Thin Solid Films 551 (2014) 174–180

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Multilevel electrochemical signal detections of metalloproteinheterolayers for bioelectronic device

Yong-Ho Chung a, Si-Youl Yoo a, Taek Lee a, Hun Joo Lee c, Junhong Min b, Jeong-Woo Choi a,c,⁎a Department of Chemical & Biomolecular Engineering, Sogang University, 35 Baekbeom-ro(Sinsu-dong), Mapo-gu, Seoul 121-742, Republic of Koreab School of Integrative Engineering, Chung-Ang University, Heukseok-dong, Dongjak-gu, Seoul 156-756, Republic of Koreac Interdisciplinary Program of Integrated Biotechnology, Sogang University, 35 Baekbeomro(Sinsu-dong), Mapo-gu, Seoul 121-742, Republic of Korea

⁎ Corresponding author. Tel.: +82 2 705 8480; fax: +8E-mail address: jwchoi@sogang.ac.kr (J.-W. Choi).

0040-6090/$ – see front matter © 2013 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.tsf.2013.11.077

a b s t r a c t

a r t i c l e i n f o

Article history:Received 31 December 2012Received in revised form 22 October 2013Accepted 18 November 2013Available online 27 November 2013

Keywords:Bioelectronic deviceMultilevel-signalProtein heterolayerCyclic voltammetryLayer by layer assembly

In the present study, we investigated the simultaneous detection of multilevel electrochemical signals from var-ious metalloprotein heterolayers for the bioelectronic devices. A layer-by-layer assembly method based on sim-ple electrostatic interaction was introduced to form protein bilayers. The gold substrate was modified with poly(ethylene glycol) thiol acid as the precursor, which introduced negative charges to the surface. Based on the iso-electric point, net-charge controlled metalloproteins by pH adjustment were sequentially immobilized on thisnegatively charged substrate. The degree of protein immobilization on the gold substrate was confirmed by sur-face plasmon resonance spectroscopy, and the surface topology changes due to the protein immobilization wereconfirmed by atomic forcemicroscopy. Redox signals in the protein layers weremeasured by cyclic voltammetry.As a result, various redox signals generated from different metalloproteins on a single electrodeweremonitored.This proposed method for the detection of multi-level electrochemical signals can be directly applied to bioelec-tronic devices that store multi-information in a single electrode.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Biomaterials have received much attention as substitutes for organicand inorganic materials in conventional electronic devices. Biomoleculesprovide important biological phenomena such as energy conversionthrough electron transfer between high and low states, and material en-trance by specific recognition and delivery [1–4]. The reactions of biomol-ecules are very effective and specific, embodying functions that cannot beoffered by nonbiological materials. Experiments using biomaterialsincludingDNA, proteins, peptides, enzymes, and antibodies have been re-ported [6–10]. In particular, Faradaic current resulting from the oxidationand reduction of biomolecules has been reported in bioelectrochemistryfor the development of biosensors that can be used to monitor biologicalphenomena in a single cells or tissues [3,5].

Recently, the fundamental concept of a biomemory device using theredox property of a metalloprotein was proposed [11]. This concept isbased on the state changes of ametal ion in the core of ametalloprotein.These changes are used in biological systems for delivering materialsand signals. The ionic valence of metal ions in a metalloprotein changesunder specific physiological conditions and can be artificially controlledby applying electrical potential. The change of an ionic state from re-duced to oxidized form can represent the ‘0’ and ‘1’ of a conventionalmemory device. Several biomemory devices have been designed and in-vestigated based on the intentional state change of a metalloprotein

2 2 3273 0331.

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[12–15]. However, these studies were conducted on a single layer com-posed of oneprotein species immobilized on a solid substrate because ofcontrol difficulties. As a result, this single layer can store only one signal.A double layer stacked with two protein species can store four pieces ofdata by combining two signals, and we already demonstrated a multi-level biomemory device using a heterolayer composed of recombinantazurin and cytochrome c [16]. However, the method for forming aheterolayer in the previous work cannot be extended to other re-searches using different kinds of heterolayers, because the recombinantDNA technique for the modification of a protein structure is difficult toapply other kinds of metalloproteins. Therefore, another fabricationmethod is required to form heterolayers more easily and effectively.

Variousmethods have been proposed to immobilize biomaterials ona solid substrate, such as theuse of the Langmuir–Blodgettfilm,moldingmethod, and layer-by-layer (LbL) assembly [17–20]. The most commonmethod for immobilizing biomaterials on a solid substrate is the use oflinker materials that can combine with the biomaterials and the solidsubstrate on each side. This immobilization method is effective in con-trolling the formation of a thin film biomaterial monolayer, although ithas some problems of inactivation and characteristic changes due tothe compulsion of the protein structure by chemical modification. TheLbL method utilizes the electrostatic interactions between positive andnegative charges to stably preserve the original characteristics of bioma-terials [21,22]. By the LbL method, a heterolayer can be formed easily,and the redox properties of immobilized biomaterials can be measureddirectly, because the LbL method is based on the natural binding be-tweenoppositely chargedmoleculeswithout theuse of chemical linkers

http://dx.doi.org/10.1016/j.tsf.2013.11.077mailto:jwchoi@sogang.ac.krhttp://dx.doi.org/10.1016/j.tsf.2013.11.077http://www.sciencedirect.com/science/journal/00406090

Fig. 2. SPRmeasurements for confirming the thickness of film deposited using LbL assem-bly. (a) Using oppositely charged metalloproteins. (b) Using various combinations ofmetalloproteins.

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or themodification of the protein structure to bindwith the linkingma-terials, in which the redox property of a metalloprotein could bechanged and inactivated due to the structural changes.

Herein, we fabricated heterolayers composed of various metal-loproteins by LbL assembly to generate multilevel electrochemical sig-nals on a single electrode simultaneously. The thickness change of theprotein layers which were combined with the various metalloproteinswas monitored by surface plasmon resonance (SPR) spectroscopy, andthe surface topologies of each layer were analyzed by atomic force mi-croscopy (AFM). The redox properties of the heterolayers were mea-sured with cyclic voltammetry (CV) for effective immobilization. Thenet charge of the biomolecules was controlled by adjusting the pH ofthe buffer solution. The surface charges of the proteins in the solutionat some pH were determined by the pI value, which is the contentratio of acidic and basic components. As shown in Fig. 1, surfacecharge-controlled metalloproteins were alternately deposited on agold substrate. The degree of immobilization was also affected by thenet charge difference between the upper and lower layers.

2. Experimental details

2.1. Materials

Azurin from Pseudomonas aeruginosa, myoglobin and cytochrome-cfrom horse heart, lactofferin from human milk, ferredoxin from spinach,and O-(2-mercaptoethyl)-O′-(2-carboxyethy) heptaethylene glycol(PEG thiol acid, MW 458) were purchased from Sigma-Aldrich(St. Louis, MO., USA). The gold electrodes were prepared by electronbeam evaporation of Cr (2 nm) as the adhesion layer and Au (43 nm)on a Si (0.5 mm)/SiO2 (300 nm) substrate and on a BK7 cover glass (Su-perior, Germany) for the SPRmeasurement. A 10 mM solution of HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and acetatebuffers containing sodium chloride were used tomake the protein solu-tion. The solution pH was adjusted with KOH and HCl. Distilled anddeionized (DI) water (resistance ≥ 18 MΩ), produced by a Milli-Q®System (Millipore Corp.).

2.2. Multi-layer formation

The gold substrates for protein immobilizationwere initially cleanedwith piranha solution consisting of 80 vol.% H2SO4 (Daejung Chemicaland Metals Co. Ltd., Korea) and 20 vol.% H2O2 (Duksan Pure ChemicalandMetals Co. Ltd., Korea) at 65 °C for 5 min. The substrates were suffi-ciently washed with pure ethanol and DI water to remove physicallyadsorbed residues, followed by drying under nitrogen gas. PEG thiolacid was dissolved in DI water to a concentration of 1 mg/ml. Andthen, the prepared gold substrates were initially dissolved in the PEGthiol acid solution to form a self-assembly monolayer (SAM), taking

Fig. 1. Schematic description of multi-layer formation by LbL assembly using electr

advantage of the covalent bonding between the gold surface and thiolgroups. This reaction was conducted for 6 h at room temperature. Fivekinds of metalloproteins (0.5 mg/ml) were dissolved in the buffer solu-tion at the appropriate pH (10 mM HEPES buffer of pH 7.0 was mainlyutilized),whichmodulates thenet charge between the positive andneg-ative charges of themetalloproteins to formmulti-layers by electrostaticbonding. The net charges of the protein solutions for the first layer werepositive, because the PEG SAMon the gold surface had a negative chargedue to the carboxyl group on its surface. The other protein solutions forthe second layerwere conversely controlled to have a negative charge. Amulti-layer was formed on the gold surface by immersing the substratealternately in the protein solutions, which had different net charges, for20 min at room temperature. The washing steps using the buffer

ostatic interaction between negatively and positively charged metalloproteins.

image of Fig.�2

Fig. 3. AFM images to analyze the changes in surface morphology, and all images fitted by the same depth level of 30 nm scale, as shown in the center of the figure. (a) Bare gold, (b) PEGthiol acid/myoglobin/cytochrome c, (c) PEG thiol acid, (d) PEG thiol acid/myoglobin, (e) PEG thiol acid/myoglobin/ferredoxin.

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solution, DI water, and nitrogen gas were also performed to remove res-idues that were physically adsorbed during the immersion steps. Afterfabricating heterolayers composed of various metalloproteins on thegold substrate, all measurements related with confirmation of the elec-trochemical properties were immediately operated to avoid the inacti-vation of the immobilized biomolecules at room temperature.

Fig. 4. Cyclic voltammograms of variousmetalloprotein heterolayers. (a) Myoglobin, (b) cytochby using mixed solution of myoglobin and cytochrome c.

2.3. The confirmation of immobilization by surface plasmon resonance(SPR) spectroscopy and atomic force microscopy (AFM)

The SPR analysis was conducted with the Multiskop system (OptrelGBR, Germany) to confirm immobilization of protein on the gold sur-face. When a material is immobilized on a gold surface, the angle of

rome c, (c) heterolayer composed ofmyoglobin and cytochrome c, (d) thin film fabricated

image of Fig.�3image of Fig.�4

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reflected light is changed by surface plasmon resonance. This methodhas very high sensitivity as it can detect nano materials immobilizedon a metal surface [23]. The laser source has a wavelength of632.8 nm, and the range of the incidence angle was 38–50°. All mea-surementswere conducted at a constant temperature of 21 °C, and reli-able results were obtained by averaging values from 10 pointmeasurements in a sample at the same interval.

The topological changes on the gold surface resulting from proteinimmobilization were monitored by AFM (Multimode, Veeco, USA),which can detect surface changes of sub-angstrom scale from frequencychanges of its cantilever. Tapping mode was utilized to prevent dam-ages of the soft immobilized metalloproteins. This measurement sup-plemented the SPR analysis, which does not show the change insurface structure due to protein immobilization. A cantilever fabricatedwith phosphorus (n) doped silicon, having a resonance frequency of257–319 kHz, was utilized for AFM measurement. The tip velocity andscan size were 2.0 μm/s and 500 nm × 500 nm, respectively.

2.4. The electrochemical measurements

The redox properties of the single and multi-protein layers wereconfirmed by cyclic voltammetry with an electrochemical analyzer(CHI 660, USA). The protein immobilized gold substrate, Pt electrode,and Ag/AgCl in saturated KCl were used as the working, counter, andreference electrodes, respectively. The scan rate was 50 mV/s, and thepotential range was controlled differently in each case, because eachprotein had different position redox signals. The volume of the electro-chemical cell was 5 mL, and the 10 mM HEPES buffer solution as the

Fig. 5. Cyclic voltammograms of metalloproteins. (a) Heterolayer composed of myoglobin andmyoglobin, cytochrome c, and ferredoxin.

electrolytewas purgedwith pure nitrogen gas for 5 min before all mea-surements to remove its oxygen content, which can react with the ma-terials immobilized on the electrode.

3. Results and discussion

3.1. SPR analysis for confirming the multi-layer

Fig. 2 shows the angle shifts according to the formation of themulti-layers composed of various metalloproteins. Film thickness was in-creased by forming a multi-layer of sequentially stacked, negativelyand positively chargedmetalloproteins, as shown in Fig. 2a. The processof LbL assembly was conducted in the HEPES buffer (pH 7.0). Cyto-chrome c and lactoferrin were positively charged at pH 7.0, becausetheir pI values are about 10.6 and 8.7, respectively [24,25], and ferredox-in was negatively charged at the same pH due to its low pI value of 3.6[26]. The charged state of myoglobin (pI, 6.8) was neutral at pH 7.0[27]. The angle shift increased gradually to about 1.15° in the multi-layer composed of PEG thiol acid, cytochrome c, ferredoxin, andlactoferrin, formed by deposition of oppositely chargedmetalloproteins.No remarkable change in myoglobin deposition was observed becauseof the neutral charge, and this phenomenon was related to the differ-ence of net charge, as confirmed by other experiments related tometalloproteins, as shown in Fig. 2b. The thickness of the multi-layerconsisting of PEG thiol acid and myoglobin was lower than heterolayerconsisting of PEG thiol acid and cytochrome c in Fig. 2a. This layer for-mation resulted from the negatively charged surface of the precursorlayer due to the carboxyl groups. Myoglobin was slightly immobilized

azurin. (b) Heterolayer composed of myoglobin and ferredoxin. (c) tri-layer composed of

image of Fig.�5

Fig. 6. Variation of redox signals due to the pH change. (a) Cytochrome c/myoglobin.(b) Cytochrome c/ferredoxin.

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on the precursor layer because of the neutral net charge at pH 7.0. Thisphenomenon was clearly monitored in the immobilization of othermetalloproteins which were negatively charged at the same pH. Whenazurin and ferredoxin, which are negatively charged at pH 7.0, were de-posited on the P/M layer, the angle shift changes were very low. Smallamounts of azurin and ferredoxinwere attached regardless of the repul-sion force between the negatively charged biomolecules, and it waslikely that the net charge of the P/M layer slightly moved to a neutralvalue due to the immobilization of myoglobin on the PEG thiol acid.The dramatic increases of angle shift were observed in the depositionof positively charged cytochrome c on the P/M and P/M/F layers, asshown in the results of the P/M/C and P/M/F/C layers.

3.2. Validation of surface topology by AFM

The grain size of the gold surface was affected by themethod of thinfilm formation such as sputtering, electron beam evaporation, and ther-mal evaporation. The gold substrate utilized in this research was pre-pared by electron beam evaporation, and the average grain size wasabout 50 nm (Fig. 3a). As shown in Fig. 2, in case of immobilizationwith the PEG thiol acid at pH 7.0, cytochrome c, which has high pIvalue, was more effective than myoglobin owing to the difference ofnet charge. However, the first metalloprotein for AFM measurementswas determined asmyoglobin because cytochrome cwas difficult to ob-serve for detection of topology changes by stage due to the high immo-bilization ratio. The topology of the gold surface was changed by thedepositions of the precursor andmetalloproteins, because biomoleculestend to aggregate into a globular shape. Fig. 3b shows the dramaticchange in surface topology due to the sequentially deposited PEG thiolacid, myoglobin, and cytochrome c. The changed grain size was about20 nm, indicating that the multi-layer was formed well by the LbL as-sembly process. The grain size due to the PEG thiol acidwas a fewnano-meters (Fig. 3c), and the clusters of the bi-layer formed by addingmyoglobin was about 10 nm, as shown in Fig. 3d. However, the surfacetopology was unchanged by the deposition of ferredoxin because of thenet charge effect (Fig. 3e), the same reason asmentioned in the descrip-tion of Fig. 2b.

3.3. Electrochemical properties of fabricated heterolayers

The redox properties of the multi-layer were measured with the CVtechnique. Fig. 4a and b show the cyclic voltammograms of single pro-tein layers consisting of myoglobin and cytochrome c on PEG thiolacid, respectively. Epc (reduction potential) and Epa (oxidation poten-tial) of the immobilized myoglobin and cytochrome c were 290 mV/320 mV, and 60 mV/230 mV, respectively. These potentials relatedwith redox reactions could be different from those of other experimentsbecause the position of the redox peak could have been affected by theproperty and length of the precursor used to bind the gold substrate tothe metalloprotein [28]. When a heterolayer was formed with myoglo-bin and cytochrome c by LbL assembly (Fig. 4c), two Epc peaks weremonitored independently on the cyclic voltammogram. The peakpositions of 300 mV for myoglobin and 60 mV for cytochrome c in thebilayer were not changed at all because they were the same as the posi-tion of the peak for the single layer (Fig. 4a, b). The Epa peak of cyto-chrome c merged with the Epa peak of myoglobin due to their similarpositions. This result shows that each protein in the bilayer interactedwith the gold surface through an individual specific potential withoutinactivation, and this interaction meant that LbL assembly was effectivefor fabricating a hetero layer composed of various metalloproteins withrespect to the stability and maintenance of redox properties in each ofthe stacked metalloproteins. The peaks of different kinds of metal-loproteins in a single layer, which could be fabricated by immobilizinga mixture of different metalloproteins, were difficult to separatedue to the signal mixing and interference between biomolecules. Thecyclic voltammogram of a single layer composed of myoglobin and

cytochrome c by immobilization of amixture did not show independentredox signals of immobilized metalloproteins (Fig. 4d). This differencebetween LbL andmixed layersmay be due to the property of the depos-ited thin film. The electrochemical property of immobilized protein wasmonitored as a continuous signal by cyclic voltammetry. It meant thatall of immobilized proteins were not reduced and oxidized at a specificpotential. Some proteins could be reacted by applying potential early orlate, according to the direction of arrangement, degree of attachment,and state of the protein. We assumed that the most important compo-nent of protein reaction by applied potential was the distance fromthe electrode surface to themetal ions in themetalloprotein. The signalsof a mixed layer would be merged by the combination of the redoxproperties of different kinds of metalloproteins, which were measuredas a continuous signal by sequentially applied potential, because the dis-tance from the gold surface to themetal ions could not be defined due tothe irregular deposition of each metalloprotein. Redox signals at Epcand Epa were generated by some metalloproteins at similar distancesfrom the solid surface in the protein layer. However, two independentsignals were well separated by LbL assembly because the distancefrom the protein to the metal surface could be divided into two partsby sequential deposition. Heterolayers composed of variousmetalloproteins for generating different redox signals in a single elec-trode were also fabricated as shown in Fig. 5a and b. The redox peakof myoglobin, as the first layer on the PEG thiol acid, was not changedafter the bilayer formation, and the Epc values of azurin and ferredoxinwere well monitored independently, whereas the Epa of azurin mergedwith the Epa of myoglobin same as cytochrome c.We utilized five kinds

image of Fig.�6

Table 1Specific results according to the metalloprotein combination and pH change.

Heterolayer pI value Net charge Epc(V) Current at Epc (pA)

1st 2nd 1st 2nd 1st 2nd

Cytochrome c/myoglobin (pH 5) +++ + 0295 0091 22.5 26.5Cytochrome c/myoglobin (pH 7) (10 6/6 8) ++ Neutral 0297 0 123 43.9 29.7Cytochrome c/myoglobin (pH 9) + − 0288 0.118 35.8 44.9Cytochrome c/ferredoxin (pH 5) +++ − 0.078 −0.226 13.2 26.6Cytochrome c/ferredoxin (pH 7) (10 6/3 6) ++ − 0.059 −0.229 10.3 27.9Cytochrome c/ferredoxin (pH 9) + − 0.056 −0.228 10.4 26.6Myoglobin/ferredoxin (pH 5) + − 0.355 −0.195 126 15.2Myoglobin/ferredoxin (pH 7) (6.8/36) Neutral − 0.33 −0.21 129 13.7Myoglobin/ferredoxin (pH 9) − − 0.353 −0.183 12.7 13.4

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of metalloproteins for this study. However, redox signals in the tri-layers were difficult to separate reliably because the deposited metal-loproteins were oxidized and reduced in a similar range of potentialsas shown in the Epa of Figs. 4c and 5a. Though the Epc and Epa valuesof myoglobin and ferredoxin were well separated as shown in Fig. 5b,most metalloproteins had similar oxidation and reduction potentials.These phenomena also resulted from the biomaterial properties,which were easy to damage and inactivate under high applied poten-tials. Representative result for the demonstration of triple redox signalswere shown in Fig. 5c. Myoglobin, cytochrome c, and ferredoxin weresequentially deposited on the PEG thiol acid layer. Cyclic voltammo-gram represented in Fig. 5c was drawn by using 4 results that operatedwith same experimental conditions (buffer pH, immobilization andmeasurement process), in which different signals were monitored,while the position of a redox signal (checked by black arrows) waswell matched with results described in Figs. 4c and 5b. We assumedthat the reason of this unstable result was the different distance fromthe electrode surface due to the imbalanced combination. The interac-tions between metalloproteins in the heterolayer would be governedwith various mechanisms (affinity, specific/non-specific binding, andhydrophobic effect) in addition to surface net charge.

3.4. pH effect in the interaction between metalloproteins

The intensity and position of redox peaks generated fromheterolayers were affected by the interaction between metalloproteins,governed by the pH value in the process of heterolayer formation. Fig. 6showed the variation of redox peaks according to the pH changes in theformation of the second layer. Cytochrome cwas deposited on PEG thiolacid as the first layer, and then myoglobin and ferredoxin dissolved inthree kinds of solution with pH 5, 7, and 9 were sequentially depositedon the first layer, respectively. In the analysis of SPR and AFM usingheterolayers fabricated by reaction solution with pH 7, the amount ofsequentially deposited metalloprotein was mainly increased by the dif-ference of pI value between two metalloproteins. However, the redoxsignals of fabricated heterolayers in Fig. 6 indicated different results,though the difference of pI value was the same, due to the fact thatthe net charge of the first layer could be also changed by the solutionpH of second layer. As shown in Fig. 6a, redox peak of cytochrome c atEpc was altered to different positions and intensities, though theamount of immobilized cytochrome cwas same because it was deposit-ed on the precursor layer before the immobilization of second layer. Thestability of electrostatic junction in the various pH conditions was al-ready reported in previous research [29,30]. This result indicated thatprotein–protein interaction by the difference of net charge affectedthe amount of redox-reactive metalloproteins. Another result using aheterolayer composed of cytochrome c and ferredoxin was shown inFig. 6b, in which a similar phenomenon related with the changes ofpeak position and intensity was monitored. Specific results accordingto the pH change in the formation of the heterolayer using two kindsof metalloproteins were arranged in Table 1.

4. Conclusion

We investigated the multilevel electrochemical signal of proteinheterolayers. The immobilization process according to the net chargeof metalloproteins at the same pH was confirmed with SPR and AFM,which proved that the efficiency of immobilization was determined bythe difference of the surface charge between upper and lower metal-loprotein layers. Using these results, the bilayers composed of variousmetalloproteins having various combinations of negative and positivecharges were fabricated, and two independent signals of differentmetalloproteins in a single electrode were well separated withoutchanging the positions of the redox peaks. These results indicated thatthe LbL assembly process was a very effective immobilization methodthat maintained the activity of the biomaterials. This proposed methodis a platform technology for formingheterolayers composed of biomate-rials, which has potential in areas such as multilevel biomemory devel-opment requiring high stability and multi-functions, biosensingsystems using electron transfer between layers, and biomimetic sys-tems based on respiration cycles.

Acknowledgments

This research was supported by the Nano/Bio Science & TechnologyProgram (M10536090001-05 N3609-00110) of the Ministry ofEducation, Science and Technology (MEST), by the National ResearchFoundation of Korea (NRF) grant funded by the Korean government(MEST) (2009-0080860), by Leading Foreign Research Institute Recruit-ment Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning (MSIP)(2013K1A4A3055268)

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Multilevel electrochemical signal detections of metalloprotein heterolayers for bioelectronic device1. Introduction2. Experimental details2.1. Materials2.2. Multi-layer formation2.3. The confirmation of immobilization by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM)2.4. The electrochemical measurements

3. Results and discussion3.1. SPR analysis for confirming the multi-layer3.2. Validation of surface topology by AFM3.3. Electrochemical properties of fabricated heterolayers3.4. pH effect in the interaction between metalloproteins

4. ConclusionAcknowledgmentsReferences