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ArticleJournal of

Nanoscience and NanotechnologyVol. 14, 2466–2471, 2014

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Electrochemical-Signal Enhanced Information StorageDevice Composed of Cytochrome c/SNP Bilayer

Jinho Yoon1, Yong-Ho Chung1, Si-Youl Yoo1, Junhong Min2, and Jeong-Woo Choi1�∗1Department of Chemical and Biomolecular Engineering, Sogang University,

35 Baekbeom-ro, Mapo-gu, Seoul 121-742, Korea2School of Integrative Engineering, Chung-Ang University, 84 Heukseok-ro,

Dongjak-gu, Seoul 507-202, Republic of Korea

The films organized with biomolecules and organic materials are important elements for develop-ing bioelectronic devices according to their electron transfer property. Until now, several conceptsof techniques have been accomplished to be used for developing biomemory devices. Howeverit is difficult to detect the current signal from the electron transfer between biomolecules and thesubstrate in these fabricated films. To enhance the current signal, the silver nanoparticle was intro-duced to the cytochrome c in this present study. The surface morphology of the fabricated film wasinvestigated by atomic force microscopy. The current signal enhancement was investigated by cyclicvoltammetry. As a result, we could obtain the redox potentials. Moreover, by chronoamperometry,we validated that this proposed layer showed the signal-enhanced memory property for biomemorydevices. This new film composed of the cytochrome c and the silver nanoparticle showed the sig-nal enhancement. Using chronoamperometry, the areas under the graphs between 0 s and 50 mswere calculated. The calculated result showed that the areas under the cytochrome c/SNP graphand cytochrome c graph were 6�93×10−7 C and 4�54×10−7 C, respectively. This numerical valueverified that the cytochrome c/silver nanoparticle hetero-layer film showed better electron chargedbiomemory performance compared to the cytochrome c monolayer. This signal-enhanced film canbe applied to the bioelectronic devices which are able to replace existing electronic devices in thenear future.

Keywords: Cytochrome c, Cyclic Voltammetry, Atomic Force Microscopy, Silver Nanoparticle,Chronoamperometry.

1. INTRODUCTIONRecently, a development of the electronic devices gave ushuge convenience and profit widely. The performance ofthe computer system is growing and the size of the com-ponents is being scaled down. Recently, even smart phoneslike iphone are widely spreading out. The developmentof the electronic technology aids these developments ofthe devices. A silicon-based memory is in almost elec-tronic devices. These memories have been developed a lotin size and density. This development approximately sat-isfies ‘Moore’s law’ and ‘Hwang’s law.’ ‘Moore’s law’ isthat the data capacity of microchips becomes double inevery 18 months. ‘Hwang’s law’ is that the capacity of

∗Author to whom correspondence should be addressed.

the semi-conductor memory becomes double in every year.But in the near future, these laws will meet the limita-tion because of technical problems.1 To overcome theselimitations, many new technics have been studied.2 Thebiomemory, one of them, is being studied to be a newmemory device for the future.3

The biomemory is the part of bioelectronics. Bioelec-tronics use electron transfer reactions in living things toapply for electronic devices.4 Electron transfer reactionsare very important part in biological process such as photo-synthesis and respiration.5�6 Making the biomolecular arti-ficial system with the electron transfer phenomena in thebiological process is essential for developing bioelectronicdevices. For these reasons, electrochemical properties ofbiomolecules have been widely studied to apply for bio-electronic devices.

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Yoon et al. Electrochemical-Signal Enhanced Information Storage Device Composed of Cytochrome c/SNP Bilayer

Kim’s group performed the work that the ferritin whichhas an iron was electrochemically immobilized on the sub-strate by the surface modification method for applying bio-electronic devices.7 Davis’s group studied the tunnelingconductance of the blue copper protein, Azurin.8 Choi’sgroup especially studied a lot in this field. They made thecystein modified azurin and immobilized it on the Au sub-strate. They investigated the biomemory performance ofthat device.9 They also studied about chemical linkers thatconnect the protein and the substrate. They used variouschemical linkers to investigate their effect in the electrontransfer of the devices.10 Above these experiments, a lotof experiments using biomolecules have been widely per-formed for bioelectronic application. On the basis of this,bioelectronic devices can be new memory devices that canovercome technical and physical limitations which presentmemory devices have now days.11�12

But until now, it is difficult to detect the current sig-nal from the electron transfer between biomolecules andthe substrate. Because the current signal is too low to bedetected easily.13 This problem is the obstacle of apply-ing bioelectronic devices to the practical area. Becauseof this problem, the focus of this study was finding aproper solution to solve this problem. The nanoparticle hasa positive effect for enhancing the current signal of theredox response.14�15 In this experiment, we used the sil-ver nanoparticle (SNP) to enhance the current signal. Thefilm which had a hetero-layer composed of biomoleculesand the SNP was fabricated. Cytochrome c was usedas biomolecules in this experiment. Cytochrome c is themetalloprotein that has an iron in the protein, so it hasthe redox property. This can be used for biomemorydevices because of the electron transfer property.16 Wecompared the cytochrome c/SNP hetero-layer film to thecytochrome c monolayer film.In this study, the cytochrome c monolayer film was

fabricated on the Au substrate by using the chemicallinker 6-Mercaptohexanoic acid (6-MHA). The chemicallinker could be immobilized on the Au substrate by self-assembly method and the cytochrome c could be attachedto the 6-MHA linker.17 The connection between the chem-ical linker and the protein was established because of theinteraction of the carboxyl group of the chemical linkerand the amine group of the protein. So the cytochrome cmonolayer was fabricated on the Au substrate. Then theSNP was added to cytochrome c using 1-Octadecanethiol(ODT).18�19 The ODT can connect the cytochrome c andthe SNP.20�21 The ODT has the methyl group and thesulfur head, the methyl group interacts with cytochrome cand the sulfur head interacts with the SNP. Finally, thecytochrome c/SNP hetero-layer film was fabricated on theAu substrate. Figure 1(a) shows the schematic diagramof the fabrication of the film and Figure 1(b) shows theschematic of the electron transfer reaction in the fabricatedfilm.

Figure 1. (a) Schematic diagram of the cytochrome c and the SNPimmobilization. (1) The bare Au without anything. (2) The 6-MHAchemical linker immobilized on the Au substrate. (3) The cytochrome cimmobilized on the Au substrate by the chemical linker. (4) The SNPimmobilized on the cytochrome c. (b) Schematic of the electron transferreaction in the signal enhanced biomemory device.

2. EXPERIMENTAL DETAILS2.1. MaterialsAu substrates composed of Au, Cr, SiO2 wafers were pur-chased from G-mek in Korea. Au substrates were used forAFM analysis and electrochemical experiments like CVand CA. The SNP was purchased from BBI internationalin UK (www.british-biocell.co.uk). cytochrome c, 6-MHA,and ODT were purchased from Sigma-Aldrich (SigmaChemical Company, St. Louis, USA). 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solutionof pH 7.0 was used as the electrochemical buffer. Distilledand deionized (DI) water was used to clean the substrate.N2 gas was used to dry the substrate.

2.2. Fabrication of Cytochrome c/SilverNanoparticle Film

For the sample preparation, first of all, the Au substratewas cleaned by Piranha solution (volume ratio was 70%of H2SO4 and 30% of H2O2) at 70

�C for 3 minutes. H2O2

and H2SO4 were purchased from Sigma-Aldrich (USA).It was used for removing impurities, dust and organicthings on the Au substrate. After cleaning, the Au substrate

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Electrochemical-Signal Enhanced Information Storage Device Composed of Cytochrome c/SNP Bilayer Yoon et al.

was cleaned by ethanol and DI water repeatedly and driedby N2 gas perfectly. The prepared Au substrate was used asa plate for immobilization of 6-MHA. 6-MHA is a chem-ical linker that can connect the Au substrate and protein.The thiol group of the 6-MHA could be connected withthe Au substrate and the carboxyl group of it could beconnected with the amine group of the protein electro-statically. The 6-MHA solution was prepared in DI water(14 �l of the 6-MHA was dissolved in 1 ml of DI water).20 �l of the 6-MHA solution was dropped on the Au sub-strate for 6 hours in 4 �C. During that time, the 6-MHAwas immobilized on the Au substrate by self-assemblymethod. So, the thiol group of the 6-MHA can be boundefficiently on the Au substrate and put the carboxyl groupfree for protein immobilization. After immobilization, theAu substrate was cleaned by DI water repeatedly and thendried by N2 gas. Then 20 �l of the cytochrome c addedon the Au substrate for 3 hours in 4 �C. cytochrome c wasimmobilized on the Au substrate by the chemical linker6-MHA. After that, the Au substrate was cleaned by DIwater and dried by N2 gas. Then cytochrome c monolayerfilm was fabricated on the Au substrate. To add the SNPon the protein, we prepared 0.5 mM solution of the ODT[(1:2) v/v, ethanol and DI water]. 12 �L of that solutionwas added to 48 �L of the SNP solution in a micro cen-trifuge tube for 2 hours. Finally, 20 �L aliquots of theSNP solution were dropped on the Au substrate for anhour in 4 �C. In an hour, the SNP was immobilized onthe cytochrome c through the ODT. And the Au substratewas washed with DI water and dried by N2 gas. Finallythe cytochrome c/SNP hetero-layer film was fabricated onthe Au substrate.

2.3. Morphology Analysis of the FabricatedFilm by Atomic Force Microscopy (AFM)

The surface morphology of the cytochrome c monolayerand the cytochrome c/SNP hetero-layer were investigatedby AFM [Digital instruments Nanoscope (R) IV, USA].AFM was performed by tapping mode. During the exper-iment, the scan size was 500 nm and scan rate was 1 Hz.To verify the immobilization of the cytochrome c and theSNP, surface morphology of the bare Au was comparedto that of the cytochrome c monolayer and that of thecytochrome c/SNP hetero-layer. Therefore, AFM verifiedthe immobilization of the cytochrome c and the SNP onthe Au substrate.

2.4. Electrochemical CharacteristicInvestigation by Electrochemical Method

The redox property of the cytochrome c and the cyto-chrome c/SNP was investigated by the electrochemicalanalyzer (CHI 660, USA). The electrochemical analyzerconsists of three electrodes which are silver/silver chloridedouble junction (Ag/AgCl) electrode as reference elec-trode, Platinum (Pt) electrode as counter electrode, and

the working electrode. The working electrode area was0.25 cm2 of the Au substrate which the cytochrome cor the cytochrome c/SNP was immobilized on. HEPESbuffer (pH = 7�0) was used as the electrolyte in theexperiment. The electrochemical analyzer was operatedby the electrochemical property analysis software. Duringthe CV experiment, the voltage range was from 600 mVto −100 mV, scan rate was 50 mV/s, sample intervalwas 1 mV/s, quiet time was 2 s and sensitivity was 5×10−7 (A/V). After using CV, we obtained two parame-ters which were the oxidation potential and the reduc-tion potential of the cytochrome c/SNP hetero-layer film.These parameters were used for estimating the biomemoryperformance. Chronoamperometry (CA) was used to com-pare the biomemory performance of the cytochrome c/SNPhetero-layer device to that of the cytochrome c monolayerdevice.

3. RESULTS AND DISCUSSION3.1. The Surface Morphology of the Fabricated Film

Analysis Using Atomic Force Microscopy (AFM)The surface morphology of the fabricated films was inves-tigated by AFM. Figure 2(a) shows the surface mor-phology of the bare Au, Figures 2(b) and (c) showsthe surface morphology of the cytochrome c and thecytochrome c/SNP. Surface morphology of the bare Aushowed that cluster size of the bare Au was between 40 nmand 50 nm irregularly. AFM image of the cytochrome cimmobilized on the Au substrate showed some aggrega-tion forms irregularly which looked like small globularforms. These globular forms were not found in the bareAu. This difference between the image of the bare Au andthat of the cytochrome c indicated the immobilization ofthe cytochrome c on the Au substrate. In case of the SNPon the cytochrome c, AFM image showed the aggregationforms which were bigger than the aggregation forms of thecytochrome c averagely. AFM experiments verified thatthe cytochrome c and the cytochrome c/SNP were wellimmobilized on the Au substrate respectively.

3.2. The Analysis of Electrochemical PropertyUsing Cyclic Voltammetry (CV)

Cyclic voltammetry was used for estimating the redoxproperty of the cytochrome c and the cytochrome c/SNP.CV experiment was performed in 10 mM HEPES buffer asthe electrolyte. The CV data showed the oxidation poten-tial and the reduction potential of the cytochrome c andthe cytochrome c/SNP. Figure 3 shows CV graphs. Fromthe results, the oxidation potential and the reduction poten-tial of the cytochrome were 22 mV and 7 mV respec-tively, and those of the cytochrome c/SNP were 265 mVand 177 mV respectively. The CV graphs showed thatthe cytochrome c with the SNP had the signal-enhancedredox currents. These results demonstrated that the SNPinduced the enhancement of the electron transfer rate of

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Figure 2. Surface morphology of the bare Au, cytochrome c and cytochrome c/SNP immobilized on the Au substrate by atomic force microscopy(AFM). (a) the bare Au. (b) the cytochrome c immobilized on the Au substrate. (c) the cytochrome c/SNP immobilized on the Au substrate.

the fabricated film. Presumably, the signal enhancementof the fabricated film by the SNP can be assumed bytwo factors: (a) The SNP facilitates the electron cou-pling between the cytochrome c and the Au substrate.So the better electron transfer reaction was induced.(b) cytochrome c has two surface lysine residues in theprotein (Lys 13 and Lys 79). Lysine residues are locatednear the hemegroup. Because of this fact, the SNP attracts

these two lysine residues of the cytochrome c, which maydock the cytochrome c in an appropriate orientation so thatthe electron tunneling distance between the hemegroup inthe cytochrome c and the Au substrate.22 These factorsmay be the reasons that induce the signal enhancementby the SNP. In conclusion, the cytochrome c/SNP hetero-layer film can be possible to develop the signal-enhancedbiomemory device.




Electrochemical-Signal Enhanced Information Storage Device Composed of Cytochrome c/SNP Bilayer Yoon et al.

Figure 3. Cyclic voltammogram of the bare Au (1), the cytochrome cmonolayer (2) and the cytochrome c/SNP hetero-layer (3) at 50 mV/sscan rate. (10 mM HEPES, pH 7.0, cytochrome c sample concentration=0�2 mg/ml, substrate area= 0�25 cm2, silver nanoparticle size= 20 nm).

Figure 4. Signal-enhanced biomemory function of the signal enhancedcytochrome c/SNP hetero-layer devices using CA. (a) the potential valueof the oxidation potential (write step) and the reduction potential (erasestep). (b) the expanded corresponding currents from 0 ms to 50 ms.Inserted small graph showed the corresponding currents as a total dura-tion of 2500 ms. Curve 1 shows the currents of the cytochrome cimmobilized on the Au substrate and curve 2 shows the currents of thecytochrome c/SNP hetero-layer immobilized on the Au substrate.

3.3. The Analysis of Biomemory PerformanceUsing Chronoamperometry (CA)

The CA method was used to estimate the signal-enhancedbiomemory performance. For CA experiment, the oxida-tion potential and the reduction potential obtained fromCV experiments were used. To acquire CA data, the oxi-dation potential of 245 mV and the reduction potentialof 177 mV were applied for the cytochrome c monolayerand the cytochrome c/SNP hetero-layer. During the exper-iment, 0.25 s of pulse width, 0.001 s of sample inter-val, 2 s of quiet time, and 1× 10−4 (A/V) of sensitivitywere applied. The Figure 4 showed the biomoemory perfor-mance of the fabricated film from 0 s to 50 ms, and insertedfigure showed the overall graphs from 0 s to 2500 ms.To evaluate the biomemory performance, the equation ‘Q=∫i ∗ dt’ was used. Using CA graphs, the areas under

the graphs between 0 s and 50 ms were calculated bythis equation, because this range showed the apparent gapbetween the graph of the cytochrome c and that of thecytochrome c/SNP. The calculated result showed that theareas under the cytochrome c/SNP graph and cytochrome cgraph were 6�93×10−7 C and 4�54×10−7 C respectively.From this result, cytochrome c with the SNP hetero-layershowed better electron charged biomemory performancethan the cytochrome c monolayer immobilized on the Ausubstrate. The current change in the oxidation potentialand the reduction potential can be a data processing rolefor a memory device, the oxidation potential value as‘write step’ and the reduction value as ‘erase step’ like‘1’ and ‘0’ values applying for the future biocomputingdevice.

4. CONCLUSIONIn the present experiment, the cytochrome c/SNP hetero-layer film was fabricated for the current signal enhance-ment. Proposed experiments using biomolecules forapplying bioelectronic devices have a problem that thecurrent signal from the electron transfer was too low tobe detected easily. To overcome this problem, the SNPwas introduced for enhancing the current signal betweenbiomolecules and the substrate.cytochrome c was immobilized on the Au substrate by

the chemical linker 6-MHA. Then, the SNP was attachedto the cytochrome c through the ODT. The immobiliza-tion and the surface morphology of the cytochrome c andthe cytochrome c/SNP were confirmed by AFM. The redoxproperty of the fabricated films was verified by CV. Thusthe redox property of the cytochrome c/SNP hetero-layerwas compared to that of the cytochrome c monolayer.It was confirmed that the electron transfer property ofthe cytochrome c/SNP hetero-layer was better than thatof the cytochrome c monolayer. Then, using the oxidationpotential and the reduction potential values from CV data,the biomemory performance of the fabricated films werecharacterized by CA. The biomemory performance of the

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cytochrome c/SNP hetero-layer was compared to that of thecytochrome c monolayer. As a result, the SNP enhanced theelectron transfer signal and the cytochrome c/SNP hetero-layer device showed better biomemory performance thanthe cytochrome c monolayer device. Thus the SNP canbe an important element for overcoming the limitationthat the present bioelectronic devices have. In conclusion,this signal-enhanced bioelectronic device composed of thecytochrome c and the SNP can contribute to overcomethe limitation of the present silicon-based memory deviceshave and can be studied for being used as a new memoryconcept.

Acknowledgments: This research was supported bythe National Research Foundation of Korea (NRF) grantfunded by the Korea government (MSIP) (2009-0080860),by the National Research Foundation of Korea (NRF)grant funded by the Ministry of Science, ICT and FuturePlanning (2005-2001333), and by the National ResearchFoundation of Korea (NRF) grant funded by the Korea gov-ernment (MEST) (2012K2A2A4021554).

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Received: 14 January 2013. Accepted: 5 March 2013.


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