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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: [email protected] (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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