1
Thermoplastic Nanofibers as Biosensors and Solid Support for Drug Synthesis
Bei Xiang1, Dong Wang2, Kit S. Lam3, Gang Sun2
1Department of Chemical Engineering & Material Sciences, UC Davis2Division of Textile and Clothing, UC Davis
3Division of Hematology/Oncology, Department of Internal Medicine, UC Davis Medical Center
RESEARCH AREAS
• Antimicrobial polymers and textiles– Rechargeable biocidal
halamine technologies on all textile materials
– Durable and rechargeable antimicrobial peroxide cotton
– Refreshable biocidal halaminepolypropylene fibers and nonwovens
– Novel super biocidal colorants• Chemical detoxifying textiles
– Halamine textiles– Radical detoxification
materials
• Nanotechnologies– Environmental benign and
high throughput fabrication of thermoplastic nanofibers
– Nanofiber applications• Biosynthesis of natural
colorants– Prodiginines produced
from marine bacteria– Fungi production of colors
• Photo-induced reactions in polymerization and functional modification of polymers
2
INTRODUCTION
• Nanofibers can be produced by a novel process using polymer immiscible blends of cellulose acetate butyrate (CAB) and thermoplastics
• Cellulose acetate butyrate (CAB), a bio-based thermoplastic polymer, can serve as a sacrificial matrix polymer and be easilyremoved by acetone after extrusion into fibers
• Poly (ethylene-co-glycidyl methacrylate) (PE-co-GMA) and poly (ethylene-co-acrylic acid( (PE-co-AA) nanofibers were prepared, chemically modified and biotinylated.
• The (PE-co-GMA) fibers were then employed as solid support materials to bind streptavidin-horseradish peroxidase (HRP).
• The streptavidin-HRP immobilized PE-co-GMA nanofibers showed high activity, efficiency, sensitivity as well as good reusability.
• Both (PE-co-GMA) and (PE-co-AA) nanofibers were used as solid support in peptide synthesis
Nanfiber Applications
ProtectiveClothing Tissue
Engineering
Sensors
BiomedicalMaterials
Polymer Nanofibers
Enormous Surface Area to Volume Ratio
FilterMedia
Fabrication of Thermoplastic nanofibers
3
Immiscible Polymer Blends
In situ Self-Reinforced or Toughen composite
Thermoplastic Nanofibers
Morphology Control
Removing Matrix
Nanofiber Productions
Immisciblity
Easy Removal
Recyclability
Wide range of Mp
Cellulose Acetate Butyrate (CAB)
Scheme of Nanofiber Production
Thermoplastics
4
Formation of Nanofibers from Single Composite Fiber
Removal of CAB
Single yarn of nanofibers
5
Formation of iPP Nanofibers from Single Composite Fiber
Removal of CAB
Single yarn of nanofibers
6
Key Steps in Formation of Nanofibers
• Dispersion of thermoplastic polymers in cellulose acetate butyrate (CAB) into micro-sized micelles– CAB/thermoplastic ratios, >70/30– Interfacial tensions
• Deformation and elongation of the micelles into nano-sized fibrillars in composite fibers– Viscosity ratios of two polymers– Interfacial tensions– Shearing speed, drawing, cooling…
• Removal of CAB from the composite– Recyclable and reusable
CAB/iPP = 60/40 CAB/iPP = 70/30
CAB/iPP = 80/20 CAB/iPP = 90/10
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Nanofibers Produced by This Process
iPP fibers made from a blend of CAB/iPP=80/20
x2500 x20000
Nanofibers Produced by This Process
PTT fibers made from a blend of CAB/PTT=80/20
x2500 x20000
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PE-co-GMA fiber made from a blend of CAB/PE-co-GMA=80/20
Nanofibers Produced by This Process
x2500 x20000
Formation of Nanofibers in Immiscieble Blends
~10050-3500.991.20CAB/PE-co-GMA
~200100-5000.792.11CAB/PTT
287100-5000.416.99CAB/iPP
Average Diameterc
(nm)
Diameter Rangec
(nm)
Viscosity Ratiob
Interfacial Tensiona
(mN/m)
Sample
a Interfacial tensions between CAB and dispersed phases, iPP, PTT and PE-co-GMA at 240°C are estimated based on the equation. b Viscosity ratio are calculated at 240°C and the apparent shear rate of 100 s-1. c same as Table 1 pp
pp
dd
dd
21
21
21
212112
44γγγγ
γγγγ
γγγ+⋅⋅
−+⋅⋅
++=
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Fabrication of Thermoplastic nanofibers
PE-co-Acrylic acid copolymer fiber
Functional Nano-fibers
COOH
H2NO
NH2
2
C
O
NH
ONH22
DIC, HOBt in DCM/DMF
O
H2NO
NH2
2OH
HN
ONH2
2
PE-co-GMA copolymer fiber
Amount of primary amino groups on fiber: 0.1mmol/g
Amount of primary amino groups on fiber: 0.15mmol/g
In EtOH, RT, overnight
10
Synthesis route of Streptavidin-HRP Immobilized PE-co-GMA Nanofibers
PE-co-GMA 10 PE-co-GMA 20
PE-co-GMA 30 Streptavidin-HRP immobilized PE-co-GMA 20
Morphology
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100 150 200 250 300 3500
10
20
30
40
50
Dis
tribu
tion
(%)
Diameter (nm)
100 150 200 250 300 350 4000
5
10
15
20
25
Dis
tribu
tion
(%)
Diameter (nm)
(a) PE-co-GMA 10
200 300 400 500 6000
5
10
15
20D
istri
butio
n (%
)
Diameter (nm) (c) PE-co-GMA 30
(b) PE-co-GMA 20
FTIR-ATR Spectra of (a) PE-co-GMA 20 nanofiber; (b) Aminated PE-co-GMA 20nanofibers; (c) Biotinylated PE-co-GMA 20 nanofibers.
12
(a) Nitrogen map
Wavelength Dispersive X-ray (WDXS) Spectra of Biotinylated PE-co-GMA 20 Nanofibers
(b) Sulfur map
Colorless reagent mixture [phosphate buffer, phenol (PhOH), 4-aminoantipyrine (4-AAP, colorless dye) and H2O2].
Streptavidin-HRP Activity Assay of Streptavidin-HRP Immobilized PE-co-GMA 20 Nanofibers
Reaction Mechanism
13
400 450 500 550 600 650 700
0
20
40
60
80
100
Rel
ativ
e In
tens
ity (%
)
Wavelength (nm)
PE-co-GMA/CAB=10/90 PE-co-GMA/CAB=20/80 PE-co-GMA/CAB=30/70
0.23359CAB/PE-co-GMA=70/30
0.37232CAB/PE-co-GMA=80/20
0.48175CAB/PE-co-GMA=90/10
N (mmol/g)Average Diameter
(nm)
Fiber diameter is determined by polymer blend ratio
Smaller diameter larger surface areas and more reactive groups
Relationship of Fiber Diameter and Activity
0 5 10 15 200
20
40
60
80
100
Rel
ativ
e In
tens
ity (%
)
Time (mins)
PE-co-GMA/CAB=10/90 PE-co-GMA/CAB=20/80 PE-co-GMA/CAB=30/70
0.23359CAB/PE-co-GMA=70/30
0.37232CAB/PE-co-GMA=80/20
0.48175CAB/PE-co-GMA=90/10
N (mmol/g)Average Diameter
(nm)
Smaller diameter larger surface areas and more reactive groups
Smaller size higher efficiency
Relationship of Fiber Diameter and Efficiency
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0.23359CAB/PE-co-GMA=70/30
0.37232CAB/PE-co-GMA=80/20
0.48175CAB/PE-co-GMA=90/10
N (mmol/g)Average Diameter
(nm)
Smaller diameter larger surface areas and more reactive groups
Smaller size higher efficiency
Relationship of Fiber Diameter and Sensitivity
1 2 3 4 5 6 7 8 9 100
20
40
60
80
100
Rel
ativ
e In
tens
ity (%
)
Circle
Reusability of Biosensors
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Solid Phase Reaction
Starting material is fixed on solid supports, and reactions are performed on solid supports by adding solution, after the reactions are finished, undesired compound and excess reagents are purified by just washing.
support support +X
A
B
C
D
Solid Support Synthesis
Solid Supports for Solid Phase Synthesis
• The most common support used in solid phase synthesis is resin (polystyrene,
polyamide) beads with tens or hundreds μm of diameter.
• Non-beaded solid supports
Pros: easily prepared by simple suspension polymerization, fairly good chemical and mechanical stability, have good reaction kinetic profiles, commercially available and easily automated.Cons: hard to handle, slow diffusion rates in high cross-linked resins, limited surface area for reaction, internal reaction site may not available for large molecules, expensive.
Multipin Polymer film
Findlay PH, Leinonen SM, Morrison MGJT, Shepherd EEA, J Mater Chem 2000, 10:2031-2034.
<http://www.krict.re.kr/~shhwang/combimd.html>
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Nanofiberous Materials As Solid Support
• Large surface areas can be achieved from nanosizedfibers
• Many functionalized polymers are available for further chemical modifications
• Nanofibers can be made into membrane forms which can be handled much more easily than microsizedbeads
• Nanofibrous membrane can be used in automatic synthesis
Examples of Nanofibers
*
x*
y
COOH
Thermoplastic functional polymers:
Polyethylene-acrylic acid copolymer (Weight Percentage of Acrylic acid : 20%)
Polyethylene-glycidyl methacrylate copolymer
*
x*
y
O
OO
CH3
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PE-co-Acrylic acid copolymer fiber
Nanofibers as Solid Support Materials
C
O
NH
ONH22
COOH
H2NO
NH2
2
DIC, HOBt in DCM/DMF
O
H2NO
NH2
2OH
HN
ONH2
2
PE-co-GMA copolymer fiber
Amount of primary amino groups on fiber: 0.1mmol/g
Amount of primary amino groups on fiber: 0.15mmol/g
In EtOH, RT, overnight
Load fiber
Couple cleavable linker
Couple first amino acid
If for on-fiber assay
Completed compounds on fiberSide chain deprotection
Peptide cleavage
Purify/Analysis
Side chain deprotection
On-fiber bio-assay
Peptide synthesis flow chart
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20 common amino acids
vVValine
yYTyrosine
wWTryptophan
tTThreonine
sSSerine
pPProline
fFPhenylalanine
mMMethionine
kKLysine
lL Leucine
iIIsoleucine
hHHistidine
gGGlycine
qQGlutamine
eEGlutamic acid
cCCysteine
dDAspartic acid
nNAsparagine
rRArginine
aAAlanine
D isomerL isomer
AbbreviationFull name
L isomer D isomer
Amino acid coupling and deprotection cycle
1- hydroxy-benzotriazole (HOBt)
NC
NN
NN
OHNH
Diisopropylcarbodiimide (DIC) Piperidin
H2N
NH
O
HO
R(protected)
NH
(Fmoc)HN
O
R(protected)
NH
H2N
O
R(protected)
CO O
Fmoc protecting group
HOBt (4 eq.), DIC (4eq.), RT, overnight
(4 eq.)
20% piperidin in DMF30mins twice
RT
(Peptide synthesis starts from C-terminal.)
19
Mechanism of Fmocdeprotection by piperidin
Primary amine on N-terminal of peptide is recovered for next aminoacid coupling after removing Fmoc.
Kaiser test (ninhydrin test)Detect ammonia or primary amines
O
O
OH
OH+ peptide NH2
O
R
peptide N
O
R
O
O
-H2O
peptide N
O
R
O
O
H
+ H2OO
O
H
NH2
peptide O
O
R
+
ninhydrin
O
O
H
N
O
O
O
H
N
O
OO
Ruhemanns Blue
ninhydrin
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OO
NH
OHO
O
OCH3
OCH3
NH2 NH
OO
NH
O
O
OCH3
OCH3
Fmoc deprotection
NH
NH2
O
O
OCH3
OCH3
peptide synthesisNH
NH
O
O
OCH3
OCH3
peptideO
TFART, 2hrsH2N peptide
O
HOBt, DIC, RT, overnight
Rink linker
Cleavable linker (rink linker) coupling and cleavage
Fmoc group
Cyclization of the peptides with cysteines on both C- and N- terminals
H2NCH
C
CH2O
SH
HN
CHC
CH2
O
N
NH
N
CO
NH
CH
C
CH2
O
H2C C NH2
O
HN
CH
C
H
O
N
C
O
N
COHN
CH
C
H2C
H2N
O
SH
H2NCH
C
CH2O
S
HN
CHC
CH2
O
N
NH
N
CO
NH
CH
C
CH2
O
H2C C NH2
O
HN
CH
C
H
O
N
C
O
N
COHN
CH
C
H2C
H2N
O
S
Oxidation in 20% DMSO pH6 HOAc/NH4Ac buffer
RT, 48 hrs
Example: Cyclization of CHPQGPPC
Cyclization confirmation: Ellman’s Test
Positive (yellow color) indicates incomplete cyclization;Negative (not yellow) indicates complete cyclization.
21
Peptides synthesizedLigands for Enzyme Immobilization
Sequence of the peptide: LHPQF (specifically bind with streptavidin)
NH2
O
HN
HN
N
O N
O
NH
O
H2N
O
NH
O
H2N
Sequence of the peptide: CHPQGPPC (specifically bind with streptavidin)
H2NCH
C
CH2O
S
HN
CHC
CH2
O
N
NH
N
CO
NH
CH
C
H2C
O
H2C C NH2
O
HN
CH
C
H
O
N
C
O
N
COHN
CH
C
H2C
H2N
O
S
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Ligand for Cell Binding
H2N
S
O
NH
O
HN
HNNH
H2N
O
HN
O
NHO
O
OH
HN
OHO
O
NH
O
HN
S
O
NH2
cGRGDdvc (specifically bind with
cancer cell integrin αvβ3 )
Ligand for Protein Binding
NH2
(S)
HN
N
O
NH
(S)
HN
O
HN
(S)
HN NH
NH2
O
HN
O
NH
(S)
HN
O
HN
(S)
O
OH
HWRGWV (bind with human Immunoglobulin G)
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On-support Characterization• Peptide sequencing
(Edman chemistry)
HPLC Next cycle
Sequencing results of LHPQF
L
H L P
L
H
Q
F
standard
Cycle 1 Cycle 2 Cycle 3
Cycle 4 Cycle 5
It’s normal that the cleavage of each residue is not complete, so some uncleaved residues from previous cycles still give signals which are significantly weakened with the sequencing going on.
24
Sequencing results of HWRGWVH
WR
G
W V
Cycle 1 Cycle 2 Cycle 3
Cycle 4 Cycle 5 Cycle 6
Biological Assay
After 4 hours shaking with ST-AP and 2 hours BCIP staining. A is the sample with ligand. B is the blank
sample without ligand.
A B
LHPQF can specifically bind with Streptavidin-AP (ST-AP). So ST-AP can be immobilized on fibers. When 5-bromo-4-chloro-3-indolyl phosphate (BCIP) is added, the fibers show blue color
Zoom Image
Biological assay provides further proof of successful peptide synthesis on modified polymer fiber
LHPQF bind with Streptavidin-AP and stained by BCIP
25
Streptavidin-AP Binding on PE-co-GMA Fibers with Cyclized Peptide CHPQGPPC
Fiber without ligand Fiber with ligand
Binding time: 2 hours and overnightStreptavidin-AP stock solution was diluted 5000 times.
Functionalized polypropylene fabric as solid support
2HCHC
CH2C* *
CH3
C
O
OH2C
H2C O C
n
CH
O
CH2
2HC CH
C
O
OH2C
H2C O C C
H
O
CH2
H2C
COOH
CH2C*
CH3
n
H
I
CH2C* *
CH3
n
2HC CH
C
O
OH2C
H2C O C C
H
O
CH2
I2 2Iheat
3HCHC C
O
OH2C
H2C O C
HC
O
CH2
HOOC HOOC
I : Initiator
CH2C* *
CH3
n
Functionalization through acrylic acid grafting
PEG Diacrylate
Polypropylene
Acrylic Acid
Surface functionalized and crosslinked polypropylene fabric
26
841.
4
950
.397
3.2
997
.7
110
4.9
1254
.5
1355
.81
377
.014
56.1
1639
.8
1735
.0
283
5.0
2840
.02
845
.22
865
.728
71.8
2876
.428
81.3
2885
.128
91.3
2896
.629
02.6
2907
.629
14.9
292
3.2
2927
.129
33.5
2937
.029
49.6
2960
.129
64.1
2969
.5
3457
.6
*blank0616*Tue May 24 11:13:03 2005 (GMT-07:00)
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Abso
rban
ce
500 1000 1500 2000 2500 3000 3500 4000 Wavenumbers (cm-1)
FTIR Spectrum of Grafted PP and Original PP
COOH
Grafting result
After grafting
Before grafting
Confirmation of the Free COOH Presence
Green coated solid support
Grafted fabric is washed with acetone 4 times, each time 30minutes.
After dyeing, wash with methanol >10 times, until methanol solution remains clear.
blank functional sample
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Peptide synthesis on functionalized fibers
1. Diamine linker coupling
COOH
H2NO
NH2
2C
O
NH
ONH22
Coupling agents
2. Peptide ligand synthesis
Peptide sppLDI, which can specifically bind with lymphoma cell wassynthesized on functionalized polypropylene fabric.
H2N
OH
O
N
ON
O
NHO
NH
O
HO
O
NH
O
NH2
20X With ligand 20X With ligand
20X With ligand 20X Without ligand
Lymphoma cell binding results
28
Acknowledgements
National Science Foundation DMI 0323409 and CTS 0424716
National Textile CenterC02-CD06 and M06-CD04
Defense Threat Reduction Agency (DOD)(HDTRA-08-05-1A)