery to this definition is that some force is required to separate the
probes held by the substrate
from the substrate).
2~. Reactions: Refers herein to chemical, physical or biological reactions
including, but not limited to,
the following: probes, targets, inhibitors, small molecules, drugs,
antagonists, antibodies, etc. The
term 'effects of reactions' or 'effects of said reactions' refers herein to
physical or chemical effects of
the probe-target reactions: for example, the probe-target reactions can
comprise a ligand-receptor
binding reaction which leads, in turn, to an ion channel opening and a change
in the surface charge
density of a cell, the latter being then detected by the nonlinear optical
technique. The effects of the
probe-target reactions, or the probe-target reactions themselves, might be
referred in some art as a
'second messenger' reaction. Also referred to herein as 'interactions'.
29. Surface layer: Refers herein to a chemical layer which functionally
derivatizes the surface 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.
30. Delivery, Illumination, Collection: In the context of manipulation of
optical radiation (e.g., light
beams), delivery and illumination refer herein to the guiding of the
fundamental beam to the interface
or regions of interest at an interface; collection refers to the optical
collection of the nonlinear light
produced at the interface (e.g., second harmonic light).
31. Inhibitor, inhibiting: Defined herein as moieties, molecules, compounds or
particles which bind to
probes in competition with targets; the probe-target interactions are
decreased or prevented in the
presence of an inhibitor compound, molecule or particle. Blocking agents
refers herein to those
compounds, molecules, moieties or particles which prevent probe-target
interactions (e.g., binding
reactions between probes and targets).
32. Agonist: Defined herein as moieties, molecules, compounds or particles
which activate an
intracellular response when they bind to a receptor.
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33. Antagonist: Defined herein as moieties, molecules, compounds or particles
which competitively bind
to a receptor on a cell surface at the same site as agonists, but which do not
activate the intracellular
response initiatied by the active form of the receptor (e.g., activated by
agonist binding), and can
thereby inhibit the intracellular responses of agonists or partial agonists.
Antagonists do not diminish
the baseline intracellular response in the absence of an agonist or partial
agaonist.
34. Partial Agonist: Defined herein as moieties, molecules, compounds or
particles which activate the
intracellular response when they bind to a receptor on the cell surface to a
lesser degree or extent than
do agonists.
35. Interactions: Defined herein as some physical or chemical reaction or
interaction between
components in a sample. For example, the interactions can be physico-chemical
binding reactions
between a probe and a target, dipole-dipole attraction or repulsion between
two molecules, van der
Waals interactions between two atomic or molecular species, a chemical
affinity interaction, a
covalent bond between molecules, a non-covalent bond between molecules, an
electrostatic
interaction (repulsive or attractive), a hydrogen bond and others.
36. Effects: Defined herein as the measurable properties of probe-target
interactions or the consequences
of the interactions (e.g., secondary reactions, ion channel opening or
closing, etc.). These include, the
following properties, for example:
i) the intensity of the nonlinear or fundamental light.
ii) the wavelength or spectrum of the nonlinear or fundamental light.
iii) position of incidence of the fundamental light on the surface .or
substrate (e.g., for
imaging).
iv) the time-course of either i), ii) or iii).
v) one or more combinations of i), ii), iii) and iv).
37. Time-course: Refers herein as the change in time of some measurable
experimental
such as light intensity or wavelength of light. Also referred to as 'kinetics'
of some probe-target
interaction, or probe-target-other component interaction for example.
38. Well-defined: In the context of 'well-defined direction', refers herein to
the deterministic
scattering of light (fundamental or nonlinear beams) from a substrate. By
contrast, for
example, fluorescence emission is emitted at somewhat random directions.
39. Sample: Contains the probes, targets or other molecules, particles or
moieties under study by
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the invention. The sample contains at least one interface capable of
generating the nonlinear optical
light, with said interface comprised of at least one surface containing
attached probes. Examples of
components of samples include prisms, wells, microfluidics, substrates, buffer
with targets, drugs in
buffers, surfaces with attached probes. The terms 'substrate' and 'surface'
are often used
interchangeably herein. In some cases, the term 'support' can be construed to
mean 'surface'.
40. Modulator, Modulates: 'This term refers herein to any substance, moiety,
molecule, biological
component or compound which influences the kinetic or equilibrium properties
of probe-target
interactions (e.g., binding reaction). Modulators may change the rate of probe-
target binding, the
equilibrium constant of probe-target binding or, in general, enhance or reduce
probe-target
interactions. Examples of modulators are the following: inhibitors, drugs,
small molecules, agonists
and antagonists.
GENERALSCHEME
A general scheme for measuring probe-target interactions or their effects
using the present invention is as
follows:
i) Illuminate the sample with light capable of undergoing second harmonic
light; in the
absence of either probes or targets, or both, the intensity and/or spectrum
and/or
timecourse of either or both intensity or spectrum of the second harmonic
light can serve
as a background or baseline.
ii) Mix probes, targets, probes and targets, drugs, etc., or other components,
which can
modulate the probe-target interactions or their effects (at the same time or
at separate
times) and measure the resulting second harmonic light intensity andlor
spectrum (or as a
function of time, i.e., timecourse). This measured information serves as the
signal for the
desired interaction.
iii) A direct, optical read-out of the measured information can be performed
or, optionally,
the measured information can be modeled to determine, for example, kinetic or
equilibrium properties of the probe-target interactions, with or without
blocking agents,
inhibitors, agonist, antagonist, etc.
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PREFERRED EMBODIMENT
In a preferred embodiment of the invention, the amine-reactive oxazole dye
(SE) 1-(3-
(succinimidyloxycarbonyl) benzyl)-4-(5-(4-methoxyphenyl) oxazol-2-
yl)pyridinium bromide (PyMPO,
SE: Molecular Probes Corp.) is reacted with a 1:1 molar r-atio of
ethylenediamine under the conditions
specified by the Molecular Probes direction and is allowed to react to
completion. The oxazole-based dye
now contains a single amine group. This product is then reacted with the 5'-
phosphate end of an
oligonucleotide using the cross-linker EDAC (ethyl dimethylaminopropyl
carbodiimide) according to
Molecular Probes protocol resulting in a phosphoramidate bond between the dye
and the oligonucleotide.
Alternatively, if the oligonucleotides are chemically synthesized, an amine
group can be incorporated at
the 5'-end and this can be reacted directly with PyMPO, SE by following
protocols published by
Molecular Probes Corp.
DNA microarrays can be obtained commercially or constructed according to
public literature (eg.,
http://cmgm.stanford.edu/pbrown/mguide/index.html). The surface chemistry to
be used is that found in
Chrisey, L.A. et al. (1996) in which oligonucleotides are attached to self
assembled monolayer silane
films on fused silica slides. Silanization is done via N (2-aminoethyl)-3-
aminopropyltrimethoxysilane.
Hybridization against labeled targets'is achieved using standard protocols
found in the prior art, for
example as found in: Ramsay, G. DNA chips - states-of the-art. Nature
Biotechnology 1998, 16(1), 40-
44; Marshall, A.; Hodgson, J. DNA chips - an array of possibilities. Nature
Biotechnology 1998, 16(1),
27-31; S.A. Fodor, Science 277 (1997), 393; M. Schena et al., Science 270
(1995), 467 and references
contained therein.
The DNA microarray chip is mounted on an x-y translation stage and driven by
personal computer (PC
control) using a motorized translator (acquired from Oriel, Inc.) or using one
of the many procedures in
the art (eg., V.G. Cheung et al., 1999).
Drawing 1 illustrates the nonlinear optical apparatus of the present
invention. A femtosecond pulsed laser
(5) (Spectra-Physics Corp.) for example, operating at 800 nm at 80 MHz with
sub-100 fs pulses at > 0.5
W average power is used as the source of the fundamental light [alternatively,
a l OW Argon ion laser
(Coherent Corp.) can be used to pump a Tiaapphire oscillator (Lexel Corp.) to
produce the femtosecond
or picosecond pulses of light]. The polarization of the fundamental can be
optionally selected using a
half wave plate (10) (Melles Griot, 16 MLB 751) and focused tightly by a lens
(15) on to a color filter
(20) (CVI Corp., LP 780) designed to pass the fundamental light but block the
nonlinear light (eg., the
second harmonic). The pass filter can be an interference filter, color filter,
etc. and its purpose is to
prevent the second harmonic light from entering the laser cavity and causing
disturbances in the lasing
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32
properties. The fundamental is then reflected from a mirror (25) and impinges
at a specific location and
with a specific angle on the sample surface (30). The beam diameter at the
substrate surface is about 100
microns. The mirror (25) is scanned using a galvanometer-controlled mirror
scanner, a rotating polygonal
mirror scanner, a Bragg diffractor, acousto-optic deflector, or other means
known in the art to allow
control of a mirror's position. For instance, the incident angle and direction
of the fundamental light on
the sample surface can be varied in a known manner by the use of a precision
adjustable mirror mount
and a polygonal mirror using a Piezoelectric mirror mount 17 ASM 001- Melles-
Griot and Mirror 02
MLQ 011/003, Melles-Griot) and driving it through the use of a stepper driven
motor (17PCS001 and
17PCC001 Melles-Griot) and PC control. A galvanometer mirror or any other PC-
controllable beam
deflection optic can be used. For example, 16-bit galvo positioners are
available from General Scanning,
Inc. and Cambridge Research which use closed loop servo-control systems to
achieve precise alignment
control.
The silica sample surface (30) is mounted on an x-y translation stage (35)
(made from stacked
linear stages, Newport Corp., PM500-L and computer controlled) to select a
specific location on the
surface for generation of the second harmonic beam. Although Fig. 1 depicts a
dry sample, the sample
surface can be enclosed and in contact with liquid or buffer. The second
harmonic beam intensity
depends on a number of factors, including the peak electric field intensity of
the fundamental which, in
turn, is related to the temporal width of the pulse. Accordingly, it can be
important to use 'fast' mirrors or
lenses to minimize the dispersion of the pulse. The fundamental and second
harmonic beams are
scattered in well-defined directions from the silica surface. Because of this,
a minimum of optics are
required to collect the second harmonic light, unlike the case with
fluorescence detection in which the
fluorescence is emitted isotropically. The fundamental light is filtered using
a color filter leaving only the
second harmonic light.
The second harmonic is reflected from mirror (40) (For example: O1 MFG
033/023, Melles-Griot
Corp.), sent through a pass-filter (45) (CVI Corp., BG 39) to pass the second
harmonic while blocking the
fundamental, and its polarization selected, if necessary, by a polarizing
optic (50), then focusing the beam
using a lens (55) onto a monochromator (CVI Laser Corp., CM 110) and on to a
photomultiplier tube
(PMT) (Hamamatsu 928P or R2658P, power supply C3830) (60). The PMT
photocurrent is fed to a
photon counting unit (Hamamatsu, C3866) which discriminates the signal and
converts the photoelectron
pulses from the PMT into 5 V digital signals which are fed, in turn, to a
photon counting board for a PC
(Hamamatsu, M7824) and controlled using Labview software and drivers. The beam
diameter will be on
the order of one element in the microarray, and the XY-location of the beam
can be determined from the
position of the scanning mirror and a feedback loop to the control PC; a map
of the intensity of nonlinear
light vs. the microarray surface location can be determined. Real-time data
can also be obtained in the
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33
same manner, either at a single region within the array, or bacle-and-forth
scanning over time between
regions so as to sample the same regions over a period of time. In this
manner, an intensity image and its
time-dependence can be acquired for any or all regions in the microarray.
Given a fixed detector and
incident angle and direction and stage position, the position of the
reflecting mirror can be used to
determine the region of the array under illumination since the nonlinear beam
will have a well-defined
direction with respect to the surface. Alternatively, a CCD array detector can
be used and controlling the
translation stage, a mirror scanner or both and correlating their positions
with the measured signal of the
photodiode elements of a CCD array is disclosed in U.S. Pat. No. 6,084,991.
By translating either the stage or changing the incident position of the
fundamental light, or some
combination thereof, an image of second harmonic intensity from the entire
array surface can be built up,
assigning intensity of second harmonic light to different regions or elements
within the array using a
standard software program such as Labview (Labview, Inc.) or other software.
'The square root of the
second harmonic intensity is proportional to the concentration of labelled
targets which are hybridized to
the probes.
ALTERNATIVE EMBODIMENTS
In an alternative embodiment, the microarray can be in contact with, attached
to, or directly
patterned on, a prism capable of allowing total internal reflection at the
interface containing the probes.
Thus, in this mode, the fundamental beam would undergo total internal
reflection at the interface
containing the probes and its evanescent wave would be used to generate the
nonlinear light. Figure 2
illustrates an embodiment of this type. In Figure 2, an index matching
material or liquid (75) is used to
couple the prism (70) to a substrate containing the microarray (80) in contact
with solution containing
targets (85), whereby total internal reflection occurs at the interface
between material (80) and solution
(85). The prism material can be, for example, BK7 type glass (Melles Griot)
and the index matching
material obtained commercially from Corning Corp. or Nye Corp.
In an alternative embodiment, the experimental set-up is as described in
Salafsky and Eisenthal,
2000 and references set forth therein. A femtosecond pulsed laser (Mail-Tai,
Spectra-Physics) is used as
the source of fundamental light at 800 nm operating at 80 MHz with <200 fs
pulses at 1 W average
power. The laser beam is focused with a concave lens (Oriel) (spot size ~ 1
mm~) on to the entrance
aperture of a Dove prism (Melles Griot, BK-7) which is mounted in a teflon
holder and in contact with
solution (10 mM phosphate buffer, pH 7) or distilled water. The beam undergoes
total internal reflection
(evanescent wave generation) within the prism and the fundamental and second
harmonic beams emerge
roughly collinearly from the exit aperture. A color filter is used to block
the fundamental light while
passing the second harmonic to a monochromator (2 nm bandwidth slit).
°The monochromator is scanned
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34
from 380 - 500 nm to detect the second harmonic spectrum. If necessary, the
fundamental light
wavelength can be tuned as well. A single photon counting detector and
photomultiplier tube are used to
detect the output of the monochromator and a PC with software are used to
record the data and control the
monochromator wavelength. A background second harmonic signal is measured.
In an alternative embodiment, a planar waveguide structure 110 is used for the
solid substrate
(Figure 3). In this embodiment, a thin layer of high index of refraction
material 115 (the waveguide),
such as Ti02 or Ta 205, is deposited on top of the substrate 110 (typically
glass). A thin diffraction
grating 115 is scribed into this waveguide and light from the laser 100 is
coupled using this grating into
the waveguide. Second harmonic light can be collected using lenses and filters
and detected with either a
PMT-type device or a CCD camera.
FIGS. 4a-4c illustrate an embodiment of a flow cell for carrying out probe-
target reactions. The flow cell
is 3220 is shown in detail. FIG. 4a is a front view, FIG. 4b is a cross
sectional view, and FIG. 4c is a back
view of the cavity. Referring to FIG. 4a, flow cell 3220 includes a cavity
3235 on a surface 4202 thereon.
The depth of the cavity, for example, may be between about 10 and 1500 µm,
but other depths may be
used. Typically, the surface area of the cavity is greater than the size of
the probe sample, which may be
about l3×l3 mm. Inlet port 4220 and outlet port 4230 communicate with
the cavity. In some
embodiments, the ports may have a diameter of about 300 to 400 µm and are
coupled to a refrigerated
circulating bath via tubes 4221 and 4231, respectively, for controlling
temperature in the cavity. The
refrigerated bath circulates water at a specified temperature into and through
the cavity.
A plurality of slots 4208 may be formed around the cavity to thermally isolate
it from the rest of the flow
cell body. Because the thermal mass of the flow cell is reduced, the
temperature within the cavity is more
efficiently and accurately controlled.
In some embodiments, a panel 4205 having a substantially flat surface divides
the cavity into two
subcavities. Panel 4205, for example, may be a light absorptive glass such as
an RG1000 nm long pass
filter. The high absorbance of the RG1000 glass across the visible spectrum
(surface emissivity of
RG1000 is not detectable at any wavelengths below 700 nm) substantially
suppresses any background
luminescence that may be excited by the incident wavelength. The polished flat
surface of the light-
absorbing glass also reduces scattering of incident light, lessening the
burden of filtering stray light at the
incident wavelength. The glass also provides a durable medium for subdividing
the cavity since it is
relatively immune to corrosion in the high salt environment common in DNA
hybridization experiments
or other chemical reactions.
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Panel 4205 may be mounted to the flow cell by a plurality of screws, clips,
RTV silicone cement, or other
adhesives. Referring to FIG. 4b, subcavity 4260, which contains inlet port
4220 and outlet port 4230, is
sealed by panel 4205. Accordingly, water from the refrigerated bath is
isolated from cavity 3235. This
design provides separate cavities for conducting chemical reaction and
controlling temperature. Since the
cavity for controlling temperature is directly below the reaction cavity, the
temperature parameter of the
reaction is controlled more effectively.
Substrate 130 is mated to surface 4202 and seals cavity 3235. Preferably, the
probe array on the substrate
is contained in cavity 3235 when the substrate is mated to the flow cell. In
some embodiments, an O-ring
4480 or other sealing material may be provided to improve mating between the
substrate and flow cell.
Optionally, edge 4206 of panel 4205 is beveled to allow for the use of a
larger seal cross section to
improve mating without increasing the volume of the cavity. In some instances,
it is desirable to maintain
the cavity volume as small as possible so as to control reaction parameters,
such as temperature or
concentration of chemicals more accurately. In additional, waste may be
reduced since smaller volume
requires smaller amount of material to perforni the experiment.
Referring back to FIG. 4a, a groove 4211 is optionally formed on surface 4202.
The groove, for example,
may be about 2 mm deep and 2 mm wide. In one embodiment, groove 4211 is
covered by the substrate
when it is mounted on surface 4202. The groove communicates with channel 4213
and vacuum fitting
4212 which is connected to a vacuum pump. The vacuum pump creates a vacuum in
the groove that
causes the substrate to adhere to surface 4202. Optionally, one or more
gaskets may be provided to
improve the sealing between the flow cell and substrate.
FIG. 4d illustrates an alternative technique for mating the substrate to the
flow cell. When mounted to the
flow cell, a panel 4290 exerts a force that is sufficient to immobilize
substrate 130 located therebetween.
Panel 4290, for example, may be mounted by a plurality of screws 4291, clips,
clamps, pins, or other
mounting devices. In some embodiments, panel 4290 includes an opening 4295 for
exposing the sample
to the incident light. Opening 4295 may optionally be covered with a glass or
other substantially
transparent or translucent materials. Alternatively, panel 4290 may be
composed of a substantially
transparent or translucent material.
In reference to FIG. 4a, panel 4205 includes ports 4270 and 4280 that
communicate with subcavity 3235.
A tube 4271 is connected to port 4270 and a tube 4281 is connected to port
4280. Tubes 4271 and 4281
are inserted through tubes 4221 and 4231, respectively, by connectors 4222.
Connectors 4222, for
example, may be T-connectors, each having a seal 4225 located at opening 4223.
Seal 4225 prevents the
water from the refrigerated bath from leaking out through the connector. It
will be understood that other
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configurations, such as providing additional ports similar to ports 4220 and
4230, may be employed.
Tubes 4271 and 4281 allow selected fluids to be introduced into or circulated
through the cavity. In some
embodiments, tubes 4271 and 4281 may be connected to a pump for circulating
fluids through the cavity.
In one embodiment, tubes 4271 and 4281 are connected to an agitation system
that agitates and circulates
fluids through the cavity.
Referring to FIG. 4c, a groove 4215 is optionally formed on the surface 4203
of the flow cell. The
dimensions of groove, for example, may be about 2 mm deep and 2 mm wide.
According to one
embodiment, surface 4203 is mated to the translation stage. Groove 4211 is
covered by the translation
stage when the flow cell is mated thereto. Groove 4215 communicates with
channel 4217 and vacuum
fitting 4216 which is connected to a vacuum pump. The pump creates a vacuum in
groove 4215 and
causes the surface 4203 to adhere to the translation stage. Optionally,
additional grooves may be formed
to increase the mating force. Alternatively, the flow cell may be mounted on
the translation stage by
screws, clips, pins, various types of adhesives, or other fastening
techniques.
In a further alternative embodiment, a suspension of beads, cells, liposomes
or other objects are
the probes (130), or comprise probes attached thereto, as shown in Figure 5.
The scattered nonlinear light
from such a sample - eg., an isotropic sample in which each individual beads
or other objects are about a
coherence length or farther apart - is generated in all directions with some
distribution in intensity.
Fundamental light is transmitted through the suspension (130) and the
nonlinear radiation collected. A
number of modes of collecting the scattered nonlinear light are available. For
example, collection of the
second harmonic can be in the forward direction (A), at a right angle to the
fundamental light (B), or
using an integrating sphere approach (C). Part C shows an integrating sphere
165 with the sample 150
placed inside. Fundamental light (145) enters the entrance port (170), passes
through the sample (150),
undergoes a reflection at the sphere wall, and is stopped by baffle (175). The
scattered second harmonic
light is collected from the sphere surface through exit port (155) and coupled
out of the sphere by a fiber
optic line (160). Beads can support phospholipid bilayers (eg., with membrane
proteins) or probes such
as proteins or nucleic acids can be attached to their surface. The beads
provide a large amount of
distributed surface area in the sample and can be a useful alternative to
planar surface geometries,
especially when the fundamental and nonlinear light is used in the
transmission mode.
In an alternative embodiment (Figure 6), the excitation light is tranformed
from a point-like shape into
some other shape using various optics. For instance, the point-like beam shape
of the fundamental beam
can be transformed into a line shape, useful for scanning the sample surface.
However, because the
intensity of the nonlinear beam depends on, among other factors, the intensity
of the fundamental
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(typically a quadratic dependence on the fundamental intensity), this
transformation will result in less
nonlinear light intensity generated at a given location. To generate a line-
shape in the fundamental
(which can typically be a round point of ~ 2 mm diameter), one can direct the
fundamental beam into a
microscope objective which has a magnification power of about 10 followed by a
150 mm achromat to
collimate the beam as well known in the prior art and as disclosed in detail
in U.S. Pat. No. 5,834,758.
As shown in Figure 6, the fundamental light 180 is a beam of typically 2-3 mm
diameter. This beam is
directed through a microscope objective 185. The objective, which has a
magnification power of 10,
expands the beam to about 30 mm. The beam then passes through a Iens 190. The
Iens, which can be a
150 mm achromat, collimates the beam. Typically, the radial intensity of the
expanded collimated beam
has a Gaussian profile. To minimize intensity variations in the beam, a mask
195 can be inserted after
lens 190 to mask the top and bottom of the beam, thereby passing only the
central portion of the beam. In
one embodiment, the mask passes a horizontal band that is about 7.5 mm.
Thereafter, the beam passes
through a cylindrical lens 200 having a horizontal cylinder axis, which can be
a 100 mm f.1. made by
Melles Griot. The cylindrical lens expands the beam spot vertically.
Alternatively, a hyperbolic lens can
be used to expand the beam vertically while resulting in a flattened radial
intensity distribution. From the
cylindrical lens, the light passes through a lens 205. Optionally, a planar
mirror can be inserted after the
cylindrical lens to reflect the excitation light toward lens 205. To achieve a
beam height of about 15 mm,
the ratio of the focal lengths of the cylindrical lens 200 and lens 205 is
approximately 1:2, thus
magnifying the beam to about 15 mm. Lens 205, which in some embodiments is a
80 mm achromat,
focuses the light to a line of about 15 mm.x 50 microns at the sample surface
210.
In an alternative embodiment shown in Figure 7, probes patterned in a two-
dimensional array (A,
top view of array on surface) where each region on the surface - f 1,35 ~ in
this example - can be a
different oligonucleotide or protein sequence (or a combination of the same
and different sequences) and
labeled targets are used to detect binding. Part B shows a side-view of the
sample surface (220) in a well
(215) containing the targets (225) shown here as protein objects with second-
harmonic-active labels (X)
attached. The well can hold liquid or buffer and serves to physically separate
the contents of the well
from other parts of the substrate or other elements in a substrate array. The
fundamental light can be
multiplexed and each resultant beam can be guided by individual mirrors to
simultaneously scan different
lines or regions within the array, thus increasing even further the potential
of the technique for high-
throughput studies.
In an alternative embodiment, the method of Levicky et al. or the method of
L.A. Chrisey et al. is
used to attach the probe DNA to the substrate. In the method of Chrisey as
illusrated in Figure 8, a fused
silica or oxidized silicon substrate is used (230) and derivatized with N (2-
aminoethyl)-3-
aminopropyltrimethoxysilane (EDA) (235). In one embodiment, the EDA-modified
surface is then
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38
treated with the heterobifunctional crosslinker (SMPB), whose succinimide
ester moiety reacts with the
primary amino group of EDA (240). A thiol-DNA oligomer subsequently (245) of
base-pair sequence
(xzzy) (where 'xzzy' represents the entire sequence) reacts with the maleimide
portion of the SMPB
crosslinker, to yield the covalently bound species shown (250).
In an alternative embodiment, elements in the surface array are physically
separated as illustrated in
Figure 9, allowing for different targets, target solutions, etc. to be added
selectively to any or all of the
elements. Part (A) is a top-view of the substrate (255) with partitions or
walls (260) separating the
different well regions - in this example, 16 wells. Part (B) shows a side-view
of a well (265) with
attached probes (270). Such arrays are commonly found in the art, such as the
96-well plates, etc. and are
commercially available (Fisher Scientific, Inc. etc.)
In an alternative embodiment, a glass substrate surface can be coated with a
layer of a reflective
metal such as silver. The metallic layer will increase nonlinear optical
generation and collection.
Biomolecules or other particles can be attached to derivatized layers built on
top of the metal. For
instance, the metal can be coated with a layer of silicon dioxide (Si02), then
with a layer of aminosilane
such as 3-amino-octyl-trimethoxysilane. Oligonucleotides or polynucleotides
can then be attached to the
aminosilane layer using linkers which connect the 3' or 5' end of the oligo to
the amine group.
Alternatively, the oligos or polynucleotides can be adsorbed to the
aminosilane layer. Figure 10
illustrates an embodiment of this type where a glass substrate (275) is
derivatized with a Ag layer (280).
A thin coat of Si02 is then deposited on top of the silver layer (285) and
derivatized with the aminosilane
(290).
In an alternative embodiment, bead-based fiber-optic arrays can be used (ref.
34) in which light
beams (eg., fundamental and second harmonic) travel via total internal
reflection along the path of the
fiber. The fundamental light is coupled into the bundle or individual optical
fibers and second harmonic
light is generated at the tip surface and collected back through the fiber. In
this embodiment, individual
optical fibers can be converted into DNA sensors by attaching a DNA probe to
the distal tip (ref. 17,18)
or by removing the cladding of the optical fiber and attaching the DNA probe
to the outside of the core
(ref. 19-22). Simple DNA arrays can be made from such optical fibers by
physically bundling multiple
fibers together (ref. 23). There are many variations on this theme, for
example by selectively etching the
distal-end cladding to create wells of different depths at the distal end of
the fiber, where the tip of the
fiber constitutes the bottom of the wells (ref. 24). Latex or silica beads can
then be loaded into the wells
(ref. 25). Fiber-optic oligonucleotide arrays can be prepared by attaching DNA
probes to microspheres
and then filling each well with a microsphere carrying a different DNA probe.
Each different type of
microsphere is tagged with a unique combination of fluorescent dyes or DNA
probes either before or after
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39
probe attachment (refs. 26,27). 'Zip codes' for universal fabrication (ref.
29) and molecular beacons
(refs. 28,30)for label-less detection can also be used with the optical sensor-
beaded arrays. Figure 11
illustrates a fiber-optic bundle array. Part (A) shows a bundle of fiber optic
cables (295) with wells at the
distals ends for placement of beads (300). Part (B) shows a close-up view of a
single optical fiber.
Fundamental light travels (c~) toward the distal end with the bead (305). Some
fundamental light is
scattered back from the bead along with second harmonic light (2c~) and
travels back through the fiber to
the proximal end where an optical train and detection system (not shown)
separates the fundamental
radiation from the second harmonic radiation. Bead (310) is covered with
attached probes.
In an alternative embodiment, the detector (65) of the nonlinear radiation in
Figure 1 is a
photomultiplier tube operated in single-photon counting mode. Photocurrent
pulses can be voltage
converted, amplified, subjected to discrimination using a Model SR445 Fast
Preamplifier and Model SR
400 Discriminator (supplied by Stanford Research Systems, Inc.) and then sent
to a counter (Model 3615
Hex Scaler supplied by Kinetic Systems). Photon counter gating and galvo
control through a DAC output
(Model 3112, 12-Bit DAC supplied by Kinetic Systems) can be synchronized using
a digital delay/pulse
generator (Model DG535 supplied by Stanford Research Systems, Inc.).
Communication with a PC
computer 29 can be accomplished using a parallel register (Model PR-604
supplied by DSP
Technologies, Inc.), a CAMAC controller card (Model 6002, supplied by DSP
Technologies, Inc.) and a
PC adapter card (Model PC-004 supplied by DSP Technologies, Inc.).
In an alternative embodiment, a bandpass, notch, or color filter is placed in
either or all of the
beam paths (eg., fundamental, second harmonic, etc.) allowing, for example,
for a wider spectral
bandwidth or more light throughput.
In an alternative embodiment, an interference, notch-pass, bandpass,
reflecting, or absorbant filter
can be used in place of the filters in the figures in order to either pass or
block the fundamental or
nonlinear optical beams.
According to another embodiment, detection of the nonlinear optical light is
achieved using a charge
coupled detector (CCD) in place of a photomultiplier tube or other
photodetector. The CCD subsystem
communicates with and is controlled by a data acquisition board installed in a
computer. Data acquisition
board may be of the type that is well known in the art such as a CIO-DAS 16/Jr
manufactured by
Computer Boards Inc. The data acquisition board and CCD subsystem, for
example, may operate in the
following manner. The data acquisition board controls the CCD integration
period by sending a clock
signal to the CCD subsystem. In one embodiment, the CCD subsystem sets the CCD
integration period at
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4096 clocle periods. by changing the clock rate, the actual time in which the
CCD integrates data can be
manipulated.
During an integration period, each photodiode accumulates a charge
proportional to the amount of light
that reaches it. Upon termination of the integration period, the charges are
transferred to the CCD's shift
registers and a new integration period commences. The shift registers store
the charges as voltages which
represent the light pattern incident on the CCD array. The voltages are then
transmitted at the clock rate to
the data acquisition board, where they are digitized and stored in the
computer's memory. In this manner,
a strip of the sample is imaged during each integration period. Thereafter, a
subsequent row is integrated
until the sample is completely scanned.
In an alternative embodiment, one is interested in finding drugs, antagonists,
agonists or other
species which block or reduce the binding of probes with targets - these
compounds may be referred to as
'inhibitors'. In this application, labeled targets are bound to probes at the
interface. The inhibitors are
added to the sample, and if the particular species being tested is successful
in blocking or reducing the
probe-target binding, the nonlinear optical light measured will change - the
background radiation in this
embodiment is due to target-probe binding; the displacement of the targets
from the probes at the
interface by the inhibitors leads to a change in the nonlinear optical light
measured, for instance as a
decrease in intensity of the nonlinear radiation generated by the interface or
a wavelength shift in the
nonlinear radiation spectrum.
In an alternative embodiment, the nonlinear spectrum of a sample is measured
by measuring the
nonlinear radiation (e.g., second harmonic radiation) at two or more spectral
points or bands, using a
monochromator, filter or other wavelength-selecting device to accomplish this.
In an alternative embodiment, a monochromator (60) can be placed before the
detecting element
in the device, in order to spectrally resolve the nonlinear optical radiation
(Figure 1).
In an alternative embodiment, nucleic acid or PNA microarrays can be obtained
commercially or
constructed according to public literature (eg.,
http://cmgm.Stanford.edu/pbrown/mguide/index.html).
The surface chemistry to be used is that found in Chrisey, L.A. et al. (1996)
in which oligonucleotides are
attached to self assembled monolayer silane films on fused silica slides.
Silanization is accomplished via
N (2-aminoethyl)-3-aminopropyltrimethoxysilane.
In other embodiments, oligonucleotides or PNAs can be attached to the solid
substrate via light-
directed synthesis (Fodor et al., 1997) or via chemical synthesis (e.g.,
Chrisey, L.A., 1996).
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In stilll other embodiments, surfaces or microarrays microarrays of
oligonucleotides or PNAs can
be obtained commercially or constructed according to public literature (eg.,
http://cmgm.Stanford.edu/pbrown/mguide/index.html).
DNA microarrays can be obtained commercially or constructed according to
public literature (eg.,
http:l/cmgm.Stanford.edu/pbrown/mguide/index.html). The surface chemistry to
be used is that found in
Chrisey, L.A. et al. (1996) in which oligonucleotides are attached to self
assembled monolayer silane
films on fused silica slides. Silanization is done via N (2-aminoethyl)-3-
aminopropyltrimethoxysilane.and Hoheisel, J.D. "Improved solid supports and
spacer/linker systems for
the synthesis of spatially addressable PNA-libraries" Nucleosides Nucleotides
18 (1999) 1289-1291 on
glass or silica. The buffer or solution in contact with the PNA
oligonucleotides can be chosen from a
range of those known in the art. Hybridization and wash solutions are found in
the art. For example, the
web site: cmgm.stanford.edu/pbrown/protocols gives detailed instructions for
probe-target hybridization.
Microarrays can be mounted on an x-y translation stage and driven by personal
computer (PC
control) using a motorized translator (acquired from Oriel, Inc.) or using one
of the many procedures in
the art (eg., V.G. Cheung et al., 1999).
In an alternative embodiment, imaging techniques described in the art (Peleg,
1999 or
Campagnola et al.) can be performed using SHG-labeled components (such as
labeled ligands or
receptors) instead of the membrane-intercalating dyes used the art. These
imaging techniques iclecan be
used to image solid surfaces, cell surfaces or other interface using SHG-
labeled components.
In an alternative embodiment, the nonlinear optical measurements can be made
in the presence of
labelled targets in solution, liquid or buffer in contact with the substrate
with attached probes (e.g., no
washing step is required to remove non-bound labelled targets).
In an alternative embodiment, channels (or microfluid) channels can be used to
introduce the
components into the sample cell via positive displacement, pumping,
electrophoretic means or other
means known in the art for manipulating the flow of components into and out of
a reaction chamber.
In an alternative embodiment, the kinetics of some probe-target binding
reaction are to be
measured at some concentration of target. In this embodiment, the timecourse
of the intensity andlor
spectrum of the nonlinear optical light are measured. The measured information
can be converted into a
timecourse of bound target concentration (e.g., probe-target concentration in
mM/s or ~M/s). Drugs or
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other enhancers or reducers, for example, of the probe-target binding
equilibrium or kinetic rate of
formation can be used so as to compare the effect of the added substance on
the probe-target reactions.
In an alternative embodiment, the apparatus can be assembled into a user-
closed product with a
user-controlled interface (an LED panel, for example, or PC-based software)
with the option of inserting
and removing disposable substrates (e.g., biochips) with the attached probes.
In an alternative embodiment, the labels can be photoactivated or
photomodulated with a beam of
light (e.g., not the fundamental) such that, upon irradiation of the sample
with the beam of light, the labels
become nonlinear optical active (or more or less nonlinear optical active).
The beam of light can, for
example, cleave a chemical bond (e.g., using UV light), well known in the art
as 'caged' compounds.
In an alternative embodiment, a photodiode, avalanche photodiode or other
photoelectric detector
(65) in Figure 1 is used as the light detection means.
In an alternative embodiment, the surface array can be in a fixed position and
the incident light
beam scanned across the surface using methods well known in the art, such as a
galvanometer mirror or a
polygonal mirror.
In an alternative embodiment, the scanning method can be a combination of both
stage translation
(x-y) and beam scanning, wherein the latter controls the incident position of
the fundamental beam on the
array surface.
In an alternative embodiment, a stop-flow mixing chamber is used to rapidly
mix the components
in the sample cell.
In an alternative embodiment, probe-target interactions with labelled targets
can be imaged on
some surface such as a tissue surface, patterned cells on a surface, surface-
attached probes (e.g.,
microarrays or arrays of DNA, protein, etc.); the imaging can occur in-vitro
or in-vivo. In cases of in-
vivo imaging, the imaging can be performed using endoscopes or other
instruments known in the art for
introducing and collect light in-vivo.
In an alternative embodiment, a biological probe-target binding reaction can
be measured in the
presence of agonists, antagonists, drugs, or small molecules which can
modulate the binding strength
(e.g., equilibrium constant) of the said probe-target binding reaction. This
embodiment can be useful in
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43
many cases, for example when one would like to know the efficacy of a drug's
ability to block a certain
probe-target reaction for medical uses or basic research.
In an alternative embodiment, the proportionality constant (calibration curve
of intensity of second
harmonic light vs. concentration of targets bound to attached probes) is
determined by measuring the
concentration of targets using another method such as radiolabeling or
fluorescence labels of the targets.
Once the calibration curve is known, for a given probe and target type (e.g.,
cDNA, RNA, size of oligos,
etc.), the concentration of bound target is determined using this relation and
the measured second
harmonic intensity.
In an alternative embodiment, the nonlinear optical, surface-selective
apparatus can comprise a
unit without the light excitation source (e.g., with sample compartment,
alters, detectors, monochromator,
computer interface, software, or other parts) so that the user can supply his
own excitation source and
adapt its use to the methods described herein.
In an alternative embodiment, the measurable information can be recorded in
real time.
In an alternative embodiment, target-probe interactions can be measured in the
presence of some
modulator of the interactions - the modulator being, for example, a small
molecule, drug, or other moiety,
molecule or particle which changes in some way the target-probe interactions
(e.g., blocks, inhibits, etc.).
The modulator can be added before, during or after the time in which the probe-
target interactions occur.
Various confi~,urations of an apparatus using the surface-selective nonlinear
optical technidue for
detection of probe-target reactions.
The apparatus for detection of the probe-target reactions or their effects can
assume a variety of
configurations. In its most simple form, the apparatus will comprise the
following:
i) a source of the fundamental light
ii) a substrate or sample with surface-attached probes
iii) a detector for measuring the intensity of the second harmonic or other
nonlinear
optical beams.
More elaborate versions of the apparatus will employ, for example: a
monochromator (for
wavelength selection), a pass-filter, color filter, interference or other
spectral filter (for wavelength
selection or to separate the fundamentals) from the higher harmonics), one or
more polarizing optics, one
or more mirrors or lenses for directing and focusing the beams, computer
control, software, etc.
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The mode of delivering or generating the nonlinear optical light (e.g., SHG)
can be based on one
or more of the following means: TIR (Total internal reflection), Fiber optics
(with or without attached
beads), Transmission (fundamental passes through the sample), Reflection
(fundamental is reflected from
the sample), scanning imaging (allows one to scan a sample), confocal imaging
or scanning, resonance
cavity for power build-up, multiple-pass set-up.
Measured information can take the form of a vector which can include one or
more of the
following parameters: {intensity of light (typically converted to a
photovoltage by a PMT or photodiode),
wavelength of light (determined with a monochromator and/or filters), time,
substrate position (for array
samples, for instance, where different sub-samples are encoded as function of
substrate location and the
fundamental is directed to various (x,y) locations}. Two general
configurations of the apparatus are:
image scanning (imaging of a substrate - intensity, wavelength, etc. as a
function of x,y coordinate) and
spectroscopic (measurement of the intensity, wavelength, etc. for some planar
surface or for a suspension
of cells, liposomes or other particles).
The fundamental beam can be delivered to the sample in a variety of ways.
Figs. 12-16 are
schematics of various modes of delivering the fundamental and generating
second harmonic beams. It is
understood that in sum- or difference-frequency configurations, the
fundamental beams will be comprised
of two or more beams, and will generate, at the interfaces, the difference or
sum frequency beams. For
the purposes of illustration, only the second harmonic generation case is
described in detail herein.
Furthermore, it shall be understood that the sample cell 3 in all cases can be
mounted on a translation
stage (1-, 2-, or 3-dimensional degrees of freedom) for selecting precise
locations of the interfacial
interaction volume. The sample cell in all cases can be fitted with flow ports
and tubes which can serve
to introduce (or flush out) components such as molecules, particles, cells,
etc.
Transmission
Fig. 12A is a schematic of a configuration relying on transmission of the
fundamental and second
harmonic beams. The fundamental 320 (cu) passes through the sample cell 330
and interacts within a
volume element (denoted by the circle) in which are contained one or more
interfaces capable of
generating the second harmonic beam 325 (2ta). The fundamental and second
harmonic beams are
substantially co-linear as denoted by beam 325. The sample cell can contain
suspended beads, particles,
liposomes, biological cells, etc. in some medium, providing interfacial area
capable of generating second
harmonics in response to the fundamental beam. As shown, the second harmonic
is detected co-linearly
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with the fundamental direction, but could alternatively be detected off angle
from the fundamental, for
instance at 90° to the fundamental beam.
Fig. 12B is a schematic of another configuration relying on transmission of
the fundamental and
second harmonic beams. The fundamental 335 is directed onto a sample cell 345
and the second
harmonic waves are generated at the top surface - this surface can be
derivatized with immobilized
probes or with adsorbed particles, liposomes, cells, etc. The second harmonic
waves 340 are generated
within a volume element denoted by the circle at the interface between the top
surface and the medium
contained within cell.
Fig. 12C is a schematic of a configuration substantially similar to the one
depicted in Fig. 2A
except that the bottom surface of the sample cell 3, rather than the top, is
used to generate the second
harmonic waves.
Total Internal Reflection
Fig. 13A is a schematic of a waveguide 4 capable of acting as a total internal
reflection
waveguide which refracts the fundamental 365 and directs it to a location at
the interface between the
waveguide 380 and a sample cell 375. At this location, denoted by the circle,
the fundamental will
generate the second harmonic waves and undergo total internal reflection; the
second harmonic beam will
propagate substantially colinearly with the fundamental and exit the prism
380. Waveguide 380 will
typically be in contact with air. In this illustration, the waveguide 380 is a
Dove prism.
Fig. 13B is a schematic of a configuration similar to the one depicted in Fig.
13A except that the
waveguide 400 allows for multiple points of total internal reflection between
the waveguide 4 and the
sample cell 395, increasing the amount of second harmonic light generated from
the fundamental beam.
Fiber Optic
Fig 14 depicts various configurations of a fiber optic means of delivering or
collecting the
fundamental or second harmonic beams. In Fig. 14A, the coupling element 410
between a source of the
fundamental wave and the fiber optic is depicted. The fundamental, thus
coupled into the fiber optic
waveguide 405, proceeds to a sample cell 415. In Fig. 14A, the tip of the
fiber can serve as the interface
of interest capable of generating second harmonic waves, or the tip can serve
merely to introduce the
fundamental beam to the sample cell containing suspended cells, particles,
etc. In Fig. 14A, the second
harmonic light is collected back through the fiber optic.
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46
Fig. 14B is identical to Fig. 14A except that a bead is attached to the tip of
the fiber optic
(according to means well known in the art). The bead can serve to both improve
collection efficiency of
the second harmonic light or be derivatized with probes or adsorbed species
and presenting an interface
with the medium of sample cell 425 capable of generating the second harmonic
light.
Fig. 14C is identical to both Figs. 14A and 14B except that collection of the
second harmonic
light is effected using a solid-angle detector 450.
Optical Resonance Cavity
An optical resonance cavity is defined between at least two reflective
elements and has an intracavity
light beam along an intracavity beam path. The optical cavity or resonator
consists of two or more
mirrored surfaces arranged so that the incident light can be trapped bouncing
back and forth between the
mirrors. In this way, the light inside the cavity can be many orders of
magnitude more intense than the
incident light. This phenomenon is well known and has been exploited in
various ways (see, for example,
Yariv A. "Introduction to Optical Electronics", 2nd Ed., Holt, Reinhart and
Winston, NY 1976, Chapter 8).
The sample cell can be present in the optical cavity or it can be outside the
optical resonance cavity.
Fig. 15 is a schematic of an optical resonance power build-up cavity
configuration. Fig. 15A is a
schematic of an optical resonance cavity in which the sample cell 465 is
positioned intracavity and the
fundamental and second harmonic beams are transmitted through it - a useful
configuration for sample
cells containing suspended particles, cells, beads, etc. The fundamental beam
455 enters the optical
resonance cavity at reflective optic 460 and builds up in power between
reflective elements 460 and 462
(intracavity beam). Mirror 460 is preferably tilted (not perpendicular to the
direction of the incident
fundamental 455) to prevent direct reflection of the intracavity beam back
into the light source. The
natural reflectivity and transmisivity of 460 and 462 can be adjusted so that
the fundamental builds up to a
convenient level of power within the cavity. The fundamental generates second
harmonic light in a
volume element within the sample cell denoted by the circle. Reflective optic
460 can reflect the
fundamental and the second harmonic, while reflective optic 462 will
substantially reflect the
fundamental but allow the pass-through of the second harmonic beam 475 which
is subsequently
detected. U.S. Pat. No. 5,432,610 (King et al.) describes a diode-pumped power
build-up cavity for
chemical sensing and it and the references it makes are hereby incorporated by
reference herein.
Fig. 15B is a schematic of an optical resonance power build-up cavity
configuration in which the
fundamental beam 475 enters the optical cavity by reflection from optic 480. A
second reflective optic
element 482 defines the optical resonance cavity. Element 490 is a waveguide
(such as a prism) in
contact with the sample cell 485 and allows total internal reflection of the
fundamental beam at the
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interface between the waveguide and sample cell surfaces, generating the
second harmonic light. Element
482 substantially reflects the fundamental beam but passes through the second
harmonic beam 495 which
is subsequently detected.
Reflection
Fig. 16A is a schematic of a configuration involving reflection of the
fundamental and second
harmonic beams. A substrate 525 is coated with a thin layer of a reflective
material 520, such as a metal,
and on top of this is deposited at layer 515 suitable for attachment of the
probes or adsorption of particles,
cells, etc. (e.g., Si02). This layer is in contact with the sample cell 510.
The fundamental 500 passes
through the sample cell 510 and generates a second harmonic wave at the
interface between layers 515
and 520. The fundamental and second harmonic waves 505 are reflected back from
the surface of layer
520.
Fig. 16B is substantially similar to Fig. 15A except that the second harmonic
and fundamental
beams are reflected 535 from the interface between the medium contained in
sample cell 540 and layer
545. Layer 545 is reflective or partly reflective layer deposited on substrate
550 and is suitable for
adsorption of particles, cells, etc. or attachment of probes.
Fig. 16C is a schematic illustrating that only the sample cell 565 need be
used for a reflective
geometry. The sample cell 565 is partly filled with some medium 570 and the
fundamental and second
harmonic beams are reflected 560 from the gas-liquid or vapor-liquid interface
at the surface of 570.
Modes of detection
Charge-coupled detectors (CCD) array detectors can be particularly useful when
information is
desired as a function of substrate location (x,y). CCDs comprise an array of
pixels (i.e., photodiodes),
each pixel of which can independently measuring light impinging on it. For a
given apparatus geometry,
nonlinear light arising from a particular substrate location (x,y) can be
determined by measuring the
intensity of nonlinear light impinging on a CCD array location (Q,R) some
distance from the substrate -
this can be determined because of the coherent, collimated (and generally co-
propagating with the
fundamental) nonlinear optical beam) compared with the spontaneous, stochastic
and multidirectional
nature of fluorescence emission. With a CCD array, one or more array elements
~Q,R} in the detector
will map to specific regions of a substrate surface, allowing for easy
determination of information as a
function of substrate location (x,y). Photodiode detector and photomultiplier
tubes (PMTS), avalanche
photodiodes, phototransistors, vacuum photodiodes or other detectors known in
the art for converting
incident light to an electrical signal (i.e., current, voltage, etc.) can also
be used to detect light intensities.
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For CCD detector, the CCD communicates with and is controlled by a data
acquisition board installed in
the apparatus computer. The data acquisition board can be of the type that is
well known in the art such
as a CIO-DAS l6lJr manufactured by Computer Boards Inc. The data acquisition
board and CCD
subsystem, for example, can operate in the following manner. The data
acquisition board controls the
CCD integration period by sending a clock signal to the CCD subsystem. In one
embodiment, the CCD
subsystem sets the CCD intregration period at 4096 clock periods. By changing
the clock rate, the actual
time in which the CCD integrates data can be manipulated. During an
integration period, each
photodiode accumulates a charge proportional to the amount of light that
reaches it. Upon termination of
the integration period, the charge is transferred to the CCD's shift registers
and a new integration period
commences. The shift registers store the charges as voltages which represent
the light pattern incident on
the CCD array. The voltages are then trasmitted at the clock rate to the data
acquisition board, where they
are digitized and stored in the computer's memory. In this manner, a strip of
the sample is imaged during
each integration period. Thereafter, a subsequent row is integrated until the
sample is completely
scanned.
Sample substrates and sample cells
Sample substrates and cells can take a variety of forms drawing from, but not
limited to, one or
more of the following characteristics: fully sealed, sealed or unsealed and
connected to flow cells and
pumps, integrated substrates with a total internal reflection prism allowing
for evanescent generation of
the nonlinear beam, integrated substrates with a resonant cavity for
fundamental power build-up, an
optical set-up allowing for multiple passes of the fundamental for increased
nonlinear response, sample
cells containing suspended biological cells, particles, beads, etc.
Data anal~is
Data analysis operates on the vectors of information measured by the detector.
The information
can be time-dependent and kinetic. It can be dependent on the concentration of
one or more biological
components, inhibitors, antagonists, agonists, drugs, small molecules, etc.
which can be changed during a
measurement or between measurements. It can also be dependent on wavelength,
etc. In general, the
intensity of nonlinear light will be transformed into a concentration or
amount of a particular state (for
example, the surface-associated concentration of a component or the amount of
opened or closed ion-
channels in cell membranes). In one example, the production of second harmonic
light follows the
equation:
(Isx)°.s ~ Ez~ - Ax(z> + B~°x(3~ (1)
where IsH is the intensity of the second harmonic light, Ez~, is the electric-
field amplitude of the second
harmonic light, A and B are constants specific to a given interface and sample
geometry, ~° is the electric
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49
surface potential, and x(2~ and x~3~ are the second and third-order nonlinear
susceptibility tensors. x~2~ is
proportional to N (surface-bound or probe-bound targets) and the
hyperpolarizability per target. Surface
binding reactions can follow a Langmuir-type equation:
dN/dt = kl(C-I~/55.5 * (Nn,az N) - k~N (2)
with N the amount of the targets binding to the surface (e.g., targets binding
to probes), NmaX the
maximum number of the binding species at the surface at equilibrium, k1 the
association rate constant, k_1
the dissociation rate constant, dN/dt the instantaneous rate of change of the
amount of surface-bound
targets and C the bulk concentration of the species. Modified Langmuir
equations or other equations used
in determining the amount of surface-bound species in the art can also be used
in the data analysis.
The details of the data analysis will depend on each specific case. If the
polarization response
due to a net charge on the surface - x~3~ - is present, it can be subtracted
out in making the measurement.
Thus, the number of surface-bound species N can be directly calculated from
the second harmonic
intensity in this manner. Kinetics or equilibrium properties can be determined
from N (at equilibrium or
in real time) according, for example, to equation 2 and procedures well known
in the art. There are a
number of relevant papers in the art which describe this process including,
for example: J.S. Salafsky,
K.B. Eisenthal, "Second Harmonic Spectroscopy: Detection and Orientation of
Molecules at a
Biomembrane Interface", Chemical Physics Letters 2000, 319, 435 and Eisenthal,
K.B. " Photochemistry
and Photophysics of Liquid Interfaces by Second Harmonic Spectroscopy" J.
Phys. Chem. 1996, 100,
12997.
For probe-target processes which result directly or indirectly in changes in
surface charge density
or potential (an example of the indirect type is in ion-channel experiments
with ion channels in a cell,
where a target binds to a probe, leading to the modulation of an ion channel's
dynamics which leads, in
turn, to the surface charge density). In this case, labels attached to the
surface of a cell are used to sense
the ion channel's dynamics or state via the effect the surface charge density
has on the nonlinear
properties of the labels.
Data analysis
Data analysis operates on the vectors of information measured by the detector.
The information can be
time-dependent and kinetic. It can be dependent on the concentration of one or
more biological
components (probes, targets, drugs, etc.), which can be changed during a
measurement or between
measurements. It can also be dependent on wavelength, etc. In general, the
intensity of nonlinear lzght
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will be transformed into a concentration or amount of a particular state. In
one example, the production
of second harmonic light follows the equation:
(IsH)°.s ~ E2~ - Ax(z> .~ B~°x(3~ (1)
where IsH is the intensity of the second harmonic light, E2~, is the electric-
field amplitude of the second
harmonic light, A and B are constants specific to a given interface and sample
geometry, (1?° is the electric
surface potential, and x(2~ and x(3~ are the second and third-order nonlinear
susceptibility tensors. Surface
binding reactions can follow a Langmuir-type equation:
dN/dt = kt(C-N)/55.5 '~' (Nn,aX N) - htN
with N the amount of the targets binding to the surface (e.g., targets binding
to probes) and dN/dt the
instantaneous rate of targets binding to the surface, NmaX the maximum number
of the binding species at
the surface at equilibrium, kt the association rate constant, k_t the
dissociation rate constant, dN/dt the
instantaneous rate of change of the amount of surface-bound targets and C the
bulk concentration of the
species. Modified Langmuir equations or other equations used in determining
the amount of surface-
adsorbed or surface-bound species in the art can also be used in the data
analysis.
The details of the data analysis will depend on the specific details of each
case. The number of
labeled, surface-bound species N can be directly calculated from the second
harmonic intensity. Kinetics
or equilibrium properties can be determined from N (at equilibrium or in real
time) according, for
example, to equation 2 and procedures well known in the art. There are a
number of relevant papers in
the art which describe this process in detail including, for example: . J.S.
Salafsky, K.B. Eisenthal,
"Second Harmonic Spectroscopy: Detection and Orientation of Molecules at a
Biomembrane Interface",
Chemical Physics Letters 2000, 319, 435 and Eisenthal, K.B. " Photochemistry
and Photophysics of
Liquid Interfaces by Second Harmonic Spectroscopy" J. Phys. Chem. 1996, 100,
12997.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 depicts one embodiment of the apparatus in which the mode of generation
and collection of the
second harmonic light is by reflection off the substrate with surface-attached
probes.
Fig. 2 depicts one embodiment of an apparatus in which the mode of generation
and collection of the
second harmonic light is by total internal reflection through a prism. The
prism is coupled by an index-
matching material to a substrate with surface-attached probes.
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51
Fig. 3 depicts one embodiment of an apparatus in which the mode of generation
and collection of the
second harmonic light is by total internal reflection through a wave-guide
with multiple reflections as
denoted by the dashed line inside the wave-guide.
Fig. 4 depicts one embodiment of a flow-cell for delivery and removal of
biological components and
other fluids to the substrate containing attached probes.
Fig. 5 depicts three embodiments of an apparatus in which the mode of
generation and collection of the
second harmonic light is by transmission through a sample. In Fig. 5A, the
second harmonic beam is co-
linear with the fundamental. In Fig. 5B, the second harmonic is collected from
a direction orthogonal to
the fundamental ('right-angle collection'). In Fig. SC, the second harmonic
light is collected by an
integrating sphere and a fiber optic line.
Fig. 6 depicts an embodiment of the transformation, using a series of optical
components, of a collimated
beam of the fundamental light into a line shape suitable for scanning a
substrate.
Fig. 7A depicts an embodiment of a substrate surface (containing attached
probes) which has been
patterned into an array format (elements 1-35). Fig. 7B depicts one element of
a substrate array in which
each element is a well with walls, with surface-attached probes, and the well
is capable of holding some
liquid and serves to physically separate the well's contents from adjacent
wells or other parts of the
substrate.
Fig. 8 depicts one embodiment of a surface chemistry used to attach
oligonucleotide or polynucleotide
samples to the substrate surface.
Fig. 9A depicts an embodiment of a substrate containing multiple wells (1-16),
each of which contains
surface-attached probes as depicted in Fig. 9B.
Fig. 10 depicts an embodiment of the apparatus substrate with the use of an
aminosilane surface-attached
layer on top of a reflective coating. The reflective coating underneath the
aminosilane layer improves
collection of the nonlinear optical Iight. The aminosilane layer is suitable
for coupling biomolecules or
other probe components to the substrate.
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Fig. 11 depicts an embodiment of an apparatus in which the mode of generation
and collection of the
second harmonic light is through a fiber optic. Fig. 1 1A depicts the use of a
bundle of fiber optic lines
and Fig. 11B depicts the use of beads coupled to the end of a fiber for
attaching probes.
Fig. 12 depicts three embodiments of an apparatus in which the mode of
generation and collection of the
second harmonic light is by transmission through a sample. Fig. 12A depicts
both the fundamental and
second harmonic beams travelling co-linearly through a sample. Fig. 12B
depicts the fundamental and
second harmonic beams being refracted at the top surface (top surface contains
attached probes) of a
substrate with this surface generating the second harmonic light. Fig 12C
depicts a similar apparatus to
Fig. 12B except that the bottom surface (bottom surface contains attached
probes) generates the second
harmonic light.
Fig. 13 depicts two embodiments of an apparatus in which second harmonic light
is generated by total
internal reflection at an interface. The points of generation of the second
harmonic Iight are denoted by
the circles. In Fig. 13A, a dove prism is used to guide the light to a surface
capable of generating the
second harmonic light (bottom surface of prism but can also be another surface
coupled to the prism
through an index-matching material). In Fig. 13B, a wave-guide structure is
used to produce multiple
points of second harmonic generation.
Fig. 14 depicts three embodiments of an apparatus in which second harmonic
light is generated using a
fiber optic line (with attached probes at the end of the fiber). Fig. 14A
depicts an apparatus in which both
generation and collection of the second harmonic light occur in the same
fiber. Fig. 14B depicts the use
of a bead containing surface-attached probes at the, end of the fiber. Fig.
14C depicts an apparatus in
which the second harmonic light is generated at the end of the fiber optic
(containing attached probges)
and collected using a mirror or lens external to the fiber optic.
Fig. 15 depicts two embodiments of an apparatus using an optical cavity for
power build-up of the
fundamental.
Fig. 16 depicts three embodiments of an apparatus in which the mode of
generation and collection of the
second harmonic light uses reflection of the light from an interface.
DESCRIPTION OF THE DRAWINGS
The drawings illustrate various embodiments of the apparatus and sample using
second harmonic
generation. The use of sum or difference frequency is not illustrated herein,
but a similar set-up is
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53
required - with the use of two fundamental beams (cal, w2) where w1 ~ w2 = S2,
with SZ the sum or
difference frequency. In the case where the sample surfaces are arrays
comprised of discrete elements, a
single element or more than one in parallel can be addressed with the
fundamental light. Furthermore,
detection can be made on a single element or many in parallel depending on the
specific apparatus set-up.
Figure 1 illustrates an embodiment wherein second harmonic light is generated
by reflecting incident
fundamental light from the surface. Light source 5 provides the fundamental
light necessary to generate
second harmonic light at the sample. Typically this will be a picosecond or
femtosecond laser, either
wavelength-tunable or not tunable, and commercially available. Light at the
fundamental frequency (t~)
exits the laser and its polarization is selected using, for example a half
wave plate 10 appropriate to the
frequency and intensity of the light (eg., available from Melles Griot, Oriel
or Newport Corp.). The beam
is then focused by lens 15 and passes through a pass filter 20 designed to
pass the fundamental light but
block the nonlinear light (eg., second harmonic). This filter is used to
prevent back-reflection of the
second harmonic beam into the laser cavity which can cause disturbances in the
lasing properties. 'The
beam is reflected from a mirror 25 and impinges at a specific location and
with a specific angle 8 on the
surface. The mirror 25 can be scanned if required using a galvanometer-
controlled mirror scanner, a
rotating polygonal mirror scanner, a Bragg diffractor, acousto-optic
deflector, or other means known in
the art to allow control of a mirror's position. The sample surface 30 can be
mounted on an x-y
translation stage 35 (computer controlled) to select a specific location on
the surface for generation of the
second harmonic beam. The surface can be glass, plastic, silicon or any other
solid surface which reflects
the fundamental or second harmonic beams. The sample surface can be enclosed
and the surface in
contact with liquid. Furthermore, the sample 30 can be fed or drained by
microcapillary or other liquid-
transporting channels (not shown), pumps or electrophoretic elements, and
these devices can be
computer-controlled. The fundamental and the second harmonic outgoing beams
(at specific angles with
respect to the surface, i.e. 91- they are typically nearly colinear in
direction) then reflected from the
surface and the fundamental is filtered using a pass-filter 45 for the second
harmonic beam, leaving only
the harmonic beam (2c~). The second harmonic is reflected from mirror 40, its
polarization selected if
necessary by polarizing optic 50, and is focused using a lens 55 onto a
detector 60. The lenses 15 and 55
can also be any combination of lenses known in the art for focusing or beam
shaping. If required, a
monochromator 60 can also be used to select a specific wavelength within the
spectral band of the second
harmonic beam. 'The detector can be a photomultiplier tube, a CCD array, or
any other detector device
known in the art for high sensitivity. For instance, a photomultiplier tube
operated in single-photon
counting mode can be used. At the detector, the light generates a voltage
proportional to its intensity.
Data is recorded for each location on the array surface as it is translated by
the stage, scanned (or a
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54
combination thereof) and an image is built up of the second harmonic intensity
generated from each
region on the surface.
Figure 2 illustrates an embodiment in which total internal reflection
(evanescent wave generation)
is used to generate the second harmonic light. Fundamental light (w) is
directed on to the surface of a
prism element 70. The beam is refracted at position (a) and passes through the
prism, through an index
matching film 75 and impinges on substrate 80. Prism 70 and substrate 80 are
made of optically
transparent materials and are preferably of the same type. Prism 70 can be a
Dove prism or any other
element which can support evanescent fields (eg., waveguides, fibers and thin
metallic films). The
refractive index matching film 75 can be an oil, but is preferably a
compressible optical polymer such as
those disclosed by Sjodin, "Optical interface means", PCT publication WO
90/05317, 1990. The prism
75 and the substrate 80 can also be a unitary, integral piece made of the same
material (i.e, without the
index matching film). An evanescent wave is generated at the interface between
80 and the medium in
sample compartment 85 according to the indices of refraction in 80 and 85 and
the angle of incidence of
the beam at their interface. The electric field amplitude decays exponentially
away from the substrate
surface with a 1/e length ranging from nanometers to microns depending on
several factors, including the
surface electric potential, the counterion density in the sample compartment
(if any). The sample
compartment can be filled with air, a gas, or a liquid such as a solution or
water. The 'x' marks on the
surface of 80 facing the sample compartment emphasize that the sample of
interest (eg., fabricated
probes) are placed on this side. Substrate 80 can be a 'chip' which can be
slid out between 75 and 85,
allowing for measurement of different substrates. Element 90 in the drawing
refers to a port in the sample
compartment for drawing liquid or gases in and out of the compartment, for
instance by pumps,
electrostatic means, etc. The entire sample assembly can be mounted on an x-y
translation stage 95 if
necessary.
Figure 3 illustrates an embodiment in which a slab-dielectric waveguide is
used to deliver the
fundamental light to the sample surface (the light beams are generated,
directed and detected as in
Drawing I with elements 1-5 and 8-13). A parallel plate or dielectric
waveguide can be used to couple the
fundamental light into a waveguide propagating mode. The drawing shows two
slabs (110 and 115) and
region (120). If the indices of refraction of slab 115 and region 120 are less
than the index of refraction of
the light (for both fundamental and second harmonic), a waveguiding mode can
be developed. This mode
produces multiple internal reflections at the substrate which can be used to
increase the amount of second
harmonic light generated by the interface. The fundamental beam 100 can be
coupled into the waveguide
110 using a diffraction grating 105 scribed or embossed on the top surface of
the waveguide, for example.
The fundamental is propagated along the length of the waveguide and makes
multiple total internal
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reflections at the top and bottom surfaces. The 'x' marks on substrate 110
denote the surface sample to be
measured (i.e., containing the probes). If this interface generates
significantly more second harmonic
light than the interface between materials 110 and 115, the light intensity
can be neglected. For example,
if SH-labeled targets are bound to immobilized probes at the 'x' locations and
the atomic structure at the
interface between 110 and 115 is epitaxially matched, the interface 110/120
will generate much more
second harmonic light than the interface 110/115.
The scope of the invention should, therefore, be determined not With the
reference to the above
description, but should instead be determined with reference to the appended
claims, along with the full
scope of equivalents to which such claims are entitled. .
References
U.S. Patent Documents
5,324,591 Jun., 1994 Georger, Jr. et al..
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