Nanoimprinting of Chitosan as a Biomaterial for Micro and Nanodevice application

Chitosan – Versatile Material for Bionanotechnology• Material properties of chitosan for nanoimprinting

Nanoimprinting Lithography of Chitosan Pattern

Bio-functionalization of Imprinted Chitosan

• Swelling properties of chitosan pattern

Inkyu Park1, Jim Cheng1, Eung-Sug Lee2, Jun-Ho Jeong2, and Albert P. Pisano1

1 Berkeley Sensor and Actuator Center (BSAC), University of California at Berkeley, Berkeley, CA, USA 947062 Korea Institute of Machinery and Materials (KIMM), Daejon, Republic of Korea 305-343

• Imprinting of microscale features • Viscosity and pressure effects on step height and residual layer

* Acknowledgement : This project has been supported by Center for Nanoscale Mechatronics and Manufacturing (Grant No. 019997), one of the 21st Century Frontier Research Programs, which are supported by Ministry of Science and Technology, Republic of Korea . We also thank Microfabrication Laboratory (Microlab) and Bioimaging Facility at the University of California at Berkeley.

• Chemistry of chitosan- Chitosan is a linear β-1,4-linked polysaccharide obtained by a partial deacetylation of chitin resulting in a copolymer of glucosamine and N-acetylglucosamine. The boundary betweenchitosan and chitin is normally set at approx. 60% degree of acetylation, based on chitosan’ssolubility in acids.- Chitin, the second most abundant polysaccharide (most abundant being celluose), is normally obtained from crab or shrimp shells, byproducts of seafood processing and thus inexpensive.- Chitosan exists in various forms in terms of molecular weight, degree of acetylation, and sequence.

• Applications of chitosan in biotechnology

• Chemical modification of chitosan- A large number of amine (-NH2) groups are available in chitosan matrix, which makes it a very useful platform for biochemical modification and immobilization of biomolecules. It can be modified with sugar molecules; dendrimers; cyclodextrin; crown-ether; vitamins; peptide; etc.

• Verification of amine groups by amine reactive fluorescein dye

- Fluidic viscosity - Thermal stability

• NHS-biotin attachment and streptavidin immobilization

OOH

O

NH2

HO

n

OOH

O

NHHO

nC=O

CH3

Chitosan Chitin

Blood-clotting bandage

Image courtesy of HemCon, Inc.

Bacteria inhibitor

w / chitosan

Control

Chung YC et al, Acta Pharmacol Sin, 2004. Yuan Y et al, Biomaterials, Vol. 25, 2004.

Cell growth scaffold

E-coli growth

Drug delivery system

Image courtesy of AICello Chemical Co, Ltd.

Virus filtering in air purifier

Image courtesy of Chungpung Co, Ltd.

Dynamic viscosity of chitosan solution with different concentration of MMW (250kDa) chitosan in 1:2 acetic acid : DI H2O measured by concentric viscometer. This result shows a strong dependence of viscosity on the chitosan mass in the solvent.

• Imprinting process flow

PR or PMMA mold for Polydimethylsiloxane (PDMS) replication

PDMS molding (nanoimprinting stamp)

Imprinting with pressure (5-25psi) & heating (90ºC), solvent gets evaporated and chitosan gets solidified.

Dispensing chitosan solution. We used MMW (250kDa) chitosan in 1:2 acetic acid / DI water solution (0.0068 g/mL-0.033 g/mL).

Chitosan line array, dried at 120ºC for 20 min

10µm

Chitosan line array, dipped in DI water for 18hrs

Thick (20 µm height)chitosan film, dry

Thick chitosan film, swollen by DI water

Chitosan is a type of hydrogel, which shows a great degree of swelling in aqueous environments. It has shown several times mass and volume expansion by absorbing H2O in its matrix. This is obvious in the thick chitosan film (left). However, no lateral expansion or significant volumetric expansion can be observed inthe thin imprinted chitosan patterns (100-400nm thick).

- Nanoscale features such as nanowire array, nanodot array, and serpentine nanowireshave been fabricated by nanoimprinting of 0.013 g/mL chitosan solution at low temperature (90ºC), low pressure (15psi) condition. First, chrome nanoscale patterns were defined by e-beam lithography and lift-off. Then PDMS replica of Cr patterns was used as a nanoimprinting mold. Reproducible imprinting results are obtained for several imprinting sessions with the same mold.

Chitosan line array, W=2µm, P= 4µm (nominal) fabricated by imprinting 0.033g/mL solution at 15 psi, 90ºC for 30min. Structure height : 900nm.

Various chitosan imprinting structures : microwell array, micropost array, checkerboard pattern

• Imprinting of nanoscale features

Array of Chitosan nanowires, W=150nm, P=500nm ; nanodots, W = 150nm, P = 400nm fabricated by 0.013 g/mL solution at 15psi, 90ºC for 30min.

- Microscale features (ex. line, microwell, and micropost arrays) with feature sizes of 2-10’s µm was fabricated by nanoimprinting of 0.033 g/mL chitosan solution at 15psi of pressure and 90ºC of temperature. PDMS was used for nanoimprintingmold. ~900 nm step height is obtained for 2µm width, 4µm pitch features.

Micro / Nanostructures of Chitosan

Succinimidyl Ester

FITC (Fluorescein Isothiocyanate)

+ →OOH

O

NH2

HO

OOH

O

NH2

HO

OOH

O

NHHO

OOH

O

NHHO

Fluorescein

Primary amine group40µm 30µm

OOH

O

NHHO

OOH

O

NHHO

+

NHS-biotinylated Chitosan Streptavidin

10µm 40µm 25µm

Fluorescence image of imprinted chitosan patterns conjugated with FITC on amine groups – checkerboard (8µm width, 16µm pitch) and trench array (2µm width, 16µm pitch)

Fluorescence image of FITC-conjugated Streptavidin immobilized on biotinlyated imprinted chitosan patterns – line array (1µm width, 4µm pitch), checkerboard (8µm width, 16µm pitch) and various microbeam structures

- NHS-fluorescein, a type of amine reactive fluorophore has been used as a marker for amine groups in imprinted chitosan patterns. This result proves abundant amine groups.

- Immobilization of streptavidin has been realized by strong binding to the biotin moiety that is bound to the amine groups of chitosan. Swelling property of chitosan (as a hydrogel) helps diffusion of streptavidin into the bulk of imprinted pattern for binding to biotin moiety.

10µm

- Swelling test of imprinted sub-2µm line array patterns (0.013 g/mL and 0.030 g/mL chitosan solution) in H2O for 1 hr does not show any significant changes of line width. However, there is a slight line height reduction (13% for 0.013 g/mL solution and 5% for 0.030 g/mL solution). This may stem from partial removal of chitosan in H2O or compression by N2 blow dry.

- Thicknesses for 0.0068g/mL – 0.020g/mL are all around 170nm for 4 µm wide line arrays. Thickness increase (170nm to 414nm) is substantial when mass changes from 0.020 g/mLto 0.033 g/mL. This is due to dramatic increase of viscosity (from 1.4kPa.s to 27kPa.s) by mass change from from 0.020 g/mL to 0.033 g/mL. - Pressure increase is found to cause slight decrease of step height and to reduce the residual layer thickness.

FTIR of Chitosan Films After Heating

2 3 4 5 6 7 8 9 10 11 12 13 14 15

Wavelength (µm)

Abs

orpt

ion

(nor

m.)

Sample 1 - Control (baked at 125°C)

Sample 2 - Heated to 205°C

Sample 2 - Heated to 250°C

Sample 2 - Heated to 300°C

N-H, O-HC-H C=O

C-H C-N, C-OC-O, C-N

C-H

FTIR spectra of chitosan films formed from 0.02 g/mL chitosan solution heated at high temperatures show that original chitosan structure is maintained up to 205 ºC. Relative absorption increase between 5.5-8 µm for samples heated at 250 ºC and 300 ºCindicates chemical or microstructural modification.

• Nature- Chitosan / Chitin is heavily used in nature at the micro/nanoscale for multiple purposes from a structural molecule in insects and crustaceans to removal of heavy metals to an agent to resist bacterial growth.

• MEMS / NEMS- Chitosan micro/nano structures have the potential to improve biocompatibility of MEMS/NEMS devices and chitosan’s wide range of material properties can be used to enhance existing MEMS/NEMS devices or for the creation of unique MEMS/NEMS bio-inspired / biomimetic devices.

- Microscale features ( microwell, micropost, and checkerboard) larger than 4µm in dimension show greater height at the feature edges (so-called “rabbit ears”). This may be due to incomplete filling of chitosan solution in PDMS mold (1.3µm feature depth). This rabbit-ear shapes could not be found in sub- 2µm features.

Crown-ether modified 4)Phenol modified 3)Peptide modified 2)Sugar modified 1)

1)Yalpani and Hall, Macromolecules, Vol. 17, pp. 272-8. 2) Nishiyama, et al., J Chem Soc Perkin Trans, 2000., I:1161-5 3) Kumar, et al., Biotechnol Bioeng, Vol. 63, 154-65. 4) Tang, et al., J. Appl Polym Sci, Vol. 83, 1886-91.

Dynamic viscosity vs. concentration of MMW (250kDa) chitosan in 1:2 HAc + H2O

100

1000

10000

100000

0 0.01 0.02 0.03 0.04

Concentration of Chitosan (g/mL)

Dyn

amic

vis

cosi

ty (P

a.s)

Effect of chitosan concentration on step height and residual layer thickness

0

50

100

150

200

250

300

350

400

450

0 0.01 0.02 0.03 0.04

Concentration of chitosan (g/mL)

Dim

ensi

on (n

m)

Effect of pressure on step height and residual layer thickness (0.020g/mL solution)

0

50

100

150

200

250

0 5 10 15 20 25 30

Pressure (psi)

Dim

ensi

on (n

m)

step heightresidual layer

step heightresidual layer

Swelling effect by DI water on the line array imprinting pattern of chitosan

(0.013 g/mL solution)

0

250

500

750

1000

1250

original swollen and dried

Dim

ensi

on (n

m)

line heightline width

Swelling effect by DI water on the line array imprinting pattern of chitosan

(0.030 g/mL solution)

0

250

500

750

1000

1250

1500

1750

2000

original swollen and dried

Dim

ensi

on (n

m)

line heightline width