자기조립기법을 이용한 GFP/Cytochrome c 복합박막제작

박 세정, 최 현구, 유 창준, 최 정우,이 원홍

서강대학교 화학공학과

Fabrication of GFP/Cytochrome c Hetero Films by self-assembly technique

Sei-Jeong Park, Hyun-Goo Choi, Chang-Joon Yoo, Jeong-Woo Choi,

and Won Hong Lee

Department of Chemical Engineering, Sogang University

C.P.O. BOX 1142, Seoul 100-611, Korea

Introduction

Ultra-thin film preparation techniques, such as Langmuir-Blodgett (LB) or self-assembly (SA), have been widely used to fabricate the highly ordered and close-packed molecular arrays of biological and/or organic molecules[1-2], and become one of the crucial technologies in the development of bioelectronic and molecular electronic devices. Recently, a novel and relatively simple technique for the preparation of ultra-thin film assembly, one of the SA techniques, has been developed using the alternate electrostatic deposition of oppositely charged polyelectrolytes, and extended to the applications to thin films fabrication of a wide range of charged molecules, such as globular proteins, enzymes, DNA, and viruses, etc.[3]. It was also reported that the several applications were demonstrated using the organized thin films by this technique[4]. This alternate electrostatic adsorption technique offers several advantages compared to the LB technique, especially for the biomolecular thin films fabrication; applicable to a large variety of water-soluble proteins, mild assembly conditions, keeping the biomaterials from the denaturation due to the spreading solvent, and not requiring the specialized instrumentation for the film preparation.

Hetero thin films, consisting of green fluorescent protein (GFP) and cytochrome c,  was fabricated by self-assembly technique. Protein formation onto the substrate was performed by the self-assembly technique. In order to make molecular assembly onto the gold substrate, cytochrome c was adsorbed onto the 11-mercaptoundecanoic acid (11-MUA) by electrostatic attractive force. 11-MUA can be spontaneously adsorbed onto the gold substrate via thiol group, leaving its one end (carboxyl group) toward surface, and thereby providing the template for cytochrome c adsorption. GFP can also be adsorbed onto the cytochrome c surface by electrostatic attractive force. The surface structure of absorbed GFP film onto the cytochrome c layer was analyzed using atomic force microscope (AFM) and scanning tunneling microscopy (STM).

Experimental

1. Materials

Cytochrome c (extracted from horse heart, type VI) and green fluorescent protein (GFP), and were used as an electron sensitizer and an electron acceptor, respectively. GFP (rEGFP) and cytochrome c were purchased from CLONTECH CO. (Palo Alto, California, USA) and Sigma Chemical Company (St. Louis, USA). 11-mercaptoundecanoic acid (11-MUA) was also purchased from Sigma Chemical Co. (St. Louis, USA) and used without further purification.

2. Methods

Electrochemically etched tungsten wire( 0.5mm diameter) was used as STM tip. STM tip etcher was TE-100 system(PSI, USA). The conditions for tip etching are that the current cutoff and voltage are 0.5AC amperes and 80V. STM microtopographic images were obtained by recording the tip height at a constant current mode (Park science Ins.) at an ambience temperature (21C). Tunnel current I=0.5nA, and a bias voltage Vbias =0.9mV.

For deposition of cytochrome c onto the gold substrate, cytochroeme c was adsorbed onto the 11-mercaptoundecanoic acid (11-MUA) by electrostatic attractive force. 11-MUA consists of thiol group and carboxyl group, thus the molecules can be easily adsorbed onto the gold substrate as shown in Fig. 1.

FIGURE 1. The schematic illustration of hetero films composed of cytochrome c and GFP

11-MUA was mixed with ethanol(0.011g/10ml) and then gold substrate was dipped into the solution for 24hours.

Result and Discussion

To verify the adsorption of the GFP molecules onto the cytochroeme c film and to find the maximum emission wavelength, fluorescence emission spectrum of cytochrome c/GFP hetero film deposited onto the gold substrate was investigated. The emission wavelength of GFP is at 510nm as shown in Fig. 2. It is given the evidence for that the GFP was deposited onto the cytochrome c film surface by electrostatic attractive force, because the net charge of cytochrome c is positive and GFP is negative.

To construct the molecular electronic devices with functional protein films, the formation of aggregated molecular films of cytochrome c and GFP have been considered as one of the most important factors dominantly affecting the device performance. The film formation of each protein was performed by self-assembly technique.

Self-assembly technique using electrostatic attractive force of molecules provided a high degree of density without loss of activity. The surface morphology of 11-MUA, cytochrome c and GFP films were verified as shown in Fig. 3. Self-assembled monolayers 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 close-packed 2-dimensional molecular array.

FIGURE 2. Characteristics of fluorescence emission spectrum of the cytochrome c/GFP hetero-film

As shown in Fig. 3(a), the height of 11-MUA film is about 20□ and the size of molecule is about 3□ in 12 □ scan size. As shown in Fig. 3(b), spherical type of aggregations of cytochrome c molecules was observed and this image indicates that the cytochrome c molecules are adsorbed onto the 11-MUA film surface. Also, as shown in Fig. 3(c), GFP molecules were observed, and this is indicated that the GFP molecules are adsorbed onto the cytochrome c film surface. The aggregation size of cytochrome c and GFP molecules is about 0.1~0.2m.

(a)                    (b)                       (c)

FIGURE 3. AFM analysis of 11-MUA film onto the gold substrate(a), cytochrome c(b), and GFP(c)

As shown in Fig. 4, the morphology of cytochrome c and GFP were observed using the STM. Imaging of proteins by STM can be problems due to high probe-sample forces, which may result in displacement or damage of the part of proteins. But a part of initially deposited cytochrome c molecules with strong adsorption onto the gold substrate by 11-MUA can be visualized by STM as shown in Fig. 4(a). Also, as shown in Fig. 4(b), the GFP molecules were well deposited onto the cytochrome c film surface. In this image, the height of cytochrome c molecule is about 80□ and diameter is 0.1□m. Also, the height of GFP molecule is about 130□ and diameter is 0.1□m. These results indicate that the cytochrome c and GFP molecules are adsorbed in solution by molecular interation and the tunneling current is dependent on the tip position over the molecule and molecule space orientation with respect to the substrate surface and the electron transport features in individual protein molecule via STM.

(a)                      (b)

. FIGURE 4. STM analysis of film surface of cytochrome c(a) and GFP(b)

This method has simple but powerful tool for deposition of bio-material onto the substrate, because of the easy handling, and no wholly heating. In this study, the authors mainly focused on the method for fabrication of biomolecular thin films composed of cytochrome c and GFP onto the substrate by self-assembly technique and it has a potential application for fabrication of bioelectronic device.

Acknowledgement

This work was supported by grants from Korea Science and Engineering Foundation (KOSEF : 20006-307-03-2)

References

[1] Ulman, A., An Introduction to Ultrathin Organic Films from Langmuir-

Blodgett to Self-Assembly, Academic Press Inc., New York, 1991.

[2] Allara, D. L., Biosensors & Bioelectronics, 10, 771,.1995

[3] Decher, G., J. D. Hong, and J. Schmitt, Thin Solid Films, 210/211, 831,            1992

[4] M□ller, W., H. Ringsdorf, E. Rump, G. Wildburg, X. Zhang, L. Angelmaier,        W. Knoll, M. Liley, and J. Spinke, Science, 262, 1706, 1993