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REVIEW Copyright © 2016 by American Scientific Publishers All rights reserved. Printed in the United States of America Science of Advanced Materials Vol. 8, pp. 1–20, 2016 www.aspbs.com/sam Electrochemical Scanning Tunneling Microscopy Analysis on Protein Based Electronic Devices Ajay Kumar Yagati 1 , Ji-Young Lee 2 , Eun-Sook Nam 3 , Sungbo Cho 1, and Jeong Woo Choi 2, 4, 1 Department of Biomedical Engineering, Gachon University, Yeonsu-gu, Incheon, 21936, Republic of Korea 2 Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, 04107, Republic of Korea 3 Sogang-Binggrae Food Advanced Analysis Research Center, Sogang University, Seoul, 04107, Republic of Korea 4 Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, 04107, Republic of Korea ABSTRACT Scanning probe microscopy (SPM) techniques demonstrate one of the most promising tools to investigate the physical and chemical properties of materials at nanoscale and become the most common and important char- acterization tools in the field of nanotechnology. Among many SPM methods electrochemical scanning tunneling microscopy (EC-STM) technique is one technique that directly provides three-dimensional real-space images with in-situ interfacial electrochemical studies and allows locally measured properties of nanostructured mate- rials at atomic resolution. Furthermore, EC-STM based studies provides information on solution covered areas of electrode surfaces, metal deposition, charge transfer, potential-dependent surface morphology, corrosion, semiconductors, and various applications such as protein conductance measurements in nanobioelectronics. Therefore, in this review, we summarize the electrochemical scanning tunneling microscopic investigation on protein based electrode structures and their applications towards novel bioelectronic devices along with recent developments in ECSTM techniques with future prospects in the field of nanobiotechnology. KEYWORDS: Review, Protein, Electrochemical, Tunneling, Azurin. CONTENTS 1. Introduction ................................. 1 1.1. Scanning Tunneling Microscopy ................. 3 1.2. Scanning Tunneling Spectroscopy ................ 3 1.3. Electrochemical Scanning Tunneling Microscopy (ECSTM): A Promising Tool for Imaging with In-Situ Electrochemical Analysis ................ 4 2. Electrochemistry with Scanning Tunneling Microscopy ..... 5 2.1. Structure of Single Crystal Surfaces, Adsorptions and Self-Assembly of Molecules ................... 5 2.2. Current Distance Spectroscopy and Break Junction Techniques ........................ 5 2.3. ECSTM Based Nanostructure Formations ........... 8 3. Application of ECSTM in Protein Electrochemistry for Nanobioelectronic Devices ..................... 10 3.1. ECSTM Studies Towards Protein Based Memory Devices, Transistors ................... 10 3.2. Single Molecule Charge Transport and Switching ...... 14 4. Summary and Outlook .......................... 18 Acknowledgment ............................. 19 References and Notes ........................... 19 Authors to whom correspondence should be addressed. Emails: [email protected], [email protected] Received: xx Xxxx xxxx Accepted: xx Xxxx xxxx 1. INTRODUCTION There is a tremendous growth in nanotechnology in recent years with innovative tools and methods that enabled us to study and analysis of atoms, molecules and larger atomic structures. 1 2 Further, with the development of new techniques and instrumentation, enabled the analy- sis and manipulation at atomic scale, which paved the path for the design and production of advanced nano- structured materials. 3 The invention of scanning tunneling microscope (STM) by Binning and Rohrer in 1982 has provided a new high resolution tool 4 5 to look at the sur- faces and their achievement was recognized by the Nobel Prize. The other form of scanning tunneling microscopy, such as atomic force microscopy 6 provides the informa- tion about the surface topography and surface forces. 7 The advancement in techniques led to various detection methods and enabled to detect surface structures in liq- uid environment with the help of an electrochemical cell with in situ electrochemical properties. 8 9 At present, STM is a powerful tool for analyzing metallic and semicon- ductor surface with real-space visualization of surface at atomic scale. By exploiting the SPM hardware in con- jugation with optical detection methods enabled to study the biological systems such as live cells and applica- tions in near-field optical systems. 10 11 SPM possess var- ious operational modes which can be tuned according to Sci. Adv. Mater. 2016, Vol. 8, No. xx 1947-2935/2016/8/001/020 doi:10.1166/sam.2016.3003 1

    Copyright © 2016 by American Scientific Publishers

    All rights reserved.

    Printed in the United States of AmericaScience of

    Advanced MaterialsVol. 8, pp. 1–20, 2016


    Electrochemical Scanning Tunneling MicroscopyAnalysis on Protein Based Electronic DevicesAjay Kumar Yagati1, Ji-Young Lee2, Eun-Sook Nam3, Sungbo Cho1,∗ and Jeong Woo Choi2,4,∗

    1Department of Biomedical Engineering, Gachon University, Yeonsu-gu, Incheon, 21936, Republic of Korea2Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, 04107, Republic of Korea3Sogang-Binggrae Food Advanced Analysis Research Center, Sogang University, Seoul, 04107, Republic of Korea4Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, 04107, Republic of Korea


    Scanning probe microscopy (SPM) techniques demonstrate one of the most promising tools to investigate thephysical and chemical properties of materials at nanoscale and become the most common and important char-acterization tools in the field of nanotechnology. Among many SPM methods electrochemical scanning tunnelingmicroscopy (EC-STM) technique is one technique that directly provides three-dimensional real-space imageswith in-situ interfacial electrochemical studies and allows locally measured properties of nanostructured mate-rials at atomic resolution. Furthermore, EC-STM based studies provides information on solution covered areasof electrode surfaces, metal deposition, charge transfer, potential-dependent surface morphology, corrosion,semiconductors, and various applications such as protein conductance measurements in nanobioelectronics.Therefore, in this review, we summarize the electrochemical scanning tunneling microscopic investigation onprotein based electrode structures and their applications towards novel bioelectronic devices along with recentdevelopments in ECSTM techniques with future prospects in the field of nanobiotechnology.

    KEYWORDS: Review, Protein, Electrochemical, Tunneling, Azurin.

    CONTENTS1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

    1.1. Scanning Tunneling Microscopy . . . . . . . . . . . . . . . . . 31.2. Scanning Tunneling Spectroscopy . . . . . . . . . . . . . . . . 31.3. Electrochemical Scanning Tunneling Microscopy

    (ECSTM): A Promising Tool for Imaging withIn-Situ Electrochemical Analysis . . . . . . . . . . . . . . . . 4

    2. Electrochemistry with Scanning Tunneling Microscopy . . . . . 52.1. Structure of Single Crystal Surfaces, Adsorptions and

    Self-Assembly of Molecules . . . . . . . . . . . . . . . . . . . 52.2. Current Distance Spectroscopy and Break

    Junction Techniques . . . . . . . . . . . . . . . . . . . . . . . . 52.3. ECSTM Based Nanostructure Formations . . . . . . . . . . . 8

    3. Application of ECSTM in Protein Electrochemistryfor Nanobioelectronic Devices . . . . . . . . . . . . . . . . . . . . . 103.1. ECSTM Studies Towards Protein Based

    Memory Devices, Transistors . . . . . . . . . . . . . . . . . . . 103.2. Single Molecule Charge Transport and Switching . . . . . . 14

    4. Summary and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . 18Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19References and Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

    ∗Authors to whom correspondence should be addressed.Emails: [email protected], [email protected]: xx Xxxx xxxxAccepted: xx Xxxx xxxx

    1. INTRODUCTIONThere is a tremendous growth in nanotechnology in recentyears with innovative tools and methods that enabled usto study and analysis of atoms, molecules and largeratomic structures.1�2 Further, with the development ofnew techniques and instrumentation, enabled the analy-sis and manipulation at atomic scale, which paved thepath for the design and production of advanced nano-structured materials.3 The invention of scanning tunnelingmicroscope (STM) by Binning and Rohrer in 1982 hasprovided a new high resolution tool4�5 to look at the sur-faces and their achievement was recognized by the NobelPrize. The other form of scanning tunneling microscopy,such as atomic force microscopy6 provides the informa-tion about the surface topography and surface forces.7

    The advancement in techniques led to various detectionmethods and enabled to detect surface structures in liq-uid environment with the help of an electrochemical cellwith in situ electrochemical properties.8�9 At present, STMis a powerful tool for analyzing metallic and semicon-ductor surface with real-space visualization of surface atatomic scale. By exploiting the SPM hardware in con-jugation with optical detection methods enabled to studythe biological systems such as live cells and applica-tions in near-field optical systems.10�11 SPM possess var-ious operational modes which can be tuned according to

    Sci. Adv. Mater. 2016, Vol. 8, No. xx 1947-2935/2016/8/001/020 doi:10.1166/sam.2016.3003 1

  • Electrochemical Scanning Tunneling Microscopy Analysis on Protein Based Electronic Devices Yagati et al.


    Ajay Kumar Yagati

    Ji-Young Lee

    Eun-Sook Nam

    Sungbo Cho

    Jeong Woo Choi

    2 Sci. Adv. Mater., 8, 1–20, 2016

  • Yagati et al. Electrochemical Scanning Tunneling Microscopy Analysis on Protein Based Electronic DevicesREVIEW

    the imaging requirements such as probe-sample interac-tions, force or surface potential mapping and to analyzethe conductance properties of redox molecules and tun-ing their tunneling properties.12 Thus, the scanning probemicroscopy tools are most advanced and powerful toolin the field of nanotechnology and led the advancementof science and technology.13 Therefore, here we outlinethe various types of scanning probe techniques, such asScanning Tunneling Microscopy (STM), scanning tunnel-ing spectroscopy (STS) and electrochemical scanning tun-neling microscopy (ECSTM) while focusing the ECSTMmethods by describing with examples on protein basedconductivity measurements.

    1.1. Scanning Tunneling MicroscopyScanning tunneling microscopy (STM) is based on theconcept of quantum mechanical tunneling. When a volt-age is applied between a sharp metal probe (tip) and thesurface of an electrically conductive material, tunnelingcurrent will be produced if the tip is positioned a fewnanometers from the surface.14�15 According to quantummechanics, an electrical current will be produced underthese circumstances without the need for the probe tipto physically touch the surface. The separation distancebetween the tip and the sample is roughly one-hundredthousandth of the thickness of the sheet of a paper. A typ-ical tunneling current is on the order of one nA and theapplied voltage is typically less than one volt.

    The magnitude of the tunneling current is very sensi-tive to any change in the tip/sample separation distanceand it is the sensitivity that makes it possible to monitorand detect changes in the separation distance. A piezo-electric tube is used to control the position of the tip inthree-dimensions relative to the sample. The probe tip canbe scanned parallel to the surface using computer control.The tip’s position perpendicular to the surface is deter-mined from the output of a feedback circuit, which sendsa voltage signal to the particular piezo element that movesthe tip towards or away from the surface in order for apreset tunneling current to be maintained. As the probetip is scanned over the surface, the topographic data werecollected by the computer. An image of the surface is thendisplayed on the computer monitor from this data.

    STM was originally designed for the investigation ofconductive or semi-conductive material. It is very usefulon high-resolution imaging or organic/biomaterials. Thereason for this is the completely different nature of thecharacteristic binding forces in the surface region of suchsubstances. STM possess following important parameterwhich are the horizontal coordinates (x� y�, the height (z�,the bias voltage (V ) and the tunneling current (I ). Based onthe utilization of these parameters, the modes of operationof STM was distinguished as(a) constant current mode, where I and V are kept con-stant, x and y are allowing to change with movement ofthe scanning tip, and z is measured;

    (b) while in constant height mode where z and V are keptconstant, x and y will change during the scan, and I ismeasured; and in(c) scanning tunneling spectroscopy (STS), which is awhole set of modes with variation of V . The most com-monly used method in STM measurements is the constantcurrent mode, in which the STM tip is allowed to scanover the surface of the sample by keeping the bias voltageand tunneling current constant. To achieve the constantvoltage and current the feedback system adjusts the verti-cal position of the STM tip by varying voltage Vz in thepiezoelectric element. This mode of operation maintains aconstant gap between the sample and the tip while scan-ning the surface topography of the sample as shown inFigure 1(a). The material surface structure height is deter-mined directly from Vz that produce a surface topographyas a function of the needle position z (x, y�.

    In constant height mode of operation, the surface isscanned with the STM tip kept at a constant voltage Vz inthe z-piezoelectric element while measuring the tunnelingcurrent (I ) as a function of the needle position (Fig. 1(b)).The voltage V between the tip and the sample is keptconstant, and the servo system feedback is turned off. Inthis case, the surface bump will be reflected in highertunneling current when passed by the needle. This modeenables to perform high scan speeds as the servo systemwas disabled. This mode of operation is suitable to studythe dynamic processes in real time perhaps the recodingthe surface structures in a video format. This mode possessdrawback as it is difficult to quantify the surface topogra-phy based on the changes in tunneling current. Scanningtunneling spectroscopy (STS) is a set of methods of scan-ning tunneling microscopy in which the voltage betweenthe tip and the sample is varied in order to obtain theinformation on the local electronic structure of the surface.

    1.2. Scanning Tunneling SpectroscopyThe scanning tunneling spectroscopy of metals, semicon-ductors or biological materials primarily focus on elastic

    Fig. 1. Schematic representation of operation of a scanning tunnelingmicroscope in (a) constant current mode and (b) constant height mode.

    Sci. Adv. Mater., 8, 1–20, 2016 3

  • Electrochemical Scanning Tunneling Microscopy Analysis on Protein Based Electronic Devices Yagati et al.


    tunneling current changes associated with the local densityof sates (LDOS),16 �s�r�E�, shown in Eq. (1). To a goodapproximation, �s�r�E� is proportional to dI/dV when thetip is far from the substrate and the density of states of thetip is reasonably smooth.

    I =∫ eV0

    �s�r�E��t�r+ eV −E�T �E� eV � r�dE (1)In a real tunneling device; such as metal-insulator-metal(M-I-M) tunnel diode, or a substrate and STM tip,17 thereare many electrons and the Pauli principle plays a keyrole. A simple model for the conduction electrons in ametal assumes that the one begins by removing the valenceelectrons, then spreads the remaining positive charge intoa uniform distribution (jelly) producing a simple con-stant potential box. Into this box the valence electrons arereturned, 2 at a time into each energy level, until the metalis just neutrally charged. The energy of the last electron togo in is the Fermi energy, (EF�, and the energy requiredto just remove it from the metal is the work function, (��.If there are no molecules in the barrier region (tunnelinggap), the current is approximately proportional to the volt-age difference between the two metals (called the bias)and exp�−Ad√��. This current is supposed to be due toelastic tunneling since the electron loses no energy to thebarrier.If the gap (barrier) between the electrodes is not a vac-

    uum, Eq. (1) must be modified in several ways. The sim-plest effect is a reduction in the effective barrier height.For an insulator or semiconductor, it may only require avolt or two of energy above EF for the electron to reachthe conduction band in the barrier, while the work func-tion may be 4 to 6 volts. In these cases, the work func-tion in I = cV exp�−d√�� is replaced by barrier height,�b where �b is approximately the difference in energybetween the bottom of the conduction band (in the insula-tor) and the Fermi energy in the electrodes at zero appliedbias. If individual molecules are present in the barrier, sev-eral new interaction mechanisms can affect the tunnelingcurrent. The best known of these is inelastic electron tun-neling and is the basis for inelastic electron tunneling spec-troscopy (IETS). In IETS the moving electronic chargeinteracts with the time varying molecular dipoles (elec-tronic or vibrational) to induce excitation of the moleculein the barrier with concomitant loss of energy by the elec-tron. This process is similar to a Raman photon processby considering vibrational motion with frequency � andenergy spacing h� as shown in Figure 2. An excitationfrom the ground vibrational state to the first excited vibra-tional state with a corresponding loss of energy by thetunneling electron. If the applied voltage is less than h�/e,the inelastic channel is closed because the final states forthe tunneling electron, the electronic levels in the metalelectrode of the appropriate energy, are already filled.To obtain an IETS spectrum we can plot d2I/dV2 versus

    V and expect to see peaks whenever the energy difference

    Fig. 2. Inelastic tunneling process and associated spectral peaks; h� isthe molecular energy level spacing, � is the barrier height in eV (approx-imately the metal work function for a symmetrical vacuum barrier), d isthe barrier width in Angstroms, I is the tunneling current, and V is theapplied bias voltage.

    between the ground and excited state (electronic or vibra-tional) just matches the applied bias voltage. It is importantto note that IETS bands appear at the same bias magnitudeindependent of sign, although the intensities may differ.

    1.3. Electrochemical Scanning Tunneling Microscopy(ECSTM): A Promising Tool for Imaging withIn-Situ Electrochemical Analysis

    Electrochemistry has been used in wide range of scienceand technology because of easy control of electrochem-ical potentials of interfaces and adsorbed molecules,18

    which initiate electron transfer and redox reactions.19 Thisprovides us a way to regulate the redox process forstudying the basic phenomena how the local environmentresponds to the electron transfer process. Thus in-situECSTM technique (in-situ electro chemical scanning tun-neling microscopy technique), which is based on the scan-ning tunneling microscopy technique used in conjunctionwith electrochemistry, is highly useful technique whichis capable of measuring the small changes in the surfacechanges20 which occur as a result of the electrochemi-cal process as shown in Figure 3. The method was usedto study the electrode surface at atomic scale resolution

    4 Sci. Adv. Mater., 8, 1–20, 2016

  • Yagati et al. Electrochemical Scanning Tunneling Microscopy Analysis on Protein Based Electronic DevicesREVIEW

    Fig. 3. Schematic representation of an ECSTM set-up with bipotentio-stat control, It represents the tunneling current which feeds the feedbacksystem for constant current imaging.

    and single molecule electron transfer in electrochemicalenvironments.21�22 The electrochemical potentials of thesample and the tip are controlled independently againstthe reference electrode by a bipotentiostat. The substratepotential determines the redox state of molecules tetheredon the substrate electrode, and the difference between thetip potential and the substrate potential sets the bias volt-age for electron tunneling.

    In the subsequent sections in this review, we focuson the developments on ECSTM experiments to studythe applications of this tool in nanotechnologicalinnovations.23�24 The control of voltage in order to achievenovel structures, and the application of oxidation andreduction potentials to study the single molecule conduc-tivity were also discussed.


    2.1. Structure of Single Crystal Surfaces, Adsorptionsand Self-Assembly of Molecules

    The formation of self-assembled monolayers (SAM)25 orself-organizing molecules are natural and spontaneous pro-cess that plays vital role in bottom-up strategies in thedevelopment of molecular electronic devices, biorecog-nition and biosensors. Mostly, thiols immobilized ongold (Au) electrodes are extensively studied and exploredtheir properties in various conditions such as ultrahighvacuum,26 air27 and in solution state.28 Several studies wereconducted to examine the SAM properties such as theiradsorption, electrical and electrochemical properties, ther-mal and chemical stabilities, however to understand themorphology of the SAM molecules, SPM were consid-ered to analyze the single molecular structures. These SPMbased methods are utilized to unravel the SAM domainsize and defect density which could affect the properties ofthe electronic devices based on the SAM materials. Several

    reports on the studies of single molecule characterizationof SAM and therefore here we consider an example onthe adsorption and assembly of 2-mercaptobenzimidazole(MBI) on Au electrode surface.29 The MBI molecules areexamined with the help of ECSTM for understanding theadsorbed layer (adlayer) structures and potential dependentliquid/solid interface charge transfer process. In-situ STMmeasurements shows that MBI molecules could form ori-ented molecular cluster lines along the reconstruction linedirection at 0.55 V. However, when the electrode poten-tial shift towards negative these molecules will undergo adisordered structural transition to form an ordered stripedadlayers on the desorption region on Au. The in-situ thecyclic voltammogram analysis on MBI modified Au elec-trode depicted two anodic peaks at 0.24 and 0.58 V whichwas attributed to the adsorption of MBI and the cathodicpeak observed at 0.18 V was attributed to the desorptionof MBI as shown in Figure 4(a).Figure 4(b) represents a typical large-scale STM image

    of an MBI-modified Au surface in 0.1 M HClO4 at0.55 V. The MBI molecules form a multilayer structurewith molecular clusters on the top layer. Interestingly,the clusters arrange along the direction of the reconstruc-tion lines. The average distance between neighboring linesis 12 nm, which is about twice the distance separatingthe pairs of reconstruction lines. When the potential isshifted negatively to 0.15 V, the disordered multilayer dis-appears and an ordered monolayer appears on the Au sur-face. The disordered-ordered transition is reversible shownin Figure 4(c). The bright spots and the dark rods inFigure 4(c) are ascribed to the thiol sulfur atoms andthe heteroaromatic rings of MBI, respectively. The sulfurgroup in thiol SAM always appears bright in the STMand the interdistance between the neighboring atoms was0.4 nm. The heteroaromtic ring was resembled as dark rodsin the STM image. Therefore, STM is a powerful tool forgathering information about the arrangement of moleculeson metals. MB SAMs observed by STM will be helpfulin understanding the mechanism of detecting metals usingMBs molecules as chelating agents and the stability ofMB-modified electrodes.

    2.2. Current Distance Spectroscopy and BreakJunction Techniques

    The bias voltage dependent STM imaging offers a possi-bility for spectroscopic studies on the sample properties.Scanning tunneling spectroscopy (STS) measurementsenable to record the electronic properties of the sam-ple on different surface sites. On the other hand, thecurrent–distance (I–s) measurements30 where the bias waskept constant and the tip-sample distance was variedand the current were measured. Therefore, in this analysisthe current decay factor � of the surface was analyzed.The surface state wave-functions of the sample materialsdecay into the vacuum with an exponential dependence on

    Sci. Adv. Mater., 8, 1–20, 2016 5

  • Electrochemical Scanning Tunneling Microscopy Analysis on Protein Based Electronic Devices Yagati et al.


    Fig. 4. (a) Cyclic voltammograms obtained on bare and MBI-SAM modified Au electrodes in 0.1 M HClO4 at a scan rate is 100 mV/s. (b) Large-scaleSTM image of MBI adsorbed on Au (111) at E = 055 V. Tunneling conditions: Vbias =−187 mV and It = 1574 nA; (c) high-resolution STM imagesof the MBI monolayer obtained at E = 015 V. Tunneling conditions: Vbias =−272 mV and It = 35 nA. Reproduced with permission from [29], B. Cui,et al., Langmuir 27, 7614 (2011). © 2011, ACS.

    the distance. The proportional conductance G = G0e−�zdepends on the constant (G0� quantum conductance andthe decay factor (��. In case of molecules adsorbed onmetal surfaces that forms a tunneling junction then thetotal conductance will be Gtotal =Ggap ∗Gfilm.The electrochemical STM results have generally been

    interpreted using a one-dimensional tunneling model.According to this model, the relationship between tunnel-ing current (It�, tunneling voltage (Vt�, and gap distance (s)is expressed by Eq. (2), if the density of states of the metalsurfaces is assumed to be structure less and the energybarrier is rectangular,31

    It ∝ Vt exp�−1025√s� (2)Here is the tunneling barrier height given in eV, ands is in Å. Experiments revealed that the dependence oftunneling current on distance follows the above equation.As model example,31 the result of the tip-approach exper-iment obtained in a NaClO4 solution of 1 mM concentra-tion was presented here. The bias voltage (Vb� was fixedat −100 mV, which is applied at a substrate against theground potential of a tip. The It − s curve shows that thetunneling current increases exponentially with decrease inthe gap distance, only when the tip and the substrate arefar apart. At small distances the It− s curve does not fol-low an exponential behavior.Tunneling conductance, Gt = It/Vt , can be calculated

    from the result of simultaneous measurements of It andVt . As can be seen in Figure 5, the lnGt versus s plotsfor the results were obtained in pure water and NaClO4solutions of 1 mM and 100 mM concentration. The plotsshow good linearity in the investigated ranges of tunnelingdistance. This observation is in conformity with the Gt− srelationship in the one dimensional tunneling model givenin Eq. (3).

    Gt =ItVt

    =G0 exp�−1025√s� (3)

    where G0 is the conductance at s = 0 Å. The resultsobtained in pure water will fall slightly below the line

    at long distances (>15 Å), but the degree of deviationfrom linearity is within the experimental uncertainty anddoes not represent a general feature. Furthermore, thelnGt−s plot is modulated with weak periodic oscilla-tions coinciding with the structure of interfacial waterlayer and those features can be disappeared after averag-ing the results of repeated tip-approach experiments.32�33

    Whereas, the lnGt − s plots are linear, the correspond-ing ln It− s plots are obviously curved due to the drop inVt at decreased tunneling distances. This indicates impor-tance to measure Gt = It/Vt , not It alone, in the elec-trochemical STM experiment, because the Vt drop couldbe a significant factor at a relatively long distance. Also,the electronic properties of single molecules were stud-ied for the development of novel molecular electronicdevices. Molecular and nanoscale structures have beenutilized for the demonstration of the developing func-tions such as rectification, negative differential resistanceand single molecule transistor behavior. Break junction

    Fig. 5. Tunneling conductance measured as a function of gap distancein pure water and NaClO4 solutions. The arrow indicates the bendingof conductance curves due to a tip-surface contact. The results shownare the average of several repeated measurements on a single system,with the data fluctuations represented by the error bars. Vb = 100 mV inNaClO4 results and Vb = 100 mV in pure water. The tip approach speedis 50 Ås−1. Reproduced with permission from [31], D. H. Woo, et al.,Surface Science 601, 1554 (2007). © 2007, Elsevier.

    6 Sci. Adv. Mater., 8, 1–20, 2016

  • Yagati et al. Electrochemical Scanning Tunneling Microscopy Analysis on Protein Based Electronic DevicesREVIEW

    techniques has been utilized to study the conductivityof single molecules as a function of redox state. So,therefore in this context, the molecule, 6-[1’-(6-mercapto-hexyl)-[4,4′]bipyridinium]-hexane-1-thiol iodide (6V6; seeFig. 6(a)), was chosen for analysis. The thiol groupsat both ends of the molecule enabled to bind with thegold substrate and the STM tip. This molecule possessesa redox group which has readily accessible energy lev-els and is symmetrically placed between defined molec-ular tunneling bridges (the two alkyl chains) at eitherend. Furthermore, these molecules are highly stable intheir redox states. When the STM tip was brought closeenough to the Au surface, by increasing the tunnel-ing current set point (I0�, there is a spontaneous for-mation of stable molecular wires between the tip andthe sample was observed. Subsequently the tip was thenlifted while keeping a constant x− y position, and the

    Fig. 6. (a) Schematic diagram of the experiment performed to studyelectrical properties of single molecules in both air and electrolyte. Theinset shows the 6V6 dication. (b) Current decay curves (I�s� scans) for6V6 on Au (111) in air; (1) Baseline for a clean Au surface and (2)in the presence of 6V6 molecular wires between the tip and the sub-strate. (c) Dependence of s1/2 on the current plateau current (IW � for117 I�s� curves taken at different locations of the substrate (filled sym-bols Ut = +02 V; open symbols Ut = −02 V; where Ut is the tip-to-substrate potential difference). I�s0�= 05 nA for all the measurements;the error bars represent the standard deviation for each class of events.Inset: Histogram of the current values from (c). Reproduced with per-mission from [34], W. Haiss, et al., JACS 125, 15294 (2003). © 2003,ACS.

    current–distance (I�s�; s = relative tip-sample distance)relation was measured.As can be seen in Figure 6(b); two unique current–

    distance (I–s) curves were observed: The first one being afast exponential decay typical of tunneling between a tipand a bare metal (curve 1 in Fig. 6(b)) and the other oneis less abrupt decay followed by a characteristic currentplateau (IW � (curve 2). From these results in can be under-stood that the plateau is related to conduction throughmolecular wires which are chemically bonded to the tipand to the substrate. Furthermore, direct tunneling cur-rent does not have significant amount of contribution inthe observed phenomena of separations. Also, the plateauis followed by another current decay at longer distances.A plot of IW versus s1/2 (s1/2 = distance for I = IW/2�for more than hundred I�s� scans were taken at differ-ent locations was presented in Figure 6(c). It was foundthat the average value of s1/2 is about 24±06 nm. Sim-ilarly, the end of the plateau is observed at approximately2 nm from the initial set point distance (s0�. Therefore, anestimate s0 places the tip-to-substrate distance at approx-imately 2.5 nm at the end of the plateau. This distanceis close to the length of 6V6 molecule, which was con-firmed by molecular modeling of the free molecule, pro-duces a distance between the two sulfur atoms of 2.4 nmfor trans oriented alkyl chains. Therefore, it clearly indi-cated the molecule was in fully extended conformationbefore disengagement from the tip as shown in Figure 6(a).It is also observed that the decrease in current for longertip-sample distance following a plateau (Fig. 6(b)) occursover a distance range not just abruptly by the extension ofmolecular assembly before the breakage at either the tipor surface end. Moreover, the molecular assembly consti-tutes the molecular wire as well as group of surface andtip atoms. At sufficiently larger tip-sample separations, thechemical contact of the molecular wire to the tip or tothe surface is broken and, thus, the current drops to zero.These events were recorded and three sets of data werepresent as shown in (Fig. 6(c)) as group 1 (squares), group2 (circles), and group 3 (triangles). From the analysis, themeasured Iw cluster values are around integer multiples ofa basic current value of (98±16) pA. Therefore, it can beunderstood that the steps in conductivity was attributed dueto the presence of a discrete number of molecules betweenthe tip and the sample. Thus, it can be concluded that thelowest conductivity unit (group 1 events) and the respec-tive subsequent steps (groups 2 and 3 events) correspondto conduction through a single molecule and the others totwo and three molecules, respectively.From the above analysis, it is concluded that the conduc-

    tivity of a single molecule (�M� at Ut = 02 V is (049±008) nS (where Ut is the tip-to-substrate potential dif-ference). The inset in Figure 6(c) shows a histogram ofthe current values observed in Figure 6(c). The obtainedresults are also compared with the conductivity results

    Sci. Adv. Mater., 8, 1–20, 2016 7

  • Electrochemical Scanning Tunneling Microscopy Analysis on Protein Based Electronic Devices Yagati et al.


    in which the wiring is realized by chemical attachmentof nanoparticles to 6V6 molecules incorporated in a hex-anethiol monolayer on gold which gave a value of (056±003) nS. Therefore, the results indicated that measurementof single molecule conductance was reliable and can beextended to examine the conductive of various other singlemolecules towards novel electronic devices.

    2.3. ECSTM Based Nanostructure FormationsMetal atomic-size nanowires and single molecular junc-tions are the main focus in nano and molecular electron-ics for their quantum transport properties. Therefore, thereis always a quest for innovative methods or proceduresfor the formation of atomic scale structures. Furthermore,electrochemical deposition of metals to form various nano-structures is a valuable route to modify the structures fornanoelectronic applications. ECSTM based technique withvariety of tip-surface interactions nanostructured surfacesunder high-vacuum conditions were prepared.35 A jump-to-contact based approach with ECSTM-break junctiontechniques enables to construct different nanostructuressuch as nanodots and nanowires. In this approach, theSTM tip was coated with material of interest and trans-ferred to the surface through jump-to-contact method. Thisprocess enables the formation of atomic sized nanowirewhen the tip retracts from the surface. This procedurewas utilized to study the conductance behavior of manymetals such as Cu, Ag, Pd and Fe, however room tem-perature measurement of metal conductance with complexelectronic structure was found difficult. So, the utiliza-tion of this technique led to tip induced formation ofnanoclusters at metal/solution interfaces, and also enabledthe formation of large-scale arrays nanopatterns with highprecision.36 Due to low decomposition potential of waterwhich is 1.23 V, there is a fundamental limit for anydeposition process in liquid environment. This limitationprevents the deposition of non-noble metals such as Feor Al from aqueous solutions. However, these limitationscan overcome by using by using ionic liquids, that is,room-temperature salt melts, which enable a potential win-dow of 4–5 V for deposition. For example, Fe clustersin a 1-butyl-3-methyl-imidazolium tetrafluoroborate (BMI-BF4� ionic liquid, was fabricated on electrode surfacewas achieved with a microprocessor-controlled pulsed tipapproach. The tip potential was chosen to be between−0.75 and −0.95 V so that Fe bulk deposition onto the tipproceeded at a fairly high rate. For the 5× 5 array of Feclusters generated at intervals of 40 nm the clusters havean average height of 0.5 nm (Fig. 7(a)).A large-scale array of 50× 50 Fe nanoclusters with

    20-nm intervals were fabricated by using 5-ms pulses at arate of 1 cluster every 300 ms (Fig. 7(b)). In this process,the total time to perform the experiment to construct thearray was about 14 min without any sign of depletion of Fesupply at the tip. In order to demonstrate the stability and

    Fig. 7. STM images of Au (111) surfaces decorated with Fe clusters.(a) 5×5 Array, scan area 200×200 nm2; (b) 50×50 Large-scale array,scan area 1× 1 mm2; (c) ring of 48 clusters with a ring diameter of120 nm; z Pulse: 0.42–0.45 V. Reproduced with permission from [35],Y. M. Wei, et al., Small 4, 1355 (2008). © 2008, Wiley.

    durability of the device a ring with a diameter of 120 nm,composed of 48 Fe clusters was also created (Fig. 7(c)),with a cluster height of approximately 0.6 nm. The Fenanoclusters possessed uniformed size however less per-fect than the Cu clusters fabricated in aqueous solution36�37

    and Zn clusters in ionic liquids.38 It is understandable thatthe potential-dependent morphology adds more complex-ity to the deposition procedures onto the tip for supplyof Fe during the jump-to-contact processes, which in turnresponsible for less uniform Fe cluster formations.

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    This approach can apply to organic molecules for con-trolled pattern of molecules with ECSTM. In an approachproposed by Song et al.,39 by controlling the substratepotential a well ordered structure of methylene blue(MB) monolayers on Au (111) surface was obtained.Methylene blue (MB) is an electron mediator in manybiological processes of living organisms and exhibits inter-esting redox behaviors. Electrochemical scanning tunnel-ing microscopy (ECSTM) is an important technique tounravel the surface-potential-induced ordering with sub-molecular resolution.40 Electrochemical scanning tunnel-ing microscopy (ECSTM) examined the monolayers ofMB on Au (111) in 0.1 M HClO4 and showed long-rangeordered, interweaved arrays of MB with quadratic symme-try on the substrate in the potential range of double-layercharging. CV of the MB-adsorbed Au (111) electrode wasperformed in 0.1 M HClO4. Figure 8 shows the CVs fora well-prepared Au (111) electrode in 0.1 M HClO4 ata scan rate of 50 mVs−1 in the absence and presence ofMB molecules. Figure 8(b) presents the CV of Au (111)electrode in the presence of 10 �M MB in 0.1 M HClO4solution. The presence of the organic molecules in thesolution resulted in a dramatic change in the CV shape,compared with that obtained in the pure HClO4 solution.Two oxidative peaks appeared at 0.39 and 0.54 V andthe corresponding reductive peaks appeared at 0.39 and0.52 V, respectively.

    A highly ordered, large area MB adsorbed layer ofhigh quality molecular resolution ECSTM images werepresented in Figure 9. The images clearly demonstratedthat the MB molecules are closely packed with internalstructure of MBs in a scan size of 7× 7 nm2. Each MBmolecule clearly showed three bright spots denoted as indi-cated by the three solid ellipses (a)–(c) on the obtainedECSTM image. From the image, it is clearly noticeablethat the structure looks elliptical rather than circular, espe-cially the spot (b). This indicated the possible drift in theECSTM measurements and abundant images were scanned

    Fig. 8. Typical cyclic voltammograms of Au (111) electrode in 0.1 MHClO4 (a) and 0.1 M HClO4 + 10 �M MB (b). The potential scan ratewas 50 mVs−1. Reproduced with permission from [39], Y. H. Song andL. Wang, Microsc. Res. Tech. 72, 79 (2009). © 2009, Wiley.

    Fig. 9. High-resolution ECSTM image (about 7×7 nm2� of MB adlayerformed on the Au (111) surface in 0.1 M HClO4 at 0.25 V versusRHE. The triple set of arrows indicates the directions of atomic rows ofAu (111) surface. Tip potential and tunneling current were 0.18 versusRHE and 2.83 nA, respectively. Reproduced with permission from: [39],Y. H. Song and L. Wang, Microsc. Res. Tech. 72, 79 (2009). © 2009,Wiley.

    and analyzed from different scan directions. However, theimages obtained all clearly showed that similar ellipticalspots which confirmed that the elliptical spots did resultfrom the MB. The bright spots resulted from ECSTMimaging was due to the strong electronic coupling betweenthe nitrogen and sulfur atoms of MB with Au having highelectron densities of phenothiazine, nitrogen of dimethy-lamide functional groups and benzene rings in the MBmolecule. The bright spots (a) and (c) are assumed to bemainly the benzene and nitrogen of dimethylamide func-tional groups in MB.The similar appearance of the spots indicated as (a)

    and (c) strongly suggests that the two functional groupsof each MB molecule are located at same adsorption sites.The bright spot (b) was assumed to be mainly the phe-nothiazine in MB. Therefore, the bright spots exhibitedelliptical forms and were positioned with a grooved shaperather than keeping along the same line. The distancebetween the outside of the spots (a) and (c) along thelong axis is 14×01 nm, which is in accordance with thelength of each individual molecule. Also, the spots (a), (b),and (c) were found to have a corrugation height of about0.1 nm, which is comparable with that observed with ben-zene, naphthalene, anthracene, and coronene.41–43 There-fore, from these analysis forms the ECSTM measurementsthe molecular plane of each MB molecule is proposed tobe parallel to the Au (111) surface. Hence, a set of threebright spots were attributed from one MB molecule with aflat-lying orientation on the Au (111) surface as shown inFigure 9. This approach of understanding the conductanceof metal with the influence of molecule-electrode interac-tions is an important issue which enables to develop singlemolecular junctions.

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    Towards the ultimate miniaturization of the electronicdevices, biomolecules have been considered as thebuilding blocks for future development of electronicdevices.44�45 Thus, single biomolecules especially theredox proteins also emerged as building blocks and hasshown great opportunity in the development of molec-ular bioelectronic devices like diodes,46 transistor47 androbust memory switching elements.48 On the other hand,ECSTM with a possibility to observe nanoscale resolutionwith combined electrochemistry enabled to develop elec-trochemical protein based devices.49

    3.1. ECSTM Studies Towards Protein Based MemoryDevices, Transistors

    The electrochemical properties of self-assembled proteinmolecules immobilized on conductive substrates whichare well swollen in electrolytic solutions has achievedmuch attention in recent days in order to study thefundamental properties, kinetics of charge transport andalso for the potential applications towards innovationbioelectronics devices based on the redox properties ofthe biomolecules. Much attention has paid for possibleimplementation of hybrid electronic devices by the self-assembly of bio molecules conjugated with other inor-ganic or organic molecules.50 Towards the development ofhybrid architectures, biomolecules such protein, RNA andDNA molecules were suited due to their size and intrinsicproperties similar to inorganic nanomaterials. Furthermore,to analyze the redox properties of protein architecturesscanning probe microscopy operated under electrochem-ical control was utilized. Recently, many works werereported based on SPM to analyze the conductivity ofthe proteins and various redox molecules adsorbed on flatmetallic surfaces.51–53 Several theoretical assumptions54

    and explanations55 were proposed for understanding theSTM signal involving resonant tunneling due to nuclearrelaxation56 or due to redox reactions.57

    The in-situ ECSTM technique (in situ electro chemicalscanning tunneling microscopy technique), which is basedon the scanning tunneling microscopy technique used inconjunction with electrochemistry, is highly useful tech-nique which is capable of measuring the small changes inthe surface changes which occur as a result of the electro-chemical process.58 For some electro-active bio or organicthin films, mass changes, which occur upon electrochemi-cal oxidation and reduction due to insertion or expulsion,respectively, of counter ions, coinons and/or solvent intoand out of the thin film have been elucidated quantitativelyusing in situ ECSTM technique.59

    A recombinant Azurin (Az) redox protein, in whichthe cysteine residues were incorporated to bind with thenoble materials without using any chemical linkers. The

    modified proteins were allowed to adsorb on Au nano-dots pattern fabricated on indium tin oxide (ITO) surface.60

    Scanning tunneling microscopy and in situ cyclic voltam-metry of protein monolayers adsorbed on the Au-dots wereperformed with a bipotentiostat. The electrochemical cellwas housed with Pt and Ag wires as counter and referenceelectrodes and filled with 50 �l of 10 mM HEPES bufferpH 7.0. The schematic diagram of the ECSTM experimen-tal set-up and immobilization of cysteine modified Az ontoAu-nanodot is shown in (Figs. 10(a, b)). Scanning tunnel-ing spectroscopy (STS) measurements were performed ata set point of 500 pA and 100 mV bias. Current–voltage(I–V ) spectra were recorded by positioning the tip onthe top of the redox protein, and the feedback loop hasbeen disengaged, the tunneling current being monitored byramping the bias in the range of ±300 mV.In-situ cyclic volammetry measurements were per-

    formed to investigate the electron transfer function ofAz monolayer on Au-dots. Representative data shown inFigure 11, reveals a robust electrochemical response witha midpoint potential of 200 mV versus Ag/Ag+. Fromthe cyclic voltammograms, redox peaks due to Az wereobserved and with no peaks at the bare Au-dot electrode,which means that the Az monolayer is well adsorbed on

    Fig. 10. Schematic representation of (a) ECSTM experimental set-upand (b) immobilization of cysteine modified Az onto Au-nanodot. Repro-duced with permission from [61], A. K. Yagati et al., Thin Solid Films518, 634 (2009). © 2009, Elsevier.

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    Fig. 11. (a) Representative cyclic voltammogram for a bare dot and (b) monolayer of the Az adsorbed on the Au-dot electrode in 10 mM HEPESelectrolyte solution. Scan rate is 50 mV/s (inset shows the bare and Az immobilized Au-dots in each case respectively). (c) I–V curves of the Az/Au-dotsystem (yellow, continuous line) and of bare Au-dots (red, continuous line) at an engaged tunneling current of 500 pA and at an engaged bias of0.1 V. Each curve has been averaged over 10 different sites; for each site an average over 10 curves has been performed. Reproduced with permissionfrom [61], A. K. Yagati et al., Thin Solid Films 518, 634 (2009). © 2009, Elsevier.

    the dots. The formal redox potential (E1/2�, calculated asE1/2 = �Epa+Epc�/2, is 195±10 mV, which appears to beshifted to more negative values than that reported for Azdirectly anchored on gold (270–280 mV).61 The separationbetween the anodic and cathodic peaks Ep, is 150 mV(at a ramp rate of 50 mV/s) and it is dependent on ramprate showing a quasi-reversible kinetics.

    STS measurements in air were conducted to examinethe conductive properties of Az adsorbed on Au-nanodots.Measurements were performed through ramping the volt-age in range of ±300 mV by disengaging the feed-back loop temporarily by positioning the STM tip on theprotein. It was observed that the shape of tunnel I–Vcurve depends substantially on the STM tip position pointover the protein globule at which the electron tunnel-ing was measured. The I–V curve shown in Figure 11(c)over the center of the protein molecules was asymmetricwith respect to that obtained for bare Au-dot with fea-tures recorded in a double-tunnel junction configuration,an STM tip-protein molecule-conducting substrate. Thisbehavior in the current profile for Az adsorbed nanodotswere expected from various factors such as if the moleculeis a donor–acceptor pair or may be conformational changesdriven by the electric field or even to schottky barriereffects, but it is mainly dominated by the air gap betweenthe tip and protein.62 Moreover, the electrical propertieswere examined in air rather than vacuum which show arectifying behavior of the metal/Az/metal tunneling junc-tion and an interesting study in view of an application innanobio-diodes.63

    Furthermore, the redox properties of Az proteins suchas oxidation and reduction potentials can be utilized forcharge storage and erase and these two states can be readby the application of open-circuit or equilibrium potentialtowards in order to demonstrate the system as a nanoscalebiomemory device. Towards this, ECSTM technique waswell suited to examine the oxidation and reduction stateswith cyclic voltammetry and simultaneous it is possible tovisualize the topography of the molecules in these threestates. Thus measurements were performed with Az onbulk Au substrate with an electrochemical cell that washoused with Pt and Ag wires as counter and referenceelectrodes and filled with 10 mm HEPES buffer at 7.0.The redox states of the protein molecule and open circuitpotential was used for charge write, read and erase func-tion of the proposed device. The redox states of the Az canbe controlled by the applied potential. Application of oxi-dation potential causes the transfer of electrons from theAz molecules to the Au surface results in storage of pos-itive charge (write) and application of reduction potentialcauses the electron back to the protein molecule thereby‘erasing’ the stored charge. These two states can be ‘read’by the application of open circuit potential or cell equi-librium potential. Application this open circuit potentialgenerally doesn’t affect the charged states hence no elec-tron transfer will occur at these states.49�62�64

    The topography for the immobilized Az molecules onAu surface was obtained with ECSTM under these threedistinct conducting states as shown in Figure 12. Tipinduced potential changes can be observed one specific

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  • Electrochemical Scanning Tunneling Microscopy Analysis on Protein Based Electronic Devices Yagati et al.


    Fig. 12. Sequence of ECSTM images obtained for immobilized azurinmolecules on Au substrate for different bias potentials for (a) OCP(80 mV) (b) oxidation (486 mV) (c) reduction (278 mV) respectively.Scan size is 50 nm (d–f) is the 3-dimensional profile of (a–c) imagesrespectively. Reproduced with permission from [49] S. U. Kim, et al.,Biomaterials 31, 1293 (2010). © 2010, Elsevier.

    region on Az molecule. Appling of an oxidization volt-age (486 mV) causes transfer of electrons from the immo-bilized Az molecules into the Au substrate, and positivecharges stored in Az molecules. On contrary, reductionvoltage (278 mV) was applied to pass electron transferreverse into Az molecules, to erase the stored charge. Forreading these charged states, open circuit potential (OCP)was utilized. Az molecules attained a stable equilibriumstate between assembled Az and electrolytes when OCPwas applied. ECSTM images were obtained at three dis-tinct conducting states as mentioned above (at 486 mV, at278 mV, at 80 mV). The typical bright spots which areseen when tuning the substrate potential in a region, appearto be strongly potential-dependent of redox potentials.65

    Such kind of behavior is consistent with a resonant natureof the current measured in STM experiments in the Auadsorbed Az molecules.According to the Marcus–Gerischer model,66 the maxi-

    mum of the density of unoccupied states (oxidized state)of a metalloprotein is at energy equal to its formal poten-tial plus the reorganization energy. To interpret an ECSTMexperiment one has to relate these quantities;(a) time-scale of the electron transition,(b) spanning from a genuine resonance process where amolecule is not reduced, to the involved potentials. In caseof single-step electron transfer, Schmickler derived the fol-lowing relationship between the working electrode poten-tial Emax corresponding to the maximum in the tunnelingcurrent, the bias voltage Ebias applied between the tip andthe substrate, the midpoint potential (Eo� of the electro-active species, and the reorganization energy (��67

    Towards the development of a nanoscale memory deviceit is expected that these results might be interrelated withcurrent flowing and charge storing defined as writing, read-ing and erasing. The results are described in Figure 12with 50 nm scale three surface images (set point current=433 nA, scan rate = 301 Hz). Figure 12(a) indicated

    assembled Az at 80 mV. There are three or four lumplittle aggregated on Au surface. Figure 12(b) pointed atassembled Az at 486 mV (in write step) and Figure 12(c)showed assembled Az at 278 mV (in erase step). Theshape and height have been changed more and more asspecific potential applied. Figures 12(d–f) shows the threedimensional profiles of the Figures 12(a–c) respectively.These studies were further extended in order to

    achieve a complete nanoscale biomemory device based onECSTM. To address, this Az protein was immobilized onAu nanopattern and with ECSTM the charge states wereexploited in order perform memory functions and alsodirect visualization of Az/Au was achieved. To achieveAu nanopattern, nanosphere lithography method68�69 wasadopted to achieve controlled 2D ordered gold nanoar-ray on ITO substrate by spin coating polystyrene spheri-cal particles followed by thermal evaporation. As a result,nanoarray of ordered hexagonal Au pattern was evolvedfrom the colloidal monolayer. With the effective immo-bilization of cysteine modified Az on the patterned Auarrays, a technique was discussed to develop a nanoscalememory based on the ECSTM experiments on the redoxreaction of Az film in aqueous solution. Well-adsorbed Azmolecules on Au hexagonal pattern on ITO surface wereobserved form electrochemical scanning probe microscopyexperiments. The application of proper bias potentials willensure the mechanism of the memory device functioning.Clear memory switching for charge storage and erase func-tions is observed, which will lead to the implementationof nanoscale bio devices and open important new perspec-tives for developing a nanoscale memory device.The schematics for the experimental set-up and the

    nanobiomemory principle are shown in Figure 13. Elec-trochemical experiments on Az/Au-ITO were conductedby ECSTM with in-situ cyclic voltammetry. Experimentswere carried out in 10 mM HEPES buffer solution, wherethe Az/Au-ITO substrate acts as working electrode (vs.Ag/Ag+� as shown in Fig. 13(a). The biomemory mecha-nism is shown in Figure 13(b).EC-STM imaging was performed in constant-current

    mode on the Az adsorbed on the Au nanotriangles. Initiallycyclic voltammetry was performed to figure out redoxproperties of Az protein. It is found that, reduction at−0.07 V and oxidation at 0.36 V for the Cu2+/1+ redoxprocess. Then, a set of images of the same sample area wasobtained at constant bias while varying the potential. It wasobserved from the imaging that there was a variation inthe conductance of the redox molecules upon varying thepotential which was reflected in a difference in apparentheight with respect to the background. Figures 13(c and d)shows the images for the applied potential of 0.36 and−0.07 V respectively for a bias voltage of 100 mV foroxidation and reduction states of Az molecules. It wasassumed that at Ebias = 0 V, the redox state of Az is vacantwith energy higher than the Fermi energy of the substrate

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    Fig. 13. Schematic diagrams for (a) Azurin on Au nano triangle with ECSTM set-up and (b) electron transfer mechanism in Az/Au nano-triangleduring oxidation and reduction potentials. Potential dependent ECSTM images of Az/Au/ITO obtained for (c) oxidation (0.36 V) and (d) reductionpotentials (−0.07 V) respectively. Reproduced with permission from [48], A. K. Yagati, et al., Biosensors and Bioelectronics 40, 283 (2013). © 2013,Elsevier.

    and tip. When a negative bias was applied to the sub-strate and the tip was kept at the positive potential, thevacant state energy and fermi energy of the substrate werematched, allowing electrons to be transferred from the sub-strate to the biomolecule, which lowers the thermal acti-vation allowing the biomolecule to transfer the electron tothe tip.

    It is understood that from the images that there weretwo conductive states for charge storage. A brighter spotemerged, which was interpreted as a small island of oxi-dized Az molecules containing a Cu center. When a volt-age of 0.36 Eox was applied, an electron tunnel formedadsorbed Az molecules on the Au substrate, and lead tostorage of positive charges in the azurin molecules. Incontrast, when a voltage of −0.07 Ered was applied, theelectrons were transferred back to Az molecules and thestored charge was erased, which was performed to erasethe stored charge. In this case, the brighter spot on the Autriangle disappeared, which confirmed that the oxidized Azmolecules were now reduced. The bright spots on the Aunanotriangles, which contained Az, were attributed to elec-tron tunneling enhancement due to the presence of the cop-per active site. These morphological changes appeared tobe strongly dependent on the redox potentials. This behav-ior is consistent with the resonant nature of the current

    measured in the STM experiments of the Au adsorbed Azmolecules.Current–time measurements were obtained on Az/Au

    and on a bare Au pattern as working electrode. Currentswere recorded by applying oxidation (0.36 V vs. Ag/Ag+�and reduction (−0.07 V vs. Ag/Ag+� potentials in 10 mMHEPES buffer solution as shown in Figures 14(a, c). Thecyclic voltammetry measurement recorded on both bareAu and Az/Au with Ag/AgCl electrode are also shown,where bare Au does not reveal any peaks however Az/Aushows broad peaks corresponds to oxidation and reductionof Az and with its currents increased linearly with increasein scan rate, which is characteristic of surface-confinedelectroactive species (Figs. 14(b, d). Sharp current transi-tions were observed for both the oxidation and reductionpotentials on the Az/Au pattern indicating that the devicecan be switched ON and OFF for charge storage. However,the bare pattern does not show any faradaic currents ratherit shows a pulsed waveform similar to the applied volt-ages which behaves as a pure conductor and cannot storeany charge. Additionally, we observed that the reducingcurrent produced the same magnitude of oxidation currentin which we assumed that the oxidized Az molecules werecompletely reduced. The switch response to a sequence forwrite and erase pulses were examined to further demon-strate the potential of the system to develop a nonvolatile

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    Fig. 14. (a) Faradaic switching currents obtained for charge write and erase (Iwrite and Ierase� on applying both oxidation and reduction voltages fora duration (twrite� of 2 ms, (b and d) CV’s obtained for Az/Au and bare Au surface in 10 mM HEPES buffer pH 7.0 at a scan rate of 0.1 Vs

    −1 insetshows the effect of scan rate on Az/Au surface and (c) currents obtained on the bare Au pattern upon the application of both oxidation and reductionvoltages for duration of 2 ms. (e) Charge storage and erase currents observed from the device with a 2 ms pulse for applied voltages of 0.36 V and−0.07 V for 15 continuous cycles. Reproduced with permission from [48], A. K. Yagati, et al., Biosensors and Bioelectronics 40, 283 (2013). © 2013,Elsevier.

    memory device. Short 2 ms write (0.36 V) and erase(−0.07 V) pulses were applied, and the readings wereobserved for 15 cycles. As shown in Figure 14(e) bothswitch states were completely stable and reversible. Theredox potentials were continuously applied to switch ONand OFF to estimate device stability, which we observedbased on the stable redox peaks by CV. The redox proper-ties were maintained for up to 103 cycles with little changein peak currents. In addition, the switching robustness atfast-voltage pulses for write and erase sequences in therange of 10−6 s did not show degraded current values.Based on this, the present device showed good endurancefor charge storage without loss of any magnitude in thefaradaic current.

    3.2. Single Molecule Charge Transport and SwitchingSingle molecule conductance between two metal junc-tions has attracted much attention in the field of molecularelectronics.70 The molecular switching between the on andoff states has been achieved where the conductance of themolecular junction can be controlled electrostatically witha third (gate) terminal.71�72 The role of the source and draincan be represented by the tip of the STM and the substrate

    (working electrode 1 and 2). Conductance measurementsin an electrochemical environment where the solid–liquidinterface forms have advantageous as it enables to con-trol the potential drop between each working electrodeand the reference electrode.73 Therefore, here the electro-chemical gate will modulate or controls the charge trans-port between the two working electrodes.74 The effectivegate-molecule distance is estimated by the double layerthickness at the electrode-electrolyte interface, which is ofsize of few solvated ions. The formation of electrochemi-cal double layers which has thickness of few nanometersacts as a separation layer for ion movements even at highconcentration levels. This double can act as reproduciblegating element for studying the conductivity of singlemolecules.75 This gate voltage can be controlled throughvariations in electrochemical potentials and can be appliedto various biomolecules, organic molecules to understandthe conductivity properties and hence to develop a singlemolecular transistor for future bioelectronic devices.76

    This concept of electrochemical gating has beenexploited to the single molecule level by varying theelectrochemical potentials and the resultant double layerin order to adjust the molecular orbital energy levels.

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    For example, in the case of redox based molecules78 ornanowires79 this methodology can be utilized to switch themolecule between their redox states. A schematic illustra-tion of electrolyte gating of a single-molecule bridge isshown in Figure 15. A single viologen molecule is cou-pled between the STM tip and the substrates in whichthe tip and substrates will act as a source and drain elec-trodes resembling to a conventional transistor. Further-more, the counter and reference electrode combination actsas a gate electrode coupled to a bipotentiostat with inde-pendent electrochemical potential control of the substrate(working electrode 1, or “source”) and the STM tip (work-ing electrode 2, or “drain”). Thus the potential differencebetween these two electrodes was applied to the molecularjunction; Figure 15(b) shows the redox group is gated bythe close proximity of electrolyte ions.

    This mechanism of electrochemical gating of singlemolecules to utilize the single molecule electrochemicaltransistor like configuration where the molecule will bridgebetween two electrodes typically made of noble materi-als. Electrolyte gating where the redox active molecule isattached to the substrate but not the STM tip, or occasion-ally vice versa.80�81 To understand this strategy, as a modelexample, viologen molecular wires were the first elec-trochemical redox system studied in this single-moleculejunction configuration, with the molecule anchored to thesource and drain electrodes through chemisorbed thiol con-tacting groups. The viologen molecules are of particu-lar interest due to the electrochemical reduction process(V 2+/V +• redox system).82

    Figure 16 shows a typical voltammetric curve resultedfrom viologen molecules having thiol groups at each end

    Fig. 15. (A) Electrochemical single molecule gating using an electro-chemical STM with bipotentiostat control of the electrochemical poten-tial of the substrate and STM tip. The single molecule bridge shownwas derived from 1,1′-bis(6-(acetylthio)hexyl)-4,4′ -bipyridinium hexaflu-orophosphate. (B) Illustration of the close proximity of the electrolyteions which are “gating” the redox group (the electrolyte gating concept).Reproduced with permission from [77], H. M. Osorio, et al. J. Amer.Chem. Soc. 137, 14319 (2015). © 2015, ACS.

    enabled to couple Au electrode (HS-6V6-SH) for a lowand a densely packed high coverage molecular adlayer.A highly ordered viologen molecules were identified in atatomic resolution which are arranged in a zig-zag mannercomposed of sulfur atoms, the alkyl chains and the violo-gen moiety. However, at high coverage the molecules weredisordered. Hence, low coverage phase was used to studythe single molecule conductance in the present study as afunction of redox state.The break junction method was performed under elec-

    trochemical potential control84�85 in which the current–distance traces were recorded by retracting the tip. Theresults indicated some characteristics plateaus of 0.2 to 0.3nm separated by abrupt steps. The obtained curves werestatistically analyzed which lead to single molecular con-ductance data of the oxidized and reduced form of the vio-logen bridge by appropriate substrate and tip polarization(Fig. 17).The single molecule conductance value of viologen

    molecule coupled to Au electrodes was constant and pos-sessed a good stability in the oxidized viologen dicationV 2+. The conductance was found increasing about 50%for more negative potentials such as at E =−07 V. Thistendency in single-molecule redox switching is credited tothe higher electron density and the higher degree of conju-gation of the radical cation as compared to the dication.86

    Recently, a large of number of metalloproteins hasevolved as possible candidate for next generation

    Fig. 16. (A) Cyclic voltammogram of an Au (111)-(1×1) electrode in0.05 M KClO4, pH∼7, modified with a low coverage (solid line) anda high coverage (dotted line) adlayer of HS-6V6-SH for the reversibleone-electron oxidation/reduction between the viologen dication V 2+ andthe radical cation V+ form. (B) Large scale in situ STM image of the lowcoverage striped phase of HS-6V6-SH, ES = −025 V, Ebias = −010 V,iT = 60 pA; (C) high resolution image of the striped phase, ES =−035 V,Ebias = 008 V, iT = 40 pA; (D) high coverage adlayer of HS-6V6-SH,ES =−025 V, Ebias =−009 V, iT = 60 pA. Reproduced with permissionfrom [83] C. Li, et al., Chimia. 64, 383 (2010). © 2010, SCS.

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    Fig. 17. Plot of the single molecule conductance currents i of Au (T) |HS-6V6-SH| Au(S) junctions versus the substrate potential ES at fixed bias(ES −ET �=−0100 V. Each data point was obtained from conductance histograms as constructed from several hundreds of individual current–distancepulling curves. The dotted line corresponds to the macroscopic current–voltage curve for the reduction of V 2+ → V +· (c.f. cyclic voltammogram inFig. 16). Reproduced with permission from [83] C. Li, et al., Chimia. 64, 383 (2010). © 2010, ACS.

    electronics to carry out many important functions towardsthe development of biomemory,62�87�88 transistor47�89 andbioprocessing devices.50 Single-protein conductance wasmeasured with an ECSTM using the STM-BJ approachon the blue copper protein Az (Az) in buffer solutionand under bipotentiostatic control90 shown in Figure 18.This redox center makes the protein capable of accept-ing and transporting electrons by switching its redox state(Cu1+/2+�.Similarly, using ECSTM in a STM-BJ approach, Az

    immobilized on Au was studied under electrochemicalcontrol in buffer solution.90 Current–distance was pre-sented in Figure 18(b) present current steps that resem-ble those reported for small organic compounds using thesame setup. These steps were absent in control experi-ments performed on a clean gold surface (compare redtraces of Figure 18(b) with black traces in the inset ofFigure 18(b), and thus they were interpreted as transientformations and ruptures of molecular junctions with Azbridging the two electrodes (the Au substrate and ECSTMprobe, see Figure 18(a). Collecting hundreds of currentsteps during one experiment allowed building conductancehistograms and then single-Az conductance was calcu-lated. By performing histograms at different sample poten-tials, conductance modulation with overpotential (“redoxgating”) was demonstrated. In Figure 18(c), shows theconductance of Az (red circles) as function of electro-chemical gate potential. It can be noticed that Az conduc-tance depends on potentials applied, while the conductanceobtained in control experiments on non-redox Zn-Az junc-tions (black squares) remains potential independent. Thisdemonstrates again that the Cu center is fundamental forthe electron transfer process and that the conductance canbe modulated with electrochemical potentials. Such con-ductance modulation is analogous to the modulation ofcurrent in a field effect transistor and possesses yields ofon/off ratio of 20 in conductance modulation (Fig. 18(b)).The in-situ behavior of single Az, was estimated by havingthe ECSTM tip at a controlled distance from the surface,

    the current was recorded in order to detect spontaneousformation of single-Az junctions. Upon the detection ofsuch single-Az junctions, the reference electrode potential(acting as gate electrode) was swept. Conductance of asingle Az determined in situ was found to be modulatedwith the electrochemical gate. These results constituteda proof of concept of a single-wired protein transistor.90

    More recently, the current–voltage characteristics of singleAz were recorded both in tunneling and wired configura-tions, which yield the lowest transition voltage reported todate (0.4 V).93

    The I–V measurements in single biomolecules such asprotein or DNA is crucial for the development of novelbioelectronic devices in which ECSTM plays an importantrole to analyze the properties in the native environment.ECSTM analysis along with other spectroscopic moleculesenabled to understand the properties of the biomolecule.Recently, transition voltage spectroscopy (TVS),93 throughwhich molecular level positions can be determined inmolecular devices without applying extreme voltages, hasbeen applied for organic monolayer and also for non-redoxproteins. TVS spectroscopy for redox proteins which isuseful for understanding voltage dependence of molecularconductance is especially relevant for the mechanism ofET in redox-active molecules.Here, azurin was covalently immobilized on Au sub-

    strate and ECSTM was employed under bipotentiostaticcontrol using Ag/AgCl as a reference electrode to examinethe redox properties of the protein. Measurements wereperformed in both tunneling configuration (no physicalcontact between the STM probe and the protein) and thewired configuration (probe is in contact with the protein).In tunneling configuration of ECSTM, like normal STS

    spectroscopy, the I–V curves were obtained by positioningthe probe over a region with a high protein surface concen-tration once the imaging is finished. The I–V curves forthe reduced azurin depicts two distinct behaviors one rela-tively linear and the other more rectifying behavior whichis not observed on bare Au electrode.

    16 Sci. Adv. Mater., 8, 1–20, 2016

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    Fig. 18. (a) Schemes of a single-Az junction formation by the STM-BJ approach. The ECSTM probe is approached to the surface and retractedafterward. Eventually a single Az bridges the two electrodes. (b) Raw data examples of I�z� plots exhibiting current plateaus or steps correspondingto single-Az junction formation. The conductance values are represented in the right axis (G0 = 2e2/h ≈ 774 �S). The inset shows curves obtainedon a clean gold surface. A horizontal offset was applied in all of them for clarity. (c) Conductance values obtained from the center of the Gaussianfit of the conductance histograms peak as a function of EC gate potential at constant –0.3 V bias for Az (red circles) and non-redox Zn-Az junctions(black squares). Upper axis represents overpotential (� = US −UAZ). Error bars indicate the full width half-maximum (fwhm) of the Gaussian fit oneach conductance peak. The red plot shows a fit of the numerical version91 corresponding to the formalism for a two-step electron transfer process.92.Control measurements with non-redox Zn—Az are indicated with a dashed black line as visual guidance. All experiments were performed in 50 mMammonium acetate solution (pH 4.55). Figure reproduced with permission from [90], J. M. Artes, I. Diez-Perez, and P. Gorostiza, Nano Lett. 12, 2679(2012). © 2011, ACS.

    This rectifying behavior is due to the applied bias volt-age such as the reduced and oxidized states of azurin.From the I–V characteristics, the conductance (G) of thetunneling gap in the presence of azurin was calculatedfrom the relation G = I/V to be between 10−6 G0 and10−5 G0. To get better understanding of the obtained I–Vcurves, TV value that can be used to describe the electro-chemical potential dependence of the azurin conductancein the context of TVS. Individual I–V curves and plot-ted ln�I/V 2� versus 1/V displayed minima in the curve,called transition voltage (T V ) value, Figure 19, which isnot visible in bare Au samples. Several ET studies ofredox molecules have shown that a transition in conduc-tance occurs when the application of an external potentialresults in the alignment of the molecular energy levelsand the Fermi level of the electrodes. Here, the low TVvalue obtained for azurin suggests that the effective barrierfor tunneling through a solution is lower than the bar-rier observed in pure tunneling processes (i.e., tunnelingthrough vacuum), in agreement with experimental and the-oretical works on tunneling through an electrochemicalenvironment. In particular, in the context of two-step ET

    in a redox molecule, a transition was predicted by theoryto occur in the range where the effective voltage in theredox center is higher than the reorganization energy ofthe molecule.For the determination of TV in the conductance of the

    redox protein, the tunneling current for the two-step elec-tron transfer mechanism, IT is given by,


    IT = ek��eUbias��






    + exp[




    in which is the electronic transmission coefficient; � is thedensity of states in the metal near the Fermi level; � isthe nuclear vibration frequency; k is Boltzmann’s constant;T is the temperature; Ubias = UP −US is the potential dif-ference between probe and sample electrodes; � is thereorganization energy; � is the overpotential, given by �=US −UAz, where UAz is the redox potential of an azurinmolecule; and � and � are two model parameters describ-ing the shifts in Ubias and � at the redox center, respec-tively. The parameters � and � are related to the electronic

    Sci. Adv. Mater., 8, 1–20, 2016 17

  • Electrochemical Scanning Tunneling Microscopy Analysis on Protein Based Electronic Devices Yagati et al.


    Fig. 19. (a) Experimental ECSTM set-up for the azurin immobilized Au structure, WE, working electrode, RE, reference electrode, CE, counterelectrode. (b) I–V curves, a triangular ramp was applied to the probe (WE1) while the feedback is off. The current signal (IT � was recorded atconstant potential (WE2). (c) 2D I–V histogram showing two populations of curves in azurin on Au. (d) Average of two I–V curves obtained in(c) corresponding the azurin (red curve) and bare Au (yellow curve). Gray error bars indicate the standard deviations. Reproduced with permissionfrom [93], J. M. Artes, et al., J. Amer. Chem. Soc. 134, 20218 (2012). © 2012, ACS.

    coupling of the molecule with the probe and substrate,respectively.To obtain values of the parameters for azurin, we fit

    the experimental I–V curves to Pobelov and Wandlowski’snumerical equation,80

    IT = 1820kUbias{exp



    + exp[973�



    in which IT is expressed in nA, potentials are in V , and �is in eV. In this expression, typical values for � in a liquidand � in a metal were used, and �, �, �, and � were leftas free model parameters. In the wired junctions, a TVSspectrum is also determined but a negative low value wasdetermined. This minimum is due to the stronger couplingwith the probe electrode, which lowers the energy barrierbetween the levels of the STM probe electrode and themolecule. Thus, in wired junctions, the TV is related to thecontact resistance, as commonly found for single-moleculejunctions. Hence, these measurements help in characteriz-ing redox proteins and understanding their performance inbiological ET chains and molecular electronic devices.

    4. SUMMARY AND OUTLOOKIn this review, we began focusing on the advancement ofscanning probe microscopic techniques such as STM, STSand ECSTM and their capabilities in atomically resolvingthe surface structures and electrical/electrochemical prop-erties. In recent years, ECSTM technique has become anincreasingly versatile tool in the advancement of molecularelectronics. With the capability to study these structuresand functions with tunneling, liquid format with nanoscaleresolution, there are many possibilities for the ECSTMtechnique to utilize in various bioelectronics applications.We discussed some of the application of ECSTM andits capabilities with selected articles of our own work oncharge transport in biomolecular structures on Au surfacein resolving the conductance of proteins and also appliedto various organic and inorganic molecules in understandthe properties and for structural analysis. This review alsopresented that many biomolecules can be imaged at single-molecule level using ECSTM, once a suitable strategy forits immobilization on an atomically flat electrode is found.However, certain factors influencing the tunneling gap androle of ionic strength needs to be considered. Further,electrochemical atomic force microscopy (ECAFM) canbe complimented in analyzing the mechanical information

    18 Sci. Adv. Mater., 8, 1–20, 2016

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    of single molecular structures and electron transport phe-nomena. Furthermore, the electron transport phenomenain complex biomolecules, cellular biology and moleculeswith multiple redox systems requires deeper understand-ing however with computer simulations and advancementin nanotechnology will provide novel concepts and appli-cations in molecular bioelectronics.

    Acknowledgment: This research was supported bythe Basic Science Research Program through theNational Research Foundation of Korea (NRF), fundedby the Ministry of Science, ICT and Future Plan-ning (2014M3A7B4051907) and Basic Science ResearchProgram through the National Research Foundation ofKorea (NRF) funded by the Ministry of Education(2016R1A6A1A03012845).

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