U.S. patent application number 11/663040 was filed with the patent office on 2009-06-04 for enhanced stability of proteins immobilized on nanoparticles.
This patent application is currently assigned to RENSSELAER POLYTECHNIC INSTITUTE a University. Invention is credited to Prashanth Asuri, Jonathan S. Dordick, Ravindra S. Kane, Sandeep S. Karajanagi.
Application Number | 20090143487 11/663040 |
Document ID | / |
Family ID | 37595566 |
Filed Date | 2009-06-04 |
United States Patent
Application |
20090143487 |
Kind Code |
A1 |
Dordick; Jonathan S. ; et
al. |
June 4, 2009 |
Enhanced stability of proteins immobilized on nanoparticles
Abstract
This invention is directed to the application of a previously
unknown property of nanomaterials--its ability to enhance protein
activity and stability at high temperatures, in organic solvents,
and in polymer composites. Nanomaterials such as single-walled
carbon nanotubes (SWNTs) can significantly enhance enzyme function
and stability in strongly denaturing environments. Experimental
results and theoretical analysis reveal that the enhancement in
stability is a result of the curvature of these nanoscale
materials, which suppresses unfavorable protein-protein
interactions. The enhanced stability is also exploited in the
preparation of highly stable and active nanocomposite films that
resist nonspecific protein absorption, i.e., inhibit fouling of the
films. The protein-nanoparticles conjugates represent a new
generation of highly selective, active, and stable catalytic
materials. Furthermore, the ability to enhance protein function by
interfacing them with nanomaterials has a profound impact on
applications ranging from biosensing, diagnostics, vaccines, drug
delivery, and biochips, to novel hybrid materials that integrate
biotic and abiotic components.
Inventors: |
Dordick; Jonathan S.;
(Schenectady, NY) ; Kane; Ravindra S.; (Niskayuna,
NY) ; Asuri; Prashanth; (Troy, NY) ;
Karajanagi; Sandeep S.; (Troy, NY) |
Correspondence
Address: |
ELMORE PATENT LAW GROUP, PC
515 Groton Road, Unit 1R
Westford
MA
01886
US
|
Assignee: |
RENSSELAER POLYTECHNIC INSTITUTE a
University
|
Family ID: |
37595566 |
Appl. No.: |
11/663040 |
Filed: |
September 7, 2005 |
PCT Filed: |
September 7, 2005 |
PCT NO: |
PCT/US2005/031652 |
371 Date: |
January 2, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60607816 |
Sep 8, 2004 |
|
|
|
Current U.S.
Class: |
514/773 ; 435/12;
435/4 |
Current CPC
Class: |
G01N 33/54346 20130101;
G01N 33/588 20130101; B82Y 15/00 20130101; G01N 33/587 20130101;
C12Q 1/001 20130101; A61P 43/00 20180101 |
Class at
Publication: |
514/773 ; 435/4;
435/12 |
International
Class: |
A61K 47/42 20060101
A61K047/42; C12Q 1/00 20060101 C12Q001/00; C12Q 1/58 20060101
C12Q001/58; A61P 43/00 20060101 A61P043/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was supported, in whole or in part, by a grant
from the National Science Foundation (DMR-0117792). The Government
has certain rights in the invention.
Claims
1. A composition comprising: (a) nanoparticles; and (b) proteins,
wherein said proteins are bound to said nanoparticles and said
nanoparticles have external surfaces whose radius of curvature is
within 2 orders of magnitude of the dimensions of each of said
proteins bound to said nanoparticles, such that the stability of
said bound proteins is greater than the stability of said proteins
bound to surfaces of the same material as that of said
nanoparticles but which forms a flat surface.
2. The composition of claim 1, wherein said protein stability is
higher than on a flat support in a liquid medium other than an
aqueous medium at neutral pH, an aqueous medium at normal salinity,
or an aqueous medium at a temperature between about 20.degree. C.
and 40.degree. C.
3. The composition of claim 2, wherein said liquid medium is
selected from the group consisting of an aqueous medium at a
temperature greater than about 40.degree. C., less than about
10.degree. C., an aqueous medium whose pH is less than about pH
6.5, an aqueous medium whose pH is greater than about pH 7.5, an
aqueous medium with a salinity of at least about 0.3 M NaCl, a
non-aqueous medium, and combinations thereof.
4. The composition of claim 1, wherein said nanoparticles are
selected from the group consisting of single-walled carbon
nanotubes, multi-walled carbon nanotubes, gold or other metallic
nanoparticles, semi-conducting nanoparticles, metal oxide
nanoparticles, quantum dots, funtionalized silica, and mixtures
thereof.
5. The composition of claim 1, wherein said proteins are bound to
said nanoparticles through hydrophobic bonding, hydrophilic
bonding, ionic bonding, covalent bonding, and non-covalent
bonding.
6. The composition of claim 1, wherein said protein is an
enzyme.
7. An article of manufacture comprising the composition of claim 1
bound to a macroscopic surface.
8. The article of manufacture of claim 7, wherein said macroscopic
surface is selected from the group consisting of a polymer, a
polymeric film, a metal, a metal alloy, and combinations
thereof.
9. The article of manufacture of claim 7, wherein said article is
incorporated in a member of the group consisting of a biosensor, a
biochip, a biofuel cell, a drug delivery system, an antimicrobial
film, a paint antifouling film, and a lubricant antifouling
film.
10. A method of making a device containing a composition which can
enzymatically act on one or more substances in a solution
comprising: (a) bonding one or more enzyme species to
nanoparticles, wherein said nanoparticles have external surfaces
whose radius of curvature is within 2 orders of magnitude of the
dimensions of each said enzyme bound to each said nanoparticle,
such that the activity of said enzymes is greater than the activity
of said enzymes bound to surfaces of the same material as that of
said nanoparticles but which forms a flat surface, thereby forming
said composition; and (b) attaching said composition to a working
surface of said device where said working surface will be in
contact with said solution when the enzyme activity of said enzymes
is desired; thereby forming the device.
11. The method of claim 10, wherein the solvent of said solution is
not an aqueous medium at neutral pH, an aqueous medium at normal
salinity, or an aqueous medium at a temperature between about
20.degree. C. and 40.degree. C. when said working surface is in
contact with said solution.
12. The method of claim 11, wherein said solvent is selected from
the group consisting of an aqueous medium at a temperature greater
than about 40.degree. C., less than about 10.degree. C., an aqueous
medium whose pH is less than about pH 6.5, an aqueous medium whose
pH is greater than about pH 7.5, a liquid hydrocarbon medium, an
aqueous medium with a salinity of at least about 0.3 M NaCl, and
combinations thereof.
13. The method of claim 10, wherein said nanoparticles are selected
from the group consisting of single-walled carbon nanotubes,
multi-walled carbon nanotubes, gold or other metallic
nanoparticles, semi-conducting nanoparticles, metal oxide
nanoparticles, quantum dots, funtionalized silica, and mixtures
thereof.
14. The method of claim 10, wherein said enzymes are bound to said
nanoparticles through hydrophobic bonding, hydrophilic bonding,
ionic bonding, covalent bonding, and non-covalent bonding.
15. A method of detecting an analyte in a solution comprising: (a)
contacting said solution containing said analyte with a composition
comprising (i) nanoparticles, and (ii) enzymes, wherein said
enzymes are bound to said nanoparticles and said nanoparticles have
external surfaces whose radius of curvature is within 2 orders of
magnitude with the dimensions of each said enzyme bound to said
nanoparticles, such that the activity of said bound enzymes is
greater than the activity of said enzymes bound to surfaces of the
same material as that of said nanoparticles but which forms a flat
surface, and further wherein said analyte is a substrate for said
enzymes; (b) allowing said enzymes to enzymatically act on said
analyte, thereby forming a product that is detectable by external
means; and (c) detecting said product by said external means,
thereby detecting said analyte.
16. The method of claim 15, wherein said enzyme activity is
maintained in a liquid medium other than an aqueous medium at
neutral pH, an aqueous medium at normal salinity, or an aqueous
medium at a temperature between about 20.degree. C. and 40.degree.
C. when said working surface is in contact with said solution.
17. The method of claim 16, wherein said liquid medium is selected
from the group consisting of an aqueous medium at a temperature
greater than about 40.degree. C., less than about 10.degree. C., an
aqueous medium whose pH is less than about pH 6.5, an aqueous
medium whose pH is greater than about pH 7.5, a liquid hydrocarbon
medium, an aqueous medium with a salinity of at least about 0.3 M
NaCl, and combinations thereof.
18. The method of claim 15, wherein said nanoparticles are selected
from the group consisting of single-walled carbon nanotubes,
multi-walled carbon nanotubes, gold or other metallic
nanoparticles, semi-conducting nanoparticles, metal oxide
nanoparticles, quantum dots, funtionalized silica, and mixtures
thereof.
19. The method of claim 15, wherein said enzymes are bound to said
nanoparticles through hydrophobic bonding, hydrophilic bonding,
ionic bonding, covalent bonding, and non-covalent bonding.
20. A method of reducing the fouling of a surface by a substance
present in a solution comprising: (a) contacting said solution
containing said substance with said surface wherein a composition
is attached to said surface, said composition comprising (i)
nanoparticles, and (ii) enzymes, wherein said enzymes are bound to
said nanoparticles and said nanoparticles have external surfaces
whose radius of curvature is within 2 orders of magnitude of the
dimensions of each said enzyme bound to said nanoparticles, such
that the stability of said bound enzymes is greater than the
stability of said enzymes bound to surfaces of the same material as
that of said nanoparticles but which forms a flat surface, and
further wherein said substance is a substrate for said enzymes; and
(b) allowing said enzymes to enzymatically degrade said substance,
thereby reducing the amount of said substance in said solution and
the fouling adherence of said substance to said surface.
21. The method of claim 20, wherein said enzyme activity is
maintained in a liquid medium other than an aqueous medium at
neutral pH, an aqueous medium at normal salinity, or an aqueous
medium at a temperature between about 20.degree. C. and 40.degree.
C. when said working surface is in contact with said solution.
22. The method of claim 21, wherein said liquid medium is selected
from the group consisting of an aqueous medium at a temperature
greater than about 40.degree. C., less than about 10.degree. C., an
aqueous medium whose pH is less than about pH 6.5, an aqueous
medium whose pH is greater than about pH 7.5, a liquid hydrocarbon
medium, an aqueous medium with a salinity of at least about 0.3 M
NaCl, and combinations thereof.
23. The method of claim 20, wherein said nanoparticles are selected
from the group consisting of single-walled carbon nanotubes,
multi-walled carbon nanotubes, gold or other metallic
nanoparticles, semi-conducting nanoparticles, metal oxide
nanoparticles, quantum dots, funtionalized silica, and mixtures
thereof.
24. The method of claim 20, wherein said enzymes are bound to said
nanoparticles through hydrophobic bonding, hydrophilic bonding,
ionic bonding, covalent bonding, and non-covalent bonding.
Description
RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/607,816, filed on Sep. 8, 2004. The entire
teaching of the above application is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] Interfacing nanomaterials, in particular carbon nanotubes,
with biomolecules are important for applications ranging from
biosensors, biorecognition probes, and molecular electronics to
drug delivery. A major obstacle in the pursuit of applications of
these conjugates stems from the poor stability of biomolecules in
harsh environments.
SUMMARY OF THE INVENTION
[0004] This invention is directed to protein compositions that
comprise biologically active proteins that are less susceptible to
degradation than normal. For example, this invention is directed to
compositions that enzymatically act on substrates where the enzymes
of the compositions are less susceptible to degradation than
normal. The proteins and enzymes of these compositions can retain
biological or enzymatic activity even when the compositions and
substrates are in normally harsh or hostile environments, such as
abnormal pHs, temperatures, high salinities, or media, including
non-aqueous media such as organic solvents, ionic liquids, gaseous
media, and supercritical fluids.
[0005] The compositions of this invention are proteins, e.g.
enzymes, bound to the external surfaces of nanoparticles. These
nanoparticles have external surfaces whose radius of curvature is
commensurate with the dimensions of each of the proteins or
enzymes, that are bound to the nanoparticles. When this size
relationship is met, the stability of the bound proteins or enzymes
is greater than the stability of these proteins when they are bound
to particles or surfaces whose radius of curvature is greater than
the dimensions of each of the bound proteins, e.g., the proteins
bound to flat surfaces. This stability difference exists even when
the material which forms the nanoparticles and the more flat
substrata are the same. The enhanced stability of the compositions
of this invention is maintained when the compositions are attached
to a macroscopic surface or are embraced within the polymer.
[0006] This invention is also directed to methods of detecting
analytes, even when the analytes are in a solution that provides a
harsh or hostile environment for enzymes. At least a portion of
these analytes is normally a substrate for the enzymes. The analyte
detection methods of this invention utilize the compositions of
this invention that contain the appropriate enzymes.
[0007] This invention is also directed to methods for preventing
fouling of surfaces by fouling agents. These fouling agents are
also substrates for enzymes and are often found in media that
constitute a harsh or hostile environment for the enzymes. In this
invention, the compositions of this invention are used to rid the
media of these fouling agents by enzymatically degrading the
agents, thereby keeping surfaces, which are often fouled by the
agents, free of these fouling agents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] The foregoing and other objects, features and advantages of
the invention will be apparent from the following more particular
description of preferred embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different views.
The drawings are not necessarily to scale, emphasis instead being
placed upon illustrating the principles of the invention.
[0009] FIG. 1(a) is a bar graph showing the percent activity of
soybean peroxidase in its native state and on various supports in
various concentrations of methanol.
[0010] FIG. 1(b) is a line graph showing the time-dependent
deactivation of soybean peroxidase on various supports in 100%
methanol.
[0011] FIG. 1(c) is a line graph showing the time-dependent
deactivation of soybean peroxidase in its native state and on
various supports in 95.degree. C. aqueous solutions.
[0012] FIG. 1(d) is a line graph showing the time-dependent
deactivation of subtilisin Carlsberg in its native state and on
various supports at various temperatures.
[0013] FIG. 2(a) is a schematic showing soybean peroxidase on a
flat support.
[0014] FIG. 2(b) is a schematic showing soybean peroxidase on a
curved support.
[0015] FIG. 2(c) is a line graph showing deactivation constants
from soybean peroxidase on various supports as a function of
surface area coverage in 95.degree. C. aqueous solutions.
[0016] FIG. 2(d) is a line graph showing deactivation constants for
soybean peroxidase on various supports as a function of surface
area coverage in 100% methanol.
[0017] FIG. 2(e) is a bar graph showing deactivation constants for
soybean peroxidase on various supports at different amounts of
surface area coverage.
[0018] FIG. 2(f) is a micrograph of signal walled nanotubes on
buckypaper.
[0019] FIG. 3(a) is a line graph showing deactivation constants for
soybean peroxidase on various supports in 95.degree. C. aqueous
solutions.
[0020] FIG. 3(b) is a line graph showing deactivation constants for
soybean peroxidase on various supports in 100% methanol.
[0021] FIG. 4(a) is a schematic showing the preparation of
biocatalytic films.
[0022] FIG. 4(b) is a line graph showing the
concentration-dependent activity of subtilisin Carlsberg on various
supports on pMMA films.
[0023] FIG. 4(c) is a line graph showing the amount of human serum
albumin adsorbed to pMMA films without and with subtilisin
Carlsberg on single-walled nanotubes attached to the films.
DETAILED DESCRIPTION OF THE INVENTION
[0024] A description of preferred embodiments of the invention
follows.
[0025] A core aspect of this invention is the formation of
nanoparticles with proteins or enzymes attached to their external
surfaces. These nanoparticles can be formed by a variety of
techniques and from a variety of materials known in the art of
nanoparticle fabrication. The nanoparticles that are suitable in
this invention generally include nanomaterials, e.g., nanotubes,
nanosheets, nanoporous materials, such as single-walled carbon
nanotubes, multi-walled carbon nanotubes, gold nanoparticles or
other metallic, semi-conducting, or metal oxide nanoparticles,
quantum dots, functionalizes silica. Single-walled carbon nanotubes
are preferred.
[0026] Proteins which can be used in this invention include
proteins which possess a biological activity. A biological activity
includes commercially relevant activities as a diagnostic,
therapeutic, enzymatic or other protein activity. Examples of
proteins include immunoglobulin-like proteins; antibodies;
cytokines (e.g., lymphokines, monokines and chemokines);
interleukins; interferons; erythropoietin; hormones (e.g., growth
hormone and adrenocorticotropic hormone); growth factors;
nucleases; tumor necrosis factor; colony-stimulating factors;
insulin; antigens (e.g., bacterial and viral antigens); DNA-binding
proteins and tumor suppressor proteins.
[0027] The enzymes of this invention can be of any type. The enzyme
species is not a critical aspect of the invention. Proteases,
peroxidases, lipases, carbohydrate cleavage enzymes, carbohydrases,
esterases, carboxylases, peroxidases, nucleases, lyases, ligases,
isomerases, transferases, etc. can be used. The only requirements
for each enzyme to be employed in the invention are that it
enzymatically acts on the substrate of interest that is present in
a solution to which the compositions of this invention are to be
exposed, and that it be bindable to the nanoparticles of the
compositions.
[0028] By way of example, transferases are enzymes transferring a
group, for example, the methyl group or a glycosyl group, from one
compound (generally regarded as donor) to another compound
(generally regarded as acceptor). For example, glycosyltransferases
(EC 2.4) transfer glycosyl residues from a donor to an acceptor
molecule. Some of the glycosyltransferases also catalyze
hydrolysis, which can be regarded as transfer of a glycosyl group
from the donor to water. The subclass is further subdivided into
hexosyltransferases (EC 2.4.1), pentosyltransferases (EC 2.4.2) and
those transferring other glycosyl groups (EC 2.4.99, Nomenclature
Committee of the International Union of Biochemistry and Molecular
Biology (NC-IUBMB)).
[0029] Oxidoreductases catalyze oxido-reductions. The substrate
that is oxidized is regarded as hydrogen or electron donor.
Oxidoreductases are classified as dehydrogenases, oxidases, mono-
and dioxygenases. Dehydrogenases transfer hydrogen from a hydrogen
donor to a hydrogen acceptor molecule. Oxidases react with
molecular oxygen as hydrogen acceptor and produce oxidized products
as well as either hydrogen peroxide or water. Monooxygenases
transfer one oxygen atom from molecular oxygen to the substrate and
one is reduced to water. In contrast, dioxygenases catalyze the
insert of both oxygen atoms from molecular oxygen into the
substrate.
[0030] Lyases catalyze elimination reactions and thereby generate
double bonds or, in the reverse direction, catalyze the additions
at double bonds. Isomerases catalyze intramolecular rearrangements.
Ligases catalyze the formation of chemical bonds at the expense of
ATP consumption.
[0031] Hydrolases are enzymes that catalyze the hydrolysis of
chemical bonds like C--O or C--N. The E.C. classification for these
enzymes generally classifies them by the nature of the bond
hydrolysed and by the nature of the substrate. Hydrolases such as
lipases and proteases play an important role in nature as well in
technical applications of biocatalysts. Proteases hydrolyse a
peptide bond within the context of an oligo- or polypeptide.
Depending on the catalytic mechanism proteases are grouped into
aspartic, serine, cysteine, metallo- and threonine proteases
(Handbook of proteolytic enzymes. (1998) Eds: Barret, A; Rawling,
N.; Woessner, J.; Academic Press, London).
[0032] Since the enzyme species is not a critical aspect of this
invention, the substrate type is also not critical. Any substrate
can be the target, provided it is enzymatically recognized by the
enzyme species on the surface of the nanoparticles of the
compositions of this invention, and that it is present in a
solution to which the compositions of this invention are to be
exposed.
[0033] The enzymes can be attached to the nanoparticles to form the
compositions of this invention by any suitable technique known in
the art. Any chemical or physical bonding can be used. Hydrophobic
bonding, hydrophilic bonding, ionic bonding, covalent and
non-covalent bonding are suitable bonding types. Of these,
hydrophobic bonding is preferred. A major consideration for the
choice of bonding process to be employed is that the specific
enzyme species bonded to the nanoparticles retain a substantial
fraction (e.g., at least about 30%, such as about 50%, at least
about 70% or more) of its native enzymatic activity after the
bonding process has been completed.
[0034] A feature of the compositions of this invention can be that
the surfaces of each of the nanoparticles to which the enzymes are
attached have a radius of curvature that is within about 2, or
preferably about 1, orders of magnitude of the dimensions of each
attached enzyme. Thus, the radius of curvature of the nanoparticles
is preferably about 100 nm or less.
[0035] The enzymes in the compositions of this invention are active
when the compositions are exposed to media, containing the
substrates that are atypical for the enzymes in an isolated or
unbound state. Although the compositions of this invention exhibit
very good enzymatic activity when the compositions are in
physiological solutions containing the enzyme substrate, they also
exhibit very good activity and stability when the media containing
the substrate are considered to present harsh or hostile
environments to the enzymes. For example, when the medium is an
aqueous medium at an elevated temperature, e.g., greater than
90.degree. C., the enzymatic activity and stability of the
compositions of this invention is maintained. If the medium is a
hydrocarbon solvent, e.g., an alcohol, the enzymatic stability of
the compositions of this invention is greater than on more
conventional (e.g., flat) surfaces.
[0036] The compositions of this invention have the advantage of
retaining activity when placed in liquid environments that are
typically noxious to the enzymes when these enzymes are not bound
to the nanoparticles that are disclosed in this invention. For
example, the compositions of this invention will be used in
non-aqueous media, e.g., organic solvents, ionic liquids, gaseous
media and supercritical fluids, or in media at abnormal
temperatures (e.g., other than 20.degree. C. to 40.degree. C.), or
media where the pH is non-physiologically acidic or basic or in
media possessing abnormal ionic strengths or salt levels (e.g.,
media with high salinity, such as sea water or a salt level of at
least about 0.3 M NaCl). Of course, it is recognized that proteins
that tolerate such conditions are known (thermophilic enzymes,
enzymes which tolerate high levels of saline, etc). Thus, one can
characterize harshness of an environment as a relative factor as
compared to the normal reaction conditions of the enzyme. Thus, an
abnormal temperature for a thermophilic enzyme could be above the
temperature at which the thermophilic enzyme is active. Likewise,
an abnormal salinity for a salt-tolerant enzyme can be above the
salinity levels at which the salt-tolerant enzyme is active.
[0037] The compositions of this invention will also be used as
antifouling agents in paints, marine paints, coatings, lubricants,
ointments, etc. These compositions are also intended for use as
antimicrobial agents in environments where the antimicrobial
activity of the bound enzymes is desired for microbial lysis or
inactivation.
[0038] Detection of analytes can be achieved by any number of
procedures known in the detection art. Formation of fluorescent
species when enzymatic action occurs, formation of absorption
species when enzymatic action occurs, liberation of fluorescent or
absorption tags when enzymatic action occurs, formation of
chemically reactive species by enzymatic action that react with
suitable target structures which thereby become detectable,
formation of an electrically charged species by enzymatic action
which can be electrically detected are examples of detection
procedures for analytes when the analytes are the targets of the
bound enzymes in the compositions of this invention. Often, the
analytes are labels that have been attached to chemical moieties
whose detection is sought. In these instances, the analytes are the
substrates for the bound enzymes in the compositions of this
invention.
[0039] The compositions of this invention can be attached to
macroscopic surfaces or spread on or embedded within a polymeric
material. The compositions of this invention can thereby be made
functional parts of useful devices. The compositions of this
invention can be added as coatings to medical instruments,
biosensors, biochips, biorecognition probes, biocatalytic films,
biofuel cells, drug delivery systems, self-cleaning materials,
resins, beads, and the like. These compositions can be integral
parts of permeable or nonpermeable membranes, sieves, tubing and
the like. When incorporated in such devices, the compositions of
this invention can be used to detect analytes that are substrates
of the enzymes bound to the nanoparticles, to monitor the presence
of substances in liquid media that are substrates of these enzymes,
to act as antimicrobial agents when the enzyme substrates are
integral constituents of viral particles, microbial membranes or
cell walls, or to prevent surface fouling by degrading substances
that form deleterious films on working surfaces of machines or
instruments. There are many utilities available to the skilled
artisan for which the compositions of this invention are
applicable. Enzymatic activity from solid materials in a liquid
environment is assumed to be one of the purposes for the
compositions of this invention. Improved enzyme stability when the
liquid environment is normally hostile or harsh to the enzymes,
when they are dissolved in the liquid, is of particular usefulness
with this invention.
[0040] The emergence of techniques to generate nanomaterials with
precise dimensions, geometries, and surface properties has resulted
in an increasingly large number of applications ranging from
electronics and high-strength, lightweight materials to sensing
elements. To date, proteins, and other biomolecules have been used
to functionalize nanomaterials and influence their properties.
However, up to now, very little is known about the ability of these
nanoscale materials to enhance protein structure and function. Such
information, however, is of fundamental importance and is also
critical for enhancing protein function and stability on
nanoparticles and therefore for designing optimal
protein-nanoparticle conjugates for use in functional materials and
surface coatings.
Materials and Methods
[0041] Enzymes and reagents. Soybean peroxidase, subtilisin
Carlsberg, and N-succinyl-L-ala-L-ala-L-pro-L-phe-p-nitroanilide
were purchased from Sigma as salt-free, dry powders and used
without further purification. Raw single-walled nanotubes, SWNTs,
were purchased from Carbon Nanotechnologies, Inc., highly oriented
pyrolytic graphite, HOPG SPI-2, was obtained from Structure Probe,
Inc, and graphite was purchased from Aldrich. All the supports were
used without further purification. All other chemicals were
purchased from Sigma and used as received.
[0042] Determination of enzyme activity. The initial rates of the
phenolic oxidations catalyzed by SBP in presence of H.sub.2O.sub.2
were monitored by spectrophotometry. SC cleaves the peptide bond in
N-succinyl-L-ala-L-ala-L-pro-L-phe-p-nitroanilide to release a
chromophore, p-nitroaniline, and the initial rates were obtained by
measuring the increase in the absorbance at 405 nm.
[0043] Enzyme immobilization on SWNTs. The enzymes, Soybean
peroxide (SBP) and subtilisin Carisberg (SC) were adsorbed on SWNTs
using hydrophobic interactions. SWNTs were first sonicated in N,
N-Dimethyl Formamide (DMF) for 20 minutes to obtain a uniform
dispersion of SWNTs in DMF (1 mg/mi). One ml of SWNT dispersion in
DMF (i.e. 1 mg of SWNTs) was then dispensed in an Eppendorf
micro-centrifuge tube and the organic phase was gradually changed
to an aqueous phase by repeated washing with pH 7 buffer (50 mM
phosphate). This gradual change from organic phase to an aqueous
phase helps in a better dispersibility of SWNTs in buffer. The
dispersion of SWNT in pH 7 buffer was then exposed to freshly
prepared solutions of enzyme in buffer (pH 7 phosphate, 50 mM).
This dispersion was shaken on Innova.TM.2000 (New Brunswick
Scientific) platform shaker for 2 h at 200 rpm at room temperature.
In the case of SC, the shaking was carried out at 4.degree. C. to
prevent autolysis of the protease during incubation. After the 2 h
incubation, the SWNTs were settled using a micro-centrifuge (Fisher
Scientific) and the supernatant was removed. Typically, 6 washes
were performed with fresh buffer to remove any unbound/loosely
bound enzyme. All supernatants were analyzed for protein content
using the BCA or the .mu.BCA assay (Pierce Biotechnology, Inc.). It
was seen that the SWNTs interfere strongly with BCA/.mu.BCA assay.
The amount of enzyme loaded on the SWNTs was, therefore, determined
by measuring the concentration of enzyme solution before and after
exposing it to the dispersion of SWNTs in buffer. The difference in
the amount of enzyme gives the amount of enzyme loaded on the
SWNTs. A stable value of enzyme loading on SWNTs was obtained by
accounting for the loss of enzyme due to leaching during the
washes.
[0044] Determination of enzyme activity. The activity of SBP was
measured using the p-Cresol assay. SBP catalyzes the oxidation of
p-Cresol by H.sub.2O.sub.2 to form oligophenol and polyphenol
products that fluoresce. For a typical solution phase assay, the
reaction mixture consisted of 0.15 .mu.g/ml solution of SBP (made
by serial dilution), 20 mM solution of p-Cresol and 0.125 mM
solution of H.sub.2O.sub.2 all solutions were made in pH 7.0 buffer
(phosphate, 50 mM). The initial rates of reaction were then
measured by tracking the increase in fluorescence of the reaction
mixture at an excitation wavelength of 325 nm and emission
wavelength of 402 nm using a HTS 7000 Plus Bio Assay Reader (Perkin
Elmer). For activity measurements in organic solvent phase, the
solvents were added during the final wash to make solutions of 0.15
.mu.g/ml solution of SBP in pH 7.0 buffer (phosphate, 50 mM)
containing the required amount of solvent in the solution. The
p-Cresol and H.sub.2O.sub.2 solutions were made in pH 7.0 buffer
(phosphate, 50 mM) containing the required concentrations of
solvent.
[0045] For measuring the activity of SBP immobilized on SWNTs
(SWNT-SBP), a well-mixed dispersion of SWNT-SBP (1 mg/ml) was made
in buffer and a known amount of SWNT-SBP was dispensed by using
serial dilution. For a typical experiment 0.5 .mu.g to 1.5 .mu.g of
SWNT-SBP was used based on the loading of the SBP. The enzymatic
activity was measured using 20 mM p-Cresol and 0.125 mM
H.sub.2O.sub.2 in PH 7.0 buffer (phosphate, 50 mM). It was observed
that some of the immobilized enzyme leached during the serial
dilutions. To account for the effect of the enzyme that would leach
during the measurement of the activity of the immobilized enzyme,
the SWNT-SBP suspension was washed 6 times more with the same
dilutions and buffer used in the final activity measurement. Since
the amount of leached enzyme during these washes was too low
(<15 ng/ml) to be reliably detected by any of the protein
measurement assays, the amount of protein was estimated by
measuring the activity of the enzyme in the washes. It was assumed
that the activity of the leached enzyme was the same as that of the
solution phase enzyme. Using the value of specific activity of the
solution phase enzyme and the initial rate of reaction for the
enzyme in the wash solution, the amount of enzyme present in the
washes was calculated. The final loading of the enzyme on the SWNTs
was corrected for this amount of leached enzyme before calculating
the specific activity of the immobilized enzyme. After all the
washes were done, the SWNT-SBP were dispersed in pH 7.0 buffer
(phosphate, 50 mM) and then exposed to the substrate solution so
that the final concentrations of the substrates were 20 mM p-Cresol
and 0.125 mM H.sub.2O.sub.2. The dispersion was shaken at 200 RPM
at all times during the reaction using Innova.TM.2000 platform
shaker to avoid problems due to diffusion limitations. At fixed
time intervals, the SWNTs were settled using a micro-centrifuge and
the fluorescence of a 200 .mu.l aliquot of the supernatant was
measured using the Bio Assay Reader. The aliquot was then replaced
in the reaction mixture. A plot of H.sub.2O.sub.2 consumed versus
time gives the initial rate of reaction and hence the activity of
the SBP immobilized on the SWNTs. For activity measurements in
solvent phase, the p-cresol and H.sub.2O.sub.2 solutions were made
in pH 7.0 buffer (phosphate, 50 mM) containing the required
concentrations of solvent. After all the washes, the SWNT-SBP were
dispersed in pH 7.0 buffer (phosphate, 50 mM) containing the
required amount of solvent and then exposed to the substrate
solution. For 100% solvent phase, aqueous SWNT-SBP phase was
gradually changed to the organic phase by repeated washing with
100% solvent. This treatment rendered the final concentration of
water in the solvent to about 1-2%.
[0046] The activity of SC was measured using
N-succinyl-L-ala-L-ala-L-pro-L-phe-pnitoranilide (tetrapeptide)
(Sigma-Aldrich) as the substrate. For a typical solution phase
assay, 1 .mu.g/ml of freshly prepared SC solution pH 8.0 buffer
(phosphate, 50 mM) was used with a 100 .mu.M solution of
tetrapeptide in pH 8.0 buffer (phosphate, 50 mM).
[0047] Subtilisin Carlsberg, which is a protease, cleaves the
peptide bond in the substrate to release the chromophore,
p-Nitroaniline, which absorbs at 405 nm. The activity of the enzyme
was measured by measuring the increase in the absorbance of the
reaction mixture at 405 nm using the Bio Assay Reader. The activity
of SC immobilized on SWNTs (SWNT-SC) was measured using the same
technique as that used for immobilized SBP (as described above).
For SWNT-SC, however, 4 .mu.g to 50 .mu.g of functionalized SWNTs
were used for the measurement of activity based on the loading of
the SC. After performing 6 washes like those done for SWNT-SBP, the
SWNT-SC were dispersed in pH 8 buffer (phosphate, 50 mM) and then
exposed to 100 .mu.M tetrapeptide solution (final concentration).
The dispersion was kept well mixed by shaking at 200 RPM at all
times during the reaction using the platform shaker. At fixed time
intervals, the SWNTs were settled using a micro-centrifuge and the
absorbance of the supernatant was measured at 405 .mu.m using the
Bio Assay Reader. A plot of concentration of p-Nitroaniline versus
time gives the initial rate of reaction and hence the activity of
the SC immobilized on the SWNTs. The activity measurements in the
organic phase were performed as explained above for SBP.
[0048] Enzyme immobilization on other supports. The enzymes were
also adsorbed on other supports including highly oriented pyrolytic
graphite (HOPG), self-assembled monolayers (SAMs) of
undecanethiolate on gold (Gold SAM), multi-walled carbon nanotubes
(MWNTs), graphite powder (1-2 .mu.m), SWNT films and MWNT films.
HOPG SPI-2 samples were obtained from Structure Probe, Inc and
fresh surfaces were exposed by peeling off the exposed layers using
a scotch tape. Self-assembled monolayers of undecanethiol were
assembled from 0.02 mM solutions in absolute ethanol for 12 h. The
samples were then removed from the solution and rinsed thoroughly
by squirting with ethanol for several seconds. This rinsing was
sufficient to remove any unbound thiols from the surface. The
synthesis of SWNT and MWNT films was performed by first dispersing
SWNTs and MWNTs in pH 7.0 buffer (phosphate, 50 mM) as explained
above and filtering the samples through a 0.8 .mu.m ATTP filter.
The filter papers with the SWNT and MWNT cakes were dried and were
attached to plastic troughs using appropriate clips. SBP and SC
were adsorbed in HOPG, Gold SAM, SWNT films, MWNT films by dipping
the supports into a solution of the enzymes and shaking the samples
on Innova.TM.2000 (New Brunswick Scientific) platform shaker for 2
h at 200 rpm at room temperature. In the case of SC, the shaking
was carried out at 4.degree. C. to prevent autolysis of the
protease during incubation. The samples were then washed 6 times
with pH 7.0 buffer (phosphate, 50 mM) to remove any loose/unbound
enzyme. The loading on MWNTs was done as described above for SWNTs.
After all the washes were done the enzyme bound supports were
exposed to the substrate solutions and their activities were
measured as outlined above for SWNTs.
[0049] The SWNT-enzyme conjugates were prepared in aqueous buffer
by adsorbing two model enzymes subtilisin carlsberg (SC) and
soybean peroxidase (SBP) onto SWNTs. The enzymes showed strong
affinity for SWNTs with saturation levels of 670 and 655 .mu.g/mg
SWNT for SC and SBP, respectively. Both SC and SBP retained a
substantial fraction of their native enzymatic activity; specific
activities of the adsorbed SC and SBP in aqueous buffer were ca.
63% and ca. 38% of the native enzyme activities respectively. FT-IR
spectroscopy analysis. revealed ca. 11.5% and ca. 13% total change
in the secondary structure of SC and SBP respectively due to
absorption onto SWNTs. AFM studies also revealed that both SBP and
SC retained their tertiary structure on adsorption on SWNTs. This
suggests that the present method employed for interfacing SBP and
SC with SWNTs results in a minimal loss in the native
structure.
[0050] Hammett analysis was used as a sensitive probe of transition
state structure and enzyme mechanism. The Hammett coefficient
(.rho.) provides a measure of the sensitivity of SBP's catalytic
efficiency to the electronic nature of substituents on phenolic
substrates. Hammett analysis revealed .rho. values of 1.7.+-.0.21
and 1.4.+-.0.12 for SWNT-SBP and HOPG-SBP, respectively, in 100%
methanol. The comparable values of .rho. suggest that the mechanism
of catalysis is similar for SBP adsorbed on the two supports; the
significantly greater retention of activity for SBP immobilized on
SWNTs than for SBP immobilized on HOPG in 100% methanol is
therefore not due to a change in the mechanism of catalysis on the
different supports.
[0051] It was also found that a variety of proteins differing in
both structure and function, including horseradish peroxidase,
subtilisin carlsberg, proteinase K, trypsin, and lipase, remain
catalytically active upon adsorption onto SWNTs, with specific
activities ranging from 40-70% relative to that of the native
protein in aqueous buffer.
[0052] Having established that a number of enzymes retain activity
on SWNTs in aqueous buffer, SWNTs were examined to determine
whether the enzymes function in strongly denaturing
environments--environments in which native enzymes show poor
retention of activity. To that end, SWNT-SBP was added to solutions
of buffer containing the denaturant methanol. FIG. 1a shows the
retention of activity in solutions containing methanol, i.e. the
enzymatic activity in solutions containing methanol normalized to
the enzymatic activity in aqueous buffer, for native SBP and SBP
adsorbed onto a variety of supports. The specific activity--the
activity normalized to the amount of enzyme--for native SBP and the
various SBP conjugates in solutions containing methanol was also
determined. Native SBP was completely inactive in 100% methanol.
However, the SWNT-SBP conjugates retained relatively high catalytic
activity, even in neat methanol (FIG. 1a). It is well known that
proteins are often stabilized by immobilization onto a support. To
assess whether the stabilization of SBP on SWNTs was simply a
result of immobilization, the enzyme was absorbed onto graphite
flakes. Since a SWNT is similar to a rolled graphene sheet,
graphite flakes represent an ideal surface for comparison. As shown
in FIG. 1a, SBP was significantly more active on SWNTs than on
graphite flakes, particularly in neat methanol. A similar trend was
also observed in isopropanol, trifluoroethanol, and acetonitrile
(data not shown).
[0053] The SWNT-SBP conjugates were also more active in methanol
than enzyme immobilized onto a variety of other flat supports,
including highly ordered pyrolytic graphite (HOPG) and
self-assembled monolayers (SAMs) of undecanethiolate on gold-coated
glass cover slips (FIG. 1a). Finally, SWNT films were prepared by
filtering a suspension of SWNTs through a 0.8 .mu.m membrane. SBP
adsorbed onto the resulting "SWNT buckypaper" was more active than
SBP adsorbed on the flat supports (FIG. 1a), suggesting
significantly different behavior under denaturing conditions on
these nanoscale supports relative to flat surfaces. Similar results
were obtained for multi-walled carbon nanotubes, and gold particles
of similar dimensions as the SWNT (data not shown).
[0054] In addition to the initial activity, the stability of the
SWNT-enzyme conjugates was evaluated in strongly denaturing
environments. The half-life of SBP adsorbed onto SWNTs in 100%
methanol was at least two-fold longer than that of the enzyme
adsorbed onto flat supports (FIG. 1b). The thermostability of the
conjugates was also tested at 95.degree. C., a temperature at which
native SBP undergoes significant and rapid denaturation. The
half-life of SBP adsorbed onto SWNTs at 95.degree. C. was
approximately 90 min, ten-fold longer than that of the native
enzyme and at least twice that of SBP adsorbed onto other supports
(FIG. 1c). A similar enhancement in stability was seen for SBP
adsorbed onto SWNT buckypaper (data not shown). These results
indicate a dramatic enhancement in stability in harsh environments
for SBP adsorbed onto SWNTs. The observed stabilization on SWNTs is
not unique to SBP, but is also seen for the unrelated protease
subtilisin Carlsberg (SC) (FIG. 1d).
[0055] To see if SWNTs render SC more resistant to degradation by
autolysis, the storage stability of SWNT-SC and native SC was
examined at two different conditions--pH 7.8, at which the protease
is most active and pH 4.5, at which the proteolytic activity of SC
is negligible. FIG. 3b shows that at both pH conditions, the loss
in activity of SC adsorbed on SWNTs is less than that of SC
adsorbed on HOPG. The half life of HOPGSC in pH 7.8 was ca. 44 h,
about two fold lower than that of SWNT-SC. Interestingly, the
activities of adsorbed SC are similar for both the pH conditions,
which shows that the loss in activity over time is not due to
autolysis, but probably due to protein-surface or protein-protein
interactions on the surface of the hydrophobic supports. This
further demonstrates the impact of the nanoscale environment on the
reported enhanced stability of SWNT-enzyme conjugates.
[0056] There are three possible hypotheses that could explain the
enhanced stability of enzymes on SWNTs. The first (hypothesis 1) is
that protein deactivation in harsh environments is primarily
mediated by protein-surface interactions, which are disfavored on
highly curved supports such as SWNTs relative to flat supports. An
alternative hypothesis (hypothesis 2) stems from the observation
that the greater stability of adsorbed enzymes relative to their
soluble counterparts is due to greater barriers to unfolding on the
supports, as a result of protein-support interactions. Therefore,
if proteins have a higher affinity for SWNTs than for other
supports, there may be greater barriers to unfolding in harsh
environments on SWNTs than on other supports, thereby explaining
the higher stability observed on SWNTs. Finally, a third hypothesis
is that lateral interactions between adsorbed proteins contribute
to protein deactivation in harsh environments, and that these
unfavorable "lateral" interactions are suppressed on highly curved
supports such as SWNTs relative to those on flat surfaces (FIGS. 2a
and b). This third hypothesis is explained in greater detail
below.
[0057] FIG. 2a depicts proteins adsorbed on a flat support, where x
and y represent the distances between adjacent proteins (measured
along the protein-substrate interface) along the X and Y axes,
respectively. Similarly, x.sub.f' and y.sub.f' represent the
center-to-center distance between adjacent proteins along the X and
Y-axes, respectively. On a flat support x=x.sub.f', and y=y.sub.f'.
Furthermore, the surface coverage of proteins is inversely
proportional to the product xy. FIG. 2b depicts proteins adsorbed
on a cylindrical support, where x and y represent the distances
between adjacent proteins (measured along the protein-substrate
interface) along the circumference (.theta.-direction) and the axis
of the cylinder, respectively. Here, the values of x and y are
identical to those in FIG. 2a. Finally, x.sub.c' and y.sub.c'
represent the center-to-center distance between adjacent proteins
along the circumference and the axis of the cylinder, respectively.
On a cylindrical support, y.sub.c'=y; however, x.sub.c' is not
equal to x, but is greater than x.
[0058] A simple geometric analysis (equation 1) reveals that,
x c ' = ( R + r ) R * x ( 1 ) ##EQU00001##
where R is the radius of the cylinder, and r represents the average
dimension of SBP. Consequently, at the same separation along the
protein-substrate interface, and the same surface coverage, the
curvature of a cylindrical support results in an increase in the
center-to-center distance between adjacent proteins (FIG. 2b). If
unfavorable interactions between adjacent proteins contribute to
their deactivation in harsh environments, then this increase in
separation should result in a decrease in the rate of deactivation,
and could contribute to the greater protein stability on SWNTs
relative to flat supports.
[0059] Both experimental data (FIGS. 2c-e) and theoretical analysis
(see discussion below and FIGS. 3a-b) are used to distinguish among
these hypotheses. The rates of deactivation were measured in
aqueous buffer at 95.degree. C. and in methanol for SBP adsorbed
onto SWNTs and graphite flakes at different fractional surface
coverages. FIGS. 2c and d reveal a strong dependence of the
enzymatic deactivation rate on surface coverage, with identical
deactivation constants on SWNTs and graphite flakes should persist
even at low coverages. Similarly, if the enhanced stability on
SWNTs is a result of a greater affinity of the protein for SWNTs
(hypothesis 2), the difference in stability should also persist at
low surface coverages. The results shown in FIGS. 2c and d are
clearly inconsistent with hypotheses 1 and 2, yet they are
consistent with hypothesis 3. If unfavorable "lateral" interactions
between adsorbed proteins contribute significantly to protein
deactivation (hypothesis 3), these interactions, and hence the rate
of enzymatic deactivation, should decrease on all supports with
decreasing surface coverage (i.e. with an increase in the average
separation between adsorbed proteins). Furthermore, hypothesis 3
also predicts that the enhancement in stability on SWNTs relative
to graphite flakes should disappear at very low surface
coverages.
[0060] Additional control experiments were performed to confirm
that the similar values of the deactivation constants on SWNT and
graphite flakes at low surface coverage are a result of a reduction
in unfavorable lateral interactions, and not due to a change in the
conformation of the adsorbed protein at low surface coverage. For
this purpose, the rates of enzymatic deactivation at 95.degree. C.
(FIG. 2e) were measured for the following sets of protein
conjugates: 1) SBP adsorbed onto SWNTs and graphite flakes at a
high fractional surface coverage (0.75); 2) SBP adsorbed onto SWNTs
and graphite flakes at a low fractional surface coverage (0.07);
and 3) SBP adsorbed onto SWNTs and graphite flakes at a low
fractional surface coverage of 0.07 (same as that for sample set
2), followed by the adsorption of catalytically inactive apo-SBP,
yielding a final fractional surface coverage of 0.75 (same as that
for sample set 1). While preparing sample set 3, the active protein
was allowed to adsorb prior to adsorbing the inactive apo-protein,
thereby allowing it to change its conformation on the support
(under conditions of low coverage). Furthermore, sample sets 2 and
3, contain the same amount of "active" protein, but differ in the
total surface coverage of protein.
[0061] As seen in FIG. 2e, the deactivation rate for SWNT-SBP
conjugate 3 at 95.degree. C. is identical to that for SWNT-SBP
conjugate 1, and is higher than that for SWNT-SBP conjugate 2.
Similar trends are seen for the graphite-SBP conjugates.
Furthermore, the deactivation rate for the SWNT-SBP conjugate 3 is
significantly lower than that for the graphite-SBP conjugate 3. In
combination, these results suggest that the decrease in
deactivation rate at low surface coverage (FIGS. 2c and d) is not
due to a change in the conformation of the adsorbed protein, and
provide further support for hypothesis 3.
[0062] Finally, a simple model has been developed that allows the
quantification of the effect of substrate curvature on the lateral
interactions between adsorbed proteins. The model assumes that the
proteins are distributed uniformly on the surface. A new variable,
S, was introduced to capture the average center-to-center distance
between adjacent proteins on the various supports. The term S is
defined to be the geometric mean of the center-to-center distances
between proteins along the two orthogonal axes; i.e.
S=(x.sub.f'.y.sub.f').sup.1/2 on a flat support, and
S=(x.sub.c'.y.sub.c').sup.1/2 on a cylindrical support (FIGS. 2a
and b). As discussed above, for the same values of x and y, the
value of x.sub.c' is greater than the value of x.sub.f', and
therefore the value of S on a cylindrical support is greater than
that on a flat support. S may be converted to a dimensionless form
(.epsilon.) by dividing it by the value of S on a flat support at
maximum surface coverage (S.sub.m); .epsilon.=S/S.sub.m. FIGS. 2a
and b indicate that S.sub.m is equivalent to the geometric mean of
the distance between adjacent proteins along the protein-substrate
interface along the two orthogonal axes, measured at maximum
surface coverage.
[0063] The deactivation rates of adsorbed SBP on the various
supports, previously plotted as a function of surface coverage
(FIGS. 2c and d), are now plotted as a function of the
dimensionless variable .epsilon. (FIGS. 3a and b). The introduction
of .epsilon., which now accounts for the curvature of the support,
allows the data for the deactivation rates on graphite flakes and
SWNTs to collapse onto a single curve, providing further evidence
in support of the "lateral-interaction" hypothesis.
[0064] The aforementioned model also predicts that the observed
enhancement in stabilization should not be unique to SWNTs.
Consistent with this prediction, an enhancement in the stability of
proteins in harsh environments on other nanostructured supports,
including gold nanoparticles, was observed in the experiments
described above. This phenomenon results from the radius of
curvature of the nanoscale support being commensurate with the
dimensions of the protein, as illustrated schematically in FIG. 2b.
Consistent with this hypothesis, a scanning electron micrograph of
SWNT buckypaper (FIG. 2f) shows SWNT bundles having an average
diameter of ca. 8 nm. Moreover, the value of SBP's saturation
loading on SWNTs (measured to be 655 mg/g) supports this value of
the average bundle diameter (16, 23). The average bundle radius is
therefore similar to the dimensions of SBP (6.1 nm.times.3.5
nm.times.4.0 nm).
[0065] These highly stable and active enzyme-nanotube conjugates
are ideally suited for designing functional nanocomposites;
composites incorporating enzymes, particularly proteases, may be
useful for designing anti-fouling or self-cleaning surfaces.
Previous applications have been limited by enzyme leaching from the
matrix, low enzyme loading, and low activity of the incorporated
enzymes because of poor stability in the harsh abiotic environment.
The stable SWNT-enzyme composites should form highly stable
biocatalytic films. To that end, SWNT-SC conjugates were dispersed
in poly(methyl methacrylate) (pMMA) (FIG. 4a) and the enzymatic
activities of the films were measured. The pMMA-SWNT-SC films
retained >90% of their initial activity over 30 days in aqueous
buffer. Furthermore, the high surface area per unit weight of the
SWNTs resulted in high enzyme loadings in the films, and
consequently, the films were over 30 times more active than those
containing identical amounts of graphite-SC conjugates (FIG. 4b).
The control pMMA-SC films exhibited significant leaching, resulting
in a nearly complete loss of activity after washing (FIG. 4b).
[0066] The proteolytic activity of the biocatalytic films will
allow the preparation of antifouling and antimicrobial surfaces,
for example those that may be used on surgical instruments,
implants, diagnostics, bioreactors, and other surfaces prone to
contamination. The attachment of bacteria to surfaces, which can
result in the buildup of biofilms, is often mediated by protein
adsorption that will most likely be prevented by making surfaces
protein-resistant. Biocatalytic nanocomposites that resist
non-specific protein adsorption were designed (FIG. 4c). To assess
the protein resistance of these materials, the biocatalytic films
were exposed to concentrated solutions (1 mg/mL) of the plasma
protein human serum albumin (HSA) continuously for 6 days.
pMMA-SWNT-SC films were able to reduce the nonspecific binding of
HSA by ca. 75% when compared to films without SC (FIG. 4c).
SDS-PAGE of HSA incubated with pMMA-SWNT-SC films revealed
proteolytic breakdown products (data not shown), suggesting that
this decrease in HSA binding is due to the proteolytic degradation
of the bound HSA and the subsequent desorption of the peptide
fragments rendering the film self-cleaning. Biocatalytic films that
also incorporated a second protease, trypsin (TRY), adsorbed onto
SWNTs to provide a broader range of proteolytic cleavage sites on
the HSA, yielded further reduction in the extent of nonspecific
protein adsorption, with as much as ca. 95% lower binding when
compared with the enzyme-free film (FIG. 4c). The biocatalytic
films demonstrate the high stability required for continuous
operation in commercial environments. When challenged with a fresh
sample of HSA (1 mg/mL) every 3 days, the pMMA-SWNT-enzyme
conjugates led to .about.50 fold decrease in HSA binding over 30
days. These films are also active in high salt buffers and at
elevated temperatures.
[0067] A thermal analysis was used to examine the pMMA films to
ensure that the changes in the physical properties of the polymer
are minimal due to the incorporation of the SWNT-enzyme conjugates.
The films were prepared as before and the glass-transition
temperature (Tg) of the films were measured. The Tg values of pMMA
and pMMA-SWNT-SC films were similar; x and y respectively.
[0068] In addition to polymeric composites, films composed solely
of SWNTs and enzymes were prepared by filtering suspensions of
SWNT-SC and SWNT-TRY conjugates through a 0.8 .mu.m membrane (FIG.
4a). These "biocatalytic buckypapers" have enzyme loadings as high
as 30% (w/w), which are among the highest loadings reported to
date. As shown in FIG. 4c, these films showed negligible protein
adsorption after 6 days (>99% reduction in the amount of protein
adsorption compared to the enzyme-free film).
[0069] The enhanced stability of proteins adsorbed on nanotubes, in
addition to the other attractive features (minimal leaching, high
surface area per unit weight, and high strength to name a few) will
thus be used in applications ranging from biosensing to biomedical
devices, which would require highly stable protein-nanotube
conjugates. The experimental results and the accompanying
theoretical analysis shown here indicate that the observed
enhancements in protein stability are not unique to nanotubes and
will also be obtained with other nanomaterials. The ability to
enhance protein function by interfacing them with nanomaterials
will have profound impact on the design of biosensors,
biorecognition probes, protein chips, biofuel cells, vaccines,
novel composites and supports for biotransformations, drug delivery
systems, and self-cleaning materials.
Figure Legends
[0070] FIG. 1 Retention of enzymatic activity when exposed to harsh
environments. (a) The initial activity (v) in solutions containing
methanol relative to the activity in aqueous buffer for native SBP
(1) and SBP adsorbed on various supports--SWNTs (2), buckypaper
made out of SWNTs (3), HOPG (4), and graphite flakes (5). The
asterisk indicates no activity of the native SBP in 100% (<0.01%
H.sub.2O) methanol. (b) Time-dependent deactivation of SBP in 100%
methanol on various supports--SWNTs (1), HOPG (2), SAM of
undecanethiolate on gold (3), and graphite flakes (4). For (b)-(d),
the activities are normalized relative to the initial activity
(activity at t=0 min), and each data point represents an average of
triplicate measurements with standard error <10%. (c)
Time-dependent deactivation of SBP at 95.degree. C. on various
supports--native SBP (1 open circles), SWNTs (2 black circles),
HOPG (3 diamonds), SAM of undecanethiolate on gold (4 open
squares), and graphite flakes (5 triangles). (d) Time-dependent
deactivation of SC on various supports in aqueous buffer--native SC
(open circles), SC adsorbed on SWNTs (black circles) at 50.degree.
C. (open circles) and 70.degree. C. (open triangles).
[0071] FIG. 2 Effect of lateral interactions on the deactivation
constants of SBP adsorbed onto different supports. (a) Schematic
depicting SBP molecules adsorbed onto a "flat" support. (b)
Schematic (drawn approximately to scale) depicting SBP molecules
adsorbed onto a cylindrical support. The curvature of the support
increases the center-to-center distance between adjacent proteins
(x.sub.c'), relative to the distance on a "flat" support
(x.sub.f'). (c) Deactivation constants for SBP adsorbed onto SWNTs
(circles) and graphite flakes (triangles) as a function of surface
coverage at 95.degree. C. In (c) and (a), error bars indicate the
standard deviation of triplicate measurements. (a) Deactivation
constants for SBP adsorbed onto SWNTs (circles) and graphite flakes
(triangles) as a function of surface coverage in 100% methanol. (e)
Deactivation constants for SBP adsorbed onto SWNTs and graphite
flakes at a fractional surface coverage of 0.75 (black bars), a
fractional surface coverage of 0.07 (hatched bars), and a
fractional surface coverage of 0.07 for SBP, with a "total" surface
coverage of 0.75 for SBP and apo-SBP (gray bars). (f) SEM image of
a typical SWNT buckypaper.
[0072] FIG. 3 Influence of the center-to-center distance between
adsorbed proteins on the deactivation rate. Deactivation constants
for SBP adsorbed onto SWNTs (circles) and graphite flakes
(triangles) as a function of the dimensionless variable .epsilon.
(a) at 95.degree. C. and (b) in 100% methanol. Error bars indicate
the standard deviation of triplicate measurements. The introduction
of the dimensionless variable .epsilon. enables the deactivation
rates on different supports to be collapsed onto a single
curve.
[0073] FIG. 4
[0074] Enzymatic activities of biocatalytic films. (a) Preparation
of biocatalytic films along with a SEM image of a typical
biocatalytic buckypaper. (b) Activities of native SC (1), SC
adsorbed on SWNTs (2), and SC adsorbed on graphite powder (3) in
pMMA films as a function of the amount of SC conjugates loaded into
the films. The activities were measured after the films were washed
extensively with aqueous buffer. (c) Protein-resistant properties
of the biocatalytic films--amount of HSA adsorbed onto plain pMMA
films (control, 1), pMMA-SWNT-SC films (2), pMMA-SWNT-SC-TRY films
(3) and SWNT-SC-TRY buckypaper (4). Error bars indicate the
standard deviation of triplicate measurements.
[0075] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *