자기조립기술로 제작된 박테리오로돕신 박막의 특성

한 건국, 최 현구, 민 준홍*, 최 정우, 이 원홍

서강대학교 화학공학과

고려대학교 생명공학원*

Characteristics of Bacteriorhodopsin Thin Films Fabricated by

Self-Assembly Technique

Keon-Kook Han, Hyun-Goo Choi, Junhong Min*, Jeong-Woo Choi, Won Hong Lee

Department of Chemical Engineering, Sogang University

Graduate School of Biotechnology, Korea University*

Introduction

Thin film preparation techniques, such as Langmuir-Blodgett (LB) and Self-Assembly (SA), have been widely used to fabricate the ordered and close packed molecular thin films of biological and/or organic molecules.

Self-assembled monolayers (SAMs) are formed at a solid-liquid interface by molecules having a head group with a strong affinity to the solid surface and a long linear alkyl chain that provides ordering on the surface. There are several bonds for binding between surface and head group. The binding affinity to the solid surface is provided by a covalent chemical reaction, as in silanes on silicon dioxide, by an ionic interaction, as in carboxylic acids on alumina, or by a charge transfer complex, as in alkanethiols on noble metals etc [2]. SAMs have several advantages such as easy film preparation and long term stability. In particular, long chain alkanethiols (HS(CH2)nX) SAMs adsorbed on gold from solutions have been recognized as a very simple and widely applicable technique to form well ordered and densely packed monolayers, in which thiols are believed to be chemisorbed on gold surface as thiolates [3].

In this study, the self-assembled monolayers of 11-mercaptoundecanoic acid (11-MUA) on gold surface were used as a template for the adsorption of bacteriorhodopsin (bR). Using poly-L-lysine (PL), as a bridging molecule for bR adsorption onto SAMs of 11-MUA, bR thin films were fabricated. The surface morphology of the molecular thin films was analyzed by atomic force microscopy (AFM).

Materials and Methods

Materials

Bacteriorhodopsin (bR), a light-driven proton pumping protein embedded in the purple membrane of Halobacterium salinarium, was purchased from Sigma Chemical Co. (St. Louis, USA). 11-mercaptoundecanoic acid (11-MUA), poly-L-lysine (PL, M.W. 70,000~150,000), ethyl alcohol, HEPES (N-[2-hydroxyethyl] piperazin-N'-[2-ethane sulfonic acid]), and other reagents were also purchased from Sigma Chemical Co. (St. Louis, USA) and used without further purification.

SAMs of 11-MUA on Gold Substrate

Gold substrate was prepared by thermal evaporation onto pre-treated Si wafer. The conditions for the evaporation of gold were as follows; 1h preheating of evaporating system, 10-4 torr pressure, and 100∼200nm thickness. The prepared gold substrate was immersed in the 2 mM  ethanolic solution of 11-MUA for 24 hrs, and then rinsed with absolute ethanol and D.I. water before nitrogen drying.

Adsorption of PL onto SAMs of 11-MUA

Prepared SAMs of 11-MUA were immersed into the 0.02w/v % PL solution (pH=8.0) for 2 hrs, and then dried for 24 hrs. Polycationic PL could be strongly adsorbed on anionic MUA surface, leaving cationic sites for binding with anionic bR molecule [4].

Figure 1. A schematic illustration of the bR thin films composed of

bR/PL/SAMs/AU films.

Adsorption of bR onto PL/SAMs/Au Films

bR was adsorbed onto the template using an electrostatic attraction. The substrate composed of PL/SAMs/Au films was treated with various concentration of bR solution, prepared by using HEPES buffer (pH, 8), for 1 hr. The bR-adsorbed thin films was rinsed with the buffer solution and finally with D.I. water to remove any salt remaining in the bR film surface, and then dried in the petri dish. A schematic illustration of the prepared thin films composed of bR/PL/SAMs/Au films was shown in Figure 1. The adsorption of each molecule was elucidated by AFM, fluorescence analysis, and ellipsometry.

Results and Discussion

SAMs of 11-MUA are spontaneously formed onto gold surface by the covalent bonding between the thiol groups of 11-MUA and the gold surface. Since 11-MUA has a long alkyl chain, which can provide van der Waals attractive force among the molecules, it can form a strong and densely packed monolayer structure. The AFM image of the SAMs of 11-MUA was shown in Figure 2(b). Although a blurred image was obtained due to the surface end group of the SAMs, carboxylic group, a fairly well organized molecular array could be observed in the nanoscale.

The structure and conformation of PL are well characterized in bulk solution, and it has been known that PL exist as a form of random coil [4]. Since the isoelectric point of PL is 9.74, the net surface charge of PL is positive in the pH below 9.74, and therefore it can be adsorbed onto the negatively charged surface of the SAMs. Because the surface end group of SAMs is a negatively charged carboxylic group, PL which has amine group as the positively charged unique R group can be adsorbed onto the surface end groups of the SAMs of 11-MUA through electrostatic interaction and/or intermolecular hydrogen bonding. These intermolecular interaction could make the thin films more stable, thus the modified surface of SAMs can be used as a template for the bR adsorption. In order to prove the adsorption of PL onto SAMs of 11-MUA, fluorescence analysis was performed. NBD-Cl, which can form a fluorescence emitting material when it is mixed with a molecule having a free amine residue, was used to form a NBD-Cl/amine complex. Since PL has amines as the unique R group, it can form a fluorescence emitting material by mixing with NBD-Cl. According to the light excitation of the NBD-Cl-treated PL layer, the characteristic peak of fluorescence emission could be observed (data not shown). In order to clarify the PL adsorption onto SAMs, the AFM analysis was also performed. The AFM image of the PL layer was shown in Figure 2(c). It could be considered that the PL molecules were adsorbed onto the SAMs with aggregated pattern.

The isoelectric point of bR is 4.85, and it is embedded in the lipid bilayer in which the head groups of the lipid can be a negatively charged ones depending on the surrounding pH. At around the neutral pH, the net surface charge of the purple membrane patches is negative, therefore it can be adsorbed onto the positively charged PL layers. Figure 2(d) shows the bR layers adsorbed onto PL layers. It can be observed that the bR-embedded purple membrane patches were adsorbed onto the PL layers, as a fairly well ordered structure.

Figure 2. AFM Images of (a) Au film, (b) SAMs of 11-mercaptoundecanoic acid,

(c) Poly-L-lysine, and (d) Bacteriorhodopsin.

It is concluded that self-assembly technique could be successfully applied to the fabrication of bR thin films. In order to prepare the more stable and organized film structures, the optimization of the fabrication conditions should be required.

Acknowledgement

This work was supported by the Korean Ministry of Science and Technology under the Brain Science Research Program (98-BR-02-04-A-03, 1998).

References

[1] Allara, David L., Biosensors & Bioelectronics, 10, 771-783, (1995)

[2] Delamarche, E., Michel, B., Thin Solid films, 273, 54-60, (1996)

[3] Tamada, K., Hara, M., Sasabe H., and Knoll, W., Langmuir, 13, 1558-1566, (1997).

[4] Jordan, C. E., Frey, B. L., Kornguth, S., and Corn, R. M., Langmuir, 10, 3642-3648, (1994).