U.S. patent application number 13/878860 was filed with the patent office on 2013-08-01 for porous fiber, methods of making the same and uses thereof.
This patent application is currently assigned to NGEE ANN POLYTECHNIC. The applicant listed for this patent is Shuchi Agarwal, Nandhini Elayaperumal, Binoy Paulose Nadappuram, Gurdev Singh. Invention is credited to Shuchi Agarwal, Nandhini Elayaperumal, Binoy Paulose Nadappuram, Gurdev Singh.
Application Number | 20130196405 13/878860 |
Document ID | / |
Family ID | 43431335 |
Filed Date | 2013-08-01 |
United States Patent
Application |
20130196405 |
Kind Code |
A1 |
Singh; Gurdev ; et
al. |
August 1, 2013 |
POROUS FIBER, METHODS OF MAKING THE SAME AND USES THEREOF
Abstract
There is provided a porous fiber having a core-shell
configuration, wherein the pores on the fiber are configured to
encapsulate and thereby retain a biological material therein.
Inventors: |
Singh; Gurdev; (Singapore,
SG) ; Nadappuram; Binoy Paulose; (Singapore, SG)
; Agarwal; Shuchi; (Singapore, SG) ; Elayaperumal;
Nandhini; (Singapore, SG) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Singh; Gurdev
Nadappuram; Binoy Paulose
Agarwal; Shuchi
Elayaperumal; Nandhini |
Singapore
Singapore
Singapore
Singapore |
|
SG
SG
SG
SG |
|
|
Assignee: |
NGEE ANN POLYTECHNIC
Singapore
SG
|
Family ID: |
43431335 |
Appl. No.: |
13/878860 |
Filed: |
November 11, 2011 |
PCT Filed: |
November 11, 2011 |
PCT NO: |
PCT/SG2011/000401 |
371 Date: |
April 11, 2013 |
Current U.S.
Class: |
435/182 ;
264/413; 428/372; 428/394 |
Current CPC
Class: |
B01D 2239/0258 20130101;
Y10T 428/2967 20150115; D01D 5/0007 20130101; B01D 2325/48
20130101; D01F 1/08 20130101; Y10T 428/2927 20150115; B01D 69/02
20130101; D01D 5/247 20130101; B01D 39/1623 20130101; B01D 2323/39
20130101; D01F 1/10 20130101; D01D 5/0038 20130101; B01D 2239/0442
20130101; D01D 5/34 20130101; B01D 2239/025 20130101; B01D
2239/0631 20130101 |
Class at
Publication: |
435/182 ;
428/394; 428/372; 264/413 |
International
Class: |
D01F 1/08 20060101
D01F001/08; D01D 5/00 20060101 D01D005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 12, 2010 |
GB |
1019119.5 |
Claims
1-33. (canceled)
34. A porous fiber having a core-shell configuration, said fiber
comprising a non-biodegradable polymer, wherein the pores on the
fiber are configured to encapsulate and thereby retain a biological
material therein.
35. The porous fiber of claim 34, wherein the fiber has a generally
longitudinal shape.
36. The porous fiber of claim 34, wherein said biological material
is dispersed throughout the length of the longitudinal fiber.
37. The porous fiber of claim 34, wherein the porosity of the
fibers is in the range of 5% to 90%.
38. The porous fiber of claim 34, wherein the pores of said fiber
have a pore size in the range of 1 nm to 1000 nm.
39. The porous fiber of claim 34, wherein said biological material
is selected from the group consisting of bacteria, viruses, algae,
fungi, cells and yeast.
40. The porous fiber of claim 34, wherein said biological material
is selected from the group consisting of enzymes, proteins and
nucleic acids.
41. The porous fiber of claim 40, wherein said biological material
is immobilized on a solid substrate.
42. The porous fiber of claim 34, wherein said fiber is an
electrospun fiber.
43. The porous fiber of claim 34, wherein the non-biodegradable
polymer is polyvinylidene fluoride.
44. The porous fiber of claim 34, wherein the polymer has a tensile
strength of at least 20 MPa.
45. The porous fiber of claim 34, wherein the polymer has a tensile
strength of at least 50 MPa.
46. The porous fiber of claim 34, wherein the polymer has a tensile
modulus of at least 400 MPa.
47. The porous fiber of claim 34, wherein the polymer has a tensile
modulus of at least 1700 MPa.
48. The porous fiber of claim 34, wherein said biological material
is a particle having a particle size in the nano-sized range or
micron-sized range.
49. The porous fiber of claim 34, wherein said biological material
is selected to have a bioremediation activity.
50. A method of forming a porous fiber having a core-shell
configuration, said fiber comprising a non-biodegradable polymer,
wherein the pores on the fiber are configured to encapsulate and
thereby retain a biological material comprising the steps of: a.
providing a core solution of the biological material; b. providing
a mixture comprising a fiber material comprised of the
non-biodegradable polymer and a pore-forming material as the shell
solution, wherein the fiber material and the pore forming material
are miscible with each other; c. forming a core-shell fiber from
said core solution and shell solution, said fiber encapsulating and
retaining said biological material in the core; and d. removing
said pore-forming material from said formed core-shell fiber to
create pores therein.
51. The method of claim 50, wherein said forming step comprises the
step of electrospinning said core solution and shell solution onto
a collector.
52. The method of claim 50, wherein said pore-forming material is a
solvent having a boiling point in the range of 30.degree. C. to
200.degree. C.
53. The method of claim 52, wherein said removing step comprises
the step of evaporating said solvent from said core-shell fiber to
form said porous fiber.
Description
TECHNICAL FIELD
[0001] The present invention generally relates to a porous fiber.
The present invention also relates to a method of making the porous
fiber and uses thereof.
BACKGROUND
[0002] Biological materials such as bacteria, virus, enzyme, algae,
cell, yeast, protein or DNA have been utilized in a wide range of
commercial applications. For example, biological material such as
bacteria and yeast are used in water, air and soil treatments for
the biodegradation and removal of organic compounds and other
contaminants such as metals and nitrates. However, the degradation
of the contaminants in the environment depends on the type of
biological material and its quantity present. When either of these
is absent, the contaminant is not efficiently degraded and removed
from the environment. This is extremely disadvantageous, in
particular, in the treatment of air and water pollution because
speedy removal of the contaminants is desired in order to reduce
the harmful impact of these contaminants on the environment.
[0003] Membrane processes are also widely used in removing
contaminants from gas and liquid streams. Membranes are used to
remove the contaminants by physical and chemical means. These
membranes have been made from polymeric materials, inorganic
materials or a combination of the two. A combination of biological
material and membranes in a reactor, or a membrane bioreactor, is a
well known device that is utilized in a wide range of commercial
applications.
[0004] However, a conventional bioreactor requires a start-up time
for the specific biological material to grow before stable removal
of the contaminant can be achieved. This start-up time can range
from days to months for some contaminant removal processes.
Furthermore, if the specific biological material that carries out
the degradation of the contaminant is absent, it can undermine the
treatment process completely. Secondly, membrane fouling is a
serious problem in all membrane processes and this is also
encountered in membrane bioreactors. Membrane fouling can
significantly reduce the performance of the membrane process and
increase energy and operating costs.
[0005] A biological material such as protein, nucleic acids, cell
or enzyme can also be used as a biosensor in which changes in an
environmental system or sample can be detected by the biological
material. A known biosensor comprises an encapsulated electrospun
nanofiber. Electrospinning is a known technique for forming
nanofibers. In order to exploit the properties of the biological
material, the biological material is encapsulated in a nanofiber.
Encapsulation occurs when the biological material is electrospun
together with the polymer forming the nanofiber. The formed
nanofiber retains the biological material in the core of the
nanofiber and does not allow interaction between the biological
material and an external environment.
[0006] Encapsulated nanofibers have not been used before in
membrane filtration processes.
[0007] There is a need to create improved membranes with biological
materials encapsulated within, that overcomes, or at least
ameliorates, one or more of the disadvantages described above.
SUMMARY
[0008] According to a first aspect, there is provided a porous
fiber having a core-shell configuration, wherein the pores on the
fiber are configured to encapsulate and thereby retain a biological
material.
[0009] The pores may be formed along the length of the fiber.
Advantageously, the pores may allow the encapsulated biological
material to be exposed to an external environment. The pores of the
fiber may be dimensioned such that they do not allow the escape of
the biological material from the fiber and at the same time,
increase the contact between the biological material and the
external environment.
[0010] In a conventional (non-porous) fiber, only the exposed ends
of the fiber allow interaction of the encapsulated biological
material with the external environment. However, in the disclosed
porous fiber, the encapsulated biological material may interact
with the external environment not only though the exposed ends, but
also along the length of the fiber via the pores. Accordingly, the
disclosed porous fiber increases the contact surface area between
the encapsulated biological material and the environment. This
increased contact area may aid in decreasing the reaction time when
the encapsulated biological material acts on a target substrate in
the external environment.
[0011] According to a second aspect, there is provided a method of
forming a porous fiber having a core-shell configuration, wherein
the pores on the fiber are configured to encapsulate and thereby
retain a biological material, comprising the steps of:
providing a core solution of the biological material; providing a
mixture comprising a fiber material and a pore-forming material as
the shell solution, wherein the fiber material and the pore forming
material are miscible with each other; forming a core-shell fiber
from said core solution and shell solution, said fiber
encapsulating and retaining said biological material in the core;
and removing said pore-forming material from said formed core-shell
fiber to create pores therein.
[0012] According to a third aspect, there is provided use of a
porous fiber having a core-shell configuration, wherein the pores
on the fiber are configured to encapsulate and thereby retain a
biological material, in the removal of contaminants from an
environmental sample.
[0013] According to a fourth aspect, there is provided use of a
porous fiber having a core-shell configuration, wherein the pores
on the fiber are configured to encapsulate and thereby retain a
biological material, as a biosensor.
[0014] According to a fifth aspect, there is provided a filtration
membrane comprising a plurality of porous fibers, each porous fiber
having a core-shell configuration, wherein the pores on the fiber
are configured to encapsulate and thereby retain a biological
material.
[0015] According to a sixth aspect, there is provided a bioreactor
comprising a membrane module, wherein said membrane module
comprises a plurality of porous fibers, each porous fiber having a
core-shell configuration, wherein the pores on the fiber are
configured to encapsulate and thereby retain a biological
material.
[0016] According to a seventh aspect, there is provided a membrane
contactor comprising a membrane module, wherein said membrane
module comprises a plurality of porous fibers, each porous fiber
having a core-shell configuration, wherein the pores on the fiber
are configured to encapsulate and thereby retain a biological
material.
DEFINITIONS
[0017] The following words and terms used herein shall have the
meaning indicated:
[0018] The term `biological material` is to be interpreted broadly
to include any substance derived or obtained from a living
organism. Examples of such biological material include, but are not
limited to, bacteria, viruses, enzymes, algae, cells, yeast,
proteins and nucleic acids.
[0019] The term "nano-sized", when referring to a nanofiber, is to
be interpreted broadly to relate to an average diameter of the
nanofiber as being less than about 1000 nm, less than about 500 nm,
or less than about 100 nm. After encapsulating a biological
material therein, the diameter of the nanofiber may be more than
about 200 nm. When the term "nano-sized" is used in relation to a
particle size, this term relates to an average particle size of the
particle of being less than about 1,000 nm. The particle size may
refer to the diameter of the particles where they are substantially
spherical. The particles may be non-spherical and the particle size
range may refer to the equivalent diameter of the particles
relative to spherical particles.
[0020] The term "micro-sized", when referring to a microfiber, is
to be interpreted broadly to relate to an average diameter of the
microfiber as being more than about 1 .mu.m, until about 10 .mu.m.
When the term "micro-sized" is used in relation to a particle size,
this term relates to an average particle size of the particle of
being between about 1 .mu.m to about 10 .mu.m. The particle size
may refer to the diameter of the particles where they are
substantially spherical. The particles may be non-spherical and the
particle size range may refer to the equivalent diameter of the
particles relative to spherical particles.
[0021] The term "nanofiber" is to be interpreted broadly to refer
to a fiber which has a diameter in the nano-sized range.
[0022] The term "microfiber" is to be interpreted broadly to refer
to a fiber which has a diameter in the micro-sized range.
[0023] The term "bioremediation" is to be interpreted broadly to
refer to a managed or spontaneous process in which microbiological
processes are used to degrade or transform contaminants to less
toxic or non-toxic forms, thereby remedying, eliminating or
removing environmental contamination. The term "bioremediation" is
also applicable to the treatment of an environmental system or
sample which contains contaminants that are not naturally present
in that system or sample such as heavy metals or chlorinated
compounds. Bioremediation may be carried out until the contaminant
is no longer detectable in the contaminated system or sample or is
reduced to an acceptable amount or concentration in the system or
sample.
[0024] The term "contaminant", and grammatical variants thereof, is
to be interpreted broadly to refer to any substance that imparts an
undesirable, but not necessarily toxic, effect on the environment
system or sample. For example, the contaminant may include, but is
not limited to, organic materials such as aliphatic hydrocarbon
compounds, aromatic-containing compounds and chlorinated compounds
as well as inorganic materials such as metals and nitrates.
[0025] The word "substantially" does not exclude "completely" e.g.
a composition which is "substantially free" from Y may be
completely free from Y. Where necessary, the word "substantially"
may be omitted from the definition of the invention.
[0026] Unless specified otherwise, the terms "comprising" and
"comprise", and grammatical variants thereof, are intended to
represent "open" or "inclusive" language such that they include
recited elements but also permit inclusion of additional, unrecited
elements.
[0027] As used herein, the term "about", in the context of
concentrations of components of the formulations, typically means
+/-5% of the stated value, more typically +/-4% of the stated
value, more typically +/-3% of the stated value, more typically,
+/-2% of the stated value, even more typically +/-1% of the stated
value, and even more typically +/-0.5% of the stated value.
[0028] Throughout this disclosure, certain embodiments may be
disclosed in a range format. It should be understood that the
description in range format is merely for convenience and brevity
and should not be construed as an inflexible limitation on the
scope of the disclosed ranges. Accordingly, the description of a
range should be considered to have specifically disclosed all the
possible sub-ranges as well as individual numerical values within
that range. For example, description of a range such as from 1 to 6
should be considered to have specifically disclosed sub-ranges such
as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6,
from 3 to 6 etc., as well as individual numbers within that range,
for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the
breadth of the range.
[0029] Certain embodiments may also be described broadly and
generically herein. Each of the narrower species and subgeneric
groupings falling within the generic disclosure also form part of
the disclosure. This includes the generic description of the
embodiments with a proviso or negative limitation removing any
subject matter from the genus, regardless of whether or not the
excised material is specifically recited herein.
DETAILED DISCLOSURE OF EMBODIMENTS
[0030] Exemplary, non-limiting embodiments of a porous fiber having
a core-shell configuration, wherein the pores on the fiber are
configured to encapsulate and thereby retain a biological material
therein, a method of making the porous fiber and uses thereof will
now be disclosed.
[0031] In the porous fiber, a plurality of pores may be present
along the length of the fiber. The pores may be present on the
surface of the fiber. The pores may extend into the fiber from the
surface of the fiber such that a three-dimensional network of the
pores may be envisaged.
[0032] The porosity of the fiber, which is defined as the ratio of
the volume of the pores in the fiber to the total volume of the
fiber, may be in the range selected from the group consisting of
about 5% to about 90%, about 10% to about 90%, about 20% to about
90%, about 30% to about 90%, about 40% to about 90%, about 50% to
about 90%, about 60% to about 90%, about 70% to about 90%, about
80% to about 90%, about 5% to about 80%, about 5% to about 70%,
about 5% to about 60%, about 5% to about 50%, about 5% to about
40%, about 5% to about 30%, about 5% to about 20%, about 5% to
about 10% and about 30% to about 60%.
[0033] In one embodiment, the porous fiber has a generally
longitudinal shape.
[0034] In one embodiment the biological material is dispersed
throughout the length of the longitudinal fiber.
[0035] In another embodiment greater than 70%, greater than 75%,
greater than 80%, greater than 85%, greater than 90%, greater than
95%, greater than 99% of the porous fibers comprise uniformly
distributed pores along the length of the fiber. This is
advantageous as the efficiency of the porous fiber is increased.
For example in membrane applications, the high percentage of pores
present in the fiber as disclosed herein results in an increase in
the number of channels that can permit the interaction of the
encapsulated biological material with the external environment of
the fiber, whilst retaining the biological material within the core
of the fiber. In one embodiment the percentage of fibers having
pores is in the range of about 5% to 90%.
[0036] The average pore size of the pores may be in the range of
about 1 nm to about 1000 nm, about 10 nm to about 1000 nm, about
100 nm to about 1000 nm, about 200 nm to about 1000 nm, about 300
nm to about 1000 nm, about 400 nm to about 1000 nm, about 500 nm to
about 1000 nm, about 600 nm to about 1000 nm, about 700 nm to about
1000 nm, about 800 nm to about 1000 nm, about 900 nm to about 1000
nm, about 1 nm to about 900 nm, about 1 nm to about 800 nm, about 1
nm to about 700 nm, about 1 nm to about 600 nm, about 1 nm to about
500 nm, about 1 nm to about 400 nm, about 1 nm to about 300 nm,
about 1 nm to about 200 nm, about 1 nm to about 100 nm, about 1 nm
to about 10 nm and about 10 nm to about 300 nm. The pores may be
substantially uniformly distributed in and along the length of the
fiber.
[0037] Depending on the size of the biological material to be
encapsulated, the average pore size of the pores may be adjusted
accordingly. The pore size should not be too small to prevent the
movement of the contaminants in and out of the fiber. However, the
pore size should not be too large such that the encapsulated
biological material can escape easily from the fiber. The
biological material may be a particle having a particle size in the
nano-sized range or in the micron-sized range. Hence, the
biological material particle may have a diameter or equivalent
diameter in the range of about 5 nm to about 10,000 nm or about 20
nm to about 5,000 nm. The pore size of the fiber may be adjusted by
adjusting the amount of humidity of the surrounding air during the
electrospinning process, the amount, molecular weight or size of
the pore-forming material added to the electrospinning mixture, the
molecular weight of the polymer making up the fiber. The pore size
of the fibers may also be altered by varying the conditions of the
electrospinning process such as voltage, tip to collector distance,
flow rate, temperature etc.
[0038] The porous fiber may be an electrospun fiber. The diameter
of the fiber may be in the range of about 10 nm to about 1000 nm,
forming a nanofiber, or the diameter of the fiber may be in the
range of about 1000 nm to 10 .mu.m, forming a microfiber.
[0039] The porous fiber may have a core-shell structure or may have
multiple cores and shells made from various materials. The pores
may be present in the core(s) and/or shell(s). The diameter of the
multiple core(s) may be in the range of about 1 nm to about 9000 nm
or about 10 nm to about 5000 nm. The thickness of the shell wall
may be in the range of about 1 nm to about 5000 nm. The biological
material(s) may be disposed in the core(s) or may be disposed in
the shell(s). Where two or more biological materials are
encapsulated, each core and shell structure may have a different
biological material therein. Advantageously, this may allow the
co-existence of two biological materials that cannot be placed
together or that need to be separated from each other. For example,
a bacteria and an enzyme that perform different functions but have
an antagonistic effect on each other when placed together can be
separated using the core-shell configuration. Further, in
bioremediation, a number of bacteria species can be used. Usually,
there are antagonistic bacteria that perform a function and other
bacteria that prevent them from performing their functions but
which also carry out a useful function. Hence, by separating these
bacteria using the core-shell configuration, this can ensure that
these bacteria are separated by a physical barrier and are not
impeded from carrying out their individual functions. The shell(s)
may act as a support for the core(s). The shell(s) may include
nutrients for the biological material(s). The shell(s) may also act
as a physical barrier to shelter the biological material present in
the core(s) from attack by harmful predatory bacteria and higher
organisms.
[0040] The biological material may be selected from the group
consisting of bacteria, viruses, algae, fungi, enzymes, cells,
proteins and nucleic acids.
[0041] When the porous fiber is used in bioremediation, the
biological material may be a bacteria, enzyme, yeast, algae or a
fungus. The bacteria, enzyme, yeast, algae or fungus may be chosen
for their bioremediation properties in which they may be able to
degrade contaminants that are present in the environment. These
bacteria, enzyme, yeast, algae or fungus may be used in water, air
and/or soil treatments for the biodegradation and removal of waste
organic materials and other contaminants. For example, the
bacteria, enzyme or fungus may be used during oil spills where the
bacteria, enzyme or fungus is able to act on the oil and degrades
it such that the oil contaminant no longer exerts a harmful effect
on the environment. In another example, the bacteria or fungus may
be able to degrade and reduce the level of heavy metals,
chlorinated compounds, nitrates or other contaminating organic
compounds in the environment. Yeast such as Yarrowia lipolytica,
may be used to degrade palm oil mill effluent and other
hydrocarbons such as alkanes, fatty acids, fats and oils. Algae may
be used to remove heavy metals and other ionic compounds from water
such as nitrogen and phosphorus containing compounds.
[0042] The biological material may also be used in industries such
as fermentation, catalysis of chemical reactions (in which enzymes
are the common biological materials to use here), food processing
(in which enzymes are the common biological materials to use here),
paper industries, biofuel industries, medical industries (such as
the use of encapsulated viruses as bacteriophages in phage
therapy).
[0043] The bacteria may include, but is not limited to, Bacillus
Lichenifonnis, Bacillus Subtilis, Bacillus Amyloliquiefaciens,
Bacillus Polymyxa, Bacillus lentus, Bacillus megaterium, Bacillus
pumilus, Bacillus cereus, Bacillus sphaericus, Bacillus
licheniformis, Acinetobacter haemolyticus, Acinetobacter baumannii,
Brevibacillus brevis, Listeria seeligeri, Alcaligenes faecalis type
II, Escherichia Hermanii, Bacillus Cereus, Bacillus Thuringiensis,
Bacillus Meg'afarium, Corynebacterium, Brevibacterium Job 5,
Alcaligines entrophus, Pseudomonas Aeruginosa, Pseudomononas
Statzeri, Pseudomonas Fluoresceni, Pseudomonas Oleovorans,
Pseudomonas putida, Pseudomonas desmolyticum, Pseudomonas
methanica, Micrococcus paraffinae, Acetobacter peroxydans,
Mycobacterium smegmatis, Mycobacterium thodochrous, Achromobacter
agile, Achromobacter centropunctatum, Arthrobacter, Bacillus
hexacarbovorum, Nocardia opacus, Nocardia corrallina, Actinomyces
oligocarbophilus, Desulfovibrio desulfuricans, Alzaligenes
eutrophus, Nitromonas sp. and Rhodopseudomonas palustris. The
concentration of the bacteria is dependent on the amount of
contaminant to be degraded.
[0044] The algae may be both freshwater and marine algal species
from the major classes of cyanobacteria, rhodophyta, chlorophyta,
dinophyta, chrysophyta, prymnesiophyta, bacillariophyta,
xanthophyta, eustigmatophyta, rhaphidophyta, phaeophyta. Specific
examples include Chlorella sp, Scendesmus sp, Chlorococcum sp,
Ulothrix sp, Pseudanabaena sp or Haematococcus sp.
[0045] Exemplary enzymes that are used in bioremediation include,
but are not limited to, mono-oxygenases, di-oxygenases, reductases,
dehalogenases, cytochrome P450 monoxygenases, enzymes involved in
lignin metabolism e.g. laccases, lignin-peroxidases,
manganese-peroxidases. Bacterial phosphotriesterases. Other
examples could also be molecularly engineered enzymes that are
being produced.
[0046] The fungus may include, but is not limited to, Candida
lipolytica, Candida tropicalis, Candida utilis, Candida
parasilosis, Candida guilliermondii, Candida rugosa, Trichosporan
cutaneum, Aureobasidiuin pullulans, Pichia spartinae, Pichia
Saitoi, Torulopsis magnoliac, Torulopsis gropengiesseri,
Trichoderma harzianum, Aspergillus versicolor, Penicillium sp and
Graphium ulni. The yeast may be Yarrowia lipolytica. The
concentration of the fungus or yeast is dependent on the amount of
contaminant to be degraded.
[0047] The contaminant may be an organic compound such as aliphatic
hydrocarbons (for example, straight or branched chain hydrocarbons
having a chain length ranging from about C.sub.5-C.sub.36 or ring
structures of saturated C.sub.5-C.sub.36 hydrocarbons) and aromatic
hydrocarbons (for example, at least one unsaturated ring structure
ranging from C.sub.9-C.sub.22 hydrocarbons). Examples of such
contaminants include, but are not limited to, dioxins, tars,
creosote, crude oil, refined oil, fuel oils (for example, Nos. 2,
4, and 6 fuel oils), diesel oils, gasoline, hydraulic oils,
kerosene, chrysene, cresol, cyclohexanone, ethylbenzene,
butylbenzene, ethyl acetate, fluorine, isoprenoids, methyl
ethylacetate, 2-butanone, methyl pentanone, methyl propylacetate,
butylacetate, petroleum oils and greases, phenanthrene,
trimethylbenzene, phenol, benzene, toluene, ethylbenzene, xylene,
Stoddard solvents, mineral spirits, naphthalene, anthracene,
acenaphthene, acenaphthylene, benzo(a)anthracene, benzo(a)pyrene,
benzo(b) fluoranthene, benzo(g,h,i)perylene, benzo(k)fluoranthene,
pyrene, terpene-based compounds, phthalates such as
bis(2)ethylhexylphthalate and/or dioctylphthalate, and/or phenolic
compounds.
[0048] The contaminant may be a halogenated aliphatic or aromatic
compound such as a chlorinated aliphatic or chlorinated aromatic
compound. Specific chlorinated hydrocarbon contaminants include,
but are not limited to, polychlorinated biphenyls, aldrin,
trichloroethylene, tetrachloroethylene, 1,2-dichloroethane, carbon
tetrachloride, chlorobenzene, chlorotoluenes, dichlorobenzene,
dichloroethanes, dichloroethylene, dichlorotoluene,
tetrachloroethane, trichloroethane, pentachlorophenol and vinyl
chloride, etc.
[0049] The contaminant may be a nitrogen containing compound such
as, but not limited to, an organic nitrogen containing compound and
an inorganic containing compound. The organic nitrogen containing
compound may be ammonia, ammonium or other materials that
contribute to the Total Kjeldahl Nitrogen in waters. The inorganic
containing compound may be Group Ia and Group IIa metal nitrates or
nitrites. The nitrate compound may be lithium nitrate, sodium
nitrate, potassium nitrate, magnesium nitrate, calcium nitrate or
ammonium nitrate. These nitrogen containing contaminants may be
removed by microalgae, nitrification and denitrification bacteria
encapsulated within the fibers.
[0050] The contaminant may be phosphorous containing compound, but
not limited to, orthophospates, polyphosphates, and organically
bound phosphates.
[0051] The contaminant may be a metal such as, but not limited to,
a transition metal, a rare earth metal, a metallic element from
Group IIIa, IVa, Va and VIa of the Periodic Table of Elements. The
metal may be, but is not limited to, cadmium, zinc, cobalt, copper,
lead, mercury, thallium, chromium and manganese in the form of
salts, either in a soluble or non-soluble state.
[0052] During bioremediation, the nanofibers or microfibers may be
collected on a membrane support to form a membrane. The membrane
support may be a polyester nonwoven support. The nanofibers or
microfibers may be collected for a sufficient period of time until
a desired membrane thickness is obtained. The thickness of the
membrane may be the same as the diameter of the nanofiber or the
microfiber or may be up to until about 300 microns. If a thicker
membrane is desired, individual layers can be stacked together. The
membrane can be prepared as individual layers or stacked nanofiber
or microfiber layers on a support or may be stacked between two
substrate materials. The substrate material may be any polymeric or
inorganic material as is known to a person skilled in the art and
may comprise openings for introducing a fluid to the membrane
layer. The membrane may be incorporated into modules such as flat
sheet, tubular, spiral wound, or hollow fibers as required. The
membrane may also be placed in a membrane housing to form a
bioreactor or contactor. Hence, the bioreactor may comprise a
membrane module in which the membrane module comprises a plurality
of porous fibers, each porous fiber encapsulating a biological
material therein. The membrane contactor may comprise a membrane
module whereby the membrane module comprises a plurality of porous
fibers, each porous fiber encapsulating a biological material
therein.
[0053] When used as membrane contactors or reactors, the material
to be acted upon or degraded by the biological material may be a
chemical compound. This chemical compound may be an inorganic
compound or an organic compound such as organic waste, enzymes
(that are used for pharmaceutical production, protein and DNA
purification), food product, starch, glucose, etc.
[0054] Since the bioreactor already contains a certain
concentration of encapsulated biological material such as bacteria,
yeast, algae or fungi, the bioreactor avoids the disadvantage of
long start-up time, which can range from a few days to months,
associated with a conventional bioreactor in which the bacteria,
yeast, algae and/or fungi needs time to grow until a certain
concentration is reached for contaminant removal. Accordingly, the
disclosed bioreactor can be used directly on a contaminated site in
which the encapsulated bacteria, yeast, algae and/or fungi can act
quickly on the contaminants.
[0055] Further, due to the pores present in the nanofiber or
microfiber, the pores offer a greater contact area between the
encapsulated bacteria, yeast, algae and/or fungi with the
contaminants. Hence, membrane fouling problems associated with the
build-up of contaminants on the surface of a membrane in a
conventional bioreactor is minimised in the disclosed bioreactor
because the encapsulated bacteria, yeast, algae and/or fungi can
act directly on the contaminants. As the contaminants are degraded
and hence removed, the source of the membrane fouling is thus
removed, leading to a decrease in the membrane fouling problem.
Another instance whereby the membrane fouling problem in membrane
bioreactors is reduced considerably is by the encapsulation of
materials and compounds in the membrane that are of anti-quorum
sensing nature, which may be from natural products or synthetically
produced. These will prevent the attachment of biofilms on the
membrane surface, which is a serious problem in membrane
applications worldwide. The anti-quorum sensing materials may
include, but is not limited to, enzymes from bacteria, furanone,
halogenated furanone, red alga Delisea pulchra, southern Florida
seaweeds, terrestrial plants, acylase, compounds containing lactone
and other poly-hydroxilated rings.
[0056] When the porous nanofiber or microfiber is used as a
biosensor, the biological material may be an enzyme, a cell, a
protein or nucleic acid. When the biosensor is to be used to detect
pathogenic organism in food stocks and supply chain, the biosensor
can be prepared from porous fibers with dye encapsulating liposomes
tagged with antibodies that are specific for the bacteria placed in
the core of the fibers. These fibers can be formed into fiber mats
or membranes. The mats or membranes can be in strip-form or
swab-form. In another embodiment, when the biosensor is to be used
to detect glucose and heavy metals, the biological material
encapsulated in the fiber may be glucose oxidase. In yet a further
embodiment, the biosensor may be comprised of a LuxAB-based
material in which the Lux gene is a gene that is responsible for
the light emitting reaction and which has been isolated from
bacteria. This biosensor can then be used for, but not limited to,
testing of antibiotic effectiveness, antimicrobial agents,
bacterial biofilms, bacterial biomass, bacterial stress response,
bacterial transport mechanisms, bioremediation process monitoring,
cell viable counts, circadian rhythms, DNA damaging agents,
environmental contaminants, foodborne pathogens, gene
expression/regulation, growth phase regulation, immunoassays,
industrial waste runoff, metabolic regulation, mutagenicity tests,
plant pathogens, toxicity assays, viral infection, xenobiotic
detection. In yet a further embodiment, the biosensor may be
comprised of a luxCDABE-based material that is used to detect 2,3
Dichlorophenol, 2,4,6 Trichlorphenol, 2,4-D, 3-Xylene,
4-Chlorobenzoate, 4-Nitrophenol, Aflatoxin B1, Alginate production,
Ammonia, BTEX (benzene, toluene, ehtylbenzene, xylene), cadmium,
chlorodibromomethane, chloroform, chromate, cobalt, copper,
hydrogen peroxide, iron, isopropyl benzene, lead, mercury, N-acyl
homoserine, naphthalene, nickel, nitrate, organic peroxides, PCBs,
p-chlorobenzoic acid, p-cymene, pentachlorophenol, phenol,
salicylate tetracycline, trichloroethylene, trinitrotoluene, zinc,
ultrasound, ultraviolet light, heat and gamma radiation.
[0057] The biological material may be a virus. The virus may
include, but is not limited to, T7, T4 bacterial viruses, Herpes
simplex, Cytomegalovirus, Papilloma virus, Adenovirus, Burkitt
lymphoma virus, Arbovirus, Arenavirus, Epstein-Barr virus,
Varicella virus, Cornavirus, Coxsackievirus, Eboli virus,
Enterovirus, Hepatitis virus, Influenza virus, Marburg virus,
Measles virus, Mumps virus, Polio virus, Rhinovirus, Rubella virus,
Smallpox virus, Rabies virus, or Rotavirus. The nanofiber or
microfiber encapsulating a virus may be used for phage therapy
(where the virus is a bacteriophage) or as a gene delivery
vector.
[0058] In one embodiment, the biological material is selected from
the group consisting of bacteria, viruses, cells and yeast.
[0059] In another embodiment, the biological material is selected
from the group consisting of enzymes, proteins and nucleic
acids.
[0060] In one embodiment, the biological material may be
immobilized on a solid substrate. This is particularly advantageous
when the biological material comprises enzymes, proteins and
nucleic acids or other small biomolecules. Accordingly, in one
embodiment, when the biological material comprises enzymes,
proteins and nucleic acids or other small biomolecules the
biomaterial may be immobilized on a solid substrate. In one
embodiment, the solid substrate may be selected from the group
consisting of gold nanoparticles, microparticles and the like. In
one embodiment, the biomaterial is covalently bonded to the solid
substrate via reactive groups, for example, --NH2, --COOH, --SH,
--CHO, --OH, peptide, carbamate linkages and the like which can
covalently bind to enzymes, proteins and nucleic acids and the
like.
[0061] In addition to the use of membrane bioreactor or membrane
contactor in bioremediation, the membrane bioreactor or membrane
contactor can also be used in the production of foodstuffs and
pharmaceuticals. For example, emulsion enzyme membrane reactors may
be used for the production of significantly high enantiomeric
excess of the (S)-naproxen acid (anti-inflammatory drugs) from
racemic mixtures of (R,S)-naproxen methyl ester in emulsion
membrane reactors where currently lipase is entrapped by physical
methods in polymeric membranes [Li; N., Giorno, L., and Drioli, E.
(2003), Effect of immobilization site and membrane materials on
multiphasic enantiocatalytic enzyme
[0062] Another use of the fiber relates to the incorporation of
quorum-quenching or quorum sensing compounds in the porous fibers
which are then formed as a membrane. This results in the prevention
or quick biofilm attachment on the membrane surface, respectively,
depending on the process required. In some water and wastewater
treatment processes, biofilm is required on the surface to carry
out degradation of the waste, for example, in biological activated
carbon or sand filtration. Here, membranes can be used as the
surface on which biological materials grow and cling to and the
water is passed through it where the contaminants are removed. In
membrane processes where water is pushed through the membrane, the
attachment of biofilm on the membrane surface is considered
detrimental as it blocks the pores and forms a cake layer. The cake
layer prevents water from flowing through the membrane, resulting
in low flux (for example, in applications such as reverse osmosis
processes, microfiltration or ultrafiltration). Here, the quorum
quenching biological materials can be encapsulated to prevent the
membrane from getting fouled with biofilm.
[0063] The porous fiber may be an electrospun fiber. The
electrospinning method will be discussed in detail further
below.
[0064] The fiber may be formed from an electrospinnable polymer
selected from the group consisting of a polyamide, a polyimide, a
polycarbamide, a polyolefin, a polyurethane, a polyester, a
polycarbonate, a polyaniline, a polysulfone, a polyacrylonitrile, a
polycarbonate, a polyanhydride, a polyorthoester, a
poly(acrylonitrile), a polybenzimidazole, a poly(siloxane), a
polysilicone, a polycaprolactone, a polyhydroxyalkanoate,
cellulose, copolymers, terpolymers and blends thereof.
[0065] The polymer may be selected from the group consisting of
poly(vinylidene fluoride), poly(vinylidene
fluoride-co-hexafluoropropylene), polyvinyl pyrrolidone,
poly(N-vinyl pyrrolidone), polymethyl methacrylate, polyacrylic
acid, polyvinyl acetate; polyacrylamide, polyethylene, cellulose
acetate, cellulose acetate butylate, polyvinyl pyrrolidone-vinyl
acetate, poly(2-hydroxy ethyl methacrylate), polyethyleneimide,
polyethersulfone, polystyrene, nylon, nylon12, nylon-6, nylon-6,6,
nylon-4,6, aramid, polydimethylsiloxane, polyvinylchloride,
poly(L-lactic acid acid-caprolactone), poly-L-lactic acid,
polyvinylalcohol, polyethyleneimine, polyethylene oxide,
poly(ethylene terephthalate), polyp-phenylene terephthalamide),
polytrimethylane terephthalate, poly(hydroxyl butyrate), polyester
urethane, polyether urethane, poly(propyleneoxide),
poly(ethylene-co-vinyl acetate), poly(ethylene glycol),
poly(methacrylic acid), polyglycolide, poly(lactide-co-glycolide),
polyanhydride, polyvinyl carbazole, polyvinyl phenol, polystyrene,
polyhydroxyacids, polysulfones, polytetrafluoroethylene,
polyacrylonitrile, polystyrene, copolymers, terpolymers and blends
thereof.
[0066] In one embodiment, the fiber may be formed from chitosan,
collagen or gelatin.
[0067] In an embodiment where the porous fiber has a core-shell
structure, the above polymer may be independently selected as the
material for the core and/or shell of the porous fiber. The same
polymer may be used for both the core and the shell structures. In
another embodiment, different polymers may be used for the core(s)
and shell(s) structures.
[0068] In one embodiment the polymer has a tensile strength
selected from the group consisting of at least 10 MPa, at least 15
MPa, at least 20 MPa, at least 25 MPa, at least 30 MPa, at least 45
MPa, at least 50 MPa and at least 60 MPa. In one embodiment the
polymer has a tensile strength in the range selected from the group
consisting of from about 10-60 MPa, about 15-60 MPa, about 20-60
MPa, about 25-60 MPa, about 30-60 MPa and about 35-60 MPa, about
40-60 MPa, about 45-60 MPa, about 50-60 MPa and about 55-60 MPa. In
one embodiment, the polymer has a tensile strength of about 52
MPa.
[0069] In one embodiment the polymer has a tensile modulus selected
from the group consisting of at least 400 MPa, at least 450 MPa, at
least 500 MPa, at least 550 MPa, at least 600 MPa, at least 650
MPa, at least 700 MPa, at least 750 MPa, at least 800 MPa, at least
850 MPa, at least 900 MPa, at least 950 MPa, at least 1000 MPa, at
least 1100 MPa, at least 1200 MPa, at least 1300 MPa, at least 1400
MPa, at least 1500 MPa, at least 1600 MPa, at least 1700 MPa. In
one embodiment the polymer has a tensile modulus in the range
selected from the group consisting of about 1000-1800 MPa, about
1100-1800 MPa, about 1200-1800 MPa, about 1300-1800 MPa, about
1400-1800 MPa, about 1500-1800 MPa, about 1600-1800 MPa and about
1700-1800 MPa. In one embodiment, the polymer has a tensile modulus
of from about 1723 MPa. In one embodiment, the polymer is
poly(vinylidene fluoride) (PVDF). In one embodiment, the polymer is
a non-biodegradable polymer.
[0070] The use of PVDF is advantageous as this polymer is
mechanically strong and as it is non-biodegradable it is ideal
candidate for applications such as waste water treatment. In
comparison, for example, fibers made of the polymer
polycaprolactone (PCL), which is a biodegradable polymer, are
significantly weaker than fibers made from PVDF. Additionally,
biodegradable polymers, such as PCL, are susceptible to degradation
by the action of aerobic and anaerobic microorganisms that are
widely distributed in various ecosystems (Tokiwa, 2009). As such,
biodegradable polymers can be easily degraded by lipases and
esterases and thus are not suitable for use with microorganisms
such as bacteria or viruses.
[0071] The method of forming the porous fiber having a core-shell
configuration, wherein the pores on the fiber are configured to
encapsulate and thereby retain a biological material may comprise
the steps of: providing a core solution of the biological material;
providing a mixture comprising a fiber material and a pore-forming
material as the shell solution, wherein the fiber material and the
pore forming material are miscible with each other; forming a
core-shell fiber from said core solution and shell solution, said
fiber encapsulating and retaining said biological material in the
core; and removing said pore-forming material from said formed
core-shell fiber to create pores therein.
[0072] The fiber may be formed using an electrospinning step.
Electrospinning is a known method in the art and typically involves
an electric charge to pull a liquid jet of a viscous solution from
the tip of a spinneret. The electrospinning technique may employ a
core-shell or coaxial electrospinning technique, or an equivalent
method known in the art, in which two solutions, one of which forms
the inner core and the other forms the outer shell. A spinneret is
used to contain the core or shell solution. The spinneret can be
further modified to contain several cores or several shells of
varying materials and properties.
[0073] During electrospinning, an electric field is applied to the
droplet by connecting a first electrode to the tip of the spinneret
which is made from a conducting material. A counter-electrode is
placed at a selected distance from the tip of the spinneret and a
high voltage current of from 1 to 30 kV is applied. The electrode
may be formed from any suitable material, such as copper or any
other conducting material. The distance between the tip of the
spinneret and the counter-electrode, known as the tip to collector
distance, is kept between 5 and 30 cm. The spinneret may be
stationary or moveable in all directions as required for the
deposition of the fibers.
[0074] When the electrostatic field applied is greater than the
surface tension of the viscous solution, a nanofiber jet is emitted
from the tip of the spinneret. Electrostatic forces associated with
mutual Coulombic interactions at different sections of the jet make
it unstable when subjected to bending perturbations. The bending
instability rearranges the jet into a sequence of connected loops,
which becomes unstable and forms secondary and tertiary loops,
leading to the stretching of the fibers to form a smaller
fiber.
[0075] A collector is placed under the tip of the spinneret. The
collector may comprise, for example, a rotating collector drum or a
moveable plate. The collector drum or plate may be formed of any
suitable material, such as aluminium, zinc, or any other conducting
material. As mentioned above, a membrane support material may be
used as the collector.
[0076] The core solution may be a mixture comprising a fiber
material, a pore-forming material and a biological material. The
core solution may be introduced into the core chamber of the
spinneret. The shell solution may comprise the same fiber material
as the core or may comprise a different fiber material compared to
that in the core. The shell solution may also comprise an inorganic
material. The shell solution may comprise a pore-forming material.
Hence, the pore-forming material may be present in one of the core
solution and shell solution or be in both the core and shell
solutions. The fiber material in the core and/or shell may be an
electrospinnable polymer as mentioned above.
[0077] The biological material may be suspended in an aqueous or
non-aqueous buffer such as a phosphate buffer solution.
[0078] The core solution may further comprise an osmolarity
regulating agent selected from the group consisting of glycerol;
glycol; polyethylene glycol; sugar such as sucrose, glucose,
fructose, lactose, etc; sugar-alcohol such as mannitol, inositol,
xylitol, and adonitol; amino acids such as glycine and arginine;
biological polymeric molecules and proteins such as albumin; as
well as Ficoll.RTM..
[0079] The core solution may further comprise additives to increase
or maintain the viability of the biological materials. The additive
may be a nutrient selected from one or more of the following
carbohydrates (such as glucose, fructose, maltose, sucrose, and
starch); other carbon sources (such as mannitol, sorbitol and
glycerol); nitrogen sources (such as urea, ammonium salts, amino
acids or crude proteins, yeast extract, peptone, casein
hydrolysates and rice bran extracts); and inorganic compounds (such
as magnesium sulfate, sodium phosphate, potassium phosphate, sodium
chloride, calcium chloride and ammonium nitrate). The core solution
may further comprise additives to increase the activity of the
biological material such as vitamins or nutrients.
[0080] The shell solution may comprise an electrospinnable polymer
as mentioned above. The shell solution may further comprise a
ceramic precursor. The shell solution may comprise the osmolarity
regulating agents and/or additives as mentioned above. The shell
solution may include inorganic material such as metallic or
metallic oxide nano-materials that increase the strength of the
fibers or increase the likelihood of interaction between the
contaminants in the water, air or solid waste to the fibers so that
they can be treated or interact better with the entrapped
biological materials.
[0081] The electrospinnable polymer may be dissolved in a suitable
solvent for the polymer. The solvent for the shell solution is
selected to have low solubility with the solvent used in the core
solution. The solvent in the core solution may be water, ethanol,
or other solvents that are somewhat incompatible with the solvents
used in the shell. The core solvent should be carefully chosen
because if it is not compatible with the polymer in the shell
solution, clogging of the needle during electrospinning may occur,
which require cleaning of the needle tip by wiping it with a cloth
or string. It is to be appreciated that a person skilled in the art
would know the type of solvent that is suitable for the core and
shell solution. A suitable solvent for the shell solution may
include, but is not limited to, hexane, cyclohexane,
dimethylformamide, acetone, acetonitrile, hexafluoroisopropanol,
dimethylacetamide, formic acid, ethanol, methanol, toluene,
m-Cresol and trifluoroethanol. The solvent may be a blend or a
combination of solvents.
[0082] In one embodiment, the boiling point of the solvent may be
in the range of about 30.degree. C. to about 200.degree. C. In
another embodiment, the pore-forming material may be a low boiling
point solvent. The boiling point of the solvent may be in the range
of about 30.degree. C. to about 80.degree. C. The low boiling point
solvent may include, but is not limited to, chloroform, acetone,
ethyl ether, benzene, cyclohexane, toluene, dimethylformamide,
tetrahydrofuran, hexafluoro-2-propanol, trifluoroethanol and other
suitable solvents that have a boiling point that falls within the
above range and can be electrospun. Due to its low boiling point,
the solvent evaporates from the formed fiber, resulting in pores
being formed in the fiber. If the biological material used is one
that can withstand a high temperature, it may be possible to apply
heat to accelerate the evaporation of the low boiling point solvent
from the fiber and to control the pore structure. Another method to
form pores in the fiber is to soak the fibers after preparation
into a non-solvent of the shell polymer, for example, water which
has a higher density than that of the solvent used in the shell.
This will cause phase separation and the formation of pores on the
shell of the fiber. A further method to remove the low boiling
point solvent is to increase the relative humidity of the
surrounding air. For example, when the low boiling point solvent
used is one of toluene, hexafluoro-2-propanol, trifluoroethanol or
dimethylformamide (that has a high vapour pressure) that is
electrospun with hydrophobic polymers such as polystyrene,
polyvinyl chloride or poly(methyl methacrylate) in the shell, pores
are formed when the relative humidity of the surrounding air is
higher than 30%.
[0083] The pore-forming material may be a polymer that is soluble
in a solvent, for example an aqueous medium. After the fiber is
formed, the fiber may be placed in a solvent such as water to allow
the polymer to dissolve. As the polymer dissolves, pores are formed
in the core-shell fiber. The polymer may include, but is not
limited to polyacrylamide, polyvinyl alcohol, polyacrylic acid,
poly (ethylene oxide), methyl cellulose, hydroxyethyl cellulose,
carboxymethyl cellulose, poly(allyl) amine, poly(diallyl dimethyl
ammonium chloride), poly(diallyl methyl amine hydrochloride),
polymethacrylic acid, sodium polyacrylate, polyvinylbenzyl
trimethylammonium chloride, poly(sodium-2-sulfoethylacrylate),
polyvinylbenzyl sodium sulfonate, poly(sodium styrene sulfonate),
polystyrene sulfonate, poly(dimethylaminoethyl methacrylate),
poly[(methacrylamido)propyltrimethylammonium chloride],
polyethylene glycol and poly(acrylonitrile).
[0084] The pore-forming material may include an inorganic, water
soluble material such as water soluble salts, sugars,
nanoparticles, nanomaterials and crystals which can be incorporated
in the shell matrix and removed by extended soaking in water. The
water soluble salt may be a water soluble metallic salt such as
Iron Salts, Lithium Salts, Calcium Salts, Sodium Salts, Magnesium
Salts, Crystals, nanoparticles or microparticles of these
materials, to generate larger pores.
[0085] The pore-forming material may be a sacrificial template
which can be organic or inorganic in nature. The sacrificial
template is added together with the biological material,
electrospun and subjected to an additional step to remove the
template in a non-surfactant template technology. For example, urea
molecules may be used as a non-surfactant template or pore-forming
agent. The urea may be removed by soaking the fiber in water or
methanol. Similarly, glucose, sucrose or other larger molecular
weight water soluble materials may be used.
[0086] In another embodiment, the pore-forming agent may be a blend
of non water soluble polymers with the water soluble polymers as
mentioned above.
[0087] The pore-forming material may be a mixture of the low
boiling point solvent, the soluble polymer, the inorganic material
and sacrificial template.
[0088] The pore size of the pores may be adjusted by controlling
the concentration, molecular weight or size of the pore-forming
material, the humidity of the electrospinning chamber, the flow
rate of the electrospun solutions and the diameter of the formed
fiber. The concentration may affect the porosity of the fibers as
the higher the concentration, the greater the number of pores
and/or the bigger the pore size. The size of the pore-forming
material in the form of a water soluble material and the amount
removed will affect the pore size. The bigger the size and amount
removed would lead to bigger pores formed. If a low boiling point
solvent is used as the pore-forming material, humidity of the
electrospinning chamber is a factor that affects the formation of
the pores. For example, if the humidity is below 20%, pores are
generally not formed. Conversely, the higher the humidity, the
bigger the pore sizes. In addition, another factor that can affect
the pore size when a low boiling point solvent is used as the
pore-forming material is the diameter of the fiber. The fiber
diameter should be larger than about 500 nm for pore formation. At
smaller diameters, not many pores are visible on the fiber.
Generally, the bigger the diameter of the fiber, the more pores are
formed and the bigger the pores are. Therefore, factors that can
affect the diameter of the fibers are injection rate of the core
and shell solutions, the applied voltage, the tip to collector
distance and the polymer concentrations.
BRIEF DESCRIPTION OF DRAWINGS
[0089] The accompanying drawings illustrate a disclosed embodiment
and serves to explain the principles of the disclosed embodiment.
It is to be understood, however, that the drawings are designed for
purposes of illustration only, and not as a definition of the
limits of the invention.
[0090] FIG. 1 is a schematic diagram of the equipment used during
electrospinning.
[0091] FIG. 2 is a graph showing the percentage conversion of
glucose by the yeast encapsulated membrane after soaking over a
number of days.
[0092] FIG. 3 is a high resolution microscope image at 100.times.
magnification of a porous fiber encapsulating an algae Chlorella
Sp.
[0093] FIG. 4 is a SEM image at 13260.times. magnification showing
a porous fiber made from Polyimide
[0094] FIG. 5 is a SEM image of thin PCL nanofibers of thickness
less than 200 nm at (a) low and (b) high magnification under
SEM.
[0095] FIG. 6 is a high magnification SEM image of PCL nanofiber
using 23% PCL nanofibers showing thick nanofibers.
[0096] FIG. 7 is a SEM image of the pores produced by using
different concentrations of Porogen (a) 20 mg/ml and (b) 30
mg/ml.
[0097] FIG. 8 shows the high magnification SEM images of PCL
nanofibers without (a) and with (b) Porogen.
[0098] FIG. 9 shows the confocal images of core-shell nanofibers of
PCL with Porogen (a) overlay image (b) X Y section image.
[0099] FIG. 10 shows the SEM high magnification images of
nanofibers electrospun using 15% PVDF solution in Dimethyl
Acetamide and Acetone.
[0100] FIG. 11 shows the SEM high magnification images of rough
nanofibers electrospun using 20% PVDF solution in Dimethyl
formamide and Water (30:1)
[0101] FIG. 12 shows the SEM high magnification images of
nanofibers electrospun using PVDF+PEO (45:8): DMF+Water (30:1) 20%
ratios, showing pores on thick fibers and beads only.
[0102] FIG. 13 shows the SEM pictures of nanofibers prepared by
using A) Higher concentration (25%) of PVDF: PEO (45:8) in DMF and
Water B) High polymer:Porogen ratio (40:15)
[0103] FIG. 14 shows SEM pictures of PVDF nanofibers with various
thickness using different percentages of polymer for spinning.
[0104] FIG. 15 shows the Optical (A) and fluorescent (B) images of
nanofibers PVDF: PEO as shell and Glycerol: Concavalin A-Alexa
Fluor as core.
[0105] FIG. 16 shows the confocal images of porous core-shell
nanofibers prepared by using PVDF and Porogen.
[0106] FIG. 17 shows the SEM images showing thick highly porous
nanofibers of electro-spinned Poly(caprolactone) with THF and
DMSO.
[0107] FIG. 18 is schematic illustration of the bioconjugation of
nanoparticles with different ligands.
[0108] FIG. 19 is a schematic to show the immobilization of
biomolecules on the nano/micro particles and beads using covalent
functionalization.
DETAILED DESCRIPTION OF DRAWINGS
[0109] Referring to FIG. 1, there is provided a schematic diagram
of the equipment used during electrospinning. The coaxial or
core-shell spinneret 2 consists of an internal chamber 4 and an
external chamber 6. The core solution denoted by the dotted line 10
is pumped from a core solution holder 8 into internal chamber 4.
The shell solution denoted by the dotted line 14 is pumped from a
shell solution holder 12 into internal chamber 6. The core solution
holder 8 and shell solution holder 12 may be a pipette or a
syringe. If syringes are used, the core and shell solution are
introduced into the respective chambers in the spinneret 2 via a
syringe pump 16.
[0110] For the spinneret 2, there are no requirements for the
volume of the internal chamber 4 and external chamber 6. However,
the internal chamber 4 and external chamber 6 should be fabricated
such that there is no mixing of the core and shell solutions except
at the tip 20 of the spinneret 2. The tip 20 of the spinneret 2 may
be a pipette tip or a needle. The diameter near the tip 20 may have
an inner diameter of about 0.05 to 3 mm. The speed by which the
core solution and shell solution exit the spinneret 2 via the tip
20 is regulated by the speed of the syringe pump 16. The speed of
the core solution and shell solution can be independently
controlled so that the speed of the core and shell solution can be
different from each other.
[0111] The core solution is a mixture of the electrospinnable
polymer and the biological material. As mentioned above, the core
solution may contain additives such as an osmolarity regulating
agent or nutrients. As mentioned above, the shell solution may
include an electrospinnable polymer, optionally mixed with one of
an inorganic material and a pore-forming material.
[0112] An electrostatic field 18 is applied to the spinneret 2. The
electrostatic field 18 can be generated between a first electrode
(not shown) and a second counter-electrode (not shown). The first
electrode is inserted in the spinneret 2 and the second electrode
is positioned at a distance of about 5 to 30 cm from the first
electrode. A high voltage of about 1 to 30 kV is applied between
the first and second electrodes to generate the electrostatic field
18.
[0113] As the electrostatic field 18 is applied to the spinneret 2,
when the electrostatic field 18 applied is greater than the surface
tension of the solutions in the spinneret 2, a nanofiber jet 22 is
emitted from the tip 20 of the spinneret 2. The nanofiber jet 22 is
then propelled towards a collector 24 which can be a rotating disc.
The second electrode can either be connected to the collector 24 or
it can be the collector 24.
EXAMPLES
[0114] Non-limiting examples of the invention and a comparative
example will be further described in greater detail by reference to
specific Examples, which should not be construed as in any way
limiting the scope of the invention.
Example 1
Materials
[0115] Nylon 6/6, Polyvinyl Alcohol (MW 85-124 kDa) and
Polyethylene glycol (Mn 570-630) were purchased from Sigma Aldrich
of St. Louis of Missouri of the United States of America. Formic
acid (98-100%) and D (+) Glucose were obtained from Merck. Bacto
Peptone and Backers yeast (Saccharomyces cerevisiae) were purchased
from Becton Dickinson of Franklin Lakes of New Jersey of the United
States of America and Gim Hin Lee Pte Ltd (Singapore)
respectively.
[0116] Methods
[0117] a) Preparation of Core-Shell Nanofiber
[0118] A 15% (w/w) Nylon spinning solution was prepared by
dissolving Nylon 6/6 in a mixture of formic acid and Polyethylene
Glycol (14:3 w/w) and used as shell solution. Baker's yeast pellets
(Saccharomyces cerevisiae) was resuspended in deionized water and
centrifuged at 7500 rpm. The residue was further resuspended in 3
ml water and used as the core solution. The shell solution was
injected into the outer coaxial needle and the core solution was
delivered into the inner coaxial needle at a constant rate of 3
ml/hr and 1 ml/hr, respectively, by using a programmable syringe
pump. A positive high-voltage supply of 23 kV was maintained
between the spinneret and the metallic grounding plate and fibers
are collected up on the plate. The tip to collector distance was
maintained at 100 mm. The nanofibrous mat obtained was then soaked
in deionized water for 15 minutes with shaking and washed for three
times with deionized water.
[0119] A control membrane of the same formulation above for the
core (except the yeast particles) and shell solutions was also
prepared under the same operating conditions. All prepared
membranes had a thickness of approximately 40 .mu.m and were cut
into circular coupons of 50 mm diameter for use in the assay of
yeast activity. The weight of the membrane was approximately 1.3
g.
[0120] b) Assay of Yeast Activity
[0121] The core-shell nanofibrous mat prepared was incubated in an
aqueous media containing 0.8% (w/v) Glucose and 0.2% (w/v) Peptone.
A quantitative analysis of Yeast activity was done by measuring the
glucose concentration spectrophotometrically at 575 nm by using DNS
as the color indicator (1). A decrease in glucose concentration
directly indicates yeast activity since yeast can metabolize
glucose into ethanol and carbon dioxide. The glucose concentrations
in the control membrane and the yeast encapsulated membrane are
shown in Table 1 below.
TABLE-US-00001 TABLE 1 Concentration of glucose remaining in the
various membranes Concentration of Glucose Remaining (mg/mL)
Membrane Day 1 Day 2 Day 5 Day 7 Control 0.7265 0.7265 0.7260
0.7269 Membrane Yeast 0.7265 0.5481 0.5180 0.1564 Encapsulated
Membrane Percentage 0 24.6 28.7 78.5 change (%)
[0122] The percentage change in glucose concentration due to the
yeast encapsulated membrane is shown in FIG. 2.
[0123] The yeast encapsulated membrane and control membrane were
removed at the end of 7 days and washed in deionised water. The
washed membranes were then placed into freshly prepared glucose
solution having an initial concentration of 0.83 mg/mL. The glucose
concentrations of the vials containing the membranes were then
monitored. It was found that the yeast encapsulated membrane was
able to convert 4% and 25.9% of glucose after Day 2 and Day 4,
respectively, of this experiment, which corresponded to Day 9 and
Day 11, respectively, of testing from the time the membranes were
first produced. The results are encouraging for the extended use of
the encapsulated membranes.
Example 2
Methods
[0124] a) Preparation of Core-Shell Nanofiber
[0125] A 15% (w/w) Nylon spinning solution was prepared by
dissolving Nylon 6/6 in a mixture of formic acid and Polyvinyl
alcohol (PVA) (14:3 w/w) and used as shell solution. Baker's yeast
pellets (Saccharomyces cerevisiae) were resuspended in deionized
water and centrifuged at 7500 rpm. The residue was further
resuspended in 3 ml water and used as the core solution. The shell
solution was injected into the outer coaxial needle and the core
solution was delivered into the inner coaxial needle at a constant
rate of 3 ml/hr and 1 ml/hr, respectively, by using a programmable
syringe pump. A positive high-voltage supply of 23 kV was
maintained between the spinneret and the metallic grounding plate
and the fibers were collected on the plate to form a nanofibrous
mat. The tip to collector distance was maintained at 100 mm. The
nanofibrous mat obtained was then soaked in deionized water for 15
minutes with shaking and washed thereafter for three times with
deionized water.
[0126] A control membrane of the same formulation above for the
core and shell solutions but without the yeast particles was
prepared under the same operating conditions.
[0127] A total of three yeast encapsulated membranes and three
control membranes were cut into circular coupons.
[0128] The mass of each pair of yeast encapsulated membrane and
control membrane was 0.27, 0.35 and 0.53 g. The yeast encapsulated
membranes and control membranes were used in the assay of yeast
activity.
[0129] b) Assay of Yeast Activity
[0130] Each of the core-shell nanofibrous mat prepared was
incubated in an aqueous media containing glucose. A quantitative
analysis of yeast activity was done by measuring the glucose
concentration spectrophotometrically at 575 nm by using DNS as the
color indicator (1). A decrease in glucose concentration directly
indicates yeast activity since yeast can metabolize glucose into
ethanol and carbon dioxide. The glucose concentrations in the
control membranes and the yeast encapsulated membranes after 4 days
of soaking are shown in Table 2 below. The starting glucose
concentration was 1.80 mg/mL.
TABLE-US-00002 TABLE 2 Glucose concentration and percentage removal
in the various membranes of different masses Weight of Membranes
(g) 0.27 0.35 0.53 Glucose concentration 1.78 1.70 1.79 remaining
in control membrane (mg/mL) Glucose concentration 1.56 1.34 1.25
remaining in yeast encapsulated membrane (mg/mL) Percentage removal
(%) 12.4 21.2 30.2
[0131] Table 2 shows a positive relationship between the amount of
glucose removed and the weight of the membrane. This is due to the
greater amount of yeast particles present in the membranes of
increasing weight.
Example 3
[0132] Algae Chlorella sp was encapsulated into a PVDF nanofiber. A
15% (w/w) Poly vinylidene fluoride (PVDF) spinning solution was
prepared by dissolving PVDF in a mixture of N,N Dimethyl acetamide
and Acetone (2:3 w/w) and used as shell solution. The algae
Chlorella sp suspended in deionized water was used as the core
solution. The shell solution was injected into the outer coaxial
needle and the core solution was delivered into the inner coaxial
needle at a constant rate of 3 ml/hr and 1 ml/hr respectively by
using a programmable syringe pump. A positive high-voltage supply
of 16 kV was maintained between the spinneret and the metallic
grounding plate and the encapsulated nanofibrous mat was collected
on an aluminium plate. Due to the presence of acetone (a
pore-forming material with low boiling point), as the mat was
collected on the aluminium plate, the acetone evaporated from the
fiber to form pores on the fiber. FIG. 3 is a high resolution image
at 100.times. magnification of the porous fiber encapsulating the
Chlorella Sp.
[0133] The total diameter of the fiber was approximately 8000 nm.
The Chlorella sp has a diameter of approximately 2500-3000 nm. FIG.
3 was obtained under a controlled flow rate to allow single algae
cells to be deposited within the fibers. More cells can be
deposited by adjusting the flow rates, concentration of the core
fluid and the electrospinning conditions.
Example 4
[0134] FIG. 4 is a SEM image of a polyimide fiber. This was made
using the following process. A 25& (w/w) Polyimide spinning
solution was prepared by dissolving Polyimide in a mixture of N,N
Dimethyl formamide and Acetone (17:3 w/w). The spinning solution
was injected through an 18 G needle at a constant rate of 2 ml/hr
by using a programmable syringe pump. A positive high-voltage
supply of 20 kV was maintained between the spinneret and the
metallic collector. The nanofibers were collected on to the surface
of the metallic collector. Due to the presence of acetone (a
pore-forming material with low boiling point), as the mat was
collected on the aluminium plate, the acetone evaporated from the
fiber to form pores on the fiber. FIG. 4 shows the porous fiber
that was collected.
Example 5
Materials
[0135] Polycaprolactone PCL (Mn45,000), poly(ethylene glycol) (PEG)
(Mw 3000-37000) and fluorescein 5 (6) isothiocynate (FITC) was
purchased from Sigma Aldrich, Singapore. Ethanol (absolute) and
glycerol was purchased from Merck, Singapore and dichloromethane
was obtained from J. T. Baker, United States of America.
Methods
[0136] Preparation of Polymer Solution
[0137] The shell solution was 23% polycaprolactone (PCL) with 20
mg/ml or 30 mg/ml of poly (ethylene glycol) (PEG) dissolved in a
mixture of 60:40 (v/v) dichloromethane: ethanol; the core solution
was a small amount of fluorescein isothiocynate mixed with 20%
glycerol.
[0138] Electrospinning Set Up
[0139] The spinning parameters were as follows: The flow rates of
both core and shell solution were 1 mL/h and 3 mL/h. The needle
gauges used for dispensing core and shell solutions were 18 G and
271/2 G. The shell solution was injected into an outer coaxial
needle and the core solution was delivered into an inner coaxial
needle. A positive high-voltage supply of 15 kV was maintained
between the spinneret and the metallic grounding plate and the
fibers were collected on the plate to form a nanofibrous mat. The
tip and collector distance was 100 mm. For confocal microscopy,
fibers were collected directly onto a microscope slide. The
nanofibrous mat obtained was then soaked in deionized water for 24
hrs to dissolve the porogen PEG and dried overnight in desiccators
prior to imaging under SEM. All the experiments were conducted at
room temperature.
Results
[0140] The size and morphology of the PCL fibers was analyzed by
scanning electron microscopy (SEM) and the core shell structure was
confirmed by confocal microscopy. A concentration of 10% of PCL was
used which produced nanofibers having a thickness of less than 200
nm (see FIGS. 5a and b) which was not suitable for bacterial
incorporation into the core. In order to increase the fiber size,
another concentration of 23% was used which produced thicker
nanofibers in the range of 1 to 1.5 .mu.m (see FIG. 6).
[0141] A porogen, PEG was used in different concentrations in
combination with PCL to create the pores of different sizes. It was
found that when 20 mg/ml porogen was used, the nanofibers showed
pores of about 100 nm size (See FIG. 7a). Whereas, when the porogen
concentration was increased to 30 mg/ml, the pore size was
increased up to 200 nm (see FIG. 7b). Pore sizes were measured
using high magnification images obtained with SEM using Cell P
Software.
[0142] No prominent changes either in the surface morphology or
fiber diameter were observed in PCL fibers formed with and without
porogen. This is represented in FIG. 8 at different
magnifications.
[0143] FIG. 9 shows the confocal image of the core shell fiber
obtained. The solution mixed with FITC was used as a core during
spinning whereas only polymer solution was used as the shell
solution without any fluorescence. Hence, the differential
interference contrast microscopy (DIC) image is overlaid with a
fluorescent image. The fluorescent material was excited with a
laser and point by point scanning was used to generate z stacking
(layer-by-layer image).
[0144] However, the DIC image was obtained using the passage of
light. As such, layer by layer scanning is not possible. FIG. 9
shows the confocal images obtained for the fibrous mat collected
after electrospinning. As the fibers are in different Z planes,
fluorescence is not observed for all the fibers. The fibers which
are at a particular plane of measurement will exhibit fluorescence.
The Z stacking is subjected to X and Y sectioning to prove the
presence of fluorescent core inside the fiber. The presence of this
feature proves that the fibers are capable of encapsulating
biological material therein.
Example 6
Methods
[0145] a) Preparation of Electrospun PVDF Fibers
[0146] Porous poly(vinylidene fluoride) (PVDF) fibers were prepared
by electrospinning from solutions in dimethylformamide,
poly(ethylene oxide) (PEO) and water.
Materials
[0147] Kynar poly(vinylidene fluoride) (PVDF) was provided by
Arkema Inc., France. Poly(ethylene oxide) (PEO) (average Mv
100,000), dimethylformamide (DMF), Dimethyl acetamide (DMAc),
Dimethyl sulphoxide (DMSO) and Acetone were obtained from Sigma
Aldrich, Inc. De-ionized water was used in the experiments for
solution preparation. All materials were used without further
purification.
Characterization of Porous Electrospun PVDF Fibers
[0148] For morphological characterization by scanning electron
microscopy (SEM) (JEOL-JSM-5200, Japan), fiber samples were
sputter-coated with a 10 nm layer of gold using a magnetron
sputtering auto fine platinum coater. SEM was used to observe the
surface structure of fibers at 30 kV acceleration voltage and 10 mm
working distance. Using the JEOL JSM-5200 SEM for image
acquisition, pores smaller than 50 nm are easily overlooked due to
the limited resolution and quality of the SEM image. A "rough
surface" is defined in this work as one having no obvious pores
larger than 50 nm on the fiber surface.
[0149] Variations in PEO concentration, solvent nature and
concentration were investigated as possible factors that may
influence the development of pores on PVDF fibers electrospun with
small amounts of PEO (additive) and water (non-solvent). All the
experimental results are shown in the following FIGS. 10 to 17. The
sample prepared using only PVDF in DMAC:Acetone was used as
benchmark to demonstrate the influence of different factors on the
final morphology of porous core-shell electrospun PVDF fibers.
Sample Preparation
[0150] PVDF and PEO were dissolved in mixtures of DMF and water, in
the amounts shown in Table 3, under gentle stirring overnight at
70.degree. C. The solutions were subsequently cooled down to room
temperature before Electrospinning. The flow rate, plate-to-plate
distance, and voltage, respectively, were 2 mL/min, 15 cm, and 16
kV for the solutions. Nanofibers were collected on a grounded
aluminum foil at room temperature. The nanofibrous mat obtained was
then soaked in deionized water for 24 hrs to dissolve the porogen
PEG and dried overnight in desiccators prior to imaging under SEM.
All the experiments are conducted at room temperature.
[0151] When 15% PVDF solution was prepared in Dimethyl Acetamide:
Acetone in (1:4) ratio the nanofibers showed very smooth surface
under high magnification SEM (FIG. 10).
Preparation of Porous Electrospun PVDF Fibers
[0152] A combination of Dimethyl formamide (DMF) and water were
used as solvents for spinning the porous nanofibers. A porogen,
poly (ethylene oxide) (PEO) was added to the polymer in order to
produce pores on the nanofiber surface. A 20% PVDF and PEO (43:10)
solution was prepared in DMF:Water (30:1). All other conditions
were same as above for Electrospinning. The nanofibers showed a
very rough surface under high magnification SEM but no pores were
seen (see FIG. 11).
[0153] In order to get porous nanofibers, different ratios of PEO
(Average Mv 100,000) and PVDF were tested as listed in Table 3
below using DMF and water. (Yang, 2011). PVDF and PEO were
dissolved in DMF and water in amounts shown in Table 3 with
continuous stirring at .about.70 C overnight. Solutions were
allowed to cool down before electrospinning.
TABLE-US-00003 TABLE 3 Different solution conditions for spinning
to obtain porous nanofibers of PVDF. PVDF/ Concentration PEO DMF/
(PVDF + PEO)/ No wt ratio H2O (DMF + H2O) Result 1 50:3 30:1 5%
Can't spin 2 50:3 30:1 8% Can't spin 3 50:3 30:1 12% Can't Spin 4
50:3 30:1 15% Can't spin, have beads 5 50:3 30:1 18% Fibers have
Beads 6 50:3 30:1 20% Fibers have pores on beads only 7 50:5 30:1
20% Can't spin 8 50:10 30:1 20% Can't spin 9 43:10 30:1 20% Fibers
without pores, roug surface 10 48:5 30:1 20% Fibers with pores on
beads only 11 45:8 30:1 20% Have pores on beads and thic fibers 12
45:8 30:1 25% Thick fibers, pores on all fibers 13 43:10 30:1 25%
Thick fibers, pores on thic fibers 14 40:13 30:1 25% Thick fibers,
pores on thic fibers 15 40:15 30:1 25% Thick fibers, bigger pores
thick fibers indicates data missing or illegible when filed
Out of these different conditions listed in Table 3, most of the
solutions could be electrospinned. However, under some conditions
pores were produced on beads only. When the total percentage
(PVDF+PEO)/(DMF+H2O) was increased from 20 to 25%, the thicker
fibers were produced (.about.>2 um) and showed pores on almost
all spun nanofibers. In this case, no beads were observed. Attempts
were also made to increase the pore size by increasing the PEO
ratio and it was found from SEM images that the size of pores
increased up to 200 nm.
[0154] FIG. 12 shows nanofibers containing pores on only thick
fibers and beads which have diameter of more than 2 .mu.m. Thin
fibers did not show any pores.
[0155] When the polymer concentration was increased to get thicker
fibers in order to obtain pores on the majority of spun fibers, it
was found that using 25% PVDF and PEO in ratio of 45:8, thicker
fibers up to 2 .mu.m could be obtained. These fibers showed well
defined pores of the size around 100 nm (see FIG. 13A) on the
surface distributed uniformly everywhere. To further increase the
size of nanofiber pores, the polymer:porogen ratio was further
increased to 40:15 which obtained bigger pores of around 200 nm
(see FIG. 13B).
[0156] Additionally, the nanofiber thickness could also be
controlled by varying the polymer concentration used to spin the
nanofibers. For example, a 15% solution obtained nanofibers have a
thickness of less than 1 .mu.m where as this thickness was
increased when polymer concentration was increased to 20% with a
further increase with 25% solution. (FIG. 14)
Preparation of Core-Shell PVDF Electrospun Nanofibers
[0157] PVDF and PEO polymer (25%) solutions were used in DMF and
water (30:1 ratio) to prepare the shell of the nanofibers and
Glycerol was used as the core. These nanofibers were fabricated
using a syringe-inside-syringe design using a NANON-03
Electrospinning set-up (MECC Co Ltd, Japan). The needle gauges used
for spinning the shell and core were 18 G and 27 G, respectively.
The flow rate for the shell solution was 2 ml/hour and 1 ml/hour
for the core solution. The voltage used was 18 KV with the
Electrospinning distance set at 10 cm. The collector drum was
rotated at about 50 rpm.
[0158] FIG. 15 depicts a phase contrast and fluorescence image of
the core-shell nanofibers obtained using glycerol and Alexa
Fluor-Concavalin A as the core. The fluorescence images clearly
showed that the protein solution was successfully encapsulated
within the core using this method.
[0159] FIG. 16 a & b are the confocal images at different Z
planes of the core-shell fiber obtained. The core solution was
mixed with fluorescent protein Concavalin A and the shell material
was non-fluorescent polymer. Hence, the differential interference
contrast microscopy (DIC) image is overlayed with fluorescent
image. The fluorescent material is excited with a laser and a
point-by-point scanning is performed to generate z stacking
(layer-by-layer image).
[0160] The DIC image was obtained using the passage of light. As
such, layer-by-layer scanning was not possible. FIG. 16 shows the
fibrous mat collected. As the fibers are at different Z planes,
fluorescence was not observed for all of the fibers. The fibers
which are at a particular plane of measurement will exhibit
fluorescence. The Z stacking was subjected to X and Y sectioning to
prove the presence of fluorescent core inside the fiber. The
presence of this feature proves that the fibers are capable of
encapsulating biological material therein.
Example 7
Methods
[0161] Poly(caprolactone) (PCL) polymer dissolved in
Tetrahydrofuran (THF) and Dimethyl sulphoxide (DMSO) was
electrospun using a NANON-03 Electrospinning set-up using a 20 KV
voltage and an 18 G gauge needle. The electrospinning distance was
set at 15 cm and the polymer flow rate was 0.6 ml/hour. No porogen
was used in this example.
[0162] THF and DMSO are non-miscible with one another and the
boiling point of THF is substantially lower than that of DMSO
causing it to evaporate first leaving pores on the surface of the
PCL fiber.
Results
[0163] Thick fibers of PCL were formed with a 29% PCL solution. 15%
and 18% PCL polymer solutions were also prepared which could not be
electrospun. However, a 27% PCl polymer solution produced
nanofibers with lots of beads. In addition, 28% and 29% PCL polymer
solutions produced uniform nanofibers without any beads (see FIG.
17) The thickness of these fibers was >5 .mu.m which is a very
good size for encapsulating bacteria or other bigger bioactive
molecules within the core of the PCL fiber. From high magnification
SEM pictures it was observed that most of the pores have a
see-through structure which in turn is beneficial in the case of
core-shell nanofibers with encapsulated biomolecules as this
structure will permit the of biomolecule to interact with the
external environment of the fiber through the pores.
Comparison of Results
[0164] Comparing Examples 5, 6 and 7, it is clear that Example 6
provides a porous fiber that is superior in many aspects to that
obtained in Example 5.
[0165] The utilization of PVDF as the main core polymer in the
formation of the porous fiber is one of the key features that is
distinctive from Example 5, which utilizes PCL as its core
polymer.
[0166] PVDF is mechanically strong and as it is non-biodegradable
it is ideal candidate for applications such as waste water
treatment.
[0167] In comparison, PCL is a biodegradable polymer which has a
significantly lower mechanical strength than PVDF. PCL has been
shown to be degraded by the action of aerobic and anaerobic
microorganisms that are widely distributed in various ecosystems
(Tokiwa, 2009). As such, it can be easily degraded by lipases and
esterases and hence is not suitable for use with microorganisms
such as bacteria or viruses.
[0168] In addition, the increased thickness of the porous fibers
(>2 .mu.m) as achieved in Example 6 is advantageous when
encapsulating bacteria within its core, given that the size of
bacteria is only about few microns (0.2-2 .mu.m) in width.
[0169] Further, the porous fiber of Example 6 also provides uniform
porous structures that improve its efficiency, when compared to the
porous fibers obtained in Examples 5 and 7. In particular, the
pores present in the fiber of Example 6 act as channels to permit
the interaction of the bacteria/bioorganism with the external
environment of the fiber, whilst retaining the bacteria/bioorganism
within the core of the fiber.
[0170] As such, the characteristics of the porous fiber of Example
6 make it suitable for use in applications such as water treatment
and biosensor technologies.
Encapsulation of Immobilized Enzymes and Proteins
[0171] Various types of particles have been used to study
antigen-antibodies, biotin-avidin, peptides, DNA/protein detections
and cellular analysis. The most common particles are gold
nanoparticles, silica nanoparticles (Knopp et al, 2009),
polystyrene beads (Lateef et. al. 2005) and the like. There are
different approaches that can be used to prepare these
micro/nanoparticles in order to modify their surface with ligands
using electrostatic interactions or physical adsorption
immobilization. However, to produce more stable linkages with
biomolecules, covalent attachment is preferred where the surface is
chemically functionalized to produce reactive groups such as --NH2,
--COOH, --SH, --CHO, --OH, peptide, carbamate linkages and the like
which can covalently bind to biomolecules (see FIG. 18).
[0172] Nanoparticles functionalized with groups that provide
affinity sites for the binding of biomolecules have been used for
the specific attachment of proteins and oligonucleotides (Li--Na
2010). Gestwicki, (2000) has used streptavidin-functionalized AuNPs
for the affinity binding of biotinylated proteins or biotinylated
oligonucleotides whereas Sergeev (2003) have used protein A
conjugate bound to AuNPs as a versatile linker to Fc fragments of
various immunoglobulins and carbohydrate-modified AuNPs to
recognize their respective binding proteins.
[0173] Such immobilized microparticles and nanoparticles can be
then encapsulated within the core of nanofibers in accordance with
the disclosure using core-shell electrospinning disclosed herein.
Leaching of biomolecules can be drastically reduced as these
particles can be easily retained within the core of the porous
fibers described herein. The entrapment of biomolecules alone is
not possible without immobilization to these particles within the
core as the size of these biomolecules is in the order of few
nanometers and the pores on the nanofibers are at least 100 nm.
Various combinations of different sizes of particles and pores on
nanofiber surface can be produced in order to retain the particles
within core and increase the efficiency of the membranes (see FIG.
19).
Applications
[0174] Advantageously, the disclosed nanofiber or microfiber can be
used to form a membrane in a bioreactor. Due to the pores present
in the nanofiber or microfiber, a greater contact surface area can
be provided between the encapsulated biological material and a
target substrate, such as a contaminant, in an external
environment. Due to the greater contact area, the biological
material can interact with the target substrate with higher
efficiency, leading to a decrease in the reaction time between the
biological material and the substrate.
[0175] Advantageously, as the encapsulated biological material is
already present in the bioreactor, the start-up time associated
with the growth of the biological material is reduced as compared
to a conventional bioreactor.
[0176] Advantageously, since the target substrate is the food
source for the biological material, the occurrence and extent of
membrane fouling in which the substrate builds up on the surface of
the nanofiber or microfiber are substantially reduced as compared
to a conventional bioreactor. The pores present in the nanofiber or
microfiber allow the biological material to interact directly with
the target substrate, leading to the removal of the target
substrate. Since the target substrate is removed by the action of
the biological material, the build-up of such target substrate is
substantially reduced. Accordingly, the problems of build-up and
the subsequent membrane fouling are substantially reduced in the
present bioreactor.
[0177] Advantageously, the disclosed membrane with biological
material encapsulated within can be used for membrane filtration
processes.
[0178] The membrane bioreactor or membrane contactor may be used in
bioremediation, bioprocess and in the production of foodstuffs and
pharmaceuticals.
[0179] Advantageously, the disclosed fiber encapsulating a
biological material therein can be used in industries such as
fermentation, catalysis of chemical reactions, food processing,
paper industries, biofuel industries, waste treatment and medical
industries.
[0180] Advantageously, the disclosed nanofiber or microfiber can be
used as a biosensor in which the biological material such as
protein, cell, enzyme or nucleic acidscan be used to detect changes
in an environment system or sample.
[0181] It will be apparent that various other modifications and
adaptations of the invention will be apparent to the person skilled
in the art after reading the foregoing disclosure without departing
from the spirit and scope of the invention and it is intended that
all such modifications and adaptations come within the scope of the
appended claims.
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