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Biological and Environmental Phenomena at the Interface
Electron Transport in Muscle Protein Collagen Jayeeta Kolay, Sudipta Bera, and Rupa Mukhopadhyay Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b01685 • Publication Date (Web): 12 Aug 2019 Downloaded from pubs.acs.org on August 13, 2019
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Electron Transport in Muscle Protein Collagen Jayeeta Kolay, Sudipta Bera and Rupa Mukhopadhyay* School of Biological Sciences, Indian Association for the Cultivation of Science, Jadavpur, Kolkata - 700 032, India
Abstract In recent times, collagen, which is one of the most abundant proteins found in animals, has appeared to be an attractive candidate for biomaterial applications, for example, in medical implants and wearable electronics. This is because collagen is water-insoluble, biocompatible and non-toxic. In addition, films of different sizes and shapes can be made using this protein as it is malleable and elastic in nature. However, its capacity of electron transport or an absence of it has remained largely untested so far. Therefore, in this work, the electron transport behaviour of collagen has been studied in both film and single fiber state in local probe configuration using current-sensing atomic force spectroscopy (CSAFS). From the CSAFS analyses, the electronic (transport) band gap of collagen has been estimated. It has been found that collagen behaves as a wide band gap semiconductor (near-insulating) in a variety of experimental conditions. The transition to a semiconducting material with low electronic band gap and nearly thousand times enhancement in the current value (pA to nA level) occurs by metal ion-treatment (here, Fe3+) of the native collagen. To the best of our knowledge, this is the first report on a molecular level study of electron transport behaviour of collagen proteins, and estimation of transport band gap values of collagen and metallated collagen.
Corresponding Author: Dr. Rupa Mukhopadhyay (Telephone: +91 33 2473 4971, Extn. 1506; Fax: +91 33 2473 2805; E-mail:
[email protected]) 1
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Introduction Collagen is the primary structural protein present in extracellular milieu in a number of connective tissues. Being the most abundant protein in mammals, it has attracted much attention over the last few decades. Consequently, varied attempts to delineate its structure, and understand the relation of its structure to function as muscle protein have been made. It has been found that collagen is composed of triple helix of polypeptides that generally consist of two identical 1 chains and an additional 2 chain, differing in composition to some extent from each other.1 The most common motif in the amino acid sequence of collagen is Gly-Xaa-Yaa triplets, where Xaa, Yaa can be any amino acid but mostly are proline and hydroxyproline. Till date, twenty eight types of collagen have been identified, out of which types I to V are the most commonly found ones.2 Approximately 90% of the total collagen content in human body are type I, typically found in bone, tendon, skin and ligament.3 The microstructure of type I collagen consists of alternating gap and overlap regions with a characteristic D period of ~ 67 nm.4 It has been observed that collagen fibrils exhibit different mechanical properties in different tissues due to varying mechanical properties of the gap and the overlap regions as verified by atomic force microscopy (AFM)-based nanoindentation experiments.5 Collagen is found to be a largely malleable and elastic material with Young's modulus of 3.75-11.5 GPa.6 As collagen is bio-derived and is therefore highly biocompatible,7 various medical applications of collagen as a biomaterial have been actively pursued over the years, for example, in tissue regeneration, bone grafts, cardiac surgery, wound dressing, dental implants etc.8 In more recent times, collagen-based films have been tested for flexible implantable electronics.9,10 Understanding electron transport via collagen is not only of physiological importance as mammalian collagen contributes to 25-35% of the whole-body protein content,11 it is important 2
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also for developing futuristic device components for in vivo and in vitro applications. Since collagen has a considerably long half-life of 117 years (cartilage collagen) or 15 years (skin collagen),12 the shelf life of collagen-based components is expected to be at least few months to few years. The capacity of electron transport in case of collagen had been demonstrated by scanning tunneling microscopy (STM) on hydrated fibrous assemblies of collagen type I, where an important role of adsorbed water in localized electronic conduction was perceived.13 The electrical conductivity of hydrated bovine achilles tendon (BAT) collagen was measured as a function of hydration over a temperature range (23-51 °C) where conductivity increased with hydration.14 Additional evidences for electronic accessibility to the collagen surface could be obtained from STM imaging of collagen I filaments and fibrils.15 However, in none of these studies, electron transport capacity of collagen could be quantified, and mostly low-resolution images were presented. The aim of the present study is to assess collagen as a bioelectronic material, with or without conjugation to a metal, in a quantitative manner, and see if metal treatment can substantially enhance its capacity for electron transport. As collagen has several functional groups on its surface like -OH, -COOH, -CONH2 and -NH2, it can bind with metal ions such as Fe(III).16 In this study, it will be shown using current-sensing atomic force spectroscopy
(CSAFS)
that
under
various
experimental
conditions,
including
hydration/dehydration, type of buffer, folded/denatured state, and the applied compressive force load, electron transport via collagen was minimally affected, whereas use of metal, here iron, enhanced the electron transport capacity of collagen to a noticeable level. Because collagen's applicability in molecular bioelectronics has remained largely untested so far, our study could provide meaningful information for its use as a bioelectronic material. 3
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Experimental Section Materials All the buffer solutions were prepared using filtered autoclaved Milli-Q water (resistivity 18.2 MΩ·cm, Millipore). Disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), sodium chloride (NaCl), sodium bicarbonate (NaHCO3), dipotassium hydrogen phosphate (K2HPO4), magnesium chloride hexahydrate (MgCl2.6H2O), potassium chloride (KCl), sodium sulphate (Na2SO4), tris(hydroxymethyl)aminomethane [(CH2OH)3CNH2] and calcium chloride (CaCl2) were purchased from Merck (purity ≥ 99%). Bovine achilles tendon (BAT) collagen was procured from Sigma-Aldrich. Preparation of collagen solution About 10 mg of BAT collagen was added in 2 mL 0.01 M H2SO4 (pH 1.7) solution (cooled at 4 °C for few min) and stored overnight at 4 °C with occasional mild shaking. The resulting dispersion was homogenized for 10 min at 0 °C to produce the individual collagen fibrils. After homogenization, a part of this solution (stock solution) was mixed with PBS, which is Naphosphate buffer (13 mM Na2HPO4, 2.5 mM NaH2PO4, 140 mM NaCl, pH 7.4), or simulated body fluid (SBF) buffer (pH 7.4) to prepare collagen solution of 0.1 mg/mL concentration. The metal-treated collagen sample was prepared by taking ~10 mg of BAT-collagen in 3.9 mL of 0.01 M H2SO4 (pH 1.8), let it swell and be stored overnight at 4 °C with occasional mild shaking. The dispersion was homogenized for 10 min at 0 °C temperature. Then 100 L of 0.01 M formic acid was added into the homogenized solution, keeping pH within 1.7-2.0, soaked for 2 h at room temperature. Then 0.071 mmol Fe2(SO4)3.xH2O (Loba chemie) was added and reacted at 30 °C with constant stirring for 6 h. Proper amount of NaHCO3 solution (15%, w/w) was gradually added within 2 h in order to increase pH of the solution to 4.0-4.2 and the reaction 4
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continued at 45 °C for another 4 h.16 The supernatant solution was filtered using 100 kDa cutoff amicon filter and washed with milli-Q water. Then the reconstituted Fe-collagen was stored at 4 °C temperature. Characterization of collagen and Fe-collagen solutions Collagen and Fe-collagen solutions were characterized by UV-visible spectrophotometry using a Varian Cary 50 Bio UV-visible spectrophotometer, and a 1 cm cuvette at 25 °C temperature. Both the solutions were also characterized by circular dichroism (CD) spectroscopy. CD spectra were collected from 190 to 400 nm wavelength with 100 nm/min scanning speed, 10 mm path length of a quartz cell cuvette using JASCO J-815 CD spectrometer. The presence of iron in Fecollagen was confirmed by Energy Dispersive X-ray spectroscopy (EDX spectra) where carboncoated copper grid was used in JEOL JEM-2010 TEM at the operating voltage of 200 keV. The amount of iron present in Fe-collagen was determined by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES using Perkin-Elmer Optima 2100 DV machine). Incorporation of iron in Fe-collagen was tested by FTIR spectroscopy using Perkin Elmer Spectrum 100 FT-IR Spectrometer at room temperature from 400-4000 cm-1 scan range at a resolution of 4.0 cm-1 with 4 scans. The FTIR spectra of protein solutions were acquired by adsorbing the protein solutions on the potassium bromide (KBr) pellet followed by subtracting the baseline spectrum. Preparation of collagen film on silicon substrate A heavily doped n-type (As) silicon(111) wafer (0.0025 - 0.004 Ω.cm 525 μm SSP Prime wafer from University Wafer, USA) was cleaned by bath sonication in ethyl acetate/ acetone/ ethanol (5 min in each), followed by 10 min of acid piranha treatment (7:3 v/v of H2SO4:H2O2) at 80 °C temperature. Then the wafer was thoroughly rinsed with milli-Q water and dipped in 2% HF solution for 2 min, followed by rinsing with milli-Q water and then dipped in a base piranha 5
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solution (NH4OH: H2O2: H2O = 1:1:5) for 1 min at 70 °C temperature. The wafer was rinsed with milli-Q water and dried under nitrogen gas. The cleaned silicon wafer was incubated into 100 g/mL of collagen solution in PBS (phosphate buffered saline)/SBF (simulated body fluid) buffer (pH 7.4) or Fe-collagen in SBF (pH 7.4) for 12 h. The silicon surface was then thoroughly rinsed using autoclaved milli-Q water to remove loosely attached collagen fibrils and salt crystals that are precipitated from the buffer solution, and dried under nitrogen gas. The preparative stages are schematically shown in Figure 1A. Characterization of collagen film Formation of collagen and Fe-collagen films onto silicon surface was characterized by AFM imaging at ambient condition. The film thickness was obtained by scratching an area of the film using contact mode AFM under tip force 190 ± 10 nN. The scratching procedure was repeated at 8-10 places to check if the film is largely homogeneous and of uniform thickness at most of the regions. In order to prepare the denatured collagen film on silicon substrate, the collagen-coated silicon piece was incubated in autoclaved milli-q water for 10 min in dry bath at 70 °C temperature, and then dried under nitrogen gas.17 The presence of iron in Fe-collagen in film condition was detected by X-ray photoelectron spectroscopy method (XPS, Omicron, model no: 1712-62-11, serial no: 0571) using Al-K (1486.7 eV) radiation. After monochromation, the radiation was focused on the sample, at an electron take off angle (TOA: angle between the analyzer and the sample surface) of 453° relative to the substrate surface. CSAFS data acquisition and analysis Current-sensing atomic force spectroscopy (CSAFS) experiments were performed in contact mode using a PicoLE AFM (5100 model, Agilent Technologies, USA) with a 10 μm scanner. Prior to starting I-V measurements, the surface coverage of each collagen sample was checked 6
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by intermittent contact AFM imaging. The AFM probes (MikroMasch, Estonia) with tip radius < 10 nm and resonant frequencies within 140-250 kHz were used for imaging. In CSAFS experiments, the AFM probes (MikroMasch, Estonia) with Ti/Pt-coated cantilevers having spring constant 2 N/m, tip radius ~35 nm, length 110 μm, and width 40 μm were used and electrical characteristics were recorded independent of the force feedback. All the CSAFS measurements were performed under ambient condition, where the temperature and the humidity levels were maintained at 25 ± 1 °C and ~45-55%, respectively. The current-voltage (I-V) response curves were acquired between ±5 V sweep range at 1 V/sec sweep rate for collagen and its denatured form; and ±1 V sweep range at 0.2 V/s sweep rate for the Fe-collagen. The I-V traces were acquired at different force loads, where the force values were calculated using the following equation: {(Force set point - cantilever deflection) cantilever spring constant}/(slope of the forcedistance curve) So, prior to obtaining the I-V curves at a specific force value, the difference between the force set point to the cantilever deflection was set accordingly. The I-V experiments were repeated for a large number of different areas, and also for different samples prepared on different days. The differential conductance plots were obtained from current-voltage response curves on collagen film using low force (5-12 nN) CSAFS data. These plots provide the position of conduction (CB) and valence band (VB) with respect to Fermi level which is considered to be aligned at 0 V. As the voltage was applied to the sample with respect to the AFM tip, the peaks in the negative voltage region of the differential conductance plot implied electrons being withdrawn from the sample (collagen) by the conductive tip and hence denoted as VB of the collagen. Similarly, the positive voltage peaks indicate the electron injected to collagen from the conductive tip and 7
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therefore provided the location of CB. From the differential conductance plot obtained from the averaged current-voltage curve, the energy difference between CB and VB was estimated as the transport band gap.
Statistical analysis Statistical analyses were performed using Prism 6 (GraphPad Software, Inc., La Jolla, CA) to find the significant difference in the band gap values for (i) collagen and denatured collagen in PBS buffer, (ii) collagen and Fe-collagen in SBF buffer, where the n value for each statistical analysis corresponds to the number of rupture events in the histogram. A p value
