thermoplastic nanofibers as biosensors and solid support ...chemgroups.ucdavis.edu/~liu/10+10/sun...

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

7

Nanofibers Produced by This Process

iPP fibers made from a blend of CAB/iPP=80/20

x2500 x20000


PTT fibers made from a blend of CAB/PTT=80/20

x2500 x20000

8

PE-co-GMA fiber made from a blend of CAB/PE-co-GMA=80/20


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+⋅⋅

−+⋅⋅

++=

9

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


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

11

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


(nm)


Smaller size higher efficiency

Relationship of Fiber Diameter and Efficiency

14

0.23359CAB/PE-co-GMA=70/30

0.37232CAB/PE-co-GMA=80/20

0.48175CAB/PE-co-GMA=90/10


(nm)


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

15

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>

16

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

17

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



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

18

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

20

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

22

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)

23

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



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

27

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)

thermoplastic nanofibers as biosensors and solid support ...chemgroups.ucdavis.edu/~liu/10+10/sun...

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