U.S. patent application number 13/144854 was filed with the patent office on 2012-05-03 for encapsulated reagents and methods of use.
Invention is credited to Lee H. Angros.
Application Number | 20120107834 13/144854 |
Document ID | / |
Family ID | 42340101 |
Filed Date | 2012-05-03 |
United States Patent
Application |
20120107834 |
Kind Code |
A1 |
Angros; Lee H. |
May 3, 2012 |
ENCAPSULATED REAGENTS AND METHODS OF USE
Abstract
The present invention contemplates use of encapsulated aqueous
and non-aqueous reagents, solutions and solvents and their use in
laboratory procedures. These encapsulated aqueous or non-aqueous
reagents, solutions and solvents can be completely contained or
encapsulated in microcapsules or nanocapsules that can be added to
an aqueous or non-aqueous carrier solution or liquid required for
medical and research laboratory testing of biological or
non-biological specimens.
Inventors: |
Angros; Lee H.; (Bethany,
OK) |
Family ID: |
42340101 |
Appl. No.: |
13/144854 |
Filed: |
January 15, 2010 |
PCT Filed: |
January 15, 2010 |
PCT NO: |
PCT/US10/21200 |
371 Date: |
September 28, 2011 |
Current U.S.
Class: |
435/7.5 ; 435/23;
436/501; 977/700; 977/902 |
Current CPC
Class: |
B01L 2300/18 20130101;
G01N 35/1002 20130101; B01L 3/52 20130101; B01L 2200/0673 20130101;
B01L 3/523 20130101; G01N 1/30 20130101; B01L 2300/1861 20130101;
G01N 2035/00237 20130101; C12Q 1/68 20130101; Y10S 435/962
20130101; C12Q 1/6841 20130101; Y10S 435/969 20130101 |
Class at
Publication: |
435/7.5 ; 435/23;
436/501; 977/700; 977/902 |
International
Class: |
G01N 21/75 20060101
G01N021/75; C12Q 1/37 20060101 C12Q001/37 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 16, 2009 |
US |
61/145269 |
Claims
1. A method of treating a biological specimen on an analytic
substrate, comprising: providing an analytic substrate having a
biological specimen disposed thereon; providing a carrier solution
having dispersed therein a quantity of microcapsules and/or
nanocapsules containing at least one encapsulated reagent, solution
or solvent wherein the microcapsules and/or nanocapsules can be
disrupted by one or more predetermined conditions to cause release
of the at least one encapsulated reagent, solution or solvent into
the carrier solution; disposing the carrier solution upon the
analytic substrate; and exposing the analytic substrate and the
carrier solution thereon to the one or more predetermined
conditions which cause disruption of the microcapsules and/or
nanocapsules thereby causing the release of the at least one
encapsulated reagent, solution or solvent into the carrier
solution, wherein the encapsulated reagent, solution or solvent
becomes available to act on or react with the biological specimen
disposed upon the analytic substrate, and/or becomes available to
react with the carrier solution and/or a component therein to form
a reagent mixture or activated reagent which acts on or reacts with
the biological specimen disposed upon the analytic substrate.
2. The method of claim 1 wherein the one or more predetermined
conditions which cause disruption of the microcapsules and/or
nanocapsules are selected from at least one of a temperature
change, electrical current, magnetism, pH change, physical
agitation, sonic agitation, a pressure change, or electromagnetic
waves such as microwaves, UV, laser, visible light, and/or
infrared.
3. The method of claim 1 wherein the carrier solution is oil-based
or oil soluble and the encapsulated reagent, solution or solvent is
aqueous-based or aqueous soluble.
4. The method of claim 1 wherein the carrier solution is
aqueous-based or aqueous soluble and the encapsulated reagent,
solution or solvent is oil-based or oil soluble.
5. The method of claim 1 wherein the carrier solution is oil-based
or oil soluble and the encapsulated reagent, solution or solvent is
oil-based or oil soluble.
6. The method of claim 1 wherein the carrier solution is
aqueous-based or aqueous soluble and the encapsulated reagent,
solution or solvent is aqueous-based or aqueous soluble.
7. The method of claim 1 wherein the encapsulated reagent, solution
or solvent, when released into the carrier solution, reacts with
the carrier solution or the component therein to form the reagent
mixture or activated reagent which reacts with or acts on the
biological specimen.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority from 35 U.S.C. 371
of International Application PCT/US2010/021200, filed Jan. 15,
2010, which claims the benefit of U.S. Provisional Application Ser.
No. 61/145,269, filed Jan. 16, 2009, the entire contents of each of
which is hereby expressly incorporated by reference herein in its
entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
BACKGROUND OF THE INVENTION
[0003] In a microscope slide treatment method known in the prior
art, a test specimen which is attached to a microscope slide is
treated using two phase-separating liquids. In this method, first,
an aqueous reagent is placed on an upper surface of the microscope
slide to which the specimen is attached. Then a layer of mineral
oil or other immiscible oil is placed over the aqueous reagent. The
two different phases remain separated even after stirring or
agitation. This is desired in this example, however, because the
purpose of the placement of the oil layer over the aqueous reagent
is to reduce the evaporation of the aqueous reagent during the
timed incubation steps (e.g., heating). However, this method
requires two separate steps to dispose the reagent and oil on the
slide. For example, in one alternative, the aqueous reagent is
placed over the biological specimen first and then, in a second
step, the oil layer is placed over the aqueous reagent.
Alternatively, one could envision first placing the oil layer over
the biological specimen, and then placing the aqueous reagent onto
the oil layer thereby wherein the aqueous reagent then submerges
through the oil layer to the microscope slide surface whereby the
oil layer floats on top of the aqueous reagent. A significant
disadvantage of this method is that the aqueous layer tends to
remain localized at the specific location where the aqueous reagent
was first placed on the slide, once the oil layer is placed
thereon. If the aqueous reagent is placed on top of the oil layer
so the aqueous reagent layer passes through the oil layer but the
aqueous reagent layer partially or entirely misses the specimen by
not covering the whole specimen area, the aqueous reagent layer is
fixed in that exact position once it passes through the oil layer
and thus the specimen is not treated with the aqueous reagent. If
one were to place, for example, a stir stick or stir device through
the oil layer and down to the aqueous layer to mix or move the
aqueous layer, the aqueous reagent tends to remain in its original
location of placement and cannot be moved to a more useful or
appropriate area upon or around the slide or specimen. This reduces
the ability of the specimen to react with the reagent in this
method. A solution to this problem to increase the efficiency of
the process and to minimize the chances of damaging the specimen is
desirable.
DETAILED DESCRIPTION OF THE INVENTION
[0004] The present invention contemplates use of encapsulated
aqueous and non-aqueous reagents, solutions and solvents and their
use in laboratory procedures. These encapsulated aqueous or
non-aqueous reagents, solutions and solvents can be completely
contained or encapsulated in microcapsules or nanocapsules that can
be added to an aqueous or non-aqueous carrier solution or liquid
required for medical and research laboratory testing of biological
or non-biological specimens (also referred to herein as "testing"
or "test" specimens). Where used herein the term "encapsulated
reagent" is intended to refer also to "microencapsulated reagents"
and/or to "nanoencapsulated reagents". Further, where used herein,
the term "capsule" is intended to refer to "microcapsules" or
"nanocapsules".
[0005] Where used herein the term "biological specimen" includes,
but is not limited to, unprocessed specimens, processed specimens,
paraffin embedded tissue, whole mounts, frozen sections, cell
preps, cell suspensions, touch preps, thin preps, cytospins, and
other biological materials or molecules including blood, urine,
cerebrospinal fluids, pleural fluids, ascites fluids, biopsy
materials, fine needle aspirates, pap smears, swabbed cells or
tissues, microbiological preps including bacteria, viruses,
parasites, protozoans, biochemicals including, but not limited to
proteins, DNA, RNA, carbohydrates, lipids, ELISA reagents and
analytes, synthetic macromolecules, phospholipids, support
structures of biological molecules (e.g., metals, beads, plastics,
polymers, glass), or any other materials attached to a biological
testing substrate for processing, examination, or observation.
[0006] The capsules of the present invention are generally
considered to have diameters in the range of from less than 0.001
angstrom (0.0001 nm) to 3000 microns (3000 .mu.m). Preferably the
range is from 1 angstrom (0.1 nm) to 1000 micrometers. The range
can also be from 1 nanometer to 1000 microns. The shells or
encapsulating material that make up the microcapsules or
nanocapsules can be gelatin, polyvinyl alcohol, urea, melamine
formaldehyde polymers, acrylics, urethanes, vinyl acetate
copolymers, oily, lipid, or non-aqueous soluble materials and
polymers, water, or aqueous based materials and polymers. The
encapsulation processes used to form the encapsulated reagents
include, but are not limited to, coacervation, vapor deposition,
fluid bed coating, entrapment/matrix, macro-emulsion,
mini-emulsion, micro-emulsion, micro-encapsulation techniques,
macro-encapsulation techniques, dispersion polymerization, in situ
polymerization, liposomal, alginate encapsulation, solvent phase
separation, and pan coating. The micro- or nanoencapsulated reagent
product can be delivered as a dry, free-flowing powder, as a
slurry, or in the form of wet filter cake.
[0007] Reagents and compounds which may be microencapsulated or
nanoencapsulated as contemplated for use in the present invention
include, by way of example only, not by way of limitation, Dextran
sulfate, formamide, SSC (sodium chloride sodium citrate solutions),
DI water, Millipore.TM. water, RNAase-free and DNAase-free water,
DAPI counter stain, propidium iodine counterstain, counterstains,
salts, buffers, chemicals, DNA probes, RNA probes, protein probes,
antibodies, monoclonal antibodies, polyclonal antibodies, probes,
detection reagents, stains, biological stains, dyes, washes,
rinses, enzymes, antigen retrieval solutions or buffers, ionic,
non-ionic, anionic, cationic, neutral detergents and surfactants,
thermoplastics, mountants, oils, lipids, phospholipids, molecular
biological building blocks, carbohydrates, sugars, lyophilized or
desiccated powder or dry reagents that can be reconstituted with
and aqueous or non-aqueous solution, preservatives, cover slip
media, liquified thermoplastic cover slip medias, xylene, toluene,
acetone, petroleum distillates, ferrofluids, magnetic particles in
a fluid, colloidal gold conjugated reagents, iron-containing
fluids, iron particles, magnetic particles, organic solvents,
inorganic solvents, aqueous solvents, non-aqueous solvents, lipid
based solvents, emulsions, liquid chemicals, Histology clearing
reagents, Histology deparaffinizing reagents, Histology hydrating
reagents, Histology dehydrating reagents, Histology fixatives,
formaldehyde, alcohols, polyols, magnetic particle powders,
powders, lyophilized reagents, lyophilized antibodies, lyophilized
molecular probes like RNA and DNA, dry chemicals, dry, powdered, or
lyophilized stains and reagents, fluorescent conjugated reagents
like antibodies, stains, and molecular probes, and detection
reagents, chromogens, DAB, hydrogen peroxide, naphthol phosphate,
fast red chromogen, acids, bases, HCL, formic acid, glacial acetic
acid, sodium hydroxide, potassium hydroxide, aqueous and non
aqueous liquids, and any other reagent and/or chemicals including
liquids, dry reagents, desiccated reagents, gel reagents, colloidal
reagents, emulsions reagents and any other reagent or chemical
known in the art of medical and research laboratory testing
reagents or chemicals. These reagents will be referred to herein as
"encapsulated reagents". The solutions or liquids to which these
encapsulated reagents can be added may be referred to elsewhere
herein as "solutions" or more particularly as "carrier solutions".
The above is exemplary only and is not intended to be an exhaustive
list of the reagents or compounds which may be encapsulated or used
herein.
[0008] Examples contemplated herein of the use of these
encapsulated reagents include for example (1) addition of
aqueous-based encapsulated reagents to non-aqueous based solutions,
(2) addition of non-aqueous based encapsulated reagents to
aqueous-based solutions, (3) addition of aqueous-based encapsulated
reagents to aqueous-based solutions, (4) addition of non-aqueous
based encapsulated reagents to non-aqueous based encapsulated
solutions, and (5) addition of both aqueous-based and non-aqueous
based encapsulated reagents to either an aqueous- or non-aqueous
solution, and wherein the resulting combination solutions contain
such encapsulated reagents in a homogenous, soluble, or colloidal,
emulsions, or at least partially soluble liquid mixture.
[0009] It is known that when adding a typical aqueous-based reagent
to a non-aqueous solution, or vice-versa, the two different phases
separate. There is therefore a need to be able to mix aqueous-based
reagents with non-aqueous-based solutions, and non-aqueous-based
reagents with aqueous-based solutions to form homogenous solutions
of both the reagent and the solution without the usual phase
separation of both. Encapsulation of the reagent as contemplated
herein enables the formation of such homogenous mixtures.
[0010] The problem in the prior art method described above in the
Background may be addressed by using a three-step procedure
involving first disposing an aqueous detergent-containing layer
over the entire area of the slide (or analytic substrate, as
defined herein) where one would like the aqueous reagent layer to
be positioned once the oil layer is added, or vice versa. In this
method, an aqueous detergent-containing layer can be placed first
on the microscope slide, followed by addition of the aqueous
reagent layer, and then followed by addition of the oil layer in a
third subsequent step. In the presence of these three layers, the
aqueous reagent layer can be moved or mixed on the slide anywhere
the aqueous detergent layer is present. However, this method is
highly inefficient in both time, materials, and cost required to
perform a staining protocol which requires an oil layer or liquid
phase/separation protocol. A further disadvantage of this
three-step method is that the aqueous detergent layer must always
be added first, though either the aqueous reagent layer, or the oil
layer can be added next. Still, whether the oil layer is added,
second or third following addition of the aqueous reagent layer, it
is obvious that the method is still requires three separate steps
and is a costly time and material consuming protocol.
[0011] The present invention provides a solution to the problems of
the prior art method. In the present invention, one or more
reagents which are encapsulated, by microencapsulation and/or
nanoencapsulation, are added to aqueous or non-aqueous solutions
for use as single ready-to-use solutions for laboratory testing of
specimens. The physical and/or chemical makeup of the encapsulated
reagents is such that the microcapsule or nanocapsule containing
the reagent is soluble, at least partial soluble, or colloidal in a
solution which has a different liquid phase or density than that of
the encapsulated reagent. In an alternative embodiment, the
encapsulated reagents have the same or similar densities or liquid
phase of the solution. Further, the microcapsule or nanocapsule
could have the same or similar density or the same or similar
liquid phase of the solution regardless if the reagent encapsulated
therein has the same or similar liquid phase or density. It is an
object of the present invention to encapsulate reagents wherein the
chemical and/or physical properties of the encapsulating material
(the outer shell of the capsule) are like or similar to that of the
solution to which the encapsulated reagent will be added thereby
allowing the capsules to be soluble, at least partially soluble, or
colloidal in or with the solution. The reagent, once encapsulated,
therefore would be soluble, at least partially soluble, or
colloidal in relation to the solution. In a preferred embodiment of
the present invention, a solution comprising at least one
encapsulated reagent which has a density different from the
solution, is provided and applied to a specimen. In an alternative
embodiment of the present invention, a solution is provided which
has at least one encapsulated reagent having a density which is the
same as or similar to the solution containing it, then the solution
is applied to a specimen.
[0012] The analytic substrates used in the present invention may be
constructed of glass, plastic, synthetic polymers, ceramics, or
metals and may be of any size or shape known in the art of
laboratory examination, for example including any laboratory
support structure or testing structure or device used in laboratory
testing or examination including, but not limited to, microscope
analytic plates, analytic substrates, medical and research
laboratory testing substrates, diagnostic substrates, biological
testing substrates, substrates, microscope slides, test tubes,
Petri dishes, micro arrays, biochips, testing plates, containers,
beads, and testing strips and any other natural or synthetic
substrate or device used in the art for medical, research,
laboratory, and diagnostic testing, in-vitro testing and/or
analysis of at least one biological specimen.
[0013] The process wherein the microcapsule or nanocapsule opens or
disintegrates upon the analytic substrate to release the reagent
contained therein is referred to herein as "disruption".
Disruption, once started, can be immediate (i.e., "immediate
release"), or can be a slow or gradual release (i.e., "controlled"
or "timed" release). The type of disruption necessary for the
capsule to release its contents is referred to herein as the
"disruption mode". The causes or stimuli of the disruption mode can
be, for example, temperature changes or differentials, high
temperature (e.g., 150.degree. C.-200.degree. C.), medium
temperature (e.g., 100.degree. C.-150.degree. C.), low temperature
(e.g., 25.degree. C.-100.degree. C.), heat, mechanical disruption,
e.g., by agitation, sonic disruption, magnetic disruption, electric
disruption, microwave disruption, UV light, infrared light, laser
light, light, other types of electromagnetic radiation or energy,
pH changes, pressure differentials, high pressure, medium pressure,
low pressure, pressure changes above or below atmospheric (e.g.,
pressures of 1 psig-5000 psig; 1-10 psig; 10-50 psig; 50-100 psig;
100-150 psig; 150-200 psig; 200-300 psig; 300-500 psig; or 500-5000
psig), vacuum, and vacuum changes, time release, time dependent,
chemical reactions, chemical changes, and physical reactions, and
physical changes. Various disruption modes can be combined, e.g.,
temperature and pressure; pH and heat; time and pressure; and
pressure and time, for example.
[0014] In an alternate embodiment, the "disruption" of the
microcapsule or nanocapsules contained within a carrier solution
occurs upon the combination of the carrier solution with at least
one other solution or compound. The carrier solution in this
embodiment has at least one reagent present in a microcapsule or
nanocapsule, and the second solution optionally has at least one
microcapsule or nanocapsule present which encapsulates a reagent.
If the second solution doesn't have any encapsulated reagent
present, the chemical activity, physical activity, or reaction when
combining with the second solution can initiate disruption of the
microcapsule or nanocapsule in the carrier solution. The
combination of at least two solutions (carrier and second solution)
each having at least one reagent present in a microcapsule or
nanocapsule or only one of the two solutions having encapsulated
reagent present when mixed can, in alternate embodiment, now
"activate" (disrupt) the microcapsule(s) or nanocapsule(s) in the
combined solutions. Activation of the microcapsule or nanocapsule
is intended to mean the ability for the microcapsule or nanocapsule
to become unstable or disruptable only after the at least two
solutions are combined, wherein if the solutions are not combined,
the microcapsule or nanocapsule are stable against disruption modes
when they are in their separate solutions. Only when the at least
two solution are mixed together are the microcapsule or nanocapsule
disruptable in this embodiment. In an alternative embodiment, there
can be one or more solutions combined to "activate" any
microcapsule or nanocapsule present in at least one of the
solutions of the combination.
[0015] A solution of the present invention may comprise an
encapsulated reagent wherein the capsule is responsive to a single
type of disruption mode, e.g., a pressure or sonic sensitive
encapsulation, or a single solution may comprise a plurality of
encapsulated reagents each wherein each type of capsule is
responsive to a different type of disruption mode. In one
embodiment, for example, a solution could contain three types of
encapsulated reagents, each used in one of three different steps of
a test protocol. For example, the first encapsulated reagent having
a capsule with a "heating" disruption mode could be released to
react with the slide specimen when the slide is heated. The second
and third encapsulated reagents could have capsules having
disruption modes which were not activated by or affected by heat
but which were activated by a pH change or pressure change, for
example, or other condition described herein.
[0016] As explained herein, embodiments of the present invention
include carrier solutions which comprise only a single type of
solute and carrier solutions which comprise multiple (two or more)
solutes. In one embodiment of the present invention, the carrier
solution can be mixed with another solution or solutions absent
encapsulated reagent(s) or with encapsulated reagents, wherein the
mixing of the two solutions may cause disruption of a encapsulated
reagent in one, both, or all of the solutions, or the mixing of the
two solutions does not disrupt the encapsulated reagent(s) but
rather the mixed solutions remain in association with each other as
a homogenous mixture, colloidal mixture, emulsion solution, phase
separated solution, suspension solution, miscible solution, or
immiscible solution, which the encapsulated reagents remain in an
intact encapsulated condition. A list of reagents which may be
encapsulated in accordance with the present invention is provided
above. This list is exemplary only and does not constitute any
limitation of the possible combinations of encapsulated reagents
and reagents or solutes present in the one or more carrier
solutions.
[0017] In one embodiment, the microencapsulated or nanoencapsulated
reagents could be manufactured by Particle Sciences, Inc. 3894
Courtney Street, Bethlehem, Pa. 18017-8920 US and/or by Microtek
Laboratories, Inc. 2400 East River Road, Dayton, Ohio 45439.
[0018] In various embodiments of the invention, exemplary methods
of producing the microcapsules and nanocapsules used herein and
descriptions of the capsular "shells" include, but are not limited
to, those disclosed in the following U.S. Patents and Published
Patent Applications, all of which are hereby expressly incorporated
by reference herein in their entireties. U.S. Patents include, but
are not limited to, U.S. Pat. Nos. 7,588,703, 7,462,365, 7,270,851,
7,052,766, 6,989,196, 6,932,984, 6,913,767, 6,881,482, 6,828,025,
6,777,002, 6,767,637, 6,716,450, 6,599,627, 6,555,525, 6,465,425,
6,458,118, 6,265,389, 6,214,300, 6,146,665, 6,113,935, 6,103,271,
6,080,412, 5,925,464, 5,863,862, 5,766,637, 5,650,102, 5,643,605,
5,552,149, 5,540,927, 5,508,041, 5,503,851, 5,503,781, 5,464,932,
5,418,010, 5,407,609, 5,403,578, 5,362,424, 5,277,979, 5,204,184,
5,164,126, 5,164,096, 5,160,529, 5,100,673, 5,091,122, 5,066,436,
5,051,306, 4,942,129, 4,895,725, 4,803,168, 4,766,012, 4,764,317,
4,711,783, 4,675,189, 4,673,595, 4,594,370, 4,521,352, 4,518,547,
4,508,760, 4,389,330, 4,269,729, 4,211,668, 4,193,889, and
4,123,382. U.S. Published Patent Applications include, but are not
limited to, 2009/0311329, 2009/0253901, 2009/0214633, 2009/0202652,
2009/0104275, 2009/0098628, 2009/0047314, 2008/0234406,
2008/0138420, 2008/0102132, 2008/0031962, 2007/0077308,
2007/0027085, 2007/0009668, 2006/0237865, 2006/0188464,
2006/0127667, 2006/0093808, 2006/0071357, 2006/0051425,
2004/0228833, 2004/0065969, 2004/0032038, 2003/0138491,
2003/0062641, 2002/0160109, and 2002/0064557.
[0019] Examples of reagent (e.g., DNA, RNA, ISH, FISH) reacting
with the biological specimen.
[0020] As noted above, the encapsulated reagent could be of a
different phase or density than that of the solution within which
the encapsulated reagent is to be disposed. For example, a
non-aqueous solution, such as an oil-based solution, could comprise
a soluble, partially soluble, or colloidal suspension, of one or
more encapsulated aqueous reagents for performing in situ
hybridization of a DNA or RNA probe to a target DNA or RNA present
in a specimen on a slide or other substrate. For example, the
specimen is present on a microscope slide and the slide is heated
to 70.degree.-110.degree. C. The oil solution with its encapsulated
reagents present therein is added to the microscope slide. Heating
at a temperature of 72.degree. C., for example would cause the
disruption of the capsule of the encapsulated reagent, for example,
and the aqueous reagents therein would thereby be released into the
oil solution. The aqueous reagents quickly separate away from the
oil layer and are deposited onto the microscope slide and onto
specimen thereon. Wherever the oil solution is present on the
microscope slide, there would now be a layer of aqueous reagents
that had separated from the oil solution and had migrated to the
surface of the microscope slide. Present on the surface of the
microscope slide therefore, would be an aqueous reagent layer, with
the oil layer of the original solution over the aqueous reagent
layer. The aqueous layer could then be agitated or stirred about
the slide because the encapsulated reagent preferably had present
as one of the reagents therein a detergent for enhancing the
dispersion and distribution of the aqueous reagents under the oil
layer and upon the slide surface and specimen thereon.
[0021] In an alternative embodiment, the microscope slide, with
biological specimen attached thereto, is first flooded with a wash
buffer that has at least one detergent and/or surfactant present.
Excess wash buffer is removed to leave only a residual layer of
wash buffer on the microscope slide and biological specimen (e.g.,
approximately 1-100 micro liters of wash buffer remaining on the
slide). An oil-based carrier solution containing the encapsulated
aqueous reagent is now added to the wet slide. The capsules of
encapsulated aqueous reagent are disrupted and the aqueous reagent
therein is released and deposited onto the residual wash buffer.
The aqueous reagent moves to the biological specimen and
surrounding areas of the microscope slide. The oil layer is above
the aqueous reagent. The aqueous reagent can be moved on the
microscope slide and biological specimen wherever the residual wash
buffer is located by agitating the aqueous reagent or moving the
oil layer which in turn would move the aqueous reagent about the
biological specimen and/or microscope slide.
[0022] It is contemplated in the present invention that each
reagent used in an in-situ hybridization process or other treatment
methods contemplated herein can be encapsulated separately, or as
mixtures that are encapsulated together. The reagents necessary for
a denaturing and hybridization step of the in-situ hybridization
protocol can be, for example, formamide, dextran sulfate, DI water,
detergents, and the required DNA or RNA probe for example. This
single solution comprising reagent capsules dispersed therein can
be used for the steps of denaturing and hybridization of the target
nucleic acid specimen is a novel one step solution which features
all the advantages of use of an oil layer to inhibit evaporation.
The use of such a single solution of the present invention is novel
because in the prior art process of co-denaturing a nucleic acid
with heat to denature and hybridize a nucleic acid to the target
DNA present in a specimen attached to a microscope slide, one would
necessarily have to use a slide having a sealed cover slip sealed
with an adhesive over the specimen with the nucleic probe mixture
placed underneath the sealed cover slip. In the method of the prior
art, after heating, usually at 72.degree. C. for 10 minutes, for
example, the attached cover slip then must be removed which can
cause damage to the specimen. Alternatively, the novel method of
the present invention, wherein a single solution that has all the
necessary reagents for hybridization present in microcapsules or
nanocapsules is used, eliminates all the multiple steps of having
to add different reagents and the manual steps of preparation
during an in-situ hybridization. The single solution method of the
present invention has all the necessary reagents, present within
capsules in the solution, so there is no waste of reagents such as
of very expensive nucleic acid probes. The single solution of the
present invention may be added to the specimen on the microscope
slide or other biological container or substrate. During the
co-denaturing step in the present invention, the heat (or other
disrupting mode contemplated herein) disrupts the microcapsules or
nanocapsules thereby causing release of the reagents onto the
specimen. There is no need for a cover slip to cover the specimen
and reagents during the hybridization process because the single
solution may be oil based while the reagents encapsulated may be
aqueous-based. The oil phase in the solution will separate from the
aqueous phase comprising the released reagents, thereby forming an
evaporation barrier over the aqueous reagents thereby inhibiting
the evaporation of the aqueous reagents during the heating or
denaturing of the DNA or RNA of the target specimen. This single
oil-based solution containing the encapsulated aqueous reagents can
be applied by a dropper bottle, pipette, or any other type of
dispenser including the dispenser described below and those noted
for example in U.S. published applications 2006/0281116,
2006/0275889, and 2006/0275861, each of which is expressly
incorporated herein by reference.
[0023] In a preferred embodiment, the encapsulated reagent is also
very stable and has a long shelf life of greater than one year at
room temperature or under 2-4.degree. C. refrigeration vs. the
non-encapsulated reagent that would normally require freezer
storage at -20.degree. C. or ultra cold freezer storage below
0.degree. C. to maintain its freshness and viability. The
encapsulation protects the temperature sensitive reagent and the
reagent, as encapsulated by the methods of the present invention
has a very long shelf life at normal refrigeration (2-4.degree. C.)
or at room temperature (25-30.degree. C.). This stability provided
by encapsulating the temperature-sensitive reagents is advantageous
during the packing and shipping of these reagents. Many of the
conventional, non-encapsulated, reagents listed herein must be
shipped via "Next Day Air" in a container that is cooled by cold
packs or dry ice. The encapsulated temperature sensitive reagents
of the present invention can instead be shipped ground in a normal
shipping container which is more cost effective without sacrificing
freshness or degradation due to insufficient packing, insufficient
temperature controlled shipping environments, and increasingly high
shipping fees of next day air shipping requirements of the
non-encapsulated temperature sensitive reagents. The encapsulation
of the present invention inhibits the degradation of these proteins
(e.g., antibodies) and chemical from bacterial or fungal attack or
degradation. The capsules preferably have anti-fungal and
anti-bacterial properties to inhibit the degradation of these
proteins and chemicals when stored at room temperature or at
refrigerated conditions and during shipping and use.
[0024] One embodiment of the invention is directed to a novel
deparaffinizing solution. In this embodiment, one or more
deparaffinizing reagents such as, but not limited to, like xylene,
petroleum distillates, or other non-aqueous deparaffinizing
solutions and solvents have dispersed therein an encapsulated
reagent such as (but not limited to) an alcohol or other water
soluble substance. This deparaffinizing solution can be placed onto
the paraffin embedded tissue section thereby causing the paraffin
associated with the biological specimen to soften and dissolve into
the deparaffinizing reagent thereby removing the paraffin from the
microscope slide and the biological specimen. Following dissolution
of the paraffin, the microscope slide and the biological specimen
must now be rinsed with a chemical solution that is miscible with
the deparaffinizing solution. In the present invention, this
"rinsing" solution or reagent is contained within the capsules
contained within the deparaffinizing solution. If the
deparaffinizing solution was xylene for example, the encapsulated
rinse agent therein could be a reagent grade alcohol, for example.
This alcohol is miscible with the xylene deparaffinizing solution.
The capsules would then be disrupted by exposure of xylene/paraffin
mixture to a predetermined disruption condition such as described
elsewhere herein causing release of the alcohol into the
xylene/paraffin mixture. The alcohol/xylene/paraffin mixture would
then rinsed or removed from the slide (or analytic substrate) and
biological specimen by an aqueous reagent (such as, but not limited
to, water) which is miscible with the alcohol/xylene/paraffin
mixture. The alcohol in this one-step deparaffinizing solution is
released from the microencapsules by a condition such as time,
heat, pressure, vacuum, mechanical disruption or combinations
thereof, or other conditions described herein. This release of the
alcohol into the deparaffinizing solution makes an
alcohol/xylene/paraffin liquid that can easily be rinsed by an
aqueous rinse buffer. The advantage of this deparaffinizing
solution which contains the encapsulated alcohol is that there are
fewer steps necessary for deparaffinization and the use of minimal
reagents that can be used effectively and efficiently with less
waste and reduced disposal cost when dealing with hazardous
deparaffinizing solutions like xylene. Although the example of
deparaffinization provided above describes the use of xylene and
alcohol, other deparaffinizing solutions are known and contemplated
for use instead. For example, deparaffinizing solutions that are
aqueous-based can be used with the above-mentioned example of the
present invention. Any deparaffinizing solution, whether aqueous or
non-aqueous based, can be used as long as there is at least one
microencapsulated reagent present in the deparaffinizing solution
that is soluble, miscible, colloidal, or at least partially soluble
in the deparaffinizing solution to prepare the
paraffin/deparaffinizing solution to be rinsed with an aqueous
buffer. An example of an aqueous-based deparaffinizing solution is
the use of detergents in water or in water and solvent compositions
(i.e., polar and non-polar solvents with detergents in water). The
deparaffinizing carrier solution whether aqueous based or
non-aqueous based can have in the alcohol-containing capsule an
additive, such as a surfactant, detergent, polyols, or other
component. Or in an alternate embodiment, these additives can be
encapsulated in their own microcapsules or nano-capsules. The
reagents may be encapsulated by any appropriate means contemplated
in any of the description, patents, or published applications
described herein.
[0025] The encapsulation material used to form the microcapsules or
nanocapsules of the present invention, in an alternate embodiment,
can also have magnetic properties and/or electrical properties.
Alternatively, the encapsulation material can be acted on by a
magnetic current or electrical current present within or adjacent
the solution for use with the magnets described elsewhere herein.
These magnetic and/or electrical properties of the capsules may be
advantageous when it becomes necessary to mix, agitate, align, or
otherwise move the capsules in relation to the solution or the
specimen. For example, capsules that have magnetic properties can
be moved toward or away from the specimen by magnets associated
with the apparatus upon which the slide is placed, such as the
instruments described in the published U.S. Patent Applications
noted above. The magnets can, for example, move certain capsules
toward the specimen just before disruption of the capsules to have
the reagents as close to the specimen as possible to reduce the
time of incubation and efficiency of the binding activity of the
reagent with the specimen. Further, in other embodiments, the
capsules can have a net charge such that they can be acted on by
electrical currents to be pulled toward or repelled from the
specimen or solution or other location on the microscope slide. For
example, an electrical current may pass through the solution
thereby pulling the charged capsules toward the specimen just prior
to disruption. Alternatively, the polarity of the electrical
current could be reversed to move the expelled or disrupted
capsules out of the way of a different capsule containing a
different reagent moving toward the specimen for the next
subsequent reagent step. The electrical and/or magnetic properties
of the capsule have further advantages of enabling the capsules to
be mobile or to move in the solution when an electrical field or
magnetic field is generated about the solution for mixing the
capsules, or mixing the reagent or reagents disrupted from the
capsules, for example, or by moving the disrupted capsules thereby
producing a swirling, moving, or agitation motion in the solution
or its respective liquid phase to mix or agitate reagents in
relation to the specimen. The capsules can be moved, or the remains
of the disrupted capsules can be moved, by a magnetic field or
electrical field or both, to agitate, move, or mobilize other
non-disrupted capsules, expelled reagents, and or the solution.
[0026] In another embodiment of the present invention, the capsules
may have magnetic or electrical properties present on or within
each capsule in the solution which can be used to evenly distribute
or dispense the capsules in the solution. Each capsule or groups of
similar capsules could have a positive or negative net charge
present therein to repel other capsules in the solution to enhance
the distribution or dispersion of different capsules within the
solution, whether or not the capsule is in a magnetic field or
electrical field. The net charge is part of the physical or
chemical properties of the capsule at all times when in the
solution, and the net charge may be changed by the chemical nature
of the solution or reagents expelled in certain embodiments. For
example, a pH change in the liquid phase of the solution or the
liquid phase of the reagent can change or maintain the net charge
of the capsule. The capsule, once disrupted, can maintain the same
net charge as prior to disruption, or can have its charge changed
after disruption. This is advantageous when placing the capsule
under an electrical current or magnetic current and having a
subsequent different result to the effects of the current on the
capsule based on the initial net charge of the capsule before
disruption or after disruption.
[0027] The encapsulation material used herein to make the
encapsulated reagents may comprise micro-iron particles, or may be
coated with an inert plastic or polymeric material.
[0028] The micro-iron particles of the encapsulation material can
be any ferro containing particle (Fe) or other metal particles that
can be moved by a magnet which is known and contemplated. The
particle can be of the size less than or equal to
1.times.10.sup.-10, 1.times.10.sup.-9, 1.times.10.sup.-8,
1.times.10.sup.-7, 1.times.10.sup.-6, or 1.times.10.sup.-5
meters.
[0029] The micro-iron particles ("micro particles") can be coated
with a ceramic, plastic or polymeric coating to help in the
stability of the particles in solutions. The coating can be
Teflon.RTM. or other fluropolymer, for example. The micro particle
can be by itself in the capsule, in the reagent diluent or attached
to a reagent in the capsule. The micro particle can be soluble, or
at least partially soluble, or colloidal in the diluent solution.
If the micro particle is not attached to a reagent element it could
be used to mix or agitate the surrounding solution. If the micro
particle is attached to the capsule or reagent in the capsule it
can be used for mixing, agitating, or moving the reagent. In an
alternative embodiment the diluent can have present therein an
electrolyte present to produce a net charge of the reagent present
and to further the effectiveness of the magnet on the reagent.
[0030] Magnets that can be used in the present invention may be
permanent magnets, superconducting magnets, or resistive magnets,
for example. The preferred embodiment is the use of a permanent
magnet that has high temperature stability for the use in high
pressure and high temperature conditions which may be used during
in situ hybridization. High temperature stable permanent magnets
which may be used herein are described, for example, in U.S. Pat.
No. 6,451,132 which is hereby expressly incorporated herein by
reference in its entirety. These high temperature permanent magnets
can be subjected to temperatures exceeding 700.degree. C. The
magnets used herein may have Tesla ratings of 0.00001 Tesla to 60
Tesla for example (one Tesla equals 10,000 Gauss). Preferably the
Gauss rating can be 1 to 20,000 Gauss.
[0031] Other magnets may be used such as Neodymium magnets which
are a type of permanent magnet that can have the ability to retain
its magnetic properties even under very high temperature
conditions.
[0032] Most permanent magnets lose their magnetic properties when
they are exposed to high heat conditions. A type of permanent
magnet contemplated for the present invention has the grade of
N42SH, the "SH" grade of Neodymium permanent magnets can be used at
temperatures over 150.degree. C. Standard "N" grade permanents
magnets have a maximum operating temperature of 80.degree. C. A
"SH" grade Neodymium permanent magnet with the dimensions of 2
inches long by 1 inch wide by one eighth inch has a Gauss rating of
3095 for its surface field strength. It also has a Brmax of 13,200
Gauss and a BHmax of 42 MGOe.
EXAMPLES
Example 1
Encapsulated IHC Fast Red Chromogen Reagents
[0033] Fast Red chromogen protocols can be utilized in one
embodiment of the present invention. It is well known in the art of
alkaline phosphatase immunohistochemistry (IHC) reactions that a
final step of the protocol is to visualize the reaction by a color
change at the antigen target site by the use of an activated fast
red substrate/chromogen solution that reacts with the alkaline
phosphatase enzyme attached to the biotinylated
antibody/streptavidin alkaline phosphatase complex. In the prior
art method, chromogen activation is accomplished by adding 2
milliliters of a substrate reagent solution (naphthol-phosphate in
a tris buffer) and 30-50 microliters of fast red chromogen reagent
solution together to form an activated chromogen. The activated
chromogen (i.e., the naphthol/tris/fast red chromogen complex) is a
time dependent mixture that has a lifespan of only 30 minutes,
after which the chromogen solution becomes inactive and must be
rinsed off the slide. It is well known in the art of fast red
chromogen chemistry that fast red chromogen is not stable at room
temperature, however, the fast red reagent is stable up to one year
if stored under 2.degree. C.-8.degree. C. refrigeration. Fast red
reagents can even degrade during shipment of the reagent from the
manufacture to the end user. In the present invention,
microencapsulated or nanoencapsulated chromagen present in a
carrier solution can be used to make the "two part" fast red
reagent system of the prior art into a single temperature stable
solution that has both reagents present in a single solution and
that is stable at room temperature and doesn't have to be
refrigerated during shipping. In a particular advantageous
embodiment of the present invention, the carrier solution is the
naphthol-phosphate/buffer reagent, and the fast red reagent is
encapsulated separately therein. When the time comes to expose the
fast red chromogen to the biological specimen, the technician can
add the novel single fast red/carrier solution to the biological
specimen. The encapsulated reagents are disrupted by one of the
methods described elsewhere herein. When the encapsulated fast red
chromagen reagents are free to mix together, the fast red chromogen
becomes activated and can now form a color change when it comes in
contact with the alkaline phosphatase enzyme. This single solution
can be any combination of the required encapsulated reagents to
render the single solution stable at room temperature as well as at
a refrigerated condition. One can envision there would be a
multitude of combinations of encapsulated reagents, in the carrier
solution, to product a stable single solution fast red chromogen
reagent. In one example of the present invention, the fast red
chromogen single solution comprises deionized water as the carrier
solution and the remaining required reagents can be encapsulated
separately or together or any combination of a single reagent type
per encapsulation or any combination of reagents in the same
encapsulation which can be disrupted to form an activate fast red
chromogen reagent which would be activated and ready for use. In
another embodiment, the deionized can have the tris buffer and
naphthol present in its solution, and only the fast red chromogen
reagent is encapsulated. In a preferred embodiment, the carrier
solution is comprised of deionized water, naphthol phosphate, and
Tris buffer. The fast red chromogen reagent comprises a desiccated,
lyophilized, dry, or powdered state and encapsulated. This solution
is very stable at room temperatures, refrigerated temperatures, and
shipping temperatures. In one example of its use, this fast red
chromogen-encapsulated solution is placed onto the biological
specimen and the solution is subject to a pressurized environment
(e.g., 0.01 psig to 5000 psig, preferably 1-200 psig) which
disrupts the desiccated, lyophilized, dry, or powdered encapsulated
fast red reagent. The fast red reagent is quickly reconstituted by
the carrier solution and further reacts with the carrier solution's
reagents to form an activated fast red chromogen able to react with
the biological specimen's labeled antigen. This method of use can
be performed using the methods and apparatuses of published U.S.
patent applications 2006/0281116, 2006/0275889, and 2006/0275861,
for example. Alternatively, the disruption of the capsule can also
be caused by any of the disruption modes listed elsewhere in this
specification. The present invention is also contemplated for use
with other chromogens such as horseradish peroxidase chromogens.
The chemicals in this case include hydrogen peroxide, deionized
water, buffer, and DAB (3,3'-diaminobenzidine). The present
invention also contemplates that any combination of two or more
reagents can be encapsulated (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, or more), either together (if
compatible) or separately encapsulated in a carrier solution. The
present invention thus contemplates there can be at least one or
more reagents encapsulated and present in a carrier solution. Any
number of combinations of encapsulated reagents and carrier
solution reagents is contemplated. A list of reagents which can be
encapsulated or present in the carrier solution is provided
elsewhere herein. This list is for example only, is not exhaustive,
and does not constitute any limitation of the possible combinations
of encapsulated reagent and reagent present in the carrier
solution.
Example 2
Encapsulated DAB Chromogen Reagents
[0034] As noted the present invention can be used with other
chromogens such as horseradish peroxidase chromogens. The chemicals
used in this embodiment are hydrogen peroxide, deionized water,
buffer, and DAB (3,3'-diaminobenzidine), and DAB enhancers. In one
example, the carrier solution comprises the buffer and hydrogen
peroxide, and DAB can be the encapsulated reagent in the carrier
solution. What in the prior art was a three part, non-waiting
system (buffer/DAB/hydrogen peroxide) necessary to obtain an
activated chromogen is now, with the present invention, a one-step,
one solution DAB chromogen. In this embodiment of the present
invention, the encapsulated DAB is released under disruption
conditions to combine with the buffer and hydrogen peroxide to
activate the DAB chromogen for use. There can be multiple
encapsulated reagents that are disrupted by different disruption
modes, an example of which is the last step of DAB chromogen,
wherein the DAB chromogen attached to the biological specimen is
"enhanced" with a copper sulfate for changing the color of DAB form
brown to a darker brown or black in color. The encapsulated copper
sulfate in the carrier solution can be disrupted and released into
the carrier solution, at the last step of the reaction, by any of
the modes listed. Now what once was a 4 part system (in the prior
art method) to activate DAB and enhance its color is now a single
solution of the present invention that has two chemicals (DAB and
Copper sulfate) encapsulated that can be released under a different
disruption modes at different times in the reaction. It is obvious
that the combination of carrier solution and encapsulated reagent
can be changed. For example the buffer and DAB could be free in the
carrier solution and the hydrogen peroxide could be the
encapsulated component which is released under disruption to
activate the chromogen for use.
Example 3
Encapsulated Pre-Treatment Enzymes
[0035] Any of the known enzymes that are used to digest the
biological specimens such as pepsin, ficin, and proteases can be
encapsulated for use as contemplated in the present invention.
These proteins can be unstable in storage and during conventional
use. The present invention contemplates encapsulated proteins in an
aqueous carrier buffered solution and wherein when the disruption
of choice occurs, the enzyme is released and able to react with the
biological specimen. The present invention protects the enzyme from
degradation during storage. These enzymes now can be stored at room
temperature if desired. The microencapsulation protects the enzyme
from degradation.
[0036] Encapsulated Antibodies.
[0037] Any of the known antibodies that are used to attach to
biological specimens during staining and/or antigen retrieval are
contemplated for use herein. Presently, these antibodies can be
unstable in storage and during use. The present invention can
encapsulate these proteins in an aqueous carrier buffered solution
and when the disruption of choice occurs, the antibody (or
antibodies) is released and able to react with the biological
specimen. The present invention protects the antibody from
degradation during storage. These antibodies now can be stored at
room temperature if desired. The microencapsulation protects the
antibodies from degradation. Antibodies of the present invention
can be encapsulated separately (one type of antibody per
encapsulation} or together to release several antibodies to react
with the biological specimen. (Several different types of
antibodies in one capsule). A further possibility is each different
antibody can be encapsulated in a different capsule separate from
the rest which can be disrupted by the same disruption condition or
by different disruption conditions. This embodiment is advantageous
when performing double stains and triple stain with antibodies
reacting with a biological specimen.
[0038] In an example of a triple stain, a single solution can have
three different primary antibodies present in the carrier solution.
Each different type of primary antibody is encapsulated into a
separate type of capsule having a different type of disruption
mode. In the method of its use, the first primarily antibody is
disrupted and released and reacts with the biological specimen.
This first primary antibody is then reacted with a detection system
(e.g., a secondary biotinylated antibody/streptavidin
label/chromogen) known in the art. The second primary antibody is
then released by a different disruption mode and then detected. The
third primary antibody is released by a different disruption mode
and then detected. The single solution can even have multiple
detection chemistries present to detect the primary antibodies
present in the carrier solution. Any combination of primary
antibodies, detection reagents, chromogens, buffers, etc. can be
encapsulated in a carrier solution in accordance with the present
invention. The antibodies are protected from degeneration by being
encapsulated. These primary antibodies and their corresponding
detection chemicals can be stored at room temperature or can be
refrigerated. The preferred embodiment is storage of these reagents
at room temperature. The encapsulation inhibits the degradation of
these proteins (antibodies) and chemicals from bacterial or fungal
attack or physical degradation. The capsules may have anti-fungal
and anti-bacterial properties to inhibit the degradation of these
proteins and chemicals when stored at room temperature or at
refrigerated conditions.
[0039] Preferably the encapsulated reagent(s) in the carrier
solutions of the present invention are stable under storage and/or
shipping conditions wherein the carrier solutions with the reagents
therein are exposed to temperature levels at low, refrigerated
temperatures (<20.degree. C.), room temperature (20.degree.
C.-25.degree. C.), or temperatures above room temperature
(>25.degree. C.). The stability of the encapsulated reagents of
the present inventions as well as the decreased processing time or
steps to activate or use reagents are primary advantages of the
present invention. The ability to store the reagents of the present
invention at room temperature is a key feature and advantage of the
present invention.
[0040] In one embodiment, the encapsulated reagents and carrier
solutions of the present invention are used in the reagent
dispensing packs and strips disclosed in U.S. Pat. Nos. 6,534,008,
6,855,292, 7,250,301, 7,476,363, 7,622,077, 7,632,467, and
Published Application numbers 2006/0281116, 2006/0275889, and
2006/0275861 and pending U.S. application Ser. No. 12/550,288 each
of which is expressly incorporated herein by reference in its
entirety. The advantage of the use of the reagents and solutions of
the present invention is that reagent dispensing packs and reagent
dispensing strips using these reagents and carrier solutions can be
shipped and stored at room temperature, as noted above and can be
stored at room temperature. This advantage is novel because the
technicians using staining apparatuses of the prior art must move
their auto stainer reagents in and out of refrigerators throughout
the day to load and unload their auto stainers with required
reagents. This refrigeration and heating up to room temperature of
the reagents shortens the life of the reagents. Further, since
these reagents of the prior art require refrigeration there is
generally a need to have more than one refrigerator to store all
the different reagents. The reagent dispensing packs and strips
which contains the reagents and carrier solutions of the present
invention can be stored at room temperature in a drawer, cabinet,
or on the counter next to the auto staining instrument for example.
The embodiment of the present invention makes storage of testing
reagents more stable and reduces the time for preparation and use.
The present invention addresses the need for room temperature
storage of reagents used in medical and laboratory testing,
increased stability of reagents at room temperature and
refrigerated conditions, decreases in the processing time to
"prepare" or "activate" reagents, and reduction of steps in the use
of auto staining instruments.
[0041] As noted above, the carrier solutions described herein,
which have encapsulated reagents therein, may be supplied in or
with or packaged in single-use or multi-use reagent containers,
packs or strips for use in microscope slide staining and antigen
retrieval processes and apparatuses such as, but not limited to,
those described in U.S. Pat. Nos. 6,534,008; 7,250,301; 6,855,292;
7,622,077; 7,632,461 and 7,476,362, and US Published application
2006/0275889, and Pending U.S. application Ser. No. 12/550,296,
each of which is expressly incorporated herein by reference in its
entirety.
[0042] The present invention is not to be limited in scope by the
specific embodiments and examples described herein, since such
embodiments and examples are intended as but individual
illustrations of one aspect of the invention and any similar or
functionally equivalent embodiments are within the scope of this
invention. Indeed, various modifications of the compositions and
methods of the invention in addition to those shown and described
herein will become apparent to those skilled in the art form the
foregoing description. Changes may be made in the construction and
the operation of the various components, compositions, elements,
methods, and assemblies described herein or in the steps or the
sequence of steps of the methods described herein without departing
from the spirit and scope of the invention as defined in the
following claims.
[0043] Each of the references, patents or publications cited herein
is expressly incorporated herein by reference in its entirety.
* * * * *