U.S. patent application number 17/038452 was filed with the patent office on 2021-03-04 for packaging material and methods of using the same.
This patent application is currently assigned to The Research Foundation for The State University of New York. The applicant listed for this patent is The Research Foundation for The State University of New York. Invention is credited to Omowunmi A. Sadik, Idris Yazgan.
Application Number | 20210061965 17/038452 |
Document ID | / |
Family ID | 1000005220326 |
Filed Date | 2021-03-04 |
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United States Patent
Application |
20210061965 |
Kind Code |
A1 |
Sadik; Omowunmi A. ; et
al. |
March 4, 2021 |
PACKAGING MATERIAL AND METHODS OF USING THE SAME
Abstract
The present disclosure is directed to films. The films can
include polyamic acid (PAA). Methods of making and using the film
for food product coverings is also included.
Inventors: |
Sadik; Omowunmi A.; (Vestal,
NY) ; Yazgan; Idris; (Johnson City, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Research Foundation for The State University of New
York |
Albany |
NY |
US |
|
|
Assignee: |
The Research Foundation for The
State University of New York
Albany
NY
|
Family ID: |
1000005220326 |
Appl. No.: |
17/038452 |
Filed: |
September 30, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15987198 |
May 23, 2018 |
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17038452 |
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62509919 |
May 23, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/02 20130101;
C08J 3/24 20130101; B65D 65/466 20130101; C08L 79/08 20130101; A01N
37/24 20130101; C08J 2379/08 20130101; C08J 5/18 20130101; A01N
25/10 20130101; C08K 5/0058 20130101; C08K 5/175 20130101; C08K
5/07 20130101 |
International
Class: |
C08J 5/18 20060101
C08J005/18; C08L 79/08 20060101 C08L079/08; A01N 37/24 20060101
A01N037/24; B65D 65/46 20060101 B65D065/46; G01N 33/02 20060101
G01N033/02; C08J 3/24 20060101 C08J003/24; A01N 25/10 20060101
A01N025/10; C08K 5/17 20060101 C08K005/17; C08K 5/07 20060101
C08K005/07 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grants
CBET 1230189 and DMR 1007900 awarded by the National Science
Foundation. The government has certain rights in the invention.
Claims
1.-37. (canceled)
38. A film comprising: polyamic acid (PAA); glutaraldehyde (GA);
and one or more molecule selected from the group consisting of
alanine (A), tryptophan (W), 2-benzoylbenzoyl (BB),
Polycaprolactone (PCl), L-Cysteine (C), D-glucosamine (DA),
dipropylene glycol (DP), p-aminobenzoic acid (pAB), L-isoleucine
(I), p-aminosalicylic acid (pAS), sulfanilic acid (SA), and
5-aminosalicylic acid (5AS).
39. The film of claim 38, wherein the one or more molecule is
selected from the group consisting of sulfanilic acid (SA),
p-aminosalicylic acid (pAS) and 5-aminosalicylic acid (5AS).
40. The film of claim 38, wherein the one or more molecule is
selected from the group consisting of alanine (A), tryptophan (W),
2-benzylbenzoyl (BB), L-Cysteine (C), D-glucosamine (DA),
dipropylene glycol (DP), p-aminobenzoic acid (pAB), L-isoleucine
(I), p-aminosalicylic acid (pAS), and 5-aminosalicylic acid
(5AS).
41. The film of claim 38, wherein the film has a thickness of about
0.02 mm to about 0.12 mm.
42. The film of claim 38, wherein the film has a modulus of
elasticity of about 2.2 GPa to about 4.1 GPa.
43. The film of claim 38, wherein the film has a tensile strength
of about 59.9 MPa to about 95.1 MPa.
44. The film of claim 38, wherein the film has an antibacterial
activity of reducing the number of colony forming units (CFUs) by
up to about 90%.
45. The film of claim 44, wherein the antibacterial activity
against one or more of gram-positive and gram-negative bacterial
species.
46. The film of claim 45, wherein the gram-positive species is
Staphylococcus epidermidis and Listeria monocytogenes.
47. The film of claim 45, wherein the gram-negative species is one
or more of Escherichia coli, Enterobacter aerogenes, Aeromonas
hydrophila, and Citrobacter freundii.
48. The film of claim 38, wherein the film is synthesized from a
solvent and does not include any petrochemical material.
49. The film of claim 38, wherein the solvent is ethanol.
50. The film of claim 38, wherein the film further comprises
water.
51. The film of claim 38, wherein the film further comprises acetic
acid.
52. The film of claim 38, wherein the concentration of GA is
between about 0.21% and about 0.35%.
53. The film of claim 38, further comprising one or more of an
adipate, a phthalate, a citrate and chitosan.
54. The film of claim 38, further comprising one or more of an
oleic acid, a palmitoleic acid, a sapienic acid, a linoleic acid, a
nitrile, a polychloroprene, a chlorinated polyethylene, an
epichlorohydrin, a sabacate, a terephthalate, a gluterate, and an
azelate.
55. A film comprising: polyamic acid (PAA); a cross-linker; and a
small molecule, wherein the small molecule is a molecule having a
molecular weight of less than about 900 Daltons.
56. The film of claim 55, wherein the cross-linker comprises one or
more of glutaraldehyde and carbodiimidazole.
57. The film of claim 55, wherein the small molecule is one or more
molecules selected from the group consisting of sulfanilic acid
(SA), p-aminosalicylic acid (pAS) and 5-aminosalicylic acid
(5AS).
58. The film of claim 55, wherein the small molecule is one or more
molecules selected from the group consisting of alanine (A),
tryptophan (W), 2-benzylbenzoyl (BB), L-Cysteine (C), D-glucosamine
(DA), dipropylene glycol (DP), p-aminobenzoic acid (pAB),
L-isoleucine (I), p-aminosalicylic acid (pAS), 5-aminosalicylic
acid (5AS).
59. The film of claim 55, wherein the small molecule is one or more
molecules selected from the group consisting of alanine (A),
tryptophan (W), 2-benzylbenzoyl (BB), Polycaprolactone(PCl),
L-Cysteine (C), D-glucosamine (DA), dipropylene glycol (DP),
p-aminobenzoic acid (pAB), L-isoleucine (I), p-aminosalicylic acid
(pAS), sulfanilic acid (SA), 5-aminosalicylic acid (5AS).
60. The film of claim 55, wherein the film further comprises
water.
61. The film of claim 55, wherein the film further comprises acetic
acid.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. application
Ser. No. 15/987,198, filed on May 23, 2018, which claims benefit of
U.S. Provisional Application 62/509,919, filed on May 23, 2017, the
contents of which are incorporated by reference.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0003] The Sequence Listing in the ASCII text file, named as
34570A_SequenceListing.txt of 33 KB, created on Sep. 29, 2020, and
submitted to the United States Patent and Trademark office via
EFS-Web, is incorporated herein by reference.
BACKGROUND OF THE DISCLOSURE
[0004] Smart packaging requires the packaging materials to provide
simultaneous active protection and intelligent communication with
food and other perishable materials. In that respect, packaging
materials should perform the dual role of sensing and
packaging.
[0005] Smart packaging requires that the packaging materials
provide active protection and intelligent communication about the
packaged food. Package materials add extra protection for the food
by providing information about time and past conditions of the
food. Intelligent packaging advances communication capabilities of
traditional packaging materials by providing information about the
integrity and quality of the packaged foods and its surrounding
environment from packaging, to storage, transport and market
shelves. Current smart packaging uses radio frequency
identification, and indicators of environmental factors such as pH
and heat. Even though these are commercially available, the cost is
still high for large scale applications.
[0006] In contrast to intelligent packaging, active packaging does
not provide information about the condition of packaged food, but
enhances the shelf-life through a variety of mechanisms, including,
but not limited to, moisture absorption, antimicrobial packaging
material, antioxidants, carbon dioxide emitters and oxygen
scavengers.
[0007] Further, most food packaging materials in use are derived
from starting materials that are either obtained from
petrochemicals or they require the use of organic toxic solvents.
The resulting polymers are not biodegradable.
[0008] Currently, there is no practical food packaging system that
integrates intelligent and active capabilities and is also
biodegradable.
[0009] Therefore, what is desired is a film and film material that
can be used for, among other uses, food packaging, that provides
intelligent and active capabilities.
[0010] Embodiments of the present disclosure provide devices and
methods that address the above and other issues.
SUMMARY OF THE DISCLOSURE
[0011] The present disclosure is directed to films. The films can
include polyamic acid (PAA). Methods of making and using the film
for food product coverings is also included.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The file of this application contains at least one drawing
executed in color. Copies of this patent with color drawing(s) will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0013] The present disclosure will be better understood by
reference to the following drawings of which:
[0014] FIG. 1a is an illustration of the synthesis of PAA and
ternary PAA copolymers.
[0015] FIG. 1b is an illustration of the synthesis of PAA.
[0016] FIGS. 2a-2q are illustrations of NMR data.
[0017] FIGS. 3a-3f are illustrations of NMR data.
[0018] FIG. 4 is an illustration of NMR data.
[0019] FIGS. 5a-5b are illustrations of NMR data.
[0020] FIG. 6 is a graphical illustration of diffusion
coefficients.
[0021] FIG. 7 is a graphical illustration of the IR spectrum of
different PAA co-polymers.
[0022] FIGS. 8a-8h are illustrations of NMR data.
[0023] FIG. 9 is illustrations of different chemical
structures.
[0024] FIGS. 10a-10aj is photographs of various PAA films.
[0025] FIGS. 11a-11l is photographs of various PAA films.
[0026] FIGS. 12m-12p is photographs of various PAA films.
[0027] FIGS. 13a-13b are graphical illustrations of absorbance and
transmittance values of various PAA films.
[0028] FIGS. 14a-14b are graphical illustrations of absorbance and
emission values of various PAA films.
[0029] FIGS. 15a-15e are graphical illustrations of fluorescence
values of various PAA films.
[0030] FIGS. 15a-15e are graphical illustrations of fluorescence
values of various PAA films.
[0031] FIGS. 16a-16b are graphical illustrations of fluorescence
values of various PAA films.
[0032] FIGS. 17a-17e are graphical illustrations of fluorescence
values of various PAA films.
[0033] FIGS. 18a-18d are graphical illustrations of fluorescence
values of various PAA films.
[0034] FIGS. 19a-19d are graphical illustrations of fluorescence
values of various PAA films.
[0035] FIGS. 20a-20d are graphical illustrations of fluorescence
values of various PAA films.
[0036] FIGS. 21a-21d are graphical illustrations of fluorescence
values of various PAA films.
[0037] FIGS. 22a-22d are graphical illustrations of absorbance and
fluorescence values of various PAA films.
[0038] FIGS. 23a-23e are graphical illustrations of fluorescence
values of various PAA films.
[0039] FIG. 24 is a graphical illustration of absorbance values of
various PAA films.
[0040] FIGS. 25a-25c are graphical illustrations of intensity
values of various PAA films.
[0041] FIGS. 26a-26g are graphical illustrations of intensity
values of various PAA films.
[0042] FIG. 27 is a graphical illustration of emission values of
various PAA films.
[0043] FIGS. 28a-28c are graphical illustrations of absorbance and
intensity values of various PAA films.
[0044] FIG. 29 is a photograph of the solubility of ternary PAA
membranes in basic solutions.
[0045] FIG. 30 is a photograph of the color changes of ternary PAA
membranes in response to alcohol exposure.
[0046] FIG. 31 is a photograph of various PAA films.
[0047] FIG. 32 is a photograph of color changes in response to
alteration in environmental conditions that can be further advanced
with pH-dependent dyes.
[0048] FIG. 33 is a photograph of an aged PAA film.
[0049] FIG. 34 is a photograph of a four-probe and Ohm meter for
characterization of electronics properties.
[0050] FIGS. 35a-35c is a photograph of PAA films.
[0051] FIGS. 36a-36b are graphical illustrations of voltage vs.
current of various PAA films.
[0052] FIGS. 37a and 37b are SEM images of PAA films.
[0053] FIGS. 38a-38h are graphical illustrations of voltage vs.
current of various PAA films.
[0054] FIG. 39 is a digital image of the oil-permeability test.
[0055] FIG. 40 is a digital image of the water-vapor permeability
test.
[0056] FIGS. 41a-4j are illustrations of NMR data.
[0057] FIG. 42 is a picture of a macroscopic and four microscopic
pictures of Trichaptum biforme.
[0058] FIG. 43 is microscopic pictures of Fusarium oxysporum.
[0059] FIGS. 44a-44b are graphical illustrations of membrane
loading in a well and cytotoxicity.
[0060] FIGS. 45a-45av are photographs of various PAA films.
[0061] FIGS. 46a-46b are SEM images of various PAA films.
[0062] FIGS. 47a-47h are photographs of various PAA films.
[0063] FIGS. 48a-48b are SEM images of various PAA films.
[0064] FIGS. 49a-49b are SEM images of various PAA films.
[0065] FIGS. 50a-50b are SEM images of various PAA films.
[0066] FIGS. 51a-51b are SEM images of various PAA films.
[0067] FIGS. 52a-52b are SEM images of various PAA films.
[0068] FIGS. 53a-53b are SEM images of various PAA films.
[0069] FIGS. 54a-54d are SEM images of various PAA films.
[0070] FIGS. 55a-55f are SEM images of various PAA films.
[0071] FIGS. 56a-56b are SEM images of various PAA films.
[0072] FIGS. 57a-57b are SEM images of various PAA films.
[0073] FIGS. 58a-58m are illustrations of different chemical
structures.
[0074] FIG. 59 is a graphical illustration of phase-inversion in
coagulation-bath.
[0075] FIGS. 60a-60p are photographs of various PAA films.
[0076] FIGS. 61a-61e are photographs of various PAA films.
[0077] FIGS. 62a-62g are photographs of various PAA films.
[0078] FIGS. 63a-63k are photographs of various PAA films.
[0079] FIGS. 64a-64d are SEM images of various PAA films.
[0080] FIGS. 65a-65d are photographs of various PAA films.
[0081] FIG. 66 is a table of various PAA films' properties.
[0082] FIGS. 67a-67e are photographs of various PAA films covering
various foods.
[0083] FIGS. 68a-68c are photographs of various PAA films covering
various foods.
[0084] FIG. 69 is a graphical illustration of a color change of a
PAA film.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0085] In the discussion and claims herein, the term "about"
indicates that the value listed may be somewhat altered, as long as
the alteration does not result in nonconformance of the process or
structure to the illustrated embodiment. For example, for some
elements the term "about" can refer to a variation of .+-.0.1%, for
other elements, the term "about" can refer to a variation of .+-.1%
or .+-.10%, or any point therein.
[0086] As used herein, the term "substantially", or "substantial",
is equally applicable when used in a negative connotation to refer
to the complete or near complete lack of an action, characteristic,
property, state, structure, item, or result. For example, a surface
that is "substantially" flat would either completely flat, or so
nearly flat that the effect would be the same as if it were
completely flat.
[0087] As used herein terms such as "a", "an" and "the" are not
intended to refer to only a singular entity, but include the
general class of which a specific example may be used for
illustration.
[0088] As used herein, terms defined in the singular are intended
to include those terms defined in the plural and vice versa.
[0089] Reference herein to any numerical range expressly includes
each numerical value (including fractional numbers and whole
numbers) encompassed by that range. To illustrate, reference herein
to a range of "at least 50" or "at least about 50" includes whole
numbers of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, etc., and
fractional numbers 50.1, 50.2 50.3, 50.4, 50.5, 50.6, 50.7, 50.8,
50.9, etc. In a further illustration, reference herein to a range
of "less than 50" or "less than about 50" includes whole numbers
49, 48, 47, 46, 45, 44, 43, 42, 41, 40, etc., and fractional
numbers 49.9, 49.8, 49.7, 49.6, 49.5, 49.4, 49.3, 49.2, 49.1, 49.0,
etc. In yet another illustration, reference herein to a range of
from "5 to 10" includes whole numbers of 5, 6, 7, 8, 9, and 10, and
fractional numbers 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,
etc.
[0090] As used herein, the term "film" refers to a thermoplastic
film made using a film extrusion and/or foaming process, such as a
cast film or blown film extrusion process. For the purposes of the
present invention, the term includes nonporous films as well as
microporous films. Films may be vapor permeable or vapor
impermeable, and function as liquid barriers under normal use
conditions.
[0091] As used herein, the term "thermoplastic" refers to polymers
of a thermally sensitive material, which flow under the application
of heat and/or pressure.
[0092] As used herein, the term "polymers" includes, but is not
limited to, homopolymers, copolymers, such as for example, block,
graft, random and alternating copolymers, terpolymers, etc. and
blends and modifications thereof. Furthermore, unless otherwise
specifically limited, the term "polymer" shall include all possible
geometrical configurations of the material. These configurations
include, but are not limited to isotactic, syndiotactic and atactic
symmetries.
[0093] Biodegradable Ternary Co-polymers of Conducting
Electroactive PAA Membranes hereby referred to as membranes or
films. For descriptive purposes, the term membrane has the same
definition as that of the term "film" discussed above.
[0094] Polyamic acid (PAA) is a polymer that has many novel
properties. PAA is electroactive, substantially biodegradable and
has free carboxyl and amide groups that can act as molecular
anchors. PAA can also be used in conjunction with both organic and
inorganic solvents due to its substantial chemical resistance. PAA
is a generic name of use for the polycondensation product of
dianilines and dianhydrides synthesized in anhydrous organic
aprotic polar solvents.
[0095] The present disclosure is directed to PAA films and PAA
films as food packaging materials that can provide both
active-packaging qualities and intelligent-packaging qualities.
These PAA films and PAA films as food packaging materials can be
formed without any petroleum based or petrochemical ingredient
and/or any ingredient formed from a hydrocarbon and/or without an
organic solvent.
[0096] The PAA films were created from compositions including
biological compounds (e.g. amino acids, sugars) and one or more of
intrinsic antimicrobial agents (e.g. sulfanilic acid,
p-aminosalicylic acid), and cross-linkers (e.g. glutaraldehyde,
carbodiimidazole) in the presence of other substances, for example
diamines and dianhydrides. Also, PAA can be further modified into
polyimide depending on the processing conditions or employed as
stabilizing agents during nanoparticles synthesis.
[0097] H DOSY NMR studies showed that the average molecular weight
of PAA films were between about 10.sup.6 and about 107 Da while
average molecular weight of regular PAA polymer was about
1.43.times.10.sup.5 Da.
[0098] PAA has advanced mechanical properties in the range of about
2.2-about 2.7 GPa modulus elasticity comparable to strong plastics
(2.4 to 3.2 GPa).
[0099] PAA also demonstrates stability in common solvents, high
optical transparency, impermeability to gas exchange, oil and water
vapor transfer.
[0100] FIG. 66 is a table of modulus elasticity, tensile strength
and elongation of six films of the present disclosure (bottom six
films on the list) as compared to other non-PAA films.
[0101] Non-crude oil-based plastic PAA films illustrate voltage
changes in response to pH change. Showing a trend in response to pH
change demonstrates the intelligent properties of the packaging
material of the present disclosure, which does not require
complicated sensor electronics to indicate food freshness/quality.
In the table below, it can be also seen that there is a voltage
change in the disclosed films as a function of their thickness.
TABLE-US-00001 TABLE A Membrane Type Thickness [mm] DC Voltage [mV]
PAA-A-GA 0.02 -0.7 PAA-W-GA 0.02 1.2 PAA-BB-GA 0.02/0.06 -0.4/0.4
PAA-PCI-GA 0.06 0.9 PAA-C-GA 0.02 1.8 PAA-DA-GA 0.03 0.1 PAA-PCI-GA
0.06/0.12 -0.6/-1.1 PAA-W-GA 0.02 2.4 PAA-A-GA 0.05 0.4 PAA-DP-GA
0.05 -0.4 PAA-pAB-W-GA 0.06 -0.9 PAA-pAB-GA 0.06 -0.5/-0.4 PAA-A-GA
0.09 -0.6
[0102] In Table A PAA: Poly (amic) acid; GA: Glutaraldehyde and the
letters in the middle refer to different small molecules such as
A-alanine and W-tryptophane. As referred to herein, the term small
molecule can refer to any organic molecule having a low molecular
weight of less than about 900 Daltons) that may regulate a
biological process, with a size on the order of about 1 nm. Type of
small molecule and concentration of glutaraldehyde affect voltage
of dry PAA membrane's potential, and its behavior against changes
as a function of pH and salt concentrations.
[0103] Microbiological tests showed that there was no bacterial
development which means that PAA copolymer films developed in
connection with the present disclosure worked as a strong active
packaging material. As a packaging material, the PAA films can be
provided on a roll, the film provided with a predetermined width
and a predetermined length. An example of an existing roll of this
type would be a roll of Saran.TM. Wrap, having a width of about
12'' and a length of tens or hundreds of feet. Rolls of the
disclosed PAA films can be wider or narrower, and can also be
longer or shorter than this example, as desired.
[0104] The disclosed PAA films can be applied so as to cover an
entire food product, or a portion thereof. The PAA films can also
be provided so as to contact the entire food product or a portion
of the food product, or so as to not contact any portion of the
food product due to an intervening material or a space between the
food product and the PAA film. The disclosed PAA films can be
applied by a user and/or the disclosed PAA films can be applied by
a packaging device/machine.
[0105] As discussed below, packaged food products did not include
color changes or fungal development, this is related to the
non-porous nature of PAA film, which did not allow air and water
vapor entry. Measured voltage (0.2 mV) did not show any changes
which means that there was no food spoilage and decomposition
during the tested times.
[0106] The utilization of organic solvents such as ethanol is
generally not preferred in the synthesis of PAA since they are
nucleophiles and can compete with the dianiline component to attack
the dianhydride resources. In the present disclosure, the use of
ethanol and even water as part of the solvent system did not show
any effect on the formation of the PAA polymer when solid
dianhydride was added to an already dissolved dianiline. This
represents a major deviation from standard chemistry of PAAs and
one that has led to the preparation of a new class of stable
polymeric compositions and novel processing procedures as reported
here. We however, observed the (FIGS. 2a-2q) formation of ester and
alteration in the repeating units of PAA. As shown in Table B, the
utilization of ethanol as part of the solvent system significantly
improved the mechanical properties of the synthesized
films/membranes.
TABLE-US-00002 TABLE B Selection of solvent and formation of
viscous PAA solution. Mixture of solvent Observation 50:50 or 35:65
High viscosity, require warning (i.e. 50.degree. C.), DMAC:EtOH
resulting in membranes that are strong but limited colors (no blue
color obtained) 25:75 Medium viscosity, require warning DMAC:EtOH
(i.e. 60.degree. C.), resulting in membranes that are strong, but
limited colors 35:50:15 or Medium viscosity, require warning
30:50:20 (i.e. 60.degree. C.), produces membranes with broad
DMAC:EtOH:Water range of colors. The color intensity is stronger
than those prepared using DMAC. 60:40 Did not form PAA viscous
solution but DMAC:Water resulted in yellow precipitate. This did
not result in any membrane formation. 60:30:10 Did not form PAA
viscous solution but DMAC:Water:AcOH resulted in yellow
precipitate. This did not result in any membrane formation. Acetic
acid limits the role of GA. indicates data missing or illegible
when filed
[0107] It should be noted that heating was not continuous; rather
it was stopped right after PMDA was added to the dissolved
4,4'-oxydianiline (ODA). Continuation of heating resulted in highly
viscous PAA solution which does not allow membrane formation.
[0108] Another important observation noted here was that the
average molecular size of PAA polymers decreased when the solvent
system changed from DMAC to DMAC/Ethanol, and further decreases
were observed for DMAC/Ethanol/Water system.
[0109] Parameters relating to the synthesis of FIG. 1 a membranes
were evaluated at four aspects(1)-(4).
(1) Formation of Amorphous, Glassy and Plasticized Membranes
[0110] When pure PAA viscous solution (either from ODA+PMDA or
PDA+PMDA) was casted on glass to form membrane, the fate of the
membrane was shown to be determined by evaporation mediated solvent
elimination and solvent-nonsolvent exchange in coagulation
bath.
TABLE-US-00003 TABLE C Effect of evaporation period on PAA membrane
preparation Incubation time (h).sup.1 Texture Character <4 h
Amorphous Similar to phase inverted PAA membrane in
coagulation-bath 4-8 h Glassy The outer surface is shiny, but not
totally plasticized. The membrane turns into brittle form within 2
h right after being taken out from the coagulation-bath 6-10 h Mix
Mostly the outer layer is fully plasticized while the inner part is
amorphous. The membranes are durable, and never turn into a glassy
form >12 h Plasticized Plastic-like transparent membrane,
durable and flexible. Coagulation-bath doesn't affect appearance of
the membrane Incubation time refers to the time-period when
membranes were incubated under a hood at 80 rpm/min face-shield. In
all cases, the coagulation bath employed was pure-water.
.sup.1Thickness and viscosity of the casted PAA solution affected
the time requirement, but 12 h or over were enough to obtain
plasticized membranes for the casted solutions at up to 2 mm
(beyond this point, thicker membranes were not tested) thickness.
For thin membranes (e.g. below 50 .mu.m), 6 h was enough to obtain
fully plasticized membranes. Evaporation of the solvent is the main
element determining the fate of the membrane's texture. This is
further discussed below. However, the most prominent parameter is
the humidity of the surrounding environment. However, pre-heating
the casted PAA solution decreases the negative effect of the
humidity, which can lead to accelerated removal of DMAC coupled
with enhanced GA activity.
(2) Crosslinker Effect on Membrane Formation
[0111] In accordance with the present disclosure, glutaraldehyde
(GA) was used as the cross-linker due to the fact that GA provided
the most pronounced effect on PAA membrane formation.
TABLE-US-00004 TABLE D Effect of evaporation period on PAA-GA
membrane preparation Incubation time (h).sup.1 Texture Character
<2 h Amorphous Similar to phase inverted PAA membrane in
coagulation-bath 4-6 h Glassy The outer surface is shiny, but not
totally plasticized while the edges are totally plastic-like. The
membrane turns into brittle form within 1 h right after taken out
from the coagulation-bath. The brittle form shows very high glassy
character. 4-8 h Mix The outer layer is fully plasticized while the
inner part is amorphous. Only thicker membranes (e.g. over 2 mm)
forms this type of membranes. These ultimately turns into glassy-
brittle form within days. >8 h Plasticized Plastic-like
transparent membrane, durable, flexible and non-soluble in common
organic solvents. Coagulation-bath does not affect appearance of
the membrane .sup.1Viscosity of the casted solution is determined
by GA activity. Other than GA, other crosslinkers were also used as
detailed below.
[0112] The time difference between transformations from amorphous
to glassy texture were linked to the degree of crosslinking. This
is attributed to the fact that the cross-linker is becoming an
element in determining the fate of the membrane in terms of color
and texture. It is not critical that the membrane loses a higher
proportion of the DMAC in order to form the plasticized PAA
membranes. This is related to cross-linking of individual PAA
membranes with crosslinker (i.e. glutaraldehyde). For example, in
the case of p-phenylenediamine (PDA)-PMDA based PAA membrane, 30
min incubation is sufficient to provide the plasticized PAA
membranes unlike the ODA-PMDA based PAA membrane that requires over
4 h incubation. This is expected because PDA has two amino groups
which enhance its cross-linking with GA. Even though, no chemical
treatment was performed in DMAC, the final forms of the membranes
even for relatively lower GA concentrations (pre-diluted in DMAC)
were still obtained in the plasticized form. This was not common
for the GA concentrations that were directly added from stock. For
example, in the case of PAA-CS-GA membrane, the same amount of GA
when dissolved in water produced an amorphous membrane while GA
that was pre-diluted in DMAC provided plasticized membrane.
Further, heat treatment to GA/PAA membranes resulted in plasticized
membranes, which can be related to the promotion of cross-linking
and faster evaporation of solvent. Further details are provided
below.
(3) Small-Molecule Effect
[0113] None of the small molecules showed any strong impact on
membrane formation when added to the casted PAA solution without
the co-addition of the cross-linker. Increase in viscosity related
to the addition of small molecule (excluding the cross-linker GA)
did not affect the overall membrane formation (amorphous, glassy or
plasticized) as detailed below. However, the use of small molecules
in the presence of the cross-linkers significantly impacts the
structure, the plasticity and other notable physical attributes of
the resulting membranes.
[0114] As shown below, while certain small molecules with PAA
copolymers ended up as plasticized membranes, others were amorphous
in nature. Similarly, the mechanical properties of the membrane
under same conditions showed direct dependence on type of the small
molecule employed.
[0115] This above chart illustrates the effect of small molecule on
membrane formation. (i) Selection of small molecule is not limited
to these small molecules; (ii) GA concentration has strong
influence on the final form of the membrane apart from the small
molecule used in the study.
[0116] The addition of glutaraldehyde is an element in the kinetics
of membrane formation. For example, at 0.5% GA concentration, the
time needed to form a stand-alone membrane diminishes. This change
is believed to be coming from the alteration in the characteristics
of the solution itself. For example, PAA alone requires 12h to form
a membrane of PAA alone; PAA-GA requires 8h while PAA-GA-SA
requires 4h to give stand-alone (FIG. 1a) membranes. The time
requirement for providing stand-alone membrane is subject to change
in response to thickness of the casted solution. However, it should
be noted that obtaining stand-alone membrane in shorter period may
not be related to faster drying.
[0117] When the stand-alone membrane is first obtained, its
mechanical property is poorer than the membranes that are fully
dried. The modulus of elasticity and tensile strength are the main
parameters that improved dramatically when the membrane is fully
dried. In contrast to this, % elongation decreases at least
two-times upon total drying, which was observed for PAA-I-GA,
PAA-K-GA, PAA-CA-GA or thicker PAA-GA (over 2 mm casted solution)
membranes; when they get dry, they show very high glassy character
which makes them as brittle as glass.
[0118] L-alanine and L-cysteine in all cases of provided
plasticized PAA membranes, and L-tryptophan-methyl ester also
provided plasticized-membranes if the conditions are controlled in
terms of humidity and heat. However, utilizing higher
concentrations of GA (i.e. 2% or higher) for any type of small
molecule co-polymerized with PAA resulted in plasticized PAA
membranes. These also affect the formation of colorful membranes.
Here, for example, L-alanine gives green membrane while PAA-GA
gives chestnut color membrane. Actually, PAA-A-GA provided the
membranes which were the best examples of plasticized membranes for
FIG. 1a membranes, which was also comparable to the membranes
obtained from FIG. 1b. Regarding color formation, even for
PAA-small molecule co-polymers, the age of GA can influence the
results.
(4) Final Step of Coagulation Bath
TABLE-US-00005 [0119] TABLE E Effect of coagulation bath on
membrane surface characteristics. Coagulation-bath
Surface-characteristics Pure-water Shiny, porous or non-porous
Methanol.sup.1,2 Sponge, nano-fabric or porous Ethanol.sup.1,3
Sponge, nano-fabric or porous Ethanol-water mixture.sup.1 Sponge
and non-porous In Table E, .sup.1For extended evaporation times,
sonication might be required in order to obtain nano-fabric and/or
sponge surfaces. .sup.2The membranes, which are giving glassy
texture/form in the case of pure-water coagulation bath, give
sponge or non-porous surfaces and durable membranes. .sup.3In the
case of pure-water coagulation bath, the membranes becomes brittle
within times.
[0120] Membranes possessing nano-porous, sponge, nano-fabric,
non-porous and featureless surfaces can be obtained using the
method depicted in FIG. 1a, which are detailed below.
[0121] Characteristic differences exist between the methods
depicted in FIG. 1b and FIG. 1a. These differences are discussed
below and throughout the application. These include (i) evaporation
is the main driving force for phase-inversion, (ii)
small-molecules, which are co-polymerized with PAA are dissolved in
PAA viscous solution or pre-dissolved in a solvent prior to being
co-polymerized with PAA, (iii) small-molecule can be cross-linked
with the cross-linker in order to adapt the overall properties of
the resulting membrane, and (iv) flexible design via combination of
small molecules and/or the order of the addition of the small
molecule or cross-linker.
[0122] GA is a cross-linker. Since GA can exist in different
chemical forms in aqueous and organic solvents (as detailed below)
it is adapted for the objectives met by the present disclosure. For
example, GA can be polymerized into a water-soluble and non-soluble
forms based on the objective. Here, GA was first aged through
incubating the solution at 70.degree. C. for hours. Optimization
was followed with .sup.1H NMR characterization. As detailed below,
the following manipulations were performed for GA to obtain the
desired membranes; [0123] i. Utilization of aged or non-aged GA.
While aged GA is used in order to obtain fluorescently active
membranes, non-aged GA was preferential to obtaining physically
strong membranes. [0124] ii. Quenching GA activity with methanol or
ethanol is needed in order to obtain physically strong membranes.
The addition of methanol results in physically strong and
non-soluble membranes. [0125] iii. Concentration of GA or
heat-treatment of GA before it is introduced to PAA solution has
strong effect on membrane formation with respect to color and
time-requirement for stand-alone membrane formation. [0126] iv.
Very high concentrations of GA (i.e. over 2%) prevent the formation
of ideal long-lasting membranes; higher GA makes PAA membranes
brittle and even in some cases disrupts proper membrane formation.
In the case of imidazole, concentrations can vary as described
below.
[0127] As used herein, the term "fresh" or "non-aged" GA refers to
GA purchased from companies, which were used as received and stored
at all times at about -20.degree. C. The term "aged" GA refers to
GA that was kept in an oven for about 1-2 hours (e.g. 50-70.degree.
C.) prior to use. The term "over-aged" refers to GA that was stored
at room temperature for about 2 weeks or longer.
[0128] The data and discussion below presents the development,
processing, characterization and novel applications of the
disclosed films. Due to the organic solvents being environmental
pollutants, replacing them with substantially environmentally
benign solvents are desired.
[0129] 1D and 2D NMR techniques indicated that DMAC/EtOH and
DMAC/EtOH/Water solvent mixtures were applicable for generating PAA
polymers synthesized in DMAC. Reducing the use of DMAC by about 75%
did not affect the PAA synthesis. However, the repeating units were
altered as cis-/trans-ratio and the average molecular weights of
the PAA polymers decreased by up to 5 times. The use of
crosslinkers, especially GA, was utilized to alter the kinetics of
the phase-inversion. GA is a component in the synthesis leading to
the formation of amorphous and plasticized membranes. Small
molecules were co-polymerized with PAA to manipulate the overall
properties of the membrane with respect to their plasticity,
antimicrobial properties and mechanical strengths, as discussed in
detail below.
[0130] The methods, apparatus and compositions of the present
disclosure will be better understood by reference to the following
Examples, which are provided as exemplary of the disclosure and not
by way of limitation.
Example 1.1-Materials and Methods
[0131] All of the reagents used in this and the following examples
were purchased from Sigma-Aldrich (St. Louis, Mo.). Escherichia
coli ATCC.RTM. 25922 Citrobacter freundii, ATCC.RTM. 8090 and
Staphylococcus epidermidis ATCC.RTM. 12228.TM. were purchased from
American Type Culture Collection (ATCC) (Manassas, Va., USA).
Dimethyl sulfoxide (DMSO)-d.sub.6, was purchased from Cambridge
Isotope Laboratories (Andover, Mass. USA). Unless otherwise
specified, phosphate saline (PBS) buffer was used as 50 mM pH 7.2.
All solutions were prepared with triply distilled Nanopure water
with resistivity of 18 M.OMEGA..
[0132] The above figure illustrates the steps in the synthesis of a
PAA Polymer of the present disclosure using optional solvent
systems: The ratio of 4,4'-oxydianiline (ODA):PMDA was tested from
1.20:1.00 to 1.00:1.05. The ratio given for each solvent was used
through the examples.
[0133] The synthesis of PAA films in accordance with the present
disclosure is shown in FIG. 1a. In the process of FIG. a,
crosslinkers serve as a reactive transforming agent that crosslinks
re-organize the kinetics of the membrane formation and define the
fate of the membrane.
[0134] The superscript numbers herein refer to the steps of the
corresponding superscript numbers in FIG. 1a. In FIG. 1a, the
preparation of PAA and Ternary PAA co-polymers is illustrated. MeOH
refers to methanol and DMAC refers to N,N'-dimethylacetamide.
.sup.1Examples include D-glucosamine, L-lysine, L-alanine and other
amino acids. .sup.2Glutaraldehyde stock was obtained in water, but
throughout the study it was added as pre-dissolved in methanol or
DMAC to possibly alter its activity by changing the working
microenvironment. .sup.3Since pre-treating GA with MeOH or DMAC
affected the activity; their incubation at room temperature was
taken at periodic intervals. But incubation time was changed just
to alter the resulting surface properties of the PAA membrane.
.sup.4Phase-inversion in ethanol/water mixture was applied to alter
the surface properties of the resulting membrane. .sup.5The last
step of phase-inversion took place in nano-pure water, followed by
drying under hood.
[0135] Further synthesis of PAA films is shown in FIG. 1b. The
superscript numbers herein refer to the steps of the corresponding
superscript numbers in FIG. 1b. .sup.1Methanol can be added to the
system at this stage; .sup.2This procedure was only used for amino
acids and glucosamine; .sup.3Methanol can be added to the system
immediately after the introduction of GA-biomolecule; .sup.4The
membrane can be sonicated in methanol/ethanol/methanol-water
mixture. .sup.5The small molecules, 4-amino-2-chlorobenzoic acid,
p-aminobenzoic acid and aminosalycilic acid, could be added to the
system during PAA synthesis. Films generated using the synthetic
method of FIG. 1b were used for food-packaging throughout the
examples.
Example 1.2-Structural Characteristics of PAA Films
[0136] The PAA film and the functionalized derivatives were
dissolved in DMSOd6 (unless otherwise stated) and then subjected to
1H Nuclear Magnetic Resonance (NMR), 13C NMR, and 1H-correlation
spectroscopy (COSY), 1H 13C Heteronuclear Single Quantum Coherence
(HSQC), 1H 15N HSQC, 1H 13C Heteronuclear Multiple Bond Coherence
(HMBC) and 1H 15N HMBC characterizations. A Bruker AM 600
spectrometer operated by Topspin.TM. 3.0 NMR software was used for
spectra measurement and analysis.
[0137] In order to fully annotate structure of phase inverted PAA
and the designed PAA, NMR and IR experiments were performed. NMR
was also used to monitor the possibility of Bisphenol A formation
in relation to heat treatment and exposure time.
[0138] To move step by step, ACD/ChemSketch (Freeware) academic
edition was used to draw the PAA structures, and PAA-GA
interaction. This was due to the fact that in all cases, GA was
used as an element in preparation of PAA films.
##STR00001##
[0139] In the above structures, the proposed structure of PAA
polymers are shown. [A].sub.a-[B].sub.b-[A]c-[B].sub.d [a, b, c and
d can be 1 or more, and can be the same or different]. In the case
of PAA synthesized in DMAC, [A].sub.2-[B].sub.3-[A].sub.2-[B].sub.3
is proposed as the possible structure.
TABLE-US-00006 TABLE F .sup.1H NMR of PAA and ternary PAA Films
Carboxyl Amino Carbonyl Aromatic Aliphatic Film group group group
protons proton PAA 13.05 10.56/10.53 N/A 8.35/8.00/7.74 N/A
7.72/7.05 PAA.sup.1 Not 10.57/10.54 N/A 8.37/8.02/7.77 N/A visible
7.73/7.06 PAA-GA 13.33 10.55/10.52 9.26 8.34/7.99/7.74 Not clear
7.71/7.05 PAA-GA.sup.2 13.54 10.65/10.54 9.30/9.22/9.1/8.75
8.33/7.97 5.00/4.92/4.87 10.52 7.70/7.04 4.68/1.77/1.23 PAA-GA- Not
10.66/10.55 9.32/9.28 8.35/8.00 4.68/3.51 SA.sup.1,2 visible 10.53
7.72/7.05 1.66/1.22 PAA-GA- 13.00 10.65/10.54 9.32/9.30
8.33/7.97/7.76 5.26/2.03/1.23 SA-pAS.sup.2 10.51 7.70/7.04
0.93/0.83 PAA-GA- 13.19 10.65/10.53 9.32/9.31/9.29 8.33/7.98/7.72
2.03/1.91/1.90 SA-pAS-A 10.51 9.06/9.04 7.70/7.04 1.39/1.23 PAA-GA-
13.19 10.65/10.53 9.39/8.74 8.33/7.97 5.74/2.08/1.23 SA-pAS.sup.2,3
10.51 7.70/7.04 PAA-GA- 13.16 10.66/10.54 9.29/9.22 8.33/7.97/7.72
6.09/5.97/4.32 SA.sup.4 10.51 7.70/7.04 3.69/3.45/1.32
1.23/1.05
TABLE-US-00007 TABLE G .sup.13C NMR of PAA and ternary PAA film
Film Carbonyl Carboxl Amide aromatic Aliphatic PAA 165.79/ 166.73/
152.95 141.20/139.08/134.81 N/A 165.69 166.42 133.12/129.83/128.86
127.56/121.53/118.83 PAA.sup.1 165.89/ 166.81/ 152.05
141.29/139.18/134.87 N/A 165.79 166.51 133.99/133.18/130.90
129.1/128.95/127.64 121.63/120.33/118.91 117.61/116.58 PAA- 165.77/
166.68/ 152.94 141.21/139.08/134.79 Not seen GA 165.66 166.38
133.06/129.05/128.83 127.53/121.51/118.81 118.29 PAA- 165.70/
166.62/ 152.86 141.14/139.01/134.74 Not seen GA.sup.2 165.59 166.31
133.71/129.02/128.77 167.59/ 127.49/121.44/121.35 167.30
118.74/118.72 PAA- 165.86/ 166.75/ 153.02 141.28/140.68/139.16
37.57 GA- 165.75 166.46 136.52/134.84/133.18 SA.sup.1, 2 167.28/
166.36 130.83/129.13/128.91 167.89 121.60/118.88 PAA- 165.72/
166.63/ 152.89 141.17/140.45/139.04 Not seen GA- 165.61 166.33
134.75/133.02/130.72 SA- 129.03/128.78/127.49 pAS-A
121.47/120.06/118.76 118.74 PAA- 165.73/ 166.63/ 152.89
141.18/140.47/139.05 37.47 GA- 165.63 166.33 134.75/131.66/130.72
SA- 167.60/ 129.04/127.49/121.48 pAS.sup.2, 3 167.27
120.05/118.77/118.74 PAA- 165.72/ 166.65/ 152.88
141.16/139.03/134.76 13.78 GA- 165.62 166.34 133.05/130.75/129.02
SA.sup.4 128.79/127.50/121.62 121.46/118.76/118.74 The following
superscript numbers refer to the tables above: .sup.1very high
concentration (150 mg/mL) of PAA; .sup.2GA used high concentration
2%. .sup.3high concentration (80-100 mg/mL) of pAS. .sup.4PAA was
synthesized in 65:35, Ethanol:DMAC. Protons of
N,N'-dimethylacetamide were not listed on the table since they are
only impurities.
[0140] Tables F and G provide a comparison for PAA alone vs.
various PAA films. Since GA and small molecules were used at very
low amount in comparison to PAA, .sup.1H and .sup.13C NMR
techniques did not provide the presence of new peaks for each
group. However, at higher amount of sulfanilic acid and
glutaraldehyde, the characteristic peaks related to these were
observed.
[0141] NMR data is shown in FIGS. 2a-2q. Specifically FIG. 2a is
NMR data for .sup.1H, FIG. 2b is NMR data for .sup.1H COSY, FIG. 2c
is NMR data for .sup.1H-.sup.13C HSQC, FIG. 2d is NMR data for
.sup.13C, FIG. 2e is NMR data for .sup.1H-.sup.13C HMBS, FIG. 2f is
NMR data for .sup.1H-.sup.15N HSQC, FIG. 2g is NMR data for
.sup.1H-.sup.15N HMBC and FIG. 2h is NMR data for co-presentation
of .sup.1H-.sup.13C HMBC and .sup.1H-.sup.13C HSQC spectra (blue
HMBC and red HSQC). Further NMR data for PAA membrane
phase-inverted in pure-water in FIG. 2i, which is NMR data for
.sup.1H, FIG. 2j is NMR data for .sup.1H COSY, FIG. 2k is NMR data
for .sup.1H-.sup.13C HSQC, FIG. 2l is NMR data for .sup.13C, FIG.
2m is NMR data for .sup.1H-.sup.13C HMBS, FIG. 2n is NMR data for
.sup.1H-.sup.15N HSQC and FIG. 20 is NMR data for .sup.1H-.sup.15N
HMBC of PAA-GA membrane prepared according to the method of FIG.
1b.
[0142] In this disclosure, NMR data is used to obtain physical,
chemical, electronic and structural information of the disclosed of
organic compounds. It is due mostly to the chemical shift on the
resonant frequencies of the nuclei present in the compound compared
to a reference magnetic field (usually tetramethylsilane or TMS).
Chemical shift is the function of the nucleus and its environment,
which is measured relative to a reference compound (i.e. TMS). As
for the specific NMR data presented in the figures of this
disclosure, PAA does not give peaks at the following region
including aliphatic region (single or double bond). So, any missing
PAA signature peak is an indication that the polymer is not present
or is degrading. The NMR images also provide how GA binds to the
PAA molecules.
[0143] The disclosed data is used to provide detailed information
on the topology, dynamics and three-dimensional structure of
molecules. The NMR data in FIGS. 2a-2o compare the NMR spectra of
PAA and PAA-GA. The figures generally illustrate the chemical
interaction of GA with PAA being due to cross linking between GA
and PAA.
[0144] .sup.1H NMR spectrum (FIG. 2a) of PAA depicts presence of
carboxyl, amino and aromatic protons. The aliphatic protons are
from residual N,N'-dimethylacetamide (DMAC). According to the
depicted .sup.1H spectrum, only one type of carboxyl group is
present in PAA polymer while two carboxyl carbons present in PAA
were revealed by .sup.13C spectrum (FIG. 2b). Since the
microenvironment of protons in the carboxyl group is more isolated,
it was observed as single carboxyl group.
[0145] However, an amino group proton was obtained as an overlap of
two peaks (FIG. 2a); according to .sup.1H-.sup.15N HSQC (FIG. 2f)
there is only one type of nitrogen, but the nitrogen locates in two
slightly distinct environments which explains the presence of the
overlapped peak. This was further supported by .sup.1H-.sup.15N
HMBC (FIG. 2g) spectrum where long-range couplings of the two
overlapped amino protons showed the same long-range couplings. The
overlapped peaks at 7.75 ad 7.72 ppm were from two different carbon
atoms which was supported by .sup.1H-.sup.13C HSQC (FIG. 2c) and
.sup.1H-.sup.13C HMBC (FIG. 2g); the peak 7.75 ppm gave cross-peak
with carbon peak at 128.92 ppm while the peak at 7.72 ppm gave
cross-peak with carbon peak at 121.57 ppm. According to
.sup.1H-.sup.13C HMBC (FIG. 2g) spectrum, both the protons gave
peak at 7.75 ppm and 7.7.72 ppm showed two distinct long range
couplings, which could not be obtained by just one proton on
proton. Further, three long-range couplings were observed for amide
carbon, which were linked to the protons on pyromellitic
dianhydride (PMDA) group. Therefore, all these results indicate
that several PAA structures can be produced in accordance with the
present disclosure.
[0146] Glutaraldehyde (GA) can bind at different positions to PAA.
According to .sup.1H (FIG. 2i) and .sup.13C (FIG. 2j), inclusion of
GA did not affect PAA structure, rather added new groups;
particularly, the presence of carbonyl proton and aliphatic protons
around 5 ppm and 1-2 ppm revealed that GA chemically bound to PAA.
Further indications that GA was chemically bound to PAA was
obtained from .sup.1H COSY, .sup.1H-.sup.13C HSQC,
.sup.1H-.sup.13C-HMBC and .sup.1H-.sup.15N HMBC NMR spectra. 1H
COSY spectrum gave new peaks related to the presence of GA. For
pure PAA, there is no long range coupling for the amino groups with
the shift at .about.8 ppm while it is strong for GA modified PAA.
Similarly, new and strong long-range couplings were observed for
carbonyl proton and the free protons on PMDA, particularly which
locates between two free carboxyl groups. .sup.1H-.sup.13C HSQC
revealed that GA interaction nearly eliminated the presence of
adjacent peaks nearby the peak at .about.7 ppm, which could be
related to that GA attacked on the phenyl ring of 4,4'-oxydianiline
(ODA).
[0147] Similarly, the long-range couplings for the adjacent peaks
got lost via GA interaction. According to .sup.1H-.sup.15N HMBC NMR
spectral data, nearly all of the long range couplings were lost
between the amide nitrogen and the protons on phenyl ring of ODA;
particularly proximal to the amino group. However, at the same
time, one of the amino peaks seen in the .sup.1H-.sup.15N HMBC NMR
spectrum was lost; this peak stayed the same for low GA
concentrations. A new amino peak was observed at .about.10.65 ppm
(FIG. 2i). Therefore, for low levels of GA, GA prefers to attach on
phenyl rings of PAA while at high levels, GA attaches on amino
groups in addition to phenyl rings.
[0148] GA preferentially binds to phenyl ring of PAA. In
particular, it binds to the ODA ring of PAA polymer. FIG. 2q
illustrates that GA interaction eliminated the presence of small
side peaks at aromatic region, which belongs to the proton of ODA.
This is because PMDA has more steric hindrance and hence, the GA
preferentially binds onto the ODA portion of the PAA molecule.
[0149] GA preferentially binds to phenyl ring of PAA. In
particular, it binds to the ODA ring of PAA polymer. This is
partially illustrated in FIG. 2p, which illustrates aromatic peaks
of PAA being at 7.07 ppm, 7.74 ppm, 8.01 ppm and 8.36 ppm. Amino
peaks of PAA are at 10.54 ppm and 10.57 ppm. Carboxyl peak of PAA
is at 13.51 ppm. ODA residue is shown in left circle and PMDA is
shown in the right circle.
[0150] FIG. 2q illustrates that GA interaction eliminated the
presence of small side peaks at aromatic region, which belongs to
the proton of ODA. This is because PMDA has more steric hindrance
and hence, the GA preferentially binds onto the ODA portion of the
PAA molecule. In FIG. 2q, the used GA concentration was less than
5% of the PAA concentration when PAA-GA membrane was prepared.
[0151] NMR data is shown in FIGS. 3a-3f Specifically FIG. 3a is NMR
data for .sup.1H, FIG. 3b is NMR data for .sup.1H COSY, FIG. 3c is
NMR data for .sup.1H-.sup.13C HSQC, FIG. 3d is NMR data for
.sup.13C, FIG. 3e is NMR data for .sup.1H-.sup.13C HMBS, FIG. 3f is
NMR data for .sup.1H-.sup.15N HSQC spectra of a PAA-SA-GA membrane
synthesized according to the method shown in FIG. 1b.
[0152] Introduction of sulfanilic acid (SA) to PAA did not produce
any additional peaks. However, some of the interactions observed in
the .sup.1H COSY spectrum of PAA-GA were not observed for
PAA-SA-GA. For .sup.1H-.sup.13C HSQC, one additional minor peak was
observed at 8.21-130.18 ppm in addition to PAA-SA-GA. Similarly,
.sup.1H-.sup.13C HMBC gave additional minor extra interactions for
the protons at 8.16 and 7.82 ppm, which were more of long-range
couplings shifted to more down-field, but simultaneously were
protected. However, the cross-peak at 7.82-167.9 ppm could be
speculated that it was from SA, rather GA. .sup.1H-.sup.15N HSQC
spectrum did not show any differences. Overall, it can be said
that, sulfanilic acid peaks were not clear in the membrane, while
minor differences were observed in 2D NMR spectra.
[0153] As can be seen in FIG. 4 PAA-GA was incubated under
sun-light for over 3 years in an airtight glass-container. Then,
the film was dissolved in DMSO. 1H-13C HSQC spectrum clearly shows
that the PAA-GA membrane lost its structural integrity, but no
Bisphenol A (BPA) formation was observed.
[0154] BPA is primarily used to make plastics such as water
bottles. There are studies showing that BPA might mimic natural
receptors in the body and thereby cause an irreversible change at
the genetic levels. Based on this potential effect, BPA and a host
of other compounds were classified as endocrine disrupting
chemicals. Certain plastics may not have BPA at the outset but with
time, they may produce BPA after extensive usage and breakdown. The
disclosed films did not produce BPA during study of their
degradation and are therefore considered substantially safe for
human health and the environment.
[0155] Further, Cabot sharp cheddar cheese was wrapped in a
PAA-pAS-SA-GA membrane of the present disclosure for three months.
Subsequently, we compared the proton .sup.1H NMR of freshly
purchased cheese (FIG. 5a) and the cheese kept in the membrane
(FIG. 5b). There was no peak related to DMAC or PAA. Before the
membrane was used to wrap the cheese, it was rinsed with tap water
10 times, and then rinsed with 70% Ethanol; in order to remove
residual ethanol, the membrane was kept in pure water for 3 h.
[0156] As seen from FIGS. 2a-2q, pure poly(amic)acid did not have
any aliphatic groups while it did possess carboxyl, amino, carbonyl
and aromatic groups. Due to the two ways of ODA-PMDA interactions,
carboxyl, carbonyl and amino groups showed two different
environments.
[0157] Insets in FIG. 2a show the cis- and trans-forms of PAA.
These two chemical environments affect proton shifts seen in NMR
spectra. They have an impact on structural characterization.
[0158] Even though two amino protons were observed, only one
carboxyl proton was observed; this difference is related to the
fact that the carboxyl proton is more isolated despite the fact
that two carboxyl carbons were observed. However, in the case of
very high amount of PAA membrane dissolved in DMSO-d.sub.6 to run
NMR, the carboxyl proton was not observed even though carboxyl
protons were present; similar results were observed for PAA-GA-SA
membranes.
[0159] Further, introduction of GA to PAA resulted in the presence
of proton peaks related to carbonyl and aliphatic groups. In
parallel to the increase in GA concentrations, the peaks became
sharper and more visible. As seen from FIGS. 3a-3f, GA can give
peaks between 4-6 ppm due to the presence of double bonds.
Therefore, the aliphatic protons provided in the Table I can be
speculated as coming from GA. GA also showed its presence via the
alterations in the aromatic region; higher concentrations of GA
eliminate presence of the peak at about 7.74 ppm while the carbon
peak related to that group remained same. As seen from FIGS. 3a-3f
.sup.1H COSY, .sup.1H .sup.13C HSQC and .sup.1H .sup.13C HMBC, the
protons peak remained same. However, the adjacent peaks around the
major PAA aromatic protons decreased, which is a sign of GA
interaction to the phenyl ring of ODA.
[0160] Amino groups did not show any change in response to GA
action while the presence of new peak at 10.65 ppm was observed in
the cases of sulfanilic acid (SA). However, .sup.1H .sup.15N HSQC
and .sup.1H .sup.15N HMBC did not show the presence of new amino
groups: there was only one type of amino group. This can be
speculated to mean that either SA content was not enough to be seen
or prior treatment of SA with GA resulted in secondary amino group
formation. .sup.1H COSY reveals the presence of aromatic proton and
amino proton of SA interacting each other. Therefore, it is clear
that SA chemically bonded to the PAA backbone.
[0161] Overall, GA chemically binds to the PAA backbone from phenyl
ring of ODA located at the edges of the individual PAA polymers.
Prior treatment of SA with GA results in the elimination of primary
amino groups, and made them visible as secondary amino groups with
PAA-SA-GA polymers.
[0162] NMR was also used to characterize the chemical stability of
PAA-GA polymer. The polymer was kept in an air-tight flask under
sun-light for over 3 years. As seen from FIG. 4, PAA polymer lost
its structural integrity, and gave fragmentation and oxidation
peaks; this was supported by presence of multiple aromatic protons
and amino protons, and loss of carboxyl proton. Besides, the
adjacent peaks, particularly, around 7 ppm gave the same integral
of the major peak which is a sign of fragmentation of individual
PAA polymers as shown in FIGS. 2a-2q.
Example 1.2.1-Molecular Weight Characterization of PAA Polymers by
NMR
[0163] Molecular weight (MW) characterization of the PAA polymers
by NMR was performed using two approaches .sup.1H DOSY and
T.sub.1-relaxation times.
[0164] .sup.1H Diffusion ordered NMR Spectroscopy (.sup.1H DOSY) is
a two-dimensional NMR technique which relies on the relation
between molecular mass of a molecule/polymer and its
self-diffusion. The technique has been shown to be useful in
determining the average molecular weight of a polymer. It is based
on the theory of the Stokes-Einstein equation. In all DOSY
experiments samples was 1.2-1.4 mg/mL in DMF-d.sub.7unless stated
otherwise. In DOSY NMR experiments (a technique giving information
about the average molecular weight of the molecules), concentration
of the molecule/polymer should be low enough (1.2-1.4 mg/mL) to
avoid viscosity related biased results.
[0165] As can be seen from FIG. 6, which illustrates the standard
graphics of .sup.1H DOSY, Polystyrene standards at 10.sup.3.114 Da,
10.sup.4.455 Da, 10.sup.5.236 Da and 10.sup.6.34 Da MWs were used
to draw the standard graphic. All standards were prepared .about.1
mg/mL in DMF-d7.
[0166] DOSY results of some PAA synthesized in the study are shown
in Table H below.
TABLE-US-00008 Polymer .sup.1H DOSY MW (Da) PAA-DA-GA (0.12M) 1.6
.times. 10.sup.5 PAA-pAB-GA (fresh) (0.12M) 1.22 .times. 10.sup.5
*Standard mixture 1 4.49 .times. 10.sup.5 .sup.#Standard mixture 2
1.01 .times. 10.sup.6 .sup.+Standard mixture 3 1.44 .times.
10.sup.5 PAA-I-W-GA (0.12M) 1.76 .times. 10.sup.5 PAA (0.14M)
40.degree. C. 3.01 .times. 10.sup.5 PAA (0.16M) 1.68 .times.
10.sup.5 PDA-PAA (0.16M) 1.49 .times. 10.sup.4 PAA (0.12M) 2.11
.times. 10.sup.5 PAA-IZ (0.12M) 1.78 .times. 10.sup.5 PAA (0.10M)
5.28 .times. 10.sup.5 PAA (0.14M) 2.33 .times. 10.sup.5 0.12M
PAA-pAS-GA (fresh) 1.36 .times. 10.sup.5 0.08M PAA (1:1.03)
40.degree. C. 5.28 .times. 10.sup.5 0.08M PAA-GA (aged) 1:1.03
40.degree. C. 3.41 .times. 10.sup.5 0.08M PAA-GA (fresh) 1:1.03
40.degree. C. 4.02 .times. 10.sup.5 0.16M PAA (1:1.03) 3.81 .times.
10.sup.5 0.12M PAA in 65:35 Ethanol:DMAC, 40.degree. C. 2.61
.times. 10.sup.5 0.12M PAA in 50:15:35 Ethanol:H.sub.2O:DMAC 1.14
.times. 10.sup.5 40.degree. C. PAA (0.14M) 30.degree. C. 2.33
.times. 10.sup.5 0.12M PAA in 60:40 Ethanol:DMAC, 40.degree. C.
1.78 .times. 10.sup.5 GA-autopolymer Less than 10.sup.3a GA-SA Less
than 10.sup.3a 0.12M PAA, 1:1, Room temperature-cleaned 1.35
.times. 10.sup.5 0.12M PAA-GA, 1:1, Room temperature- 6 .times.
10.sup.5 cleaned 0.12M PAA-GA-SA, 1:1, Room temperature- 7.35
10.sup.5 cleaned In Table H, *Polymer mixture 1 [23% of
10.sup.3.114; 58% of 10.sup.5.236 and 19% of 10.sup.6.34];
.sup.#Polymer mixture 2 [14% of 10.sup.6.34, 2.6% of 10.sup.3.114];
.sup.+Polymer mixture 3 [38% of 3.11, 14% of 10.sup.4.455, 19% of
10.sup.5.236, 29% of 10.sup.6.34]. IZ: Carbodiimizole; 1:1.03
refers to ODA:PMDA ratio; I: isoleucine; W: L-tryptophane
methylester; pAS: p-aminoscalicylic acid; PDA-PAA refers to
p-phenylenedianiline + pyromellitic dianhydride PAA; SA: sulfanilic
acid. .sup.aRefers to the value was below lowest MW of standard, so
it was not calculated.
[0167] .sup.1H DOSY is a technique to identify average MW of
polymer mixtures. Four individual polystyrene standards and three
mixtures of them were used in order to generate the standard
graphic shown in FIG. 6, and evaluate the parameters of .sup.1H
DOSY experiments. As seen from the standard graphic, .sup.1H DOSY
has less than 0.01% uncertainty. Table 3 shows that .sup.1H DOSY
provides highly satisfactory results for revealing the average MW
of the polystyrene polymer mixtures.
[0168] Typically, crosslinked PAA polymers are supposed to show
higher molar masses (MS). .sup.1H DOSY experiments showed that even
individual PAA polymers showed higher MS than glutaraldehyde (GA)
crosslinked PAA. Further tests include aged GA-crosslinked PAA,
(fresh) GA-cross-linked PAA, GA autopolymers, and GA-small molecule
co-polymers gave more clues about the size of the membranes. Among
the cross-linked PAA polymers, fresh GA-PAA gave the highest value
while PAA-W-GA (aged) gave the lowest MW. Since it is not possible
to apply a strict control on the activity of GA, there can be a
variety of co-polymers which could be generated from just the GA
autopolymer-PAA, GA autopolymer, GA-small molecule copolymer,
PAA-GA-PAA copolymers etc.
[0169] Comparison of different concentrations of PAA and the
solvent systems showed that the average PAA size was not changed.
However, heat treatment and ODA:PMDA ratio affected the MW. Based
on .sup.1H DOSY data along with the observed viscosity, 0.12 M PAA
prepared with Ethanol/DMAC mixture at 40-50.degree. C. was employed
as the standard film condition for any type of application
described throughout the present disclosure.
[0170] NMR data provided additional information about the MW of
polymers based on T.sub.1-relaxation times, which relies of
spin-lattice relaxation. Due to the fact that PAA polymers possess
aromatic protons, T.sub.1 relaxation times were compared in order
to compare the MWs of the synthesized polymers. According to
T.sub.1 relaxation time test, heavy crosslinking by GA increases
the MW of PAA polymers in accordance with the present
disclosure.
[0171] IR Characterization--Functional groups on PAA and
PAA-copolymers were determined with a Spectrum 65 FT-IR
spectrometer [Perkin Elmer, Waltham, Mass.]. Membranes at
solid-state was used to perform IR study. The results are tabulated
in Table I.
TABLE-US-00009 TABLE I Effect of GA on shifts in IR functional
groups Film O--H NH.sub.2/NH C.dbd.O C--N C.dbd.C Phase 3688/3222/
3422/3161/ 1692/1769 1352/1287/ 1352/1452/ inverted- 2680 1624/1578
1306 1520/1580 PAA All of 3224/2700 3432/3164/ 1812/1170/
1352/1308/ 1636/1444/ the 1578 1668 1289 1464/1526/ modified
1574-1578 PAAs
[0172] Stand-alone membranes were directly used for
IR-characterization; the membranes were not crushed into powder or
located onto IR cards.
[0173] Functional groups on PAA and PAA-copolymers were determined
with a Spectrum 65 FT-IR spectrometer [Perkin Elmer, Waltham,
Mass.]. Membranes at solid-state were used to perform IR study.
[0174] FIG. 7 illustrates the IR spectrum of different PAA
co-polymers. Series: 1: PAA-A-GA; 2: PAA-pAB-GA; 3: PAA-PCI-GA; 4:
PAA-PCI-GA (direct hood); 5: PAA-DPC-GA; 6: PAA-C-GA; 7: PAA-BB-GA;
8: PAA-W-GA; 9: PAA-A-GA (direct hood); 10: PAA-A-GA (partially
dissolved A); 11: PAA-pAB-GA (direct hood). Stand-alone membranes
were directly used for IR-characterization; the membranes were not
crushed into powder or located onto IR cards.
[0175] As seen from Table I, GA modification shifted the IR peaks
to slightly higher frequencies for a majority of the PAA functional
groups which is a sign of increases in mass of the polymers, which
was depicted by .sup.1H DOSY results as GA increased MW of PAA
polymers up to 5 times. Besides, abundant peaks for C.dbd.C and
C.dbd.O bonds were observed while O--H and --NH showed less peaks.
Due to some groups overlapping in these polymers, characteristics
of certain added groups were not observed in IR spectrometry. As
seen from NMR characterization, introduction of GA and small
molecules reveal more peaks correlated to --C.dbd.O and --C.dbd.C--
groups, so it implies that the extra peaks seen are from GA and the
small molecules. Decreases in O--H and --NH peaks could be related
to the data that shows that cross-linking with GA might be shifting
the amino groups resulting in overlapped and/or non-differentiable
in IR spectra, whose spectrums are provided in FIG. 7.
[0176] FIG. 8a is NMR data for .sup.1H, FIG. 8b is NMR data for
.sup.1H COSY, FIG. 8c is NMR data for .sup.1H .sup.13C HSQC and
FIG. 8d is NMR data for .sup.13C NMR spectra of the aged GA while
FIG. 8e is NMR data for .sup.1H, FIG. 8f is NMR data for .sup.1H
COSY, FIG. 8g is NMR data for .sup.1H .sup.13C HSQC and FIG. 8h is
NMR data for .sup.13C NMR spectra of stock GA did not show
characteristic alterations in groups.
[0177] .sup.1H COSY showed that the interaction at 0.9-0.9 ppm,
1.45-2.43 ppm, 2.47-9.65 ppm and 4.08-6.47 ppm were only seen for
stock GA. Actually, the interaction at 0.9 ppm shows that the peak
at 0.9 ppm of stock GA was not seen in the aged GA.
[0178] Comparison shows that .sup.1H .sup.13C has some differences
as well such as the aged GA has more interaction at 1.2-1.7 (H)
13-35 (C) ppm and 4.6-5.2 (H) -93-97 (C) ppm ranges.
[0179] Integration of the characteristic peaks in .sup.1H showed
that aging decreased free available carbonyl groups. GA can have
different forms in aqueous solutions, some of them are shown in
FIG. 9. The peaks at 12 ppm, 9.6 ppm, 6.0-6.5 ppm range, 4.5-5.2
ppm range, 1.0-2.0 ppm were accepted as that these peaks are from
hydroxyl groups, carbonyl groups, cyclic groups, the protons of
double bond containing C groups and hydrogen of saturated carbons,
respectively. Carbonyl group has the function of GA to show its
cross-linking potency; that's why, its integration was calibrated
to 1, and the rest was calculated relative to the carbonyl
integrals. For the aged integrations were obtained as 0.057 (--OH),
1 (HC.dbd.O), 3 (H-cyclic), 16.1 (HC.dbd.C) and 50 (--CH.sub.3)
while the integrations of the stock (fresh) GA were obtained as
0.04 (--OH), 1 (HC.dbd.O), 2.14 (H-cyclic), 14.26 (HC.dbd.C) and 44
(--CH.sub.3). This shows the aging decreased the percentage of free
carbonyl group around 30% in comparison to stock GA. Presence of
doublet C.dbd.C bonds and cyclic C-residues increased. This could
be the reason of getting colored and fluorescent active PAA with
aged GA in comparison to the stock GA. However, it should be
mentioned that it is not required to use aged GA to get colorful
and fluorescent active PAA; the stock GA can be dissolved in DMAC,
followed by introduced to PAA or PAA-small molecule mixture to get
colorful and fluorescent active membranes.
Example 1.3--Scanning Electron Microscopy/Optical
Characterization
[0180] Characterization of the PAA membrane morphology was carried
out on a Zeiss Supra 55 VP field emission scanning electron
microscope (SEM). The membranes were imaged both before and after
filtration. All samples were coated with 2-5 nm gold layers for SEM
imaging.
[0181] Only the membranes produced according to FIG. 1b were
characterized for optical properties. Uv-vis properties were
evaluated using HP Agilent 8452 spectrometry while Shimadzu RF 6000
fluorometer was utilized to characterize fluorescence properties.
Uv-vis characterization was only performed for the stand-alone
films while both stand-alone membranes and their dissolved forms
were utilized for fluorescence characterizations.
[0182] Digital images of ternary PAA membranes from FIGS. 1a and
1b. a--PAA; b--PAA-DA; c--PAA-A; d--PAA-A was incubated in 30 min
at 70.degree. C. in addition to overnight incubation; e--PAA-A
similar to d but higher GA concentration; f--PAA-A same GA
concentration to e, but just incubated in room temperature; FIG.
1a. GA was applied at different concentrations to the PAA
solutions. g-PAA-A with % 0.3 GA; h-PAA-A with % 0.9 GA; i-PAA-CA
with % 0.3 GA; j-PAA-CS with % 0.3 GA; k-PAA with % 0.3 GA; l-PAA
with % 0.9 GA, and m-PAA-DA with % 0.9; FIG. 1a i. n-PAA-A 3h
incubation; o-PAA-A; p-PAA-C; q-PAA and r-PAA-DA. FIG. 1a iii with
0.9% GA from 70% GA stock. s-PAA with % 0.3 GA; FIG. 1a ii. This
showed woven-like surface as shown by SEM imaging. The images "t"
and "u" are synthesized with FIG. 1a iii with 0.35% GA
concentration. In 6h, PAA-CS gave green membrane [t] which could be
peeled off from glass surface, which gave gel-like structure. The
gel like membrane [t] was then phase-inverted in pure water and
incubated overnight under hood [u]. The following membranes were
prepared according to FIG. 1b; v: PAA-5AS-GA, w: PAA-4AS-GA-MeOH,
x: PAA-AcOH-CA-GA, y: PAA-pAB-GA, z: PAA-AcOH-Ser-GA-MeOH, aa:
PAA-PCl-GA, ab: PAA-AcOH-A-GA-MeOH, ac: PAA-5AS-GA but this is just
incubated in RT, ad: PAA-5AS-GA but direct hood evaporation, ae:
PAA-PCl-GA-MeOH [right after GA], af: PAA-MeOH-Ammonium Nitrate-GA
[direct hood], ag:PAA-PCI-GA, ah:PAA-5AS-GA, ai:PAA-A-GA. Even
though the images v, ac, ad and ah are made out of PAA-5AS, 5AS
content and incubation procedure affect the color formation; ah has
the lowest 5AS concentration. aj-PAA-I-GA.
[0183] Digital images of some films from FIG. 1b are shown in FIGS.
11a-11l. All the films were prepared according to FIG. 1b, and GA
concentration was 0.1% while PAA was 0.12 M; a: PAA phase inverted
under hood; b: pAB-GA-PAA; c: W-GA cross-linking for 15 min then
introduced into PAA solution; d: pAB dissolved in DMAC incubated
with GA for 15 min, followed by introduced into PAA solution; e:
pAB was added to PAA solution, followed by addition of GA; f:W-GA
cross-linking for 5 min then introduced into PAA solution; h::pAB
dissolved in DMAC incubated with GA for 30 min, followed by
introduced into PAA solution; i:W was crosslinked with aged GA,
followed by introduced into PAA; j: pAS and W were added into PAA
solution, followed by addition of GA; k::pAS was dissolved in DMAC,
and then added into PAA solution, followed by added 0.2% pAB-GA (at
that moment the incubation was passed already 30 min); j: W
cross-linked with fresh GA (stock 70%), followed by added into
PAA.
[0184] A discussion of FIGS. 10a-10aj and FIGS. 11a-11l
follows.
[0185] As seen in FIGS. 10a-10aj, PAA-DA gave some blue region but
the rest is yellowish due to the fact that high amount of GA
stacked in localized places because of high viscosity-related
quenched stirring. Similarly, FIG. 10ac, FIGS. 10ah-10ai and FIG.
11b and FIG. 11h possessed uneven surfaces. Interestingly,
increased incubation time and high GA concentration form colorful
plastic like membranes in FIG. 1a, these parameters didn't show any
significant effect on formation of different colored membranes with
plastic-like structures. However, for both FIGS. 1a and 1b,
treating GA with DMAC alters the formed color as well as affecting
on the other parameters such as contact angle and mechanical
strength. For example, the membranes FIGS. 10 m and 10r were from
PAA-DA. Even though, the membrane FIG. 10m is plastic-like
transparent while the membrane FIG. 10r is opaque and dark-blue
color with possessing higher contact angle; top/bottom contact
angles of the membranes FIGS. 10m and 10r were 62.35/55.7
45.3/47.3, respectively. Due to the aggressive nature of GA, it can
make excessive cross-linking in PAA solution.
[0186] Comparing FIG. 1a i and iii for same GA concentration and
incubation periods, it was shown that pre-dissolving GA in DMAC
makes it much more active; this can be resulted from that
dissolving GA from stock in dry DMAC partially or totally altering
GA microenvironment, which then might change binding preferences
and/or rate of binding. Formation of transparent membrane also
strongly depends on the small molecule added to the PAA solution.
For example, L-Alanine added PAA membranes always formed
transparent plastic like membranes if a special treatment was not
applied even for FIG. 1a.
[0187] However, L-Tryptophan methyl ester, L-Isoleucine and some
other small molecules resulted in opaque membranes. Individual PAA,
PAA-DA and PAA-CS are the ones gave distinctly different membrane
formations by just shifting the procedure, FIG. 1a i to iii. It
should be noted that the membranes of FIG. 1a were partially or
totally formed before rinsing step. Unless the membrane is totally
formed, rinsing with water forms partially or totally opaque
membranes, which can be explained with the model proposed
elsewhere. However, further drying (after rinsing step) turns the
opaque membranes into transparent forms within 24 h under hood for
relatively higher GA concentrations such as % 1; but mostly these
are brittle. For example, the membrane FIG. 10u couldn't be turned
into a transparent membrane even at 48 h incubation. This could be
related to the high DMAC content formed thicker interacted with
non-solvent.
[0188] In contrast to this, it is possible to synthesize
substantially transparent and durable membranes of FIG. 1a for all
of PAA-small molecules even PAA-I if the GA concentration is higher
2% with pre-dissolved GA in DMAC. Using low GA concentration as
0.35% still can provide substantially transparent and durable
membranes, but the incubation time should be 12 h at room
temperature and 12 h under hood. 12 h incubation does not totally
remove DMAC, but further rinsing does not cause any
opaque-structure formation.
[0189] FIG. 12m is photographs of PAA-SA (warmed)-pAS-5AS-GA, FIG.
12n PAA-SA-pAS-5AS-GA, FIG. 12o PAA-SN-GA, FIG. 12p PAA-SN-pAS-GA.
Color change of the same films can also be manipulated by heating
the small molecule, or introducing other small molecules at very
low quantity. Warming up SA before it was pre-treated with GA
changed the resultant film color from yellow to brown while FIG.
12p has only 0.1 mg/mL pAS in addition to FIG. 12o, but the color
did changed.
[0190] Color formation in FIG. 1b is distinctly different from FIG.
1a. Pretreatment of small molecule with GA, GA condition (aged or
fresh) and presence of cross-linking quenchers are the predominant
parameters which can be even confirmed by only FIG. 10al. For
instance, pretreatment pAB with aged GA provided blueish membrane
formation while adding pAB directly into PAA-GA mixture formed
slightly maroon color membrane. Another example is that using fresh
GA instead of aged GA resulted in shifting the color from green
(FIG. 11i) to yellow (FIG. 11l).
Example 1.3.1--UV-Vis Spectra of PAA and Ternary PAA Membranes
[0191] UV-Vis spectroscopic properties of PAA membranes were
evaluated to determine the effect of small molecule and GA on
formed membranes. PAA phase-inverted membranes that were processed
in the hood were compared with the PAA that were synthesized
according to FIG. 1a.
[0192] FIG. 13a illustrates UV-vis of some of the synthesized
membranes. PAA: Poly(amic)acid; A: L-alanine; GA: glutaraldehyde;
5AS: 5-aminosalycylic acid; I: L-isoleucine; pAB: p-aminobenzoic
acid; W: L-tryptophan-L-methylester; pAS: p-aminosalycylic
acid.
[0193] FIG. 13b illustrates ransmittance of some of the membranes
synthesized in the study. PAA: Poly(amic)acid; A: L-alanine; GA:
glutaraldehyde; 5AS: 5-aminosalycylic acid; I: L-isoleucine; pAB:
p-aminobenzoic acid; DA; glucosamine; pAS: p-aminosalycylic acid;
SN: sulfanilamide.
[0194] In FIG. 13a, there is no PAA peak from 400 to 700 nm range.
The peaks are related to a small molecule being introduced to a PAA
molecule. Even though the overall color of the membranes showed
strong dependence on the condition of GA and GA pretreatment of
small molecule, UV-Vis characterization did not provide any
significant difference.
[0195] Transmittance of the membranes is important for food
packaging material applications. All of the membranes showed over
65% transmittance between 450 to 700 nm. The used membranes (i.e.
PAA-I-GA, PAA-I provided good visibility for monitoring food
conditions. However, PAA-SA-pAS-5AS-GA and PAA-SN-pAS-5AS-GA have
lower % transmittance at certain wavelengths such as .about.510 nm
and 650 nm. It should be mentioned that these are not affecting the
overall visibility of the packaged food.
[0196] Unlike UV-Vis properties, fluorescence characteristics of
PAA membranes showed strong dependence on GA condition, incubation
period, GA pretreatment with small molecules and the presence of
methanol and ethanol. However, it should be noted that optimizing
the conditions are challenging due to the fact that GA can
crosslink a variety of other groups including primary/secondary
amino groups, thiol groups, hydroxyl groups of sugars and aromatic
carbons.
[0197] The Fluorescence Characteristics of several films are
discussed below. FIGS. 14a and 14b illustrate Rhodamine 6G
standards.
[0198] FIGS. 15a-15e illustrate several spectra. The spectra seen
in "a" and "b" belong to yellowish PAA-A-GA membranes while the
spectrums seen in "c" and "d" belong to the greenish PAA-A-GA
membrane. The spectrum "e" belongs to PAA-GA. All of the membranes
were synthesized according to FIG. 1a, and standalone membranes
were used during fluorescence run. Excitation wavelengths were 581
nm, 598 nm, 608 nm and 596 nm for a-d membranes, respectively.
Emission ranges were 592-648 nm, 610-698 nm, 619-699 nm and 600-640
nm for a-d membranes, respectively. Absorbance was kept below 1 for
all, and during fluorescence measurement sensitivity was kept high.
As it is seen, for all membranes, fluorescence intensity started
with a decreasing trend, followed by increases in the intensity.
However, PAA-GA showed an increasing trend for fluorescence
intensity from the starting point, whose excitation was 612 nm
while the emission range was between 620 nm and 700 nm. More than
one maximum-emission peak was observed for all. The fluorescence
quantum yields of these membranes were below 0.1.
[0199] FIGS. 16a and 16b illustrate fluorescence intensity vs.
emission and emission wavelengths. The membrane seen in FIG. 16a
was dissolved in DMF at three different concentrations as 1, 0.67
and 0.5 for spectrum "a" while 1, 0.75 and 0.5 for spectrum "b".
Excitation/Emission wavelengths were 608 nm/619-699 nm range and
621 nm/632-700 nm range for the spectrum "a" and "b", respectively.
Similar to FIG. 16b of solid membrane, more than one maximum
emission peaks were observed. Dilution enhanced the observed
fluorescence intensity, while the dilutions decreased the UV-Vis
absorbance of the corresponding solutions.
[0200] FIGS. 17a-17e are wavelength illustrations. In these figures
2 mg/mL pCl was introduced into 10 mL of PAA viscous solution,
followed by 200 .mu.L GA from aged 25% stock was introduced to the
PAA-pCl solution. The mixture was casted on glass to prepared
PAA-pCl-GA membrane according to FIG. 1b. (a) Ex 485 nm/Em 486-700;
(b) Ex 505 nm/Em 506-700 nm; (c)Ex 523 nm/Em 524-700 nm(d) Ex 550
nm/Em 551-700; (e) Ex 602/Em 603-700 nm. The best quantum yield was
0.2 (Ex 523/Em 524-700), for the rest was between 0.17-0.19.
[0201] FIGS. 18a-18d are wavelength illustrations. In these figures
20 mg pCl and 200 .mu.L of aged 25% GA were simultaneously
dissolved in 2 mL DMAC, and mixed for 5 min. The solution was then
added to 8 mL PAA solution, which was mixed for 10 min before
casting on the glass to prepare the membrane according to FIG. 1b.
(a) Ex 598 nm/Em 599-700 nm; (b) Ex 602/Em 603-700 nm; (c) Ex 657
nm/658-700 nm; (d) Ex 621 nm/Em 620 nm/Em 621-627 nm. The best
quantum yield was 0.1 (Ex 598 nm/Em 599-700 nm).
[0202] FIGS. 19a-19d are wavelength illustrations. In these figures
20 mg pAB and 200 .mu.L of aged 25% GA were simultaneously
dissolved in 2 mL DMAC, and mixed for 10 min. The solution was then
added to 8 mL PAA solution, which was mixed for 10 min before
casting on the glass to prepare the membrane according to FIG. 1b.
(a) Ex 485 nm/Em 486-650 (but shown 486-526 nm); (b) Ex 523 nm/Em
524 nm-700 nm (shown 524-600 nm); (c) range between 535 nm to 600
nm of b; (d) Ex 619/Em 620-700 nm. Series 1 always refer to the
thicker PAA-pAB-GA while series 2 depicts the thinner PAA-pAB-GA
(aged). The best quantum yield obtained was 0.1 (Ex 485 nm/Em
486-650 nm of Series 2).
[0203] FIGS. 20a-20d are wavelength illustrations. In these figures
10 mg I was dissolved in 200 .mu.L of aged 25% GA, which was then
vortexed for 10 min. I-GA mixture was then introduced to 10 mL PAA
viscous solution, and mixed for 10 min, followed by casted on glass
surface to prepare PAA-I-GA membrane according to FIG. 1b. (a) Ex
567 nm/Ex 568-700 nm; (b) Ex 587 nm/Em 588-700 nm; (c) Ex
590/591-700 nm; (d) Ex 601 nm/Em 602-700 nm. The best quantum yield
obtained was 0.08 (Ex 590/591-700 nm).
[0204] FIGS. 21a-21d are wavelength illustrations. In these figures
20 mg pAS and 200 .mu.L of aged 25% GA were simultaneously
dissolved in 2 mL DMAC, and mixed for 10 min. The solution was then
added to 8 mL PAA solution, which was mixed for 10 min before
casting on the glass to prepare the membrane according to FIG. 1b;
(a) Ex 485 nm/Em 486-600 nm; (b) Ex 523 nm/Em 524-600 nm; (c) Ex
550 nm/Em 551-600 nm; (d) Ex 598 n/Em 599-700 nm. The best quantum
yield obtained was 0.08 (Ex 523 nm/Em 524-600 nm).
[0205] FIGS. 22a-22d are wavelength illustrations. In these figures
10 mg I was dissolved in 200 .mu.L of aged 25% GA, which was then
vortexed for 10 min and 2 min heated at 70.degree. C. sequentially.
I-GA mixture was then introduced to 10 mL PAA viscous solution, and
mixed for 10 min, followed by casted on glass surface to prepare
PAA-I-GA membrane according to FIG. 1b. (a) UV-Vis of the solid
membrane; (b) Ex 523 nm/Em 500-550 nm; (c) Ex543/Em 544-610; (d)
Ex598/Em 590-610 nm. The best quantum yield obtained was 0.1 (Ex543
nm/Em 544-610 nm).
[0206] FIGS. 23a-23e are wavelength illustrations. In these figures
10 mg DA was dissolved in 200 .mu.L of aged 25% GA, which was then
vortexed for 10 min. DA-GA mixture was then introduced to 10 mL PAA
viscous solution, and mixed for 10 min, followed by casted on glass
surface to prepare PAA--GA membrane according to FIG. 1b. (a)
PAA-DA-GA (aged) UV-Vis of the solid membrane; (b) Ex450/Em451-500;
Ex487/Em488-510; (c) Ex523/Em524-590; (d) Ex601/Em602-700. Ex543/Em
544-610. The best quantum yield obtained was 0.24 (Ex487/Em488-510)
while at the other excitations quantum yields were observed between
0.1-0.17.
[0207] Then a series of films were synthesized to test the
fluorescence properties with Synchronous Fluorescence Spectroscopy.
PAA-pAS-GA, PAA-pAB-GA, PAA-W-GA and PAA-W-GA combined with GA
treated pAS.
[0208] In FIG. 24, Series 1-4: (1) PAA-pAB-GA (aged); (2)
PAA-pAS-GA (aged)-purplish; (3) PAA-pAS-GA (aged)-greenish; (4)
PAA-W-GA (aged)-reddish.
[0209] FIGS. 25a-25c are wavelength illustrations. In these figures
PAA-pAB-GA (aged) greenish. First, pAB was cross-linked with aged
GA in DMAC for 20 min, followed by introduced to PAA solution which
was then strongly mixed for 10 min. The solution was then casted on
glass, and incubated at room temperature for 6 h, followed by
incubated under hood for 6 h. Then, the resulted standalone
membrane was rinsed with nano-pure water. Experimental conditions
for fluorescence was as described follow; room temperature, quartz
fluorescence cuvette, standalone membrane itself, 1.5 slit width,
excitation filter auto, emission filter auto, at synchronous mode,
delta 0 nm, run mode was slowest (30 nm/min), start 220 nm, stop
700 nm. The instrument was Cary Eclipse run with Eclipse software.
PAA-pAB-GA, slit 1.5, speed normal, delta 0.1 (b) and 1 nm (c).
[0210] FIGS. 26a-26g are wavelength illustrations. In these figures
Synchronous fluorescence of modified PAA and Rhodamine B. (a)
PAA-pAS-GA (purplish); (b) PAA-pAS-GA (greenish); (c) PAA-W-GA
(reddish); (d) PAA-W-pAS-GA (purplish); Rhodamine B with delta 0 nm
(e) and 10 nm (f); (g) formed PAA-pAS-GA membrane was dissolved in
DMF, and 0.1 mg/mL 2-aminopyridine was added to the dissolved
PAA-W-pAS-GA, which was then casted on glass for evaporation
mediated membrane formation. In all cases experiments were
performed for standalone membranes in quartz fluorescence cuvette
at room temperatures. The fluorimeter was set to auto mode for
excitation filter and open-mode for emission filter while scanned
region was kept between 250 nm and 700 nm under slowest run which
was 30 nm/min for the instrument. Slit width for excitation and
emission were selected as 1.5 for "a", "c", "d" and "g" while they
were set to 2.5 for "b", "e" and "f". Delta was selected 0 nm if
not specified otherwise. All of the membranes were prepared
according to FIG. 1b. To make PAA-pAS-GA green, pAS and GA were
simultaneously dissolved in DMAC where GA cross-linked pAS for 20
min. Then, mixture of PAA-pAS-GA was introduced to viscous PAA
solution containing 0.5 mg/mL pAS for 20 min mixing which was then
casted on glass to form membrane according to FIG. 1b. Reddish
PAA-W-GA was synthesized through introducing 20 min GA-crosslinked
W into viscous PAA solution.
[0211] In the above reference figures, increasing delta from 0 to
10 nm, improved fluorescence intensity was observed as expected
since possessing too close excitation and emission wavelengths
causes decreases in fluorescence intensity. However, for the
membranes increasing delta from 0 to 1 nm, disrupted the observed
fluorescence intensity. This was most probably from that over-load
of fluorophore in the medium; fluorophores within the membranes can
show FRET which then diminished the observed fluorescence
intensity. This idea was supported through adding more diverse
fluorescent active molecules in the membrane. Introduction of pAS
into PAA-W-GA decreased the observed fluorescence. Similarly,
introduction of GA cross-linked pAS into PAA-pAS diminished the
fluorescence intensity. PAA-pAS-W-GA did not provide enough
fluorescence intensity when the slit width was 1.5; the highest
peak was around 0.5 (not shown). However, dissolving PAA-pAS-W-GA
in DMF, and introduction of 2-aminopyridine to the membrane gave a
visible spectrum even at 1.5 slit width. However, if the PAA
membrane was used without dilution, the intensity was not visible
at 1.5 slit width.
[0212] As seen from FIGS. 14a and 14b, Rhodamine 6G provides very
smooth excitation and emission curves while the PAA membranes did
not provide any smooth curves. PAA films can be synthesized as
fluorescence active, but the best quantum yield was obtained around
0.1 leaving 10 nm gap between excitation and the starting emission
wavelengths. These figures were used to compare two things; (i)
that the dyes can be introduced into the PAA membrane, where their
presence is strong and similar to the original dyes molecules, (ii)
FIGS. 14a and 14b represent the graphical illustrations of
absorbance and emission values of various PAA film and these were
used to calculate the quantum yields of the dye-modified PAA
membranes.
[0213] Besides the selection of the emission range, the conditions
must be controlled for reproducible results:
[0214] (1)Glutaraldehyde must be aged to obtain fluorescence active
membranes, which was determined experimentally in the study. NMR
spectroscopy can be used to keep the best conditions. (2) The
presence of methanol end ethanol during membrane formation prevents
fluorescence active membrane formation, which can be overcome with
addition of water. (3) Humidity and longer incubation at room
temperature disrupts UV-Vis and Fluorescence properties of PAA
membranes. The humidity was not measured experimentally. (4) Care
required during the addition of GA into the PAA-small molecule
mixture. It should be drop by drop; sudden addition of high amount
of GA prevents membrane formation.
[0215] Due to possessing unique properties and low cost, conjugated
polymers are preferred as a sensor support material or direct
sensing agent from electrical to optical sensors.
[0216] pAS was introduced into different polymers, and its
fluorescent activity was shown dependent on the chemical and
physical properties of the polymer, including ionization of side
groups within the polymer, allowed volume and polarization
properties of the groups within the molecules. Above, pAS is
becoming part of the membrane itself. So, it is quite normal that
fluorescence characteristics of pAS will show change. Besides the
environment itself, the solvent also possesses strong effect on
excitation and emission spectra, and quantum yield as well. For
example, pAS itself gives only one emission in aprotic solvents, it
gives more than one peak in protic solvents.
[0217] PAA itself is inherently not fluorescence-active, but
introducing side groups to PAA was shown as a method to add
fluorescence character to PAA such as grafted PAA with
toluene-2,4-diisocyanate and straight alkyl chains showed
fluorescence properties. Maximum absorption and excitation
wavelengths of the high purity Rhodamine 6G in anhydrous ethanol
were obtained at .about.527 nm and .about.550 nm, which refers to
the stoke shift is over 20 nm which matched the literature. This
shows there was only one fluorophore in the medium, and the working
conditions only allowed possessing one broad peak. However, this
was not valid for the stand-alone membranes and the dissolved
membranes due to the fact that the membranes were composed of more
than one fluorophore. As detailed in the examples below, GA can
cross-link individual PAA polymers as well as it can cross-link
PAA-small molecule and small molecule-small molecule, which can
allow possessing fluorophore. FIGS. 26a-26g depict synchronous
fluorescence run of Rhodamine B and PAA-pAB-GA, PAA-W-GA (reddish
membrane), PAA-pAS-GA, respectively. SRB, as expected, provide a
sharp peak which depicts that SRB in anhydrous ethanol has only one
characteristics excitation and emission peaks while PAA-pAB-GA and
PAA-pAS-GA have more than one.
[0218] As seen from FIGS. 26a-26g, when there is 0 nm difference
(delta), under same conditions, between excitation and emission
wavelengths for SRB, the fluorescence intensity is lower than the
one in the case the delta was 10 nm. However, this situation is
distinctly different for the membranes.
[0219] As seen from the FIGS. 26a-26g, when the delta is 0, the
fluorescence intensity is the highest while the intensity decreased
400 times when delta was 1 nm. When the delta was selected 0.1 nm,
the intensity decreased 20 times in comparison to the case of delta
was 0 nm.
[0220] Quantum yield of a fluorophore is more influenced by the
environment in comparison to molecular extinction coefficients.
Fluorescence quenching is the process of reduction in fluorescence
quantum yield, which can be resulted from collisional quenching
and/or occurrence of vibrations of non-fluorescent ground-state
species. Self-quenching is, also, a common problem seen in
fluorescence, which is arisen from over-load of fluorophores
presence. Fluorescence resonance energy transfer (FRET) is a
fluorescence technique in which emission of a fluorophore is
absorbed by another fluorophore as the excitation. The technique is
distance-dependent, and the yield of FRET is proportional to the
distance, which makes it sensitive.
[0221] FIG. 1b films can provide fluorescent active under strictly
controlled conditions. However, current results are not allowing
them to be used as a sensor material for FRET applications due to
the fact that extensive fluorophore presence results in loss of
quantum yield. However, studies related to metals and oxygen
concentrations, the synthesized fluorescent active membranes can be
a candidate for metal and oxygen sensors.
[0222] FIG. 27 illustrates excitation data for several films.
Specifically, Series 1-6, respectively; PAA-Sulfanilamide-GA
(Ex-Ee=1 nm); PAA-Sulfanilamide-GA (Ex-Em=5 nm); pAS in PAA
(Ex-Em=5 nm); pAS in PAA (Ex-Em=10 nm); pAS+W in PAA (Ex-Em=5 nm);
W in PAA (Ex-Em=5 nm).
[0223] FIGS. 28a-28c illustrate excitation and wavelength data.
Specifically, UV-vis, Synchronous fluorescence of Rhodamine 6G
(R6G) embedded PAA membranes. (a) UV-vis of PAA-R6G, (b)
Synchronous fluorescence of PAA-R6G and (c) Emission of
PAA-R6G.
[0224] Even though UV-vis gave peak at 570 nm, the best quantum
yield was obtained for Ex 533 nm. While R6G in ethanol solution as
shown in FIGS. 14a and 14b provides very smooth emission curve, R6G
in PAA shows repeating ups and downs. This could be related to that
R6G within the PAA might have different microenvironments where R6G
behaves different than how it behaves in solution. The obtained
quantum yield for PAA-R6G varies between 0.78 and 0.82; keeping 90%
of quantum yield in comparison to pure R6G can make this type of
membranes usable for fluorescent labeled membrane applications.
Since, the membrane was rinsed with excess water, it can be said
that there was no fluorescence come from R6G stays on the
membrane.
1.4 Solvent Resistance Properties
[0225] Film lengths and widths were 2.5 cm while thicknesses were
between 0.00128-0.00512 cm. The following buffers were used; 50 mM
Acetate (pH 4.5) buffer, 50 mM PBS buffers (pH 6.8/7.0/7.4), 50 mM
(pH 8.0) Tris-HCl and 50 mM (pH 9.6) carbonate buffer. All the
buffers were prepared from their salts and pH was adjusted with 1M
HCl and/or 1M NaOH. Glacial acetic acid, 30% hydrochloric acid,
100% sulfuric acid, 1 M sodium hydroxide and 37% NH4. The following
organic solvents were employed for solubility testing": ethanol,
methanol, tetrahydrofuran, hexane, ethylacetate, dimethyl
formamide, dimethyl acetamide, dichloromethane, p-xylene,
aceticanhydride, dimethyl-sulfoxide and acetone. The solvent
resistance properties of various films is discussed in the data
below.
[0226] PAA films are soluble in complex media such as protein and
carbohydrate containing media and polar organic solvents.
Therefore, it is essential to show the material will not live a
problem of dissolvation during contact to food and possible
chemical contaminants or the chemicals from food-itself.
[0227] Solvent resistance of the synthesized PAA membranes showed
close relation to mostly GA condition and the small molecule. The
findings are listed as below.
[0228] Aged GA made the membranes soluble for all small molecules
PAA-copolymers in the case of FIG. 1bi/ii in strong organic polar
solvents include DMF, DMAC, DMSO and buffers pH over 7.4. However,
FIG. 1b ii membranes showed different character such as
L-Tryptophan methyl ester individually, or the other amino acids
combined with sulfanilic acid make the membranes non-soluble in
DMAC and all the buffers tested in the study.
[0229] Fresh GA made the membranes of the small molecules PAA
co-polymers synthesized according to FIG. 1b non-soluble in all the
solvents tested here except the membranes containing pAS, pAB, 5AS,
PCl over 1 mg/mL.
[0230] Sulfanilic acid (SA) advanced solvent resistance character
of all the membranes synthesized according to FIG. 1b. However,
extra-GA must be added to the PAA solution prior to SA
addition.
[0231] Tryptophan methyl ester-PAA-GA copolymers formed non-soluble
membranes in the cases of that GA was fresh; methanol addition is
required, or GA addition to the PAA solution could be beneficial to
make the membranes non-soluble.
[0232] Membranes synthesized according to FIG. 1 a except PAA-A-GA
were soluble in strong polar organic solvents include DMAC, DMSO,
DMF, and the buffers include 50 mM pH 8.0 PBS. Also, the most
aggressive solvent for the most resistant membranes were
ammonium-hydroxide.
[0233] Solubility of all the membranes did not show dependence on
pH as shown in the figures and discussed below.
[0234] FIG. 29 shows the solubility of ternary PAA membranes in
basic solutions. Solubility is not related to pH, because pH 4.5 50
mM Acetate buffer, 1M NaOH, 1M HCl, Glacial acetic acid, pH 8.00 50
mM PBS buffer, pH 6.0 1M PBS did not dissolve the membrane. But 29%
NH.sub.4OH totally dissolved the Ile-PAA-GA membrane in less than 1
h under no agitation. This is important because ammonia is produced
during food-purification.
[0235] Color changes of ternary PAA membranes in response to
alcohol exposure is shown in FIG. 30. Color change related to
exposure to alcohol using ternary PAA membranes. Ile-PAA; normally
the membrane is greenish yellow. Alcohol turns the color into
chestnut color as it was shown for Ethanol, Methanol and Ethylene
Glycol.
[0236] Color changes of PAA membranes in response to alcohol
treatment is shown in FIG. 31. All of the membranes change their
color different intensities of red in response to alcohol
treatment. Color change is accompanied with new peak formation in
UV-Vis Spectrum. While peak change in response to Methanol and
Ethylene Glycol is more characteristics at around 520-550 nm, the
new peak formation is less characteristics at around 560 nm and 250
nm. Speed of color changes is dependent on the membrane
composition. In the case of L-isoleucine enhanced membranes, the
color change becomes visible in less than 6 h while L-alanine and
p-aminosalicylic acid conjugated PAA membranes show the color
change within 12 h and 120 h, respectively. The synthesized hybrid
PAA films can determine the onset of alcohol (or microbial
degradation) production. The intelligent properties of the PAA were
assessed by monitoring changes in reversible and irreversible
properties of the membranes. Changes in voltage and color in
response to microbial development and/or food decomposition would
provide real-time monitoring of food condition. Color changes by
alcohols are important findings for intelligent food packaging
material of the synthesized membranes. Ethanol is the most common
byproducts of microbial development, and can be seen from FIG. 31,
color changes can be seen with bare eyes and does not need to be
monitored using any sophisticated tools.
[0237] Color change in response to exposure alcohol is achieved in
all types of PAA films, but when the membrane is very dark (low
transparency), the change takes a comparatively longer time. Among
the membranes, Ile containing membranes gave the response fastest;
while Ile enhanced PAA-GA membrane gave color change within 30 min,
pAS enhanced PAA-GA membrane required 2-4 h to give color
change.
[0238] Color changes in response to alteration in environmental
conditions can be further advanced with pH-dependent dyes. Here
bromophenol blue (BPB) was simply tested, with the results shown in
FIG. 32.
[0239] In FIG. 32, pH dependence of bromophenol blue supported PAA
ternary membrane. PAA-BPB-GA (aged) was prepared according to FIG.
1b. All of the membranes were 4 cm.times.2 cm.times.0.05 cm
(length/width/thickness). (a) 50 mM pH 6.00 PBS; (b) 50 mM pH 6.5
PBS; (c) 50 mM pH 7.1 PBS; (d) 50 mM pH 7.6 PBS and (e) 50 mM pH
8.0 PBS buffer. Color changes started within 30 min for c, d and e
vials while color change was seen for a and b for up to 6h (test
was completed in 6h). End of 6h incubation, the membranes were
removed from the vials, and no visible color change was seen for a
and b (f and g, respectively) while the color has changed for c, d
and e (h, f and k, respectively). The membranes were also tested
for 50 mM pH 4.5 Acetate buffer, and no color change was
observed.
[0240] Further color change is shown in FIG. 33. In FIG. 33 at the
end of 6h incubation, PAA-BPB-GA (aged) membrane was taken out from
the vial. As it is seen, the top part kept its yellowish color,
because the part was not submerged into the buffer, while the rest
of the membrane was turned into pale greenish form.
[0241] This section was to entrap BPB within PAA-GA, and release
the BPB into the medium when there is a change in pH and/or ionic
strength; when the PAA is treated with aged GA it doesn't have
insoluble form for harsh environment such as high pH, strong
organic solvents and so on. The color change of the membrane itself
might be resulting from certain amount of BPB crosslinked to PAA.
Actually, using fresh GA can advance BPB cross-linking to PAA,
which could also a possible way of monitoring pH changes.
1.5 Mechanical Characterizations
[0242] The mechanical strengths of the membranes were determined
using Instron.RTM. Tension Testers run by Merlin Project Software
(Norwoon, Mass.). The strength tests were evaluated using maximum
load, tensile strength and modulus of elasticity.
[0243] Mechanical properties of food-packaging materials are a
parameter in assessing their relevance as packaging materials. This
is because packaging materials must protect the containment against
possible exposure to physical pressures during transportation and
storage. Maximum load, modulus elasticity, break elongation and
tensile strength are common parameters to evaluate mechanical
properties of a packaging material. Different approaches have been
applied to advance the properties such as combination of
biopolymers and chemicals, composites or nanomaterials (i.e.
chitosan and graphene, whey protein-zein).
TABLE-US-00010 TABLE J illustrates Mechanical properties of FIG. 1a
Films Maximum Modulus Break Tensile load elasticity Elongation
Strength Film-Type (kg) (MPa) % (MPa) Composite.sup.a 12.75 180
24.3 13.79 PAA-C-GA.sup.b 5.13 406.89 13.4 32.41 PAA-A-GA.sup.b
3.86 1096.55 10.9 24.14 PAA-GA.sup.c 1.81 13.79 29.6 7.07 PAA-CS-GA
7.39 37.24 139.5 6.21 PAA-G-GA 6.08 45.52 83.1 8.97 PAA-W-GA 2.95
31.03 42.9 4.14 PAA-I-GA 5.67 24.14 181.3 8.97 PAA-R-GA 4.68 26.89
79.8 6.89 PAA-K-GA 6.21 35.86 73.9 9.66 PAA-T-GA 2.31 164.14 28.8
1.45 PAA-E-GA 5.67 58.62 67.9 8.28 PAA-CA-GA 10.61 86.89 115.4
15.86 PAA-DA-GA 6.4 121.3 7.2 11.73 PAA-GA.sup.d 8.62 485.52 38.8
43 .45 PAA-GA.sup.e 9.03 232.41 63.6 45.52 PAA-GA.sup.f 12.47
737.41 126.4 63.49 PAA-A-GA.sup.h 9.48 821.38 8.8 28.97
PAA-S-GA.sup.h 5.72 697.24 30 43 .45 PAA-A-CS-GA 4.04. 72.41 8.56
6.21 PAA-C-GA 14.51 920 48.3 73.79 All of these films were at 0.18M
PAA concentration, but some of the membranes were at 0.20M
concentrations. .sup.aPAA-GA was casted onto glass, and then non-GA
treated W was added onto the PAA-GA. .sup.bGA treated PAA-C
incubated overnight after being casted on the glass; 3-week old
membrane. .sup.cGA treated PAA incubated overnight after being
casted on the glass; 2h old membrane. .sup.dGA was first diluted in
dry DMAC and then applied to PAA solution. PAA membranes were
casted on clean glass and incubated overnight. After incubation,
DMAC was eliminated from the membranes by soaking them in pure
water. The thickness of PAA membrane is important for its modulus
elasticity and % break elongation; .sup.ewhile thicker one has
higher (4) break elongation, its modulus elasticity is lower [i.e.
thicker one has %63.6 and 273.79 MPa while one was %38.8 and 485.2
MPa. Altering the incubation time was also an important factor as
seen in .sup.fwhich has lower incubation time. .sup.hGA was
dissolved in dry DMAC and then directly applied to PAA-A solution.
The length and width of all membranes were 2.52 cm while thickness
of the membranes was between 0.025-0.1 mm.
[0244] Maximum-load bearing capacities of the membranes mostly
showed relation with the thickness of the membrane. For example,
PAA-CA, Composite membrane, PAA.sup.f, PAA-CS were relatively
thicker than PAA-T, PAA-A.sup.b and PAA-R which showed lower
maximum-load bearing potential. However, PAA-C, PAA.sup.c and
PAA.sup.th which were relatively thinner but could withstand high
load, which is a sign of how small molecule affects the membrane's
mechanical properties. Modulus elasticity and break elongation were
dependent on the procedure and the small molecule combined with
PAA. L-Cysteine and L-Alanine were by far best membranes in FIG.
1a. In terms of L-alanine, two different concentrations (1 mg/mL
and 2 mg/mL) were tested; we however found that the concentration
was not so important ion but the thickness of the crystal L-alanine
residues applied on the membrane makes a difference in terms of
modulus of elasticity. Larger crystals make the membrane weaker.
However, manipulations in the procedure results in alterations of
the mechanical properties.
[0245] New membranes were prepared according to FIG. 1b, and the
mechanical results are discussed below.
TABLE-US-00011 TABLE K Mechanical properties of FIG. 1b films
Tensile Break Break Modulus Maximum strength strength, Elongation
elasticity Film Type load (kg) (MPa) (MN) (%) (Mpa) Combined.sup.1,
2 5.25 26.89 24.68 3.4 1043.87 PAA-A.sup.1, 3, 4 3.13 47.57 22.27
6.1 1750.58 PAA-A.sup.1, 3, 5 2.54 19.3 16.55 2.2 930.79 PAA.sup.1,
3, 6 4.49 68.26 41.85 11.6 1872.62 PAA.sup.1, 3, 7 4.13 62.74 19.72
8 2244.93 PAA.sup.1, 8 2.86 86.18 30.34 16.4 1552.69 PAA.sup.1, 3,
4, 8 2.18 66.19 41.58 23.8 1101.51 PAA-CA.sup.1, 3, 4, 8 4.13 62.74
55.85 24.7 1221.06 PAA-A.sup.4, 9, 10, 11 2.9937072 45.51724138
44.62068966 61.7 235.862069 PAA-A.sup.9, 10, 12 2.3586784
35.86206897 21.5862069 11.2 961.3793103 PAA.sup.7, 9, 13 2.79412672
84.82758621 65.65517241 13.8 2639.310345 PAA-A.sup.4, 9, 10, 14
3.9916096 30.34482759 27.86206897 9.1 1038.62069 PAA-A.sup.7, 9,
10, 14 1.8597272 57.24137931 47.37931034 8.4 2062.068966
PAA-A.sup.7, 9, 10, 11 1.7236496 52.4137931 1.103448276 11.9
1678.62069 PAA-A.sup.7, 9, 10, 15 3.9008912 29.65517241 29.44827586
7.4. 1031.724138 PAA-A.sup.7, 9, 10, 15 4.3544832 33.10344828
27.31034483 8.6 866.2068966 PAA.sup.8, 9, 11 2.2226008 33.79310345
25.86206897 6.7 1193.793103 PAA-A.sup.7, 16, 17 2.5854744
39.31034483 34.89655172 6.3 1382.758621 PAA-A.sup.7, 13, 18
4.4905608 33.79310345 33.03448276 14.6 1264.137931 PAA-A.sup.7, 8,
18 1.9504456 29.65517241 27.24137931 4.9 1400 PAA.sup.7, 8, 19
3.2205032 48.96551724 41.31034483 14.2 1397.931034 PAA.sup.7, 8, 19
3.40194 51.72413793 48.13793103 29.4 1823.448276 PAA- 2.1772416
65.51724138 35.86706897 9.7 2185.517241 DC.sup.8, 9, 20, 21
PAA-A.sup.8, 9, 20 2.2226008 66.89655172 48.27586207 5.8
2762.068966 PAA-W.sup.8, 9, 20 1.4968536 46.20689655 42.48275862 9
1848.275862 PAA-W.sup.8, 9, 20 1.7690088 53.79310345 43.31034483
12.7 1880 PAA-W.sup.8, 9, 20 2.9029888 55.17241379 49.79310345 37.4
1615.172414 PAA-BB.sup.8, 9, 22 2.26796 68.96551724 58.68965517
40.9 2177.241379 PAA-DA.sup.8, 9, 20 2.0865232 63.44827586
10.48275862 30.4 1725.517741 PAA- 0.8164656 31.03448276
-12.27586207 11.8 1315.172414 PCl.sup.8, 9, 23, 24 PAA- 2.4493968
46.89655172 32.48275862 6.6 1368.275862 PCl.sup.8, 9, 23, 25
PAA-pAS- 6.25 95.147 88.942 58 4101.62 SA (4 mg/mL).sup.26, 27
PAA-T-pAS- 4.8 48.95 47.44 63.3 1793.33 SA.sup.26, 27 PAA.sup.26,
27, 28 3.22 48.95 48.95 5.7 2255.28 PAA-SA.sup.26, 29 3.26 33.1 31
63.8 1245.88 PAA-pAS- 3.49 35.16 31.44 37.3 1531.3 SA-A.sup.26, 29
PAA-pAS- 3.76 38.61 31.78 27.1 1490 W.sup.26 PAA-SA.sup.26, 30 9.66
85.49 68.12 10.4 2590 PAA- 4.35 32.6 33.1 18.4 1530 SA.sup.26, 30,
31 PAA-IZ.sup.26 4.94 30.34 24.2 51.6 1170 PAA- 3.99 30.34 23.99
54.7 1400 SA.sup.26, 28, 30, 31 PAA-SA-SN- 5.3 37.4 37.4 45.3 2367
pAS.sup.26, 28, 30, 31 PAA- 5.6 91.2 84.76 57 3850 SA(4 mg/mL)-
pAS-5AS- GA.sup.25, 26, 28 All GA concentrations were
betweenn0.035-0.1% if not otherwise mentioned. In the cases of
Sulfanilic acid (SA), GA was directly added to the SA for
pre-crosslinking, flowed added to the PAA solution. .sup.1PAA
concentrations were 0.18M with FIG. 1b; .sup.2Viscous PAA was
casted on Glass, followed by 0.1 mg/mL GA added PAA introduced on
top of the already casted PAA; .sup.3MeOH was directly added to the
system right after GA addition, or GA-small molecule addition;
.sup.440 .mu.L/mL MeOH added; .sup.580 .mu.L/mL MeOH added;
.sup.610 .mu.L/mL MeOH added; .sup.720 .mu.L/mL MeOH added;
.sup.8FIG. 1b ii; .sup.9PAA concentration is 0.16M; .sup.10FIG. 1b
i; .sup.11Glass-surface was rinsed with dry DMAC; .sup.1210 min
incubation at 72.degree. C.; .sup.134 mg/mL imidazole was used as
cross-linker; .sup.14Glass-surface is rinsed with dry MeOH;
.sup.15Glass-surface and membrane surface were rinsed with DMAC;
.sup.16FIG. 1a iii; .sup.17Glass-surface and membrane surface were
rinsed with MeOH; .sup.18Glass-surface and membrane surface with
EtOH; .sup.19Glass-surface was rinsed with EtOH; .sup.2020 .mu.L/mL
MeOH was added to the PAA solution before cross-linker addition;
.sup.214 mg/mL Diphenolcarbazide [DC]; .sup.224 mg/mL
2-Benzylbenzoyl; .sup.234 mg/mL 4-amino-2-chlorobenzoic acid;
.sup.24The casted solution directly incubated under-hood for
overnight; .sup.254 h incubation in room temperature, and then
incubated under-hood for overnight; .sup.260.12M PAA; .sup.274
months old; .sup.28PAA prepared in 35% DMAC in Ethanol; .sup.291-3
days old; .sup.307-10 days old; .sup.31GA dissolved in DMAC was
added in addition to GA in water. In Table K, BB refers to
2-benzylbenzoyl.
[0246] Among the synthesized films, sulfanilic acid supported
membranes provided the highest mechanical property. Increasing the
concentration of sulfanilic acid from 2 mg/mL to 4 mg/mL improved
the advanced mechanical properties by up to 3 times as shown in
Table K. Among the small molecules incorporated to PAA, sulfanilic
acid is the only small molecule containing --SOOOH group, this
could be the main reason of why sulfanilic acid improved mechanical
properties. The compound readily forms diazo compounds and is used
to make dyes and sulpho-drugs.
1.6 Contact Angle Characterization
[0247] Pure-water contact angles of PAA membranes were tested with
CAM Contact Angle Meter [KSV Instrument, CT] run by CAM100 image
recorder software.
[0248] Contact angle is the parameters typically used to evaluate
hydrophilicity of food-packaging materials. It provides information
on the tendency of the material to absorb water. A good contact
angle, which refers to over hydrophobic range (i.e. over
65.degree.), can substantially eliminate water-vapor absorption
that may trigger microbial development on or within the packaging
material.
TABLE-US-00012 TABLE L Contact angle of PAA membranes Procedure of
Procedure of Membrane FIG. 1a Membrane FIG. 1b Type Top/Bottom Type
Top/Bottom PAA-GA 55.87/51.27 PAA-GA.sup.b 61.73/74.66 PAA-CS-
62.07/53.6 PAA-PCl-GA 80.54/72.10 GA PAA-DA- 62.35/55.7; PAA-pAB-GA
91.49/73.63 GA 45.3/47.3 PAA-CA- 87.26/95.16; PAA-AN-GA.sup.c
57.74/59.21 GA 81.09/79.77* PAA-K-GA 47.81/54.89 PAA-pAB-
73.45/50.75 GA.sup.d PAA-A-GA 80.88/57.02; PAA-CA- 65.91/66.78
56.58/63.7* AcOH-GA PAA-W-GA 47.05/65.32 PAA.sup.e-AcOH-
88.19/80.56 GA PAA-R-GA 44.03/51.54 PAA.sup.f-AcOH- 82.84/79.88 GA
PAA-C-GA 57.07/41.89 PAA.sup.g-AcOH- 80.98/78.43 67.37/51.2* GA
PAA-T-GA 63.91/84.67 PAA-GA.sup.g 80.23/79.20 PAA-I-GA 47.23/55.65
PAA-GA.sup.h 62.20/66.25 PAA-G-GA 66.53/69.1; PAA-A-pAS-
75.60/74.20 65.11/62.5* GA.sup.h PAA-E-GA 78.78/59.09 PAA-I-pAS-
77.25/78.30 GA.sup.h PAA-S-GA 97.33/62.48; PAA-S-GA 52.62/60.53
74.27/61.5* Composite.sup.a 95.27/80.7 PAA-SA-pAS- 82.56/84.3 GA In
Table L * refers to 0.5-1% GA concentration and overnight
incubation. The remaining membranes were consistent with 0.1-0.2%
GA concentration and 6 h incubation: .sup.aGA treated PAA casted on
the glass, and then non-GA treated PAA-W and PAA-CA was added onto
the PAA-GA and left 6 h incubation. .sup.bmembrane was
phase-inverted under hood; .sup.cAN refers to Amonium Nitrate;
.sup.dFormaldehyde was used for cross-linking; .sup.e20 .mu.L/mL
olive oil; .sup.f40 .mu.L/mL olive oil; .sup.g100 .mu.L/mL olive
oil; .sup.h0.12M PAA. Standard deviations out of 3-runs were less
than 6% for all membranes developed.
[0249] Contact angle of the membranes synthesized according to
FIGS. 1a and 1b did not indicate a substantial difference.
[0250] For FIG. 1a membranes, when the membranes includes a shiny
surface with having amorphous inner part gave the highest contact
angle. However, the membranes containing L-lysine (K) and
L-arginine (R) gave the lowest contact angle even though they were
fully amorphous; these K and R containing membranes were somewhat
porous.
[0251] For FIG. 1b membranes, adding acetic acid to PAA before
introducing GA advanced the contact angle from 65 to 88, but
increasing olive-oil concentration into the PAA decreased the
observed contact angle. Typically, oil is thought of as
hydrophobic; probably acetic-acid making oil causing pore-opening
which results in enhances water-membrane interaction. However,
adding oil into the PAA without acetic acid, it advances the
contact angle. Among FIG. 1b membranes, p-aminobenzoic acid (pAB)
containing glutaraldehyde (GA) treated PAA membranes gave the
highest contact angle; this could be related to that GA eliminate
free amino-groups on pAB, which decreases hydrophilic properties of
PAA.
[0252] In all cases, the present films contain sulfanilic acid,
p-aminosalicylic acid and glutaraldehyde The obtained contact angle
was over 65.degree., thus, the small molecule incorporated within
the PAA film can serve as good-packaging materials due to the
contact angle data.
1.7 Electrochemical Characterization
[0253] Four-probe and Ohm meters were utilized for characterization
of the electronics properties of the membranes, as shown in FIG.
34. In this figure a Jandel-brand four-point probe is shown. The
technique is widely used to measure resistivity of thin conducting
layers. In this system, current and voltage were measured
simultaneously. The system has two current and two voltage probes;
it gives information about probe resistance, contact resistance and
semiconductor resistance. The results obtained from the four-probe
was more accurate than simple voltmeter reading because the contact
resistance is negligible in the four-point probe systems.
[0254] According to 4-probe and ohmmeter, none of the films was
found to be conductive. The multimeter can go up to 200 M.OMEGA.
and the scale was not sufficient to measure the resistance of the
membranes, therefore the membranes were accepted as insulators.
[0255] Then the films were utilized as support material for
gold-coating, ash shown in FIGS. 35a-35c. FIGS. 35a-35c include
digital images of E-beamed gold on PAA ternary films and
Whatman.RTM. paper. As seen from "a" and "b", 100 nm gold layer was
coated on two different PAA membranes via E-beam under the current
of 0.104 A. Similarly, 100 nm gold layer was coated on Whatman.RTM.
paper (c) under same conditions.
[0256] Four-point probe resistivity of gold e-beamed PAA ternary
membrane and Whatman.RTM. paper. FIG. 36a shows the current-voltage
graphic of the coated gold from 4-probe test at 4-different
sections on the paper; FIG. 36b shows current-voltage graphic of
the coated gold from 4-probe test on PAA-SA and PAA-pAS-SA
membranes. The obtained voltages demonstrates the difference of
over 50% for the paper electrode, while the coated gold on
different PAA membranes did show difference of less than 15%.
However, the difference from different sections of the coated gold
on same-membrane was obtained at less than 5%. When a multimeter
was used to determine the resistance of the coated gold from one
end to other end, the measured conductivity was 7.4.OMEGA.. The
membrane itself was found to be a total insulator; the multimeter
reading went beyond the calibrated level of 200 M.OMEGA., and the
instrument showed that the resistance was beyond the limit.
[0257] SEM images at 10000 and 100000 magnification of gold
e-beamed PAA ternary membrane and Whatman.RTM. paper are shown in
FIGS. 37a and 37b. FIGS. 37a and 37b indicate that the coated gold
on the PAA-SA was uniform and even. This could be the reason of the
characteristic stable resistance recorded. The obtained resistance
was 15-times larger than that noted on the Whatman.RTM. paper,
which could be related to the fact that some gold could be embedded
into the PAA membrane.
[0258] Since the membranes were determined to be non-conductive
according to the 4-probe conductivity measurement,
cyclic-voltammetry was further utilized to determine any possible
electro-activity of the PAA membranes. All of the experiments were
performed in 50 mM pH 7.4 PBS buffer. Platinum and silver wires
were used as auxiliary and reference electrodes, respectively.
Working electrodes were 200 nm gold-coated (via e-beam)
Whatman.RTM. papers. Scanning rate was 50 mV, and the range was
200-600 mV.
[0259] Cyclic voltammetry results of ternary PAA membranes is shown
in FIGS. 38a-38h. (38a) Series 1-7 are PAA, PAA-GA (aged GA),
PAA-PDA, PAA-W-GA, PAA-pAS-GA (longer drying), PAA-pAS-GA, PAA-GA
(aged) longer drying, respectively. (38a a) overlapped of all types
of PAA membranes; (38a b) PAA, (38a c) PAA-GA (aged GA), (38a d)
PAA-PDA, (38a e) PAA-W-GA, (38a f) PAA-pAS-GA (longer drying), (38a
g) PAA-pAS-GA, (38a h) PAA-GA (aged) longer drying, respectively.
Ternary PAA membranes were loaded on gold surface on e-beamed
Whatman.RTM. paper. Longer drying refers to over 12 h drying, and
while the rest were dried at than 6 h. All the tests were performed
in pH 7.0 (50 mM) phosphate buffer in the presence of silver-wire
as reference electrode and platinum wire as auxiliary
electrode.
TABLE-US-00013 TABLE M Summary of the Electrochemical
Characterization of PAA Membranes Peaks for Peaks for oxidation
reduction Membrane (mV) (mV) Reversibility PAA -50 150
Quasi-reversible PAA-GA -50/100/295 130/320/470 Quasi-reversible
PAA-PDA 55 410 Irreversible P AA-W-GA 100 220 Quasi-reversible
PAA-pAS-GA -75 34 Quasi-reversible PAA-pAS-GA No peak No peak NA
PAA-GA No peak No peak NA
[0260] Conductivity of the PAA co-polymers showed dependence on
aged GA and the small molecule type, including the time of
incubation. PAA-PDA-GA provided the highest oxidation reduction
potentials (see the scale on the y-axis for FIG. 38b), followed by
PAA-W-GA and PAA-GA. Overnight incubation under hood made PAA-GA
non-conductive under the tested conditions. Similarly, PAA-pAS-GA
(8 h incubation under hood) lost conductivity when it was incubated
overnight under the hood. Even though there was no peak for
PAA-pAS-GA, recorded current seems to be 10-times higher than PAA.
As seen from Table M, PDA and W additions masked the peaks coming
from PAA itself. PAA-PDA-GA provided the highest
oxidation/reduction peaks while PAA-W-GA gave the closest distance
for oxidation and reduction potentials, 110 mV. GA added two extra
reduction and oxidation peaks to the membrane, and pulled PAA's
reduction to 130 mV from 150 mV which could be a sign that the
electroactive nature of the aged GA and/or GA-crosslinked PAA
requires less potential for reduction. GA, as shown elsewhere
affects the oxidation/reduction peaks of PAA and CS, and showed the
existence of additional new peak that was not sharp in PAA-GA and
PAA-CS-GA because of that the GA was fresh and much diluted.
[0261] Even though PDA provides good conductivity, PDA-PMDA based
PAA membranes did not provide strong mechanical properties and
durability, so GA was subsequently introduced. PDA was first
dissolved in DMAC, followed by cross-linking with GA for 30 sec;
the polymerized PDA was then directly introduced to PAA solution.
The resulting PAA-PDA-GA was casted on gold-coated Whatman.RTM.
paper, followed by drying under hood for 6 h with further rinsing
in pure water. Similarly, all of the PAA coated electrodes were
rinsed in pure water before they were exposed to cyclic
voltammetry.
1.8 Water, Water-Vapor and Oil Permeability
[0262] FIG. 39 is a digital image of the oil-permeability test used
in gathering the below data. This test was performed with the
protocol of 5 mL Extra virgin olive oil was put into a vial whose
interior diameter and outer diameter were 20 mm and 24 mm,
respectively. The tested membranes were used to cover the open part
of the vial; the vial was placed upside down on top of a
Whatman.RTM. paper [Sigma-Aldrich, MO] as shown in the digital
image in FIG. 39. The membranes were incubated for 14 days at
37.degree. C. with 95% humidity. The experiment was discontinued
after 14 days.
[0263] FIG. 40 is a digital image of the water-vapor permeability
test used in gathering the data below. This test was performed by
the following method; measuring the weight changes of the vial
containing 5 g dry desiccant. The tested membranes were used to
cover the vial entrance, and the vial was incubated at 37.degree.
C. incubator, 5% CO.sub.2 and 95% humidity. Incubation was
conducted for 7 days. In addition to this, water permeability of
the membrane was tested with Millipore Lab-scale TFF System 115V
(Millipore, Billerica Mass.); the pressure was kept under 30 psi,
and eluent-side was observed for wetness testing.
[0264] Resistance to oil and water-vapor penetration are important
for keeping a containment fresh and for preventing the loss of
taste and flavor. Different approaches have been applied to provide
resistant surface to oil and water vapor transfer.
[0265] Due to the good mechanical properties, FIG. 1b membranes
were used in these tests. PAA-A-GA and PAA-GA were used for both
tests. It was proven that oil does not pass through the membranes.
The tests were carried out in buffers and strong polar and apolar
solvents including PBS buffer, DMF and hexane.
[0266] The water vapor permeability takes thickness as a parameter
to determine the power of membrane against vapor permeability by
which both the quality of membrane and the importance of thickness
can be evaluated more objectively.
[0267] Certain membranes can be used to cover the top of the vial
without requiring an adhesives for which only thin string or
parafilm is enough. However, in some cases such as sulfanilic acid
enhanced or 2BB co-polymerized PAA membranes are not easily
attached with a thin string or parafilm since they are harder
plastic.
[0268] Further, PAA-I-GA, PAA-I-pAS-GA and PAA-A-pAS-GA were
tested, and no transfusion was observed. These results showed that
the membranes synthesized according to FIG. 1b were better than
those reported ones such as glycerol enhanced-cellulose
sulfate.
[0269] Since sulfanilic acid (SA), sulfanilamide (SN),
p-aminosalicylic acid (pAS) and 5-aminosalicylic acid (5AS) were
used as the main molecules in the developed membranes,
PAA-pAS-5AS-SA-GA and PAA-pAS-5AS-SN membranes were tested as well.
Membranes at different thickness were tested. Thickness and texture
did not affect the membranes resistant to oil-permeability.
1.9 Biodegradability and Toxicity Characterization
[0270] The synthesized membranes were heavily cross-linked with
glutaraldehyde, and co-polymerized with intrinsic antimicrobial
agents (i.e. sulfanilic acid, p-aminosalicylic acid). The
biodegradability of these films is determined below. However, the
introduction of these antimicrobial agents made the resulting
membranes to be biodegradable. Therefore, it was required to test
biodegradability of the PAA membranes.
[0271] For the below data microorganisms were obtained from rotten
sticks from American Elm (Ulmus americana) in the University's
garden, Binghamton-NY. .sup.1H NMR, .sup.1H-correlation
spectroscopy (COSY), and .sup.1H-.sup.13C-heteronuclear single
quantum coherence (HSQC). The fungus chunk was directly introduced
to the bioreactor without any selection. Pre-selected fungus and
fungus chunk were characterized with molecular biological
techniques. The plasticized PAA membranes showed dramatic
differences in terms of physical characteristics than the membranes
designed elsewhere, so it required further biodegradation testing
of the new membranes.
[0272] Characterization of the isolated fungi species were done in
the Department of Sustainable Bioproducts, College of Forest
Resources, Mississippi State University, Starkville, Miss.,
USA.
[0273] FIG. 1a/1b membranes were used in biodegradation studies
because if these membranes can be degraded, the rest of PAA
membranes synthesized in this study can be degraded as they have
lower degree of cross-linking. PAA-GA and PAA-CS-GA have been shown
to degrade in less than two months by the ascomycete fungus.
Fusarium oxysporum.
[0274] The bioreactor contained 25 mg/mL YPD medium, 0.1 mg/mL
D-glucose, 1% L-glutamine, 25 .mu.L/mL trace-metal solution and 5
mg/mL Peptone, which only contains the fungi chunk that was later
identified as Trichaptum biforme. The pH of the medium was adjusted
to pH 5.7 before autoclave. The bioreactor volume was 100 mL, and
the membranes were from 50 mg PAA-A-GA of FIG. 1a i and 50 mg FIG.
1a ii PAA-GA. It also includes 50 mg PAA-A-pAS-GA and 50 mg
PAA-SA-GA of FIG. 1b.
[0275] The membranes were not crushed, and put into the medium as
they were. The blank bioreactor was cultured under same conditions
without the membranes. Disintegration was monitored by visual
decreases in the membrane size while structural degradation was
monitored via NMR. Disintegration was not able to be monitored when
microbial biomass totally covered the membrane surface. In this
experimental design, there were four main differences from previous
designs: [0276] The starting fungus inoculum was prepared by
dissecting the fungi chunk, and taken the inner part as the
starting biomass, which was later on characterized as Trichaptum
biforme. [0277] Cells were not acclimatized before they were used
to degrade the films of FIGS. 1a and 1b. [0278] Trace metal
solution was used at 25 .mu.L/mL to enhance overall activation of
laccases and manganese peroxidases to advance PAA degradation
[0279] Fed-batch process was used instead of combination of
fed-batch and continuous process
[0280] For each NMR run, 1 mL of solution from the bioreactors was
put into 1.5 mL polypropylene microcentrifuge tube. The sample was
kept in -20.degree. C. overnight, and then lyophilized for 24 h.
The resulted solid sample was dissolved in 0.9 mL D.sub.2O. The
dissolved sample was then left for precipitation of non-dissolved
sample for 15 min; the final volume was between 0.75-0.8 mL.
Degradation of the membranes was monitored via .sup.1H NMR, .sup.1H
COSY and .sup.1H .sup.13C HSQC NMR techniques.
[0281] "The trace metals solution" contained 20 mM
FeSO.sub.47H.sub.20, 2 mM CuSO.sub.45H.sub.20, 5 mM ZnCl.sub.2, 20
mM MnSO.sub.4 H.sub.20, 6 .mu.M CoCl.sub.2 6H.sub.20, 1 mM
NiCl.sub.26H.sub.20, and 1 mM MoCl.sub.3.
[0282] Both Trichaptum biforme, a white rot fungus, belonging to
Basidiomycota division of Fungi kingdom and Trichaptum biforme,
similar to Fusarium oxysporum can degrade aliphatic and cyclic
organic pollutants were used. Basidiomycetes are among the higher
fungi that can develop multicellular mycelium. They are mainly
found in rotten trees where they degrade lingo-cellulosic polymers
via extracellular enzymes including manganese peroxidases, laccase
peroxidase and so on.
[0283] Full .sup.1H spectra of the PAA for peptone-yeast medium
(FIG. 41a), at day 20.sup.th (FIG. 41b), 30.sup.th (FIG. 41c),
35.sup.th (FIG. 41d), 40.sup.th (FIG. 41e) and 50.sup.th (FIG.
41f), and (FIG. 41g) 120.sup.th. Degradation of the plasticized PAA
membranes were monitored by examining the aromatic peaks belonging
to PAA. The three peaks between 2-3 ppm [2.13, 2.53 and 3.10 ppm]
were observed which could be thought to belong to DMAC. However, as
seen in FIG. 41j, DMAC gave three peaks in .sup.1H .sup.13C HSQC;
at 120.sup.th day .sup.1H .sup.13C HSQC, these peaks were confirmed
as also belonging to DMAC. This could be related to that addition
of high amount of PAA membranes resulted in partial dissolution of
the polymers which were surrounded by the metabolites or
peptides/proteins released into the medium. This could then prevent
the release of DMAC into the medium.
[0284] DMAC is a volatile organic-solvent, and is supposed to
evaporate from the system upon overnight lyophilization. NMR sample
was prepared via freeze- and thawing procedure where
overnight-lyophilization was applied to eliminate all the solvents
coming from the biodegradation media. Disintegration of the
membranes was completed within 20 days. That was why the 20.sup.th
day was selected as the first day of sampling. Aromatic regions in
I H NMR spectra at 20.sup.th and 30 days showed strong
similarities; the doublets and triplets are quite similar. Aromatic
regions in .sup.1H NMR spectra at 35.sup.th and 40.sup.th days
showed largely triplets in contrast to 20.sup.th and 30.sup.th
days; 35.sup.th day still showed some doublets and singlets which
signifies the presence of PAA. However, the aromatic regions in
.sup.1H NMR spectrum of 40.sup.th day did not show any clear
evidence of the presence of PAA. Interestingly, the aromatic
regions in .sup.1H NMR spectra at 20.sup.th and 40.sup.th days
showed strong similarities for the presence of triplets. For the
20.sup.th and 30.sup.th days, a doublet was seen at 5.82 ppm which
could be a sign of fragmentation of the PAA molecule, which was
then consumed because at 35.sup.th and later-days the peak
disappeared.
TABLE-US-00014 TABLE N Characteristic peaks related to PAA
degradation Day Aliphatic range, incubation Aromatic range, ppm ppm
20 Doublets of PAA, 6.9-8.1 30 Doublets of PAA, 6.9-8.1 35
Triplets, no sign of PAA 40 Triplets, no sign of PAA 50 Doublets of
PAA (6.9-8.1), and triplets from degradation/metabolites 120
Overwhelmingly doublets Peaks of DMAC of PAA, 6.9-8.1 (1.95, 2.7
and 3.2)
[0285] Since PAA is composed of aromatic groups, it is not expected
to show any triplet. The triplets at aromatic region are a strong
sign of partial or total saturation of the rings found in PAA. Then
50 mg of PAA-pAS-GA and PAA-SA-GA were added to the system at
40.sup.th day, which was then analyzed at 50.sup.th day. During
this period, only 0.2 mg/mL sugar was added to the medium at
40.sup.th day. Then, the system was run in continuous mode. From
6.5 to 8.5 ppm range of 50.sup.th and 120.sup.th day was overlapped
to see the changes in PAA degradation. Analyzing the 50.sup.th day
data we found that the aromatic region in .sup.1H NMR revealed the
peaks of PAA and the triplets which are the signs of saturation of
aromatic groups in PAA and/or the newly formed cyclic groups. Then,
100 mg of PAA-pAS-GA were added to the medium, and the mixture was
incubated for additional 70 without adding any sugar or peptone to
the system. Then, at 120.sup.th day, .sup.1H NMR of the bioreactor
was carried out; but only the PAA related peaks were found.
[0286] When the .sup.1H NMR spectra were compared the followings
were observed; Peptone yeast .sup.1H NMR possesses doublets and
triplets between 3.27-3.94 ppm, which is not found in any other; a
singlet at 3.10 ppm, which is slightly larger at 20th and 30th
days, while very small at 35th and 50th days; a singlet at 3.30 ppm
is relatively larger at 20.sup.th, 30.sup.th and 35.sup.th days
while it is smaller at 40.sup.th and 50.sup.th days; the peak is
not clear for peptone-yeast medium; a singlet at 3.93 ppm probably
found in all of them but not found in peptone-yeast medium; three
singlets between 2.75-2.78 ppm in all, but not in peptone yeast;
the triplet at 2.43 ppm found in all; a singlet 2.12 found in
20.sup.th and 30.sup.th days, might be in 50 day; but it was not
found in peptone-yeast medium, 35.sup.th and 40.sup.th days; a
singlet at 2.01 ppm is found in 20th, 30th, 35th and 50th days, but
not in peptone yeast and 40th days, this peak is relatively bigger
for 20th and 30th days; a peak at 1.95 ppm for 20.sup.th,
30.sup.th, 35.sup.th days which is very small for 50th day while it
was not found in 40.sup.th day and peptone-yeast medium; a singlet
found in all, except 30th day at 8.49 ppm; the singlets found at
8.37 and 8.29 ppm only for peptone-yeast medium; the singlet at
7.84 is only for peptone-yeast and 40th days; they are quite
similar; the two singlets at 7.6 and 7.7 are only for peptone-yeast
medium; there is a triplet at 7.46 ppm found in peptone yeast
medium, 20.sup.th, 35.sup.th and 40.sup.th days; slight presence of
the peak is in 50.sup.th day while it was not in 30th day; the
singlet at 7.10 ppm is only for peptone yeast medium; the doublet
(for 35.sup.th and 40.sup.th days) or the broad peak (for
20.sup.th, 30.sup.th and 50.sup.th days) at 7.22 ppm where peptone
yeast medium has a triplet. However, the doublet is relatively
bigger for 30.sup.th day. There are 4 broad (or doublet) and a one
singlet on upper field of this peak. These are the sign of presence
of PAA. However, it is not easy to say that they are present in
30th and 35th days; the broad peak at 7.61 ppm for 20.sup.th,
30.sup.th and 50.sup.th days, which is not included in 35.sup.th
and 40.sup.th days; the singlet peaks between 6.5 to 8.5 ppm
shifted during from 20 to 50.sup.th days, could be related to the
biodegradation of PAA; the triplet at 7.56 ppm for 20.sup.th,
30.sup.th and 35.sup.th days where 40.sup.th day has nothing;
peptone yeast medium has a doublet and 50.sup.th day has a singlet.
This shows there is a degradation of PAA, but at 50.sup.th day
intact PAA polymers or PAA polymers protected their back-bone
structure are present in the media; the two triplets and a doublet
between 7.3-7.5 ppm found in 20.sup.th, 30.sup.th, 35.sup.th and
40.sup.th day look similar, but that is not possible to say they
are from peptone or sugar since the peaks between 2-4 ppm showed no
similarity. Actually, the shapes of these triplets don't look
alike; when 50.sup.th and 120.sup.th days compared, there is no
improvement rather the triplet at 7.46 and the doublets 7.41 and
7.35 went away for 120.sup.th day, and the singlet at 8.49 ppm went
away as well.
[0287] Biodegradation of the membranes (PAA-CS-GA and PAA-GA)
showed some-differences such as PAA was fragmented into little
fragment which was seen as that integrals of the four-peaks between
6.9 to 7.2 ppm became similar with time. For example, at 15.sup.th
day, the major peak was overwhelmingly larger while at 30.sup.th
day they were all the same. Also, at 30.sup.th day, the triplets
appeared; the sign of saturation of the double bonds in PAA and/or
conversion of the groups. However, for 15.sup.th to 27.sup.th days,
the triplets were not seen or could be very low. Therefore, it can
be said that the fungi degraded the PAA-CS-GA and PAA-GA polymers
into two steps; first degraded the larger PAA polymers into small
PAA polymers. In the second step, the fungi quickly degraded the
smaller-sized PAA polymers. 10287 In contrast to this, as seen from
the FIGS. 41a-41j, consumption of the PAA membranes synthesized in
this chapter were done without requiring fragmenting the larger PAA
polymers into smaller PAA polymers. These results are summarized in
Table N above, as a function of time of degradation. As seen from
Table N, at the 35.sup.th day, all the PAA was consumed by the
fungi.
Characterization of the Fungi Responsible for PAA Degradation Using
Molecular Biological Techniques
[0288] Genomic DNA Isolation-Genomic DNA was isolated from dried
mycelium by use of the NucleoSpin.RTM. Plant II Kit (MACHEREY-NAGEL
GmbH & Co. KG). The dried weight of the mycelium was 0.05 g for
sample A and 0.07 g form sample B. Mycelium was washed with 95%
ethanol for 2 hours. The mycelium was transferred to tubes with 2
mm glass bead sand homogenized with CTAB lysis buffer (2%
cis-trimethyl ammonium boric acid, 100 mM Tris, 20 mM Na2 EDTA, 1.4
M NaCL, and 1% polyvinylpyrolidine). The extract was treated with
RNase A (200 ng/ul, incubate at 65.degree. C. for 10 min) followed
by Proteinase-K and incubated at 65.degree. C. for 1 hour. The
remainder of the extraction followed the kit instructions for
isolation of DNA from fungi. DNA was eluted with 50 .mu.l buffer PE
heated to 65.degree. C. The concentration was measured on a
Nanodrop 1000 spectrophotometer. The purity of the DNA was
evaluated in gel electrophoresis on a 1% agarose gel in 1.times.SBA
(Sodium Boric Acid).
PCR Amplification
[0289] The Internal transcribed spacer (ITS) region of the fungi
was amplified using primers ITS1-F (5'-CTT GGT CAT TTA GAG GAA GTA
A-3') (SEQ ID NO: 1), which is specific for the higher fungi
(Gardes et al. 1993), and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3')
(SEQ ID NO: 2), the universal primer. Amplifications were performed
in Eppendorf Mastercycler.RTM. with the following settings: an
initial hot start at 98.degree. C. for 2 min (DNA template only),
melting at 95.degree. C. for 45 s, annealing at 52.degree. C. for
45 s, and extension at 72.degree. C. for 2 min, and final extension
at 72.degree. C. for 10 min for 35 cycles. After the initial hot
start, a master mix containing 10 mM reaction buffer, 25 mM
MgCl.sub.2, 10 mM Forward and 10 mM Reverse primers, 10 mM
deoxynucleotide triphosphates (dNTPs), 10 mg/ml Bovine Serum
Albumin (BSA), between 100-200 ng/.mu.l total DNA isolated from
samples, deionized water, and 2.5 U/.mu.l Tag polymerase was added
to each sample.
[0290] PCR products were separated by electrophoresis in 1%
(wt/vol) agarose gels in 1.times.SBA buffer (Sodium Boric Acid)
with RedGel (100 ng/ml) and running buffer; DNA bands were
visualized by the fluorescence of the intercalated RedGel under UV
light and photographed.
Sequence Analysis
[0291] The amplified fragments were inserted into the pGEM-T easy
vector (Promega, Madison, Wis.) for sequencing, and the sequence of
ITS regions were confirmed by sequencing at least three individual
recombinant colonies using a Beckman Coulter (Brea, Calif.) CEQ8000
DNA sequencer. The sequence data were assembled and analyzed by the
use of CEQ sequencing analysis software and MegAlign
(Lasergene.RTM.) and were then searched by using the ITS-1F and
ITS4 primer sequences to define the ITS region. Each sequence was
analyzed into the ITS region and was then separately used to
perform the individual nucleotide-nucleotide searches with the
BLASTn algorithm at the NCBI website. The outputs from the BLAST
searches were sorted on the basis of the maximum identity and were
recorded. Sequence-based identities with a cutoff of 99% or greater
were considered significant in this study, and the best hit was
defined as the sequence with the highest maximum identity to the
query sequence.
TABLE-US-00015 TABLE O The consensus sequence of sample Aand sample
B. Consensus sequences Sample
AGTTGGGGTTTAACGGCGTGGCCGCGACGATTACCAGTAA A
CGAGGGCTTTACTACTACGCTATGGAAGCTCGACGTGACC
GCCAATCAATTTGAGGACAGGCATGCCCGCCAGAATACTG
GCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGA
ATTCTGCAATTCACATTACTTATCGCATTTTGCTGCGTTCTT
CATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTT
GATTTATTTATGGTTTTACTCAGAAGTTACATATAGAAACA
GAGTTTTAGGGGTCCTCTGGCGGGCCGTCCCGTTTTACCGG
GAGCGGGCTGATCCGCCGAGGCAACAAGTGGTATGTTCAC
AGGGGTTTGGGAGTTGTAAACTCGGTAATGATCCCTCCGC TGGTTCACCAACGGAGACCT (SEQ
ID NO: 3) Sample CCCGGGGCAAGGGGCGGGCGGCGTTGGATTTTGCGGGACC B
CTTAACACCCGCTTCCAGCCGCGCGGGCGCCGCCGCCCCG
AGGCCCGGCGCCGATCTAACAAGTAATACATCTCAAAGGT
GTCCAACCGTATCCAACCAGTGGACGTCCGAGGGTCGCGC
CGTTTGAGTGTCATGTTAATATCAACTCTGATGGTTTTTTG
TTAATCATTGGATGTTGGACTTGGGGATCCCGTCACAGTCG
ACTACTGATGAGTACTATAGACTACGCATCGCGCAGCTGA
TATATTTAATGTCTACGTATATCAATCCATTAATAAA (SEQ ID NO: 4)
[0292] FIG. 42 shows pictures of a macroscopic and four microscopic
pictures of Trichaptum biforme (Picture of 1000.times. Oil
Immersion).
[0293] FIG. 43 is microscopic pictures of Fusarium oxysporum
(Picture of 1000.times. Oil Immersion).
Raw sequence data for the samples are listed below:
TABLE-US-00016 Sample A-1 Forward (SEQ ID NO: 5)
CCGCGNGGAGGTTTCTGGACCGCTGTCCGACCGCGCCGCTCCGTTC
GGCGCCGAGTTCCACTTTGTCCCCTCATTNATATTGTCAATTACGCGGGTA
TTCCACCGATTCCAGCTCACTTCGAAGTTGGGGTTTAACGGCGTGGCCGCG
ACGATTACCAGTAACGAGGGTTTTACTACTACGCTATGGAAGCTCGACGTG
ACCGCCAATCAATTTGAGGAACGCGAATTAACGCGAGTCCCAACACCAAG
CTGTGCTTGAGGGTTGAAATGACGCTCGAACAGGCATGCCCGCCAGAATA
CTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCA
ATTCACATTACTTATCGCATTTTGCTGCGTTCTTCATCGATGCCAGAACCA
AGAGATCCGTTGTTGAAAGTTTTGATTTATTTATGGTTTTACTCAGAAGTT
ACATATAGAAACAGAGTTTTAGGGGTCCTCTGGCGGGCCGTCCCGTTTTAC
CGGGAGCGGGCTGATCCGCCGAGGCAACAAGTGGTATGTTCACAGGGGTTT
GGGAGTTGTAAACTCGGTAATGATCCCTCCGCTGGTTCACCAACGGAGACC
TGTNACAACTTTNACTCCCTCTAATGACAAAATCACTANTGAATCCCGCCG
CCGCAGTCACATATGGGAGAGCTCCCACGCGTGGATCTANCTGAGTATCTA
TANGTCACCTAATACTGGCGTATCTGGTATACCGTCCCGGTAATGTTATCC
CCCATTCCCCACTCACCGAACTAATGTAACGGGTCA Sample A-1 Reverse (SEQ ID NO:
6) CCCTCTTTNAAATTCTTTTTAGGGGGGGGCGACTTCCCGGCGGGGCT
ACTCAGTCATGGATCTCTGGATGCAATAANATATTAGCGATCTTCGCCNGT
GAACCACGAGGAGGATCACNAGTGCAACCCCAAACCCCTGTGAACATCC
ACTTGTTGCCGCGCCGATNCGNCCGCCCCCGTAAAACGGGACGGCCCGCC
AGAGGACCCCTAAAACTCTGTTTCTATATGTAACTTCTGAGTAAAACCATA
AATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAG
AACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCA
TCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCT
GTTCGAGCGTCATTTCAACCCTCAAGCACAGCTTGGTGTTGGGACTCGCGT
NAATTCGCGTNCCCTCAAATTGATTGGCGGTCACGTCAAGCTTCCATAGCG
TAATAGTAAAAACCCTCGTTACTGGTAATCTCCGGCCACGCCGTAACCCCA
CTTTGAATGTGACCCGATCGGTAGGATACCGCGAACTAACTATATACGAG A Sample A-2
Forward (SEQ ID NO: 7)
CCGGGCGGGAGGTTTNGTTAGGGATCCCGTCGCTCGACGCGCGCCG
CGCCGGTCGGCGCGCGAGTGGCCATCGGTGTCCGCCTCATTCAGTATNGTC
AAGTGTGACGCGGGTATTCCTCACCCGATTCCAGGTGCACTTCCAGAAGTT
GGGGTTTAACGGCGTGGCCGCGACGATTACCAGTAACGAGGGCTTTACTA
CTACGCTATGGAAGCTCGACGTGACCGCCAATCAATTTGAGGAACGCGAA
TTAACGCGAGTCCCAACACCGAGCTGTGCTTGAGGGTTGAAATGACGCTC
GAACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAAAGA
TTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTTGCTG
CGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATT
TATTTATGGTTTTACTCAGAAGTTACATATAGAAACAGAGTTTTAGGGGTC
CTCTGGCGGGCCGTCCCGTTTTACCGGGAGCGGGCTGATCCGCCGAGGCAA
CAAGTGGTATGTTCACAGGGGTTTGGGAGTTGTAAACTCGGTAATGATCCC
TCCGCTGGTTCACCAACGGAGACCTTGTTACGACTTTTACTTCCTCTAAAT
GACCAAGAATCACTAGTGAATTCGCGGCCGCCTGCAGGTCAACATATGGA
GAGCTCCACCCGTGGATGCATANCTGAGTATCTATAGTGTCCCTAATACTT
GGCGTATCATGGCATACCGGTTCCGTGTGAAATGTTATCGCTCACCATCCA
ACAAATACNACCCGAAACTTAANGTTAACCGGGGGTCCTAATAGTGACCA
CCCATTANTGCNTTGCC Sample A-2 Reverse (SEQ ID NO: 8)
CGGAGGTTTTTTGGGNCNCCGTCGCGACNAGGGCCCTCACTTGGAG
CTCCGACCGGNCGCGCCAATTAACTCATGGATTTCGGGGATTTAGAGGAA
GTAAAAAGTTTTAACAGGTGTCCCGTTGGTGAACCAGCGGAGGGATCTTAC
CGAGTTTACACTCCCAAACCCCTGTGAACATACCACTTGTTGCCTCGGCGG
ATCAGCCCGCTCCCGGTAAAACGGGACGGCCCGCCAGAGGACCCCTAAAA
CTCTGTTTCTATATGTAACTTCTGAGTAAAACCATAAATAAATCAAAACTT
TCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATG
CGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACG
CACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTT
CAACCCTCAAGCACAGCTCGGTGTTGGGACTCGCGTTAATTCGCGTTCCTC
AAATTGATTGGCGGTCACGTCGAGCTTCCATAGCGTAGTAGTAAAGCCCTC
GTTACTGGTAATCGTCGCGGCCACGCCGTTAAACCCCAACTTCTGAATGTT
GACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGC
GGAGGAAATCGAATTCCGCGGGCGCCATGGCGGCCGGAACATCAACTTCG
GCCAATCCCCTATATATGTATACATCCTGGCGNTTNACAACTGGACGGGAA
ACGCGTACCACTATCCTGCNCATCCCTTCCCCGGCTATTCAAGCCCCCACC
CTCCAATGCCCCAATGG Sample A-3 Forward (SEQ ID NO: 9)
CCGGAGGTNAGNCAGCACCCGCCCCTNGGAACCCNCCCATATTCTA
CCTGTNACCCATTTAGGCATACAATTGGGTGAACGCTGGCCCACATACCTA
ACAGGGCTACACTACCATGGAAGCCACTGACCGCCATCATTTGAGGAACG
CAATTAACGCGAGTCCCAACACCGAGCTGTGCTTGAGGGTTGAAATGACG
CTCGAACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAA
AGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATTCGCATTTT
GCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTT
GATTTATTTATGGTTTACTCAGAAGTTACATATAGAAACAGAGTTTTAGGG
GTCCTCTGGCGGGCCCGTCCCGTTTTACCGGGAGCGGGCTGATCCGCCNAG
CAACAAGTGGTATGTTACAGGGGTTGGGAGTTGTAACCGTAAT Sample A-3 Reverse (SEQ
ID NO: 10) GGGCGTTATATCTTGTGGTCTCCCGCGCTTGAGGAGCTCTCCCATAT
GTGTCGACCTGCAGGCGGCCGCGAATTCACTAGTGATTCTTGGTCATTTAG
AGGAAGTAAAAGTCGTAACAAGGTCTCCGTTGGTGAACCAGCGGAGGGAT
CATTACCGAGTTTACAACTCCCAAACCCCTGTGAACATACCACTTGTTGCC
TCGGCGGATCAGCCCGCTCCCGGTAAAACGGGACGGCCCGCCAGAGGACC
CCTAAAACTCTGTTTCTATATGTAACTTCTGAGTAAAACCATAAATAAATC
AAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAG
CAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATC
TTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAG
CGTCATTTCAACCCTCAAGCACAGCTCGGTGTTGGGACTCGCGTTAATTCG
CGTTCCTCAAATTGATTGGCGGTCACGTCGAGCTTCCATAGCGTAGTAGTA
AAGCCCTCGTTACTGGTAATCGTCGCGGCCACGCCGTTAAACCCCAACTTC
TGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATC
AATAAGCGGAGGAAATCGAATTCCGCCGGCCGCCATGGCGGCCGGGAGCA
TGCGAAGTCGGGCCCAATTCGCCCTATAGTGAGTTTTATTACAATTCACTG
GCCCGTCTTTTACAAACNTTGTGACTGGG Sample B-1 Forward (SEQ ID NO: 11)
GGATCGCGCCGGGGGTGGGGCGGGGCCTTAAGATTTTACGAGAATT
AGGTTAGAGATTTTGTCTTAGATCGAGACAGACTCAAGAATAGTTCATGGT
CAAGAGTAGGATCTAACAAGTAATACATCTCAAAGGTGTCCAACCGTATC
CAACCAGTGGACGGATCTTNACCGAGTTGGTGCGCAGGGGGCGCATCCCCT
TGTCGAACCCACTACCCCTGGATGGCTCGTAGCTCCATCGGACGGGTGCCG
GGGGGGGATCGCGTCACTGTCGANTACTGATGNGAACTATAGACTATNGA
TCCGGGCAGCTGATATATCCNANATCTATGTATATNAATCCATNAATAAA Sample B-1
Reverse (SEQ ID NO: 12)
NNNNTGTTTTTCGGGCGCGTCGCGCGGGGCCCTCTCTGGGGAGCGT
CCGCCGGNCGTCCGCCGNTTACACTAAGATGNATTTGCGAGCACGNGCTA
ACATGAGATAGTTATAGGCGTTNCGAGTCTTTCTACGNGAGCTCAAATCCC
CTAGNTCACTGAGNCTCCCCAGCACGNGCTACAGNCCTCCTTGCAGAGAG
GGGCGCTCTCTTTCGGGATCAGAATATNTACACGGGCGAAAAAAGAGGGC
CCCCNTNATANCNANACNCGAGACAGTGCGACAGNCTGGACNCNGNTACA
CAGGTTCTGAGAGTCGNTGGNGNGGAAGAGAGTGAGACGGGNCAAACAG
GGAAAACCANANAGNTCGAGTTTGTNCNGCNGTGGTNCNCNATNGGAAA
AANCTCATCCCGTNGAAGGGCCCACCGANGAGCCCCCNACNAAAATNCTN
GGGGTTGGGCCCGGCNCTNGTTCCNACCAAAAANGTNATNGTTCTNCTTGT
AATNTCTGGGGGGGGNGTGCCCGCCCCCCNGTNCANGAATTNTANCANTA
NGANCGNAANAGNNTGNTGGGCAAAAACGGAGGTTCCCTCNACNCTNGA
ATATTAACATATTTCCCCCCCCACCAAAATATTGGTTCCTCCCACCCCGCC
CCCCTTTTTGTGGGGCCCCCGCGGGTTTGGGGTTTCCAATTCCCTCGGCCT
NTNTTGGCCAGAAGGAAGGTGGGGGGCNGCNGANGAAAAAAANTCCGCAAA
NANGGCCANGTNCAAGTTGCNACNGCNAATNGTGGGGCCTNATTTTTGGA AACCANCAATTGGGGT
Sample B-2 Forward (SEQ ID NO: 13)
CCGGGGCNGCCGGGGCGCGTCGCCGGCNGNCNGCGGCNCNTNGGC
NGCCCGCNGCGCGAGCGCAGCGNGCCGGTGGTGCNCGCGCNCACCTCCCG
TCCCACCTCCTTCGCGCTCGNTGCGCNCANCTCTATANTANGTNAGAGNAG
ATNGAATACTAGNACTATACNTATACNTATAGCACGTAGGACGANGNAAG
NGANTCNCGANATTTTTATTTGGCCGATTNTCCTATANTGNANANGGGGA
AAANGGNAGNAATTTTTGAA Sample B-2 Reverse (SEQ ID NO: 14)
CGGCGGTGGGTTTGGCTCGTGGGCCNCCCGTGCGCGGGGGGCGCCG
CCTCCCTTTTTGCGGACGCGTCCNGCCCGGGCGCGCCCGNCGCGGTANACG
GCTANAGTGGAGTGTGTTGCAGTGCACGNGCTATACATGGTAGTAGTTAT
AGGCAGTTGGGCNTGAGTACTGCTCTGTACNGGGAGNCTCAAATCCCATG
AGTCCCGTGGAGGCTCCCCGACACGGGCGTACAGGCCCTCCTTTGAGAGA
GGGGGCGCTCTCTTTTCCGGACAGANATATACGCGGGCGAAAANGAGGGCC
CNCNTTTNTNTCGGNACNCNAGGGTCANGTNCNGAGCANGNTCNTAGNAC
CCCCCGGGGAAACAACANGGTTTTNCTCGACGAAAGTNCGNGTGGGGGCG
GGGGGAAAGAACCAAGTNGAAAGAACGGGGGCCCANTAACAGGAGGAAA
AACCCAAAGANGANTCNGAATTTGTNCCNCNGTGGTNAACCNATNGGAAN
GANCTTATNCNGTNGAAGGGCCNAGNGANGAGCCCCCNACNGACATNCTT
GGGGGTTGGGCCCGGCNCTNGTTCCCAACCAANACCGGTTAATNGTTCCTC
CCTTGTTTAATNTCTGGGGGGGGGGTNNGTGCCCCGGCCCCCCCCTCGGTT
CAAAAGAAATTTNTAACCAAANAAGGAACGCAAAAAAGNNTGNGTGCCA
AAAACCGNAGGTTCCCTCNACNCTNGAATTANACNNATTCCCNCGCCACC
AAANATTTGTTCCTCAACNCGGCCCCCTTTTGTGGGGCCCCCGGGGTTTGG
TGTTTCTAAATTCCTTGGC Sample B-3 Forward (SEQ ID NO: 15)
CCCGGCGAATGTTTTATGGGGTCATGTTCGACCGCGCCGTCCGGTTG
GCGGAGTTNCATTTTCGTGATCTANAAGAGATAAAATGGCTAAACAGGTT
TACCGTAGGTTATTANCCGCGGAAGGATCTTAACAGTTTTGAAGTGGGCTT
GATGCTGGCTTGTAACAGAGCACTGTGCTCAGTCCCGCTCCAATCCATTCA
ACCCCTGTGCACTATTCGGAGTGTTGCAAGCTAAGACAATGTGGGGAGTG
GTCCCGGTTGTATTTCTAATGCGACTTGGGCTTACTTTCAAACGGTCAAGG
CTTGTCCTCCGGTTTATATACAAACACTTTTATTGTCTTGTCGAATGTATT
AGCCTCTCGTTAGGCGAAATTTAAATACAACTTTCAACAACGGATCTCTTG
GCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTG
CAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTTGGCT
ATTCCGAGGAGCATGCCTGUTGAGTGTCATGTTAATATCAACTCTGATGG
TTTTTTGTTAATCATTGGATGTTGGACTTGGAGGTTCGTGCTGGCTGCAAA
GTCGGCTCCTCTTGAATGCATTAGCTTGGACCTGTGCGCGTTTGCTAGCGG
TGTAATACATTTAATTCACCACGGGCCGTGTCACTATTAGGGTCTGCTTCT
ATTCGTCCTACCGGACAATAATAACTTATGACCTGACTCAATAGGTAGACA
CCCCGACTAACTTAATACCGAGAATCANTATCCGCCCGCGTACATGAAA Sample B-3
Reverse (SEQ ID NO: 16)
CCAGAAGGATTTNATGAAACAAGATAAGCAGAGGTCCCTCATCTTN
GGACTCCGACGGCGNCGCCATATAACTCATGATTTCCCGCTCTATTGATAT
GCTAAGTTTTTAGCGGGTAGTCCACCGATTTGAGGTCAGAGTCATAAAGTT
TATTATTGTCCGGTAAGGACGATTAGAAGCAGACCCTAATAGTGACACGG
CCCGTGGTGAATAAAATGTATTACACCGCTAGCAAACGCGCACAGGTCCA
AGCTAATGCATTCAAGAGGAGCCGACTTTGCAGCCAGCACGAACCTCCAA
GTCCAACATCCAATGATTAACAAAAACCATCAGAGTTGATATTAACATGA
CACTCAAACAGGCATGCTCCTCGGAATAGCCAAGGAGCGCAAGGTGCGTT
CAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATT
TCGCTGCGTTCTTCATCGATGCGAGAGCCAAGAGATCCGTTGTTGAAAGTT
GTATTTAAATTTCGCCTAACGAGAGGCTAATACATTCGACAAGACAATAA
AAGTGTTTGTATATAAACCGGAGGACAAGCCTTGACCGTTTGAAAGTAAG
CCCAAGTCGCATTAAAAATACAACCGGGACCACTCCCCACATTGTCTTAGC
TTGCAACACTCCGAATAGTGCACAGGGGTTGAATGATGGAACGGACTGAC
ACAGTGCTCTGTACAGCCACATAAGCCACTCAACTCGTATGATCTTCCGCA
GTACTACGAACTGTACATTTATTCCCTATACA
[0294] The Sample B fungus used in this study was taken from a
rotted-elm tree in Binghamton University Garden, Binghamton-New
York. Sample A fungus was isolated semi-selectively from sample B
which was grown on Nutrient Broth Medium, Trametes defined medium,
and Candida albicans selective medium. The candidate fungi A and B
were submitted for molecular characterization to the Molecular
Biology lab in the Department of Sustainable Bioproducts at
Mississippi State University.
TABLE-US-00017 TABLE P Comparison of Gen-Bank top hits for the ITS
region No. of ITS matches/ Organism ITS % ITS no. identified
Isolate identified identity in GenBank Sample A Fusarium 100%
328/328 oxysporum Sample B Trichaptum 99% 551/558 biforme
[0295] The characterization results showed that the fungi selection
in the previous study lead selection of Fusarium oxysporum over
Trichaptum biforme; it should be noted the fungi chunk can have
some impurities.
[0296] Polycyclic organics can be degraded by Fusarium oxysporum
and Trichaptum biforme, as well as, Penicillium italicum (P.
italicum), Glomerella cingulata (G. cingulata), Aspergillus flavus,
Colletotrichum alatae, Fusarium solani, Ceriporiopsis carnegieae,
and Xenoacremonium falcatus. The type of extracellular enzymes
released to the medium by these fungi, and their growth pattern and
performance have an impact on their PAA degradation, which might be
the reason for the time required to achieve full degradation of the
membranes.
[0297] Full degradation refers to when the fungi have completely
consumed the PAA membrane. As seen from the Table N above, 1H NMR
did not show any peak related to PAA polymers and the polymer has
been totally, or nearly totally, degraded.
Cytotoxicity Characterization of Ternary PAA Membranes
[0298] PAA membranes did not show any cytotoxic effects on
non-cancerous and cancerous cell lines. Since the membranes
synthesized here are different, their cytotoxicity on non-cancerous
IEC6 and cancerous A549 cell lines were tested as well. Two
different membranes from FIG. 1a and 4 membranes from FIG. 1b were
used.
[0299] FIG. 44a is an illustration of membrane loading in a well.
As seen from the graphics in FIG. 44b, PAA-GA of FIG. 1a, and
PAA-A-pAS-GA, PAA-I-pAS of FIG. 1b did not show any significant
cytotoxicity, while PAA-5AS-GA of FIG. 1b showed significant
cytotoxicity, for which viability decreased at nearly 40%. However,
it should be mentioned that low concentration of 5AS (below 0.3
mg/mL) was not toxic to the cells.
[0300] PAA-A-GA of FIG. 1a and PAA-W-GA of FIG. 1b membranes showed
unexpected results; cells did not only grow in the wells, but also
they grew on the membranes. SEM images of the membranes are seen
below. The surfaces were either woven-like or micro-porous, which
can allow cells to grow on them. 3D cell culture provides unique
environment for seeded cells to recapitulate in vivo conditions.
Biocompatibility, porosity and hydrophilicity are important for 3D
cell culture materials. High-mass transport, ease of process,
flexibility and good-mechanical strength is the desired properties
of 3D cell culture material, which limit choice of 3D cell
culturing materials.
[0301] Good 3D cell culturing supports possess high transparency
and low-background fluorescence ability for high quality light
microscopy and fluorescence imaging. Stiffness of the support
material is a factor for proper cell migration because cells must
apply cytoskelatal forces for movement instead of passive-movement
driven by fluidity of the support material or the system. This is
possible by providing solid stiff support materials.
Antimicrobial Activity of the Films
[0302] The featureless membranes synthesized according to FIG. 1b
were utilized characterized for antimicrobial studies.
Staphylococcus epidermidis ATCC.RTM. 12228.TM., Escherichia coli
ATCC.RTM. 25922.TM. and Citrobacter frenduii ATCC.RTM. 8090 were
cultured in Mueller-Hinton broth at room temperature for 24 hrs.
The viable cell number was determined by conventional agar plate.
The resulting figures were Detailed explanations were given under
related figures.
[0303] Antibacterial activity of ternary PAA membranes. FIG. 45a
0.2 mg/mL Ile was dissolved in GA and then added to 0.16 M PAA.
Overall GA concentration was % 0.1. FIG. 45b 3 mg/mL 5AS was
dissolved in 0.16 M GA, and then % 0.2 GA was added to the system.
Both membranes were incubated at RT for 12, and then membranes were
peeled off. 10.sup.4 cfu/mL for one area and 10.sup.6 cfu/mL for
the three sections were inoculated for both membranes. While Ile
did not show any antibacterial activity, 5AS did not allow
bacterial development. Incubation was 72h. FIG. 45c 0.2 mg/mL PCAM
sugar was dissolved in 0.16 M PAA, followed by 0.2 mg/mL Ile was
dissolved in GA and then added to 0.16 M PAA-PCAM. Overall GA
concentration was % 0.2. FIG. 45d 3 mg/mL PAS was dissolved in 0.16
M GA, and followed by W addition [1 mg/mL W dissolved GA was added
to the system]. Overall GA concentration was % 0.2. Both membranes
were incubated at RT for 12, and then membranes were peeled off.
1500, 150, 15 and 1.2 cfu/mL added to the different area. While
Ile-PCAM did not show high antibacterial activity [600 colonies
formed out of 1500 cfu, and 26 cfu out of 150 cfu], PAA-W/GA-PAS
did not allow bacterial development. Incubation was 24 h for FIGS.
45c and 72h for FIG. 45d.
[0304] The introduction of pAS or 5AS was found to advance the
antibacterial activity of the disclosed films. Antibacterial
activity refers to the fact that the disclosed film will not cause
the growth of bacterial and hence will preserve contained food.
[0305] To visualize bacterial development, plate counting method
was utilized. Incubations were made up to three days, and no
bacterial colonies were observed. The disclosed films provide about
a 99.999% reduction of bacterial growth.
[0306] Similarly, 5AS, pAS and pAB enhanced PAA membranes showed
good antibacterial activity against Aeromonas hydrophila,
Pseudomonas aeruginosa, Escherichia coli DH5alfa, Listeria
monocytogenes strains F2365 and HCC7. Good antibacterial activity
of the PAA membranes can also be displayed against other
gram-positive and/or gram-negative bacterial species. Gram-positive
species other than Listeria monocytogenes, can include
Staphylococcus epidermidis. Gram-negative species other than
Escherichia coli, Aeromonas hydrophila, can include Enterobacter
aerogenes and Citrobacter freundii.
[0307] Virulent type strain L. monocytogenes were grown in a rich
medium such as brain heart infusion (BHI). Lysogeny broth (LB), a
nutritionally rich medium agar also used for the maintenance of the
tested E. coli. The bacteria were taken from the culture collection
unit -80.degree. C. freezer in department of Basic Science, College
of Veterinary Medicine, Mississippi State University
Mississippi-USA.
[0308] Even though 5AS enhanced-PAA membranes showed that good
antimicrobial activity includes showing antifungal activity, it
disrupted membrane mechanical properties with over 0.5 mg/mL usage
while pAS can be used up to 2 mg/mL for PAA membrane preparation.
So, the packaging membrane can contain 0.5 mg/mL pAS and 0.1 mg/mL
5AS, which provides good physical and antimicrobial properties.
However, it should be mentioned that selection of antibacterial
molecule is also affecting the color, so instead of pAS/5AS, pAS
and 5AS can be used independently at different concentrations.
[0309] As seen from FIGS. 45a-45k, pAB can be used instead of pAS
since it showed similar antibacterial activity. 2.times.10.sup.7
cfu of E. coli and S. epidermidis in 20 .mu.L were dropped on the
agars shown in FIGS. 45e-45h and FIGS. 45j-45k. 0.2 mg/mL
Ampicillin were dissolved in agar placed in well FIG. 45e while
same amount of ampicillin was put on left side of well FIG. 45h;
combination of 0.1 mg/mL 5AS and 0.2 mg/mL pAS were dissolved in
agar placed in well FIG. 45f while same composition was put on left
side of well FIG. 45 j; combination of 0.1 mg/mL 5AS and 0.2 mg/mL
pAB were dissolved in agar placed in well FIG. 45 g while same the
composition was put on left side of well FIG. 45k.
[0310] For well FIG. 45h, slight bacterial growth was observed in
comparison to well FIG. 45j and FIG. 45k; this could be related to
that ampicillin dissolved in agar while combination of 5AS and pAS
or 5AS and pAB did not dissolve. When ampicillin was dissolved in
agar, no bacterial formation was observed. Similarly, combination
of 5AS and pAS eliminated all the bacteria while combination of 5AS
and pAB did only wipe out 99.99%. Therefore, the good antibacterial
activity of 5AS/pAS and 5AS/pAB modified membranes was obtained in
comparison to non-antibiotic containing PAA membranes. However, in
the case of sulfanilamide based membranes, utilization of pAS, 5AS
or pAB are not required at higher levels since sulfanilamide
membranes showed antibacterial activities.
[0311] For the well in FIG. 45l, a control of Pseudomonas
aeruginosa in agar is shown, while FIG. 45m contains
PAA-SA-pAS-5AS-GA and FIG. 45n PAA-SA-pAS-GA and FIG. 45o
PAA-SA-pAS-W-GA for the membranes prepared according in DMAC. FIG.
45p PAA-pAS-5AS-GA and FIG. 45r PAA-SA-pAS-GA membranes prepared in
60:40, Ethanol/DMAC, solvent. All of the membranes showed strong
suppressing effect (cidal effect was well) on Pseudomonas
aeruginosa; synthesizing the membranes in ethanol:DMAC mixture did
not show any negative effect on membrane's duty. Here, another
important results were observed that introduction of 5AS to the
membrane enhanced its antibacterial activity which was observed as
smaller colony formation in contrast to only pAS containing
membranes.
[0312] Aeromonas hydrophila was tested on membranes for 24h and 48h
incubation. For FIGS. 45s-y, incubation period was 24h. FIG. 45s is
control for 24 incubation period; FIG. 45t PAA-SA-pAS-5AS-GA; FIG.
45u PAA-pAS-5AS-GA; FIG. 45v PAA-SA-pAS-GA (solvent 65:35,
Ethanol:DMAC); FIG. 45w PAA-SA-pAS-5AS-GA (solvent 60:40,
Ethanol:DMAC); FIG. 45y PAA-SA-pAS-GA (solvent 60:40,
Ethanol:DMAC). For the membrane FIG. 45y, the bacteria did not grow
on membrane, but around the membrane microbial colonies were
observed. For FIG. 45z-ae, incubation period was 48 h. FIG. 45z is
control for 48 h incubation; FIG. 45aa PAA-SA-pAS-5AS-GA membrane;
FIG. 45ab PAA-SA-pAS-W-GA membrane: in the cases of losing
membranes integrity causing A. hydrophila grow on membrane while
the protected area of the membrane did not allow bacterial growth.
Thus, it appears that the antibacterial activities of the membranes
were not coming from releases of pAS or 5AS into the media.
[0313] As a note, as seen from FIGS. 45f and g, free pAS and 5AS
were wiped out both gram (+) and gram (-) bacteria. So, it can be
said that GA might cross-link pAS and 5AS, and when they were
released into the media, they do not show strong antibacterial
activity while when they were bonded to the PAA, they were more
active.
[0314] FIG. 45ac PAA-SA-pAS-5AS-GA membrane; likewise, FIG. 45ab,
when the membrane lost its integrity, the membrane allowed
bacterial growth. FIG. 45ad PAA-SA-pAS-5AS-GA membrane while FIG.
45ae PAA-SA-pAS-GA membrane. Membranes eliminated A. hydrophila
development strongly as long as the membrane protected its
structure.
[0315] FIG. 45af is a control of Listeria monocytogenes for 24h
incubation; FIG. 45ag PAA-SA-pAS-W-GA: L. monocytogenes showed
growth on some sections of the membrane where the membrane
integrity got lost; FIG. 45ah PAA-SA-pAS-5AS-GA FIG. 45aj
PAA-pAS-SA-GA; FIG. 45ak PAA-pAS-5AS-GA; FIG. 45al PAA-SA-pAS-GA
(solvent 65:35, Ethanol:DMAC).
[0316] Antifungal activity of some ternary PAA membranes is shown
in FIGS. 45am-45av. The fungi used here was Aspergillus nidulans.
Incubation period was 6 days for all these membranes. FIG. 45am
PAA-SA-pAS-W-GA; FIG. 45an PAA-SA-pAS-GA; FIG. 45ap
PAA-SA-pAS-5AS-GA; FIG. 45ar PAA-pAS-5AS-GA; FIG. 45as
PAA-SA-pAS-GA; FIG. 45at PAA-SA-pAS-5AS-GA (65:35, Ethanol:DMAC
solvent); FIG. 45au PAA-SA-pAS (60:40, Ethanol:DMAC solvent); FIG.
45av PAA-SA-pAS-5AS-GA. For all the membranes, where there was
disintegration, the fungi showed growth pattern. However, the
growth was not all over the membrane. For FIG. 45ar, S. aerugenosa
and A. nidulans were inoculated together; while the fungus showed
growth at the edge of the membrane, the bacteria did not show any
growth. For FIG. 45av, when there is distortion on the membrane,
development of fungus was observed while the protected side of the
membrane did not allow fungus development.
[0317] Poly(amic)acid polymer has been synthesized from
4,4-oxydianiline (ODA) and pyromellitic dianhydride (PMDA) in
anhydrous N,N-dimethylacetamide. Three procedures have been applied
to develop antibacterial and antifungal PAA membranes and thin
films. (i) entrapping p-aminosalyclic acid (1 mg/mL) into PAA thin
film, (ii) polymerizing p-aminosalyclic acid (1 mg/mL) via
glutaraldehyde, followed by introduced to the PAA thin-film, and
(iii) entrapping p-aminosalyclic acid (1 mg/mL) in PAA membrane.
Thin films were prepared via controlled solvent evaporation method
while membrane was prepared via coagulation-based phase inversion
where 2 h controlled evaporation under hood was applied to increase
pore-size. 1 mg/mL p-aminosalicyclic acid was shown to eliminate
both E. coli and S. epidermidis under the testing conditions.
[0318] Scanning Electron Microscope (SEM) images of FIG. 46a PAA
thin film and FIG. 46b PAA membrane which contains p-aminosalyclic
acid cross-linked with GA and p-aminosalyclic acid molecules, are
shown respectively.
[0319] Antibacterial activity of p-aminosalyclic acid supported PAA
thin films and membranes on Escherichia coli ATCC 25922 (E. coli,
gram-) and Staphylococcus epidermidis ATCC 12228 (S. epidermidis,
gram+) are shown in FIGS. 47a-47h. FIG. 47a is a control of E. coli
and S. epidermidis; FIG. 47b E. coli and FIG. 47c S. epidermidis
inoculated onto the PAA film which contains p-aminosalicylic acid
was cross-linked with glutaraldehyde, followed by rinsed and dried
in anhydrous methanol to quench further cross-linking; FIG. 47d E.
coli and FIG. 47e and FIG. 47f S. epidermidis inoculated onto the
PAA film which entraps p-aminosalyclic acid; FIG. 47g E. coli and
FIG. 47h S. epidermidis inoculated onto the PAA porous membrane
which entraps p-aminosalyclic acid.
[0320] The results indicate that capturing p-aminosalicylic acid in
PAA membranes did not show a descent antibacterial activity towards
both E. coli and S. epidermidis. However, as seen from FIG. 47f,
suppression of bacterial development is shown; the film was thinner
in comparison to the film of FIG. 47e, which depicts that thinner
film was more prone to release its containment to show its
antibacterial activity. As seen from FIG. 47b and FIG. 47c,
bacterial growth for E. coli and S. epidermidis were suppressed,
particularly the thin film didn't allow much bacteria to grow on
the thin film, but still S. epidermidis grew on the membrane. In
contrast to the thin films, nanostructured PAA membrane showed high
antibacterial activity (i.e. up to about 90%) towards both E. coli
and S. epidermidis.
[0321] The results indicate that thickness of PAA thin film and
eligibility of the molecule to transfer from inside of the membrane
to the media (PAA membrane), and possible non-covalent
modifications done on thin film enhances their antibacterial
capability.
1.10 Surface Characterization
[0322] Surface characterization of the membranes synthesized
according to FIGS. 1a and 1b are provided together. Description of
the membranes and procedures are given under the related
figures.
[0323] SEM image of PAA membrane of FIG. 1a are shown in FIGS. 48a
and 48b. FIG. 48a top and FIG. 48b bottom phases of PAA. This
membrane synthesized as followed; the viscous solution from 0.18 M
PAA solution was casted on glass and incubated in air-tight cabinet
for 6h, and then phase-inverted under hood for 12h (FIG.
1a-ii).
[0324] SEM image of PAA-A-GA membrane of FIG. 1 a are show in FIGS.
49a and 49b. FIG. 49a top and FIG. 49b bottom phases of the
membrane. This membrane synthesized as followed; 0.25 mg/mL GA was
added to 0.20 M PAA containing 1 mg/mL A, and stirred for 2 min.
Then the viscous solution was casted on glass and incubated in
air-tight cabinet for 6h, followed by 3h incubation in hood and
then phase-inverted in pure water for 3h. It is clear that the
localized pores or deposits possibly from L-alanine couple to PAA
via GA; this is characteristics of the membranes prepared according
to FIG. 1a. However, when the molecules totally dissolved in PAA
viscous solution of FIG. 1 a, they provided featureless
surface.
[0325] SEM image of PAA-A-GA membrane of FIG. 1a-ii are shown in
FIGS. 50a and 50b. FIG. 50a top and FIG. 50b bottom of the
membrane. This membrane synthesized as followed; 0.25 mg/mL GA was
added to 0.20 M PAA containing 1 mg/mL A, and stirred for 2 min.
Then the viscous solution was casted on glass and incubated in
air-tight cabinet for 6h, and then phase-inverted in anhydrous
ethanol for 2h, followed by 3h phase-inversion in pure water. FIG.
1a-ii, this membrane did not require sonication, however sonication
is possible.
[0326] SEM image of PAA-A-GA of FIG. 1b-ii are shown in FIGS. 51a
and 51b. FIG. 51a is top face, and FIG. 51b is bottom face of the
membrane. 2 mg/mL Alanine was added to the PAA solution. 100
.mu.L/mL GA from 25% GA was introduced to 5 mL PAA solution. 20
.mu.L/mL methanol was introduced to the 0.18 M PAA solution The
glass surface was then wetted with dry methanol, and then the
solution was casted on the glass on which methanol was then
spreaded, then incubated at room temperature for 6 h. Finally, the
membrane was dried under hood for 12 h. Both faces have no pores.
FIG. 1b-ii, the major difference of this PAA-A-GA membrane from
other PAA-A-GA membranes was that A was pre-dissolved and treated
in GA-water, which resulted in elimination of localized porous-area
formations.
[0327] SEM image of PAA-CA-GA of FIG. 1b are shown in FIGS. 52a and
52b. FIG. 52a is the top face and FIG. 52b is the bottom face of
the membrane. 2 mg/mL cellulose acetate was added to the 0.18M PAA
solution. 100 .mu.L/mL GA from 25% GA was introduced to 5 mL PAA
solution. 40 .mu.L/mL ethanol was introduced to the PAA solution
The glass surface was then wetted with dry ethanol, and then the
solution was casted on the glass on which ethanol was then
spreaded, then incubated at room temperature for 6 h. Finally, the
membrane was dried under hood for 12 h. Both faces have no pores.
FIG. 1b-ii.
[0328] SEM image of PAA-SA-pAS-GA membrane of FIG. 1b are shown in
FIGS. 53a and 53b. FIG. 53a top and FIG. 53b bottom phases of the
membrane. Both of the surfaces are featureless.
[0329] SEM images of PAA-W-GA membrane are shown in FIGS. 54a-54d.
10 mg Trp (W) is dissolved in 100 .mu.L GA from 25% GA stock. The
Trp was incubated for 20 sec for polymerization with the help of
GA. Then, the Trp-GA solution was introduced to 0.16 M PAA (or 0.12
M) solution at 5 drops, and 5 second interval was followed each
consecutive drops. 3 min stirring after last drop of Trp-GA, the
PAA solution was casted on glass. 4 h incubation at room
temperature, followed by 12 h incubation under-hood. Finally, the
membrane was sonicated in anhydrous methanol for 20 min, which was
then dried under hood. FIGS. 54a and 54b are images were taken with
inlens detector, while FIG. 54c and FIG. 54d images were taken with
SE2 detector. Sonication of the same membrane in pure water, 20%
MeOH and 50% MeOH did not provide any pore-formation. However, 80%
EtOH allows pore-formation.
[0330] SEM images of PAA-W-GA and PAA-SA-GA membranes are shown in
FIGS. 55a-55f. FIG. 55a top and FIG. 55b bottom of the PAA-SA was
incubated at room temperature for 4 h, followed by incubated in 70%
Ethanol in water for 2h. FIG. 55c top and FIG. 55d bottom of the
PAA-W was incubated at room temperature for 4h, followed by 2h
incubation in 70% Methanol. FIG. 55e top and FIG. 55f bottom of the
PAA-SA was incubated at room temperature for 4h, followed by 2h
incubation in 70% Methanol. PAA-SA gets solidified faster than
PAA-W; so it is normal to see less porous surface for PAA-SA. As
seen from all of the FIGS. 54a-54d and FIGS. 55a-55f, the surfaces
of the membrane are porous enough for cell adhesion; since both
sides showed pores, transfer of wastes resulted from cellular
metabolism is possible.
[0331] Surface characteristics of all synthesized PAA membranes did
not show any difference in response to alteration in PAA
concentration (from 0.08 M to 0.20 M range), GA concentration and
small molecule and its concentration. However, at macro-scale all
parameters affected the eye-visible membrane surface. Addition of
organic solvents include methanol, ethanol and tetrahydrofuran did
not make any difference on surface characteristics. Here, the most
dramatic change in surface characteristics were seen in parallel to
changes in procedure. Here, three major surface types were
obtained; featureless, macro-porous and woven-like surfaces. Nearly
all the procedures provided featureless membrane surface; pore-free
surfaces can be a good barrier against penetration of oil,
water-vapor and gas transfers.
[0332] SEM image of PAA-GA are shown in FIGS. 56a and 56b; FIG. 56a
top and FIG. 56b bottom phases of PAA-GA membrane. This membrane
synthesized as followed; 0.25 mg/mL GA from stock was added to
0.18M PAA solution and stirred for 3 min, and then the viscous
solution was casted on glass and incubated in air-tight cabinet for
12h, and then further incubated under hood for 6h. The membranes
synthesized according to FIG. 1a-i.
[0333] The right figure is top face FIG. 57a, and left one FIG. 57b
is bottom face of the membrane. 5 mg Alanine was dissolved in 100
.mu.L of 25% GA solution. The cross-linked L-Alanine was then
introduced to 5 mL 0.18 M PAA solution. 40 .mu.L/mL methanol was
introduced to the system. The glass surface was then wetted with
dry DMAC, the PAA solution was casted on the glass and then
incubated at room temperature for 6 h. Finally, the membrane was
dried under hood for 12 h. Bottom is nearly featureless while the
top has recognizable numbers of defects. FIG. 1b-ii.
[0334] As illustrated and discussed above, both FIG. 1a and FIG. 1b
can provide featureless surface if the small molecule or
cross-linker are dissolved in PAA solution properly.
1.10 PAA Concentrations
[0335] PAA concentration was selected as 0.16, 0.18, 0.20 and 0.25
M based on the pore size of pure PAA membrane obtained via
phase-inversion. PAA solution's viscosity was not measured with an
instrument; if the viscosity is low enough to be stirred with
stirring magnet at high speed, the concentration was accepted as
good. The following optimizations were obtained from FIG. a. 0.18 M
was selected as best concentration for FIG. 1 a subsequent membrane
preparation.
[0336] In addition to viscosity, other parameters were considered
including the ratio of ODA to PMDA, temperature of the medium and
the speed of stirring showed great impact on PAA formation; in
optimum conditions, the PAA concentration is 0.08 M in the cases of
1.00:1.03 ODA:PMDA ratio at 40.degree. C. under mild mixing (i.e.
between 400-600 rpm). The mixing should be enough to totally
dissolve PMDA at less than 120 seconds but no earlier than 30 sec.
In the case of 0.12 M, ODA:PMDA a good ratio was found to be
between 1.00:1.04 at 40.degree. C. under mild mixing to obtain
viscous PAA solution. However, the formed PAA solution was highly
viscous.
[0337] In the case of high temperature such as 70.degree. C., PMDA
was dissolved in seconds and the resulting 0.12 M PAA solution was
not as viscous at 1.1:1.0 ODA:PMDA ratio. Similar results were
observed for 1:1 ODA:PMDA ratio at high temperatures. At 40.degree.
C., 1.00:1.10 ODA:PMDA ratio, 0.12 M PAA became less viscous than
1:1 ratio. So, it can be concluded that a good PAA concentration is
0.08 M or 0.12 M for FIG. 1b membranes.
TABLE-US-00018 TABLE Q Summary of optimization of ODA:PMDA ratio,
stirring speed and temperature needed to produce viscous PAA
solution. Observation ODA:PMDA Stirring viscosity low, ratio speed
Temperature Time high, same? 1.00:1.00 400 25.degree. C. Overnight
Mild 1.00:1.01 400 25.degree. C. Overnight Mild 1.00:1.02 400-600
40.degree. C. Overnight High 1.00:1,02 400 50.degree. C. Overnight
Very high 1.00:1.02 400 60.degree. C. Overnight Mild 1.00:1.02 1200
70.degree. C. Overnight Very low 1.00:1,02 400-600 70.degree. C.
Overnight Low 1.00:1.04 400 40.degree. C. Overnight Mild 1.00:1.05
400 40.degree. C. Overnight Low 1.2:1.0 400 25.degree. C. Overnight
Very low
[0338] The choice of appropriate solvent depends on three
parameters as (i) environmental-friendliness, (ii) chemistry such
as reaction yield and (iii) engineering which is more of
scalability and ease of down-stream process. Environmental aspects
of solvents are regulated by US Environmental Protection Agency
(EPA). Chemistry and engineering are defined by the process itself
and aim of the study.
[0339] The solvents aim to meet the following requirements: the
solvent must be neutral to all members of the reaction mixture,
including reactants, products as well as catalysts; if the solvent
is a provider of any group such as --O, --H, that should be just a
carrier; the solvent must be liquid at the reaction condition such
as if the reaction is happening at room temperature the solvent
should not require higher degrees to be liquid; if the phase split
is required for the solvent that should be preferred; the solvent
should provide the desirable solubility for the reactants as well
as products if it is required; the solvent should not undergo
association or dissociation; and the solvent should selectively
dissolve reactants or the possible products if it is desired.
[0340] In order to obtain "greener" membranes (those with a smaller
ecological impact), DMAC was combined with ethanol at varying
ratios. Ethanol is generally accepted as a "green" solvent.
However, the criteria listed above were taken into account in the
selection. The goal was to use as high a percentage of ethanol as
possible while minimizing the volume of DMAC employed. Ultimately,
an optimum condition is sought that will provide the highest
benefit. Determining the possible highest Ethanol:DMAC ratio was
done using only two parameters; (i) the physical properties of the
membrane and (ii) the aim of application of the membrane.
[0341] The observed characteristics of the membrane were (i)
durability, (ii) resistance against solvents and (iii) mechanical
properties. 65:35 Ethanol:DMAC ratio was accepted as a good ratio
to develop 0.12 M PAA membrane with 1.00:1.03 ODA:PMDA ratio at
50.degree. C. medium temperature under mild mixing. However, the
PAA polymer formed in 65:35 Ethanol:DMAC ratio did not form
fluorescent active membrane, so it is not advised for fluorescent
active membrane formation. Actually, introducing ethanol into PAA
solution prepared in only DMAC still disrupting formation of
fluorescent active membrane formation.
TABLE-US-00019 TABLE R Determining good conditions for Ethanol:DMAC
ratios in membrane preparation. Mixture Observation 50:50,
DMAC:EtOH Viscosity high, require warming up (i.e 50.degree. C.),
and forming membranes that are strong but hard to obtain different
colors 35:65, DMAC:EtOH Viscosity mild, require warming up (i.e
50.degree. C.), and forming membranes that are strong, but hard to
obtain different colors 35:65, DMAC:EtOH Viscosity mild, require
warming up (i.e 50.degree. C.), and forming membranes are strong,
require the addition of 2% water to obtain desired colors 25:75,
DMAC:EtOH Viscosity acceptable, require warming up (i.e 60.degree.
C.), and forming membranes that are strong, but hard to obtain
different colors 35:50:15, Viscosity acceptable, require warming up
(i.e DMAC:EtOt:Water 60.degree. C.), and require special care to
form good membranes, but provide desired different colored
membranes 30:50:20, Viscosity low, require warming up (i.e
60.degree. C.), DMAC:EtOH:Water and require care to form good
membranes, but provide desired different colored membranes 60:40,
DMAC:Water Did not form PAA viscous solution 60:30:10, Did not form
PAA viscous solution DMAC:Water:AcOH *PAA concentration employed
was 0.12 for all experimental conditions indicated.
[0342] Water:Ethanol:DMAC and Ethanol:DMAC mixtures were tested as
well. In all cases, PAA concentration was kept constant at 0.12 M.
The following solvent mixtures were obtained a good reachable ratio
as 15:50:35 (water:ethanol:DMAC) and 75:25 (Ethanol:DMAC). However,
it was shown that 20:50:30 (water:ethanol:DMAC) ratio is possible,
but the PAA solution should be used within 2 days, otherwise PAA
precipitates in the solution due to presence of high water content
and low DMAC ratio.
[0343] However, it was noted that introduction of water to the
solvent system eliminates the ethanol effect of preventing colorful
and fluorescent PAA membrane formation. Methanol was not tested
with combination of DMAC; ethanol is less toxic in comparison to
methanol. However, methanol, ethanol and water containing 0.1 M
hydrochloric acid (HCl) were tested individually, but viscous PAA
solutions were not obtained.
Optimization of Small Molecule Concentrations
[0344] Optimization of the small molecule concentrations were
performed for the membranes synthesized according to FIG. 1 a.
Since the small molecules used were mostly insoluble in viscous PAA
solution. Therefore, it was a goal to find good concentrations of
the molecules in order to form uniform and stand-alone membranes.
Changes in viscosity related to small molecule addition was not
tested with an instrument, rather the viscosity was described as
low, mild, high and very high in relation to membrane preparation;
and ease of spread onto the glass substrate prior to phase
inversion. low and very high viscous PAA-small molecules were not
be able to cast on glass surface to form even membranes.
[0345] However, for the membranes synthesized according to FIG. 1b,
the small molecules were pre-dissolved before being introduced to
the viscous PAA solution, hence optimization of the concentrations
was performed mostly with respect to the aspect of mechanical
strength and antimicrobial activity.
[0346] Structures of the small molecules used in FIG. 1a membranes
are shown in FIGS. 58a-58m. FIG. 58a Chitosan, FIG. 58b cellulose
acetate, FIG. 58c glucosamine, FIG. 58d L-alanine, FIG. 58e
L-lysine, FIG. 58f L-cysteine, FIG. 58g L-isoleucine, FIG. 58h
L-tryptophane methylester, FIG. 58j glycine, FIG. 58k glutamic
acid, FIG. 58l L-arginine and FIG. 58m L-threonine.
[0347] For all PAA concentration, 2 mg/mL chitosan (CS)
[Low-molecular weight chitosan, Sigma-Aldrich] concentration can be
used. CS mediated increase in the viscosity of PAA solution showed
distinct characteristics; At 0.5 mg/mL, 1 mg/mL and 3 mg/mL of CS,
PAA solution became highly viscous. From observations, CS is
insoluble in DMAC: so it is clear that yellowish CS flanks are in
PAA solution, which causes un-uniformity.
[0348] Cellulose acetate (100 kDa molecular weight) completely
dissolved resulting in a clear solution when mixed with PAA
solution despite the fact that cellulose has similar structure to
CS. PAA and PAA-CA solutions exhibit similar color and uniformity
with no air bubble in PAA-CA unlike PAA-CS mixture. The formation
of the air bubbles resulted from combination of insolubility of the
small molecule and high viscosity. When the PAA-small molecule
mixture was stirred at 100 rpm for 10-30 min, the air-bubbles
disappeared. Interestingly, CA at 1 mg/mL concentration makes PAA
solution highly viscous which is like solid, so it is concentration
should be used less than 0.5 mg/mL. Even at 0.5 mg/mL
concentration, PAA-CA solution became viscous which made it
eligible for membrane preparation by water-bath mediated phase
inversion.
[0349] D-glucosamine (DA) is one of the two monomers in chitosan
molecule, so they were expected to possess similar properties.
Similar to CS, DA was not soluble in DMAC. However, when 1 mg/mL
concentration of DA was dissolved in PAA solution, the resulting
mixture became highly viscous resulting in even higher viscous
solution than PAA-CA. Hence the optimum concentration of DA needed
should be less than 0.5 mg/mL concentration in 0.20 M PAA, but 1
mg/mL works for 0.18 M PAA.
[0350] L-Alanine (A) can be used at 1 mg/mL concentration; higher
concentrations were not tested because 1 mg/mL gave desirable
viscosity even at 0.20M PAA solutions. A does not dissolve in DMAC,
its insoluble crystals are visible in PAA-A solution; however it is
possible some of A dissolved because in terms of mechanical
properties PAA-A membrane was good. Its plastic-like structure did
not turn brittle even at six months' exposure in the hood. Similar
characteristics were observed for 3 weeks with PAA/PAA-W/PAA-CA
composite membranes; when prepared using layer-by-layer casting on
glass. In order to develop more durable membranes based on FIG. 1a,
PAA was incubated with GA, and PAA-W and PAA-CA were sequentially
casted on PAA. The aim was to observe if GA moves to PAA-CA
solution.
[0351] Based on contact angle measurements data, this composite
produced the highest hydrophobicity among FIG. 1a membranes. This
layer-by-layer casting is not similar to the technique used to
prepare polyelectrolyte multilayer membranes which relies on
charge-charge interactions of different layers.
[0352] L-lysine (K) shows similar pattern to A, but it makes PAA
solution much more viscous at the same concentration. A good
concentration should be less than 1 mg/mL at 0.18 M PAA. At 0.20 M
PAA concentration, 0.5 mg/mL of K produced a highly viscous PAA-K
solution.
[0353] L-Cysteine (C) was selected because it has a free --SH
group. Introducing C to CS membrane via GA makes CS membrane very
flexible. Like-wise K, C is not soluble in DMAC. Interestingly, it
was observed that C forms fibrils in DMAC and during membrane
preparation these fibrils resulted in blocks within the PAA-C
membrane shown in FIGS. 62a-62g. Therefore, highly heterogeneous
PAA was formed, but these membranes protect the plasticized form
[FIG. 1a i] more than the others except PAA-A [stable for around 3
months]. However, stirring PAA-C solution at very high speed
minimized the fibril formation. It was observed that cysteine makes
wrinkled PAA membranes, however its mechanical strength was still
high in comparison to phase-inverted PAA membranes. When GA
concentration was 0.21% in PAA-C solution, it did not result in
plasticized PAA-C membrane but it resulted in shiny and relatively
stronger membranes than individual PAA membranes. So, for
preparation of PAA-C membranes, the optimum GA concentration should
be between about 0.21 to about 0.35%.
[0354] For the amino-acids of FIG. 66 and the other molecules
utilized to prepare the disclosed PAA films, the GA concentration
can be up to about 2%. However, the concentration of GA depends on
the formula and composition of the membrane.
[0355] L-isoleucine (I) can be used at less than 1 mg/mL
concentration, at which concentration desirable PAA-I viscosity can
be obtained. In this study, 0.5 mg/mL-3 mg/mL were tested at 0.20M
PAA solution, and in all cases the extremely high viscosity of the
mixture did not allow membrane formation. However, 0.5 mg/mL I was
a good concentration at 0.18 M PAA solution for desired fluidity
during membrane preparation.
[0356] Likewise, L-Tryptophane-methyl ester (W) can be used at less
than 0.5 mg/mL concentration, at which concentrations desirable
PAA-W viscosity could be obtained. In this study, 0.2 mg/mL-1 mg/mL
were used at 0.20 M PAA solution, and in all cases extremely high
viscosity did not allow membrane preparation. However, 0.5 mg/mL W
could be used at 0.18 M PAA solution to get the desired fluidity.
For the same concentrations, PAA-W gave the highest viscosity. W,
gave unexpected results for FIG. 1b membranes as well.
[0357] Glycine (G) and L-Aspartic Acid (D) exhibit similar pattern
to A, but makes PAA solution much less viscous at the same
concentration, hence the optimum concentration should be at about 1
mg/mL at 0.18 M PAA.
[0358] After PAA-W, PAA-R (L-Arginine (R)) has the second highest
viscosity [these are just based on observations]. 0.2 mg/mL R can
be used with 0.20 and 0.23 M PAA; 0.25 M PAA. 0.5 mg R was observed
to give a good concentration at 0.18 M PAA solution. However, the
formed membranes did not provide durable membranes; keeping the
membrane at room temperature for 2 days made the membrane
brittle.
[0359] L-Threonine (T) can be used at 0.5 mg/mL for 0.20 M PAA and
0.18 M PAA solutions. However, similar to L-arginine, T made the
PAA membrane brittle within 3 days after drying.
[0360] All these amino acids were introduced to the PAA solution
immediately after the formation of PAA solution as described in
FIG. a. However, these biomolecules were added to the system at the
same time with ODA. The resulting PAA solutions showed similar
characteristics. All these characterizations were made for FIG. 1a
membranes; the concentrations of these amino acids can be increased
up to 2 mg/mL in FIG. 1b. However, thiol containing amino acids and
other molecules did not lead to the formation of membranes when
FIG. 1b were employed.
Optimization of Cross-Linker Concentrations
Optimization of GA Concentration:
[0361] For desirable PAA membranes, the following parameters were
found to be factors: Concentration of GA stock [Sigma-Aldrich, 70%
Glutaraldehyde] solutions, the final concentration of GA when mixed
with PAA solutions, age and temperature of GA added to the PAA
solutions are highly important in terms of obtaining desirable PAA
membranes. Age of GA is a term used here to describe what type of
pre-treatment was applied to GA before it was introduced to PAA
solution. Temperature of GA refers to that at which temperature GA
was incubated before it was introduced to PAA solution.
[0362] There are similar procedures to age GA, but the way of aging
in this study is not related to time, rather it is related to
temperature. Aged GA, in general, provides distinct results than
fresh GA during crosslinking. General rules in optimization of GA
can be listed as below: concentration of GA stock is a factor in
terms of how much water is introduced to the PAA solution; even
though water provides a working microenvironment to GA, it can
disrupt the cross-linked PAA membrane formation and also cause
localized phase inverted PAA membrane formation in the PAA
solution's vial, or during the membrane formation. So, it is
advisable to use high stock concentration if the FIG. 1a iii is
used to prepare the membranes.
[0363] Concentration of GA added to the PAA solutions should not be
over the concentration at which it makes pure 0.16 M or 0.12 M PAA
solutions solid less than 15 min and 30 min, respectively. Beyond
this point, the leads to the formation of easily breakable PAA
membranes. Similar observation was shown for PAA-CS.
[0364] Further optimization can be performed for each molecule
accompanied with PAA.
[0365] However, it should be noted that incubation time also
matters in defining the degree of cross-linking. When GA is less
than 0.35%, the resulting PAA membranes are in between pure water
phase inverted PAA membrane resulting in plasticized form.
[0366] However, higher GA concentrations such as 2% GA convert PAA
solution into completely non-fluidic within 3 min, which was
observed in FIG. 1a iii and FIGS. 1bi and 1bii.
[0367] Age of GA defines its active individual GA molecule and
degree of auto-polymerization. NMR characterization and further
explanations are provided above.
[0368] GA stock vial should be kept in the hood until before being
placed at room temperature for approximately 10-20 min; when the GA
stock has been left at 4.degree. C. needs 10 min, and it is then
transitioned into room temperature, its viscosity decreases and
becomes highly fluidic (for 25% or less forms), which was concluded
based on observations through preparing different concentrations
from 70% stock. When it is introduced directly from the
refrigerator, it forms localized phase-inverted PAA membranes using
the PAA solution.
[0369] Another point with respect to GA is that it must be
thoroughly mixed with dry-DMAC before introducing it to PAA viscous
solution. Moreover, there are two observations in this application
as (i) adding GA into DMAC containing vial causes heat formation.
In that respect, DMAC should be added slowly to the GA containing
vial [note: the resulting heat may not increase the temperature up
to flash point of DMAC but being cautious is advisable because
mixing 25% stock GA with DMAC releases heat causes over 40.degree.
C.]. Increase in heat is possibly related to water-DMAC interaction
since the same amount of GA in less water content added to the DMAC
released less heat. The second observation (ii) is that using the
stock solution of GA is different than GA that has been mixed in
water. Pretreating GA with DMAC gives better membranes and causes
no local membrane formation in PAA viscous solution. The PAA
membranes prepared using FIG. 1a ii were found to be more stable
for months when even kept under hood and at room temperature.
Simple-treatment of GA in DMAC prior to the introduction into the
PAA solution can bring a change in the resulting membrane.
[0370] The overall result of addition of GA to PAA viscous
solutions from stock GA [FIG. 1a iii] makes the PAA membranes
[except PAA-Cys and PAA-A] brittle after drying. This could be
related to the kinetics of membrane formation according to FIG. 1a.
It should be noted that the membranes described here were
synthesized according to the procedure described in FIG. 1a iii.
However, the PAA membranes can be stored in pure water which
protects their mechanical properties or prevents them from being
brittle. However, PAA-A and PAA-Cys membranes were found to be
strong and more durable even up to months. Addition of pre-diluted
GA stock with dry DMAC to PAA solutions make PAA membranes strong
and durable.
Optimization of 1, 1'-diimidazole
[0371] 1, 1'-diimidazole (IZ) was employed as a solid by slowly
adding the solid particles into PAA solutions. Introducing over 1
mg/mL IZ to the PAA solutions at once causes localized orange color
solid formation. However, IZ's original color is pale-yellow.
Besides; IZ-treated PAA forms heterogeneous membrane, for instance
the inner part looked similar to the non-cross-linker treated PAA
membranes while the outer layer looks more like a plastic
[Synthesized as described in FIG. 1 a iii]. In order to eliminate
formation of non-even membranes, IZ should be used at low amounts
and under good mixing.
[0372] IZ works slower than GA for membrane formation; while GA
requires less than 3 min to increase the viscosity up to the
desired level, IZ requires 15 min at 0.16 M PAA while 45 min
requires at 0.12M PAA. The final membrane is physically similar to
GA-treated PAA membrane. In order to prepare a totally
plasticized-membrane, 3 mg/mL IZ should be added to the system
under FIG. 1a iii. Besides, imidazole was dissolved in anhydrous
DMAC and then applied to the PAA and PAA-small molecule viscous
solutions. However, there was no difference between direct addition
of solid imidazole and DMAC-dissolved imidazole. Beside
1,1'-diimidazole, N,N'-dicyclohexylcarbodiimide [DCC] was tested,
and similar membranes were obtained with longer incubation times.
DCC mediated membranes gave similar colored membranes as obtained
with IZ. However, EDC/NHS did not work either direct solid
addition, or dissolved in water.
[0373] Likewise, EDC/NHS, the use of glutaric acid [Sigma-Aldrich,
MO] did not provide any plastic-like membranes. However, glutaric
acid simply enhanced the IZ's activity to make PAA solutions'
viscosity high enough to be prepared membrane relatively quick. It
should be emphasized that plastic-like structure does not mean just
being a transparent membrane, which is common for evaporation
mediated phase inversion, but that means formation cross-linker
mediated membranes. Neither carbodiimidazoles nor glutaraldehyde
required EDC/NHS to modify PAA molecules. These combinations did
not require EDC/NHS for crosslinking. Extensive tests were
performed for GA, and NMR data show that GA covalently binds to PAA
without the use of any EDC/NHS intermediate (discussed above).
[0374] Treating PAA with DCC until the PAA solution became solid
resulted in vibrating solid which was seen for GA treated CS
solution. This effect was not seen for GA treated PAA, and was
observed for relatively lower in the cases of IZ treated PAA. For
IZ, it could be related to lower and localized solubility of IZ in
viscous PAA solution. The vibration property was not tested with an
instrument: it was visually observed.
Time-Dependent Alterations in Membrane Physical
Characteristics.
[0375] Incubation of PAA with a cross-linker alters its physical
and chemical characteristics. These changes both depend on the type
of cross-linker and the biomolecule used to modify PAA.
Glutaraldehyde is highly active molecule, and it is not easy to
control its diverse binding to PAA and other small molecules.
Immediately after the phase-inversion, most of the PAA membranes
protect their flexible natures. The membranes other than
L-cysteine, L-alanine and chitosan modified PAA membranes did not
protect their plasticized nature after about a week. However, the
mentioned three membranes protected their plasticized nature well
over 6 months. Measurement did not exceed 6 months. However, in
parallel to increase in phase inversion incubation in water they
become brittle, which takes weeks to months [Synthesized as
described in FIGS. 1ai and 1aii].
[0376] Phase inversion or the process of transformation from
solution state to solid state is a technique in preparation of
membranes. It is used from micro-filtration to gas separation
applications. Four main approaches have been described for phase
inversion; (i) coagulation bath mediated phase inversion, (ii) heat
triggered phase inversion, (iii) precipitation from vapor phase and
(iv) evaporation in non-solvent.
[0377] Pore formation on the top layer of membrane through
coagulation-bath mediated phase inversion forms due to flow of
non-solvent into the membrane while the thickness of membrane's
skin layer depends on flow of solvent from inner part of membrane
into the coagulation bath. However, open-pore formation also
requires coalescence in the polymer-poor area which is right under
the top-layer. The relation between solvent and polymer itself, and
solvent and non-solvent are also important in determining the
characteristics of the pores. For example, in the presence of high
affinity solvent and non-solvent, macrovoids are formed.
[0378] FIG. 59 is an illustration of phase-inversion in
coagulation-bath (immersion precipitation).
[0379] Solvent refers to the solvent used to prepare membrane
solution while non-solvent refers to the solvent used in the
coagulation bath (which is also not supposed to be main solvent for
the membrane). The solvent can be any suitable solvent capable of
dissolving other substances, such as ethanol, methanol, and
combinations thereof. The non-solvent can be any suitable material
that is not capable of substantially dissolving other substances,
such as water.
[0380] Besides altering the non-solvent, addition of non-solvent
into solvent (casting solution) also makes difference in pore
formation. While a very fast desolvation of solvent into
non-solvent forms finger like structures and cause no or rare pore
formation, addition of solvent into non-solvent allows porous
surface formation because this situation reduces solvent
desolvation into the non-solvent. However, addition of non-solvent
into the solution before it is casted, less porous surface with a
less dense surface forms. The solvation power of solvent also
possesses strong effect on pore formation for instance a less
solvation power provides more pores on membrane surface.
[0381] The membrane formation process here is mostly driven by
combination of glutaraldehyde, evaporation and
coagulation-bath/glutaraldehyde evaporation. When there is no
glutaraldehyde in the system, even the speed of stand-alone
membrane formation through evaporation is reduced. However, it
should be mentioned that this does not mean that GA enhances
solvent evaporation. Faster formation of stand-alone membrane
implies that GA cross-links individual polymers leading into
macro-polymeric systems by which stand-alone membrane formation
eliminates high percentage of DMAC removal from the system.
[0382] In the method used herein glutaraldehyde is not only
functioning as a cross linker, it is also transforming the resulted
PAA into optically active form such as fluorescence active forms.
These bindings alter the membrane formation; the very basic
alteration is the rate of solvent evaporation. Here,
encrossslinking was used to refer extensive crosslinking.
[0383] As explained for PAA-GA, PAA-CS-GA, PAA-A-GA, PAA-DA-GA and
PAA-C-GA, the concentration and activity of GA (i.e. heat treatment
and pre-treatment with DMAC) showed dramatic effect on the
resulting PAA membranes synthesized according to FIG. 1a, in which
coagulation bath was used as the final step of the
phase-inversion.
[0384] For some small molecules, the membrane showed thin inner
solid layer which was then disappeared when the membrane was
incubated under hood for 6 h or more, which refers to a continuing
progress of membrane formation. However, in the cases of higher GA
concentrations and/or enhanced activity of GA, formation of
plastic-like and transparent membrane surface didn't show any
requirement to sonication in methanol/ethanol or drying under hood.
It is evident that GA alters the kinetics of membrane formation;
additives were shown a role-player in the kinetics. In the cases of
the membranes synthesized according to FIG. 1b, all of the
membranes are plastic-like and transparent, and don't form any
solid amorphous layer even they are submerged into water or other
solvents.
[0385] In FIG. 1 a membranes, there is no pore formation in the
cases of water bath becomes the final phase-inversion step.
Therefore, the speed of DMAC releases into water bath is too fast
to form porous surface, but the surface becomes shiny and
plastic-like which can be assigned to the activity of GA. However,
when the final step of phase-inversion was performed in methanol,
the surface lost its shiny structure and formed woven-like
structures (as discussed above) which can be explained through DMAC
having a relatively lower tendency to diffuse into methanol.
[0386] Similarly, losing the shiny outer surface appearance can be
explained by the quenching activity of methanol on the activity of
GA crosslinking. However, when methanol/ethanol bath was
accompanied with sonication, membranes then can be synthesized
porous. In the cases of high GA concentrations, the membranes can
also be made porous if they are sonicated in methanol/ethanol bath.
In essence, surface patterns of FIG. 1a membranes depend on the
final step of phase-inversion while the texture of them is mostly
dependent on concentration and treatment of GA. However, it should
be noted that small molecule also makes difference in formation of
the membrane such as PAA-C-GA, PAA-W-GA and PAA-A-GA are more of on
the plastic-like surface forming co-polymers while chitosan and
glucosamine urge the membranes become more of amorphous.
[0387] Another test was performed to determine how glutaraldehyde,
small molecule and the final step of phase-inversion affect the
final texture of PAA membranes. The co-polymers were left in a vial
overnight and the resulted PAA co-polymers were sticky gels. Then,
they were exposed to evaporation under hood, phase-inversion in
water-bath, methanol, methanol-water mixture, and ethanol and
ethanol-water mixtures. PAA-W-GA and any other PAA co-polymers
enhanced with sulfanilic acid provided plastic-like membrane
surfaces under all final phase inversion conditions, but only
sonication in organic solvent (i.e. methanol and ethanol) made the
membranes fully plastic-like; as shown for FIG. 1b membranes, high
amount of residual DMAC in PAA makes it possessing solid amorphous
layer.
[0388] This is evidence that the speed of solvent removal from the
inner part of the membrane is one of the parameters in order to
form totally plastic-like membranes. Besides, sonication allowed
the membranes possess porous surfaces in organic solvents; even
though ethanol works better such as 80% ethanol is enough for
porous surfaces while methanol can be 100%, methanol bath treated
membranes showed better durability. Besides, introduction of
organic solvents such as ethanol, hexane and others into
co-polymerization media did not affect the pore formation as well.
Introducing solvent into coagulation bath was shown a parameter to
make flat membranes porous, but the approach did not work for the
membranes synthesized according to FIG. 1a.
[0389] Classical pore preserving agents include PEG 400 or any
other agents weren't tested to make the membranes porous since the
goal was just to understand how membrane formation progresses under
different conditions.
Preparation of Ternary PAA Membranes
[0390] Images of several films are shown in FIGS. 60a-60h. FIG.
60a: PAA-CS-GA; FIG. 60b: PAA-A-GA; FIG. 60c: PAA-A-GA; FIG. 60d:
PAA-A-GA; FIG. 60e: PAA-GA; FIG. 60f: PAA-A-GA; FIG. 60g: PAA-GA
and FIG. 60h: PAA-CS-GA. All of these digital images were taken
between 30-60 min right-after castings on the glasses. Steps of the
phase-inversion according to FIG. 1a resulted in different
surface-properties.
[0391] The casted solutions shown in FIGS. 60a-60h underwent
different final phase-inversion process, by which different surface
properties of same types of membranes were obtained. FIGS. 60i-60p
illustrates-PAA-CS-GA FIG. 60a was incubated for 6 h at room
temperature, followed by incubated at 70.degree. C. for 20 min. The
resulted membrane turned into brownish colored transparent and
brittle membrane FIG. 60i. PAA-A-GA FIG. 60a was incubated at room
temperature for overnight FIG. 60j. PAA-A-GA FIG. 60c was incubated
at room temperature for 6h, followed by incubated at 70.degree. C.
for 1h.
[0392] The resulted membranes showed quite-similar characteristics
with PAA-CS-GA membrane FIG. 60i. PAA-A-GA FIG. 60d was incubated
at room-temperature for 3h, followed by incubated in 100% methanol
for 1 h. The resulted membrane FIG. 60l showed opaque and non-shiny
surface with possessing plastic-like edges. PAA-CS-GA FIG. 60h was
incubated overnight at room temperature, and the resulted membrane
possessed slightly shinny surface FIG. 60m. Since the thickness was
over 0.3 mm, total transparency was not obtained.
[0393] PAA-GA FIG. 60e was incubated at room temperature for 6h,
and the resulted PAA-GA FIG. 60n showed shinny surface. PAA-A-GA
was incubated at room temperature for overnight, and the resulted
membrane FIG. 60o showed slightly shiny color. PAA-GA FIG. 60g was
incubated at room temperature overnight at room-temperature, and
the resulted membrane FIG. 60p showed shiny surface. This membrane
did not show any plastic-like surface how it was seen for the
membrane FIG. 60n, which can be attributed that the edges of FIG.
60n was thinner while the center was thicker. However, for the
PAA-GA membrane FIG. 60p, the solution was evenly distributed. All
of these membranes were prepared according to FIG. 1a, which means
the last step of phase-inversion took place in pure water if not
specified otherwise.
[0394] FIGS. 61a-61e includes several images. FIG. 61a PAA-A-GA
(0.25%); FIG. 61b PAA-A-GA (0.75%); FIG. 61c PAA-C-GA (0.25%); FIG.
61d PAA-CS-GA (0.3%), FIG. 61e PAA-GA (0.25%). 70% stock GA was
pre-dissolved in DMAC if not mentioned otherwise. All of the
membranes prepared according to FIG. 1a.
[0395] FIGS. 62a-62g includes several images. FIG. 62a--PAA-GA;
FIG. 62b--PAA-DA-GA; FIG. 62c--PAA-A-GA; FIG. 62d: PAA-DA-GA
(direct from 70% stock); FIG. 62e--PAA-A-GA was first incubated in
70.degree. C. for 30 min, followed by overnight incubation at room
temperature; FIG. 62f--PAA-A-GA similar to c but higher GA
concentration (% 0.9); FIG. 62g--PAA-C-GA. 70% stock GA was
pre-diluted in DMAC if not mentioned otherwise, followed by
introduced to the membrane formation processes at the concentration
of 0.3% if not mentioned otherwise.
[0396] GA concentration and its form are important in terms of
membrane characteristics. Besides, viscosity of PAA solution is
important. As seen in FIG. 60g, PAA-DA gave some blue region but
the rest is yellowish. Interestingly, increased incubation time and
high GA concentration make the membranes plastic like-structures
and colorful. However, this is not clear during phase-inversion.
When they are getting dry, their transparent natures become
visible. At low GA concentration and short-incubation time, the
membranes don't turn into transparent form, but a thin layer forms
on top of the membranes. The bottom of the membrane is mostly
not-shinny. It should be mentioned that the way of GA application
is important in membranes' mechanical and optical properties. For
example, while PAA-CS forms strong-green color membrane with FIG.
1aii, it does form pale chestnut color with FIG. 1ai. For example,
FIG. 13a illustrates UV/Visible spectroscopy for several disclosed
films. The Y-axis in the data is referred to as Optical Density, or
DO, with a comparatively good number between 0.1 and 0.9. The
larger the number the stronger the color intensity.
[0397] FIGS. 63a-63k are images of the following films: FIG. 63a:
PAA-pAS-GA; FIG. 63b:PAA; FIG. 63c: pAB-PAA-GA; FIG. 63d:
pAB-DMAC-GA-PAA; FIG. 63e: W-GA (long incubation)-PAA: FIG. 63f:
W-GA-PAA; FIG. 63g: pAB-DMAC-GA-PAA (30 min incubation); FIG. 63h:
PAA-pAS-W-GA; FIG. 63j: pAS-DMAC-GA-PAA; FIG. 63k: PAA-pAB-GA. All
of the membranes prepared according to FIG. 1b.
[0398] 0.32 M highly viscous (no fluidity) was treated with 0.25%
GA for 6 h at room temperature, followed by casted on glass and
phase-inverted according to FIG. 1a. The resulting films are shown
in FIG. 64a final steps of phase inversion were in 100% Ethanol,
FIG. 64b 50% Ethanol:water and (FIGS. 64c/d) and nano-pure water.
All of the membranes prepared according to FIG. 1a.
PAA-GA
[0399] As seen from FIG. 60e and FIG. 60g, GA (aged) treated PAA
showed similar transparent-view while they ended up showing
distinct surfaces in response to alteration in the final step of
phase-inversion (FIGS. 60n and 60p respectively). Both of the
membranes were synthesized according to FIG. 1a iii, but completion
of the phase inversion was performed in methanol (FIGS. 60 g/p) in
addition to water-bath (membrane shown in FIGs. e/n). Thinner
regions of the membrane FIG. 60n showed fully-plastic-like
structure while the thicker regions showed two different
characters; the outer part was fully plasticized while the inner
part was amorphous. However, the membrane shown in FIG. 60p was
more of amorphous membrane and no plastic-like outer layer was
seen. Contact angles of the membranes showed difference as well;
they were 67 and 61 for the membranes shown in FIGS. 60n and 60p,
respectively. However, when PAA-GA showed total plastic-like such
as the edges of the membrane FIG. 60n was 58. When the GA was
diluted in DMAC before it was applied to PAA solution brought
strong impact on membrane formation (shown in FIG. 60a). GA from
70% stock was pre-dissolved in DMAC, followed by introduced to the
PAA solution. The membrane was prepared according to FIG. 1a ii
with 0.9% GA. The resulted PAA gave durable and fully plastic-like
membrane. Even though, the PAA-GA (0.25%) membrane shown in FIG.
61e was prepared according to FIG. 1a ii, the resulted membrane
showed totally different character; outer layer and thinner regions
were plastic like while the center was amorphous because of the
thickness.
PAA-CS-GA:
[0400] Here three different PAA-CS-GA were shown how glutaraldehyde
affected color formation and formation of plastic-like structure.
As seen from FIGS. 60a and 60h, both PAA-CS-GA were transparent.
(FIG. 60a) PAA-CS-GA (% 0.25) was first incubated under hood for
6h, followed by incubated at room temperature for overnight
(.about.14h). However, (FIG. 60h) PAA-CS-GA (% 0.5) was only
incubated at room temperature for overnight (.about.20h).
Phase-inversion of both membranes was finalized in water-bath,
followed by dried under hood for 2h. As seen from FIG. 60i, the
membrane (FIG. 60a) resulted in a brownish plastic-like membrane
while the membrane (FIG. 60h) resulted in greenish amorphous
membrane (FIG. 60m). For both of these membranes, aged-GA was
directly introduced to the membrane formation process. However, GA
from 70% stock was pre-diluted in DMAC, followed by warmed for 10
min at 70.degree. C., which was then introduced to PAA-CS solution
seen in FIG. 61d. The membrane (FIG. 61d) was incubated at room
temperature for 15 h, and phase inversion was completed in
water-bath for 2 h followed by dried under hood for 2h. The
resulted PAA-CS-GA membrane (FIG. 61d) was durable, strong and
resistant to the organic solvents while the membrane (FIG. 61m)
became brittle within three days. The membrane (FIG. 61m) did not
turn into plastic-like membrane within 2 weeks, but rather the
membrane became brittle. The membrane (FIG. 61d) did not form a
transparent greenish membrane at once, rather it took over 2 h to
form the transparent membrane; at first it was opaque and within
time it turned into transparent form.
PAA-A-GA
[0401] FIGS. 60b, 60c, 60d and 60f are showing the membranes before
they underwent final step of the phase-inversion. Even though same
GA (stock 25%) was used for these four membranes, heat treatment
and alteration in final step of phase-inversion showed strong
impact on physical properties of final membrane. Heat treatment at
70.degree. C. for 1 h was applied to the casted PAA-A-GA solution
before it underwent incubation at room temperature. The resulted
membrane showed fully plastic-like reddish structure (FIG. 60k),
but it was not durable and became brittle within a week. When the
completion of phase-inversion took place in methanol/water mixture,
the resulted PAA-A-GA (FIG. 61) was opaque. In contrast to this,
outer layer of the PAA-A-GA (FIG. 60k) was plastic-like and shiny.
However, when the membrane was thick, the outer layer did not
provide similar plastic-like view, rather it gave a shiny surface
(FIG. 60o). When 70% GA was dissolved in DMAC, followed by
introduction to PAA-A solution, it affected membrane formation;
before the membrane formation underwent incubation at room
temperature, it was warmed at 70.degree. C. for 10 min. The
resulted membrane (FIG. 61a) showed thicker plastic-like outer
layer in comparison to the membrane shown in FIG. 61b. Similarly,
low GA concentration and short incubation (6h total) resulted in
non-plastic like membrane formation (FIG. 62c). The edges of
PAA-A-GA (FIG. 61a) are totally transparent, and the overall
durability of the membrane is better than FIG. 60j membrane. When
GA concentration was increased (0.75%), the resulted PAA-A-GA
became transparent and brownish (FIG. 62b); the membrane was
durable and strong. Similarly, when the GA concentration was
increased from 0.75% to 0.9%, the resulted membrane became
yellowish (FIG. 62f) and better transparency in comparison to the
PAA-A-GA membrane shown in FIG. 61b. Heat treatment allowed
PAA-A-GA (FIG. 62e) totally shiny and plastic-like view, which was
also durable in comparison to heat treated-PAA-GA (has similar
color).
PAA-C-GA and PAA-DA-GA
[0402] Dilution of GA in DMAC totally altered the view of PAA-DA-GA
even though same GA concentration was used. PAA-DA-GA (FIG. 62d)
showed plastic-like outer layer and stronger and durable membrane.
However, as shown in FIG. 60m, fully plastic-like membrane can be
obtained if PAA-DA-GA was heated for 20 min as of first step
incubation at 70.degree. C. Heat treatment allowed PAA-C-GA (FIG.
60i) membrane became totally plastic-like structure, but not very
transparent. When there is no heat treatment and high GA level,
PAA-C-GA (FIG. 60m) did not end up forming transparent membrane.
Glucosamine might react with GA first over C1-OH in addition to
amino group, followed by introduced to PAA through amino groups on
PAA. This could be a type of Maillard reaction, which gives
brown-color formation (FIG. 62d).
PAA-pAB-GA, PAA-W-GA, PAA-pAS-GA
[0403] pAB is also another small molecule altered overall view of
PAA. In all cases, aged GA was used for preparation of PAA-pAB-GA
membranes. As seen from the FIG. 63c, PAA-pAB-GA is bluish with
0.25% GA concentration while the PAA-pAB-GA (FIG. 63k) is more of
brownish with 0.5% GA, which unexpectedly became brittle within a
month. Similar to high GA concentration, incubation of pAB with GA
in DMAC before they were introduced to PAA solution resulted in
color changes of PAA-pAB-GA membranes; pAB was pre-treated with GA
in DMAC for 10 min and 20 min for the membranes seen in FIGS. 63d
and 63g. Similarly, W was pre-treated with GA for 20 min and 30 min
before they were added to PAA solutions resulted in yellowish (FIG.
63f) and reddish membrane formation (FIG. 63e), respectively. pAS
was pretreated with GA in DMAC, followed by added to PAA solution,
gave yellowish view (FIG. 63j) while PAA-pAS-GA (FIG. 63a) gave
light purple view. However, when GA treated pAS and GA treated W
were simultaneously added to PAA solution, the membrane gave light
yellowish view (FIG. 63h) which resembles to PAA membrane
phase-inverted under hood (FIG. 63b). All of these membranes were
synthesized according to FIG. 1b.
[0404] FIGS. 65a-65d are images of: FIG. 65a PAA (ODA+PMDA)-GA-SA 1
h incubation at room temperature, followed by phase-inversion in
pure water; FIG. 65b PAA (PDA+PMDA)-GA-SA 1 h incubation at room
temperature, followed by phase-inversion in pure water; FIG. 65c
PAA (ODA+PMDA)-GA-W 1h incubation at room temperature, followed by
phase-inversion in pure water; FIG. 65d PAA (PDA+PMDA)-GA-SA 30 min
incubation at room temperature, followed by phase-inversion in pure
water. In order to get transparent membranes, in the cases of PDA
as amine sources less time required due to the fact that PDA has
more reactive amino groups than ODA because of conjugation. Small
molecule such as W resulted in bluish non-transparent while SA
provided highly transparent membrane; PAA-SA-GA gave glassy brittle
membrane while PAA-W-GA gave amorphous durable membrane.
1.13 Application of PAA Membranes for Food Packaging
[0405] Cheese, pepperoni, apple and walnut were used to test
packaging properties of the membranes.
[0406] The formed films are shown in FIGS. 67a-67e, with FIG. 67a
POLLY-O part-skim mozzarella cheese (Campbell, N.Y.); FIG. 67b
Merve pepperoni (NJ); FIG. 67c Cabot extra sharp cheddar cheese
(Cabot, Vt.); FIG. 67d Green apple (WalMart, Jonson City N.Y.) and
FIG. 67e Diamond walnut (CA). PAA-A-GA, PAA-A-pAS and PAA-I-pAS
membranes were used for packaging, respectively.
[0407] The films were sterilized rinsing 70% Ethanol, followed by
rinsing with excess pure water (18.2 M.OMEGA.), which were finally
treated with 1 h UV light. Food samples were kept in fridge at
4.degree. C. Cheeses and pepperoni protected their stability for
the tested period, 3-6 months.
[0408] FIGS. 68a-68c are images of the stored foods of FIGS.
67a-67c. FIGS. 68a and 68c are images of cheeses stored for 10-11
months. As it is seen, the microbial growth is localized in FIG.
68a, which was intentionally pierced.
[0409] FIG. 68c is an image of pepperoni after 15 months
incubation. No microbial growth was observed.
[0410] FIG. 69 illustrates the disclosed film concept for both
detection and packaging. Step 1: air packed or vacuum packed food
sample produces volatile organic compounds or other compounds; (ii)
the VOC interacts with PAA, (ii) this results in color-change that
is visually detected or electronically detected.
[0411] The disclosed films can be used as a packaging material but
at least a portion of the film makes no direct contact with the
food sample. Any volatile or semivolatile organic vapor that is
produced as a result of food spillage is drawn on to the
sensor/packaging PAA. The sensor responds via a visible color
change and a measurable change in conductivity using an optional
conductivity monitor that is placed on the package. The
concentration of the emission of volatile organic compounds
(VOCs--e.g., sulfur compounds, acetone, methyl ethyl ketone,
toluene, ethylbenzene, m,p-xylene, styrene, and o-xylene) largely
increased over the storage time and should be correlated with the
total number of microbial numbers. This should allow a rapid
detection of food spoilage and may also allow consumers to visually
determine food freshness. The PAA film can also detect pH-related
changes in the air around the food (e.g. ammonia, alcohol).
1.14 Thermoplastic Examples
[0412] This example includes formulations that are capable of
forming a PAA film that i) softens when heated (thus allowing the
film to be molded to different shapes and sizes); ii) is flexible
and undergoes crystallization transitions by incorporating
sulfur-containing monomers, fatty acids, ionic salts and liquids,
and plasticizers between the different functional groups; and iii)
is resistant to shrinking while retaining good strength and
chemical stability.
[0413] In this example, these films can exclude, wholly or
partially, the formation of covalent bonds while increasing ionic
properties, mechanical strength and dissolution. The resultant film
in this example is referred to as "Thermoplastic PAA".
[0414] Unlike a "thermoset" PAA polymer that is held together via
irreversible chemical bonds, Thermoplastic PAA is relatively weakly
held together through electrostatic interactions and Van der Walls
forces. These relatively weak bonds in the thermoplastic polymers
allow them to be re-usable, relatively soft when heated, and to be
molded and remolded one or more times.
[0415] This ability to reuse thermoplastic typically means a higher
recyclability. Also, other properties such as good strength and a
tendency to resist shrinking is realized by these Thermoplastic PAA
films.
[0416] These Thermoplastic PAA films can be made more thermoplastic
by reacting sulfur containing monomers (e.g. 4,4'-thiodianiline; an
analogue of 4,4'-oxydianiline) in a stoichiometric ratio of
acid/amine functionality and other additives. Examples of
Thermoplastic PAA Films include but are not limited to those shown
in Table S.
TABLE-US-00020 TABLE S Examples of Proposed Thermoplastie PAA Film
Formulations ##STR00002## ##STR00003## ##STR00004## ##STR00005##
##STR00006## ##STR00007## ##STR00008## ##STR00009## ##STR00010##
##STR00011## adipate (DMAD) to PAA ##STR00012## ##STR00013##
##STR00014##
[0417] Various Thermoplastic PAA films can be developed using
various calculated concentrations of acid/amine, plasticizers, and
monomers. These concentrations can be derived using the concept of
critical branching coefficient. Mixtures of Thermoplastic PAA films
can incorporate plasticizers (e.g. adipates, phthalates, and
citrates) and/or two polymer chains (e.g PAA and chitosan)
interacting via hydrogen bonding and electrostatic forces. The main
polymer chains can move freely using these formulations.
[0418] Additional formulations can include the use of shorter or
longer alkyl chains and a range of other dicarboxylic acids (e.g.
oleic acids, palmitoleic acid, sapienic acid, and linoleic acid).
Other materials to be added for the formation of the Thermoplastic
PAA films can include low to high polarity esters (e.g. nitriles,
polychloroprene, chlorinated polyethylene and epichlorohydrins) in
order to decrease the attraction between polymer chains to make
them more flexible. A range of esters (e.g. sabacates,
terephthalates, gluterates and azelates) are options. These
polymers can be synthesized in environmentally-friendly
solvents.
[0419] The Thermoplastic PAA films can be analyzed in several ways,
for example their structures can be characterized using 1H and 13C
Nuclear Magnetic Resonance (1H NMR) Spectroscopy and Heteronuclear
Single Quantum Coherence (HSQC) spectroscopy of the 1H-13C system.
The polymerization can be validated via Infrared Spectroscopy (IR)
by analyzing changes in the functional groups. The molecular
weights can be determined via size exclusion chromatography. Also,
Differential Scanning Calorimetry (DSC) can be used to study the
thermal transitions of the polymers.
[0420] These Thermoplastic PAA films exhibit a decrease in the
glass transition temperature (Tg) for the films containing
plasticizers in their DSC curves. For all formulations, the
appearance of a crystallization temperature (Tc) peak is evident in
the DSC curves. This peak indicates the crystallinity of the
polymers upon cooling, which also suggests thermoplasticity. Also,
these films have an increase in plasticity and a reduction in
rigidity due to an absence or a relatively low amount of covalent
crosslinking.
[0421] Throughout the application the following acronyms are used
when discussing PAA films. The meaning of these abbreviations
appears below:
PAA: Poly(amic) acid
GA: Glutaraldehyde
A-alanine
W-tryptophane
CS-Chitosan
[0422] SA-Sulfanilic acid
I-isoleucine
K-L-Lysine
[0423] CA-cellulose acetate pAS-p-aminoscalicylic acid
PDA-PAA-p-phenylenedianiline+pyromellitic dianhydride PAA
IZ-Carbodiimizole
[0424] pAB-: p-aminobenzoic acid PCL-3-chloro-4-aminobenzoic
acid
C-cysteine
[0425] BB-2 benzoylbenzoic acid 5AS-5-aminosalycylic acid
4AS-p-aminoscalicylic acid
Ser-L-Serin
DA-D-glucosamine
SN-sulfanilamide
T-L-Threonine
[0426] The described embodiments and examples of the present
disclosure are intended to be illustrative rather than restrictive,
and are not intended to represent every embodiment or example of
the present disclosure. While the fundamental novel features of the
disclosure as applied to various specific embodiments thereof have
been shown, described and pointed out, it will also be understood
that various omissions, substitutions and changes in the form and
details of the devices illustrated and in their operation, may be
made by those skilled in the art without departing from the spirit
of the disclosure. For example, it is expressly intended that all
combinations of those elements and/or method steps which perform
substantially the same function in substantially the same way to
achieve the same results are within the scope of the disclosure.
Moreover, it should be recognized that structures and/or elements
and/or method steps shown and/or described in connection with any
disclosed form or embodiment of the disclosure may be incorporated
in any other disclosed or described or suggested form or embodiment
as a general matter of design choice. Further, various
modifications and variations can be made without departing from the
spirit or scope of the disclosure as set forth in the following
claims both literally and in equivalents recognized in law.
Sequence CWU 1
1
16122DNAArtificial Sequenceprimer 1cttggtcatt tagaggaagt aa
22220DNAArtificial Sequenceprimer 2tcctccgctt attgatatgc
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aagattcgat gattcactga attctgcaat tcacattact 180tatcgcattt
tgctgcgttc ttcatcgatg ccagaaccaa gagatccgtt gttgaaagtt
240ttgatttatt tatggtttta ctcagaagtt acatatagaa acagagtttt
aggggtcctc 300tggcgggccg tcccgtttta ccgggagcgg gctgatccgc
cgaggcaaca agtggtatgt 360tcacaggggt ttgggagttg taaactcggt
aatgatccct ccgctggttc accaacggag 420acct 4244319DNAFusarium
oxysporum 4cccggggcaa ggggcgggcg gcgttggatt ttgcgggacc cttaacaccc
gcttcagccg 60cagcgggcgc cgccgccccg aggcccggcg ccgatctaac aagtaataca
tctcaaaggt 120gtccaaccgt atccaaccag tggacgtccg agggtcgcgc
cgtttgagtg tcatgttaat 180atcaactctg atggtttttt gttaatcatt
ggatgttgga cttggggatc ccgtcacagt 240cgactactga tgagtactat
agactacgca tcgcgcagct gatatattta atgtctacgt 300atatcaatcc attaataaa
3195794DNAFusarium oxysporummisc_feature(6)..(6)n is a, c, g, or
tmisc_feature(76)..(76)n is a, c, g, or tmisc_feature(609)..(609)n
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tmisc_feature(644)..(644)n is a, c, g, or
tmisc_feature(696)..(696)n is a, c, g, or
tmisc_feature(710)..(710)n is a, c, g, or t 5ccgcgnggag gtttctggac
cgctgtccga ccgcgccgct ccgttcggcg ccgagttcca 60ctttgtcccc tcattnatat
tgtcaattac gcgggtattc caccgattcc aggtcacttc 120gaagttgggg
tttaacggcg tggccgcgac gattaccagt aacgagggtt ttactactac
180gctatggaag ctcgacgtga ccgccaatca atttgaggaa cgcgaattaa
cgcgagtccc 240aacaccaagc tgtgcttgag ggttgaaatg acgctcgaac
aggcatgccc gccagaatac 300tggcgggcgc aatgtgcgtt caaagattcg
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tcttcatcga tgccagaacc aagagatccg ttgttgaaag 420ttttgattta
tttatggttt tactcagaag ttacatatag aaacagagtt ttaggggtcc
480tctggcgggc cgtcccgttt taccgggagc gggctgatcc gccgaggcaa
caagtggtat 540gttcacaggg gtttgggagt tgtaaactcg gtaatgatcc
ctccgctggt tcaccaacgg 600agacctgtna caactttnac tccctctaat
gacaaaatca ctantgaatc ccgccgccgc 660agtcacatat gggagagctc
ccacgcgtgg atctanctga gtatctatan gtcacctaat 720actggcgtat
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780aatgtaacgg gtca 7946605DNAFusarium
oxysporummisc_feature(9)..(9)n is a, c, g, or
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a, c, g, or tmisc_feature(118)..(118)n is a, c, g, or
tmisc_feature(167)..(167)n is a, c, g, or
tmisc_feature(170)..(170)n is a, c, g, or
tmisc_feature(453)..(453)n is a, c, g, or
tmisc_feature(463)..(463)n is a, c, g, or t 6ccctctttna aattcttttt
aggggggggc gacttcccgg cggggctact cagtcatgga 60tctctggatg caataanata
ttagcgatct tcgccngtga accacgagga ggatcacnag 120tgcaacccca
aacccctgtg aacataccac ttgttgccgc gccgatncgn ccgcccccgt
180aaaacgggac ggcccgccag aggaccccta aaactctgtt tctatatgta
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tggttctggc atcgatgaag 300aacgcagcaa aatgcgataa gtaatgtgaa
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agtattctgg cgggcatgcc tgttcgagcg tcatttcaac 420cctcaagcac
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480ggtcacgtca agcttccata gcgtaatagt aaaaaccctc gttactggta
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taccgcgaac taactatata 600cgaga 6057873DNAFusarium
oxysporummisc_feature(16)..(16)n is a, c, g, or
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tmisc_feature(864)..(864)n is a, c, g, or
tmisc_feature(868)..(868)n is a, c, g, or t 7ccgggcggga ggtttngtta
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cctcattcag tatngtcaag tgtgacgcgg gtattcctca 120cccgattcca
ggtgcacttc cagaagttgg ggtttaacgg cgtggccgcg acgattacca
180gtaacgaggg ctttactact acgctatgga agctcgacgt gaccgccaat
caatttgagg 240aacgcgaatt aacgcgagtc ccaacaccga gctgtgcttg
agggttgaaa tgacgctcga 300acaggcatgc ccgccagaat actggcgggc
gcaatgtgcg ttcaaagatt cgatgattca 360ctgaattctg caattcacat
tacttatcgc attttgctgc gttcttcatc gatgccagaa 420ccaagagatc
cgttgttgaa agttttgatt tatttatggt tttactcaga agttacatat
480agaaacagag ttttaggggt cctctggcgg gccgtcccgt tttaccggga
gcgggctgat 540ccgccgaggc aacaagtggt atgttcacag gggtttggga
gttgtaaact cggtaatgat 600ccctccgctg gttcaccaac ggagaccttg
ttacgacttt tacttcctct aaatgaccaa 660gaatcactag tgaattcgcg
gccgcctgca ggtcaacata tggagagctc cacccgtgga 720tgcatanctg
agtatctata gtgtccctaa tacttggcgt atcatggcat accggttccg
780tgtgaaatgt tatcgctcac catccaacaa atacnacccg aaacttaang
ttaaccgggg 840gtcctaatag tgaccaccca ttantgcntt gcc
8738822DNAFusarium oxysporummisc_feature(16)..(16)n is a, c, g, or
tmisc_feature(18)..(18)n is a, c, g, or tmisc_feature(29)..(29)n is
a, c, g, or tmisc_feature(57)..(57)n is a, c, g, or
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tmisc_feature(739)..(739)n is a, c, g, or
tmisc_feature(774)..(774)n is a, c, g, or t 8cggaggtttt ttgggncncc
gtcgcgacna gggccctcac ttggagctcc gaccggncgc 60gccaattaac tcatggattt
cggggattta gaggaagtaa aagttttaac aggtgtcccg 120ttggtgaacc
agcggaggga tcttaccgag tttacactcc caaacccctg tgaacatacc
180acttgttgcc tcggcggatc agcccgctcc cggtaaaacg ggacggcccg
ccagaggacc 240cctaaaactc tgtttctata tgtaacttct gagtaaaacc
ataaataaat caaaactttc 300aacaacggat ctcttggttc tggcatcgat
gaagaacgca gcaaaatgcg ataagtaatg 360tgaattgcag aattcagtga
atcatcgaat ctttgaacgc acattgcgcc cgccagtatt 420ctggcgggca
tgcctgttcg agcgtcattt caaccctcaa gcacagctcg gtgttgggac
480tcgcgttaat tcgcgttcct caaattgatt ggcggtcacg tcgagcttcc
atagcgtagt 540agtaaagccc tcgttactgg taatcgtcgc ggccacgccg
ttaaacccca acttctgaat 600gttgacctcg gatcaggtag gaatacccgc
tgaacttaag catatcaata agcggaggaa 660atcgaattcc gcgggcgcca
tggcggccgg aacatcaact tcggccaatc ccctatatat 720gtatacatcc
tggcgnttna caactggacg ggaaacgcgt accactatcc tgcncatccc
780ttccccggct attcaagccc ccaccctcca atgccccaat gg
8229494DNAFusarium oxysporummisc_feature(9)..(9)n is a, c, g, or
tmisc_feature(12)..(12)n is a, c, g, or tmisc_feature(27)..(27)n is
a, c, g, or tmisc_feature(35)..(35)n is a, c, g, or
tmisc_feature(52)..(52)n is a, c, g, or tmisc_feature(449)..(449)n
is a, c, g, or t 9ccggaggtna gncagcaccc gcccctngga acccncccat
attctacctg tnacccattt 60aggcatacaa ttgggtgaac gctggcccac atacctaaca
gggctacact accatggaag 120ccactgaccg ccatcatttg aggaacgcaa
ttaacgcgag tcccaacacc gagctgtgct 180tgagggttga aatgacgctc
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ttcgatgatt cactgaattc tgcaattcac attacttatt cgcattttgc
300tgcgttcttc atcgatgcca gaaccaagag atccgttgtt gaaagttttg
atttatttat 360ggtttactca gaagttacat atagaaacag agttttaggg
gtcctctggc gggcccgtcc 420cgttttaccg ggagcgggct gatccgccna
gcaacaagtg gtatgttaca ggggttggga 480gttgtaaccg taat
49410785DNAFusarium oxysporummisc_feature(774)..(774)n is a, c, g,
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tcgacctgca 60ggcggccgcg aattcactag tgattcttgg tcatttagag gaagtaaaag
tcgtaacaag 120gtctccgttg gtgaaccagc ggagggatca ttaccgagtt
tacaactccc aaacccctgt 180gaacatacca cttgttgcct cggcggatca
gcccgctccc ggtaaaacgg gacggcccgc 240cagaggaccc ctaaaactct
gtttctatat gtaacttctg agtaaaacca taaataaatc 300aaaactttca
acaacggatc tcttggttct ggcatcgatg aagaacgcag caaaatgcga
360taagtaatgt gaattgcaga attcagtgaa tcatcgaatc tttgaacgca
cattgcgccc 420gccagtattc tggcgggcat gcctgttcga gcgtcatttc
aaccctcaag cacagctcgg 480tgttgggact cgcgttaatt cgcgttcctc
aaattgattg gcggtcacgt cgagcttcca 540tagcgtagta gtaaagccct
cgttactggt aatcgtcgcg gccacgccgt taaaccccaa 600cttctgaatg
ttgacctcgg atcaggtagg aatacccgct gaacttaagc atatcaataa
660gcggaggaaa tcgaattccg ccggccgcca tggcggccgg gagcatgcga
agtcgggccc 720aattcgccct atagtgagtt ttattacaat tcactggccc
gtcttttaca aacnttgtga 780ctggg 78511348DNATrichaptum
biformemisc_feature(167)..(167)n is a, c, g, or
tmisc_feature(272)..(272)n is a, c, g, or
tmisc_feature(281)..(281)n is a, c, g, or
tmisc_feature(296)..(296)n is a, c, g, or
tmisc_feature(319)..(319)n is a, c, g, or
tmisc_feature(321)..(321)n is a, c, g, or
tmisc_feature(334)..(334)n is a, c, g, or
tmisc_feature(342)..(342)n is a, c, g, or t 11ggatcgcgcc gggggtgggg
cggggcctta agattttacg agaattaggt tagagatttt 60gtcttagatc gagacagact
caagaatagt tcatggtcaa gagtaggatc taacaagtaa 120tacatctcaa
aggtgtccaa ccgtatccaa ccagtggacg gatcttnacc gagtggtgcg
180cagggggcgc atccccttgt cgaacccact acccctggat ggctcgtagc
tccatcggac 240gggtgccggg gggggatcgc gtcactgtcg antactgatg
ngaactatag actatngatc 300cgggcagctg atatatccna natctatgta
tatnaatcca tnaataaa 34812813DNATrichaptum
biformemisc_feature(1)..(4)n is a, c, g, or
tmisc_feature(54)..(54)n is a, c, g, or tmisc_feature(64)..(64)n is
a, c, g, or tmisc_feature(78)..(78)n is a, c, g, or
tmisc_feature(92)..(92)n is a, c, g, or tmisc_feature(119)..(119)n
is a, c, g, or tmisc_feature(134)..(134)n is a, c, g, or
tmisc_feature(152)..(152)n is a, c, g, or
tmisc_feature(161)..(161)n is a, c, g, or
tmisc_feature(174)..(174)n is a, c, g, or
tmisc_feature(182)..(182)n is a, c, g, or
tmisc_feature(225)..(225)n is a, c, g, or
tmisc_feature(252)..(252)n is a, c, g, or
tmisc_feature(254)..(254)n is a, c, g, or
tmisc_feature(258)..(258)n is a, c, g, or
tmisc_feature(260)..(260)n is a, c, g, or
tmisc_feature(262)..(262)n is a, c, g, or
tmisc_feature(265)..(265)n is a, c, g, or
tmisc_feature(282)..(282)n is a, c, g, or
tmisc_feature(289)..(289)n is a, c, g, or
tmisc_feature(291)..(291)n is a, c, g, or
tmisc_feature(293)..(293)n is a, c, g, or
tmisc_feature(314)..(314)n is a, c, g, or
tmisc_feature(318)..(318)n is a, c, g, or
tmisc_feature(320)..(320)n is a, c, g, or
tmisc_feature(339)..(339)n is a, c, g, or
tmisc_feature(356)..(356)n is a, c, g, or
tmisc_feature(358)..(358)n is a, c, g, or
tmisc_feature(361)..(361)n is a, c, g, or
tmisc_feature(372)..(372)n is a, c, g, or
tmisc_feature(374)..(374)n is a, c, g, or
tmisc_feature(377)..(377)n is a, c, g, or
tmisc_feature(383)..(383)n is a, c, g, or
tmisc_feature(385)..(385)n is a, c, g, or
tmisc_feature(387)..(387)n is a, c, g, or
tmisc_feature(390)..(390)n is a, c, g, or
tmisc_feature(398)..(398)n is a, c, g, or
tmisc_feature(409)..(409)n is a, c, g, or
tmisc_feature(424)..(424)n is a, c, g, or
tmisc_feature(433)..(433)n is a, c, g, or
tmisc_feature(436)..(436)n is a, c, g, or
tmisc_feature(442)..(442)n is a, c, g, or
tmisc_feature(445)..(445)n is a, c, g, or
tmisc_feature(461)..(461)n is a, c, g, or
tmisc_feature(464)..(464)n is a, c, g, or
tmisc_feature(470)..(470)n is a, c, g, or
tmisc_feature(479)..(479)n is a, c, g, or
tmisc_feature(482)..(482)n is a, c, g, or
tmisc_feature(485)..(485)n is a, c, g, or
tmisc_feature(491)..(491)n is a, c, g, or
tmisc_feature(500)..(500)n is a, c, g, or
tmisc_feature(512)..(512)n is a, c, g, or
tmisc_feature(526)..(526)n is a, c, g, or
tmisc_feature(529)..(529)n is a, c, g, or
tmisc_feature(532)..(532)n is a, c, g, or
tmisc_feature(538)..(538)n is a, c, g, or
tmisc_feature(541)..(541)n is a, c, g, or
tmisc_feature(544)..(544)n is a, c, g, or
tmisc_feature(547)..(547)n is a, c, g, or
tmisc_feature(550)..(550)n is a, c, g, or
tmisc_feature(553)..(553)n is a, c, g, or
tmisc_feature(556)..(556)n is a, c, g, or
tmisc_feature(559)..(560)n is a, c, g, or
tmisc_feature(563)..(563)n is a, c, g, or
tmisc_feature(587)..(587)n is a, c, g, or
tmisc_feature(590)..(590)n is a, c, g, or
tmisc_feature(593)..(593)n is a, c, g, or
tmisc_feature(697)..(697)n is a, c, g, or
tmisc_feature(699)..(699)n is a, c, g, or
tmisc_feature(724)..(724)n is a, c, g, or
tmisc_feature(727)..(727)n is a, c, g, or
tmisc_feature(730)..(730)n is a, c, g, or
tmisc_feature(739)..(739)n is a, c, g, or
tmisc_feature(748)..(748)n is a, c, g, or
tmisc_feature(750)..(750)n is a, c, g, or
tmisc_feature(756)..(756)n is a, c, g, or
tmisc_feature(759)..(759)n is a, c, g, or
tmisc_feature(768)..(768)n is a, c, g, or
tmisc_feature(771)..(771)n is a, c, g, or
tmisc_feature(774)..(774)n is a, c, g, or
tmisc_feature(778)..(778)n is a, c, g, or
tmisc_feature(788)..(788)n is a, c, g, or
tmisc_feature(803)..(803)n is a, c, g, or t 12nnnntgtttt tcgggcgcgt
cgcgcggggc cctctctggg gagcgtccgc cggncgtccg 60ccgnttacac taagatgnat
ttgcgagcac gngctaacat gagatagtta taggcgttnc 120gagtctttct
acgngagctc aaatccccta gntcactgag nctccccagc acgngctaca
180gncctccttg cagagagggg cgctctcttt cgggatcaga atatntacac
gggcgaaaaa 240agagggcccc cntnatancn anacncgaga cagtgcgaca
gnctggacnc ngntacacag 300gttctgagag tcgntggngn ggaagacagt
gagacgggnc aaacagggaa aaccananag 360ntcgagtttg tncngcngtg
gtncncnatn ggaaaaanct catcccgtng aagggcccac 420cgangagccc
ccnacnaaaa tnctnggggt tgggcccggc nctngttccn accaaaaang
480tnatngttct ncttgtaatn tctggggggg gngtgcccgc cccccngtnc
angaattnta 540ncantangan cgnaanagnn tgntgggcaa aaacggaggt
tccctcnacn ctngaatatt 600aacatatttc cccccccacc aaaatattgg
ttcctcccac cccgcccccc ttttgtgggg 660cccccgcggg tttggggttt
ccaattccct cggcctntnt tggccagaag gaaggtgggg 720ggcngcngan
gaaaaaaant ccgcaaanan ggccangtnc aagttgcnac ngcnaatngt
780ggggcctnat ttttggaaac cancaattgg ggt 81313266DNATrichaptum
biformemisc_feature(8)..(8)n is a, c, g, or
tmisc_feature(28)..(28)n is a, c, g, or tmisc_feature(30)..(30)n is
a, c, g, or tmisc_feature(32)..(32)n is a, c, g, or
tmisc_feature(38)..(38)n is a, c, g, or tmisc_feature(40)..(40)n is
a, c, g, or tmisc_feature(42)..(42)n is a, c, g, or
tmisc_feature(46)..(46)n is a, c, g, or tmisc_feature(53)..(53)n is
a, c, g, or tmisc_feature(68)..(68)n is a, c, g, or
tmisc_feature(80)..(80)n is a, c, g, or tmisc_feature(86)..(86)n is
a, c, g, or tmisc_feature(116)..(116)n is a, c, g, or
tmisc_feature(122)..(122)n is a, c, g, or
tmisc_feature(125)..(125)n is a, c, g, or
tmisc_feature(133)..(133)n is a, c, g, or
tmisc_feature(136)..(136)n is a, c, g, or
tmisc_feature(139)..(139)n is a, c, g, or
tmisc_feature(144)..(144)n is a, c, g, or
tmisc_feature(149)..(149)n is a, c, g, or
tmisc_feature(159)..(159)n is a, c, g, or
tmisc_feature(167)..(167)n is a, c, g, or
tmisc_feature(173)..(173)n is a, c, g, or
tmisc_feature(191)..(191)n is a, c, g, or
tmisc_feature(193)..(193)n is a, c, g, or
tmisc_feature(197)..(197)n is a, c, g, or
tmisc_feature(200)..(200)n is a, c, g, or
tmisc_feature(203)..(203)n is a, c, g, or
tmisc_feature(207)..(207)n is a, c, g, or
tmisc_feature(226)..(226)n is a, c, g, or
tmisc_feature(234)..(234)n is a, c, g, or
tmisc_feature(237)..(237)n is a, c, g, or
tmisc_feature(239)..(239)n is a, c, g, or
tmisc_feature(241)..(241)n is a, c, g, or
tmisc_feature(250)..(250)n is a, c, g, or
tmisc_feature(253)..(253)n is a, c, g, or
tmisc_feature(256)..(256)n is a, c, g, or t 13ccggggcngc cggggcgcgt
cgccggcngn cngcggcncn tnggcngccc gcngcgcgag 60cgcagcgngc cggtggtgcn
cgcgcncacc tcccgtccca cctccttcgc gctcgntgcg 120cncanctcta
tantangtna gagnagatng aatactagna ctatacntat acntatagca
180cgtaggacga ngnaagngan tcncganatt tttatttggc cgattntcct
atantgnana 240nggggaaaan ggnagnaatt tttgaa 26614817DNATrichaptum
biformemisc_feature(26)..(26)n is a, c, g, or
tmisc_feature(70)..(70)n is a, c, g, or tmisc_feature(86)..(86)n is
a, c, g, or tmisc_feature(94)..(94)n is a, c, g, or
tmisc_feature(102)..(102)n is a, c, g, or
tmisc_feature(126)..(126)n is a, c, g, or
tmisc_feature(160)..(160)n is a, c, g, or
tmisc_feature(178)..(178)n is a, c, g, or
tmisc_feature(184)..(184)n is a, c, g, or
tmisc_feature(272)..(272)n is a, c, g, or
tmisc_feature(290)..(290)n is a, c, g, or
tmisc_feature(299)..(299)n is a, c, g, or
tmisc_feature(301)..(301)n is a, c, g, or
tmisc_feature(305)..(305)n is a, c, g, or
tmisc_feature(307)..(307)n is a, c, g, or
tmisc_feature(312)..(312)n is a, c, g, or
tmisc_feature(315)..(315)n is a, c, g, or
tmisc_feature(317)..(317)n is a, c, g, or
tmisc_feature(325)..(325)n is a, c, g, or
tmisc_feature(328)..(328)n is a, c, g, or
tmisc_feature(330)..(330)n is a, c, g, or
tmisc_feature(336)..(336)n is a, c, g, or
tmisc_feature(338)..(338)n is a, c, g, or
tmisc_feature(341)..(341)n is a, c, g, or
tmisc_feature(345)..(345)n is a, c, g, or
tmisc_feature(365)..(365)n is a, c, g, or
tmisc_feature(372)..(372)n is a, c, g, or
tmisc_feature(385)..(385)n is a, c, g, or
tmisc_feature(388)..(388)n is a, c, g, or
tmisc_feature(415)..(415)n is
a, c, g, or tmisc_feature(433)..(433)n is a, c, g, or
tmisc_feature(457)..(457)n is a, c, g, or
tmisc_feature(460)..(460)n is a, c, g, or
tmisc_feature(463)..(463)n is a, c, g, or
tmisc_feature(472)..(472)n is a, c, g, or
tmisc_feature(475)..(475)n is a, c, g, or
tmisc_feature(477)..(477)n is a, c, g, or
tmisc_feature(483)..(483)n is a, c, g, or
tmisc_feature(488)..(488)n is a, c, g, or
tmisc_feature(491)..(491)n is a, c, g, or
tmisc_feature(496)..(496)n is a, c, g, or
tmisc_feature(499)..(499)n is a, c, g, or
tmisc_feature(505)..(505)n is a, c, g, or
tmisc_feature(507)..(507)n is a, c, g, or
tmisc_feature(510)..(510)n is a, c, g, or
tmisc_feature(519)..(519)n is a, c, g, or
tmisc_feature(522)..(522)n is a, c, g, or
tmisc_feature(525)..(525)n is a, c, g, or
tmisc_feature(534)..(534)n is a, c, g, or
tmisc_feature(537)..(537)n is a, c, g, or
tmisc_feature(543)..(543)n is a, c, g, or
tmisc_feature(563)..(563)n is a, c, g, or
tmisc_feature(566)..(566)n is a, c, g, or
tmisc_feature(579)..(579)n is a, c, g, or
tmisc_feature(590)..(590)n is a, c, g, or
tmisc_feature(609)..(609)n is a, c, g, or
tmisc_feature(624)..(625)n is a, c, g, or
tmisc_feature(661)..(661)n is a, c, g, or
tmisc_feature(670)..(670)n is a, c, g, or
tmisc_feature(687)..(688)n is a, c, g, or
tmisc_feature(691)..(691)n is a, c, g, or
tmisc_feature(705)..(705)n is a, c, g, or
tmisc_feature(716)..(716)n is a, c, g, or
tmisc_feature(719)..(719)n is a, c, g, or
tmisc_feature(722)..(722)n is a, c, g, or
tmisc_feature(729)..(729)n is a, c, g, or
tmisc_feature(732)..(733)n is a, c, g, or
tmisc_feature(740)..(740)n is a, c, g, or
tmisc_feature(751)..(751)n is a, c, g, or
tmisc_feature(766)..(766)n is a, c, g, or t 14cggcggtggg tttggctcgt
gggccncccg tgcgcggggg gcgccgcctc cctttttgcg 60gacgcgtccn gcccgggcgc
gcccgncgcg gtanacggct anagtggagt gtgttgcagt 120gcacgngcta
tacatggtag tagttatagg cagttgggcn tgagtactgc tctgtacngg
180gagnctcaaa tcccatgagt cccgtggagg ctccccgaca cgggcgtaca
ggccctcctt 240tgagagaggg ggcgctctct ttccggacag anatatacgc
gggcgaaaan gagggcccnc 300ntttntntcg gnacncnagg gtcangtncn
gagcangntc ntagnacccc ccggggaaac 360aacanggttt tnctcgacga
aagtncgngt gggggcgggg ggaaagaacc aagtngaaag 420aacgggggcc
cantaacagg aggaaaaacc caaagangan tcngaatttg tnccncngtg
480gtnaaccnat nggaanganc ttatncngtn gaagggccna cngangagcc
cccnacngac 540atncttgggg gttgggcccg gcnctngttc ccaaccaana
ccggttaatn gttcctccct 600tgtttaatnt ctgggggggg ggtnngtgcc
ccggcccccc cctcggttca aaagaaattt 660ntaaccaaan aaggaacgca
aaaaagnntg ngtgccaaaa accgnaggtt ccctcnacnc 720tngaattana
cnnattcccn cgccaccaaa natttgttcc tcaacncggc ccccttttgt
780ggggcccccg gggtttggtg tttctaaatt ccttggc 81715808DNATrichaptum
biformemisc_feature(56)..(56)n is a, c, g, or
tmisc_feature(72)..(72)n is a, c, g, or tmisc_feature(113)..(113)n
is a, c, g, or tmisc_feature(787)..(787)n is a, c, g, or t
15cccggcgaat gttttatggg gtcatgttcg accgcgccgt ccggttggcg gagttncatt
60ttcgtgatct anaagagata aaatggctaa acaggtttac cgtaggttat tanccgcgga
120aggatcttaa cagttttgaa gtgggcttga tgctggcttg taacagagca
ctgtgctcag 180tcccgctcca atccattcaa cccctgtgca ctattcggag
tgttgcaagc taagacaatg 240tggggagtgg tcccggttgt atttctaatg
cgacttgggc ttactttcaa acggtcaagg 300cttgtcctcc ggtttatata
caaacacttt tattgtcttg tcgaatgtat tagcctctcg 360ttaggcgaaa
tttaaataca actttcaaca acggatctct tggctctcgc atcgatgaag
420aacgcagcga aatgcgataa gtaatgtgaa ttgcagaatt cagtgaatca
tcgaatcttt 480gaacgcacct tgcgctcctt ggctattccg aggagcatgc
ctgtttgagt gtcatgttaa 540tatcaactct gatggttttt tgttaatcat
tggatgttgg acttggaggt tcgtgctggc 600tgcaaagtcg gctcctcttg
aatgcattag cttggacctg tgcgcgtttg ctagcggtgt 660aatacattta
attcaccacg ggccgtgtca ctattagggt ctgcttctat tcgtcctacc
720ggacaataat aacttatgac ctgactcaat aggtagacac cccgactaac
ttaataccga 780gaatcantat ccgcccgcgt acatgaaa 80816784DNATrichaptum
biformemisc_feature(13)..(13)n is a, c, g, or
tmisc_feature(46)..(46)n is a, c, g, or tmisc_feature(61)..(61)n is
a, c, g, or t 16ccagaaggat ttnatgaaac aagataagca gaggtccctc
atcttnggac tccgacggcg 60ncgccatata actcatgatt tcccgctcta ttgatatgct
aagtttttag cgggtagtcc 120accgatttga ggtcagagtc ataaagttta
ttattgtccg gtaaggacga ttagaagcag 180accctaatag tgacacggcc
cgtggtgaat aaaatgtatt acaccgctag caaacgcgca 240caggtccaag
ctaatgcatt caagaggagc cgactttgca gccagcacga acctccaagt
300ccaacatcca atgattaaca aaaaccatca gagttgatat taacatgaca
ctcaaacagg 360catgctcctc ggaatagcca aggagcgcaa ggtgcgttca
aagattcgat gattcactga 420attctgcaat tcacattact tatcgcattt
cgctgcgttc ttcatcgatg cgagagccaa 480gagatccgtt gttgaaagtt
gtatttaaat ttcgcctaac gagaggctaa tacattcgac 540aagacaataa
aagtgtttgt atataaaccg gaggacaagc cttgaccgtt tgaaagtaag
600cccaagtcgc attaaaaata caaccgggac cactccccac attgtcttag
cttgcaacac 660tccgaatagt gcacaggggt tgaatgatgg aacggactga
cacagtgctc tgtacagcca 720cataagccac tcaactcgta tgatcttccg
cagtactacg aactgtacat ttattcccta 780taca 784
* * * * *