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I. BACKGROUND OF THE INVENTION
A. Field of the Invention
This invention entails a novel method, apparatus, and materials for the
measurement of binding between molecules which have mutual affinity. The
affinity of binding displayed by certain molecules (referred to here as
binding molecules) towards other specific molecules (referred to here as
ligands) is used commonly as the basis of assays to measure the quantity
of a particular binding molecule or ligand in a sample.
The two molecules involved in forming a binding molecule-ligand complex are
also referred to as a specific binding pair. One member of a specific
binding pair is referred to as a specific binding member. This invention
includes methods for performing the assays using specific binding pairs of
binding molecules and ligands, with Raman light scattering as the method
of detecting binding. This invention also includes materials used in
performing the assays and instrumentation used to perform the assays.
An assay is a test (1) to detect the presence of a substance in a sample,
(2) to identify a substance in a sample, or (3) to measure the amount of a
substance in a sample. In the terminology of this field, the substance
that the assay is designed to detect (or identify or measure) is called an
"analyte" (a glossary of some of the terms used herein is included as an
appendix to this application).
Ligand binding assays are especially relevant to medical diagnostics. In
modern medical practice, ligand binding assays are routinely run on
patients' blood, urine, saliva, etc. in order to determine the presence or
levels of antibodies, antigens, hormones, medications, poisons, toxins,
illegal drugs, etc.
New, better, cheaper, and faster assays can advance the level of health
care. Such assays can provide a physician with more and better information
about a patient and do so consistent with reasonable cost. In addition, by
making assays easier and cheaper, a higher level of health care can be
extended to less developed parts of the world. Ligand binding assays are
also being used to monitor groundwater contamination, toxins and
pesticides in foods, industrial biological processes, and in many areas of
biological research.
B. Present Ligand Binding Assays
For many assays it is required that minute quantities of a certain
substance (the analyte) be detected and measured in the presence of much
larger quantities of other substances. This is possible because the high
affinity a binding molecule can have for a ligand can result in a high
degree of specificity of binding for that particular ligand, irrespective
of the presence of other substances. The most common ligand binding assays
are immunoassays.
In an immunoassay, an antibody serves as a binding molecule which
specifically binds an antigen, which serves as the ligand, thereby forming
a specific binding pair. In order to measure the extent of the
antibody/antigen binding, one member of the specific binding pair is
tagged or labeled with a traceable substance. The unique properties of the
traceable substance allow its presence, and hence the presence of the
specific binding member to which it is attached, to be detected or
measured. The labeled member of the specific binding pair is referred to
as the indicator reagent.
In a direct immunoassay, the quantity of indicator reagent bound to the
other member of the specific binding pair is measured. In an indirect
immunoassay, the degree of inhibition of binding of the indicator reagent
to the other member of the specific binding member by the analyte is
measured.
The individual members of a specific binding pair do not have to be
antigens or antibodies, however. Any two molecules having affinity for
each other may comprise a specific binding pair and may form the basis of
a ligand-binding assay. Other examples of such specific binding pairs are:
lectins and the complex carbohydrates to which they bind, hormones and
their receptors, any effector molecule and its receptor, binding molecules
designed through molecular modeling and synthesized specifically to bind
another molecule, and other molecules with mutual affinity such as avidin
and biotin.
Two commonly-used immunoreaction techniques are radioimmunoassay (RIA) and
enzyme immunoassay (EIA), both of which employ a labeled specific binding
member as an indicator reagent. RIA uses a radioactive isotope as the
traceable substance attached to a specific binding member. Because the
radioactive isotope can be detected in very small amounts, it can be used
to detect or quantitate small amounts of analyte. There are, however, a
number of substantial drawbacks associated with RIA. These drawbacks
include the special facilities and extreme caution that are required in
handling radioactive materials, the high costs of such reagents and their
unique disposal requirements.
EIA uses an enzyme as the label attached to a specific binding member. This
enzyme-labeled specific binding member then serves as the indicator
reagent, and enzymatic activity is used to detect its binding. While EIA
does not have some of the same disadvantages as RIA, EIA techniques
require the; addition of substrate materials to elicit the detectable;
enzyme reaction. In addition, enzyme stability and rate of substrate
turnover are temperature sensitive, the former decreasing and the latter
increasing with temperature.
A drawback common to all of these assay configurations is the necessity of
separating unbound labeled reagent from that bound to the analyte. This
usually entails wash steps which are tedious when the assays are performed
manually and require complicated robotics in automated formats.
Immunoassays may also be performed by automated instruments. Examples of
such instruments are the TDx and IM.sub.x analyzers which are commercially
available from Abbott Laboratories, Abbott Park, Ill. The TDx and Im.sub.x
are used to measure analyte concentrations in biological fluids such as
serum, plasma and whole blood.
Other types of assays use the so-called "dipstick" and "flowthrough"
methods. With these methods, a test sample is applied to the "dipstick" or
"flowthrough" device, and the presence of the analyte is indicated by a
visually detectable signal generated by a color forming reaction.
Flowthrough devices generally use a porous material with a
reagent-containing matrix layered thereon or incorporated therein. Test
sample is applied to the device and flows through the porous material. The
analyte in the sample then reacts with the reagent(s) to produce a
detectable signal on the porous material. Such devices have proven useful
for the qualitative determination of the presence of analytes.
More recently, assay techniques using metallic sol particles have been
developed. The specific binding member to be labeled is coated onto the
metal sol particles by absorption and the metal sol particles become the
label. Localization of these labeled binding members on a solid support
via an immunoreaction can produce a signal that is visually detectable, as
well as measurable by an instrument.
Fluorescent and visible dyes and spin labels have also been used as labels
in ligand binding assays.
All of these binding molecule-ligand assays have inherent drawbacks. The
use of Raman light scattering as a means of detecting or measuring the
presence of a labeled specific binding member, avoids some of these
drawbacks, as explained below.
C. Rayleigh Light Scattering
For many years, it has been known that when certain molecules are
illuminated by a beam of light, for example ultraviolet, visible, or near
infrared, a small fraction of the incident photons are retained
momentarily by some of the molecules, causing a transition of the energy
levels of some of those molecules to higher vibrational levels of the
ground electronic state. These higher vibrational levels are called
virtual states. Most of the time, these are elastic collisions, and the
molecules return to their original vibrational level by releasing photons.
Photons are emitted in all directions at the same wavelength as the
incident beam (i.e., they are scattered). This is called Rayleigh
scattering.
D. Raman Light Scattering
In 1928, C. V. Raman discovered that when certain molecules are
illuminated, a small percentage of the molecules which have retained a
photon do not return to their original vibrational level after remitting
the retained photon, but drop to a different vibrational level of the
ground electronic state. The radiation emitted from these molecules will
therefore be at a different energy and hence a different wavelength. This
is referred to as Raman scattering.
If the molecule drops to a higher vibrational level of the ground
electronic state, the photon emitted is at a lower energy or longer
wavelength than that retained. This is referred to as Stokes-shifted Raman
scattering. If a molecule is is already at a higher vibrational state
before it retains photon, it can impart this extra energy to the remitted
photon thereby returning to the ground state. In this case, the radiation
emitted is of higher energy (and shorter wavelength) and is called
anti-Stokes-shifted Raman scattering. In any set of molecules under normal
conditions, the number of molecules at ground state is always much greater
than those at an excited state, so the odds of an incident photon hitting
an excited molecule and being scattered with more energy than it carried
upon collision is very small. Therefore, photon scattering at frequencies
higher than that of the incident photons (anti-Stokes frequencies) is
minor relative to that at frequencies lower than that of the incident
photons (Stokes frequencies). Consequently, it is the Stokes frequencies
that are usually analyzed.
The amount of energy lost to or gained from a molecule in this way is
quantized, resulting in the scattered photons having discrete wavelength
shifts. These wavelength shifts can be measured by a spectrometer. Raman
scattering was considered to have the potential to be useful as an
analytical tool to identify certain molecules, and as a means of studying
molecular structure. However, other methods, such as infrared
spectroscopy, proved to be more useful.
E. Resonance Raman Scattering
Interest in Raman spectroscopy was renewed with the advent of the laser as
a light source. Its intense coherent light overcame some of the
sensitivity drawbacks of Raman spectroscopy. Moreover, it was discovered
that when the wavelength of the incident light is at or near the maximum
absorption frequency of the molecule, and hence can cause electronic as
well as vibrational transitions in the molecules, resonance Raman
scattering is observed. With resonance Raman scattering, the re-emitted
photons still show the differences in vibrational energy associated with
Raman scattering. However, with resonance Raman scattering, the electronic
vibrational absorption is approximately 1000 times more efficient. Even
with the increased signal from resonance Raman scattering, its usefulness
as an analytic tool was limited due to its still comparatively weak
signal. The relatively recent discovery of the surface enhancement effect,
however, has provided a means to further dramatically enhance Raman
scattering intensity.
F. Surface Enhanced Raman Scattering
A significant increase in the intensity of Raman light scattering can be
observed when molecules are brought into close proximity to (but not
necessarily in contact with) certain metal surfaces. The metal surfaces
need to be "roughened" or coated with minute metal particles. Metal
colloids also show this signal enhancement effect. The increase in
intensity can be on the order of several million-fold or more. In 1974 Dr.
Richard P. Van Duyne was the first to recognize this effect as a unique
phenomenon and coined the term "surface enhanced Raman scattering" (SERS).
The cause of the SERS effect is not completely understood; however, current
thinking envisions at least two separate factors contributing to SERS.
First, the metal surface contains minute irregularities. These
irregularities can be thought of as spheres (in a colloid, they are
spheroidal or nearly so). Those particles with diameters of approximately
1/10th the wavelength of the incident light are considered to contribute
most to the effect. The incident photons induce a field across the
particles which, being metal, have very mobile electrons.
In certain configurations of metal surfaces or particles, groups of surface
electrons can be made to oscillate in a collective fashion in response to
an applied oscillating electromagnetic field. Such a group of collectively
oscillating electrons is called a "plasmon." The incident photons supply
this oscillating electromagnetic field. The induction of an oscillating
dipole moment in a molecule by incident light is the source of the Raman
scattering. The effect of the resonant oscillation of the surface plasmons
is to cause a large increase in the electromagnetic field strength in the
vicinity of the metal surface. This results in an enhancement of the
oscillating dipole induced in the scattering molecule and hence increases
the intensity of the Raman scattered light. The effect is to increase the
apparent intensity of the incident light in the vicinity of the particles.
A second factor considered to contribute to the SERS effect is molecular
imaging. A molecule with a dipole moment, which is in close proximity to a
metallic surface, will induce an image of itself on that surface of
opposite polarity (i.e., a "shadow" dipole on the plasmon). The proximity
of that image is thought to enhance the power of the molecules to scatter
light. Put another way, this coupling of a molecule having an induced or
distorted dipole moment, to the surface plasmons, greatly enhances the
excitation probability. The result is a very large increase in the
efficiency of Raman light scattered by the surface-absorbed molecules.
The SERS effect can be enhanced through combination with the resonance
Raman effect. The surface-enhanced Raman scattering effect is even more
intense if the frequency of the excitation light is in resonance with a
major absorption band of the molecule being illuminated. The resultant
Surface Enhanced Resonance Raman Scattering (SERRS) effect can result in
an enhancement in the intensity of the Raman scattering signal of seven
orders of magnitude or more.
G. Application of SERS to Immunoassays
The SERS effect has been used by physical and analytical chemists to follow
chemical reactions on electrode surfaces in order to study molecular
surface structure and dynamics. Recently, the technique has also been
applied to biological molecules containing Raman-active prosthetic groups,
such as hemes.
Up until now, there has been no application of the SERS effect to
immunodiagnostics.
Utilization of this technology in immunodiagnostics offers several unique
advantages. Because of the extraordinary dependence of the SERS signal
upon close association with a suitable surface, only those reporter
molecules which have become immobilized on the SERS-active surface will
contribute a significant signal, while the signal contribution of those
remaining in solution will be negligible. Molecules bound in different
environments or different orientations can exhibit differences in their
Raman scattering characteristics. As a result, it is potentially possible
to differentiate among the Raman scattering spectra of free species in
solution, bound species in solution, surface absorbed species and species
attached to a surface via a specific ligand binding reaction.
In view of these unique characteristics Raman-based ligand binding assays
can be designed which eliminate the need to remove excess unbound
scattering molecules.
II. SUMMARY OF THE INVENTION
According to one feature of the present invention there is provided a
method for assaying an analyte in a test sample by first combining the
test sample with a specific binding pair having an affinity for the
analyte being assayed and in which one specific binding pair member
includes a Raman-active reporter. Then, the Raman spectra of the resultant
is measured.
According to another feature of the present invention, there is provided a
method for assaying an analyte in a test sample by combining the test
sample with a specific binding pair having an affinity for the analyte
being assayed and in which one specific binding pair member includes a
Raman-active reporter and the other specific binding pair member is bound
to a metallic surface. Then, the Raman spectra of the resultant is
measured.
According to another feature of the present invention, there are provided
surfaces, reporter labels, and excitation sources for use in a method for
assaying an analyte in a test sample in which the test sample is first
combined with a specific binding pair having an affinity for the analyte
being assayed and in which one specific binding pair member includes a
Raman-active reporter and the other specific binding pair member is bound
to a metallic surface after which the Raman spectra of the resultant is
measured.
According to yet another feature of the present invention, there is
provided an analyzer for assaying an analyte in a test sample in which the
test sample is combined with a specific binding pair having an affinity
for the analyte being assayed and in which one specific binding pair
member includes a Raman-active reporter and the other specific binding
pair member is bound to a metallic surface after which the Raman spectra
of the resultant is measured.
III. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a profilometer tracing of an intact, chemically deposited silver
film surface.
FIG. 2 is Raman spectra of (A)2,4-dinitrobenzene solution, 10.sup.-3 M, in
the presence of chemically deposited silver film, (B)
2,4-dinitrophenyl-BSA conjugate, 10.sup.-7 M with respect to DNP moieties,
in the presence of a chemically deposited silver film, and (C)
2,4-dinitrobenzene, 10.sup.-3 M, in the absence of a silver film (ordinate
expanded fourfold relative to A and B to enhance features). Spectra
acquisition conditions: acquisition time, 19 s; power, 41 mW; excitation
wavelength, 457.9 nm.
FIG. 3 is a SERRS spectrum obtained from a chemically deposited silver film
incubated in (A) a 3 mM solution of HABA and (B) a 2.5.times.10.sup.-5 M
solution of avidin subsequently made 0.3 mM in HABA. No discernible
spectrum was observed in this region from surface-absorbed. avidin in the
absence of HABA (C). Spectra acquisition conditions: acquisition time, 100
s; power, 50 MW; excitation wavelength, 457.9 nm.
FIG. 4 is a combined plot of typical SERRS spectra obtained in a "sandwich"
immunoassay for TSH antigen using a DAB-anti-TSH antibody conjugate.
Silver electrodes coated with anti-TSH capture antibody were incubated
with various concentrations of TSH antigen and then transferred to a 40
.mu.g/ml solution of DAB-anti-TSH antibody conjugate. (A) SERRS spectrum
of a 40 .mu.g/ml solution of DAB-anti-TSH antibody conjugate in the
absence of a silver surface. Plots (B), (C), (D), (E), and (F) show
spectra obtained by incubating capture antibody-coated electrodes in
solutions containing 0, 4, 10, 25 and 60 .mu.IU of TSH antigen,
respectively, followed by transfer to a 40 .mu.g/ml solution of
DAB-anti-TSH antibody conjugate.
FIG. 5 is a plot of average SERRS intensity at 1141 cm.sup.-1 as a function
of TSH antigen concentration for known TSH standards. Values were obtained
at five different places on the silver electrode and averaged. One
electrode was used for each concentration of TSH antigen measured. Numbers
in parentheses are the coefficients of variation (standard deviation/mean)
for each concentration of analyte measured.
FIG. 6 is absorbance (492 nm) vs. TSH antigen concentration obtained using
reagents from a commercial enzyme immunoassay kit (Abbott Labs No. 6207).
Each data point represents the average of four determinations. The numbers
in parentheses are the coefficients of variations (standard
deviation/mean) for each concentration of TSH antigen measured.
FIG. 7 is a SERS spectra using near IR excitation for A) spectrum of a
blank silver film determined separately and added to solution state
spectrum done in the absence of a silver surface, of the
p-dimethylaminoazobenzene bovine serum albumin conjugate at 20 mg/ml, B)
spectrum obtained by immersing the blank silver film in the aforementioned
solution of the p-dimethylaminoazobenzene bovine serum albumin conjugate.
IV. DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS
A. Alternative Preferred Embodiments
1. Surfaces
Many metallic materials and configurations may be used for the SERS active
surface. These materials (for example, silver, gold, copper, platinum
etc.) could take the form of flat. surfaces (electrodes, strips, slides,
etc.) or dispersed colloids, particles, droplets, i.e. mercury, or inert
support structures of silica, plastic, glass, paper, or other materials
which may be in the form of macroscopically flat or textured (ruled,
etched, dimpled, or molded) pieces, slides, strips or spheroids, or fibers
which are coated with the active material (e.g., silver, gold, etc.) such
that they will support the surface enhancement of Raman scattering
described above. The surface or layer giving the enhancement can also be
coated with another material (silica, plastic, oxide, etc.) to which the
specific binding member is attached.
The presence of photoexcitable surface plasmons is generally considered
necessary for surface enhancement. In order for surface plasmons to
surface so that plasmon emission can occur. SERS effect, its surface
plasmons must be localized so that their resident energy is not dispersed.
This can be accomplished by preparing a roughened surface composed of small
particles. In practice, the surface of a solid piece of metal can be
electrochemically "roughened". As in the examples which follow silver
particles can be precipitated from solution onto a support, or silver can
be deposited on a support by evaporation or sputter coating. Silver coated
replica gratings also give strong SERS enhancement as do silver coated
surfaces which have been textured with bumps or posts, or coated with
spheres, then coated with silver.
An attractive surface for SERRS based assays is metal colloid. A metal
colloid combines a very strong SERS activity with the advantage of a
liquid medium that can readily be handled. The combination of a SERS
readout and e colloidal reagent would allow assays to be run in a manner
similar to that used for present clinical chemistry analysis.
Another surface that may be used in the present invention is a glass,
impregnated with metal particles. This surface may be a silver-impregnated
glass, used as a substrate upon which an improved surface enhancement of
Raman scattering can be achieved. Certain glasses are commercially
available which have a percentage of particulate silver embedded into
their formulations. One glass in particular has been developed by Corning
Glassworks which may be particularly applicable to SERRS. This product,
Corning No. 8612 Polarcor, has elongated crystalline silver embedded into
its surface to a depth of approximately 35 micrometers. The crystals are
oriented in such a fashion so as to capitalize on the plasmon or resonant
absorption effects of the silver conduction band electrons. This
distribution and orientation of the silver in this product is intended to
behave as a polarizer. Light of random polarization whose waves are
aligned parallel to the long axes of the particles will be absorbed by the
particles. Light waves whose polar orientation is perpendicular to the long
axes will be transmitted unattenuated. The former case is the same
condition that must exist to produce the pronounced enhancement as seen
with SERRS. The SERRS enhancement is considerably greater on a
microscopically toughened surface than on a polished surface. This
roughening provides for a certain percentage of the total surface area to
have proper angular and distance components to absorb the correspondingly
polarized component of the impinging light waves. The aforementioned
filled glass product satisfies the required conditions without the need
for secondary processes like roughening. Side reactions such as oxidation,
sulfide formation and photodegradation, which are known to occur on
conventional pure silver surfaces, such as electrodes, are also avoided.
The product is produced with particle dimensions and spacial distributions
to accommodate a relatively broad band of wavelengths. Several discrete
band passes throughout the red and near infrared portion of the optical
radiation spectrum are available.
A specific binding member coupled to a Raman active label and bound near
the surface of this glass can potentially exhibit an even more pronounced
SERRS effect than in conventionally used surfaces for the following
reasons: (a) the spacings between the silver surface and the label are
more uniform (since the particle orientation is far less random); (b) the
orientation of the silver particles with respect to the polarization of
the light waves can be made optimal by physically moving the glass and (c)
the incident light and the Rayleigh scattered light should be almost
totally absorbed into the filled glass, simplifying the removal of
reflected or Rayleigh scattered light from the Raman signal.
2. Attachment of Specific Binding Members to SERS-Active Surfaces
A specific binding member can be attached to the SERS-active surface by
direct absorption, absorption through a linker arm covalently attached to
the specific binding member, or by the covalent attachment of the specific
binding member to a coating on the SERS-active surface directly or through
a linker arm or by intercalation of the distal portion of a linker arm
into the enhancing surface.
3. Reporter Molecules or Labels
The SERS-active reporter groups or labels can be any one of a number of
molecules with distinctive Raman scattering patterns. Unlike the enzymes
used in enzyme immunoassays, these labels species can be stable, simple,
inexpensive molecules which can be chemically modified as required.
The following attributes enhance the effectiveness of the label in this
application:
(a) A strong absorption band in the vicinity of the laser excitation
wavelength (extinction coefficient near 10 to the four power).
(b) A functional group which will enable it to be covalently bound to a
specific binding member.
(c) Photostability.
(d) Sufficient surface and resonance enhancement to allow detection limits
in the subnanogram range.
(e) Minimal interference in the binding interaction between the labeled and
unlabeled specific binding members.
(f) Minimal exhibition of strong fluorescence emission at the excitation
wave length used.
(g) A relatively simple scattering pattern with a few intense peaks.
(h) Labels with scattering patterns which do not interfere with each other
so several indicator molecules may be analyzed simultaneously.
Potential candidates for this reporter can be
4-(4-Aminophenylazo)phenylarsonic acid monosodium salt, arsenazo I, basic
fuchsin, Chicago sky blue, direct red 81, disperse orange 3, HABA
(2-(4-hydroxyphenyl- azo)-benzoic acid, erythrosin B, trypan blue, ponceau
S, ponceau SS, 1,5-difluoro-2,4-dinitrobenzene, and
p-dimethylaminoazobenzene. The chosen labels may be covalently attached to
the specific binding members of interest.
4. Excitation Sources
In the preferred embodiment, a laser serves as the excitation source. The
laser may be of an inexpensive type such as a helium-neon or diode laser.
An operating lifetime of such lasers may be in excess of 50,000 hours.
In one embodiment, a diode laser is used to excite at or at the near IR
spectrum, minimizing fluorescence interference. The excitation sources
used need not necessarily be monochromatic and they also need not
necessarily have to be of high intensity. Lamps may also be used.
The SERS effect can be excited by direct illumination of the surface or by
evanescent waves from a waveguide beneath the plasmon-active surface.
5. Conjugates
Several different conjugates could be prepared from specific binding
members having different specificities, each type with a different Raman
active label having a distinctive scattering pattern. Mixing these
conjugates in an assay would allow the simultaneous analysis of several
different analytes in the same sample.
6. Detection
Several methods are available for detecting Raman scattering. These
generally can be used with different types of spectrometers. In SERS, the
primary measurement is one of light scattering intensity at particular
wavelengths. SERS requires measuring wave-length-shifted scattering
intensity in the presence of an intense background from the excitation
beam. The use of a Raman-active substance having a large Stokes shift
simplifies this measurement.
Several concepts for further simplifying the readout instrument have been
proposed. These include the use of wavelength selective mirrors or
holographic optical elements for scattered light collection.
Neither the angle of the incident light beam to the surface nor the
position of the detector is critical using SERS. With flat surfaces
positioning the surface of the laser beam at 60 degrees to the normal is
commonly done and detection at either 90 degrees or 180 degrees to the
beam are standard. SERS excitation can be performed in the near infrared
range which would suppress intrinsic sample fluorescence. It may also be
possible to perform SERS-based ligand binding assays using evanescent
waves produced by optical waveguides.
No signal development time is required as readout begins immediately upon
illumination and data can be collected for as long as desired without
decay of signal unless the excitation light is extremely intense and
chemical changes occur. The signal cannot overdevelop as in systems
dependent on optical absorbance. Unlike fluorescent readout systems, SERS
reporter groups will not self-quench so the signal can be enhanced by
increasing the number of Raman reporter groups on the probe molecule.
Fluorescent molecules near the SERS-active surface will actually be
surface-quenched.
7. Instrumentation
The present invention is adaptable for use as an automatic analyzer. Since
the instrument would monitor discrete Stokes shifted spectral lines, the
need for an elaborate monochromator system is not necessary. Recent
advances in state-of-the-art optics technology, such as holographic
optical elements, allow the design of a suitable spectrometer with cost
and complexity below that of the laboratory grade device.
Optical readout energies as result of SERS are above that which require
ultra-sensitive photon counting devices. In fact, some SERRS spectrometers
now in use incorporate silicon photodiode detectors. The optical efficiency
of a typical monochromator used in a laboratory grade spectrometer is less
than 10%. The advances in optical materials and components mentioned above
should make possible two to three-fold increases in optical efficiency for
a simple spectrometer dedicated to only a few specific spectral lines.
This also addresses one of the previously major concerns, blocking of the
Rayleigh scattering line. With blocking capabilities of newer filters on
the order of 10.sup.-9, substitution of filters for one or more stages of
the typical monochrometer system should be possible with significant cost
savings.
B. EXAMPLES
Example 1
Preparation Of Silver Surfaces
Support surfaces--Supports for the silver films were either flat, frosted
glass pieces cut from microscope slides or quartz pieces cut from 4
in..times.4 in..times.20 mil. quartz substrates (General Electric type
124).
Chemical deposition--Silver was deposited on support surfaces by chemical
reduction of silver nitrate as previously described by Ni and Cotton.
Anal. Chem., 58, 3159, 1986. Tollens reagent was used to deposit the
silver. Tollen's reagent was prepared in a small beaker by adding about 10
drops of fresh 5% NaOH solution to 10 mL of 2-3% AgNO.sub.3 solution,
whereupon a dark-brown AgOH precipitate is formed. This step is followed
by dropwise addition of concentrated NH.sub.4 OH, at which point the
precipitate redissolves. The beaker containing the clear Tollen's reagent
was then placed in an ice bath. The frosted slides, which had been cleaned
with nitric acid and distilled water, were placed into a Teflon frame,
which could accommodate up to 15 slides, and placed into the Tollen's
reagent. Three milliliters of 10% D-glucose was added to the solution with
careful swirling to ensure mixing. The beaker was then removed from the ice
bath and the solution allowed to reach room temperature. The beaker was
placed into a water bath (55.degree. C.) for 1 min followed by
sonification for 1 min (Branson Sonicator, Model B22-4, 125 W). Finally,
the silver-coated slides were rinsed several times with distilled water
and again sonicated in distilled water for 30 s. The slides were then
stored in distilled water for several hours prior to exposure to the
adsorbate solution. By use of this procedure, slides were found to be
stable in distilled water for up to 1 week.
The surfaces were yellow by transmitted light and demonstrated a coarse,
granular appearance by scanning electron microscopy. A profilometer probe
traversing the surface revealed many prominences, some approaching
10.sup.3 nm in height (FIG. 1). A cross section of the silver layer
generated by scratching the surface with a stylus revealed it to be
composed of partially fused spheroids approximately 100 nm in diameter.
The step produced by scratching the silver off the substrate was found to
be approximately 130 nm thick by profilometry.
Sputter coating--Quartz pieces were coated with a 75A layer of silver by
sputter coating using a Perkin-Elmer Randex Model 2400-85A while being
rotated at 2.25 rpm for 4.5 min at a distance of 6.75 cm from the silver
target. A forward power of 200 W and an argon flow rate of 12.25 cc/min
were used. The silver film was transparent, blue by transmitted light.
Scanning electron microscopy at a 2500-fold enlargement showed a
fine-grained featureless surface.
Silver electrodes--Silver electrodes were prepared as previously described
by Ni and Cotton, J. Raman Spectroscopy, 19, 429, 1988. They were
constructed by sealing a flattened silver wire into a glass tube with Torr
Seal. The exposed surface was rectangular with dimensions of approximately
2.times.10 mm. The electrode was polished with a slurry of 0.3 .mu.m
alumina in water on a mechanical polishing wheel. It was then rinsed and
sonicated in distilled water to remove any alumina which might have
adhered to the surface. This step was followed by roughening the electrode
by an oxidation-reduction cycle (ORC), consisting of a double potential
step from an initial potential of -550 mV to +500 mV and back to -550 mV
in 0.1M Na.sub.2 SO.sub.4 solution. An Ag--AgCl electrode was used as the
reference electrode and a Pt electrode as the auxiliary electrode. The
total charge passed during the oxidation step was equivalent to 25 mC
cm.sup.-2.
Silver colloids--Silver colloids were prepared by a modification of the
procedure of Lee and Meisel, J. Phys. Chem. 86, 3391, 1982. 90 mg of
silver nitrate was dissolved in 500 ml of distilled water and brought to
boiling. A 10 ml solution of 1% sodium citrate was added all at once and
the solution was stirred for 45 minutes, during which the silver colloid
formed. The colloid was cooled to room temperature and stored for use
without further purification. Typical particle sizes resulting from such
preparations ranged from 20 to 80 nm.
Example 2
Preparation of Dye-Antibody Conjugates
Antibody (2 mg) was dissolved in 2 ml of 1% NaHCO.sub.3, pH 8.6, and a
20-.mu.l aliquot of a solution of 1 mg/ml
4-dimethylaminoazobenzene-4'-isothiocyanate in dimethylformamide (DMF)
added. The mixture was stirred overnight, then desalted on a Sephadex G-25
(coarse) column (1.times.30 cm). The ultraviolet and visible spectrum of
the conjugate was compared to that of DAB and antibody alone, to determine
the degree of substitution. The erythrosin-antibody conjugate was prepared
the same way, except the concentration of the erythrosin-isothiocyanate in
DMF was 2.5 mg/ml.
Example 3
Nitration of Bovine Serum Albumin
A solution of 2 ml of 2,4-dinitrofluorobenzene in 150 ml of ethanol was
mixed with a solution of 200 mg of bovine serum albumin of 10 g Na.sub.2
CO.sub.3 in 100 ml distilled water. The mixture was stirred for 24 h and
centrifuged at 3000 g for 20 min to remove precipitated material and the
supernate was dialyzed against 6 liters of phosphate-buffered saline (PBS)
for 23 h, then against two changes of 2 liters of PBS for 6 h each, and
finally against two changes of 2 liters of distilled water, 6 h each.
Dialysis Was carried out at room temperature with 0.02% sodium azide
present in all solutions except the final 2 liters of water. The contents
of the dialysis bag were then lyophilized to dryness, yielding 136 mg. A
sample was compressed into a potassium bromide pellet and its infrared
spectrum. recorded on a Nicolet 60 SX FT infrared spectrometer. A strong
vibrational band at 1340 cm.sup.-1, not inherent to native BSA, indicated
introduction of nitro groups (data not shown). The degree of substitution
of the BSA was determined by comparing the degree to which BSA and the
nitro-BSA conjugate could be derivatized with
2,4,6-trinitrobenzenesulfonic acid (TNBSA). After reaction with TNBSA, the
average absorbance at 330 nm of a 1 mg/ml solution of native BSA increased
from 0 to 1.5 as the result of the derivitization of free amino groups.
The same concentration of the DNP-BSA conjugate had an initial absorbance
at 330 nm of 1.2 (from the DNP groups) which did not increase after
incubation with the TNBSA reagent. It can be concluded that essentially
all the available amino groups in the DNP-BSA conjugate had been
derivatized with DNP by the Sanger's reagent.
Example 4
Generation of SERS Spectra by DNP-BSA Conjugate Absorbed to Silver Films
Freshly prepared silver-coated slides (chemically deposited) were incubated
in buffer (pH 8.6) containing free DNB (FIG. 2A) or DNP-BSA conjugate (FIG.
2B), and SERS spectra obtained in both cases. Similar peak intensities were
observed with free DNB at 10.sup.-3 M and DNP-BSA at 10.sup.-7 M with
respect to DNP moieties (2.times.10.sup.-9 BSA), respectively. The four
orders of magnitude difference in the specific intensity of
surface-enhanced Raman light scatter observed between the free DNB and the
DNP moieties of the DNP-BSA conjugate represents the greater ability of the
latter to adsorb to the island film surface, thereby enabling its DNP
moieties to display the SERS enhancement. A10.sup.-3 M solution of DNB in
the absence of an island film gave a very weak Raman spectrum (FIG. 2C).
Example 5
Use of a Raman-Active Dye to Demonstrate Surface-Enhanced Resonance Raman
Spectroscopy
An avidin molecule will also bind four molecules of the dye HABA, with an
affinity constant of K.sub.a =5.8.times.10.sup.6 liter/mol at pH 7.0.
Because this dye has a major spectral absorption at a wavelength which can
be used to excite Raman light scattering (absorption maximum =495 nm when
bound to avidin at pH 7) it is capable of SERRS.
Chemically deposited silver films, with and without prior coating with
avidin, were incubated in a 3 mM solution of HABA. The films were then
removed from the HABA solution and washed with PBS, and their Raman
spectra taken. FIG. 3A is the spectrum obtained when HABA is adsorbed
directly onto the surface of a silver film. A single major peak of light
scattering intensity is observed at 1406 wavenumbers, with a shoulder at
1459 and minor peaks at 1188 and 1139 cm.sup.-1. The spectrum shown in
FIG. 3B was obtained when a silver film was first incubated for 20 min at
room temperature in a 2.5.times.10.sup.-5 M solution of avidin, then HABA
added to a final concentration of approximately 0.3 mM, and incubation
continued for an additional 20 min. Under these conditions, the major peak
of Raman scattering intensity is observed at 1610 cm.sup.1, with several
smaller peaks appearing between 1160 and 1491 cm.sup.-1. The large peak at
1406 cm.sup.-1, seen in the absence of avidin, is no longer observed. In
the absence of HABA, an avidin-coated silver film gave no discernible
spectrum in this region (FIG. 3C).
Example 6
Dye-Antibody Conjugates and Raman Readout in a Sandwich Immunoassay
Silver electrodes were incubated at 37.degree. C. for 1 h in a 1 ml
aliquots of a solution of 20 .mu.g/ml anti-TSH antibody in 1%
NaHCO.sub.3,pH 8.6, and then overcoated for an additional hour in 1% BSA
in PBS at 37.degree. C. The films were then incubated for 1 h at
37.degree. C. in the 0, 4, 10, 25 or 60 .mu.IU/ml TSH antigen standards
from the Abbott TSH EIA kit, Abbott No. 6207. After being washed three
times with PBS, the films were transferred to test tubes containing 1 ml
of the DAB-anti-TSH antibody conjugate at a concentration of 40 .mu.g/ml,
incubated for an additional hour at 37.degree. C., washed again, and the
SERRS spectra obtained.
SERRS spectra were obtained at five different places along each electrode
and the results recorded. A combined plot of typical spectra obtained is
shown in FIG. 4 for the five concentrations of TSH antigen studies. The
averaged peak intensities at 1151 cm.sup.-1 were used to generate a signal
vs. concentration curve (FIG. 5). The same standards were also assayed
using a modified commercial enzyme immunoassay (Abbott No. 6207, FIG. 6).
Comparison of the two plots shows that the response obtained using the
SERRS readout is similar to that given by the enzyme immunoassay, except
for an anomalously high value for the zero antigen standard. This high
zero reading was consistent upon reassay and must reflect a difference in
composition between the zero standard and the other standards | | |
