STM을 이용한 Fusion Protein으로 구성된 자기조립박막의 정류특성 분석

박 세정, 최 정우, T. Nagamune*, 이 원홍

서강대학교 화학공학과

도쿄대학교 화학·생물공학과*

Investigation of Rectifying Property of Self Assembled Monolayer Composed of Fusion Protein with STM

Sei-Jeong Park, Jeong-Woo Choi, T. Nagamune* and Won Hong Lee

Department of Chemical Engineering, Sogang University

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

Department of Chemistry and Biotechnology Graduate School of Engineering

The University of Tokyo 7-3-1, Japan*

Introduction

The substantial interest recent years was in the field of supramolecular architecture with protein immobilization and formation of protein mono- and multilayers on solid surface via Langmuir-Blodgett (LB) and self-assembling technique[1]. Manufacturing and study of such supramolecular protein structures and investigation of long-range electron transfer reactions in redox proteins is important for biomaterials science, biomedical and biotechnological applications, for development of micro- and nanocomponents of biosensor and bioelectronics system[2]. Recently, immobilized proteins monolayers were investigated with modern high resolution methods as Atomic Force Microscopy (AFM)[3] and Scanning Tunneling Microscopy (STM)[4]. STM allows to visualized the molecular nanostructure and to study redox processes in single molecules and clusters.

In this study, the fusion protein, composed of cytochrome b562 and GFP which used as an electron acceptor and an electron sensitizer, was deposited onto Au substrate by self assembly technique. The unidirectional electron flow from GFP to cytochrome b562 was confirmed by current-voltage characteristic with STM.

Experimental

1. Materials

The fusion protein was designed by genetic engineering. In vector construction, the linker sequence between cytochrome b562 and GFP is Gly-Ser and the fusion protein was expressed in E. coli strain. The cytochrome b562 part of fusion protein was expressed mainly the apo form, however, it could be easily reconstituted with exogenous hemin. Figure 1 shows the Expression vector of cytochrome b562-GFP fusion protein.

FIGURE 1. Expression vector of cytochrome b562-GFP fusion protein

2. Methods

STM microtopographic images were obtained by the tip height at a constant tunneling current in a modified Nanoscope STM device (Digital Instruments) as an ambient temperature. Tunneling current I = 135pA and 250pA, and a bias voltage Vbias = 500mV. The images were stable and reproducible. The measurement procedure consisted in obtaining a topographic image, and then positioning the tip above an isolated protein molecule for the tunneling spectroscopy measurements. I-V curves were recorded while temporarily disconnecting the feedback circuit, and therefore, with constant tunneling junctions parameters. During the I-V measurements topographic images were monitored regularly to ensure that the tip did not drift apart. Electrons flowed from the tip to the substrate for positive V value.

Result and Discussion

To create stable fusion protein arrays on Au substrate for STM measurements, we have used a method based on self assembly technique. The fusion protein have a thiol group, so this protein is adsorbed onto the Au substrate. Figure 2 shows STM topographic images of fusion protein monolayer deposited by self assembly technique onto the surface of gold substrate. STM images of the film surface were reproducible and revealed globular structures about 500Å diameter. Imaging of proteins by STM can be a problems due to high probe-sample forces which may result in displacement of the part of physisorbed protein. It is possible that only a part of initially deposited fusion protein molecules with most strong adsorption to substrate surface can be visualized by STM in our experiments, and weakly adsorbed molecules were removed by STM tip as shown in figure 2(a).

(a)                                 (b)

FIGURE 2. STM investigation of fusion protein adsorbed onto the gold substrate

(a : 3□m, b : 1□m)

Typical I-V curves in different STM tip positions and step bias over the protein molecule are shown on figure 3. These curves are typically observed for single tunnel junctions. The complex features of electron tunneling in substrate/fusion protein/STM tip system observed can be determined by the tunnel junctions parameters, energy separation in molecular orbital system and its combination with Coulombic single electron tunneling effects.

(a)                                  (b)

FIGURE 3. Current-Voltage characteristics of fusion protein with STM tip

(Step bias a : 0.1V, b : 0.05V)

The I-V curve can be complicated also by molecular orbital anisotropy and effects of pathways of intraprotein electron transfer in protein when tunneling current is correspondingly dependent on the tip position over the molecular and on the molecule space orientation with respect to the substrate surface.

In this study, molecular photodiode composed of fusion protein based on biological electron transport in the initial process of photosynthesis were investigated with STM system. The proposed method for investigation of rectifying property by the STM system can provide the applicability of molecular photodiode composed of biomolecules to the electronic device.

Acknowledgement

This work was supported by grants from Korea Science and Engineering Foundation (KOSEF : 20006-307-03-2) and the National Program for Tera-level Nanodevices of the Ministry of Science and Technology as one of the 21 century Frontier Programs

References

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[2] Swalen J.D., Allara D.L., Andrade J.D., Chandross E.A., Garoff S., Israelchvili J.,     McCarthy 7.T.J., Murray R., Pease R.F., Rabolt J.F., Wynne K.J., Yu H.,           Langmuir, 932, 3 (1987)

[3] Patel N., Davies M.C., Heaton R.J., Roberts C.J., Tendler S.J.B., Williams P.M.,     Appl. Phys., S569, A66 (1998)

[4] Welland M.E., Miles M.J., Lambert N., Morris V.J., Coombs J.H., Pethica J.B.,       Int. J. Biol. Macromol., 29, 11 (1989)