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Optical Materials 27 (2005) 1175–1177

Optical fabrication of nano-structured biopolymer surfaces

P.S. Ramanujam *

Department of Optics and Fluid Dynamics, Risoe National Laboratory, DK-4000 Roskilde, Denmark

Received 14 May 2004; accepted 10 August 2004

Available online 27 October 2004

Abstract

A maskless nano-patterning of the surface of a biocompatible polymer that can be employed for tissue engineering and cell

growth is described. The technique is based on holographic diffraction grating recording with a UV laser in a biodegradable polymer

containing various amino acids.

� 2004 Elsevier B.V. All rights reserved.

PACS: 61.14.L; 42.40.E; 42.70Keywords: Biopolymers; Nano-patterning; Holography

1. Introduction

Biocompatible and biodegradable polymers are of

great interest for their use in tissue engineering. Accord-

ing to American investigations, approximately 5 billion

dollars per year are used for knee and hip implantations.

This figure is expected to rise, as the population getsolder [1]. Currently metal implants are utilized for repair-

ing bone decay. There is great interest in developing tech-

nology using biological cells, which can be grown on the

bones under decay. After this implantation, the cells are

expected to grow and form a bone-mass that will grow

together with the bones to form a solid structure. The

biological cells are grown on substrates that are pat-

terned. Typically this nano-patterning consists of a grat-ing with peaks and valleys with periods varying between

10 and 1000nm. The cells attach themselves to the rough

surface, and divide and grow. In order for the process to

be efficient and bioresorbable, the substrate must be

made of a biocompatible polymer. The light-sensitive

additives in the polymer should also be biocompatible,

and biodegradable. Furthermore, the solvents used in

0925-3467/$ - see front matter � 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.optmat.2004.08.079

* Tel.: +45 4677 4507; fax: +45 4677 4588.

E-mail address: p.s.ramanujam@risoe.dk

the process must be harmless both for the patient and

environment in general. Finally, the process of fabrica-

tion of the substrates must be economically viable. Here

I demonstrate a fabrication method based on hologra-

phy in biopolymers with UV laser light.

2. Experimental

As a matrix, dextran is chosen; this is a biopolymer

based on sugar with no toxic effects, with a molecular

weight of 40,000. Starch can also be used, although it

is not soluble to the same extent as dextran. Dextran

is completely water-soluble.

One hundred milligrams of LL-tryptophan is added to1g of dextran (Mol. wt. 40,000) and dissolved in 6ml of

water (environmentally friendly). A few drops of this

solution are then cast on a microscope slide. For com-

parison, a film with only dextran is also made. The films

are initially dried at room temperature for 48h, followed

by 48h of drying in an oven at a temperature of 80 �C.After evaporation, the film formed is clear, and homoge-

neous. For measurement of absorption spectra, twospin-coated films are also fabricated. The spin coating

was performed for 120s at 800rev/s. The films were

257 nm laser

S HWP PBS

M

M

QWP

QWP QWP

POL

635 nm laser

FILM

D

Fig. 1. Holographic set-up for the fabrication of nano-period gratings

on a biopolymer surface.

1176 P.S. Ramanujam / Optical Materials 27 (2005) 1175–1177

dried in an oven at 80 �C for 24h before running the

spectra. Film thicknesses were measured with a Filmet-rics reflectometer and with a Dektak profilometer. The

thickness of the solution cast film was 32lm; the thick-

ness of the spin-coated dextran film was 0.9lm, while

that of the tryptophan/dextran film was 1.1lm.

UV–visible absorption spectra were recorded with a

Shimadzu UV-1700 spectrophotometer. A holographic

set-up shown in Fig. 1 is used to investigate the optical

behaviour of the film [2]. The two writing beams at257nm have the same circular polarization. A laser

beam at 257nm at an intensity of approximately

100mW/cm2 is used as the source. The diffraction grat-

ing so formed is then read out with a red diode laser with

an output power of 3mW. A TopoMetrix atomic force

microscope (AFM) was utilized for investigating surface

relief in the film.

3. Results and discussion

Tryptophan has an absorption band extending all the

way to 300nm and is known to undergo deamination on

irradiation with UV light. Fig. 2 shows the UV–visible

absorption spectra of dextran alone (curve a) and a dex-

200 250 300 350

0.0

0.5

1.0

1.5

2.0

2.5

c

b

a

abso

rban

ce

wavelength (nm)

Fig. 2. Absorption spectra of (a) dextran, (b) dextran with 10% w/w of

LL-tryptophan before irradiation with UV light at 257nm and (c) after

irradiation at 257nm.

tran film containing 10% LL-tryptophan (curve b). Dex-

tran does not display any absorption in the

investigated area of the UV spectrum. The peaks in

curve (b) are due to tryptophan. The film was then irra-

diated at 257nm at an intensity of 5mW/cm2 for 2h. The

resulting spectrum is shown as curve (c) in Fig. 2. Thedecrease in the intensity of the bands is due to the deam-

ination process.

Diffraction gratings were recorded in films of dextran

and dextran containing LL-tryptophan using the experi-

mental set-up shown in Fig. 1. Fig. 3 shows the diffrac-

tion efficiency of the films as a function of time. With

dextran alone, no diffraction is observed. This is consist-

ent with the fact that there is no absorption in dextran at257nm. With a tryptophan containing film, more than

1% diffraction efficiency at 633nm can be achieved. Thus

tryptophan is necessary for the recording of the gratings.

Photolysis of amino acids has been known for a long

time. Neuberg [3] studied solutions of amino acids after

exposure to sunlight in the presence of small amounts of

uranyl salts. Neuberg discovered that the effect of radi-

ation on amino acids was deamination, resulting in a re-lease of ammonia. This effect may be expected to

produce a dimensional change in the irradiated areas

of the film. Fig. 4 shows an AFM scan of the irradiated

area. It is seen that a nano-pattern consisting of peaks

and valleys with a peak height of approximately

120nm, and a period of 680nm is formed. The grating

period can be varied by varying the angle between the

interfering beams. The minimum period that can be gen-erated using technique is approximately 125nm. Larger

periods, up to several microns can be fabricated by

choosing a small angle between the beams. Typical size

of the grating in the present case is on the order of a few

square millimeter. The surface relief gratings have been

stable under ambient conditions for a year.

0 500 1000 1500 2000 2500 30000.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

diffr

actio

n ef

ficie

ncy

(%)

time (s)

Fig. 3. Diffraction efficiency in a film of (a) pure dextran and (b)

tryptophan in dextran as a function of time.

Fig. 4. Atomic force microscope scan of 10lm · 10lm area of the

irradiated film. The period of the grating is 680nm and the height of

the surface relief is 110nm.

P.S. Ramanujam / Optical Materials 27 (2005) 1175–1177 1177

I propose that large area gratings can be fabricatedthrough the use of a mask with the appropriate period.

Even sub-wavelength period metallized gratings can be

used. It has been shown that such sub-wavelength metal-

lized gratings actually lead to higher transmission (than

predicted by classical optics) through surface plasmon

effects [4]. A grating master, several square cms in size

can be placed on the polymer substrate, and irradiated

with UV light from a mercury lamp. It must be pointedout that all the materials used in this process are quite

inexpensive. As light sensitive chromophores, most

other amino acids, and in particular histamine and tyr-

osine with a large NH3 split-off [5] can be used. Serine

due to its cluster forming and chirality amplifying prop-

erties [6] can also be of potential use. Recently, Ponce-

Lee et al. [7] have recorded computer holograms insugar crystals. The mechanism of recording is different

from the one proposed in this article. In their case,

UV light is used for photopolymerizing the sugar

molecules.

In conclusion, I have demonstrated the recording of

stable surface relief grating in a LL-tryptophan/dextran

system with UV holography. Experiments on the bio-

compatibility of the films before and after irradiationare in progress.

References

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[3] C. Neuberg, Biochem. Z. 13 (1908) 304.

[4] W.L. Barnes, W.A. Murray, J. Dintinger, E. Devaux, T.W.

Ebbesen, Phys. Rev. Lett. 92 (2004), Art. No. 107401.

[5] J.P. Greenstein, M. Winitz, Chemistry of the Amino Acids—vol. 1,

John Wiley, New York, 1961.

[6] Z. Takats, S.C. Nanita, R.G. Cooks, Angew. Chem. Int. Ed. 42

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[7] E.L. Ponce-Lee, A. Olivares-Perez, I. Fuentes-Tapia, Opt. Mater.

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