U.S. patent application number 10/177194 was filed with the patent office on 2003-01-23 for lyophilization of colloidal metals for surface enhanced raman scattering.
Invention is credited to Carron, Keith T., Ray, Bryan Hubert, Sulk, Roberta A..
Application Number | 20030017620 10/177194 |
Document ID | / |
Family ID | 26873026 |
Filed Date | 2003-01-23 |
United States Patent
Application |
20030017620 |
Kind Code |
A1 |
Carron, Keith T. ; et
al. |
January 23, 2003 |
Lyophilization of colloidal metals for surface enhanced Raman
scattering
Abstract
An assay and method of making same for use in SERS spectroscopy.
The assay includes colloidal particles of a metal, which have been
lyophilized. The lyophilized particles of metal produce a SERS
active solution when reconstituted. The lyophilized particles of
metal may be provided in a container in an assay system.
Inventors: |
Carron, Keith T.;
(Centennial, WY) ; Ray, Bryan Hubert; (Laramie,
WY) ; Sulk, Roberta A.; (Laramie, WY) |
Correspondence
Address: |
Duane Morris LLP
Suite 100
100 College Road West
Princeton
NJ
08540
US
|
Family ID: |
26873026 |
Appl. No.: |
10/177194 |
Filed: |
June 21, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60300270 |
Jun 21, 2001 |
|
|
|
Current U.S.
Class: |
436/525 ;
422/400; 436/164; 436/172; 436/174 |
Current CPC
Class: |
G01N 21/658 20130101;
Y10T 436/25 20150115; G01N 33/54373 20130101; G01N 33/54393
20130101 |
Class at
Publication: |
436/525 ;
436/164; 436/174; 436/172; 422/61; 422/102 |
International
Class: |
G01N 001/00; G01N
021/00 |
Claims
What is claimed is:
1. An assay for use in SERS spectroscopy, the assay comprising
lyophilized colloidal particles of a metal, which when
reconstituted, produce a SERS active solution.
2. The assay of claim 1, wherein the metal is a noble metal.
3. The assay of claim 1, wherein the assay is used in SERS
spectroscopy to determine the concentration of an unknown
solution.
4. The assay of claim 1, further comprising a material for
preventing the particles from sticking to one another.
5. The assay of claim 4, wherein the material comprises a wax-like
material.
6. The assay of claim 4, wherein the material comprises
polyethylene glycol.
7. An assay system for use in SERS spectroscopy, the assay system
comprising: a container; lyophilized colloidal particles of a metal
disposed in a first section of the container; wherein
reconstitution of the lyophilized colloidal particles of a metal
produce a SERS active solution.
8. The assay system of claim 7, wherein the metal is a noble
metal.
9. The assay system of claim 7, further comprising a closure for
sealing the container.
10. The assay system of claim 7, further comprising a reagent
disposed in a second section of the container, the reagent for
coupling an analyte to a SERS active metal.
11. The assay system of claim 10, wherein the second section of the
container is remote from the first section of the container.
12. The assay system of claim 7, further comprising a material for
preventing the particles from sticking to one another.
13. The assay system of claim 12, wherein the material comprises a
wax-like material.
14. The assay system of claim 12, wherein the material comprises
polyethylene glycol.
15. The assay system of claim 12, further comprising a pretreatment
disposed on an inner surface of the container, the pretreatment for
preventing the particles from sticking to one another and sticking
to the inner surface of the container.
16. The assay system of claim 15, wherein the pretreatment
comprises a wax-like material.
17. The assay system of claim 15, wherein the pretreatment
comprises polyethylene glycol.
18. The assay system of claim 7, further comprising a pretreatment
disposed on an inner surface of the container, the pretreatment for
preventing the particles from sticking to one another and sticking
to the inner surface of the container.
19. The assay system of claim 18, wherein the pretreatment
comprises a wax-like material.
20. The assay system of claim 18, wherein the pretreatment
comprises polyethylene glycol.
21. The assay system of claim 7, wherein the assay is used in SERS
spectroscopy to determine the concentration of an unknown
solution.
22. The assay system of claim 7, wherein the first section of the
container includes a plurality of wells and lyophilized colloidal
particles of a metal disposed in each of the wells.
23. The assay system of claim 22, wherein the lyophilized colloidal
particles of metal include an antibody.
24. The assay system of claim 23, wherein the assay system forms a
Surface Enhanced Raman ImmunoAssay.
25. A method of producing an assay for use in SERS spectroscopy,
the method comprising: providing a suspension of colloidal
particles of metal; lyophilizing the suspension of colloidal
particles of metal to produce the assay, which when reconstituted,
produce a SERS active solution.
26. The method of claim 25, wherein the metal is a noble metal.
27. The method of claim 25, further comprising the step of mixing
the colloidal particles of metal with a material prior to the
lyophilizing step, the material preventing the particles from
sticking to one another.
28. The method of claim 27, wherein the material comprises a
wax-like material.
29. The method of claim 27, wherein the material comprises
polyethylene glycol.
30. A method of making an assay system for use in SERS
spectroscopy, the method comprising the steps of: providing a
container; placing a suspension of colloidal particles of a metal
in a first section of the container; lyophilizing the suspension of
colloidal particles of the metal to produce an assay, which when
reconstituted, produce a SERS active solution.
31. The method of claim 30, wherein the metal is a noble metal.
32. The method of claim 30, further comprising the step of mixing
the suspension of colloidal particles of metal with a material
prior to the placing step, the material preventing the particles
from sticking to one another.
33. The method of claim 32, wherein the material comprises a
wax-like material.
34. The method of claim 32, wherein the material comprises
polyethylene glycol.
35. The method of claim 32, wherein the container providing step
includes applying a pretreatment to an inner surface of the
container, the pretreatment for preventing the particles from
sticking to one another and sticking to the inner surface of the
container.
36. The method of claim 35, wherein the pretreatment comprises a
wax-like material.
37. The method of claim 35, wherein the pretreatment comprises
polyethylene glycol.
38. The method of claim 30, further comprising the step of placing
a reagent in a second section of the container, the reagent for
coupling an analyte to a SERS active metal.
39. The method of claim 38, wherein the lyophilizing step further
includes lyophilizing the reagent.
40. The method of claim 38, wherein the second section of the
container is remote from the first section of the container.
41. The method of claim 30, where the container providing step
includes applying a pretreatment to an inner surface of the
container, the pretreatment for preventing the particles from
sticking to one another and sticking to the inner surface of the
container.
42. The method of claim 41, wherein the pretreatment comprises a
wax-like material.
43. The method of claim 41, wherein the pretreatment comprises
polyethylene glycol.
44. A method of analyzing a material, the method comprising the
steps of: providing a container; placing a suspension of colloidal
particles of a metal in a first section of the container;
lyophilizing the colloidal particles of the metal to produce an
assay; simultaneously reconstituting and mixing the assay with the
material to be analyzed to produce a SERS active solution;
performing SERS spectroscopy on the SERS active solution.
45. The method of claim 44, wherein the metal is a noble metal.
46. The method of claim 44, further comprising the step of mixing
the suspension of colloidal particles of metal with a material
prior to the placing step, the material preventing the particles
from sticking to one another.
47. The method of claim 46, wherein the material comprises a
wax-like material.
48. The method of claim 46, wherein the material comprises
polyethylene glycol.
49. The method of claim 46, wherein the container providing step
includes applying a pretreatment to an inner surface of the
container, the pretreatment for preventing the particles from
sticking to one another and sticking to the inner surface of the
container.
50. The method of claim 49, wherein the pretreatment comprises a
wax-like material.
51. The method of claim 49, wherein the pretreatment comprises
polyethylene glycol.
52. The method of claim 44, further comprising the step of placing
a reagent in a second section of the container, the reagent for
coupling an analyte to a SERS active metal.
53. The method of claim 52, wherein the lyophilizing step further
includes lyophilizing the reagent.
54. The method of claim 52, wherein the second section of the
container is remote from the first section of the container.
55. The method of claim 44, where the container providing step
includes applying a pretreatment to an inner surface of the
container, the pretreatment for preventing the particles from
sticking to one another and sticking to the inner surface of the
container.
56. The method of claim 55, wherein the pretreatment comprises a
wax-like material.
57. The method of claim 55, wherein the pretreatment comprises
polyethylene glycol.
58. The assay of claim 1, further comprising an antibody.
59. The assay system of claim 7, further comprising an
antibody.
60. The method of claim 25, further comprising the step of mixing
the suspension of colloidal particles of metal with an antibody
prior to the lyophilizing step.
61. The method of claim 30, wherein the first section of the
container includes a plurality of wells and lyophilized colloidal
particles of a metal are placed in each of the wells.
62. The method of claim 61, wherein the lyophilized colloidal
particles of metal include an antibody.
63. The method of claim 62, wherein the assay system forms a
Surface Enhanced Raman ImmunoAssay.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/300,270, filed Jun. 21, 2001.
FIELD OF THE INVENTION
[0002] This invention relates to Raman spectroscopy and Surface
Enhanced Raman Scattering (SERS). More specifically, this invention
relates to a stable form of colloidal metal particles that when
reconstituted, produces a SERS active solution.
BACKGROUND OF INVENTION
[0003] Raman spectroscopy stems from the inelastic scattering of
light by molecular vibrational energy levels. Raman spectroscopy as
an analytical tool has been known for decades, and is particularly
popular for several reasons. For example, molecular composition can
be determined in the presence of water. Visible light can be
employed for analysis allowing for the use of conventional fiber
optics. Unique spectral fingerprints allow for identification and
quantification of a wide variety of solids, liquids, and gases. One
of the significant disadvantages of Raman spectroscopy is the
inadequate sensitivity for trace or ultratrace analysis. This stems
from the inherently weak nature of Raman scattering.
[0004] In the early 1970s several researchers found an anomalous
enhancement of Raman scattering at the surface of certain metals.
It has subsequently been found that the metals that have both
practicality and strong enhancing properties are silver, gold, and
copper. The enhancement is believed to generally come from an
electromagnetic effect and in some cases, an enhancement due to the
nature of the chemical bond to the metal surface has also been
found. The reported enhancement for SERS depends on the structure
of the surface and ranges from about 105 to 108. This discovery
immediately made it possible to detect very small amounts of
material adsorbed to these surfaces. The SERS effect is limited to
molecules attached to or in very close proximity with the
surface.
[0005] The drawback to conventional SERS is that it is limited to
analytes that will naturally adsorb to a SERS active metal surface.
Thus, while in special cases SERS provides sensitive detection, in
most cases it suffers from the inability of the molecule to adsorb
to the surface and to benefit from the SERS effect.
[0006] A method to overcome the lack of adsorptivity to SERS
surfaces by an analyte, is to provide surface coatings that have an
affinity for the analyte. An example is an early publication which
describes using a surface bound coating in the detection of
hydrogen ions at a surface using SERS (Determination of pH with
SERS Fiber Optic Probes. Ken I. Mullen, DaoXin Wang, L. Gayle
Hurley, and Keith Carron Anal. Chem., 64, 930, 1992). This
publication showed that it was possible to permanently attach a
coating to a SERS surface and to have the coating provide the
affinity for the analyte.
[0007] More recently, it has been demonstrated that an irreversible
covalent bonding reagent could be used to achieve even more
sensitive detection. Furthermore, it was shown that the surface
need not be coated with the surface bound reagent, but rather, the
reagent could have two reactive sites. One site is analyte specific
and the other is surface binding specific. This produces a high
affinity permanent bond to the analyte and a high affinity
permanent bond to the surface. An example of a dual binding reagent
for trace detection is a reagent that binds bilirubin and which has
an argentiphillic sulfide group to bind to silver (Surface Enhanced
Raman Assays (SERA): Measurement of Bilirubin and Salicylate,
Roberta Sulk, Collin Chan, Jason Guicheteau, Cieline Gomez, J. B.
B. Heyns, Robert Corcoran, and Keith Carron, J. Raman Spectrosc.,
1999, 30, 853-859).
[0008] Accordingly, an assay is needed which is capable of
producing a SERS active solution that is sensitive to a specific
analyte or group of analytes.
SUMMARY OF THE INVENTION
[0009] An assay and method of making same is disclosed herein for
use in SERS spectroscopy. The assay comprises lyophilized colloidal
particles of a metal, which have been lyophilized. The lyophilized
particles of metal produce a SERS active solution when
reconstituted.
[0010] An assay system and method of making same is further
disclosed herein for use in SERS spectroscopy. The assay system
comprises a container with lyophilized colloidal particles of a
metal disposed in a first section thereof. The lyophilized
colloidal particles of a metal contained in the container produce a
SERS active solution when reconstituted.
[0011] A method of analyzing a material is further disclosed
herein. The method comprises the steps of: providing a container;
placing colloidal particles of a metal in a first section of the
container; lyophilizing the colloidal particles of the metal to
produce an assay; simultaneously reconstituting and mixing the
assay with the material to be analyzed to produce a SERS active
solution; performing SERS spectroscopy on the SERS active
solution.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1A is a spectrum of reactive blue (RB) 15 dye in fresh
colloid suspension.
[0013] FIG. 1B is a spectrum of a RB 15 dye with reconstituted
lyophilized colloidal suspension.
[0014] FIG. 2A is a spectrum of a Partial Least Squares (PLS)
correlation model using a reconstituted lyophilized colloid and RB
15 dye.
[0015] FIG. 2B is a correlation plot between actual concentration
and predicted concentration for a reconstituted lyophilized colloid
and RB 15 dye solution.
[0016] FIG. 3A is a spectrum of a Partial Least Squares (PLS)
correlation model using a reconstituted lyophilized colloid with
0.05% w/w polyethylene glycol (PEG) and RB 15 dye.
[0017] FIG. 3B is a schematic representation of an exemplary
apparatus used for producing the lyophilized, colloidal noble metal
particles and PEG according to the present invention.
[0018] FIG. 3C is a correlation plot between actual concentration
and predicted concentration for a reconstituted lyophilized colloid
solution with 0.05% w/w PEG and RB 15 dye.
[0019] FIG. 4A is a spectrum of a Partial Least Squares (PLS)
correlation model using a reconstituted lyophilized colloid with
0.05% PEG, RB 15 dye, and a reagent of Analyte Reactive Coating
soluble in Water (ARCW).
[0020] FIG. 4B is a diagrammatic representation of the chemical
structure of the Analyte Reactive Coating soluble in Water
(ARCW).
[0021] FIG. 4C is a correlation plot between actual concentration
and predicted concentration for a reconstituted lyophilized colloid
solution with 0.05% w/w PEG and RB 15 dye and a reagent of
ARCW.
[0022] FIGS. 5 and 6 are PLS cross-validation plots of actual
versus predicted hGH concentration levels for a Surface Enhanced
Raman ImmunoAssay (SERIA) analysis using a lyophilized silver
colloidal base according to the present invention.
[0023] FIG. 7 is a schematic representation of an assay system
according to a first illustrative embodiment of the present
invention using lyophilized colloids.
[0024] FIG. 8 is a schematic representation of an assay system
according to a second illustrative embodiment of the present
invention using lyophilized colloids.
DETAILED DESCRIPTION OF THE INVENTION
[0025] One aspect of the present invention includes an assay and an
assay system for use in Surface Enhanced Raman Scattering (SERS)
spectroscopy. Another aspect of the present invention includes
methods for preparing the assay and assay system. Still another
aspect of the present invention includes a method of analyzing a
material using the assay system of the present invention and SERS
spectroscopy. Another aspect of the present invention includes a
method of analyzing a material using the assay system of the
present invention and Surface Enhanced Raman ImmunoAssay
(SERIA).
[0026] The assay of the present invention comprises a colloidal
suspension of noble metal particles, such as silver, gold, or
copper particles, which have been lyophilized to dryness using
conventional lyophilizing techniques. The lyophilized, colloidal
particles of the present invention have long term stability, i.e.,
the colloidal particles can be stored for a long period of time,
and are sensitive to a specific analyte or group of analytes. The
lyophilized, colloidal noble metal particles of the present
invention produce a SERS active solution, when reconstituted to a
colloidal suspension. A specific advantage of having a SERS active
solution is that the SERS phenomenon exhibits a signal from
material localized near the particle surface. This phenomenon
precludes the need for removing excess analyte, impurity, or
reagent, that indicates the presence of an analyte, from the sample
mixture. This aspect combined with the aspect of a coated particle
with long-term stability makes the assay of the invention
commercially important.
[0027] A particularly important aspect of the assay of the present
invention is that the amount of colloidal particles is determined
very accurately through a volumetric delivery of a known
concentration of colloidal suspension, or delivery of a known mass
of colloidal suspension. The mass delivery is enabling to an assay
since a large mass of diluted colloid can be used to accurately
deliver a small amount of colloid into the chamber of a sample
container.
[0028] The lyophilized, colloidal particle assay of the present
invention may further comprise one or more reagents. The type or
types of reagents used will depend upon the particular application
or sample to be analyzed. Further, the lyophilized, colloidal
particle assay may further comprise one or more antibodies, thereby
forming a Surface Enhanced Raman ImmunoAssay (SERIA).
[0029] The assay system of the present invention comprises a sample
container which contains the lyophilized, colloidal noble metal
particles of the present invention. The container typically
includes a pretreatment which prevents the lyophilized, colloidal
particles from binding to the surface of the chamber of the
container, or from binding with each other, thus inhibiting the
lyophilized, colloid's ability to be reconstituted to a colloidal
suspension. The pretreatment may comprise a wax-like material, such
as polyethylene glycol (PEG), or combination of materials, which is
applied to the chamber of the container. It should be understood,
however, that the pretreatment can be omitted when the sample
container is made from a material which possesses the ability to
contain the lyophilized, colloid without inhibiting its ability to
be reconstituted.
[0030] In addition or alternative to pretreating the chamber
surface(s) of the container, a material or combination of materials
may be added to the colloidal particles, prior to lyophilization,
which also immobilizes the lyophilized, colloidal particles within
the container and prevents the colloidal particles from binding to
the surface of the chamber of the container, or from binding with
each other. This pretreatment may also be omitted. This material
may also comprise a wax-like material, such as PEG. Another example
would be a surfactant material such as sodium dodecylsulfate (SDS).
In view of the above, it should be apparent that the assay system
of the present invention affords long term stability such that a
pretreated assay can be provided for a customer for later use.
[0031] In certain applications, one or more reagents must be added
in sequential fashion to the assay of the present invention. FIG. 7
shows an assay system according to a first illustrative embodiment
of the present invention, which enables sequential addition of such
reagents to the assay via physical separation of the reagent and
colloid. As shown, a lyophilized assay reagent 16 is located in a
chamber 11 of a tube-style container 10 adjacent to a tapered open
end 12 thereof, and a lyophilized, colloidal particle assay 18 of
the present invention is in the chamber 11 adjacent to an opposing
open end 14 of the container 10. This enables one to introduce a
sample into the chamber 11 of the container 10 and have it
reconstitute and mix with the reagent 16 first by capillary action.
Mixing of the sample and reagent is accomplished with a mixing rod
13 disposed in the chamber 11 of the container 10, after which the
reagent and sample mixture reconstitutes the lyophilized, colloidal
particle assay 18 and mixes therewith. A closure 17, such as a
Critoseal, is provided and is inserted in the tapered open end 12
of the container 10 after the sample is collected. It should be
understood, that containers of other configurations can be utilized
in the assay system of the present invention.
[0032] Assays are typically performed either individually or
multiply. Multiple assays have an advantage that many of the steps
involved in the assay can be performed in parallel, thus decreasing
the time of the assay. Accordingly, the assay system of the present
invention may also comprise a sample container with multiple sample
chambers, which enables multiple assays to be performed in
parallel, if desired. FIG. 8 shows an system according to a second
illustrative embodiment of the invention constructed as a
multiformat assay 40 which forms a Surface Enhanced Raman
ImmunoAssay (SERIA) where the lyophilized colloid is treated with
antibodies and reconstitution is performed in a microwell plate 42
with added sample and reagents. As shown, the microwell plate 42
includes a plurality of wells 44 containing identical lyophilized
colloidal particles 46 (colloidal solid) treated with one or more
antibodies for analyzing multiple assays at one time. Each
lyophilized colloidal solid 46 is reconstituted to a SERS active
colloidal solution by the reagent liquids of the assay.
[0033] Additionally, as the assay of the present invention takes
special advantage of the SERS effect to produce a one-step assay,
the sample chamber or chambers include closures, e.g. Critoseal 17
(FIG. 7), which are used for sealing the chambers after
introduction of a sample, to prevent contamination of the sample or
more importantly, to prevent potential spread of the sample, which
may be hazardous to testing personnel or the test facility.
[0034] FIGS. 1A and 1B represent the spectral results of an initial
study of lyophilized, colloidal noble metal particles. FIG. 1A is a
spectrum of reactive blue (RB) 15 dye in fresh colloid suspension
having a pH of 6.8. Ten milliliters of this colloid was lyophilized
to dryness using conventional lyophilization techniques. The
leftover powder was dark gray and very light and susceptible to air
currents. The gray powder was then reconstituted with 10 ml of
H.sub.2O and 2 .mu.L of an aqueous 1% sodium hydrogen carbonate
solution was added to raise the pH from 5.7 to that of the fresh
colloid (6.8). Note that no polyethylene glycol (PEG) was added to
the reconstituted colloid. The reconstituted colloid was then
ultrasonicated for 5 min to produce a cloudy gray solution. The
reconstituted colloidal suspension was made to be 60 .mu.m RB 15.
FIG. 1B is a spectrum of this reconstituted colloidal suspension
with RB 15 dye.
[0035] FIG. 2A represents a spectrum of a Partial Least Squares
(PLS) correlation model obtained from running a concentration curve
of a reconstituted colloid and RB 15 dye. A stock RB 15 solution
was prepared by dissolving 0.01283 grams of RB 15 into 10 ml
Millipore H.sub.2O. Concentrations were prepared from the stock
solution ranging from 1.0-6.18.times.10.sup.-6 M RB 15 using
Millipore H.sub.2O. The reconstituted colloid (200 .mu.L) was
placed into separate 1 ml auto sampler vials. A 20 .mu.L aliquot of
each of the RB 15 concentrations was added to separate vials filled
with reconstituted colloid. Each mixture was shaken for 20 sec to
facilitate an even distribution of RB 15 dye in the colloid. A
spectrum of each mixture was taken and recorded, after which PLS
was performed on the data set. FIG. 2B shows the result in a
correlation plot between actual concentration and predicted
concentration with a R.sup.2=0.9793. This shows possibilities for
lyophilized colloids. However, the lyophilized colloid is a very
fine powder that is very susceptible to air currents. Accordingly,
in the discussion which follows, polyethylene glycol (PEG) 900 will
be introduced into the lyophilizing process to alleviate problems
with containing the fine powder and preventing the lyophilized
colloidal particles from sticking to the surface of a
container.
[0036] FIG. 3A represents a spectrum of a PLS correlation model
using 0.05% w/w PEG 900 m.w./colloid and RB 15 dye. In a 20 mL vial
0.200 g of PEG 900 was diluted to 4.00 g with colloid. A 370 .mu.L
Caraway blood collecting tube was submerged into liquid nitrogen
until the nitrogen ceases to boil then 100 .mu.L of the solution
was injected into the frozen tube. Approximately 40 tubes can be
prepared from 4.00 g of PEG 900/colloid solution. As shown in FIG.
3B, the tubes, denoted by numeral 30, were placed in a high vacuum
vial 32 with a stopper 33 purchased from Labconco. The vial 32 and
stopper 33 was placed in a Styrofoam cooler 34 packed in dry ice
and attached to a lyophilizer (not shown), via a steel tube 36
which extends through the stopper, for 24 hours. A stock solution
of 10.0.times.10.sup.-6 M RB 15 was prepared. A range of
concentrations from 1.0-10.0.times.10.sup.-6 M RB 15 was prepared
by diluting with Millipore H.sub.2O. In separate lyophilized tubes
100 .mu.L of each concentration of RB 15 was injected and a
0.5.times.5 mm Teflon coated rod was inserted into each tube. A
magnet was used to stir the solution with the rod until the
lyophilized colloid was completely dissolved, this taking
approximately 2 min. The solution was then allowed to flow to the
narrow end of the blood collecting tube, a plug of Critoseal was
inserted and the tube was placed, plug side down, into the Raman
sample holder. A spectrum was taken and recorded for all the
different concentrations, after which PLS was performed on the data
set. FIG. 3C shows the result in a correlation plot between actual
concentration and predicted concentration with a R.sup.2=0.9956.
When the spectrum of FIG. 2A is compared to the spectrum of FIG.
3A, there are identical peaks and correlation. This indicates that
the PEG has little effect on the Raman scattering and it aids in
the containment of the lyophilized colloid eliminating
susceptibility of the colloid powder to disruption by air currents
and/or vibrations in a container. The PLS model from this
experiment shows that PEG/colloidal solutions have great potential.
Using this model, predictions of 40 samples at the same
concentration (100 .mu.L of 4.0.times.10.sup.-6 RB 15) were made to
show reproducibility, i.e., STD=1.99; AVG=4.53; % Corr.=3.6%.
[0037] FIG. 4B shows a reagent of Analyte Reactive Coating soluble
in Water (ARCW). Using this reagent in blood collecting tubes, a
PLS model was made with bilirubin. FIG. 4A represents a spectrum of
this PLS correlation model. The blood collecting tubes were
prepared using the same procedure described above. In addition to
lyophilizing the colloid in the tubes, 1 .mu.L of 0.00299 M
Diazonium ARCW was added to the opposite end from the colloid of
each tube and also lyophilized. The tubes were then placed in a
vacuum to remove the water from the colloid suspension and ARCW
solution. A 2.99.times.10.sup.-4 M stock solution of bilirubin was
prepared by dissolving 0.00175 g of bilirubin in a 5:4:1 ethanol,
water, saturated bicarbonate solution. A 2.99.times.10.sup.-6 M
solution of bilirubin was prepared by diluting with Millipore
H.sub.2O, 100 .mu.L of the stock solution to 9.8 mL H.sub.2O+100
.mu.L 0.05 M KOH. A concentration range 5.98.times.10.sup.-7 to
2.99.times.10.sup.-6 M bilirubin was prepared. In the lyophilized
tubes 100 .mu.L of each concentration and a Teflon coated iron rod
was added to the end of the tube, which contained the ARCW. This
was allowed to set for 5 min, after which the solution was mixed
with the colloid and a spectrum was taken. FIG. 4C shows the result
for 0.05M KOH 100 .mu.L in 9.8 mL H.sub.2O and 100 .mu.L bilirubin,
in a correlation plot between actual concentration and predicted
concentration with a R.sup.2=0.9367.
[0038] FIG. 5 shows a PLS cross-validation plot of actual versus
predicted human Growth Hormone (hGH) concentration levels for a
Surface Enhanced Raman ImmunoAssay (SERIA) analysis using a
lyophilized silver colloidal base. A Lee and Meisel silver
colloidal suspension (R) was prepared to which was added a
1:400,000 dilution of polyclonal hGH antibody (Ab). This addition
is accomplished by slowly adding small aliquots of the antibody
solution (140 .mu.L total) to 1 mL of the colloidal suspension with
gentle agitation with continued mixing for 15 min. A stabilizer of
polyethylene glycol (PEG, ave. 15000 MW) was added to the solution
to make a 5% solution of PEG/RhGHAb. A mini-microwell plate
(NUNC.RTM.) was used as the sample container for the analysis. To
each of 10 wells of the plate was added 20 .mu.L of the PEG/RhGHAb
solution. The plate was placed in liquid nitrogen to `flash` freeze
the wells of solution and then placed in a high vacuum vial set in
a cooling container of dry ice. The vacuum vial was then attached
to the lyophilizer for 24 hr. Each well of lyophilized colloidal
base appeared as a light tan colored waxy semi-solid. The addition
of liquid immediately reconstituted the base to produce a light tan
colored colloidal suspension resembling the original solution. The
analysis was completed by the addition of varying concentration of
hGH antigen in an aqueous sodium bicarbonate solution (1%),
followed by the addition of a reporter molecule solution. The
solution of antigen was used to reconstitute the lyophilized
PEG/RhGHAb for the assay.
[0039] FIG. 6 shows a PLS cross-validation plot of actual versus
predicted hGH concentration levels for a SERIA analysis similar to
that described above. In this instance, the lyophilized colloidal
base was a 0.5% PEG/RhGHAb. After lyophilization, the wells of
colloidal base appeared as tan colored nugget with a puckered
surface. Addition of the antigen solution caused complete
reconstitution of the colloidal base.
[0040] While the foregoing invention has been described with
reference to the above embodiments, various modifications and
changes can be made without departing from the spirit of the
invention. Accordingly, all such modifications and changes are
considered to be within the scope of the appended claims.
* * * * *