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Current Applied Physics 6 (2006) 760–765

Charge trap in self-assembled monolayer of cytochromeb562-green fluorescent protein chimera

Jeong-Woo Choi a,*, Yun-Suk Nam a, Bum Hwan Lee b,Dong Jun Ahn c, Teruyuki Nagamune b

a Department of Chemical and Biomolecular Engineering, Sogang University, 1 Shinsu-dong, Mapo-gu, Seoul 121-742, Republic of Koreab Department of Chemistry and Biotechnology, The University of Tokyo, Tokyo, Japan

c Department of Chemical and Biological Engineering, Korea University, Seoul 136-701, Republic of Korea

Received 21 June 2004

Available online 4 June 2005

Abstract

The self-assembly layer consisting of fusion protein is investigated in molecular-scale for the construction of bioelectronic device.

Cytochrome b562 and green fluorescent protein were used as an electron acceptor and a sensitizer in the molecular layer by mim-

icking the photosynthesis. Self-assembled monolayer of fusion protein was formed on Au coated glass. The formation of fusion pro-

tein layer onto the Au substrate was observed by the surface plasmon resonance measurement. The surface of fusion protein layer

was observed and analyzed by the scanning tunneling microscopy observation. For embodiment of the molecular electronic device,

molecular arrays of fusion protein SA layer were formed by micro contact printing. Surface charge distribution of fusion protein SA

layer was measured to confirm an electrical conductivity by electrostatic force microscopy observation.

� 2005 Elsevier B.V. All rights reserved.

PACS: 85.65.+h

Keywords: Bioelectronic device; Cytochrome b562; GFP; Self-assembly layer

1. Introduction

The transfer of an electron from one side of a mole-cule to the other or between molecules is one of the most

fundamental and ubiquitous processes in electronic

materials and biological systems [13]. The control and

exploitation of this process in organized molecular sys-

tems is a major proposition for molecular electronics

and bioelectronics [14]. Progress in molecular electronic

devices engineering is still rather modest due to prob-

lems associated with the elucidation and effective control

1567-1739/$ - see front matter � 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.cap.2005.04.035

* Corresponding author. Tel.: +82 2 705 8480; fax: +82 2 711 0439.

E-mail address: jwchoi@ccs.sogang.ac.kr (J.-W. Choi).

of such structures and interactions at the nanometer

level.

Photoinduced electron transport processes in nature,such as photoelectric conversion and long-range elec-

tron transfer in photosynthetic organisms, are known

to be occurred not only very efficiently but also unidirec-

tionally guided by biomolecular functional groups

[13,8]. The concepts for the development of new func-

tional bioelectronic devices can be inspired from the bio-

logical systems such as the electron transfer chain or the

photosynthetic reaction center. By mimicking the orga-nization of the functional molecules in a biological elec-

tron transfer system, the biomolecular electronic devices

can be realized artificially. In the initial process of

photosynthesis, a biological electron transfer system,

Fig. 1. (a) Electron transfer diagram of GFP/cytochrome b562 fusion

protein and (b) structure of fusion protein.

J.-W. Choi et al. / Current Applied Physics 6 (2006) 760–765 761

photoelectric conversion occurs and then long-range

electron transfer takes place very efficiently in one direc-

tion through the biomolecules [10]. The specific energy

and electron transfer take place on a molecular scale

due to the redox potential difference as well as the elec-

tron transfer property of functional molecules, espe-cially the electron acceptor, sensitizer, and electron

donor [9].

Molecular thin films fabricated by the appropriate

techniques can be used as model systems of the corre-

sponding photosynthetic reaction center in the biologi-

cal system. Substantial interest in recent years has

focused upon thin film fabrication or the formation of

biomaterials mono- and multi-layers on solid surfaces,by using the Langmuir–Blodgett (LB) film technique

or SA technique [17,2].

Based on these techniques, various artificial molecu-

lar devices have been fabricated to mimic the electron

transport function of biological photosynthesis. Fujihira

et al. have reported the electrochemical LB photodiode

consisting of three functional organic molecules or as

an aligned triad on the electrode, which worked inelectrolyte solution [9,18]. Isoda et al. investigated the

optical and electrical characteristics of a molecular

photodiode consisting of flavin-porphyrin hetero-LB

films [12]. The authors investigated the molecular diode

consisting of hetero organic LB film of four functional

organic molecules ferrocene, flavin, viologen, and

TCNQ used as an electron donor, sensitizer, relay and

acceptor, respectively [1]. Further we have investigatedthe fabrication of biomolecular photodiodes consisting

of hetero proteins/organic molecular layers such as

GFP, viologen, TCNQ and cytochrome c, in which het-

ero layers were formed by LB film technique [3,4]. The

photoinduced electron transfer of the biomolecular

photodioe with metal/insulator/metal (MIM) structure

was observed [5,6]. Nagamune et al. reported two types

of fusion protein which consisted of two functional elec-tron transfer protein GFP and Cytochrome b562 [19].

Also, Nagamune et al. reported the biophotodiode con-

sisting of fusion protein (DM type) layer fabricated by

SA technique without including organic molecular lay-

ers [16]. Photoswitching and rectifying function of

fusion protein SA layers have been investigated in our

previous study [16]. However, molecular scale pattern

formation of fusion protein SA layer and its electricalproperty for molecular device has not been investigated

yet.

In this study, GFP/Cytochrome b562 structured fu-

sion protein SA layer was formed. SA layer was verified

by surface plasmon resonance (SPR) and scanning tun-

neling microscopy (STM) observation. To form the

molecular scale biodevice, molecular pattern forma-

tion of fusion protein SA layer was investigated by mi-cro contact printing and its electrical property was

observed.

2. Materials and methods

2.1. Fusion protein SA layer

The fusion protein layer consisted of GFP and cyto-

chrome b562, which functioned as a sensitizer and anelectron acceptor, was synthesized by genetic recombi-

nation [19]. Fig. 1 shows the electron transfer diagram

of a molecular array consisting of GFP/cytochrome

b562 fusion protein SA layer. By the adsorption of

fusion protein onto Au substrate, molecular film was

fabricated. To deposit the fusion protein onto the Au

substrate, thiol (–SH) functional group was adsorbed

on Au surface. Au substrate was dipped into thefusion protein solution for 24 h, and then SA layer

was formed on Au substrate due to the thiol group on

its surface.

To confirm the fusion protein adsorption on Au sub-

strate, SPR measurement and STM observation of

fusion protein SA layer was investigated. SPR was mea-

sured using a Multiskop SPR spectrometer (Optrel

GbR, Germany). STM observation was performedusing the EasyScan STM (Nanosurf, Swiss). The typical

set-point, external bias, and scan rate of STM measure-

ment were 0.1 nA, 1 V, and 3 Hz, respectively.

420 440 460 480 500 520 540 560 580 6000.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Abs

Wavelength

Fig. 2. Absorption spectrum of fusion protein.

Fig. 3. SPR spectrum of adsorbed fusion protein on Au substrate.

762 J.-W. Choi et al. / Current Applied Physics 6 (2006) 760–765

2.2. Pattern formation by micro contact

printing method

A micro patterned PDMS stamp was used for micro

contact printing. A conventionally fabricated silicon

master was replicated by pouring liquid prepolymer ofPDMS onto the master and then curing it. After its re-

lease, the stamp served as the vehicle to transfer the

‘‘ink,’’ in this case, fusion protein to a substrate by con-

tact. The stamp was covered with a solution of fusion

protein for inking and was subsequently dried in air.

The stamp was then placed on the Au substrate for

about 1 min without any extra force. Then, the fusion

protein molecules were transferred from the stamp tothe substrate. After the stamp was displaced, substrate

on which the pattern of fusion protein was formed was

rinsed by DI water. This procedure is necessary to re-

move fusion protein non-specifically adsorbed by weak

physical interactions [15]. Pattern formation was con-

firmed by STM measurement.

2.3. Electrical property measurement

Electrostatic force microscopy (EFM) is capable of

measurement of topography and other physical proper-

ties simultaneously at the same sample positions. In

addition, EFM is capable of measuring variations in

surface potential and capacitance gradient on the sam-

ple surface. The efforts have revealed a much broader

potential for the measurement of local mechanical prop-erties and electrical properties such as surface potential,

surface charge by EFM [11].

The electrical property of fusion protein SA layer was

measured by surface charge distribution under light illu-

mination. Surface charge distribution of fusion protein

SA layer was measured to confirm an electrical conduc-

tivity by EFM. The conventional non-contact mode

EFM is used to maintain constant tip-sample distance,thus producing topographic images. In addition, Vac

and Vdc are applied between the conducting tip and

sample. The frequency f, time constant, sensitivity,

amplification, and harmony are 17 kHz, 10 ms, 20 mV,

5 V, and 1, respectively.

3. Results and discussion

3.1. Verification of fusion protein SA layer

To verify the electron transfer property from GFP to

cytochrome b562, absorption spectrum of fusion protein

was measured and shown in Fig. 2. It shows a sharp sor-

et band at around 420 nm and two broad bands around

540 nm of cytochrome b562, with an additional charac-teristic band of GFP at 488 nm [16]. Absorptions at 528

and 558 nm indicate reduced form of cytochrome b562.

Generally, cytochrome b562 absorbs around 540 nm.

However, if cytochrome b562 got electron, absorption

band of cytochrome b562 is separated into two broad

bands at 528 and 558 nm. From the absorption feature,

it was concluded that the fusion protein had been suc-

cessfully constructed, and preserved its electron transfer

property.

To verify the fusion protein SA layer, SPR and STMmeasurement was investigated. Fig. 3 shows the change

of SPR minimum position by adsorption of fusion

protein deposited on Au substrate. As the fusion protein

adsorbed, the value of SPR minimum position was shift

to higher angles. SPR minimum reflectivity position was

shifted significantly from 43.15� ± 0.02� to 43.85� ±

0.02� by the fusion protein adsorption on Au substrate.

This result suggests that the fusion protein were wellimmobilized on Au substrate by thiol adsorption process.

Surface morphology of fusion protein SA layer was

shown in Fig. 4 by STM observation.

Fusion protein molecules were well deposited onto

the Au substrate. In the STM image, the size of ad-

sorbed fusion protein aggregates were about 10–25 nm.

Fig. 4. STM image of fusion protein SA layer.

J.-W. Choi et al. / Current Applied Physics 6 (2006) 760–765 763

Coverage of fusion protein adsorption on Au substrate

is about 75%. Fusion protein molecules were chemically

immobilized onto Au substrate by thiol functionalgroup. It can be suggested that the fusion protein mole-

Fig. 5. (a) Schematic diagram of pattern formation of fusion protein by dire

formed on Au by direct micro contact printing, and (c) line profile of STM

cules were aggregated before SA layer formation. Never-

theless, fusion protein molecules were fairly uniformly

adsorbed on Au substrate via thiol functional group.

Also the STM image of fusion protein SA layer shows

that the electron transfer takes place from GFP to the

cytochrome b562. Thus the electron transfer throughthe molecular array consisting of GFP/cytochrome

b562 fusion protein was verified.

3.2. Pattern formation of fusion protein SA layer

Fig. 5(b) shows that the fusion protein dot arrays

were formed directly by micro contact printing on the

Au substrate in micrometer-scale. The size of the fusionprotein dots was 2 lm in diameter and the spacing dis-

tance was 7 lm, which is in good agreement with the

geometry of the original PDMS stamp having protruded

dot regions of 3 lm in diameter and 10 lm in spacing

[15]. After rinsing the substrate with DI-water, the aver-

age height of protein is slightly decreased since non-

specifically attached fusion protein molecules to the

substrate were removed [15]. The patterns of fusion

ct micro contact printing, (b) STM images of fusion protein SA array

image.

Fig. 6. Topography of patterned fusion protein SA layer; (a) non-contact mode AFM image and (b) EFM image.

764 J.-W. Choi et al. / Current Applied Physics 6 (2006) 760–765

proteins obtained by micro contact printing method had

high contrast and resolution. This indicates that the

protein pattern has the mechanical stability and the

adsorbed proteins had no surface diffusion.

3.3. Surface charge distribution of fusion protein

SA layer

The fusion protein layer was formed by the SA

technique since these methods provide a high density

without activity loss. To verify electrical property in

molecular-scale, EFM morphology was observed. Theelectron flow from GFP to cytochrome b562 can be

observed in the molecular level.

Fig. 6 shows the EFM topography of fusion protein

adsorbed onto the Au substrate where the bright region

consisted of fusion protein SA layer and the dark region

consisted of Au substrate. In the image that was ob-

tained by non-contact mode EFM for charge distribu-

tion on fusion protein adsorbed layer, the brightregion indicated charge trapped fusion protein regions

and the dark region indicated the non-charge trapped

Au region. This result indicated that the electron is

transferred from GFP to cytochrome b562, and then

the electron is trapped into cytochrome b562.

In the fusion protein SA layer consisting of GFP/cyto-

chrome b562 on Au aubstrate, the electrons of GFP (sen-

sitizer, S) molecules are initially excited from theirground state to the excite state (S*) by the 488 nm light

illumination. The photo-excited electrons return to their

ground state and in so doing the green fluorescence emits

at 510 nm. However the electron acceptor, cytochrome

b562 (A), was exposed to excited sensitizer (S*), some

of photoexcited electrons of S* are separated and trans-

ferred to A as S+/A� [7]. Thus the photoinduced one-way

charge transfer could be achieved and the photocurrentcan be generated. The sensitizer function of GFP in

macroscale was proved by the transient photocurrent

measurement of hetero films of GFP/organic mole-

cules/protein molecules in MIM structured device [5].

Also the one-way electron transfer property of fusionprotein SA layer was observed by the measurement of

rectifying property [16]. In Fig. 6(b), Surface potential

of fusion protein adsorbed region is about �29 mV.

However, non-fusion protein adsorbed region is about

�91 mV. The potential gap of 62 mV indicates a trapped

charge in fusion protein. These results suggest that

charges were transported from GFP to cytochrome

b562 only and trapped in cytochrome b562. Thus thehetero structure fusion protein of GFP/cytochrome

b562 can be functioned as the molecular memory.

4. Conclusions

In this study, fusion protein SA layer consisting of

GFP/cytochrome b562 was investigated, which was de-signed based on the biological photosynthesis system.

GFP and cytochrome b562 were used as a sensitizer

and an electron acceptor, respectively. The fusion pro-

tein SA layer had the charge trapping in molecular scale

due to the photoinduced electron transfer. It can be con-

cluded that the proposed fusion protein SA layer was

functioned as the molecular memory with charge trap-

ping in molecular-scale. The proposed molecular layer,which mimics the biological photosynthesis system,

can be suggested that the charge trapping of biomole-

cules such as proteins can be applied to construct the

nano-scale biomolecular memory.

Acknowledgment

This work was supported by grants from the contribu-

tion of Advanced Backbone IT Technology Development

J.-W. Choi et al. / Current Applied Physics 6 (2006) 760–765 765

Project (IMT2000-B3-2) of the Ministry of Information

and Communication.

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