U.S. patent application number 17/592271 was filed with the patent office on 2022-06-09 for polycationic quaternary ammonium polymer coatings for immobilizing biological samples.
The applicant listed for this patent is TriPath Imaging, Inc.. Invention is credited to William A. Fox, William Carl Ray, III.
Application Number | 20220178797 17/592271 |
Document ID | / |
Family ID | 1000006164762 |
Filed Date | 2022-06-09 |
United States Patent
Application |
20220178797 |
Kind Code |
A1 |
Fox; William A. ; et
al. |
June 9, 2022 |
POLYCATIONIC QUATERNARY AMMONIUM POLYMER COATINGS FOR IMMOBILIZING
BIOLOGICAL SAMPLES
Abstract
The present invention is directed to a pre-coated substrate,
such as a slide, that is useful for immobilizing a sample. The
invention is further provides methods of preparing such pre-coated
substrates and methods of analyzing biological samples immobilized
on such pre-coated substrate. The substrate is coated with a
polycationic polymeric coating material specifically selected such
that that coated substrate exhibits increased stability and
prolonged shelf-life. Preferred polymeric coating materials include
allylic or vinylic polymers having cationic groups thereon and
having no more than a small percentage of peptidic monomeric
linkages, particularly polydiallyldimethylammonium (PDDA).
Inventors: |
Fox; William A.;
(Burlington, NC) ; Ray, III; William Carl;
(Durham, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TriPath Imaging, Inc. |
Burlington |
NC |
US |
|
|
Family ID: |
1000006164762 |
Appl. No.: |
17/592271 |
Filed: |
February 3, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14104364 |
Dec 12, 2013 |
11274999 |
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17592271 |
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11233496 |
Sep 22, 2005 |
8617895 |
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14104364 |
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60612391 |
Sep 23, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y10T 436/173845
20150115; G01N 33/54393 20130101; G01N 1/405 20130101; Y10T
436/108331 20150115; Y10T 436/10 20150115 |
International
Class: |
G01N 1/40 20060101
G01N001/40; G01N 33/543 20060101 G01N033/543 |
Claims
1.-48. (canceled)
49. A coated substrate for immobilizing a biological sample for
analysis, comprising: a substrate, wherein the substrate is a glass
slide; and a coating comprising a non-peptidic quaternary ammonium
polymeric material having an average molecular weight greater than
350,000 Da, wherein the coating allows cells in a biological sample
to be immobilized directly thereon when brought into contact with
the biological sample.
50. The coated substrate of claim 49, wherein the non-peptidic
quaternary ammonium polymeric material comprises a monomer selected
from the group consisting of diallyldimethylammonium,
methylacrylamidopropyltrimethylammonium,
methacryloyloxyethyltrimethylammonium, 4-vinyl-benzyltrimethyl
ammonium, acryloxyethyldimethylbenzyl ammonium,
acryloxyethyltrimethyl ammonium, dimethylaminoethylmethacrylate,
methacryloxyethyldimethyl ammonium,
methacryloxyethyltrimethylbenzylammonium,
trimethyl-2-methacryloylethylammonium,
trimethyl-2-methacrylaminopropylammonium, or a mixture thereof.
51. The coated substrate of claim 49, wherein the non-peptidic
quaternary ammonium polymeric material is a homopolymer.
52. The coated substrate of claim 49, wherein the non-peptidic
quaternary ammonium polymeric material is hydrophilic.
53. The coated substrate of claim 49, wherein the non-peptidic
quaternary ammonium polymeric material is
polydiallyldimethylammonium (PDDA).
54. The coated substrate of claim 50, wherein the non-peptidic
quaternary ammonium polymeric material has an average molecular
weight of about 400,000 Da to about 500,000 Da.
55. The coated substrate of claim 49, wherein the coating does not
comprise poly-L-lysine (PLL).
56. The coated substrate of claim 49, wherein the substrate is a
microscope slide.
57. The coated substrate of claim 49, wherein the coating has a
layer thickness of about 0.005 .mu.m to about 500 .mu.m.
58. The coated substrate of claim 49, wherein the coating has a
layer thickness of about 1.mu.m to about 50 .mu.m.
59. The coated substrate of claim 49, wherein the biological sample
comprises a fluid sample, a tissue sample, or a mixture
thereof.
60. The coated substrate of claim 49, wherein the cells comprise
human cells.
61. The coated substrate of claim 49, wherein the cells comprise
cervical carcinoma cells.
62. The coated substrate of claim 49, wherein the non-peptidic,
quaternary ammonium polymeric material is applied to the substrate
as a solution, wherein the non-peptidic, quaternary ammonium
polymeric material is present in the solution at a concentration of
about 0.01% (w/v) to about 10% (w/v).
63. The coated substrate of claim 62, wherein the non-peptidic,
quaternary ammonium polymeric material is present in the solution
at a concentration of about 0.25%.
64. The coated substrate of claim 62, wherein the solution has a pH
of about 8 to about 14.
65. The coated substrate of clam 62, wherein the solution has a pH
of about 9.2.
66. The coated substrate of claim 49, wherein the coating is
capable of allowing adherence of the cells in the biological sample
following at least five weeks of storage.
67. The coated substrate of claim 65, wherein the coating is
capable of allowing adherence of the cells in the biological sample
following 16 weeks of storage.
68. The coated substrate of claim 65, wherein the storage comprises
storage at about room temperature to about 45.degree. C.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 11/233,496, filed Sep. 22, 2005, which claims the benefit of
U.S. Provisional Application No. 60/612,391, filed Sep. 23, 2004,
which is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The present invention is directed to a method for preparing
a coated substrate for immobilizing a biological sample thereon,
preferentially for analysis thereof. The present invention is
further directed to a pre-coated substrate prepared according to
the above method. The substrate is coated with a polycationic
polymer providing a stable polymer layer capable of ionic
interaction with anionic biological components.
BACKGROUND
[0003] Various biological preparatory techniques require
immobilization of sample materials, such as cells, tissue,
proteins, or nucleic acids, to a substrate prior to subsequent
processing. Many of these biological materials of interest are
anionic in nature, exhibiting net negative charge sites. One method
of immobilizing these materials is to coat the target substrate
with a chemical solution containing active ingredients that are
cationic in nature, exhibiting a net positive charge. As the
biological materials of interest to be immobilized exhibit net
negative charge sites, the biological materials bind to the surface
of the substrate through interaction with the net positive charge
sites of the coating solution. This adhesion property of the
coating solution allows the immobilization of sample material for
subsequent processing.
[0004] The immobilization effect described above can be created
through the use of coatings containing various active ingredients
currently known in the art. For example, it is currently known to
use coating agents, such as poly-1-lysine, 3-aminopropyl
triethoxysilane, chrome alum gelatin, and egg white albumin. One of
the most widely used of these known immobilization agents is
poly-1-lysine (PLL).
[0005] PLL is a large polycationic homopolymer that exhibits a
strong positive charge produced by the terminal amino groups of the
lysine residue side chains all along the polymer. L-Lysine
[(S)-2,6-diaminohexanoic acid] is an amino acid of the chemical
structure shown below in formula (1).
##STR00001##
The polymer PLL is a chain of 1-lysine monomer units attached
through peptide bonds. The chemical structure for PLL is provided
below in formula (2), wherein n is an integer representing the
number of monomer units in the polymer chain.
##STR00002##
[0006] While PLL is widely used as a polycationic polymer coating,
substrates coated with PLL tend to lose their immobilization
effectiveness over a relatively short time period. This decline in
effectiveness over time is generally thought to be due to oxidation
of the PLL side chain amine groups. The oxidized groups do not
exhibit the net positive charge required for proper adhesion to the
biological materials to be immobilized.
[0007] The effectiveness of PLL as an immobilization agent is also
limited by its inherent chemical structure shown above in formula
(2). As previously noted, the amino acid residues of the polymer
are connected by peptide bonds (--CO--NH-- bonds). These peptide
bonds are highly vulnerable to cleavage by proteolytic enzymes,
such as trypsin, and to general hydrolytic cleavage, such as
through attack from a nucleophilic substance. Cleavage of the
peptide bonds results in PLL molecules of substantially shorter
chain length, as measured by the average molecular weight of the
polymer. As the molecular weight of the PLL molecule is reduced
through proteolytic cleavage, the immobilization capability of the
molecule (i.e., its adhesive property) becomes greatly reduced.
[0008] Known immobilization agents, such as PLL, exhibit limited
usefulness as a result of the chemical instabilities described
above. Accordingly, substrates coated with the known agents also
exhibit limited usefulness, particularly for long-term use or use
after significant storage time. Given the limited stability of
substrates coated with the known immobilization agents, it would be
highly useful to have a pre-coated substrate that is coated with an
immobilization agent that exhibits increased stability,
particularly being useful for immobilizing a biological sample for
observation.
SUMMARY OF THE INVENTION
[0009] The present invention provides a coated substrate
preferentially adapted for immobilizing a biological sample. The
substrate is coated with a polycationic polymer exhibiting
increased stability in comparison to the immobilization agents
previously known in the art. Accordingly, the substrate coated with
the stable polycationic polymer is useful for immobilizing
biological samples having net a negative charge, and the coated
substrate maintains such usefulness for an extended time
period.
[0010] In one embodiment of the present invention, there is
provided a method for preparing a coated substrate. Preferentially,
the coated substrate is adapted for immobilizing a biological
sample. According to one embodiment, the method comprises providing
a substrate having a surface comprising a plurality of anionic
groups, and contacting the substrate with a composition comprising
a solution of a non-peptidic polymeric material to form a coating
of the non-peptidic polymeric material on at least a portion of the
surface of the substrate. The solution comprising the non-peptidic
polymeric material can be an aqueous solution or an organic
solution, preferably having a pH of at least about 6.
[0011] In one preferred embodiment of the invention, the method
further comprises the steps of drying the substrate coated with the
non-peptidic polymeric material. Preferentially, the coated
substrate with the dried non-peptidic polymeric material thereon is
rinsed.
[0012] In another preferred embodiment of the invention, the method
is characterized by the absence of substrate cleaning. In
particular, the method excludes subjecting the substrate to a
cleaning process prior to contacting the substrate with the
non-peptidic polymeric material.
[0013] According to another aspect of the present invention, there
is provided a pre-coated substrate, such as a microscope slide,
that is preferentially adapted for immobilizing a biological sample
for analysis. According to one embodiment, the substrate comprises
a surface having a plurality of anionic groups for providing a net
negative charge, and the substrate is coated with a non-peptidic
polymeric material comprising a plurality of cationic groups.
[0014] The pre-coated substrate, according to this aspect of the
invention, is characterized by its capability of immobilizing an
average number of cells per surface area of the substrate. In one
particular embodiment, the pre-coated substrate is capable of
immobilizing an average number of cells per surface area of at
least about 20,000 cells/cm.sup.2 when the pre-coated substrate is
contacted with 1 mL of a suspension of cells from the SiHa cell
line.
[0015] According to another embodiment of the invention, the
non-peptidic polymeric material used for coating the pre-coated
substrate comprises an allylic polymer, a vinylic polymer, or a
combination thereof, preferentially comprising cationic groups
selected from the group consisting of primary amines, secondary
amines, tertiary amines, and quaternary amines. In one preferred
embodiment, the non-peptidic polymeric material comprises
polydiallyldimethylammonium (PDDA). In another preferred embodiment
of the invention, the non-peptidic polymeric material comprises
polyallylamine (PAH).
[0016] The substrate according to the present invention can be any
item or apparatus useful or necessary for observing or analyzing a
biological material. In one preferred embodiment, the substrate is
selected from the group consisting of slides, plates, beads, test
tubes, cuvettes, dipsticks, swabs, and gauze. In a further
embodiment, the substrate could be a device useful as a contaminant
gathering device. For example, the substrate could be a glove, a
towel, or a medical drape.
[0017] The coated substrates according to the present invention are
adapted for immobilizing materials that are at least partially
anionic in nature. Preferably, the materials for immobilization
have a net negative charge. Accordingly, the coated substrates are
useful for immobilizing various materials, particularly being
adapted for immobilizing biological material, such as cells,
tissue, fluids, DNA, RNA, proteins, and similar biological material
having anionic groups available for interaction with the cationic
groups of the non-peptidic polymeric material used in preparing the
coated substrate of the present invention.
[0018] According to another aspect of the present invention, there
is provided a method of analyzing a biological sample. In one
embodiment according to this aspect of the invention, the method
comprises the following steps: providing a pre-coated substrate
adapted for immobilizing a biological sample, the substrate
comprising a surface having a plurality of anionic groups, wherein
the substrate is coated with a non-peptidic polymeric material
comprising a plurality of cationic groups; applying a biological
sample to the pre-coated substrate to immobilize the biological
sample on the substrate; and analyzing the biological sample
immobilized on the pre-coated substrate. In a particular
embodiment, the pre-coated substrate is capable of immobilizing an
average number of cells per surface area of at least about 20,000
cells/cm.sup.2 when the pre-coated substrate is contacted with 1 mL
of a suspension of cells from the SiHa cell line.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention now will be described more fully
hereinafter. However, this invention may be embodied in many
different forms and should not be construed as limited to the
embodiments set forth herein; rather, these embodiments are
provided so that this disclosure will satisfy applicable legal
requirements. As used in this specification and the claims, the
singular forms "a," "an," and "the" include plural referents unless
the context clearly dictates otherwise.
[0020] The pre-coated substrate of the present invention is
characterized by the use of a polymeric coating material preferably
demonstrating immobilization capabilities at least equivalent to
the coating agents presently known, but also demonstrating extended
stability of this immobilization effect after coating of the
substrate. Further, in a preferred embodiment, the polymeric
coating material is of a chemical structure that is less
vulnerable, or invulnerable, to proteolytic or hydrolytic
degradation as compared to conventional coating agents, such as
PLL.
[0021] The polymeric coating material according to the present
invention comprises a plurality of cationic groups that are
available for interaction with anionic groups, such as on a
substrate to be coated and within a biological sample to be
immobilized. The cationic groups can be an integral component of
the polymeric backbone of the polymeric coating material or present
as side chain groups. The cationic groups can be any group having a
net positive charge and being capable of ionic interaction with
oppositely charged particles or groups. Particularly preferred
cationic groups include amine groups and ammonium groups, which can
be primary, secondary, tertiary, or quaternary amine groups or
ammonium groups. Cationic groups, particularly an ammonium group,
often have a negatively charged counterion associated with the
group, such as chloride.
[0022] Cationic groups exhibiting greater degrees of substitution
are particularly preferred. As previously noted, simple amine
groups, such as primary amines, are highly susceptible to
oxidation. Substituted amines are less susceptible to such
oxidative attack and therefore exhibit increased stability. It is
believed substitution of the hydrogen groups on the amine with more
complex groups, such as methyl groups, provides protection against
oxidation, the more complex groups being less susceptible to
substitution. Accordingly, higher degrees of substitution are
believed to yield amines of increased stability. Quaternary
ammonium groups are particularly preferred for their increased
stability.
[0023] The polymeric coating material of the present invention is
preferably formed by polymerization of one or more allylic or
vinylic monomers. Allylic polymers are understood to be polymers
derived from monomers comprising at least one allylic group, which
is illustrated below in formula (3).
##STR00003##
Vinylic polymers are understood to be polymers derived from
monomers comprising at least one vinylic group which is illustrated
below in formula (4).
##STR00004##
Acrylic acid, methacrylic acid, and various esters thereof are
examples of vinylic monomers useful in the present invention. Both
allylic and vinylic monomers result in formation of non-peptidic
polymer backbones and, as a result, exhibit greater resistance to
proteolytic degradation than PLL.
[0024] In one embodiment of the present invention, a particularly
preferred polymer derived from an allylic monomer for use as the
polymeric coating material is polydiallyldimethylammonium (PDDA),
which is generally available as the chloride salt of the polymer.
Like PLL, PDDA is a large polycationic homopolymer that exhibits a
strong net positive charge. The strong net positive charge on the
PDDA molecule is produced by side chain dimethylated ammonium
groups on the residues all along the polymer. The chemical
structure of the polymer PDDA is provided below in formula (5),
wherein n is an integer representing the number of monomer units in
the polymer chain.
##STR00005##
[0025] PDDA is a particularly stable immobilization agent for use
as the polymeric coating material of the present invention. The
cationic groups on PDDA are quaternary ammoniums, meaning they are
much less susceptible to oxidation as described above. The polymer
backbone of PDDA is derived from allylic groups and contain no
peptide bonds, such as those found in the PLL molecule. This
absence of peptide bonds makes PDDA resistant to attack by
proteolytic agents, such as trypsin, that have been proven to break
down the PLL polymer chain and reduce immobilization
capabilities.
[0026] The increased stability of substrates coated with a
polymeric coating material comprising PDDA has been substantiated
by laboratory testing. In one test, a substrate was coated with a
polymeric coating material comprising PDDA, allowed to dry, and
rinsed with deionized water. Accelerated stability studies
comparing PLL coated substrates against the PDDA coated substrates
at 45 .degree. C. predicted exceptional performance stability in
excess of 15 months. This comparison is further illustrated in
Example 2.
[0027] In addition, PDDA is advantageous for use in the polymeric
coating material of the present invention because of its inherent
hydrophilicity. Surprisingly, substrates coated with PDDA exhibit
increased hydrophilicity in comparison to substrates coated with
the known coating agents, such as PLL. This is an advantageous
effect because small aqueous analytical samples will spread more
evenly across the substrate coated with PDDA. This allows for a
more uniform distribution of the immobilized sample, which
facilitates better observation of the immobilized sample.
[0028] In another embodiment of the present invention, the polymer
used in the polymeric coating material is polyallylamine (PAH),
which is generally available as the hydrochloride salt
(polyallylamine hydrochloride). As with PDDA, PAH is an allylic
polymer having no peptide bonds. The amine group of PAH is not
highly substituted, such as with PDDA; however, PAH is still useful
as a polymeric coating material according to the present invention.
The chemical structure of the polymer PAH is provided below in
formula (6), wherein n is an integer representing the number of
monomer units in the polymer chain.
##STR00006##
[0029] In addition to PDDA and PAH, the polymeric coating material
can be a polymer derived from polymerization of one or more various
monomers, particularly allylic or vinylic monomers. Accordingly,
the polymeric coating material can be a homopolymer, copolymer, or
terpolymer. Additionally, the polymeric coating material can be a
physical mixture of one or more homopolymers, copolymers, or
terpolymers. When the polymeric coating material comprises a
homopolymer, the monomers are preferably all cationic monomers;
however, when the polymer is a copolymer, terpolymer, or physical
polymer mixture, it is not necessary for all monomers to be
cationic. In one preferred embodiment, the polymeric coating
material comprises a mole percentage of about 5% to about 100%
cationic polymer or monomers. More preferably, the polymeric
coating material comprises a mole percentage of about 30% to about
100% cationic polymer or monomers, most preferably about 50% to
about 100% cationic polymer or monomers.
[0030] The cationic monomer used in the polymeric coating material
can be cationic in its normal state or can be derivatized from a
non-cationic state to a cationic state. Such derivatization can be
through any method generally known in the art, such as through
addition of an ionic functionality, such as an amine or ammonium
group. That is to say, the monomers used to form the polymeric
coating material may contain native cationic groups, as in the case
of the monomers used to form PDDA and PAH, or can contain side
groups that can be derivatized to form cationic side groups.
[0031] Preferably, the polymeric coating material is derived from
at least one monomer selected from the group consisting of
diallyldimethylammonium, allylamine,
methylacrylamidopropyltrimethylammonium, acrylamide, acrylic acid,
methacryloyloxyethyltrimethylammonium,
4-vinyl-benzyltrimethylammonium, methacrylic acid,
hydroxyethylacrylate, methacrylate, methylmethacrylate,
hydroxyethylmethacrylate, 4-vinylpyridinium,
4-vinyl-1-methylpyridinium, methyl acrylate, ethyl acrylate, butyl
acrylate, 2-ethyl hexyl acrylate, dimethylaminoethylacrylate,
dimethylaminoethylacrylate methyl chloride quaternary,
dimethylaminopropylacrylamide, dimethylaminopropylacrylamide methyl
chloride quaternary, acryloxyethyldimethylbenzyl ammonium,
acryloxyethyltrimethyl ammonium, dimethylaminoethylmethacrylate,
methacryloxyethyldimethylammonium,
methacryloxyethyltrimethylbenzylammonium, ethene, ethyleneimine,
propene, styrene, vinyl chloride, isobutylene,
trimethyl-2-methacryloylethylammonium,
trimethyl-2-methacrylaminopropylammonium, and mixtures thereof.
[0032] In another preferred embodiment of the invention, the
polymeric coating material comprises a copolymer of a cationic
monomer and at least one additional monomer.
[0033] Preferentially, the polymeric coating material comprises a
copolymer of diallyldimethylammonium and at least one additional
monomer. Most preferably, the at least one additional monomer
comprises a vinylic monomer. In one embodiment, the polymeric
coating material comprises a copolymer comprising
diallyldimethylammonium and acrylic acid monomer units. In another
embodiment, the polymeric coating material comprises a copolymer
comprising diallyldimethylammonium and acrylamide monomer units. In
another embodiment of the invention, the polymeric coating material
comprises a terpolymer comprising diallyldimethylammonium, acrylic
acid, and hydroxyethylmethacrylate monomer units.
[0034] Preferably, the polymeric coating material according to the
present invention is "non-peptidic", meaning the linkages between
monomer units are predominately and, preferably substantially,
non-peptidic in nature. Preferably, the polymeric coating material
comprises no greater than about 25% peptidic monomeric linkages,
meaning no more than about 25% of the linkages between monomer
units comprise peptide bonds. More preferably, no greater than
about 10% of the monomeric linkages are peptidic linkages, and most
preferably no greater than about 5% of the monomeric linkages are
peptide bonds. In certain preferred embodiments, the polymer
coating material is completely free of peptidic linkages.
[0035] The polymer used in the polymeric coating material of the
present invention is preferably of relatively high molecular
weight. High molecular weight polymers are preferred because of the
high charge density associated with such high molecular weight.
Accordingly, while high molecular weights are preferred, polymers
having lesser molecular weights than as described herein would also
be useful according to the present invention if the lesser
molecular weight polymers exhibited a charge density sufficiently
high to be considered equivalent to the charge density of the high
molecular weight polymers described herein.
[0036] The polymer used in the polymeric coating material
preferably has a molecular weight of greater than about 75,000 Da,
more preferably greater than about 100,000 Da. In particular
embodiments, the polymer has a molecular weight in the range of
about 250,000 to about 750,000 Da, most preferably in the range of
about 400,000 Da to about 500,000 Da. Unless otherwise noted,
molecular weight is expressed herein as weight average molecular
weight (MO, which is defined by formula (7) below
NiMi 2 NiMi , ( 7 ) ##EQU00001##
wherein Ni is the number of polymer molecules (or the number of
moles of those molecules) having molecular weight Mi.
[0037] In one preferred embodiment, the polymeric coating material
comprises PDDA, as shown above in formula (5), wherein n is an
integer between about 500 and 6,000, preferably between about 2,000
and about 5,000, more preferably between about 3,000 and about
4,000. In another preferred embodiment, the polymeric coating
material comprises PAH, as shown above in formula (6), wherein n is
an integer between about 1,000 and about 15,000, preferably between
about 5,000 and about 12,000, more preferably between about 8,000
and about 10,000.
[0038] In one aspect of the invention, there is provided a method
for preparing a coated substrate that is preferentially adapted for
immobilizing a biological sample. Generally, the method comprises
providing a substrate having a surface comprising a plurality of
anionic groups, contacting the substrate with a composition
comprising a solution of anon-peptidic polymeric coating material,
as described above, to form a coating of the polymeric coating
material on at least a portion of the surface of the substrate, and
drying the polymeric coating material coated on the substrate
surface.
[0039] The substrate used according to the method of the invention
can be any substrate comprising a surface having a plurality of
anionic groups and exhibiting a net negative charge and that would
be useful for immobilizing a sample thereon. Preferably, the
substrate is an item useful as a diagnostic tool, an observation
tool, an anti-contamination tool, and other similar tools, the use
of which would be apparent to one of skill in the art.
Preferentially, the substrate used in the method of the invention
comprises glass, metals, ceramics, natural or synthetic polymers,
natural or synthetic fibrous materials, and mixtures thereof.
Specific, non-limiting examples of substrates useful in the method
include slides, beads, test tubes, cuvettes, dipsticks, swabs,
gauze, and the like.
[0040] In one embodiment of the invention, the substrate to be
coated with the polymeric coating material is a plate or slide,
such as a microscope slide. The slide can comprise any material
generally accepted in the art as being useful as such. For example,
the slide can be constructed of glass, ceramic, or a polymer
material. When glass is used, the glass can be any kind of standard
glass comprising primarily silicon dioxide, such as standard soda
lime glass. Alternately, the glass can be specialty glass, such as
borosilicate glass.
[0041] When the slide is comprised of a polymer, it is preferred
that the polymer, in its normal state, comprises anionic groups
capable of interaction with the cationic groups of the polymeric
coating material. In the absence of such groups, however, the
polymer can be derivatized to enhance cell adhesion. Examples of
polymer useful as slides according to this embodiment of the
present invention include, but are not limited to, polystyrene,
polyhydroxy methacrylate, polyethylene terephthalate,
polytetrafluoroethylene, fluorinated ethylene, and
polydimethylsiloxane. The polymer can be a homopolymer, copolymer,
terpolymer, or physical polymer mixture.
[0042] In a preferred embodiment of the method of the invention,
the polymeric coating material is in solution, which can be in an
aqueous solution or an organic solution. Any suitable solvent known
in the art can be used to solubilize the polymeric coating
material, such as deionized water to make an aqueous solution or an
alcohol to make an organic solution. The solution can have a
concentration of the polymeric coating material ranging from about
0.001% (w/v) to about 50% (w/v). Preferably, the polymeric coating
material concentration in the solution is about 0.01% to about 10%,
more preferably about 0.05% to about 2%, still more preferably
about 0.1% to about 1%, and most preferably about 0.15% to about
0.75%.
[0043] Particularly surprising according to the present invention,
in certain embodiments, lower concentration solutions can be used
to prepare a coated substrate having an immobilization capability
superior to a coated substrate prepared using a higher
concentration solution. For example, in particular embodiments,
solutions having a polymeric coating material concentration of
about 0.25% have been shown especially advantageous for preparing a
pre-coated substrate according to the invention.
[0044] As noted above, the polycationic polymers useful in the
invention can exist in a neutral state being coupled with a
counterion (for example, chloride in the case of PDDA and
hydrochloride in the case of PAH). When in solution, the ions tend
to disassociate. Accordingly, the polymer is in its cationic state,
ready for use as an immobilizing agent according to the present
invention.
[0045] The present invention also encompasses facilitating the
activation of binding sites on the substrate, thereby increasing
the number of anionic sites available for interacting with the
cationic groups on the polymeric coating material. Any method known
in the art for activation of anionic binding sites on a substrate
would be useful according to the present invention.
[0046] According to one embodiment of the present invention, the pH
of the polymeric coating material can be adjusted. Such adjustment
of the pH of the polymeric coating material can be to raise or
lower the pH and can take place preceding or following the coating
of the substrate with the polymeric coating material. This ability
to adjust the pH of the polymeric coating material is particularly
advantageous for increasing the number of anionic binding sites on
the substrate through deprotonization of the substrate when
contacted with the polymeric coating material. Generally,
increasing the pH at the substrate surface will promote
deprotonization and increase the number of available anionic
binding sites.
[0047] Preferentially, the pH of the solution comprising the
polymeric coating material is adjusted to a preferred pH. In one
embodiment, the pH of the solution is at least about 6. In other
words, the solution pH is about 8, about 9, about 10, about 11,
about 12, about 13, or about 14. In one preferred embodiment, the
pH of the solution comprising the polymeric coating material is
about 8 to about 14, preferably about 8 to about 10.
[0048] After contacting the substrate with the polymeric coating
material, the polymeric coating material coated on the substrate is
preferably dried prior to further processing or use. Dryness of the
polymeric coating material can be evaluated by any method generally
known in the art. In one embodiment of the invention, the polymeric
coating is at least dried to a point of visual dryness. The visual
difference between a wet polymeric material and a dry polymeric
material would be easily recognizable to one of skill in the
art.
[0049] Drying of the polymeric coating material can be achieved by
any method generally accepted in the art and can comprise passive
drying or active drying (e.g., forced air, such as a fan). The
polymeric coating material coated on the substrate can be dried at
ambient temperature or at an elevated temperature. Ambient
temperature, as used herein, is understood to refer to the
temperature of the surrounding environment. In one embodiment,
ambient temperature is an average room temperature, generally
considered to be in the range of about 20.degree. C. to about
25.degree. C. (about 68.degree. F. to about 77.degree. F.).
[0050] Of course, temperatures below about 20.degree. C. are not to
be excluded by the present invention. In fact, drying could be
performed at temperatures as low as about the freezing temperature
of the polymeric coating material.
[0051] Drying of the polymeric coating material coated on the
substrate can also be carried out at an elevated temperature. The
temperature can be elevated up to about the temperature wherein
further increase would cause degradation of the polymeric coating
material. Accordingly, the polymeric coating material coated on the
substrate can be at least partially dried at a temperature elevated
to about 35.degree. C. to about 120.degree. C. (about 95.degree. F.
to about 248.degree. F.), more preferably about 45.degree. C. to
about 80.degree. C. (about 113.degree. F. to about 176.degree. F.),
most preferably about 50.degree. C. to about 60.degree. C.
(122.degree. F. to about 140.degree. F.).
[0052] The period of time over which the polymeric coating material
coated on the substrate is dried can vary depending upon the
temperature and method of drying. Generally, the period of time for
drying can vary from about 1 minute to about 1 hour, or longer. For
example, when drying of the polymeric coating material coated on
the substrate is carried out at ambient temperature, such drying is
preferably carried out for a period of time of up to about 1 hour,
more preferably for a period of time of about 5 minutes to about 1
hour, and most preferably for a period of time of about 10 minutes
to about 30 minutes. Drying at ambient temperature can be continued
in excess of 1 hour without detriment to the polymeric coating
material.
[0053] When drying of the polymeric coating material coated on the
substrate is carried out at an elevated temperature, such drying is
preferably carried out for a period of time of about 1 minute to
about 20 minutes, more preferably for a period of time of about 2
minutes to about 10 minutes. Drying at elevated temperatures can
take place for a period of time in excess of about 20 minutes so
long as the time and temperature combination would not lead to
polymer degradation.
[0054] The substrate with the dried polymeric coating material
applied thereto is preferentially rinsed, such as with deionized
water, prior to use for immobilizing a sample. Such rinsing is
useful for removing disassociated counterions as well as excess
polymeric coating material that has not ionically interacted with
the substrate. Drying the polymeric material coated on the
substrate prior to rinsing is preferred since failure to perform
the drying step prior to the rinsing step can result in a coated
substrate wherein the coating is incomplete (i.e., "patchy").
Rinsing immediately after coating leads to washing away of
excessive amounts of the polymeric coating material leaving a
coated substrate with limited ability for later immobilization of a
sample. Drying the polymeric coating material coated on the
substrate prior to rinsing (as described above), however,
facilitates maximum ionic interaction between the polymeric coating
material and the substrate, which provides a coated substrate
having a maximum amount of polymeric coating material applied
thereto (i.e., maximum charge density) and therefore having a
maximized ability for later immobilization of a sample.
[0055] Maximization of immobilization ability is further possible
according to the present invention in that there is provided a
method for application of the polymeric coating material to the
substrate in a controlled manner such that the rinsing step is
completely eliminated. Rinsing is generally included in the coating
method to remove excess polymeric material that has not been
immobilized on the substrate through ionic interactions. This is
economically undesirable. First, the rinsing step increases the
time necessary to prepare the coated substrates, particularly in
mass production, such as with microscope slides. Second, rinsing
represents a loss of material. Excess polymeric material applied to
the substrate (i.e., polymeric material that does not adhere to the
substrate) is lost in the rinse. Again, in mass production, the
amount of polymeric material lost in rinsing can add up to a
substantial cost.
[0056] The present invention solves these problems, however. In one
embodiment, the invention provides a method for controlled
application of a polymeric material to a substrate. In this method,
the volume of polymeric material needed for maximum ionic
interaction with the ionic groups on the surface of the substrate
is calculated, and only the amount of polymeric material necessary
is applied to the substrate. Accordingly, the substrate is coated
with the polymeric material, and there is no excess volume present
to require a rinsing step. Preferably, the polymeric material is
still dried prior to use of the coated substrate for immobilization
of a sample.
[0057] Another surprising aspect of the present invention
heretofore unrecognized in the art is that when the method of the
invention specifically excludes subjecting the substrate surface to
a cleaning process prior to contacting the substrate with the
polymeric coating material, the resulting coated substrate exhibits
improved immobilization properties. It is generally accepted in the
art that substrates used for immobilizing samples thereon undergo a
vigorous cleaning prior to the immobilization step. For example,
when the substrate is a microscope slide, common practice is to
take the slide, as received from the manufacturer, and wash the
slide prior to proceeding with any immobilization steps. Multiple
examples of cleaning, or washing, processes are provided by Cras,
J. J., et al., Biosensors & Bioelectronics, 14 (1999)
683-688.
[0058] Cleaning processes to be avoided according to the present
invention are processes comprising the use of chemical recognized
as useful for removing organic compounds from substrate surfaces.
Exemplary of the cleaning processes to be avoided are processes
including the use of acids (e.g., hydrochloric acid, sulfuric acid,
nitric acid, chromic acid, and chromosulfuric acid), bases (e.g.,
ammonium hydroxide, sodium hydroxide, and potassium hydroxide), and
organic solvents (e.g., methyl alcohol, ethyl alcohol, propyl
alcohol, toluene, acetone, methylene chloride, and mineral
spirits). Further cleaning processes to be avoided include
silanization processes designed to expose silane groups on
substrates, such as glass. Processes such as described above (and
further described by Cras, J. J., et al.) include a mechanism of
action beyond simple rinsing or wiping of a substrate surface.
Accordingly, process steps, such as rinsing a substrate with
deionized water or wiping the surface of a substrate with a cloth,
are not excluded according to the invention. In other words, the
present invention encompasses processes wherein a substrate is
wiped free of dust or rinsed with water prior to coating with the
non-peptidic polymeric material.
[0059] Cleaning processes, such as described above, are time
consuming and can include the use of toxic chemicals. The method of
the present invention, therefore, is particularly useful in that
such cleaning steps are completely excluded in preferred
embodiments. Accordingly, a microscope slide, for example, can be
used as received from the manufacturer without including any
cleaning steps. In other words, in the method of the invention, the
method excludes subjecting the substrate to a cleaning process
prior to contacting the substrate with the non-peptidic polymeric
coating material.
[0060] In another aspect of the invention, there is provided a
pre-coated substrate particularly useful for immobilizing a
biological sample thereon. In one embodiment, the pre-coated
substrate is prepared according to the method described above.
[0061] A pre-coated substrate according to the invention utilizing
the polymeric coating material described herein is advantageous in
that even when the coating layer of the non-peptidic polymeric
coating material is relatively thin, the pre-coated substrate is
still useful and effective for immobilizing a biological sample. Of
course, the effectiveness of the coating is not limited to such
relatively thin coatings, and the polymeric coating material is
also effective with relatively thick coatings. The ability to
prepare a per-coated substrate according to the invention, however,
is particularly advantageous in terms of cost of preparation of
such slides. In other words, the ability to prepare substrates for
immobilizing a sample thereon using only a thin coating of the
polymeric coating material is economical in that a reduced amount
of the polymeric material can be used.
[0062] The polymeric coating material, when coated on a substrate,
can have a thickness of about 0.005 .mu.m to about 500 .mu.m.
Preferably, the polymeric coating material has a coating thickness
of about 0.5 .mu.m to about 100 .mu.m, more preferably about 1
.mu.m to about 50 .mu.m. Preferably, the polymeric coating material
is coated onto the substrate as a single layer, meaning there are
no intervening layers of a different material sandwiched between
two or more layers of the polymeric coating material of the
invention. However, multi-layer coatings are also envisioned by the
present invention.
[0063] The pre-coated substrate of the present invention is
particularly useful not only in terms of increased shelf-life, but
also in terms of ability to immobilize an increased amount of a
biological sample. For example, in one embodiment, the pre-coated
substrate of the invention can be characterized as being capable of
immobilizing an increased average number of cells per surface area
over other previously known pre-coated substrates.
[0064] In one particular embodiment, the average number of cells
per surface area immobilized on the pre-coated substrate of the
present invention is at least about 10% greater than the average
number of cells per surface area over the same area of a substrate
not coated according to the methods of the present invention that
has been contacted with the same cell sample. Preferably, the
average number of cells per surface area immobilized on the
pre-coated substrate of the present invention is at least about 15%
greater than the average number of cells per surface area over the
same area of a substrate not coated according to the methods of the
present invention, most preferably at least about 20% greater.
[0065] The increased cell count of biological material associated
with a pre-coated substrate according to the present invention can
be determined using various equipment and methods that would be
recognized by one of skill in the art. For example, it is well
known in the art that a hemacytometer can be used to count cells
manually over a representative number of fields of view and
thereafter extrapolate a total number of cells per area.
[0066] Cell counts can also be obtained through use of
computer-controlled, automated equipment, such as the
FOCALPOINT.TM. Cell Profiler automated slide reading system
(available from TriPath Imaging, Inc.). The FOCALPOINT.TM. Cell
Profiler uses specific algorithms to limit the number of cells
included in the cell count to a population of diagnostically
significant value (i.e., counts only actual cells and disregards
artifacts). This cell count is accumulated from approximately 950
to 1,000 images taken at high resolution in fields of cells
evaluated as highest potential.
[0067] Preferably, methods used to obtain cell counts, such as
described above, are capable of providing reproducible results and
are capable of providing results that can be evaluated in a
statistically significant manner. Accordingly, a biological sample
applied to a substrate can be processed using standardized
equipment such that samples processed using the equipment can be
comparatively evaluated. One example of such processing equipment
is a PrepStain Slide Processor (available from TriPath Imaging,
Inc.). The PrepStain Slide Processor allows for preparation of a
slide with a consistently applied volume of a biological sample,
such that the area of the slide to which the sample is applied is
consistent and reproducible. Such processing is particularly useful
for evaluating a biological sample applied to a substrate based on
an average cell count per surface area of the substrate.
[0068] One particular embodiment of the invention provides a
pre-coated substrate adapted for immobilizing a biological sample
for analysis. The substrate can be any substrate as described
herein that is coated with a non-peptidic polymeric coating
material, such as described above. The pre-coated substrate in this
embodiment of the invention is characterized in that it is capable
of immobilizing an average number of cells per surface area. Such
immobilization ability can be evaluated based on immobilization of
a standard cell line. For example, the ability of a pre-coated
substrate to immobilize cells can be evaluated using human cervical
carcinoma cells, commonly known as a SiHa cell line. SiHa cells are
readily available, such as from American Type Culture Collection
(ATCC) identified by ATCC Number HTB-35.
[0069] According to one embodiment of the invention, a pre-coated
substrate is provided wherein the pre-coated substrate surface is
capable of immobilizing an average number of cells per surface area
of at least about 20,000 cells/cm.sup.2 when the pre-coated
substrate is contacted with 1 mL of a suspension of cells from the
SiHa cell line. Preferably, the pre-coated substrate surface is
capable of immobilizing an average of at least about 21,000
cells/cm.sup.2 when the pre-coated substrate is contacted with 1 mL
of a suspension of cells from the SiHa cell line, more preferably
at least about 22,000 cells/cm.sup.2, most preferably at least
about 23,000 cells/cm.sup.2.
[0070] The improved immobilizing ability of the pre-coated
substrate according to the present invention can be observed
through further analytical methods as well. One method suitable for
use in quantifying charge density of the coated substrate would
directly measure charge density in terms of charge density per area
of coated substrate. Another method would indirectly measure charge
density by correlation to another measurable property. For example,
the charge density of the polymeric coating material coated on the
substrate can be quantified through spectrographic measurement of a
dye associated with the coated substrate (e.g., adsorbed
thereon).
[0071] As previously noted, the ability of a coated substrate for
adhering a biological sample (generally being negatively charged)
is directly related to the quantity of excess positive charge on
the slide surface. When a negatively charged dye is associated with
the slide surface, the excess positive charge on the slide surface
can be quantified through spectrographic analysis of the dye. It is
well known in the art that the absorption of electromagnetic
radiation at a given wavelength by a dye is directly proportional
to the concentration of the dye. Therefore, given a proportional
relationship between the anionic dye and the cationic coating
material, a measurement of absorbance of the dye associated with
the coating material is a reliable indicator of the quantity of
excess positive charge on the surface of the coated substrate. In
other words, the greater the charge density, the greater the
concentration of the dye adsorbed on the coated substrate, and the
greater the dye's absorption of electromagnetic radiation at a
given wavelength. Such a measurement technique is described by
Tadao Sakai and Akihiko Hirose (Talanta 59 (2003) 167-175), which
is incorporated herein by reference.
[0072] Multiple dyes known in the art are useful in an analytical
technique for quantifying the charge density of a polymeric coating
material coated on a substrate according to the present invention.
A class of dyes particularly useful for quantifying charge density
of a coated substrate in the present invention is xanthene dyes,
such as eosin and tetraiodofluorescein. A particularly useful dye
according to the present invention is Eosin Y (shown below in
formula 8) which is, in a neutral aqueous solution, di-anionic. As
a di-anionic species, the dye binds to a mono-cationic species in a
1:2 relationship.
##STR00007##
[0073] Eosin Y adsorbed onto a cationic polymer, such as PDDA, has
a maximum absorption wavelength (.lamda..sub.max) of about 542 nm.
Therefore, absorption measurements at this wavelength are effective
for quantification of excess positive charge on the surface of a
coated substrate. Such measurements can be taken on any analytical
device known in the art as useful for such measurements, such as a
UV-Vis spectrophotometer. An example of the measurement of the
charge density of a substrate coated with a polymeric coating
material according to the present invention is provided below in
Example 4.
[0074] A coated substrate according to the present invention coated
with a polymeric coating material has an excess of positive charge
sites, such excess being of a quantity to effectively bind a
biological sample. The polymeric coating material on the coated
substrate is effective for binding a biological sample according to
the present invention when the polymeric coating material exhibits
at least a minimally acceptable charge density. The charge density
of a pre-coated substrate prepared according to the present
invention, through quantitative measurement, can easily be seen to
be much greater than the charge density of a pre-coated substrate
that is not prepared according to the present invention. When Eosin
Y dye is used in a quantification method as described above, a
pre-coated substrate according to the present invention would
exhibit an absorbance that is at least twice as great as the
absorbance on a substrate not prepared according to the present
invention. More preferably, the absorbance exhibited by a substrate
according to the present invention is at least about three times
greater than the absorbance on a substrate not prepared according
to the present invention. Even more preferably, the absorbance
exhibited by a substrate according to the present invention is at
least about four times greater than the absorbance on a substrate
not prepared according to the present invention.
[0075] According to one embodiment of the present invention, there
is provided a pre-coated glass slide adapted for immobilizing a
biological sample for analysis. The glass slide has a plurality of
anionic groups, and the slide is coated with a non-peptidic
polymeric coating material comprising a plurality of cationic
groups. The pre-coated glass slide has a charge density such that
when Eosin Y dye is adsorbed on the coated slide and is thereafter
subjected to electromagnetic radiation at a wavelength of 542 nm,
the dye exhibits an absorbance of at least about 0.05, which is
indicative of a minimally acceptable charge density (i.e., excess
positive charge) on the polymeric coating material coating the
glass slide. Preferably, the absorbance is at least about 0.1. Most
preferably, the absorbance is at least about 0.15.
[0076] As would be known to one of skill in the art, the choice of
substrate could affect the measured absorbance of the dye adsorbed
on the coating material used to coat the substrate. Accordingly, if
a substrate other than a glass slide was used according to the
present invention, absorbance values could vary from the range
provided above. Nevertheless, as previously noted, a substrate
coated according to the present invention would be would exhibit an
absorbance that is at least about two times greater than the
absorbance exhibited when the same substrate is coated by a method
that is not according to the present invention, preferably at least
about three times greater, most preferably at least about four
times greater.
[0077] The sample for immobilization on the substrate coated with
the polymeric coating material can be any sample having anionic
groups capable of interacting with the cationic groups of the
polymeric coating material and thereby being immobilized
thereon.
[0078] Preferably, the sample comprises a biological component.
Examples of biological samples for immobilization on the coated
substrate according to the present invention include, but are not
limited to, cells, tissue, fluids, nucleic acids, including
polynucleotides and oligonucleotides (e.g., DNA, RNA and fragments
thereof), polypeptides, and proteins.
[0079] In one embodiment according to the present invention, a
single layer of the biological sample is immobilized on the
substrate coated with the polymeric coating material. The phrase
"single layer" is intended to mean that only one layer of material
is deposited on, and immobilized on, the coated substrate.
Accordingly, no further layers are immobilized in addition to,
particularly over, the biological sample, such as would obstruct
viewing and hinder analysis of the biological sample immobilized
directly on the polymeric coating material on the substrate.
[0080] The biological sample can be immobilized on the coated
substrate for a variety of uses. Preferentially, the use is a
diagnostic use. For example, the biological sample could be
immobilized for the purposes of extraction from a greater sample,
for additional processing or testing, and for various analytical
methods. Specific, non-limiting examples of uses for the coated
substrate include, tissue micro-arrays (TMA), cytological
micro-arrays (CMA), nucleic acid micro-arrays, and other
cytological or histological diagnostics. In addition to such
specific uses, the pre-coated substrates of the present invention
could also be used for immobilization of various biological samples
for manual or automated diagnostic assay kits.
[0081] The pre-coated substrate of the invention can be used in a
variety of diagnostic methods. For example, the pre-coated
substrate could be used to immobilize an antibody that is selective
for a particular protein. In another example, the pre-coated
substrate could be used to immobilize a reactive substrate, which
would be particularly useful for isolating a particular protein
that is an enzyme capable of acting on the reactive substrate.
Other similar diagnostic uses are also encompassed by the present
invention.
[0082] In one embodiment of the present invention, there is
provided a pre-coated bead. Preferably, the pre-coated bead is
adapted for extracting a biological component from a sample. The
bead in this embodiment has a surface comprising a plurality of
anionic groups capable of interacting with the cationic groups of a
non-peptidic polymeric coating material, such as described above.
Accordingly, the bead has a non-peptidic polymeric coating material
overlaying and ionically attached to the surface of the bead. The
coated bead therefore has a plurality of exposed cationic groups
for interacting with the anionic groups of the biological component
of interest in the sample. The biological component can then be
immobilized on the surface of the bead and extracted from the
sample.
[0083] The pre-coated bead preferentially comprises a material
selected from the group consisting of glass, polymers, silicas,
metals, metal oxides, and ceramics. In one particularly preferred
embodiment, the bead comprises a polymer selected from the group
consisting of polystyrene, polyacrylate, polymethacrylate,
polyethylene, polypropylene, polyester, polyurethane, polyamide,
polycarbonate, polydimethylsiloxane, polydialkylsiloxane,
cellulose, derivatives thereof, co-polymers thereof, and
combinations thereof.
[0084] Pre-coated beads according to the present invention can be
used in a variety of extraction and separation methods, such as
have been previously described, and as would be readily envisioned
by one of skill in the art. For example, the pre-coated beads could
be used in various chromatographic separatory methods.
Additionally, the pre-coated beads could be inserted into a sample
and selectively removed to extract a biological component
therefrom.
[0085] Of course, the present invention also encompasses multiple
other embodiments wherein a pre-coated substrate as described
herein can be used in a diagnostic method, and invention is not
limited by the present disclosure. For example, embodiments wherein
the pre-coated substrate is a microscope slide have previously been
described herein.
[0086] According to another aspect of the present invention, there
is provided a method of analyzing a biological sample. The method
generally comprises providing a pre-coated substrate adapted for
immobilizing a biological sample, wherein the substrate is coated
with a polymeric coating material as described herein, immobilizing
the biological sample on the pre-coated substrate, and analyzing
the biological sample immobilized on the pre-coated substrate.
[0087] In one particular embodiment, the step of analyzing the
biological sample immobilized to the pre-coated slide is performed
through use of a diagnostic instrument; however, the present
invention also contemplates analysis of the immobilized sample by
an individual without the aid of additional instrumentation (i.e.,
through use of the senses alone). Examples of diagnostic
instruments useful in the analysis of the immobilized sample
according to the present method include, but are not limited to,
microscopes (such as light microscopes or electron microscopes),
chromatographs, spectrometers, and imaging devices (such as digital
cameras, video cameras, and charge-coupled device (CCD)
cameras).
[0088] The present invention also encompasses various embodiments
wherein the pre-coated substrate of the invention has uses other
than diagnostic uses, as previously described. For example, in one
embodiment, there is provided a device useful for gathering one or
more biological contaminants. In this embodiment, as before, the
substrate comprises a material having a plurality of anionic groups
and is coated with a non-peptidic polymeric coating material.
Particularly preferred embodiment in this embodiment, the substrate
comprises a fibrous material. The fibrous material can be natural
or synthetic and can be woven or non-woven. Non-limiting examples
of fibrous materials useful as the biological contaminant gathering
device include cotton, cellulose, and polyethylene.
[0089] In one preferred embodiment, the biological contaminant
gathering device is selected from the group consisting of gauze,
towels, and medical drapes. Accordingly, the biological contaminant
gathering device can be used in transferring samples, in medical
procedures, and in other situations wherein it is useful to collect
or gather possible or known biological material to prevent the
biological material from contaminating a material or area. For
example, the biological contaminant gathering device could be used
for holding a slide with a DNA sample thereon. Accordingly,
extraneous DNA, such as from the individual handling the slide, is
prevented from contaminating the DNA sample on the slide.
Similarly, the contaminant gathering device could be used around a
surgical site to collect biological material to prevent the
material from contaminating the surgical site.
[0090] In a particularly preferred embodiment of the invention, the
biological contaminant gathering device is a glove, such as a
surgical glove. The glove could be comprised of a fibrous material
coated with a polymeric coating material as described above.
Alternately, the glove could be comprised of a natural or synthetic
polymer (e.g., a "rubber" glove).
[0091] Further embodiments of the present invention are more
distinctly described according to the following experimental
examples.
EXPERIMENTAL The present invention is more fully illustrated by the
following examples, which are set forth to illustrate the present
invention and are not to be construed as limiting thereof.
Example 1
Pre-Coating Glass Microscope Slide with PDDA
[0092] In the preparation of a pre-coated glass microscope slide, a
solution of PDDA in deionized water was prepared such that the
final concentration of the solution was 1% PDDA (+/-0.05%) w/v. The
pH of the solution was then adjusted to 9.0 (+/-0.2) through
addition of 1N NaOH.
[0093] The pH adjusted PDDA solution was placed into a manual slide
staining bath. A manual slide staining rack was loaded with glass
microscope slides, and the rack with the glass microscope slides
was added to the PDDA solution in the bath, with the solution
covering the slides up to the frosted edge of the slides. The
slides were allowed to rest in the PDDA solution for approximately
10 seconds. The rack was removed from the bath, the slides were
removed from the rack, and the slides were placed in an upright,
slightly angled position and allowed to dry at ambient temperature.
The slides with the PDDA solution applied thereto were allowed to
dry to visual dryness then rinsed with deionized water. The rinsed
slides were again allowed to dry providing PDDA-coated slides ready
for use in analyzing anionic samples.
Example 2
Comparison of PDDA-Coated Microscope Slide with PLL-Coated
Microscope Slide
[0094] A PDDA solution was prepared and multiple glass microscope
slides were coated using the solution according to Example 1.
Multiple additional microscope slides were coated similarly using
PREPSTAIN.TM. Slide Coat Reagent (PLL) (available from TriPath
Imaging, Inc.).
[0095] The PDDA-coated slides and the PLL-coated slides were each
separated into two groups. One group of PDDA-coated slides was
stored for 16 weeks at ambient temperature. Likewise, one group of
PLL-coated slides was stored for 16 weeks at ambient temperature. A
second group of PDDA-coated slides, and a second group of
PLL-coated slides were stored for 5 weeks at room temperature and
were stored for 9 additional weeks at 45.degree. C., for a total of
16 weeks of storage. At the end of the 16 weeks, all four groups of
slides were removed from storage for further testing, as described
below. As a comparative in the experimentation, a fresh set of
PLL-coated slides was prepared according to the same method
previously described for comparison with the Stability Groups.
[0096] Slides from each of the five groups described above were
subjected to a single pool of cytological material and subsequently
stained using the PREPSTAIN.TM. method. The slides from all five
groups were then compared on the basis of the amount of cytological
material that adhered to the coated surface.
[0097] The PLL coated slides coated were noted as having reduced
stability, which decreases with time and temperature. This reduced
stability was visibly recognizable from the decreased amount of
adhered and stained cytological material present on the PLL coated
slides. Slide 1, the control slide, was freshly coated with PLL at
the time of evaluation and was not subjected to the 16 week
testing. Slide 2, coated with PLL, was allowed to sit at ambient
temperature for 16 weeks. A comparison of slide 2 with slide 1
indicated less stained cytological material adhered to slide 2,
which indicated a degradation of the polymer coating over the
16-week period. Slide 4, also coated with
[0098] PLL, sat for 5 weeks at ambient temperature and 9 weeks at
45.degree. C. The polymer degradation in this slide was even more
apparent. Visual inspection of slide 4 indicated only minimal
cytological material adhered to the slide surface (i.e.,
practically no visible stained cytological material).
[0099] Slide 3 was coated with PDDA and stored at ambient
temperature for 16 weeks. Slide 5 was coated with PDDA, stored at
ambient temperature for 5 weeks, and then stored at 45.degree. C.
for 9 weeks. Both of slides 3 and 5 indicated little to no coating
degradation. This was visibly apparent by the complete and even
distribution of stained cytological material adhered to the coated
slides. Further, in comparison with the freshly coated PLL control
slide (slide 1), the PDDA slides, even after sitting for 16 weeks,
exhibited deeper staining of the coating, indicating an increased
concentration of polymer (and thus cationic binding sites) on the
PDDA-coated slides in comparison with the PLL-coated slides.
Example 3
Comparison of Microscope Slides Coated Using Varying Coating
Methodologies
[0100] Two sets of microscope slides were coated with PDDA
according to the method of the present invention and a previously
described method. A comparison of the immobilization capabilities
of the slides is provided below.
[0101] Twelve glass microscope slides were coated with PDDA
according to the present invention. Particularly, a 0.25% (w/v)
solution of PDDA in deionized water was prepared and pH adjusted
using NaOH to a final pH of 9.2. The 12 microscope slides were
removed from the packaging as received from the manufacturer and
were specifically not subjected to any cleaning process prior to
coating with the PDDA solution. Next, the uncleaned slides were
manually dipped in the PDDA solution, removed, allowed to dry at
ambient conditions to visual dryness, and then rinsed with
deionized water to remove any residual PDDA. The rinsed slides were
allowed to dry prior to use.
[0102] Twelve additional microscope slides were prepared according
to known preparation methods for comparison with the PDDA-coated
slides of the invention. First, the 12 slides, taken new from the
same packaging and manufacturer, were cleaned according to Method 2
disclosed by Cras, J. J., et al., Biosensors & Bioelectronics,
14 (1999) 683-688. The cleaned slides were then coated with a 1.0%
aqueous PDDA solution according to the method provided by Seyfert,
S., et al., Biomaterials, 16 (1995) 201-207. Particularly, the
cleaned slides were manually dipped in the 1% PDDA solution,
removed from the solution, and immediately rinsed to remove any
residual PDDA solution (i.e., no drying of the coating was
performed prior to rinsing). The rinsed slides were allowed to dry
prior to use.
[0103] To prepare the cell sample for immobilization of the
microscope slides, three bottles of SiHa control cells (from
TriPath Imaging, Inc.) were obtained. The SiHa cells used in the
experimentation were from a single cell line first described in
Friedl, F., Proc. Soc. Exp. Biol. Med., 135 (1970) 543-545. The
contents of the three bottles were centrifuged (800 g for 10
minutes) to compact the cells into a pellet. The supernatant was
discarded and the cells resuspended in deionized water. The cells
were then recompacted into a pellet by centrifugation (800 g for 10
minutes). The supernatant was discarded, and the cells were
resuspended into approximately 30 mL deionized water. One mL of the
cell suspension was transferred into each of 24 conical tubes, and
the samples were processed according to the standard protocol using
a TriPath Imaging PREPSTAIN.TM. Slide Processor instrument (the
samples were applied to the slides and stained).
[0104] After the slides were stained and coverslipped, the slides
were evaluated with a TriPath Imaging FOCALPOINT.TM. Slide
Profiler. The number of cells on each slide was counted by the
instrument, and the cell count for each microscope slide was
extracted from the instrument's database (cell count being directly
proportional to the number of objects registered by the Slide
Profiler.
[0105] The cell sample on each microscope slide was prepared as a
uniform circle having a known diameter of 1.3 cm (13 mm).
Accordingly, the sample area on each slide was 1.33 cm.sup.2 (132.7
mm.sup.2). The number of cells immobilized on each slide is shown
below. Table 1 provides the number of cells immobilized on the
slides prepared according to the methods of the present invention,
and Table 2 provides the number of cells immobilized on the slides
prepared according to previously described methods.
TABLE-US-00001 TABLE 1 Slide No. Cell Count 1 28,997 2 27,914 3
25,992 4 24,398 5 22,525 6 39,738 7 41,413 8 36,446 9 38,968 10
26,200 11 27,023 12 35,053 Average 31,222
TABLE-US-00002 TABLE 2 Slide No. Cell Count 13 25,521 14 24,361 15
24,712 16 25,086 17 26,011 18 25,419 19 23,528 20 29,788 21 28,230
22 22,035 23 25,843 24 24,146 Average 25,390
[0106] Comparison of the cell counts provided above in Table 1 and
Table 2 using Student's t-distribution reveals a significance level
of less than 0.005. Accordingly, the cell counts illustrate with
statistical significance that the PDDA coated slides prepared
according to the present invention immobilize a greater average
number of cells than PDDA coated slides prepared according to
previously known methods. In particular, the PDDA coated slides of
the invention immobilized an average number of cells 22.97% greater
than the average number of cells immobilized on the PDDA coated
slides prepared according to the previously known methods.
[0107] As noted above, the cell sample area on each slide was 1.33
cm.sup.2 (132.7 mm.sup.2). Accordingly, it is possible to calculate
the average number of cells immobilized on a given surface area.
With the slides prepared according to the present invention, the
average number of cells per surface area immobilized was 23,475
cells/cm.sup.2 (235.2 cells/mm.sup.2). By contrast, the slides
prepared according to previously known methods had an average
number of cells per surface area immobilized of only 19,090
cells/cm.sup.2 (191.3 cells/mm.sup.2).
Example 4
Analysis of Slides Coated with PDDA by UV Absorption of Adsorbed
Eosin Y dye
[0108] Fifteen ESCO microscope slides (catalog number 2951) were
obtained. Three slides were set aside for use as control slides.
The remaining twelve slides were divided into four groups of three
slides each. Group 1 slides were coated with a 1% solution of PDDA
at a pH of approximately 9.2. The coated slides were allowed to dry
for 1 hour, were rinsed with deionized water, and allowed to dry
for an additional 1 hour. Group 2 slides were coated with a 1%
solution of PDDA at a pH of approximately 9.2. The coated slides
were immediately rinsed with deionized water (no drying of the PDDA
coating), and the rinsed slides were allowed to dry for 1 hour.
Group 3 slides were coated with a 1% solution of PDDA at a pH of
approximately 5.3. The coated slides were allowed to dry for 1
hour, were rinsed with deionized water, and allowed to dry for an
additional 1 hour. Group 4 slides were coated with a 1% solution of
PDDA at a pH of approximately 5.3. The coated slides were
immediately rinsed with deionized water (no drying of the PDDA
coating), and the rinsed slides were allowed to dry for 1 hour.
Group 5 slides (the control slides) were not coated.
[0109] All slides in the above 5 groups were prepared for treatment
by placing each slide into a Hettich microscope slide-holder base
and positioning a Hettich settling chamber on the slides to isolate
a portion of the slide. The isolated portion of the surface of each
slide was treated with 200 .mu.L of a 5% w/v Eosin Y solution in
deionized water for 1 minute. The dye solution was removed with
vacuum suction, and each slide was treated twice with 2.5 mL of
deionized water, allowing each rinse to stand for 1 minute before
removing with vacuum suction. Each slide was then treated twice
with 2.5 mL isopropanol, allowing each rinse to stand for 1 minute
before removing with vacuum suction. Each slide was then removed
from the slide holder and allowed to dry for at least 10 minutes.
Each slide treated with the dye had a circular stained portion
having an area of about 240 mm.sup.2, the center of the circular
stained portion being approximately 17.5 mm from the unfrosted
short end of the slide.
[0110] Spectrographic analysis was performed using a UV-Vis
spectrophotometer as 542 nm. The instrument was zeroed using a
plain, untreated, uncoated glass slide. The measured absorbance for
each slide (provided below in Table 6) indicated a significant
difference between slides coated with no drying of the polymeric
coating prior to rinsing and those coated by the method of the
present invention. Eosin Y adsorption onto the positively-charged
surfaces of the PDDA-coated slides was much greater on the slides
which were allowed to dry at ambient temperature for about 1 hour
prior to being rinsed with deionized water.
TABLE-US-00003 TABLE 3 Average Method of Slide Treatment Absorbance
Group 1-PDDA coated (1%), 0.158 pH 9.2, dried prior to rinsing
Group 2-PDDA coated (1%), 0.037 pH 9.2, not dried prior to rinsing
Group 3-PDDA coated (1%), 0.109 pH 5.3, dried prior to rinsing
Group 4-PDDA coated (1%), 0.023 pH 5.3, not dried prior to rinsing
Group 5-Uncoated 0.002
[0111] A direct comparison of the slides coated without drying
(Group 2) and the slides coated according to the methods of the
present invention (Group 1), both at pH 9.2, indicates that the
excess positive charge was about 4.3 (+/-0.8) times greater on the
slides prepared according to the present invention. Similarly, a
direct comparison of the slides coated without drying (Group 4) and
the slides coated according to the methods of the present invention
(Group 3), both at pH 5.3, indicates that the excess positive
charge was about 4.7 (+/- 3.0) times greater on the slides prepared
according to the present invention. The contribution of the glass
itself to the adsorption of Eosin Y dye is negligible, as indicated
by the near-zero absorption values for the uncoated slides (control
slides). The adsorption of Eosin Y dye can therefore be attributed
solely to the positive charges carried by the PDDA coated on the
slides.
[0112] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing description. Therefore, it is to be
understood that the inventions are not to be limited to the
specific embodiments disclosed and that modifications and other
embodiments are intended to be included within the scope of the
appended claims. Although specific terms are employed herein, they
are used in a generic and descriptive sense only and not for
purposes of limitation.
* * * * *