Reviewnbel.sogang.ac.kr/nbel/file/국제 324. Recombinant... · 2019-06-03 · In this review, we summarize recent research towards devel- oping nanoscale biomemory devices including

Copyright © 2014 American Scientific PublishersAll rights reservedPrinted in the United States of America

ReviewJournal of

Nanoscience and NanotechnologyVol. 14, 433–446, 2014

www.aspbs.com/jnn

Recombinant Protein-Based Nanoscale

Biomemory Devices

A. K. Yagati1, J. Min3�∗, and J.-W. Choi1�2�∗1Research Center for Integrated Biotechnology, Sogang University, Seoul 121-742, Korea

2Department of Chemical and Biomolecular Engineering, Sogang University, Seoul 121-742, Korea3School of Integrative Engineering, Chung-Ang University, Seoul 156-756, Korea

Biomolecular computing devices that are based on the properties of biomolecular activities offera unique possibility for constructing new computing structures. A new concept of using variousbiomolecules has been proposed in order to develop a protein-based memory device that is capableof switching physical properties when electrical input signals are applied to perform memory switch-ing. To clarify the proposed concept, redox protein is immobilized on Au nanoelectrodes to catalyzereversible reactions of redox-active molecules, which is controlled electrochemically and reversiblyconverted between its ON/OFF states. In this review, we summarize recent research towards devel-oping nanoscale biomemory devices including design, synthesis, fabrication, and functionalizationbased on the proposed concept. At first we analyze the memory function properties of the proposeddevice at bulk material level and then explain the WORM (write-once-read-many times) nature ofthe device, later we extend the analysis to multi-bit and multi-level storage functions, and then wefocus the developments in nanoscale biomemory devices based on the electron transport of redoxmolecules to the underlying Au patterned surface. The developed device operates at very low volt-ages and has good stability and excellent reversibility, proving to be a promising platform for futurememory devices.

Keywords: Recombinant Protein, Cyclic Voltammetry, Nanoscale Biomemory, Nanofabrication,Nanobiochip, Biomemory Devices.

CONTENTS1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433

2. Device Structure and Fabrication of

Protein-Based Memory Devices . . . . . . . . . . . . . . . . . . . . . . . . . 435

2.1. Basic Concept of the Memory Device . . . . . . . . . . . . . . . . 435

2.2. Thin Film Fabrication Methods . . . . . . . . . . . . . . . . . . . . . 435

2.3. Characterizations on the

Proposed Biomemory Device . . . . . . . . . . . . . . . . . . . . . . . 437

3. Development of a Protein-Based Memory

Device and Its Worm Nature . . . . . . . . . . . . . . . . . . . . . . . . . . . 438

4. Enhancement in Data Storage Capacity in

Protein-Based Memory Devices . . . . . . . . . . . . . . . . . . . . . . . . . 440

4.1. Multi-Bit Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440

4.2. Multi-Level Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440

5. Nanobiomemory Device Composed of Recombinant

Azurin on Nanostructured Surfaces . . . . . . . . . . . . . . . . . . . . . . 442

6. Future Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445

Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445

References and Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445

∗Authors to whom correspondence should be addressed.

1. INTRODUCTIONSince the invention of first junction transistor one of

the most important issues has been to reduce the size

of the transistor. Many engineers and scientists have

focused their research on the manipulation of structures

at micro/nano-scale.1 Relentless decrease in the size of

silicon-based microelectronics devices is posing prob-

lems. The most important among these are the limitations

imposed by quantum-size effects and instabilities intro-

duced by the effects of thermal fluctuations.2 Also, as

the size of the structures has been reduced, the complex-

ity and cost of fabrication have increased exponentially.3

The continuity of this miniaturization trend is becom-

ing difficult due to the limitations of conventional lithog-

raphy, complexity of integration, physical phenomena,

and other aspects.4 For decades, semiconductor technol-

ogy has advanced at an exponential rate as stated by

Moore’s law, in which the number of features in a given

J. Nanosci. Nanotechnol. 2014, Vol. 14, No. 1 1533-4880/2014/14/433/014 doi:10.1166/jnn.2014.9006 433

Recombinant Protein-Based Nanoscale Biomemory Devices Yagati et al.

area of a substrate doubles every 18 to 24 months, and

the computing power and capabilities will increase with

each generation of device while the cost per function

decreases.5–7 Also, semiconductors have served as basic

materials for constructing electronic devices for the past

50 years with tremendous growth. Despite the vast growth

of semiconductors, it is uncertain whether devices that

depend on the bulk properties of materials will retain the

required characteristics to function when feature size ulti-

mately reach nanoscale dimensions.8–10 These problems

prompted scientists to focus on the use of biomolecules

as they have the advantages of functionality and speci-

ficity. Much work has been done in the bioelectronics field,

but since the 1990s, it’s been focused on creating bet-

ter bioelectronic devices by integrating biomolecules with

semiconductors.11

On the other hand, nature consists of perfectly con-

trolled and manipulated nano-scale components using

molecular recognition of various biological materials such

as deoxyribonucleic acid (DNA), ribonucleic acid (RNA),

protein, and cell. The knowledge obtained from nature

A. K. Yagati obtained his B.Sc. and M.Sc. in Electronics from Andhra University, India

(1999 and 2001), M.Tech. in Bioelectronics from Tezpur University, Assam, India (2006)

and Ph.D. in Integrated Biotechnology from Sogang University, Korea in 2011, respec-

tively. Currently he is working as a post-doctoral researcher at nanobioelectronics lab,

Sogang University with Professor Jeong Woo Choi. He also worked as a Senior Project

Assistant at the School of Medical Science and Technology (SMST), Indian Institute of

Technology, Kharagpur, India with Professor Sujoy K. Guha. His research interests include

electrochemistry; sensors based on chemically modified electrodes and working on the

development of nano-scale biomemory devices.

J. Min obtained his B.S., M.S., and Ph.D. in Chemical Engineering from Sogang Uni-

versity, Korea (1992, 1994, and 1998). Currently he is an Associate Professor at Chung-

Ang University Korea. He was a Post-doctoral Research Associate at Environmental and

Biological Engineering, Cornell University, NY, USA (2000–2001), Senior Scientist at

Innovative Biotechnologies, International, NY, USA (2002–2003), and Project Leader at

Samsung Advanced Institute of Technology, Korea (2003–2006). His research has been

mainly focused on the fields of bio-nanodevices, especially for protein-based memory,

RNA-based microsensors, and cell-based microchips.

J.-W. Choi obtained his B.S. and M.S. in Chemical Engineering of Sogang University,

Korea (1982, 1984). He received a Ph.D. in Chemical and Biochemical Engineering of

Rutgers University, USA (1990), D.Eng. In Biomolecular Engineering of Tokyo Institute

of Technology, Japan (2003), and MBA in Business School of University of Durham,

UK (2006). He is a Professor of Chemical and Biomolecular engineering, and Director

of the Institute of Integrated Biotechnology; Sogang University, and Director of Sogang-

Binggrae Research Center for Advanced Food Analysis. Professor Choi’s research has

been mainly focused on the fields of nanobioelectronics, especially for biomemory, protein

chips, and cell chips. He has published more than 300 journal papers in bioelectronics

and biotechnology field. He is also interested in doing research about management of

innovation, especially for open innovation in fused biotechnology area.

can be used to solve the miniaturization issues. Moreover,

engineers and scientists have applied this knowledge to

implement artificial nano/microstructures for developing

bioelectronic devices.12–14 Bioelectronics is a rapidly pro-

gressing interdisciplinary research field that aims to inte-

grate biomaterials and electronic elements into functional

devices.15–17 Genetic engineering and chemical methods

can optimize the binding, catalytic, and electron transport

properties in the nanometer-sized biomaterials. As a con-

sequence, there has been an intense interest in developing

molecular-based electronic materials for use in both mem-

ory architectures and circuit elements.18–20

In storage technology, the increase in density of stor-

age devices based on silicon and inorganic materials

is becoming increasingly difficult. Hence biomaterials

such as protein and DNA with their inherent size and

electronic properties could be alternatives to succeed

the silicon-based technologies in developing bioelectronic

devices.21 A bioluminescent molecular switch for glu-

cose was developed22 by inserting glucose-binding pro-

tein (GBP) into the structure of the photoprotein aequorin

434 J. Nanosci. Nanotechnol. 14, 433–446, 2014

Yagati et al. Recombinant Protein-Based Nanoscale Biomemory Devices

(AEQ). In the presence of glucose, GBP undergoes a con-

formational change, bringing the two segments of AEQ

together, “turning on” bioluminescence and detecting glu-

cose. A photonic DNA memory was developed23 based

on the positional information of DNA for scaling up the

address space of the DNA memory. Use of the optical

techniques is useful in controlling positional addresses in

parallel. Multiple logic gates based on electrically wired

surface-reconstituted enzymes was developed,24 and a mul-

tifunctional logic gate25 based on folding/unfolding transi-

tions of a protein. Further, a lithography technique was pro-

posed based on a protein pattern on a solid surface that can

perform write, read, and erase operations.26 Also, viruses

show their good side in the field of bionanoelectronics.

Being viruses, they carry a bad reputation regardless of

wherever we find them, but they exhibit memory phenom-

ena when they are coupled with nanoparticles.27 Further,

inversion switch using the fim and hin inversion recom-

bination systems was used to create a sequential memory

switch.28 This switch is capable of transitioning from one

state to another in a manner analogous to a finite state

machine, while encoding the state information into DNA.

In this review, we analyze and focus on using geneti-

cally engineered recombinant protein to store information

as a charge storage element in the design and construction

of a nanoscale biomemory device. Redox reactions of pro-

teins are utilized for information transfer and energy stor-

age in major natural mechanisms. This is because redox

reactions in proteins are of great interest in various fields

including biology and chemistry29–31 particularly proteins

with the redox property have been introduced in molecular

electronic devices.32–34

2. DEVICE STRUCTURE AND FABRICATIONOF PROTEIN-BASED MEMORY DEVICES

2.1. Basic Concept of the Memory DeviceElectron transfer through proteins, a fundamental ele-

ment of many biochemical reactions, has been studied

extensively in solution and is one of the most fundamental

processes in biological systems,35 crucial for different bio-

logical energy conversion processes from respiration to

photosynthesis, and prominent in diverse metabolic cycles.

Electron transfer reactions are also performed by a range

of proteins in which electron tunneling occurs over long

distances.36 While these reactions proceed in the proteins,

when present in their natural, usually aqueous environ-

ment, there have been attempts to explore the electronic

conductance of various proteins in both wet electrochem-

ical and solid state conditions. Hence, redox reactions of

proteins or enzymes are utilized for information transfer

and energy storage in major natural mechanisms. There-

fore, redox reactions are of great interest to develop bio-

based devices by mimicking the biomechanisms.

Recently, Yang’s group developed a digital mem-

ory device composed of tobacco mosaic virus (TMV)

conjugated with platinum nanoparticles (TMV-Pt).37 The

TMV-Pt nanocomposite has properties that show electrical

bistability depending on the voltage controlled for con-

ductance states. The mechanism of this memory device,

due to charge transfer from the RNA to Pt NPs under the

high electric field and the charge trapped in the nanoparti-

cles, subsequently changes the conductivity of the material

system. This conjugate system was one of the innovative

candidates for future electronic devices. The Rinaldi group

also developed a transistor based on the properties of met-

alloprotein, in which they explain the influence of the gate

voltage on the current flow when source to drain is inter-

connected with protein molecules. It is observed that the

device behaves both like p-channel and n-channel MOS-

FET, which is an innovative investigation for future elec-

tronic devices.38 Ghadiri group demonstrated molecular

logic circuits based on DNA molecules and showed a com-

plete set of modular DNA-based Boolean logic gates. This

approach is based on solid-supported DNA logic gates

that are designed to operate with single-stranded DNA

inputs and outputs. As the solution-phase serves as the

communication medium between gates, circuit wiring can

be achieved by designating the DNA output of one gate

as the input to another thus facilitating the construction

of multi-level circuits.39 These works pave the path for

developing enzyme-based bioelectronic computers. Choi’s

group also demonstrated a new concept of biomemory

device using metalloprotein as a charge storage element.

The fundamental principle of the proposed memory device

is shown in Figure 1(a). The two basic stages (charge writeand erase) are required for achieving the memory con-

cept. Metalloprotein possesses two distinct electrochemical

states (oxidation and reduction) that can be controlled by

applied potential.

The application of oxidation potential to the protein

molecule on the Au substrate causes the transfer of an

electron from the protein molecule to the Au substrate

resulting in storage of positive charge (write) in the

molecule as shown in Figure 1(b). On the other hand,

the application of a reduction potential causes the transfer

of an electron back to the protein molecule thereby eras-

ing the stored charge (erase). The charge confined in the

two states can be determined (read), when an open circuit

equilibrium potential is applied to the system. The contin-

uous application of oxidation, OCP, and reduction poten-

tial provide a parameter for constructing protein-based

biomemory devices in which write, read, and erase charges

can manipulate information like a conventional inorganic

memory device. It is a key mechanism to retain and han-

dle the electrochemical properties of a metalloprotein for

mimicking and developing a biomemory system.40–42

2.2. Thin Film Fabrication MethodsProtein adsorption on surfaces in a biological environ-

ment is the first step in biofilm formation. Different

J. Nanosci. Nanotechnol. 14, 433–446, 2014 435


Figure 1. (a) A cyclic voltammogram showing the potentials used

for developing protein-based memory devices. (b) The electron transfer

mechanism of ferredoxin on Au electrode: (1) application of oxidation

voltage causes an electron to move from the protein to Au electrode

(leads to positive charge); (2) application of reduction voltage leads an

electron back to the protein (leads to charge neutral).

physical surface parameters are influencing the pro-

tein adsorption on surfaces such as topography, wetting

behavior, surface charge, and mechanical stiffness. To

form a well-ordered, self-assembled monolayer (SAM)

consisting of biomolecules on solid surfaces, three

main issues have to be considered: (i) orientation,

(ii) address, and (iii) surface coverage. Many researchers

are working to fabricate monolayers of biomolecules

with high orientation. A successful immobilization scheme

requires:

(1) reproducibility and stability of the protein layer,

(2) uniformity of the surface structures

(3) the ability to control the immobilization density of the

immobilized molecules,

(4) if possible, higher coverage/density is preferred, and

(5) orientation of molecules so that maximum binding

density can be achieved.

SAMs, by using chemical linker materials, have been used

to covalently attach proteins on solid supports. The applica-

tion has been used in developing bioelectronic devices, as

well as studying protein/surface and ligand/receptor inter-

actions. Since the emphasis of this review is on memory

functioning, the focus will be mainly on protein immo-

bilization. Here three different types of adsorption meth-

ods are discussed for protein immobilization; however,

the techniques described here apply to other biomolecules

as well.43

2.2.1. Physical Adsorption (van derWaals Adsorption)

Proteins adsorb onto a variety of surfaces. It is gen-

erally believed that the adsorption takes place due to

hydrophobic, ionic, and Van der Waals interactions. The

phenomenon of adsorption is essentially an attraction of

adsorbate molecules to an adsorbent surface. The prefer-

ential concentration of molecules in the proximity of a

surface arises because the surface forces of an adsorbent

solid are unsaturated. Both repulsive and attractive forces

become balanced when adsorption occurs, and the process

is nearly always an exothermic process.44

Physical adsorption is the simplest way to immobilize

proteins on surfaces. However, the binding to a surface is

usually not stable, and the activity of the protein is usually

lost. For this reason, adsorption has to be controlled care-

fully. Concentrated protein solutions should be avoided

since additional layering will occur. Additional adsorp-

tion usually involves protein-protein adsorption instead of

surface–protein adsorption. Such adsorption is inherently

unstable, and the protein may gradually desorb from the

surface.

2.2.2. Chemisorption �Activated Adsorption�In chemisorption, there is a transfer or sharing of electrons,

or breakage of the adsorbate into atoms or radicals that are

bound separately. Here, the formation of chemical bonds

between the adsorbate and adsorbent is a monolayer, often

releasing more heat than the heat of condensation. Cova-

lent immobilization of proteins on surfaces is based on the

linkage between surface and protein functional groups.45

Generally there are three different approaches to attach

proteins to a surface other than physical adsorption. The

first approach is to modify the substrate, then attach pro-

teins to the substrate. The second approach is to link

the protein of interest to sulfur-containing molecules. The

modified protein is then self-assembled onto gold substrate

through gold-thiol linkage. The third approach is to immo-

bilize the protein through a lock and key interaction, or

ligand/receptor mechanism.46

2.2.3. Site-Directed ImmobilizationSite-specific covalent immobilization, via a unique natural

or non-natural residue, affords a uniformly oriented pro-

tein array. One such method is protein recombination to

generate cysteine residues on specific parts of the protein

to bind at the gold surface. When protein has cysteine

residues in the right regions, protein could be immobi-

lized and addressed on the gold surface with good orienta-

tion and coverage. The regenerated recombinant ferredoxin

with cysteine residues by site-directed mutagenesis47�48 is

demonstrated in Figure 2.



Figure 2. Construction of recombinant plasmids and DNA sequence analysis. (a) Schematic representations of fdxA gene variants for expression.

(b) Analyses of PCR products of the fdxA gene variants. (line 1) size marker, (line 2) PCR product of fdxA gene, (line 3) size marker, (line 4) PCR

product of mfdxA gene. (c) Verification by DNA sequencing. (d) Design of expression vector for fdxA gene variants.

2.3. Characterizations on theProposed Biomemory Device

The formation of biofilm on electrode surfaces requires

morphological and physical measurements to confirm the

layer characteristics. To achieve this, scanning probe

microscopy (AFM and STM), surface plasmon resonance

(SPR), and other optical detection methods are normally

used. Surface plasmon resonance spectroscopy has been

demonstrated as a technique to monitor biomolecular

interactions between ligands immobilized on a thin metal

substrate with other biomolecules adsorbed on it. One

advantage to this technique is the lack of sample solution

interference with the spectroscopic measurement. The light

in SPR is not passed through the sample solution, eliminat-

ing many problematic matrix effects that take place with

other spectroscopic methods. Also, many researchers ana-

lyzing biological problems need to develop recombinant

proteins, and it is important to show that the recombinant

protein has the same structure as its native component.

Here, the intensity of the reflected light is measured as

a function of the angle of incidence. At a critical angle

of excitation of surface plasmon resonance, a minimum

in the intensity of the reflected light is observed. Also to

determine the optimum concentration of the protein to be

immobilized on the Au surface, SPR spectrums at vari-

ous concentrations were observed. As the concentration

of the protein is increased, the value of the SPR angle is

shifted to higher values and saturated at a concentration of

0.1 mg/ml shown in Figure 3.

Figure 3. (a) Change of SPR angle shift for the increasing concentra-

tion inset shows SPR characterization of the immobilization of 0.1 mg/ml

ferredoxin molecules on gold substrate comparative to Au surface.



Figure 4. STM topology of (a) bare Au surface and (b) ferredoxin-immobilized Au surface respectively obtained at a scan rate of 1 Hz.

To construct the molecular electronic device with func-

tional protein film, the formation of aggregated pro-

tein molecules has been considered as one of the most

important factors that dominantly affects the device per-

formance. The film formation was achieved on the Au

substrate by self-assembly technique where the protein is

immobilized and the samples were treated with Tween 20

a non-ionic surfactant. The protein treated with non-ionic

surfactant allowed well-distributed protein molecules with-

out loss of its activity. The surface morphology of bare Au

surface and protein film formed on Au surface are shown

in Figure 4.

3. DEVELOPMENT OF A PROTEIN-BASEDMEMORY DEVICE AND ITSWORM NATURE

Electron transport in nature, such as photoelectric con-

version and long-range electron transfer in photosynthetic

conversion, is one of the most efficient mechanisms

to control information and energy in natural and arti-

ficial systems because of its unidirectional transport

and high conversion yields.49–53 Particularly, sequential

redox reactions by proteins or enzymes are utilized for

information transfer and energy storage in major natu-

ral mechanisms. Therefore, redox reactions are of great

interest in various fields including biology and chem-

istry. Currently, these redox reactions by proteins or

enzymes are divided mainly into two fields, alternative

energy and next-generation electronic devices to enhance

their energy conversion efficiency and information con-

trol efficiency by mimicking biomechanisms using redox

reactions.54�55 Bioelectronic devices, mimicking biomech-

anisms, have recently emerged to combine biomaterial

functions into conventional electronic format.56�57 Several

methods have been proposed for developing new concept-

memory devices to overcome the physical and techni-

cal limitations of the conventional silicon-based memory

devices.58–61 A memory device using conducting polymers

also has been reported,62 but this device can be writ-

ten only once and cannot be used as programmable

memory. Recently, a flash memory with protein-mediated

assembly has been proposed,63 and an electrochemi-

cal memory device using conducting polymer has been

fabricated.64

Even though programmable electrical bi-stability was

observed in a memory device made from polymer

thin film65 and logical circuits have been developed

using biomolecules and organic molecules as active

components,66 electronic memory using these organic

materials and biomolecules is still in the early stages

of demonstrating the function of new concept-memory

device. The concept of biomemory device consisting of

metalloprotein by their electrochemical reaction67 has been

reported, but here we review the multiple reading of the

stored charge and temperature dependency of the stored

charge.

Ferredoxin is one of the promising biomolecules

applicable to biomemory systems because of its redox

properties and electron transport mechanisms in nature.

Ferredoxins are ubiquitous in nature and play a vital

role in electron transport mechanisms. These proteins use

oxidation-reduction chemistry to transfer an electron from

a donor site to an acceptor site. The spinach ferredoxin

belongs to 2Fe–2S class of ferredoxin (fdx) that is sim-

ple iron-sulfur proteins containing a 2Fe–2S cluster. Ferre-

doxin is a small, soluble, generally very acidic protein

involved mainly as an electron carrier of low oxidation-

reduction potentials in fundamental metabolic processes.

Ferredoxin is the most abundant form of photosynthetic-

type ferredoxin present in spinach chloroplasts. The 2Fe–

2S cluster of fdx contains two atoms of iron, bridged by

two acid-labile, inorganic sulfide atoms, and coordinated

to four cysteinyl sulfurs in the polypeptide chain.68 Since

ferredoxin is a constituent in the acting site as an electron

transfer protein, it could be used as an electron accep-

tor in the development of molecular electronic devices by

mimicking biological mechanisms. Therefore, ferredoxin



Figure 5. Cyclic voltammogram obtained for ferredoxin immobilized

Au electrode showing the oxidation and reduction potentials for charge

write and erase functions of a memory device. Inset shows the open

circuit potential (OCP) for ferredoxin immobilized Au electrode, which

is used to read the stored charge.

proteins can be applied directly to verify this new concept

of memory function with biomaterial combined electronic

devices, due to their charge transfer and trap function as

well as their thermal69 and chemical stability.70

Cyclic Voltammetry (CV) for Spinach ferredoxin on

gold electrode in 10 mM Tris-HCl buffer (pH 7.4) in

potential range of 0.1 to −0�45 V versus Ag/AgCl at

a scan rate 50 mV/s was performed. Well-defined redox

peaks with an anodic redox wave at Epa =−0�12 V versus

Ag/AgCl and cathodic wave at Epc = −0�2 V Ag/AgCl

resulted as shown in Figure 5. This corresponds to the

redox process of [2Fe–2s]2+/1+ cluster in fdx. These redoxstates were used to charge storage and erase in the protein

molecule to perform memory functions. The open circuit

potential observed was −0�05 V, which is used to read the

charged states.

Figure 6. (a) Arrhenius plot obtained for conductivity with increase in

temperature clearly shows two distinctly different regions; (b) �E� versusT plot of ferredoxin on Au electrode showing the variation in standard

potential with increase in temperature.

Temperature-dependent conductance measurements

were carried out to investigate the influence of temperature

on the switching mechanism of the device. The conduc-

tivity (�) was determined at a given temperature from the

plot of current (I) versus voltage (V ). Figure 6(a) indicatesthat the conductance greatly depends on temperature.

By increasing the temperature from 289 to 338 K, progres-

sive changes in structural and functional properties have

been monitored. Up to 320 K, the conductance showed a

linear, positive shift with increasing temperature but inter-

estingly enough, from 320–338 K the conductance begins

to decrease. Conductance of the protein increases with

the rise in temperature because it acquires extra thermal

energy, but after 320 K the conductance decreases due to

the thermal unfolding of the 2Fe–2S cluster in the protein.

Thermally induced unfolding and denaturation of ferre-

doxin were monitored by differential scanning calorime-

try (DSC) with 5 �C/min scan rate going from 20 to

80 �C. The complete denaturation of this protein was esti-

mated by differential scanning calorimetry at 342 K. The

thermally induced unfolding reaction of the ferredoxin

is irreversible71 due to the degradation of the cluster in

the unfolding state.72 Also, E� potentials were decreas-

ing with increasing temperature but up to 320 K, this

change in E� potential could be the structural fluctuations

that occur within the protein73 as shown in Figure 6(b).

At 320 K, slope change in entropy (�S� = dE�/dT ) dueto the non-spontaneous disruption of the Iron-sulfur clus-

ter is followed by the spontaneous complete unfolding of

the protein. This behavior is because of loss of iron-sulfur

cluster and denaturation of protein. Therefore, the mem-

ory function of this device is stable is up to 320 K above

which the device’s working performance will change, with

no information being stored.

For the analysis of the memory function, electrical volt-

age pulses for write-read-erase cycles were applied as

shown in Figure 7(a). In each memory cycle, an oxida-

tion voltage of −120 mV was applied to store the charge

in the protein layer; reduction voltage of −200 mV was

applied to erase all the stored charge in the device. Two

pulses of −50 mV (open circuit voltage) were applied

after oxidation and reduction voltages, to read the charged

states. To read the stored charge multiple times an OCP

of −50 mV was applied, and the current reveals that

the stored charge can be read multiple times. Figure 7(b)

shows the oxidation voltages of −120 mV can repro-

ducibly charges on the device. A set of three voltage

pulses of −50 mV (OCP) with small disconnecting times

showed the current switching, thereby maintaining the oxi-

dized state of the memory device. Finally, a voltage pulse

of −200 mV erases all the stored charge. The detailed

change of current (I) is a function of time for two mem-

ory cycles. Also, the device switching speed was tested

with fast write pulse in the write-read-erase-reset sequence

in 10−6∼10−9 sec duration without degradation in perfor-

mance. Notably, it has less than 20% decay in its charged



Figure 7. (a) Current response obtained upon application of sequential

redox potentials for operation of memory function (b) multiple times

reading test shows the current response upon multiple times of OCP

where Iwrite and Ierase correspond to write and erase currents and Ireadrepresenting the multiple times reading of the stored charge.

state after 2 weeks, but almost no change in 2 weeks con-

firms the nonvolatile nature of biomemory device.

4. ENHANCEMENT IN DATA STORAGECAPACITY IN PROTEIN-BASEDMEMORY DEVICES

4.1. Multi-Bit ApproachLogic and memory are the essential functions in normal

electronic products. All the developments of these memory

cells are driven by the market requirement for low cost. For

the memory cells, the traditional way of achieving low cost

per bit is by scaling size or multilevel storage, and great

developments have been achieved in this areas.74�75 Multi-

bit storage is attracting more and more research attention

due to the scaling method limited by photolithography.76

The strategy to increase memory density imposes a multi-

bit approach wherein the charge storage element contains

molecules with multiple redox states.77�78 Here, an efficient

way of azurin thin film formation on four Au electrodes

is introduced where each will act as a memory structure

to achieve the basic memory function with multiple bit

storage capacity.

The schematics for the experimental system for multi-bit

and multi-level systems are shown in Figure 8. The redox

properties of 4 different azurin variants (Ni type; Cu type;

Mn type; Fe type) were examined as each modified azurin

having different metals would affect the electrochemical

properties of the azurin variants. Cyclic voltammetry (CV)

of each self-assembled azurin variant layer showed differ-

ent redox potentials for each metal substitute, which can

be utilized for memory function.

Direct immobilized azurin variant layers have three dis-

tinct conducting states and each variant has different redox

potentials. Briefly, in the case of the Cu-type azurin, apply-

ing of an OP resulted in the transfer of electrons from the

immobilized azurin layer into the Au substrate, and pos-

itive charges were stored in the azurin layer. The reverse

process occurred during the reduction step. When the RP

was applied, the electrons were transferred back into the

azurin layer and the stored charge was erased. The open

circuit potential (OCP) was then used to read these charged

states. Using these charged states, different logic perfor-

mance can be realized by the application of these poten-

tials in a sequential manner. In the case of Fe-type azurin,

simple “write” and “erase” functions are studied. Also,

in the case of Ni type Azurin, apart from these two charged

states, an open circuit voltage will confirm (read) both

charged states making one kind logical sequence of write,

read, and erase functions. Apart from these, the Mn-type

azurin also displayed a WORM-type current response with

three defined parameters. In this case, when an OCP was

applied at three very small disconnecting times to read

the stored information, the observed current responses of

the stored charge could be read three times. Finally, in the

case of Fe-type azurin, potentials were applied to per-

form WORM-type memory with an OCP to confirm the

erase state of the biomemory. These results are shown in

Figure 9.

4.2. Multi-Level ApproachAn alternative approach for multi-bit functionality is by

mixing different redox active molecules whose potentials

are well separated. This multi-level approach is aimed

to store more than 1 bit of information. Here in this

approach, recombinant azurin containing cysteine residues

was produced by site-directed mutagenesis and immo-

bilized directly on Au surface; then, cytochrome c was

adsorbed onto the immobilized azurin layer by electrostatic

bonding. This heterolayer is composed of recombinant

azurin and cytochrome c, which is capable of storing the

two pairs of information, referred to as the multistate

memory. The electrons were flowing into the immobi-

lized azurin molecule when oxidation potential (OP) was

applied to the fabricated electrode, which was assigned

an information value of ‘1’. In contrast, when reduction

potential (RP) was applied, which was assigned an infor-

mation value of ‘0’, the stored electrons were released

from the azurin.



Figure 8. (a) Schematic diagram shows the immobilization of 4 different variants of azurin on Au electrodes for the basic operating mechanisms of

4-bit operation of the fabricated biomemory chip. (b) The electron-transfer mechanism of a cytochrome c/recombinant azurin layer on Au surface.

Cyclic voltammetry of the recombinant azurin/

cytochrome c heterolayer immobilized on the Au surface

shows two anodic waves at Epa of 0.062 V versus Ag/AgCl

and cathodic wave at Epc of 0.184 V versus Ag/AgCl,

which correspond to the redox process of recombinant

azurin center Cu2+/1+. In addition, the anodic wave at Epa

of 0.131 V versus Ag/AgCl and cathodic wave at Epc of

0.324 V versus Ag/AgCl were observed, which correspond

to the redox process of the cytochrome c center Fe3+/2+

shown in Figure 10(a).

Figure 10(b) shows the faradaic currents obtained for a

multi-level operation of a protein-based memory device.

Faradaic currents were obtained when the heterolayer was

applied with an oxidation potential of 0.324 V versus

Ag/AgCl and the cytochrome c layers became oxidized.

The electrode was left open for a small duration of time,

and again when connecting the OCP of 0.104 V versus

Ag/AgCl, large amplitude currents were monitored in the

absence of background currents. When a reduction poten-

tial 0.131 V was applied, the stored charge was erased.

This was evident by the small amplitude currents. In the

same way, faradaic currents were obtained when an oxida-

tion potential of 0.184 V versus Ag/AgCl was applied to

the heterolayer. Under these conditions, the recombinant

azurin layers became oxidized, which was then reduced

when a reduction potential of 0.062 V was applied to the

recombinant azurin layer. Then, the electrode was left open

for a small duration of time and again connecting the OCP

of 0.150 V versus Ag/AgCl resulted in the appearance

of small amplitude currents in the absence of background

currents. Here, applying 0.184 V was another writing step

whereas applying 0.150 V was another reading step with

respect to the measurement of currents. Finally, the appli-

cation of 0.062 V erased the stored charge in the recom-

binant azurin layer. Similarly, when two pairs of redox

potentials were applied to the protein film for duration of

320 ms transient currents for the two pairs of the charge

to write, read, and erase functions were clearly monitored,

which are prerequisites for any molecular memory storage

device.



Figure 9. (a) Verification of multi-functional memory performance;

(a) simple write and erase function on Cu-type azurin (b) Ni-type azurin

with read confirmation of the charged states (c) Fe-type azurin with mul-

tiple reading performance (d) Mn-type azurin of multiple read and also

confirmation of its erased states.

Figure 10. (a) Cyclic voltammogram showing the behavior of

cytochrome c and azurin hetero conjugates onto the gold surface;

(b) characteristics of multistate protein-based biomemory device; two

pairs of input pulse potentials of oxidation (charge write 1, 2), open-

circuit (charge read 1, 2), and reduction (charge erase 1, 2) potentials

were applied to gold electrodes simultaneously, having a pulse width of

20 ms. (ii) The corresponding charging currents were measured for a

total duration of 320 ms.

5. NANOBIOMEMORY DEVICE COMPOSEDOF RECOMBINANT AZURIN ONNANOSTRUCTURED SURFACES

Due to continuously increasing demand for ultimate

miniaturization of electronic systems, molecular electronic

devices are currently thriving as alternative technologies

because of their promising potential in writing, read-

ing, and processing of information at the nanoscale.79�80

Recently, colloidal or nanosphere lithography has emerged

as an effective and inexpensive method for patterning

nanostructures over a large area in which the colloidal

nanospheres act as a mask. Regular pattern of desired

material can be obtained after deposition and the removal

of mask.81–83 Electrical conduction in the self-assembled

electro-active protein on nanostructured electrode surfaces

that are well swollen with electrolyte/solvent has been

a subject of great interest for the fundamental studies

on the mechanism and kinetics of charge transport as

well as potential applications (such as optical/electronic

devices, biosensors, and bioelectronics) based on their

redox properties.84–87 Development is occurring towards

a nanoscale protein-based memory device, in which the

recombinant protein is self-assembled on Au pattern-

formed indium tin oxide (ITO) coated glass plate. By using

electrochemical scanning probe microscopy with in-situcyclic voltammetry, memory function experiments were

performed. To realize a practical biomemory device, a sin-

gle detection system is not enough. To ensure the possi-

bility of multiple detection systems to read the stored and

erased charges states, optical and magnetic detection sys-

tems were introduced.

The schematics for the experimental setup and

the nanoscale biomemory principle are shown in

Figure 11. Electrochemical experiments on Az/Au–ITO

were conducted by ECSTM with in-situ cyclic voltamme-

try. Experiments were carried out in 10 mM HEPES buffer

solution, where Az/Au–ITO substrate acts as the work-

ing electrode (vs. Ag/Ag+� as shown in Figure 11(a). The

operation mechanism of the proposed nanoscale biomem-

ory is depicted in Figure 11(b). To examine the redox

properties for analyzing the reduction and oxidation prop-

erties of azurin at nanoscale, ECSTM with in-situ cyclic

voltammetry was performed. Obviously, the cyclic voltam-

mogram of bare Au nanopattern did not reveal any redox

peaks; however, the CV for azurin on the Au nanopat-

tern clearly depicted the peaks for reduction and oxida-

tion of Az molecules at −0�04 V and 0�26 V respectively

at a scan rate of 50 mV/s, which corresponded to the

redox process of the Cu2+/1+ center in azurin, as shown in

Figure 11b(i), (ii). The formal reduction potential (E1/2�,calculated as E1/2 = �Epc +Epa�/2, was 0.11 V. A set of

images of the same sample area of protein on Au triangle

was obtained at constant bias while varying the potential.

It can be seen that there was a variation in the conduc-

tance of the redox molecules upon varying the potential,



Figure 11. (a) Schematic diagrams for azurin on Au nanodots with ECSTM setup and (b) electron transfer mechanism in Az/Au nanodots during

oxidation and reduction potentials. In-situ CV’s for (i) bare and (ii) Az/Au/ITO at a scan rate of 50 mV s−1.

which was reflected in a difference in apparent height with

respect to the background. Figure 12 shows the images for

the applied potential of 0.26 and −0�04 V, respectively for

a bias voltage of 100 mV for oxidation and reduction states

of azurin moelcules. It was assumed that at Ebias = 0 V, the

redox state of azurin is vacant with energy higher than the

fermi energy of the substrate and tip. When a negative bias

was applied to the substrate and the tip was kept at the

positive potential, the vacant state energy and fermi energy

of the substrate were matched, allowing electrons to be

transferred from the substrate to the biomolecule, which

lowers the thermal activation allowing the biomolecule to

transfer the electron to the tip.88�89

Figure 12. ECSTM images obtained for immobilized azurin molecules

on Au substrate for different bias potentials for (a) oxidation and

(b) reduction, respectively.

The topography for the immobilized azurin molecules

on Au surface was obtained with ECSTM at these two

distinct conducting states as shown in Figure 12. How-

ever, bias potentials have induced only specific regions

between tips and substrates. The typical bright spots seen

when tuning the substrate potential in a region, appear to

be strongly potential-dependent of redox potentials. Such

kind of behavior is consistent with a resonant nature of the

current measured in STM experiments in the Au-adsorbed

azurin molecules. Figure 12(a) points at assembled azurin

at oxidation voltage (Write/Store) and Figure 12(b) shows

assembled azurin at reduction voltage (Erase). The shape

and height have been changed more and more as specific

potentials were applied. From these images, it is assumed

that there were two conductive states for charge storage.

A brighter spot emerged, which was interpreted as a small

island of oxidized azurin molecules containing a Cu cen-

ter. When a voltage of 0.26Eox was applied, an electron

tunnel formed adsorbed azurin molecules on the Au sub-

strate, and lead to a storage of positive charges in the

azurin molecules. In contrast, when a voltage of −0�04Ered

was applied, the electrons were transferred back to azurin

molecules and the stored charge was erased, which was per-

formed to erase the stored charge. In this case, the brighter

spot on the Au triangle disappeared, which confirmed that

the oxidized azurin molecules were reduced.

The possibility of an optical detection system was ana-

lyzed where an LSPR-based optical detection system90�91



Figure 13. (a) Absorption spectra of Au pattern on ITO, Az/Au–ITO clearly shows a broad peak at 732 nm and a shoulder peak at 400 nm (b) UV

spectra obtained upon sequential application of oxidized and reduced potential clearly shows the variations in the peak intensity in both oxidized and

reduced forms and (c) zoomed portion of the spectra for the shoulder peak at 400 nm and (d) zoomed portion of the image at the 732 nm peak.

is utilized. Figure 13(a) shows the absorption peak of the

Au nanopattern with azurin immobilized on Au nanopat-

tern, which clearly depicts that azurin due to Cu2+

results in the absorbance at �max = 400 nm (small peak)

and at �max = 730 nm (broad peak). The absorbance at

400 nm is attributed to His → Cu charge transfer transi-

tion, which has a low extension coefficient.92�93 The tran-

sition at 730 nm arises from the d–d transitions, which

also has a low extinction coefficient. So upon applica-

tion of the oxidation and reduction potential and simul-

taneous observation of UV signal, the variation in the

peak intensity shifts down and up at every step shown

in Figure 13(b). This observation leads to optical detec-

tion of read-out mechanism for the operation of memory

device. Figures 13(c), (d) shows the zoomed portions indi-

cating the variations of the shoulder and intense peaks

upon application of oxidation and reduction potentials. All

these spectral changes are a clear indication of the trans-

formation of Az and its redox states, and the process was

reversible.

Earlier reports reveal that reduced azurin has diamag-

netic and oxidized azurin behaves as paramagnetic.94 From

this magnetic behavior, there is a chance for another

read-out mechanism of the charged states. Hence, mag-

netic output can also be used to perform the molec-

ular switch for the read-out mechanism of the switch.

Thus, ESR on the oxidized and reduced forms of azurin

could be detected and magnetic output can also be used

for reading the state of the molecular switch. Based on

these findings, azurin on a hexagonal nanopattern can

be used as a nanoscale switch on binary logic circuits.

The applied electrochemical voltage can be considered

the input; when oxidation voltage was applied, the azurin

turned to its oxidized form (Input 1), and when reduc-

tion voltage was applied the azurin turned back to original

form (Input 0). Four output mechanisms can be detected

from these inputs: (a) an absorption peak at 400 nm, (b) an

absorption peak at 732 nm, (c) the ESR signal, and the

(d) electrochemical output signals. So, a binary logic gate

can be realized from these four outputs.

Based on the electrochemical nature of the protein,

it was reversibly converted between its redox states (oxi-

dized and reduced forms) for memory switching oper-

ations. Additionally, the optical and magnetic responses

can be used as a readout mechanism. Because the azurin

was immobilized on an Au pattern, it can be addressed

locally, operates at very low voltages with its stability,

robustness, and reversibility, making the system a very

promising memory device. The proposed approach will

directly impact the implementation of recent advances in

biotechnology such as biocomputing systems which use

biomolecules, such as DNA/proteins, to perform compu-

tational calculations involving storing, retrieving, and pro-

cessing data.



6. FUTURE PERSPECTIVEThe discussed results back the conviction that proteins

are interesting materials in developing nanoscale mem-

ory devices. Not only for developing memory devices,

but also due to their structural and functional versatility,

they are potentially suited to a wide range of applications.

Further development toward commercial applications can

be envisaged as (at least) tenfold. On the edge of

molecular electronics, protein-based circuits can be con-

ceived of as an interesting solution, once appropriate

techniques for patterning and interconnecting devices are

made available.95�96 The nanoscopic size of the comput-

ing units, down to a single molecule, and their possibly

low cost can be the strengths of new molecular machines.

Conversely, new architectures and computing paradigms

may be required to meet the constraints of this novel

biohardware.97�98 In the memory device operation, memory

characteristics should be obtained without the operation

of STM, which demands the proper external connecting

circuitry mandatory for developing a new generation of

nanoscale memory devices. Also these molecules coupled

to Au nanopattern are of great interest and could be the

possible candidate for next-generation memory devices.

Using the self-assembly properties of proteins and possi-

bly other molecules (e.g., DNA) can lead to very complex

macroscopic structures, achieving finer-than-lithographic

resolution without using traditional lithography.99–101 This

is, in fact, what is needed to build memories and com-

plex computing circuits; that is, this may be the milestone

marking the beginning of the true post-silicon era.

ABBREVIATIONSAEQ Aequorin

AFM Atomic force microscope

AZ Azurin

CV Cyclic voltammetry

DNA Deoxyribonucleic acid

DSC Differential scanning calorimetry

ECSTM Electrochemical scanning tunnelling

microscope

ESR Electron spin resonance

GBP Glucose binding protein

ITO Indium tin oxide

MOSFET Metal oxide semiconductor field effect

transistor

OCP Open circuit potential

OP Oxidation potential

RNA Ribonucleic acid

RP Reduction potential

SAM Self-assembled monolayer

SPR Surface plasmon resonance

STM Scanning tunnelling microscope

TMV Tobacco mosaic virus

UV Ultra violet

WORM Write-once-read-many times.

Acknowledgment: This research was supported

by the Nano/Bio Science and Technology Program

(M10536090001-05N3609-00110) of the Ministry of Edu-

cation, Science and Technology (MEST), by the National

Research Foundation of Korea (NRF) grant funded by the

Korean government (MEST) (2009-0080860), and by the

Ministry of Knowledge Economy (MKE) and Korea Insti-

tute for Advancement in Technology (KIAT) through the

Workforce Development Program in Strategic Technology.

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Received: 3 August 2013. Accepted: 26 August 2013.


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