Negative Differential Resistance Behavior of the Iron Storage Protein

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Negative differential resistance behaviour of the iron storage protein ferritin Jayeeta Kolay, Sudipta Bera, Tatini Rakshit, and Rupa Mukhopadhyay Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b04356 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 8, 2018

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Negative differential resistance behaviour of the iron storage protein ferritin J. Kolay, S. Bera, T. Rakshit † and R. Mukhopadhyay* Department of Biological Chemistry, Indian Association for the Cultivation of Science, Kolkata700 032, India

Abstract Realization of useful nanometre length scale devices in which metalloproteins are junctionconfined in a distinct molecular arrangement for generating practical electronic signals (for example, in bioelectronic switch configuration), is elusive till date. This is mostly due to difficulties in observing electronically appropriate signal (i.e., reproducible and controllable), when studied under junction-assembled condition. A useful 'ON' - 'OFF' behaviour, based on the negative differential resistance (NDR) peak characteristics in the current-voltage response curves, acquired using metal-insulator-metal (MIM) configuration, has been observed only in case of a few proteins, namely, azurin, cytochrome C and bacteriorhodopsin, so far. The case of NDR in ferritin, an iron-storage protein having a semiconducting iron core consisting of few thousands of iron atoms connected in an oxide network, has not been studied in MIM configuration where single (or a few) molecule(s) are junction-trapped, for example, as in the case of local probe configuration of scanning probe microscopy. The present study by scanning tunnelling microscopy (STM), using the naturally occurring iron-containing ferritin (human liver), as well as differently iron-loaded ferritins, provides clear indication of the capability of ferritins to be NDR-capable, at varying sweep conditions. As ferritin can be tailor-made in a structurally conserved manner, metal core reconstituted ferritins, i.e., Mn(III)-ferritin, Cu(II)ferritin and Ag-ferritin, were prepared. A correlation between the NDR peak signatures, as observed in the respective current-voltage response curves of these reconstituted ferritins, and the nature of the metal core, is demonstrated. In support of our earlier proposition, here we affirm that ferritin protein behaves as a conductor-insulator (metal core-polypeptide shell) composite, where the overall electronic structure of the material can alter as a function of the nature of the conducting filler placed inside the insulated matrix.

†

Present address: Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892-5055, USA. * Corresponding author: [email protected] 1

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Introduction Negative differential resistance (NDR) behaviour, i.e., a sharp decrease in current with progressively increasing voltage, in the current vs. voltage response of a semiconductor, although widely observed in quantum well structures, is rarely found in proteins. The application of NDR was first exemplified six decades back in Esaki diode using germanium p-n junction.1 Since then, its relevance has been realized in different device configurations such as resonant tunnelling diode,2 oscillator,3 amplifier,4 RAM device,5 analog-to-digital signal converter etc.6,7 The NDR peak signature has been observed in a variety of materials like nitro-substituted conjugated

molecules,8-10 iodide-bridged

platinum complex,11

polyanion

derivatives,12

heteropolyacid,13 boron-nitride nanoribbons,14 organic molecules possessing intra-molecular hydrogen bonds,15 titanium oxide16 etc. Interestingly, NDR characteristics could be identified only in a handful of redox metalloproteins like yeast iso-1-cytochrome C,17 azurin18,

19

and

ferritin20 so far. For construction of stable tunnel junctions in the solid-state metal-insulator-metal configurations (Scheme 1A), the redox metalloproteins are found to be attractive candidates because of their intrinsic capability of transferring single electrons over long distances (10-20 Å)21 in a fast directional manner.21-23 During the past decade, considerable progress has been made in developing methods for preparing surface-assembled protein adlayer that is electronically addressable. One seminal work by Maruccio and Rinaldi et al. demonstrated positioning of azurin protein molecules within a 5 nm junction space, and thereby incorporated the azurin adlayer in a device geometry.24 They studied electron transport across such a molecular tunnel junction at the single molecule level, and reported observation of negative differential resistance in the current-voltage characteristics in support of electron transport through the protein molecules. 2


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The present study is performed using ferritin protein, which is an iron-storage protein found in both the prokaryotes and the eukaryotes. It consists of 24 subunits arranged in a symmetrical fashion, resulting in generation of an internal space made available for iron accumulation, and formation of eight 3-fold ion channels for the movement of Fe2+ ions to/from the iron core region (Scheme 1B). It can retain its structural integrity when immobilized onto a solid substrate, for example, via the exposed cysteine thiols on the protein surface (see Fig. S1A in Supporting Information) and functions well up to 85 °C temperature in aqueous media within a pH range of 4.0-9.0.25 The presence of an iron core inside ferritin makes this protein a potential bioelectronic material, since the iron (in form of oxide, see Fig. S1B in Supporting Information) core is semiconducting in nature.26-28 Importantly, as the metal centre of ferritin can be substituted with other metals in a structurally conservative manner (Scheme 1C),29-32 its electron transport characteristics can be tuned as a function of the metal type.33,34 However, in these earlier reports, only a preliminary account of the electron transport properties, in terms of current-voltage response variation, was presented. Herein, we report unique NDR-rich behaviour of the ferritins, which is dependent on the metal core, and its implications.

Materials and Methods Preparation of ferritin solutions Apoferritin (equine spleen) and holoferritin (human liver) were obtained from Sigma-Aldrich and used as is. Preparation of ferritins with reconstituted iron cores having varying iron content was carried out using 0.05 M MOPS, 0.1 M NaCl buffer (pH 7.5) [MOPS(3Morpholinopropane-1-sulfonic acid)] from Sigma-Aldrich (purity ≥ 99%).35 In order to prepare 800 Fe containing ferritin, 25 L Mohr salt solution (10 mM in 1 mM HCl) was added to 0.3 M 3


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apoferritin solution in MOPS buffer at stirring condition. Stirring was continued for 30 min in air to allow oxidation of Fe2+. Then one additional aliquot of Mohr salt solution was added following the identical procedure. In order to prepare 1600 Fe containing ferritin, four additional aliquots of Mohr salt solution were added following the identical procedure.35 In both cases, the resulting solution was passed through Microcon ~ 100 KDa cutoff filter to remove the unbound iron. All the reconstituted Fe-ferritin solutions were stored at 4 °C temperature. The metal core reconstituted ferritins with different types of metals, i.e., Mn-ferritin, Cuferritin and Ag-ferritin, were synthesized from apoferritin using procedures previously reported.29-32 Manganese (II) chloride tetrahydrate (MnCl2. 4H2O) of purity ≥ 99% was procured from Sigma Aldrich. Silver nitrate GR (AgNO3) of purity ≥ 96% were purchased from Merck. Copper(II) sulphate Pentahydrate (CuSO4.5H2O) of purity ≥ 99% was purchased from Fluka. All the reconstituted ferritin solutions were stored at 4 °C temperature. Characterization of ferritin proteins The protein solutions were characterized by UV-visible spectrophotometry (using a Varian Cary 50 Bio UV-vis spectrophotometer and a 1 cm cuvette at 25 °C), high-resolution transmission electron microscopy (HR-TEM) (using a JEOL JEM-2010 TEM at the operating voltage 200 keV and standard preparation protocol) (see Fig. S2-S4 in Supporting Information). The protein concentrations were determined by UV-visible spectrophotometry using the molar extinction coefficient value of apoferritin (ε280=468000 M-1 cm-1).30 Preparation of ferritin film on gold(111) substrate Gold (150-200 nm thick film) on mica substrate (Phasis, Switzerland) was flame annealed in a butane flame until a reddish glow appeared. The procedure was repeated 7-8 times, and after a short period (2 s) of cooling in air, the substrate was immersed into the desired ferritin solution. 4


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The stock solutions of the ferritin proteins were diluted to 20-30 pM concentration (apo- and holoferritin diluted with 0.3 mM Tris 0.15 M NaCl buffer, pH 7.4 [Tris [2-Amino-2(hydroxymethyl)-1,3-propanediol and NaCl (Sodium Chloride) from Merck (purity ≥ 99%)]; Mn-ferritin with 50 mM AMPSO, 50 mM NaCl, pH 8.9 [AMPSO [N-(1, 1-Dimethyl-2hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid from Sigma Aldrich (purity ≥ 99%]; Agferritin with 7.5 mM Ammonium hydroxide solution pH 8.3 (NH4OH from Rankem); and Cuferritin with 100 mM Tris, 150 mM NaCl, pH 8.0. All the buffer solutions were prepared using autoclaved Milli-Q water (resistivity 18.2 MΩ.cm, Millipore) and filtered using 0.22 m filter (Millex-GV, Millipore) prior to use. The freshly annealed gold(111) substrate was immersed into the respective ferritin solution at room temperature (24 ± 1 °C) and incubated for 12 hrs. Then it was washed with 1 mL (4250 µL) of respective buffer solution and dried under a weak stream of nitrogen gas prior to imaging experiment. Protein film formation onto gold(111) surface (Fig. 1) was checked by STM imaging in ambient condition under pre-established scan conditions.28 STM/STS data acquisition and analysis STM and STS experiments were performed using the PicoLE STM/AFM microscope of Agilent Corp. (USA) with a 1 m scanner. Mechanically cut Pt-Ir tips were always prepared fresh and tested by imaging freshly cleaved HOPG (highly oriented pyrolytic graphite) at atomic resolution to check for their suitability for high-resolution imaging prior to protein imaging and the STS experiments. Imaging was carried out in the constant current mode (to obtain height data) and constant height mode (to obtain current data) with tunneling current 100-200 pA and bias voltage (tip positive) 0.7-1.4 V. The current-voltage (I-V) response was recorded by positioning the tip on top of a protein molecule followed by disengaging the feedback loop, and then by monitoring the tunnel current in varying bias voltage ranges, varying sweep directions and sweep durations. 5


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I-V curves acquired from protein molecules of different images as well as from different areas of the sample were the average over five bias sweeps. All the STS experiments were performed in ambient condition where temperature and humidity were maintained at 24 ± 1 °C and 35-40 %, respectively. The negative differential resistance values were obtained by drawing a tangent on the NDR region between the peak and the valley position of the I-V curve, and then calculated from the inverse slope by applying the equation, rdiff = (valley voltage-peak voltage)/(valley current-peak current). For statistical analysis, we analyzed the entire set of STS I-V curves for the different ferritins, considering only the complete, symmetric and the noise-free traces (200300 I-V curves acquired from experiments done at least three times on different days for each type of ferritin). CSAFS data acquisition and analysis CSAFS experiments were performed in contact mode using the PicoLE AFM microscope (5100 model, Agilent Technologies, USA) with a 10 m scanner. The AFM probes (masch, Estonia) with platinum coating on tip and the backside of the cantilever, having spring constant 0.17 N/m, tip radius < 20 nm, length 500 μm, and width 30 μm were used in the CSAFS experiment, where the electrical characteristics were recorded independent of force feedback. I-V curves were taken at ±3 V for human liver holoferritin film on gold(111) substrate at different force loads starting from 6 nN to 42 nN (bias applied to sample with respect to tip at -1 V). I-V curves were acquired from different areas of the sample and also from the other samples prepared on the different days. Each I-V spectrum acquired on holoferritin layer was an average over five sweeps. All CSAFS measurements were performed in ambient condition where temperature and humidity were maintained at 24 ± 1 °C and 35-40 %, respectively.

6


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Results and Discussion The current-voltage (I-V) response from the ferritin proteins immobilized onto gold(111) surface was recorded using Scanning Tunnelling Spectroscopy (STS) at room temperature. The protein layers prepared onto gold(111) surface were mostly dense and of uniform coverage (Fig. 1). Formation of self-assembled monolayer (SAM) was confirmed by performing AFM contact mode scratching experiments on a number of areas of the protein film (Fig. S5). In all the scratched areas, the depth value was found to be less than the ferritin molecular dimension (i.e., 12 nm) that could be ascribed to the usual effects of AFM tip-induced compression of the soft polypeptide exterior. Current-sensing atomic force spectroscopy (CSAFS) were performed under different force loads, as control experiments, in order to confirm that the ferritin proteins are NDR-capable. Molecule-independent influence like that of the STM tip condition36 in developing NDR characteristics in the I-V trace acquired by STS could be ruled out by such control CSAFS experiments. Negative differential resistance (NDR) behaviour in holoferritin In Fig. 2, is shown the representative I-V trace (and the differential conductance response shown in the inset) for holoferritin, which is the naturally occurring ferritin containing ~2000 Fe atoms. Two distinct maxima in current, corresponding to NDR, one each in the positive (0 to +2 V) and in the negative (0 to -2 V) voltage range, were observed. The peak positions showed some fluctuations (within -1 to -1.5 V, and 1 to 1.5 V), most likely due to variability in the contacts formed between the tunnel gap-immobilized protein molecules and the substrate, as there could be one or multiple S-Au contact(s) formed per protein molecule depending on the location on the protein surface that was involved in substrate-anchoring. The unequal potential drop generated across each protein molecule could also be due to a variation in the nature of amino acid residues 7


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exposed immediately under the STM tip. In order to assess whether the NDR behaviour originated from the iron core of holoferritin, the I-V trace of apoferritin, which is the iron-free form, was acquired (Fig. 2). This trace was devoid of any NDR peak and showed a wide band gap of ~2.0 eV. Significantly higher current was sensed in case of holoferritin, than apoferritin, at the peak current regions, i.e., 1.3 V (positive voltage region) and -1.4 V (negative voltage region). It is only outside the voltage range of -1.5 V to + 1.5 V, the tunnel current decreased after reaching the peak current region and gradually up to the valley current, which is almost comparable with the current from apoferritin. So only in the terminal voltage sweep range (approximately +2 voltage region and -2 voltage region) tunnel current was found to be comparable for both apoferritin and holoferritin, due to positioning of the valley current region for holoferritin. The observation of NDR peaks in case of holoferritin, and its absence in case of apoferritin, clearly indicates through-molecule electron transport and a role of iron core in the development of NDR response. The NDR behaviour in case of holoferritin, i.e., both the NDR peaks as appeared upon a full-scale sweep, was reproducibly observed. However, the NDR peak voltage values were found to be largely different from those observed in other types of electron transfer/transport experiments on ferritins. In an experimental configuration like the SWNTferritin-SWNT device on Si-SiO2 substrate, the NDR peaks arose at 5 V and -5 V (with a considerable variation within 3-7 V in the peak position). Here, the origin of NDR behaviour in ferritins is thought to be due to the reversible electrochemical redox chemistry which involves reduction of the Fe(III) redox state and oxidation of Fe(II) during I-V scan.20 But, in our case, the ferritin molecules were immobilized onto gold(111) substrate and the I-V curves were taken using conductive Pt/Ir tip in the scanning local probe configuration using the STS approach. The drastic deviations in the NDR peak voltages as observed in our solid-state configuration, from 8


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the above-mentioned case, could be related to the fact that if there is any redox change involving Fe2+/Fe3+ couple leading to protein conformational change(s), such a change can be more conveniently accommodated in the electrochemical configuration as the protein molecules are in a substantially hydrated state. In case of other proteins too, for example, yeast iso-1-cytochrome C, a wide variation between the NDR peak position as obtained from conductive probe atomic force microscopy (CP-AFM) experiments and the equilibrium redox potential as measured using in situ cyclic voltammetry has been reported.17 A rational consequence of the observation of considerable variation in the NDR peak voltages from the reported values was to test if the sweep conditions had any effect on the NDR behaviour of ferritins. In Fig. 3A-C, are shown the effects of different sweep durations, i.e., 1s, 2s and 5s, and sweep made in both the upwards (-ve voltage to +ve voltage) and the downwards (+ve voltage to -ve voltage) directions, where the sweep range was kept fixed at ± 3V. The case of half sweep range (3V to 0V, 0V to -3V; and the reverse) was also monitored (Fig. 3D-E), which depicts similar NDR response as in case of sweeping over the full range of ± 3V (Fig. 3AC). Almost similar NDR responses were observed when compared between the varying sweep ranges as well (Fig. 4). In all cases, were observed the distinct NDR regions, one in the positive voltage region (+1 to +2.2 V), and the other in the negative voltage region (-1 to -2 V), meaning that the electron transport took place via the protein's accessible molecular state(s), irrespective of the applied set of sweep conditions. In order to confirm whether development of NDR signals was due to the Fe species, dependence of NDR peak characteristics on the iron content of ferritins was explored by obtaining I-V traces for two different ferritins (containing 1200 and 800 Fe atoms) apart from holoferritin (containing ~2000 Fe atoms) (Fig. 5A-C). In all cases, two NDR peaks, one each in 9


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the positive (0 to +2 V) and in the negative (0 to -2 V) voltage range were observed, with peak voltage variation within a range of 1.3 to 1.6 V (for the positive peak) and -1.3 to -1.6 V (for the negative peak). The two NDR peaks were found to be increasingly more separated as the iron content reduced from 2000 to 1200 and to 800. The NDR peak current values were also found to reduce as the iron content decreased. Such iron content dependence of the NDR peak positions and the peak current values confirms the origin of the observed NDR to be the Fe species present inside the ferritin core structure. The observations of the NDR peaks both in the positive and the negative voltage regions, and of the variations in the NDR peak current values as a function of iron content, probably also reflect a role of redox chemistry of iron to be operative in the development of NDR response in ferritins. Numerous mechanisms for the NDR observation in molecular systems have been proposed in the last one decade. These are primarily 1) polaron-mediated charge transport through the molecular junction, where the charge can be trapped inside the molecule,37-39 2) intra-molecular redox processes,18-20 3) charge transfer from a high-mobility to a low-mobility valley,40,41 and 4) resonant tunneling between localized states.42,43 The mechanism of resonant tunneling is probably the most pertinent here, since the increase in the current followed by a decrease, against an applied voltage sweep, can be understood in terms of resonance between the tip electronic state and the molecular energy band (as ferritin contains few thousands of iron atoms connected in an oxide network, energy bands rather than localized states could be more appropriate situation to consider), as the tunnel current can increase to a maximum value, when the tip and molecular density of states come in complete resonance [Fig. 6]. In case of ferritin, an analogy of the metal-loaded protein can be made to a conductor-insulator composite, where a change in the overall electronic structure of the material can occur when the 10


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metal core is present within the protein shell, just as if some conducting filler is placed inside an insulated solid matrix, the resulting material starts to behave like a conducting material after crossing a critical conducting filler concentration.44 In the present work, some local density of states (LDOS) of the ferritins, were observed to be missing in both the valance band (VB) and the conduction band (CB) in the differential conductance spectra [Fig. 2 inset figure]. Almost no tunnel current at the sweep voltage (Vapp ≠ 0) inside the band gap region of ferritin could be recorded, and the current increased in the positive and negative voltage region after crossing the band gap region. The tunnel current reached a high value only when a large number of surface DOS could overlap with the STM tip electronic state. When the Fermi level of STM tip was aligned with the DOS missing region of conduction band of ferritin layer, tunnel current decreased with increase in tip bias (negative voltage). Similarly, the tunnel current decreased with increase in tip bias (at positive voltage), only when the DOS missing valence band region was aligned with just the above of Fermi energy level of the STM tip [Fig. 6]. These kinds of DOS missing regions could be treated as the forbidden energy levels of the ferritin layer that led to the off-resonance condition. The mechanism of electron transport from a high-mobility to a low-mobility site leading to NDR is not unlikely in case of ferritin, since ferritin core is an amorphous heterogeneous semiconducting electronic material, which is surrounded by a polypeptide shell. Electron transport through the protein molecule could accelerate inside the metal core as the metal core offers high-mobility sites, and then retard within the protein shell as the protein shell offers lowmobility sites. In ferritins, some non-uniform aperiodic potential (unlike Bloch potential for pure crystalline material) could be operative due to different types of the amino acid residues and their random orientations one after another inside the protein shell, and the dissimilar sites (Fe, O, H) 11


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and structures of the iron deposits within the metal core. The resulting electron mobility fluctuations might be a cause of the variations observed in the NDR peak position. Indeed, the non-uniform electron flow could be largely influenced by the orientation of the protein molecules on gold substrate and the positioning of the electron entry site on ferritin surface below the STM tip as well. It is worth noting that in STM experiments, despite the tip-protein separation (tunnel gap), an appreciable current signal can be recorded. In fact, it has been reported by Andolfi et al. that a considerable amount of current can be recorded in STS for a wide range of tip-protein vertical distances and that the applied voltage has only a small influence on the protein-gap barrier.45 In case of STM, the tunnel current appears due to overlapping between the surface-exposed allowed electronic states (LDOS) of protein layer and the STM conductive tip. The DOS missing zone or the energy forbidden zone inside both the valence and the conduction band could be the cause of NDR in STM configuration. However, in order to verify that the observation of NDR was not related to any non-specific like that of the STM tip condition,36 we employed current-sensing atomic force spectroscopy (CSAFS). At force values less than 2-3 nN, no reliable electrical contact to the protein molecules could be established and transport was found to be dominated by dielectric breakdown of the water film as present on the protein surface under ambient condition. The NDR behaviour in CSAFS configuration could be observed within the force range 6-29 nN (Fig. 7A-D), meaning that the corresponding tip-substrate distances could ensure no direct tipsample mechanical contact, thereby reducing the probability for direct tunnelling, and allowing resonant tunnelling to occur via the accessible molecular states. The NDR peak features disappeared, presumably indicating loss of resonance, at the higher force value of 35-36 nN (Fig. 7E). It is worth mentioning here that no NDR observation was made for a comparable force 12


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range of 24-29 nN and above this range in another CSAFS study on ferritins.27 We propose that low loading force is suitable for addressing the NDR behaviour for ferritin layer under CSAFM configuration, and ~ 30 nN is the borderline force value for NDR and non-NDR I-V response. Negative differential resistance behaviour in the metal core-reconstituted ferritins In some metalloproteins, for example the ferritins, metal substitution can be structurally conservative, allowing experiments in which the role of metal site in facilitating electron transport can be investigated.33 We prepared three metal core-reconstituted ferritins, namely Mn(III)-ferritin, Cu(II)-ferritin and Ag-ferritin, and acquired current-voltage (I-V) response curves under similar conditions as in case of holoferritin. The NDR behaviour could be observed in the I-V plots obtained from all the three reconstituted ferritins, as shown in Fig. 8A1-C1. The NDR peaks were found to develop at ~1.5 V and ~ -1.82 V [for Mn(III)-ferritin], at ~1.64 V and ~ -1.6 V [for Cu(II)-ferritin], and at ~ 1.6 V and ~ -1.4 V [for Ag-ferritin], with small shifts in the peak position observed upon repeated scanning. In most of the I-V traces, the NDR peaks appeared moderately sharp. Since the sharpness of a resonant tunneling peak is related to the width of the allowed energy bands that can be broadened under environmental influences, the observation indicates that the metal redox centres having low-lying energy bands could be coupled to the environmental fluctuations. In case of ferritins, since the metal centres are connected in an oxide network, such a situation may prevail, and the observed peak broadening can be attributed to fluctuations in the nuclear configuration, which shift the energy levels of the redox centres. Also, since all these metal core-reconstituted ferritins are like conductor-insulator composites, it is likely that the observed broadened NDR signals are the manifestation of core (high-mobility electron transport metal sites) - shell (low-mobility electron transport amino acid sites) interactions, where electron mobility fluctuations can get influenced by molecular 13


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orientation and therefore positioning of the electron entry site on ferritin surface under the STM tip. To illustrate the fine structure of the transport data, the differential conductance plots [dI/dVV] were obtained, wherefrom the NDR responses could be distinctively delineated (Fig. 8A2C2). However, as the information contained in the dI/dV-V plots do not directly correlate with the sample local density of states (LDOS) due to convolution between the sample and the tip LDOS, the normalized conductance [(dI/dV)/(I/V)] plots46 were obtained [Fig. 9]. The negative differential resistance values for the Cu(II)-ferritin and Mn(III)-ferritin systems were found to be within the range 0.2-0.5 GΩ, whereas for the Ag-ferritin and holoferritin, lower resistance values of 0.02-0.05 GΩ were observed. A high percentage of NDR characteristics could be observed in the I-V traces acquired for all the four metal-containing ferritins, i.e., holoferritin (75%), Mn-ferritin (78%), Cu-ferritin (84%) and Ag-ferritin (79%), clearly supporting data reproducibility for the NDR observation. The variation in the NDR peak voltage (sweep voltage corresponding to the peak current) was found to be as following: -0.97 to -1.63 V (negative voltage region) and 1.12 to 1.76 V (positive voltage region) for holoferritin (Holo-f); -1.7 to -1.92 V (negative voltage region) and 1.4 to 1.6 V (positive voltage region) for Mn-ferritin (Mn-f); -1.3 to -1.93 V and 1.25 to 2.0 V for Cuferritin (Cu-f); and -1.17 to -1.90 V and 1.23 to 1.96 V for Ag-ferritin (Ag-f). As the peak voltage positions follow normal distribution, the most probable peak position could be considered as mean peak position. Therefore, the deviation of NDR peak voltage position with respect to the most probable peak position is represented as the error bar for both positive and negative voltage region as “mean NDR peak voltage ± standard deviation” (Figure 10). The statistical data variability of the different types of ferritins are found to be -1.44 ± 0.19 V and 1.3 ± 0.15 V for Holo-f, -1.82 ± 0.08 V and 1.53 ± 0.12 V for Mn-f, -1.6 ± 0.25 V and 1.64 ± 0.29 V 14


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for Cu-f and lastly -1.4 ± 0.12 V and 1.6 ± 0.1 V for Ag-f (Figure 10). The observed NDR peak voltage variation could be influenced by the defects generated during SAM formation, inhomogeneities on the ferritin surface due to different amino acid arrangements, and also positioning of the protein molecule in different orientations under the tip when anchored via one or more of the available surface-exposed cysteine thiols. One implication of the outcome of the present study could be signal amplification, since while a positive resistance consumes power from current passing through it, a negative resistance produces power. The most obvious application of NDR could however be imparting switchability to a device, where the gating current flow is precisely controlled. 47-49 If tunnel junctions can be established where a dominant proportion of current flow occurs via the redox site then the efficiency of this will be highly dependent on the bias applied. In a previous study,28 we observed that the ferritin molecules could be imaged by STM only when the bias voltage was about 0.7-1.0 V (tip positive), indicating tuneable resonance in case of the ferritin proteins, and therefore electron transport being predominantly coherent in nature. In the same study, we also observed a temperature dependence of electron transport as the band gap value reduced with an increase in temperature till 40 ˚C. Since band gap reduced with increasing temperature in case of apoferritin as well, it is tempting to conclude that the observed temperature dependence had arisen from electron transport within the polypeptide exterior, via hopping mechanism, and therefore the dominant electron transport being incoherent in nature. Ferritin protein being considerably large in size (~12 nm), along with a complex and extended network of iron-oxide core (~8 nm), therefore offers an interesting case to study solid-state electron transport in proteins, which has remained perplexing so far.

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An important class of bioelectronic materials, the metalloproteins are nature-derived and nature-tuned for electron transfer reactions that sustain life. Their use in solid-state devices like tunnel diodes has therefore been envisaged over long periods of time. However, except in few cases, observation of NDR could not be made, most likely due to preparation difficulties of junction-assembled electronically addressable protein films. Coupling of the protein surface residues to the electrode surface has remained an important concern, since covalent coupling without distorting the active conformation of the protein, so that the electron transport routes remain available, can be a challenge. Protein fouling at the surface may be a reason for lack of reproducibility in most of the studies. Herein, success with ferritins probably lies in the protein’s intrinsic capacity of being surface-stable. Ferritin is a large protein, the sub-units being all arranged in an ordered and symmetric fashion leading to a tight spherical shape. This protein is probably one of the most evolved proteins that survived nature-induced challenges over many years. Whether or not, it’s stability and electron transport capacity in a solid-state configuration can be effectively utilized in a device, is yet to be seen.

Conclusions The high percentage of NDR observation in the solid-state electron transport characteristics of the junction-confined ferritin molecules (the naturally occurring holoferritin and the metal core reconstituted proteins) means a step ahead towards obtaining utilizable electronic signals from metalloproteins. Potential applications of ferritins as molecular switches, where ON-OFF signal generation is required, and/or in power generation, where signal amplification is an obvious requirement, can be pursued forward. Since an influence of the metal type inside ferritin core on the electron transport characteristics has been observed, novel types of ferritin core using even 16


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bi- or multimetallic systems can be designed and new electronic applications can be explored. Although, ferritins are robust proteins, it is also necessary to assess whether these are stable enough in device-integrated form over considerable period of storage time. The author's group is working on such issues at present.

Acknowledgements R.M. acknowledges financial support from IACS, Kolkata, and DST-SERB, Govt. of India [Grant no. SB/SO/BB-33/2014]; and the research fellowships of J. Kolay, S. Bera and T. Rakshit from CSIR, Govt. of India; DST-INSPIRE, Govt. of India; and CSIR, Govt. of India, respectively.

References 1. Esaki, L. New Phenomenon in Narrow Germanium p-n Junctions. Phys. Rev. 1958, 109, 603604. 2. Sollner, T. C. L. G.; Goodhue, W. D.; Tannenwald, P. E.; Parker, C. D.; Peck, D. D. Resonant Tunneling through Quantum Wells at Frequencies up to 2.5 THz. Appl. Phys. Lett. 1983, 43 (6), 588-590. 3. Brown, E. R.; Söderström, J. R.; Parker, C. D.; Mahoney, L. J.; Molvar, K. M.; McGill, T. C. Oscillations up to 712 GHz in InAs/AlSb Resonant-Tunneling Diodes. Appl. Phys. Lett. 1991, 58 (20), 2291-2293. 4. McWhorter, A. L.; Foyt, A. G. Bulk GaAs Negative Conductance Amplifiers. Appl. Phys. Lett. 1966, 9 (8), 300-302. 5. Van Der Wagt, J. P. A. Tunneling-Based SRAM. Proc. IEEE 1999, 87 (4), 571-595. 17


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6. Broekaert, T. P. E.; Brar, B.; Van Der Wagt, J. P. A.; Seabaugh, A. C.; Morris, F. J.; Moise, T. S.; Beam III, E. A.; Frazier, G. A. A Monolithic 4-Bit 2-Gsps Resonant Tunneling Analog-toDigital Converter. IEEE J. Solid-State Circuits 1998, 33 (9), 1342-1349. 7. Mathews, R. H.; Sage, J. P.; Sollner, T. C. L. G.; Calawa, S. D.; Chen, C. L.; Mahoney, L. J.; Maki, P. A.; Molvar, K. M. A New RTD-FET Logic Family. Proc. IEEE 1999, 87 (4), 596-605. 8. Rawlett, A. M.; Hopson, T. J.; Nagahara, L. A.; Tsui, R. K.; Ramachandran, G. K.; Lindsay, S. M. Electrical Measurements of a Dithiolated Electronic Molecule via Conducting Atomic Force Microscopy. Appl. Phys. Lett. 2002, 81 (16), 3043-3045. 9. Chen, J.; Wang, W.; Reed, M. A.; Rawlett, A. M.; Price, D. W.; Tour, J. M. RoomTemperature Negative Differential Resistance in Nanoscale Molecular Junctions. Appl. Phys. Lett. 2000, 77 (8), 1224-1226. 10. Le, J. D.; He, Y.; Hoye, T. R.; Mead, C. C.; Kiehl, R. A. Negative Differential Resistance in a Bilayer Molecular Junction. Appl. Phys. Lett. 2003, 83 (26), 5518-5520. 11. Iguchi, H.; Takaishi, S.; Jiang, D.; Xie, J.; Yamashita, M.; Uchida, A.; Kawaji, H. Negative Differential Resistance in MX- and MMX-Type Iodide Bridged Platinum Complexes. Inorg. Chem. 2013, 52, 13812-13814. 12.Watson, B. A.; Barteau, M. A.; Haggerty, L.; Lenhoff, A. M.; Weber, R. S. Scanning Tunneling Microscopy and Tunneling Spectroscopy of Ordered Hetero- and Isopolyanion Arrays on Graphite. Langmuir 1992, 8 (4), 1145-1148. 13. Song, I. K.; Barteau, M. A. Correlation of Negative Differential Resistance (NDR) Peak Voltages of Nanostructured Heteropolyacid (HPA) Monolayers with One Electron Reduction Potentials of HPA Catalysts. Langmuir 2004, 20 (5), 1850-1855.

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14. An, Y.; Wang, K.; Jia, G.; Wang, T.; Jiao, Z.; Fu, Z.; Chu, X.; Xu, G.; Yang, C. Intrinsic Negative Differential Resistance Characteristics in Zigzag Boron Nitride Nanoribbons. RSC Adv. 2014, 4, 46934-46939. 15. Sadek, M.; Wierzbowska, M.; Rode, M. F.; Sobolewski, A. L. Multipeak Negative Differential Resistance from Interplay between Nonlinear Stark Effect and Double-Branch Current Flow. RSC Adv. 2014, 4, 52933-52939. 16. Du, Y.; Kumar, A.; Pan, H.; Zeng, K.; Wang, S.; Yang, P.; Wee, A. T. S. The Resistive Switching in TiO2 Films Studied by Conductive Atomic Force Microscopy and Kelvin Probe Force Microscopy. AIP Advances 2013, 3, 082107. 17. Davis, J. J.; Peters, B.; Xi, W. Force Modulation and Electrochemical Gating of Conductance in a Cytochrome. J. Phys. Condens. Matter 2008, 20, 374123. 18. Mentovich, E. D.; Belgorodsky, B.; Richter, S. Resolving the Mystery of the Elusive Peak: Negative Differential Resistance in Redox Proteins. J. Phys. Chem. Lett. 2011, 2, 1125-1128. 19. Axford, D.; Davis, J. J.; Wang, N.; Wang, D.; Zhang, T.; Zhao, J.; Peters, B. Molecularly Resolved Protein Electromechanical Properties. J. Phys. Chem. B 2007, 111 (30), 9062-9068. 20. Tang, Q.; Moon, H. K.; Lee, Y.; Yoon, S. M.; Song, H. J.; Lim, H.; Choi, H. C. RedoxMediated Negative Differential Resistance Behavior from Metalloproteins Connected through Carbon Nanotube Nanogap Electrodes. J. Am. Chem. Soc. 2007, 129 (36), 11018-11019. 21. Willner, I.; Willner, B. Biomaterials Integrated with Electronic Elements: En Route to Bioelectronics. Trends Biotechnol. 2001, 19 (6), 222-230.

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22. Willner, I. Protein Hinges for Bioelectronics. Nat. Biotechnol. 2001, 19, 1023-24. 23. Rosi, N. L.; Mirkin, C. A. Nanostructures in Biodiagnostics. Chem. Rev. 2005, 105 (4), 1547-1562. 24. Maruccio, G.; Marzo, P.; Krahne, R.; Passaseo, A.; Cingolani, R.; Rinaldi, R. Protein Conduction and Negative Differential Resistance in Large-Scale Nanojunction Arrays. Small 2007, 3 (7), 1184-1188. 25. Granick, S. J. Physical and Chemical Properties of Horse Spleen Ferritin. J. Biol. Chem. 1942, 146 (2), 451-461. 26. Xu, D.; Watt, G. D.; Harb, J. N.; Davis, R. C. Electrical Conductivity of Ferritin Proteins by Conductive AFM. Nano Lett. 2005, 5 (4), 571-577. 27. Axford, D. N.; Davis, J. J. Electron Flux through Apo-and Holoferritin. Nanotechnology 2007, 18, 145502-145508. 28. Rakshit, T.; Banerjee, S.; Mukhopadhyay, R. Near-Metallic Behavior of Warm Holoferritin Molecules on a Gold(111) Surface. Langmuir 2010, 26 (20), 16005-16012. 29. Meldrum, F. C.; Douglas, T.; Levi, S.; Arosio, P.; Mann, S. Reconstitution of Manganese Oxide Cores in Horse Spleen and Recombinant Ferritins. J. Inorg. Biochem. 1995, 58, 59-68. 30. Zhang, B.; Harb, J. N.; Davis, R. C.; Kim, J. W.; Chu, S. H.; Choi, S.; Miller, T.; Watt, G. D. Kinetic and Thermodynamic Characterization of the Cobalt and Manganese Oxyhydroxide Cores Formed in Horse Spleen Ferritin. Inorg. Chem. 2005, 44 (10), 3738-3745. 31. Ga ́lvez, N.; Sa ́nchez, P.; Domínguez-Vera, J. M. Preparation of Cu and CuFe Prussian Blue Derivative Nanoparticles Using the Apoferritin Cavity as Nanoreactor. Dalton Trans. 2005, 2492-2494.

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32. Shin, Y.; Dohnalkova, A.; Lin, Y. Preparation of Homogeneous Gold-Silver Alloy Nanoparticles Using the Apoferritin Cavity as a Nanoreactor. J. Phys. Chem. C 2010, 114 (13), 5985-5989. 33. Rakshit, T.; Mukhopadhyay, R. Tuning Band Gap of Holoferritin by Metal Core Reconstitution with Cu, Co, and Mn. Langmuir 2011, 27 (16), 9681-9686. 34. Rakshit, T.; Mukhopadhyay, R. Solid-State Electron Transport in Mn-, Co-, Holo-, and CuFerritins: Force-Induced Modulation is Inversely Linked to the Protein Conductivity. J. Colloid Interface Sci. 2012, 388, 282-292. 35. Snow, C. L.; Martineau, L. N.; Hilton, R. J.; Brown, S.; Farrer, J.; Boerio-Goates, J.; Woodfield, B. F.; Watt, R. K. Ferritin Iron Mineralization proceeds by Different Mechanisms in MOPS and Imidazole Buffers. J. Inorg. Biochem. 2011, 105, 972-977. 36. Xue, Y.; Datta, S.; Hong, S.; Reifenberger, R.; Henderson, J. I.; Kubiak, C. P. Negative differential resistance in the scanning tunneling spectroscopy of organic molecules. Phys. Rev. B 1999, 59 (12), R7852-R7855. 37. Galperin, M.; Ratner, M. A.; Nitzan, A. Hysteresis, Switching, and Negative Differential Resistance in Molecular Junctions: A Polaron Model. Nano Lett. 2005, 5 (1), 125-130. 38. Galperin, M.; Ratner, M. A.; Nitzan, A.; Troisi, A. Nuclear Coupling and Polarization in Molecular Transport Junctions: Beyond Tunneling to Function. Science 2008, 319, 10561060. 39. Yeganeh, S.; Galperin, M.; Ratner, M. A. Switching in Molecular Transport Junctions: Polarization Response. J. Am. Chem. Soc. 2007, 129 (43), 13313-13320. 40. Hess, K.; Morkoç, H.; Shichijo, H.; Streetman, B. G. Negative differential resistance through real-space electron transfer. Appl. Phys. Lett. 1979, 35 (6), 469-471. 21


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41. Bigelow, J. M.; Laskar, J.; Kolodzey, J.; Leburton, J. P. Observation of tunnelling real-space transfer in pseudomorphic MODFETS at T=300 K. Semicond. Sci.Technol. 1991, 6, 1096-1099. 42. Guisinger, N. P.; Greene, M. E.; Basu, R.; Baluch, A. S.; Hersam, M. C. Room Temperature Negative Differential Resistance through Individual Organic Molecules on Silicon Surfaces. Nano Lett., 2004, 4 (1), 55-59. 43. Fan, F. R. F.; Yang, J.; Cai, L.; Price, D. W.; Dirk, Jr. S. M.; Kosynkin, D. V.; Yao, Y.; Rawlett, A. M.; Tour, J. M.; Bard, A. J. Charge Transport through Self-Assembled Monolayers of Compounds of Interest in Molecular Electronics. J. Am. Chem. Soc. 2002, 124 (19), 55505560. 44. Bera, S.; Kolay, J.; Banerjee, S.; Mukhopadhyay, R. Nanoscale on-silico electron transport via ferritins; Langmuir 2017, 33 (6), 1951−1958. 45. Andolfi, L.; Bizzari, A. N.; Cannistraro, S. Electron Tunneling in a Metal-Protein-Metal Junction Investigated by Scanning Tunneling and Conductive Atomic Force Spectroscopies. Appl. Phys. Lett. 2006, 89, 183125. 46. Stroscio, J. A.; Feenstra, R. M.; Fein A. P. Electronic Structure of the Si(111)2 x 1 Surface by Scanning-Tunneling Microscopy. Phys. Rev. Lett. 1986, 57 (20), 2579-2582. 47. Du, Y.; Pan, H.; Wang, S.; Wu, T.; Feng, Y. P.; Pan, J.; Wee, A. T. S. Symmetrical Negative Differential Resistance Behavior of a Resistive Switching Device. ACS Nano 2012, 6 (3), 25172523. 48. Nozaki, D.; Lokamani; Santana-Bonilla, A.; Dianat, A.; Gutierrez, R.; Cuniberti, G. Switchable Negative Differential Resistance Induced by Quantum Interference Effects in Porphyrin-based Molecular Junctions. J. Phys. Chem. Lett. 2015, 6, 3950-3955.

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49. Chen, S. L.; Griffin, P. B.; Plummer, J. D. Negative Differential Resistance Circuit Design and Memory Applications. IEEE Transactions on Electron Devices, 2009, 56 (4), 634-640.

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Conductive probe

C

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e-

Protein film

Apoferritin

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B

M0-ferritin M = Ag

M(III)-ferritin M = Fe, Mn

M(II)-ferritin M = Cu

Scheme 1: A. Local probe tunneling configuration to study electron transport through a protein film, B. X-ray structure of human L-chain holoferritin (PDB ID 2FFX), four locations of 3-fold ion channels are shown by solid black arrows, C. A schematic diagram for reconstitution of the ferritin core with different types of metals.


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Figure 1: STM height and current images for (A1, A2) bare gold(111) surface, (B1, B2) apoferritin, (C1, C2) holoferritin, (D1, D2) Mn(III)-ferritin, (E1, E2) Cu(II)-ferritin and (F1, F2) Ag-ferritin films on gold(111). Set point tunnel current: 1 nA for gold(111) surface; 150-200 pA for different ferritins. Applied STM tip bias: 0.1 V for bare gold(111) surface; 0.75-0.8 V for apoferritin, Mn-ferritin, Cu-ferritin; 1.0 V for holoferritin; 1.4 V for Ag-ferritin, scale bar: 20 nm, z-range: 0-10 nm.


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Figure 2: Negative differential resistance (NDR) peaks (the NDR regions shown with solid black arrows) as observed in the STS-derived current (I) vs. voltage (V) curve for holoferritin (blue line) on gold(111) surface. I-V trace for apoferritin (red line) is devoid of the NDR peaks. Differential conductance response of holoferritin is shown in the inset figure.


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Figure 3: Overlay of current-voltage response curves for both the up and down sweep traces of human liver holoferritin at (A) 1 s, (B) 2 s, (C) 5 s sweep duration on gold(111) surface over the sweep range ±3V. Overlay of current-voltage response curves of human liver holoferritin for (D) -3V to 0V, 0V to 3V (up sweep), (E) 3V to 0V, 0V to -3V (down sweep) on gold(111) surface.


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Figure 4: Overlay of current-voltage response curves of human liver holoferritin for (A) up sweep, and (B) down sweep on gold(111) surface for ±3V, ±2.5V, ±2V sweep range, and fixed sweep duration of 1 s.


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Figure 5: Negative differential resistance (NDR) peaks (the NDR regions shown with solid black arrows) as observed in the current (I) vs. voltage (V) curve for (A) holoferritin (~2000 Fe) on gold(111) surface. The NDR peaks are observed also in case of (B) 1200 Fe-ferritin, (C) 800 Fe-ferritin on gold(111) substrate by STS study for 1s sweep duration, down sweep direction.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

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E Figure 6: (A) Non-zero Vapp results in zero tunnel current at the sweep voltage inside the ferritin band gap region, (B) high tunnel current from STM tip to conduction band of ferritin in the negative voltage region, (C) decreased tunnel current (negative voltage NDR zone) at the higher negative voltage, (D) high tunnel current from the valance band of ferritin to STM tip at positive voltage region, and (E) the decreased tunnel current (positive voltage NDR zone) at the higher positive voltage.


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Figure 7: Overlay of current (I) vs. voltage (V) curves for holoferritin at (A) 6-8 nN, (B) 13-15 nN, (C) 21-22 nN, (D) 28-29 nN and (E) 35-36 nN force loads showing NDR peaks at positive voltage (blue), negative voltage (black) on gold(111) surface for ± 3 V sweep range, 5 s sweep duration, up sweep direction by Current Sensing Atomic Force Spectroscopy (CSAFS).


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Figure 8: Overlay of current (I) vs. voltage (V) curves and corresponding conductance curves for (A1, A2) Mn(III)ferritin, (B1, B2) Cu(II)-ferritin having NDR peaks at positive voltage (purple) and negative voltage (black); (C1, C2) Ag-ferritin having NDR peaks at both positive and negative voltage on gold(111) substrate via STS study. The NDR peaks are shown with solid black arrows.


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Figure 9: Normalized conductivity (dI/dV)/(I/V) vs. voltage (V) curves for (A) Mn(III)-ferritin, (B) Cu(II)-ferritin having NDR peak at positive voltage (purple), negative voltage (black); (C) Ag-ferritin and (D) holoferritin having NDR peaks at both positive and negative voltage film on gold(111) substrate via STS study. The NDR peaks are shown with solid black arrows.


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%NDR

Sweep Voltage (Volt)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

75%

78%

84%

Page 34 of 35

79%

2

2

1

1

0

0

-1

-1

-2

-2

Holo-f

Mn-f

Cu-f

Ag-f

Figure 10: Data variability of NDR peak position (mean NDR peak voltage ± standard deviation) and data reproducibility as the percentage of current voltage curve with NDR characteristic of different ferritins on gold(111) substrate in STM configuration.


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Conductive probe Scanning Tunnelling Spectroscopy (STS) protein film holoferritin

eM-ferritin [M= Mn, Cu, Ag]

Conductive substrate Metal-Insulator-Metal Configuration

12 6 I (nA)

Page 35 of 35

Current-voltage response curves

0 -6

NDR zone apoferritin holoferritin

-12 -2

-1

0 1 2 V(volt) Negative Differential Resistance (NDR) in Ferritin


Negative Differential Resistance Behavior of the Iron Storage Protein

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