Iwamoto M., Kwon Y.-S., Lee T. (Eds.) Nanoscale Interface for Organic Electronics

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Nanoscale Bioelectronic Device Consisting of Biomolecules 349

nanotube and Seeman et al. invented DNA mechanical device. Also,

Tseng et al. developed a memory device which was composed of tobacco

mosaic virus. Fugibayashi et al. makes DNA-based algorithm systems

and computing device. Kershner et al. also developed placement and

orientation of individual DNA shapes on lithographically patterned

surfaces for making integrated chip. The other genius researchers

developed valuable concepts and ideas regarding bioelectronics.

8-17

Thus,

electronic switching for electronic device and for increasing electronic

device density is known to exist in a wide variety of biomaterials.

To make the bioelectronic device which was composed of

biomaterials, it needs an important consideration of thin film preparation

methods. Recently, thin layer preparation technology is regarded as

important role in the fabrication of bioelectronic device. Conventionally,

when immobilizing the biomaterial on an inorganic surface, immobilizing

methods are generally classified two types. One is langmuir-blodgett

Fig. 1. Schematic diagram of Moore’s Law and diversification concerning semiconductors

taken from the international technology roadmap.

J.-W. Choi, T. Lee and J. Min 350

(LB) technique and another is self-assembly monolayer (SA) technique.

These two methods are comprehensively used for immobilizing

biomolecules on a substrate. The LB technique can be very powerful for

making nanoscale film which contains biomolecules. Because, it is

available to process in biological amphiphilic layers, and it can be

considered biological membranes. Also, this membrane has been

shown to its activity when transferred to inorganic surface. In these

reasons, there are many researchers studied this technique for making

bioelectronic device such as photodiode and biosensor.

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The SA

technique is also promising tool for constructing nanoscale layer which is

composed of organic molecules for various applications such as field-

effect transistor (FET), single-electron transistor (SET) and the others.

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Generally molecule which is used for self-assembly contains a head

group, and a terminal group and its body group. Head group which is

consisted of alkanethiols is used to react with solid surface and terminal

group is reacted with biomolecules. The body group constitutes

hydrocarbon chain structure. In many case, this self-assembly molecule

was regarded as a chemical linker for immobilizing the biomaterial. In

many case, it has been shown that sulfur compounds coordinate strongly

many metal substrates, for example, Au, Ag, Ti, Cu and Pt. In nowadays,

Au substrate has been widely used to make SAM. It is hard to oxidize in

the ambient conditions.

In previous time, our group proposed some bioelectronic device

composed of biomolecular hetero langmuir-blodgett (LB) film. The

purpose of this electronic device was to accomplish electronic functions

of the molecular photodiode, switching device for photocurrent

generation and rectifier function.

30-33

Recently, our group introduced the

redox protein for making biomemory device by self-assembly technique

and the principles of this protein based biomemory device were

demonstrated in a previous study by our research group.

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Here, we review the working principle, fabrication technologies, and

electronic function proofs of biomolecules based on bioelectronic device

and also briefly survey nanobio elelctronic device which could be one of

alternative standard device format in human-mechanics interfacing

system.

Nanoscale Bioelectronic Device Consisting of Biomolecules 351

2. Nanoscale Biomolecule Layer Fabrications

2.1. Langmuir-Blodgett (LB) Technique

The Langmuir-Blodgett (LB) technique is named after Irving Langmuir

and Katharine B. Blodgett. The Langmuir-blodgett film is composed of

layers of organic molecules, deposited from the substrate surface of a

liquid onto a solid by dipping the solid surface into the liquid. And then,

the organic molecular layer is adsorbed with each dipping step. The thin

films are assembled vertically and are usually contained amphiphilic

molecules with a hydrophilic head group and a hydrophobic tail group

such as fatty acids.

40-44

2.1.1. Optimal Deposition Condition of Chlorophyll a

Langmuir-Blodgett Film

To determine the optimal deposition conditions, the p–a isotherm of

chlorophyll a was analyzed at various temperatures and pH conditions.

The pressure/area isotherm (p–a isotherm) is called the dependence of

the surface pressure on the unit area per molecule. This isotherm usually

depicts the formation of the thin layer and phase transitions.

The p–a

isotherm of chlorophyll a is described in Fig. 2. When it reached about

Fig. 2. p–a isotherms of chlorophyll a langmuir-blodgget layers at various pH conditions.

Reprinted with permission from Ref. 48 Y. S. Nam et al., Optimal deposition condition

of chlorophyll a Langmuir–Blodgett film, Mater. Sci. Eng. C., 24, 35 (2004), Copyright

Elsevier (2009).

J.-W. Choi, T. Lee and J. Min 352

25-35 mN/m as the monolayer was compressed, the isotherm depicts

that the surface pressure constantly increased. This first condensed

monolayer region was originated by a transition region, which showed a

rapid increase in compressibility, when it formed a second condensed

monolayer region. At first time, compression didn’t converse the surface

pressure. In this case, the molecules are nearly removed from each other

and do not interact. This area is referred as a two dimensional (2D)

gaseous state (GS). As compression is continued, surface pressure

increases. This area is called as the 2D liquid state (LS). In this state,

each molecule begins to interact with each other. A 2D solid-like (SL)

state will be reached when the compression of the thin layer is continued.

Molecules are closely condensed and the thin layer can be regarded as

a 2D crystal. The p–a isotherm of Fig. 2 at various phase pH conditions

are presented to this phenomena in Fig. 2. Here, GS appeared in the

area above 110-170 Å

/molecule, LS from 110 to 170 Å

/molecule to

around 50 Å

/molecule and SL in the area below 50 Å

/molecule. The

area/molecule and the surface pressure of the transition regions of the

chlorophyll a 2D monolayer of the GS, LS and SL states were all

increased when increasing the pH condition from 5 to 10. It is provided

that chlorophyll a molecules offer a promising basis for the applications

of LB layers due to characteristics with respect to pH. Since the diameter

of chlorophyll a head group is around 8-10 Å

46,47

The region of

chlorophyll a single molecule on water phase is about 50-75 Å

. The

chlorophyll a LB layer deposited at area/molecules under 50 Å

forms

the aggregates. In this research, the p–a isotherms of chlorophyll a were

investigated at various pH conditions and temperatures. Thus, it is

demonstrated that stable deposition conditions for the chlorophyll a LB

layer is 25°C and pH 7.0.

2.2. Self-Assembly (SA) Technique

The self-assembly (SA) could be defined as the spontaneous organization

of molecular units into specific ordered structures by non-covalent

interactions. The interactions were responsible for the formation of the

self-assembled system act on a strictly local level in the nanostructure

builds itself.

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Nanoscale Bioelectronic Device Consisting of Biomolecules 353

2.2.1. ph Effect of Recombinant Protein Monolayer on Au Surface by

Self-Assembly Technique

The redox property of cysteine-modified protein layer was investigated

by cyclic voltammetry method. The redox reaction was reversible,

suggesting that the self-assembled protein maintain the reduction-

oxidation properties.

We take an optimal pH conditions by pH-control

experiments from pH 5.0 to the pH 9.0. The result was described in

Fig. 3(A). As seen in Fig. 3(B), the reduction potential was 146 mV and

Fig. 3. Cyclic voltammogram of cysteine-modified azurin. (A) a: pH 5.0, b: pH 6.0,

c: pH 7.0, d: pH 8.0, e: pH 9.0 (10 mM HEPES, pH 7.0, azurin sample concentration =

0.1 mg/ml, electrode area = 0.25 cm

). (B) Cyclic voltammetry at pH 7.0. Reprinted with

permission from Ref. 56 S.-U. Kim et al., Direct immobilization of cupredoxin azurin

modified by site-directed mutagenesis on gold surface, Ultramicroscopy, 108, 1390

(2008), Copyright Elsevier (2009).

J.-W. Choi, T. Lee and J. Min 354

the oxidation potential was 265 mV. That is matched with our previous

results and reference.

The obtained results were remarkable. These

stable electrical properties of azurin layer by the direct immobilization of

cysteine-modified azurin imply that the direct immobilization technology

can offer very stable and well oriented protein layer. In case of the use

of the linker in the process of protein immobilization, not-completed

immobilized protein existed in the protein layer and this protein seems to

be detached under a harsh condition.

2.2.2. Nanoscale Film Formation of Recombinant Azurin Variants with

Various Cysteine Residues on Gold Substrate

The orientation of recombinant azurin with various cysteine residues

immobilized on the Au substrate was analyzed by fluorescence

microscope, scanning tunneling microscope.

The binding ability of

recombinant azurin variants on Au substrate was also examined by

fluorescence imaging analysis. As shown in Fig. 4(A), the relative

fluorescence signal intensity enhanced with increasing number of thiol

group on cysteine residue because of strong affinity between thiol of

cysteine and Au surface.

The surface topography of the prepared metalloprotein layer was

obtained by commercially available scanning probe microscopy. In order

to measure the increased relative coverage, we calculated the average

surface roughness (Ra) and number of protein for each STM 2D image

with provided software of the instrument. STM analysis was performed

to examine the surface morphologies of recombinant azurin layers on a

gold surface. The data might provide direct immobilization on how each

recombinant azurin layer with cysteine residue forms on the Au substrate.

The surface coverage was calculated based on the changes of the surface

roughness following each protein immobilization. For recombinant

protein immobilization, the areas showing lower Ra values than the Ra of

bare gold are considered to be covered. Figure 4(B) shows the STM

images of the recombinant layers and the changed surfaces by covalent

bond with corresponding Ra (nm) and coverage (number of protein)

values. We calculated the coverage (number of protein) for each STM

image with provided software of the instrument.

Nanoscale Bioelectronic Device Consisting of Biomolecules 355

2.2.3. Temperature Dependent Redox Reaction of Recombinant Protein

Thin Film

Thermally induced unfolding and denaturation of ferredoxin was

monitored by differential scanning calorimetry because biomolecules has

affected by heat or thermal turbulence.

The conductivity (σ) was

determined, at a given temperature from the plot of current (I) versus

voltage (V) or from log I versus log V when the current and voltage

range was large in Fig. 5(A).

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As observed in general electrochemical

phenomena, the increase in conductance of the protein with increase in

temperature is because it acquires extra thermal energy but after 320 K

the conductance decreases and this due to the thermal unfolding of the

Fe-S cluster in the protein.

The complete denaturation of this protein was estimated by

differential scanning calorimetry at 342 K (Fig. 5(B)). The thermally

induced unfolding reaction of the recombinant protein is irreversible.

Fig. 4. (A) Fluorescence imaging analyses of recombinant azurin variants immobilization

on gold surface. (B) STM topography images of the recombinant azurin variants on gold

surface.

J.-W. Choi, T. Lee and J. Min 356

Since the irreversibility is most likely due to degradation of the cluster in

the unfolding state.

The temperature dependence of standard potential (E°) was

recognized were shown in Fig. 5(C). Initially, the E° potentials were

observed to decrease with increasing temperature because protein itself

has some resistance but as the temperature increases its resistance

decreases and consequently the E° decreases up to 320 K, this change in

E° potential could be the structural fluctuations that occur within the

protein.

Nevertheless, at 320 K, slope change in entropy (∆S° = dE°/dT)

Fig. 5. Temperature dependent working performance of protein-based device: (A)

Arrhenius plot for the increase in temperature with two distinctly different regions.

(B) DSC thermogram of recombinant protein. The sample pans contained 0.1 mg/ml

solution, DSC heating rate was 5°C/min. (C) E° versus T plot of recombinant protein on

Au electrode in 10 mM Tris-HCl buffer solution (pH 7.4) respectively. Dotted lines

are least square fit to the data points. Reprinted with permission from Ref. 36. A. K.

Yagati et al., Write-Once–Read-Many-Times (WORM) biomemory device consisting

Elsevier (2009).

Nanoscale Bioelectronic Device Consisting of Biomolecules 357

is due to the non-spontaneous disruption of the Iron-sulfur cluster is

followed by the spontaneous complete unfolding of the protein. Finally,

this behavior can be understood because of the loss of Iron-sulfur cluster

and denaturation of protein.

2.2.4. Surfactant CHAPS Effect of Bioelectronic Device

We showed previously that this protein layer was formed as cohesive

clusters on Au surface through physical adsorption.

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To reduce the

formation of these cohesion clusters, a zwitterionic surfactant, (3-[(3-

cholamidopropyl) dimethylammonio]-1-propanesulfonate) (CHAPS) was

introduced to modify the surface properties.

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The surface morphology

of the fabricated electrode surface was investigated by AFM to determine

the effect of CHAPS treatment.

Figure 6(a) depicts the recombinant azurin layer immobilized on a Au

surface without any treatment. As shown in this image, in the absence of

CHAPS the surface contains several azurin aggregates. Treatment with

10.0 mM CHAPS resulted in recombinant azurin clusters that were

highly segregated and existed as near single molecules (Fig. 6(d)).

However, any further increase in CHAPS concentration over 10.0 mM

Fig. 6. Surface morphology of the recombinant azurin layer after CHAPS treatment by

atomic force microscopy. (b): 2.5 mM CHAPS treatment, (c): 5.0 mM CHAPS treatment,

(d): 10.0 mM CHAPS treatment, (e): 15.0 mM CHAPS treatment, (f): 20.0 mM CHAPS

treatment.

J.-W. Choi, T. Lee and J. Min 358

did not result in an increased reduction in protein aggregation, as shown

in Figs. 6(e) and 6(f).

3. Nanoscale Bioelectronic Devices based on Biomolecules

3.1. Nanoscale Biophoto Diode

In a biological system, an electron transfer from one side of a molecule

to the other parts or between molecules is one of the most fundamental

and general processes.

The control and study of this electron transfer

phenomena in organized molecular systems are major destination for

bioelectronics.

Progress in bioelectronic devices engineering is still

rather moderate, owing to some serious problems associated with the

effective control of such as its structures and interactions at the nanoscale

level.

The electron transport from photoinduced processes in nature

is known to occur very efficiently as well as undirectionally such

as photoelectric conversion and long-range electron transfer in

photosynthetic organisms guided by molecular functional groups.

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The notions for the advance of bioelectronic devices can be originated

from biological systems, for example, a TCA cycle or the electron

transfer chain or the photosynthetic reaction center. An artificial

bioelectronic device can be achieved by mimicking the organization of

the functional molecules in a biological photosynthetic system. At the

first time, an initial process of photosynthesis in the electron transfer

system occur photoelectric conversion followed by long-range electron

transfer, which replaces very efficiently in specific direction through the

biomolecules.

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The specific energy and electron transfer occurs on a

molecular scale, because of the redox potential difference and electron

transfer property of the functional molecules which are the electron

acceptor, sensitizer and electron donor. Molecular films, fabricated by

LB techniques, can be used as in vitro biological systems for the

response to photosynthetic reaction center in the biological system.

Recently, a considerable interest has focused on nanoscale layer

fabrication or the formation of biomaterials thin layers on solid

surfaces.

Based on these techniques, various bioelectronic devices have