U.S. patent application number 15/381699 was filed with the patent office on 2017-04-27 for hydrogel adhesion to molded polymers.
The applicant listed for this patent is Becton, Dickinson and Company. Invention is credited to Javier ALARCON.
Application Number | 20170114200 15/381699 |
Document ID | / |
Family ID | 49261233 |
Filed Date | 2017-04-27 |
United States Patent
Application |
20170114200 |
Kind Code |
A1 |
ALARCON; Javier |
April 27, 2017 |
HYDROGEL ADHESION TO MOLDED POLYMERS
Abstract
Methods for adhering a hydrogel matrix to a molded polymer
substrate and its use as a biosensor, e.g., a continuous or
episodic glucose monitor, are disclosed. The presently disclosed
subject mater provides a method for adhering a hydrogel matrix to a
molded polymer substrate, the method comprising; (a) molding a
polymer comprising one or more polymer chains with an oxidizer to
form a molded polymer substrate; (b) providing a hydrogel matrix
comprising a hydrogel, a component comprising one or more acrylate
groups or another functional group that can form one or more
radicals upon polymerization in the molded polymer substrate, and a
photo initiator; (c) combining the molded polymer substrate and the
hydrogel matrix; and (d) curing the combined molded polymer
substrate and hydrogel matrix for a period of fime.
Inventors: |
ALARCON; Javier; (Durham,
NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Becton, Dickinson and Company |
Franklin Lakes |
NJ |
US |
|
|
Family ID: |
49261233 |
Appl. No.: |
15/381699 |
Filed: |
December 16, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14388886 |
Sep 29, 2014 |
9574057 |
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PCT/US2013/034290 |
Mar 28, 2013 |
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15381699 |
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61616735 |
Mar 28, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C08J 2323/12 20130101;
C08J 7/18 20130101; A61B 5/14556 20130101; C08J 2369/00 20130101;
C07K 17/04 20130101; C08J 2367/02 20130101; A61B 2562/028 20130101;
C08J 2367/03 20130101; A61B 5/14532 20130101; G01N 33/66 20130101;
C08J 2325/06 20130101 |
International
Class: |
C08J 7/18 20060101
C08J007/18; G01N 33/66 20060101 G01N033/66 |
Claims
1. A biosensor comprising a hydrogel matrix covalently bound to a
molded polymer substrate, wherein the hydrogel matrix comprises a
protein-reporter group.
2. The biosensor of claim 1, wherein the hydrogel matrix comprises
polyethylene glycol dimethacrylate.
3. The biosensor of claim 1, wherein the molded polymer substrate
comprises a material selected from the group consisting of a
polycarbonate, a polystyrene, a polyethylene, a copolyester, and a
polypropylene.
4. The biosensor of claim 1, wherein the protein comprises a
glucose binding protein (GBP).
5. The biosensor of claim 1, wherein said hydrogel matrix is a
polyhydroxy acid selected from the group consisting of polylactic
acid, polyglycolic acid, polycaproic acid, polybutyric acid,
polyvaleric acid, and copolymers thereof.
6. The biosensor of claim 1, wherein said polymer substrate is
polyethylene terephthalate.
7. The biosensor of claim 1, wherein said protein-reporter group is
labeled with a fluorescent dye.
8. The biosensor of claim 1, wherein said protein-reporter group is
a horseradish peroxidase or an antibody.
10. The biosensor of claim 1, wherein said molded polymer substrate
comprises a mixture of a polymer having one or more polymer chains
and an oxidizer, and said hydrogel matrix comprises a hydrogel, a
photoinitiator, and a component comprising one or more functional
groups for polymerizing with the molded polymer substrate.
11. The biosensor of claim 10, wherein said one or more functional
groups is an acrylate group.
12. The biosensor of claim 1, wherein said biosensor is obtained by
a method of molding a mixture of a polymer comprising one or more
polymer chains and an oxidizer to form said molded polymer
substrate, combining the hydrogel matrix comprising a hydrogel, a
component comprising one or more acrylate groups or other
functional group that can form one or more radicals upon
polymerization in the molded polymer substrate, and a
photoinitiator, and curing the combined molded polymer substrate
and hydrogel.
13. A biosensor comprising a molded polymer substrate and a
hydrogel matrix having a protein-reporter group covalently bonded
to the molded polymer substrate, wherein said molded polymer
substrate comprises a polymer having one or more polymer chains and
an oxidizer that can break the one or more polymer chains and where
the one or more polymer chains can recombine while retaining one or
more putative radicals in the molded polymer substrate, and where
the hydrogel matrix comprises a hydrogel, a component comprising
one or more acrylate groups or other functional group forming a
radical upon polymerization in the molded polymer substrate and an
initiator.
14. The biosensor of claim 13, wherein said biosensor is obtained
by a method of combining said molded polymer substrate and hydrogel
and curing to covalently bond the hydrogel matrix to the molded
polymer substrate.
15. The biosensor of claim 13, wherein the hydrogel matrix
comprises polyethylene glycol dimethacrylate.
16. The biosensor of claim 13, wherein the molded polymer substrate
comprises a material selected from the group consisting of a
polycarbonate, a polystyrene, a polyethylene, a copolyester, and a
polypropylene.
17. The biosensor of claim 13, wherein said hydrogel matrix is a
polyhydroxy acid selected from the group consisting of polylactic
acid, polyglycolic acid, polycaproic acid, polybutyric acid,
polyvaleric acid, and copolymers thereof.
18. The biosensor of claim 13, wherein said polymer substrate is
polyethylene terephthalate.
19. The biosensor of claim 13, wherein said protein-reporter group
is labeled with a fluorescent dye.
20. The biosensor of claim 13, wherein said protein-reporter group
is a horseradish peroxidase or an antibody.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/388,886, filed Sep. 29, 2014, which is based on PCT
Patent Application No. PCT/US13/034290, filed Mar. 28, 2013, and
which claims the benefit of U.S. Provisional Application Ser. No.
61/616,735, filed Mar. 28, 2012, the entire contents of which are
incorporated herein by reference.
BACKGROUND
[0002] In the past, physical entrapment typically was used to
immobilize detection matrices on a molded polymer substrate in
analytical systems designed for the diagnostic testing of liquid
samples, e.g., biological samples from a patient or subject. In
such systems, the molded substrate physically holds the detection
matrix in place. Analytical testing, however, is not feasible in
such systems, especially with dense or colored liquids, because the
detection matrix has a tendency to slide or move away from its
original position on the substrate. Under these circumstances, the
liquid sample can infiltrate between the substrate and the
detection matrix and potentially interfere with optical
measurements. Further, in optical measurements, the matrix can move
out of the focal point of the interrogating light source, causing
inaccurate or even false negative readings.
SUMMARY
[0003] In some aspects, the presently disclosed subject matter
provides a method for adhering a hydrogel matrix to a molded
polymer substrate, the method comprising: (a) molding a polymer
comprising one or more polymer chains with an oxidizer to form a
molded polymer substrate, wherein the oxidizer breaks the one or
more polymer chains, and wherein the one or more polymer chains can
recombine while retaining one or more putative radicals in the
molded polymer substrate; (b) providing a hydrogel matrix
comprising a hydrogel, a component comprising one or more acrylate
groups or another functional group that can form one or more
radicals upon polymerization in the molded polymer substrate, and a
photoinitiator; (c) combining the molded polymer substrate and the
hydrogel matrix; and (d) curing the combined molded polymer
substrate and hydrogel matrix for a period of time to covalently
bind the hydrogel matrix to the molded polymer substrate, thereby
adhering the hydrogel matrix to the molded polymer substrate.
[0004] In some aspects, the hydrogel matrix further comprises a
protein-reporter group, which in some aspects comprises a glucose
binding protein (GBP). In certain aspects, the reporter group
comprises a fluorescent dye.
[0005] In further aspects, the presently disclosed subject matter
provides a biosensor comprising a hydrogel matrix covalently bound
to a molded polymer substrate, wherein the hydrogel matrix further
comprises a protein-reporter group. In certain aspects, the protein
comprises a glucose binding protein (GBP) and the reporter group
comprises a fluorescent dye. In particular aspects, the biosensor
is a glucose sensor that is used to determine the presence or
amount of glucose in a biological sample.
[0006] Certain aspects of the presently disclosed subject matter
having been stated hereinabove, which are addressed in whole or in
part by the presently disclosed subject matter, other aspects will
become evident as the description proceeds when taken in connection
with the accompanying Examples and Figures as best described herein
below.
BRIEF DESCRIPTION OF THE FIGURES
[0007] Having thus described the presently disclosed subject matter
in general terms, reference will now be made to the accompanying
Figures, which are not necessarily drawn to scale, and wherein:
[0008] FIG. 1 shows (left panel) a representative substrate
(polycarbonate) and (right panel) matrix after dry process; and
[0009] FIG. 2 shows (left panel) a representative substrate
(polystyrene) and (right panel) matrix after dry process.
DETAILED DESCRIPTION
[0010] The presently disclosed subject matter now will be described
more fully hereinafter with reference to the accompanying Figures,
in which some, but not all embodiments of the inventions are shown.
Like numbers refer to like elements throughout. The presently
disclosed subject matter 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. Indeed, many
modifications and other embodiments of the presently disclosed
subject matter set forth herein will come to mind to one skilled in
the art to which the presently disclosed subject matter pertains
having the benefit of the teachings presented in the foregoing
descriptions and the associated Figures. Therefore, it is to be
understood that the presently disclosed subject matter is 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.
I. Hydrogel Adhesion to a Molded Polymer Substrate
[0011] The presently disclosed subject matter provides methods for
adhering a detection matrix, e.g., a hydrogel matrix, to a moldable
polymer substrate. In methods known in the art, physical entrapment
is used to immobilize the detection matrix onto the polymer
substrate. In such systems, the molded substrate physically holds
the detection matrix in place. The detection matrix, however, has a
tendency to slide or move away from its original position on the
substrate in such systems. Under these circumstances, analytical
testing is not feasible, especially in dense or colored liquids. In
such cases, the liquid under test can infiltrate between the
substrate and the matrix, potentially interfering with optical
measurements.
[0012] Generally, the presently disclosed methods for adhering a
hydrogel matrix to a molded substrate include two stages. The first
stage includes molding a polymer mix with an oxidizer to form a
molded polymer substrate. The oxidizer breaks the polymer chains,
which can recombine while retaining some putative radicals in the
molded part. The second stage includes polymerizing hydrogels with
acrylate groups or other materials that can form radicals upon
polymerization in the molded substrate.
[0013] When the material prepared in the first stage, i.e., the
molded polymer substrate, is combined with the material prepared in
the second stage, i.e., the polymerized hydrogel, and cured, e.g.,
under ultraviolet (UV) light or heat, the hydrogel binds covalently
to the molded polymer substrate, thereby promoting adhesion of the
hydrogel to the substrate.
[0014] Accordingly, the final product, i.e., a molded substrate
having a hydrogel matrix covalently bound thereto, can undergo
various chemical and physical processes without the bound hydrogel
moving away from its original position on the substrate. Thus, the
presently disclosed methods and materials facilitate the
development of new processes and combinations of materials.
[0015] Another feature of the presently disclosed system is that
other methods of binding materials to a plastic substrate, such as
with plasma treatment, are not as efficient. As provided herein
below, hydrogels provide binding to the substrate with plasma
treatment, but with time, the hydrogel often separates from the
substrate. In contrast, the presently disclosed subject matter
provides a covalent attachment of the hydrogel matrix to the
substrate. Such features make the presently disclosed methods and
materials attractive for storage stability.
[0016] Accordingly, in some embodiments, the presently disclosed
subject matter provides a method for adhering a hydrogel matrix to
a molded polymer substrate, the method comprising: (a) molding a
polymer comprising one or more polymer chains with an oxidizer to
form a molded polymer substrate, wherein the oxidizer breaks the
one or more polymer chains, and wherein the one or more polymer
chains can recombine while retaining one or more putative radicals
in the molded polymer substrate; (b) providing a hydrogel matrix
comprising a hydrogel, a component comprising one or more acrylate
groups or another functional group that can form one or more
radicals upon polymerization in the molded polymer substrate, and a
photoinitiator; (c) combining the molded polymer substrate and the
hydrogel matrix; and (d) curing the combined molded polymer
substrate and hydrogel matrix for a period of time to covalently
bind the hydrogel matrix to the molded polymer substrate, thereby
adhering the hydrogel matrix to the molded polymer substrate.
[0017] Generally, a "hydrogel" is a three-dimensional network of
crosslinked hydrophilic polymers that are typically insoluble or
poorly soluble in water, but can swell to an equilibrium size when
dispersed in excess water. Hydrogel compositions can include,
without limitation, for example, poly(ethylene glycol),
poly(ethylene oxide), poly(vinyl alcohol), polyvinylpyrrolidone,
polyacrylates, such as poly(hydroxyethyl methacrylate),
poly(esters), poly(hydroxy acids), poly(lactones), poly(amides),
such as poly(acrylamide), poly(ester-amides), poly(amino acids),
poly(anhydrides), poly(ortho-esters), poly(carbonates),
poly(phosphazines), poly(thioesters), polysaccharides, sol gels and
or polymers generated out of siloxanes, and tetraalkylammonium, or
other polymers or copolymers having an abundance of hydrophilic
groups, and mixtures thereof.
[0018] Hydrogels also can include, for example, a poly(hydroxy)
acid, including poly(alpha-hydroxy) acids and poly(beta-hydroxy)
acids. Such poly(hydroxy) acids include, for example, polylactic
acid, polyglycolic acid, polycaproic acid, polybutyric acid,
polyvaleric acid, and copolymers and mixtures thereof. One of
ordinary skill in the art would recognize that the constituents
making up the hydrogel can be modified to change the hydrophobicity
and charge of the hydrogel if so desired.
[0019] Moldable polymers suitable for use with the presently
disclosed methods include, but are not limited to, polycarbonates,
polystyrenes, polyethylenes, copolyesters, such as polyethylene
terephthalate (PET), and polypropylenes.
[0020] The oxidizer can be a peroxide, including, but not limited
to t-butyl phenyl peroxide, lauroyl peroxide, and dicumyl peroxide.
In particular embodiments, the perioxide is t-butyl phenyl
peroxide. The raw polymer can be mixed with a weight percent of
peroxide ranging from about 0.1% to about 0.4%. In particular
embodiments, the weight percent of peroxide is about 0.2%.
[0021] In some embodiments, the photoinitiator is selected from the
group consisting of 2-hydroxy-2-methylpropiophenone,
2,2-dimethyl-2-phenylacetophenone, p-(octyloxyphenyl)phenyliodonium
hexafluoroantimonate, bis-acyl-phosphine oxide (BAPO) in water, and
a liquid mixture of an oligomeric .alpha.-hydroxyketone (oligomeric
2-hydroxy-2-methyl-1,4-(1-methylvinyl)-phenylpropanone) and
2-hydroxy-2-methyl-1-phenyl-1-propanone. In particular embodiments,
the photoinitiator is 2-hydroxy-2-methylpropiophenone.
[0022] The presently disclosed methods provide for analytical or
diagnostic testing on such hydrogels bound to a moldable polymer
substrate, e.g., a plastic. The materials prepared by the presently
disclosed methods can be incorporated into well plates, individual
laminate sensors, or any other format suitable for episodic or
continuous testing. Further, because the hydrogel can bind an
interrogating analyte, an analytical sensor can be made from this
platform. Such sensors can be tested with water, buffers, or any
biological fluids.
[0023] The presently disclosed biosensors can be used in
combination with binding protein assays to detect physiologically
important molecules, including metabolites, such as glucose, fatty
acids, and lactates, in biological samples.
[0024] Of particular interest is testing of viscous and/or
non-transparent samples, because the test article can be poured on
top of the hydrogel and it will not interfere with the analytical
interrogation of the analyte. Systems known in the art only allow
for the use of clear liquids that did not interfere with the
optical path of the sensing unit. With the matrix bound firmly to
the molded substrate, the presently disclosed methods and materials
allow the use of any kind of dense or colored liquid without
interference with optical measurements.
[0025] In some embodiments, the hydrogel can incorporate a
protein-reporter group, e.g., a glucose binding protein labeled
with a fluorescent dye, which can function as a biosensor. In such
embodiments, the binding domains of the protein-reporter group are
either physically entrapped in and surrounded by the hydrogel or
the domains are covalently bound to and surrounded by the hydrogel.
One of ordinary skill in the art would recognize that any protein
can be included in the hydrogel matrix. In representative
embodiments, the protein can be a horseradish peroxidase or an
antibody.
[0026] In particular embodiments, the protein comprises a glucose
binding protein (GBP). In some embodiments, the GBP is from E.
coli. Such GBPs also are referred to as a D-galactose/D-glucose
binding protein (GGBP). One of ordinary skill in the art would
recognize that various GBPs can be used in the presently disclosed
methods. For example, thermophilic GBPs, such as tmGBP from
Thermotoga maritime, are even more stable at higher temperatures
than the E. coli GBP using the methods of the presently disclosed
subject matter.
[0027] More particularly, the term "glucose/galactose binding
protein" or "GGBP" as used herein refers to a type of protein
naturally found in the periplasmic compartment of bacteria. These
periplasmic proteins are naturally involved in chemotaxis and
transport of small molecules (e.g., sugars, amino acids, and small
peptides) into the cytoplasm. For example, GGBP is a single chain
protein consisting of two globular domains that are connected by
three strands to form a hinge. The binding site is located in the
cleft between the two domains. When glucose enters the binding
site, GGBP undergoes a conformational change, centered at the
hinge, which brings the two domains together and entraps glucose in
the binding site. The wild type E. coli GGBP DNA and amino acid
sequence can be found at www.ncbi.nlm.nih.gov/entrez/accession
number D90885 (genomic clone) and accession number 23052 (amino
acid sequence).
[0028] As used herein, a "derivative of a protein" is a protein
that shares substantial sequence identity with the wild-type
protein. Derivative proteins of the presently disclosed subject
matter can be made or prepared by techniques well known to those of
skill in the art. Examples of such techniques include, but are not
limited to, mutagenesis and direct synthesis. Derivative proteins
also can be modified, either by natural processes, such as
post-translational processing, or by chemical modification
techniques which are well known in the art. Such modifications are
well described in basic texts and in more detailed monographs, as
well as in voluminous research literature. Modifications can occur
anywhere in the polypeptide chain, including the peptide backbone,
the amino acid side-chains and the amino or carboxyl termini. It
will be appreciated that the same type of modification can be
present in the same or varying degrees at several sites in a given
polypeptide or protein. Also, a given polypeptide or protein can
contain more than one modification. Examples of modifications
include, but are not limited to, glycosylation, acetylation,
acylation, ADP-ribosylation, amidation, covalent attachment of
flavin, covalent attachment of a heme moiety, covalent attachment
of a nucleotide or nucleotide derivative, covalent attachment of a
lipid or lipid derivative, covalent attachment of
phosphotidylinositol, cross-linking, cyclization, disulfide bond
formation, demethylation, formation of covalent cross-links,
formation of cystine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins such as arginylation, and ubiquitination.
Polypeptides or proteins can even be branched as a result of
ubiquitination, and they can be cyclic, with or without branching.
(See, e.g., T E. Creighton, Proteins--Structure And Molecular
Properties, 2nd ed., W.H. Freeman and Company, New York (1993);
Wold, F., "Posttranslational Protein Modifications: Perspectives
and Prospects," in Posttranslational Covalent Modification Of
Proteins, B. C. Johnson, Ed., Academic Press, New York (1983);
Seifter et al., Methods in Enzymol, 182:626-646 (1990) and Rattan
et al., Ann NY Acad Sci., 663:48-62 (1992), each of which is
incorporated herein by reference. Examples of derivative proteins
include, but are not limited to, mutant and fusion proteins.
[0029] A "mutant protein" is used herein as it is known in the art.
In general, a mutant protein can be created by addition, deletion
or substitution of the wild-type primary structure of the protein
or polypeptide. Mutations include, for example, the addition or
substitution of cysteine groups, non-naturally occurring amino
acids, and replacement of substantially non-reactive amino acids
with reactive amino acids. A mutant protein can be mutated to bind
more than one analyte in a specific manner. Indeed, the mutant
proteins can possess specificity towards its wild-type analyte and
another target ligand. Likewise, a mutant protein can be able to
only bind an analyte or analytes that the wild-type binding protein
does not bind. Methods of generating mutant proteins are well-known
in the art. For example, Looger, L. L. et al., Nature 423 (6936):
185-190 (2003), which is incorporated herein by reference, disclose
methods for redesigning binding sites within periplasmic binding
proteins that provide new analyte-binding properties for the
proteins. These mutant binding proteins retain the ability to
undergo conformational change, which can produce a directly
generated signal upon analyte-binding. By introducing between 5 and
17 amino acid changes, Looger, et al. constructed several mutant
proteins, each with new selectivities for TNT (trinitrotoluene),
L-lactate, or serotonin. For example, Looger et al. generated
L-lactate binding proteins from GGBP. Other mutations to GGBP are
found in Looger L. L. et al., Nature 423: 185-190, (2003), which is
incorporated herein by reference in its entirety.
[0030] Examples of mutations of a GGBP protein, for example the
GGBP protein of GenBank Accession No. P02927 without the 23 amino
acid leader sequence (i.e., the mature chain), include, but are not
limited to, having a cysteine substituted for lysine at position 11
(K11C), a cysteine substituted for aspartic acid at position 14
(D14C), a cysteine substituted for valine at position 19 (V19C), a
cysteine substituted for asparagine at position 43 (N43C), a
cysteine substituted for glycine at position 74 (G74C), a cysteine
substituted for tyrosine at position 107 (Y107C), a cysteine
substituted for threonine at position 110 (T110C), a cysteine
substituted for serine at position 112 (S112C), a double mutant
including a cysteine substituted for serine at position 112 and
serine substituted for leucine at position 238 (S112C/L238S), a
cysteine substituted for lysine at position 113 (K113C), a cysteine
substituted for lysine at position 137 (K137C), a cysteine
substituted for glutamic acid at position 149 (E149C), a double
mutant including a cysteine substituted for glutamic acid at
position 149 and an arginine substituted for alanine at position
213 (E149C/A213R), a double mutant including a cysteine substituted
for glutamic acid at position 149 and a serine substituted for
leucine at position 238 (E149C/L238S), a double mutant including a
serine substituted for alanine at position 213 and a cysteine
substituted for histidine at position 152 (H152C/A213S), a cysteine
substituted for methionine at position 182 (M182C), a cysteine
substituted for tryptophan at position 183 (W183C), a cysteine
substituted for alanine at position 213 (A213C), a double mutant
including a cysteine substituted for alanine at position 213 and a
cysteine substituted for leucine at position 238 (A213C/L238C), a
cysteine substituted for methionine at position 216 (M216C), a
cysteine substituted for aspartic acid at position 236 (D236C), a
cysteine substituted for leucine at position 238 (L238C) a cysteine
substituted for aspartic acid at position 287 (D287C), a cysteine
substituted for arginine at position 292 (R292C), a cysteine
substituted for valine at position 296 (V296C), a triple mutant
including a cysteine substituted for glutamic acid at position 149
and a serine substituted for alanine at position 213 and a serine
substituted for leucine at position 238 (E149C/A213S/L238S), a
triple mutant including a cysteine substituted for glutamic acid at
position 149 and an arginine substituted for alanine at position
213 and a serine substituted for leucine at position 238
(E149C/A213R/L238S), a cysteine substituted for glutamic acid at
position 149 and a cysteine substituted for alanine at position 213
and a serine substituted for leucine at position 238
(E149C/A213C/L238S). Another example includes a mutated GGBP having
the following substitutions: N391, G82E, Q83K, N84D, Q175E, Q177H,
L178M, W183C, N259E and N260S (referred to as "SM4"). Additional
embodiments include mutations of GGBP at Y10C, N15C, Q26C, E93C,
H152C, M182C, W183C, L255C, D257C, P294C, and V296C. Other mutated
GGBPs are disclosed in U.S. Patent Publication No. 2008-0044856,
which is incorporated herein by reference in its entirety.
[0031] The mutation can serve one or more of several purposes. For
example, a naturally occurring protein can be mutated to change the
long-term stability, including thermal stability, of the protein,
to conjugate the protein to a particular encapsulation matrix or
polymer, to provide binding sites for detectable reporter groups,
to adjust its binding constant with respect to a particular
analyte, or combinations thereof.
[0032] The analyte and mutated protein can act as binding partners.
The term "associates" or "binds" as used herein refers to binding
partners having a relative binding constant (Kd) sufficiently
strong to allow detection of binding to the protein by a detection
means. The Kd can be calculated as the concentration of free
analyte at which half the protein is bound, or vice versa. When the
analyte of interest is glucose, the Kd values for the binding
partners are between about 0.0001 mM and about 50 mM. Mutations of
binding proteins are described in U.S. Pat. No. 7,064,103 to Pitner
et al., issued Jun. 20, 2006, U.S. Pat. No. 6,855,556 to Amiss et
al., issued Feb. 15, 2005, and U.S. Patent Application Publication
No. 2006/0280652, filed May 18, 2005, each of which is incorporated
by reference in its entirety.
[0033] In addition to changing binding characteristics, derivative
proteins also can be used to incorporate a fluorophore, e.g., a
fluorescent dye, onto or within the binding member.
[0034] As used herein, the term "fluorophore" is meant to include a
moiety of a larger molecule or conjugate that can be induced to
emit fluorescence when irradiated, i.e., excited, by
electromagnetic radiation of an appropriate wavelength. More
particularly, a fluorophore can be a functional group of a molecule
or conjugate that absorbs light of a certain wavelength and emits
light at different wavelength. The intensity and the wavelength of
the light emitted, as well as other fluorescence properties
including, but not limited to, fluorescence lifetime, anisotropy,
polarization, and combinations thereof, depend on the identity of
the fluorophore and its chemical environment. A fluorophore can
include a fluorescent molecule, such as the presently disclosed
fluorescent dyes.
[0035] Representative fluorophores suitable for use with the
presently disclosed subject matter include those disclosed in U.S.
Patent Application Publication No. 2011/0184168, for Long
Wavelength Thiol-Reactive Fluorophores, to J. B. Pitner, et al.,
published Jul. 28, 2011; U.S. Patent Application Publication No.
2010/0167417, for Long Wavelength Thiol-Reactive Fluorophores, to
J. B. Pitner, et al., published Jul. 1, 2010; U.S. Patent
Application Publication No. 2010/0003763, for Long Wavelength
Thiol-Reactive Fluorophores, to J. B. Pitner, et al., published
Jan. 7, 2010; U.S. Patent Application Publication No. 2008/0311675,
for Dyes Having Ratiometric Fluorescence Response for Detecting
Metabolites, to J. Thomas, et al., published Dec. 18, 2008; U.S.
Patent Application Publication No. 2006/0280652, for Long
Wavelength Thiol-Reactive Fluorophores, to J. B. Pitner, et al.,
published Dec. 14, 2006; U.S. Pat. No. 8,129,525, for Long
Wavelength Thiol-Reactive Fluorophores, to J. B. Pitner, et al.,
issued Mar. 6, 2012; U.S. Pat. No. 8,071,794, for Long Wavelength
Thiol-Reactive Fluorophores, to J. B. Pitner, et al., issued Dec.
6, 2011; U.S. Pat. No. 7,767,821, for Long Wavelength
Thiol-Reactive Fluorophores, to J. B. Pitner, et al., issued Aug.
3, 2010; and U.S. Pat. No. 7,563,891, for Long Wavelength
Thiol-Reactive Fluorophores, to J. B. Pitner, et al., issued Jul.
21, 2009, each of which is incorporated herein by reference in its
entirety.
[0036] The fluorophores can be used to indicate a change in the
binding member, including, but not limited to, three-dimensional
conformational changes, changes in orientation of the amino acid
side chains of proteinaceous binding domains, and redox states of
the binding member. With the addition/substitution of one or more
residues into the primary structure of a protein, some of the
labeling moieties used in the current methods and compositions can
be attached through chemical means, such as reduction, oxidation,
conjugation, and condensation reactions. Examples of residues
commonly used to label proteins include, but not are limited to,
lysine and cysteine. For example, any thiol-reactive group can be
used to attach a fluorophore to a naturally occurring or engineered
cysteine in the primary structure of the polypeptide. Also, for
example, lysine residues can be labeled using succinimide ester
derivatives of fluorophores. See Richieri, G. V. et al., J. Biol.
Chem., 267: 23495-501 (1992), which is incorporated herein by
reference. The fluorophore can be attached at a site on the binding
protein so that the conformational change maximizes the change in
fluorescence properties.
[0037] The fluorophore can be attached to the mutated protein, for
example a GGBP, by any conventional means known in the art. For
example, the reporter group can be attached via amines or carboxyl
residues on the protein. Other embodiments include covalent
coupling via thiol groups on cysteine residues of the mutated or
native protein. For example, for mutated GGBP, cysteines can be
located at position 10, at position 11, position 14, at position
15, position 19, at position 26, at position 43, at position 74, at
position 92, at position 93, position 107, position 110, position
112, at position 113, at position 137, at position 149, at position
152, at position 154, at position 182, at position 183, at position
186, at position 211, at position 213, at position 216, at position
238, at position 240, at position 242, at position 255, at position
257, at position 287, at position 292, at position 294, and at
position 296.
[0038] As used herein, the term "binding protein" or "binding
member" refers to a protein, that when conjugated with a
fluorophore, interacts with a specific analyte or ligand in a
manner capable of producing a detectable florescence signal
differentiable from when a target analyte or ligand is present or
absent, or when a target analyte or ligand is present in varying
concentrations over time. The term "producing a detectable signal"
refers to the ability to recognize a change in a property of a
reporter group, e.g., a fluorophore, in a manner that enables the
detection of ligand-protein binding. Further, the producing of a
detectable signal can be reversible or non-reversible. The
signal-producing event includes continuous, programmed, and
episodic means, including one-time or reusable applications. The
reversible signal-producing event can be instantaneous or can be
time-dependent, so long as a correlation with the presence or
concentration of analyte is established.
[0039] The binding member may be at least one fragment of a
receptor. As used herein, the term "receptor" refers to a protein
macromolecule having an active site capable of binding a ligand.
More particularly, a "receptor" refers to an antibody, a hormone or
bacterial receptor, an affinity protein, a transport protein, a
viral receptor, or any polypeptide having a specific affinity for a
ligand.
[0040] As used herein, the term "ligand" refers to any molecule
capable of binding to the receptor via an active site. For example,
the ligand may be a protein, a peptide or hapten antigen, such as a
bacterial antigen, or a hormone, a cytokine, an interleukin, a
tumor necrosis factor (TNF) a growth factor, a viral protein, or a
peptide or nucleotide sequence.
[0041] As used herein, the term "active site" when referring to,
for example, "an active site of the receptor or of the receptor
fragment" refers to amino acid residues of the receptor or receptor
fragment that contribute to the binding of the ligand. This active
site also can be referred to as a "binding site" or "paratope."
[0042] The term "proximity" as used herein refers to the position
of amino acid residues of the receptor that are in direct contact
with the ligand, those that are in contact by water molecules, and
those for which the solvent accessible surface area (ASA), as that
term in known in the art, see, e.g., Creighton, T. E., "Proteins:
Structure and molecular properties," 2nd ed., (W.H. Freeman &
Co., New York) 227-229 (1993), is modified by the binding of the
ligand.
[0043] The change in the detectable characteristics of GBP can be
used in a biosensor. As used herein, the term "biosensor" and
"biosensor compound" refers to a compound that undergoes a
detectable change in specific response to a ligand or target
analyte. Without wishing to be bound to any one particular theory,
the binding protein comprising the biosensor can adopt two
conformations: a ligand-free open form and a closed form when bound
to a ligand. These two conformations can interconvert, for example,
via a global hinge-binding mechanism upon ligand binding or changes
in ligand concentration. By positioning environmentally-sensitive
dyes in locations that undergo local conformational changes in
concert with these global conformational changes, such
ligand-mediated conformational changes can be exploited to couple
ligand binding to a color change. Accordingly, these engineered
conformational coupling mechanisms enable reagentless optical
biosensors to be formed from selected binding proteins and
environmentally-sensitive dyes.
[0044] Fluorescent dyes in biosensors exhibit a change in intensity
of the fluorescence signal, a shift in the emission wavelength of
the maximum fluorescence emission, a change in fluorescence
lifetime, a change in anisotropy, a change in polarization, or a
combination thereof, when the binding protein undergoes a
conformational change as a result of changes in the glucose
concentration. An energy source, such as a laser or LED, is applied
to the biosensor to excite the fluorescent dye, and a fluorescence
property is detected. Due to either a conformational change in the
binding protein, subsequent changes in the microenvironment of the
dye, or both, the detected fluorescence property or change of the
detected fluorescence property can be correlated to the presence of
an analyte or a analyte concentration. The fluorescence and
detection can be carried out continuously or intermittently at
predetermined times. Thus, a biosensor can have episodic or
continuous sensing of analyte, for example, glucose.
[0045] The fluorescent dye can be covalently attached to the
binding protein in a site-specific manner to obtain the desired
change in the fluorescence. The fluorescent dye is attached at a
site on the binding protein so that the conformational change
maximizes the change in fluorescence properties. In other
embodiments, the fluorescent dyes may have a thiol-reactive group
that can be coupled to the thiol group on a cysteine residue of the
binding protein. Fluorescent dyes include, but are not limited to,
derivatives of the squaraine nuclei, benzodioxazole nuclei, Nile
Red nuclei, coumarin nuclei and aza coumarin nuclei. Fluorophores
that operate at long emission wavelengths (for example, about 575
nm or greater) are used when the molecular sensor is to be used in
vivo, for example, incorporated into an implantable biosensor
device (the skin being opaque below about 575 nm).
[0046] To accurately determine glucose concentration in biological
solutions, such as blood, interstitial fluids, occular solutions or
perspiration, and the like, it may be desirable to adjust the
binding constant of the sensing molecule of a biosensor so as to
match the physiological and/or pathological operating range of the
biological solution of interest. Without the appropriate binding
constant, a signal may be out of range for a particular
physiological and/or pathological concentration. Additionally,
biosensors may be configured using more than one protein, each with
a different binding constant, to provide accurate measurements over
a wide range of glucose concentrations as disclosed by Lakowicz
(U.S. Pat. No. 6,197,534).
[0047] Embodiments that exhibit a shift in emission wavelength upon
ligand binding enable a biosensor comprising a fluorophore to be
self-referencing. In such embodiments, the fluorophore exhibits an
increase in a first emission wavelength in the presence of a
metabolite, such as glucose, and a decrease in a second emission
wavelength. A ratio between the first emission wavelength and the
second wavelength can be calculated to determine the concentration
of glucose in the sample under test. Such self referencing can
correct for variations in excitation source intensity and other
sources of noise and instability in the biosensor without requiring
a reference dye. Thus, a single excitation wavelength can be used
to observe a ratiometric response in the fluorescence output of the
biosensor. As used herein, the term "ratiometric response" means
that the intensities of the first emission wavelength and the
second emission wavelength vary independently such that the ratio
of the two emission wavelengths (the "ratiometric quotient" or
"QR") can be used to indicate the presence and/or amount, e.g.,
concentration, of the ligand or analyte in a sample. See U.S.
Patent Application Publication No. 2009/0104714, for Visual Glucose
Sensor and Methods of Use Thereof, to J. Thomas, et al., published
Apr. 23, 2009, which is incorporated herein by reference in its
entirety. Thus, in some embodiments, the biosensor is a glucose
sensor that is used to determine the presence or amount of glucose
in a biological sample. A glucose sensor can be used in vitro or in
vivo to follow the kinetics of biological reactions involving
glucose, as well as to monitor the amount of glucose in a sample.
The glucose sensors of the presently disclosed subject matter are
capable of measuring or detecting micromolar to molar glucose
concentrations without reagent consumption.
[0048] As used herein, the term "biological sample" includes any
liquid or fluid sample, including a sample derived from a
biological source, such as a physiological fluid, including whole
blood or whole blood components, such as red blood cells, white
blood cells, platelets, serum and plasma; ascites; urine; saliva;
sweat; milk; synovial fluid; peritoneal fluid; amniotic fluid;
percerebrospinal fluid; lymph fluid; lung embolism; cerebrospinal
fluid; pericardial fluid; cervicovaginal samples; tissue extracts;
cell extracts; and other constituents of the body that are
suspected of containing the analyte of interest. In addition to
physiological fluids, other liquid samples, such as water, food
products and the like, for the performance of environmental or food
production assays are suitable for use with the presently disclosed
subject matter. A solid material suspected of containing the
analyte also can be used as the test sample. In some instances it
might be beneficial to modify a solid test sample to form a liquid
medium or to release the analyte.
[0049] The presently disclosed biosensor devices are suitable for
use in various settings, including in vivo, in vitro and in situ.
Such devices include medical devices or implants for monitoring
metabolic substrate levels in a subject. When such devices are
implanted in a subject, the implants should be biocompatible such
that they produce little or no detectable inflammation/rejection
reaction. One approach for rendering the implants more
biocompatible comprises coating the implants with biocompatible
polymers, such as poly(urethane) elastomers, poly(urea) and
poly(vinylchloride). Exemplary biosensor devices are described in
U.S. Patent Application Publication No. 2006/0078908, filed Jun. 7,
2005, and U.S. Patent Application Publication No. 2005/0113657,
each of which is incorporated herein by reference in its entirety.
The presently disclosed biosensors can be used or adapted for use
in strips, implants, micro- and nano-particles, and the like.
[0050] Although specific terms are employed herein, they are used
in a generic and descriptive sense only and not for purposes of
limitation. Unless otherwise defined, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this presently described
subject matter belongs. While the following terms in relation to
the methods of the presently disclosed subject matter are believed
to be well understood by one of ordinary skill in the art, the
following definitions are set forth to facilitate explanation of
the presently disclosed subject matter. These definitions are
intended to supplement and illustrate, not preclude, the
definitions that would be apparent to one of ordinary skill in the
art upon review of the present disclosure.
[0051] Following long-standing patent law convention, the terms
"a," "an," and "the" refer to "one or more" when used in this
application, including the claims. Thus, for example, reference to
"a molecule" includes a plurality of molecules, unless the context
clearly is to the contrary (e.g., a plurality of molecules), and so
forth.
[0052] Throughout this specification and the claims, the terms
"comprise," "comprises," and "comprising" are used in a
non-exclusive sense, except where the context requires otherwise.
Likewise, the term "include" and its grammatical variants are
intended to be non-limiting, such that recitation of items in a
list is not to the exclusion of other like items that can be
substituted or added to the listed items.
[0053] For the purposes of this specification and appended claims,
unless otherwise indicated, all numbers expressing amounts, sizes,
dimensions, proportions, shapes, formulations, parameters,
percentages, parameters, quantities, characteristics, and other
numerical values used in the specification and claims, are to be
understood as being modified in all instances by the term "about"
even though the term "about" may not expressly appear with the
value, amount or range. Accordingly, unless indicated to the
contrary, the numerical parameters set forth in the following
specification and attached claims are not and need not be exact,
but may be approximate and/or larger or smaller as desired,
reflecting tolerances, conversion factors, rounding off,
measurement error and the like, and other factors known to those of
skill in the art depending on the desired properties sought to be
obtained by the presently disclosed subject matter. For example,
the term "about," when referring to a value can be meant to
encompass variations of, in some embodiments, .+-.100% in some
embodiments .+-.50%, in some embodiments .+-.20%, in some
embodiments .+-.10%, in some embodiments .+-.5%, in some
embodiments .+-.1%, in some embodiments .+-.0.5%, and in some
embodiments .+-.0.1% from the specified amount, as such variations
are appropriate to perform the disclosed methods or employ the
disclosed compositions.
[0054] Further, the term "about" when used in connection with one
or more numbers or numerical ranges, should be understood to refer
to all such numbers, including all numbers in a range and modifies
that range by extending the boundaries above and below the
numerical values set forth. The recitation of numerical ranges by
endpoints includes all numbers, e.g., whole integers, including
fractions thereof, subsumed within that range (for example, the
recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as
fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and
any range within that range.
EXAMPLES
[0055] The following Examples have been included to provide
guidance to one of ordinary skill in the art for practicing
representative embodiments of the presently disclosed subject
matter. In light of the present disclosure and the general level of
skill in the art, those of skill can appreciate that the following
Examples are intended to be exemplary only and that numerous
changes, modifications, and alterations can be employed without
departing from the scope of the presently disclosed subject matter.
The synthetic descriptions and specific examples that follow are
only intended for the purposes of illustration, and are not to be
construed as limiting in any manner to make compounds of the
disclosure by other methods.
Example 1
Substrate Molding
[0056] Representative results were obtained with a polycarbonate
(LEXAN.RTM., HF 1110, SABIC Innovative Plastics, Pittsfield, Mass.)
substrate, which offered high flow and low density. The polymer was
dried at 250.degree. F. for 4 hours before use. The raw polymer was
mixed 0.2% by weight with t-butyl phenyl peroxide (Aldrich).
Lauroyl peroxide (Aldrich Product # 290785) and dicumyl peroxide
(Fluka Product # 36590) also are suitable for use with the
presently disclosed methods. Benzoyl peroxide (Fluka Product #
33581), however, did not produce satisfactory results.
[0057] The weight percent of peroxide could range from about 0.1%
to about 0.4%. The polymer was allowed to cool before mixing,
because the peroxide could react with the hot polymer. Once mixed,
the polymer was fed to the molding machine, purged, and parts were
molded as rapidly as possible. Cycle times longer than one minute
produced defective parts. The molding machine (Ferromatik Milacron
K100, Braunform GmbH, Bahlingen, Germany) conditions for a
polycarbonate substrate are provided in Table 1. The best parts
were 1.5 mm or less in thickness as determined by minimum
autofluorescence.
TABLE-US-00001 TABLE 1 Molding Machine Conditions for Polycarbonate
Substrate Barrel Barrel Barrel Mold Injection Injection Back Nozzle
Front Barrel 1 Barrel 2 Rear Temp Press Speed Press 520.degree. F.
560.degree. F. 560.degree. F. 540.degree. F. 480.degree. F.
210.degree. F. 2400 bar 400 mm/sec 80 psi
[0058] A polystyrene (e.g., BASF, PS145D) substrate also can
provide binding to the matrix (see FIG. 2) when treated by the
presently disclosed methods. The molding machine conditions for a
polycarbonate substrate are provided in Table 2.
TABLE-US-00002 TABLE 2 Molding Machine Conditions for Polystyene
Substrate Barrel Barrel Barrel Mold Injection Injection Back Nozzle
Front Barrel 1 Barrel 2 Rear Temp Press Speed Press 440.degree. F.
490.degree. F. 490.degree. F. 440.degree. F. 380.degree. F.
170.degree. F. 2400 bar 400 mm/sec 80 psi
[0059] Other polymers suitable for use with the presently disclosed
methods include, but are not limited to the copolyester
polyethylene terephthalate (PET) (DuraStar.TM. DS1010, Eastman
Chemical Co., Kingsport, Tenn.) and polypropylene (PH592,
LyondellBasell, Rotterdam, Netherlands)
Example 2
Matrix Preparation
[0060] Materials. The following materials were used as received
unless indicated otherwise: polyethylene glycol 1000 dimethacrylate
(PEGDMA 1000) (Degussa Product # 6874-0);
2-hydroxy-2-methylpropiophenone photoinitiator (PI) (Aldrich
Product #405655); 6-arm polyethylene glycol (PEG) with acrylate
terminations, molecular weight (MW) 10,000/arm (Biolink Product #
BLS-018-083); Sorbitol (Aldrich Product # S1876); phosphate buffer
(PBS); glucose binding protein (GBP) GBP=3MNBD solution (1 mg/mL)
(Paragon Bioservices, Baltimore, Md.); and UV light, 200-500 Watts
Hg Arc lamp (Oriel Instruments, Stratford, Conn.).
[0061] Other photoinitiators suitable for use with the presently
disclosed methods include 2-2-Dimethyl-2 phenyl acetophenone
(Aldrich Product # A6118); p-(octyloxyphenyl)phenyliodonium
hexafluoroantimonate, Gelest, Inc., Morrisville Pa.);
bis-acyl-phosphine oxide (BAPO) in water (Irgacure.RTM. 819DW, Ciba
(BASF)); and a liquid mixture of an oligomeric
.alpha.-hydroxyketone (oligomeric
2-hydroxy-2-methyl-1,4-(1-methylvinyl)-phenylpropanone) and
2-hydroxy-2-methyl-1-phenyl-1-propanone (SarCure.TM. SR1129,
Sartomer USA, LLC, Exton, Pa.). Neither a blend of
2,4,6-trimethylbenzoyldiphenyl phosphine oxide,
2,4,6-trimethylbenzophenone, 4-methylbenzophenone and oligo
(2-hydroxy-2-methyl-1-(4-(-methylvinyl)phenyl) propanone)
(SarCure.TM. SR1135 (Sartomer)) nor anthraquinone 2 sulfonic Na
salt monohydrate (Aldrich) produced satisfactory results.
[0062] Preparation of Hydrogel Containing Protein-Reporter Group.
In a glass container, 200 .mu.L of PEGDMA1000 MW, 2.5 .mu.L of
6-arm PEG with acrylate terminations, 1 .mu.L of photo initiator,
and 8.25 mg of sorbitol were mixed with 450 .mu.L of PBS. The
mixture was vortexed for 15 seconds and then 150 .mu.L of GBP-3MNBD
protein-reporter group (2 mg/dL) were added.
[0063] Once the matrix was prepared, it was dispensed (1.2 .mu.L)
into each well of the molded polycarbonate plates and then UV-cured
for 2 min. After curing, the plate-matrix device can be tested wet.
Alternatively, the wet matrix was dispensed into each well of the
molded polycarbonate plates, as above, and then placed in an oven
under vacuum for 15 min to dry the matrix (see FIG. 1). The matrix
stayed in place and several analytical tests could be performed on
the plate-matrix device. Representative results using polystyrene
as the substrate in the dry process are provided in FIG. 2.
REFERENCES
[0064] All publications, patent applications, patents, and other
references mentioned in the specification are indicative of the
level of those skilled in the art to which the presently disclosed
subject matter pertains. All publications, patent applications,
patents, and other references are herein incorporated by reference
to the same extent as if each individual publication, patent
application, patent, and other reference was specifically and
individually indicated to be incorporated by reference. It will be
understood that, although a number of patent applications, patents,
and other references are referred to herein, such reference does
not constitute an admission that any of these documents forms part
of the common general knowledge in the art.
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[0094] Although the foregoing subject matter has been described in
some detail by way of illustration and example for purposes of
clarity of understanding, it will be understood by those skilled in
the art that certain changes and modifications can be practiced
within the scope of the appended claims.
* * * * *
References