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Structural Analysis and Protein Functionalization of Electroconductive Polypyrrole Films Modified by Plasma Immersion Ion Implantation Alexey Kondyurin, Kostadinos Tsoutas, Quentin-Xavier Latour, Michael J Higgins, Simon E. Moulton, David R. McKenzie, and Marcela M.M. Bilek ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00369 • Publication Date (Web): 15 Aug 2017 Downloaded from http://pubs.acs.org on August 18, 2017

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ACS Biomaterials Science & Engineering

Structural Analysis and Protein Functionalization of Electroconductive Polypyrrole Films Modified by Plasma Immersion Ion Implantation

Alexey Kondyurin1*#, Kostadinos Tsoutas1#, Quentin-Xavier Latour1, Michael J. Higgins2, Simon E. Moulton2, David R. McKenzie1, Marcela M.M. Bilek1*

1

Applied and Plasma Physics, School of Physics, A28 Physics Road, University of Sydney, Sydney, NSW 2006, Australia 2

ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer

Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia #

equal first authors

* Corresponding Authors. Tel: +61 2 9351 2484, Fax: +61 2 9351 7725, Emails:[email protected], [email protected]

1 ACS Paragon Plus Environment


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Abstract Conducting polymers are good candidates for electronic biomedical devices such as biosensors, artificial nerves and electrodes for brain tissue. Functionalizing the conducting polymer surface with bioactive molecules can limit adverse immune reactions to the foreign body and direct tissue integration. In this work, we demonstrate a simple one-step method to attach biomolecules covalently to a conductive polymer. Electrochemically synthesized polypyrrole was activated using plasma immersion ion implantation (PIII) in nitrogen. A short treatment with relatively low ion fluence (20 seconds) was found to enable direct covalent immobilization of protein upon incubation in a protein solution, while the protein is easily removed from untreated polypyrrole by washing in buffer. The covalent nature of the protein immobilization was demonstrated by its resistance to elution when repeatedly washed with SDS detergent. Changes in the surface properties and their evolution with time after PIII activation were studied by a combination of attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), Raman spectroscopy, x-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), cyclic voltammetry and water contact angle measurements. Notable changes in the chemistry of the modified layer in polypyrrole include the appearance of nitrile groups that gradually disappear with time and oxidation of the surface that increases over time in air. The kinetics of surface energy are consistent with the generation of radicals in the modified layer that are lost predominantly through oxidation. The conductivity of the modified surface layer (64 nm in thickness) decreases for low fluence treatments and is partially restored after high fluence treatment. This simple surface modification process opens up the possibility of creating biologically active


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interfaces for electro-stimulating biomedical devices and electrical sensing of neurological processes.

Keywords: polypyrrole; plasma immersion ion implantation; covalent protein immobilization; surface functionalization; conductive polymer; electrodes for neuroscience

1 Introduction

Unraveling the secrets of brain and mind function is an important endeavor in science and medicine which is expected to accelerate greatly through the application of powerful new approaches over the coming decades 1-4. Neuroscience studies often utilize external sensors to monitor electrical activity in the brain. Typically these external electrodes receive signals from only a few neuron circuits over a short time interval. More holistic investigations of the future will require new generation electrodes that make good and permanent electrical contact directly to neurons. There have been a number of attempts to develop electrodes for brain tissue that can provide enduring signals from multiple neurons. However, the blood-brain barrier and foreign body response of the brain tissues to the implanted device cause dramatic changes in the neural tissue microenvironment that modify the electroactivity and signal propagation of the neural cells 5. New electrode materials which do not cause adverse



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reactions in brain tissue are required to advance the field. Conducting polymers such as polypyrrole are promising materials for enabling these developments. 6-9

Prospective electronic biomedical devices that could be enabled by the unique properties of conductive polymers include artificial nerves, biosensors, integrated bionics, and drug delivery vessels 10-15. Conductive polymers are ideal for these applications due to their high chemical stability, inherent electrical properties and compatibility with a range of cell lines 16-21. A problem that must be overcome before these materials are considered for long term neural use, however, is the associated chronic inflammatory immune response, or foreign body response, leading to fibrotic encapsulation 22. Polymer surface functionalization using bioactive molecules is a promising approach for limiting this immune response by delivering specific biological signals to local tissue to promote integration with a particular cell phenotype 23. Functionalization can be achieved through a number of methods, including physical adsorption, incorporation of molecular species into the polymer matrix, and covalent conjugation through wet chemical approaches 24. Physical adsorption has the disadvantage that the protein may be easily removed and replaced by other proteins adsorbing from the physiological environment of the host, while incorporation into the polymer tends to bury the active fragments of the biomolecule, inhibiting its activity and interaction with cells. Covalent conjugation is therefore the preferred method of biofunctionalization 25.

Plasma immersion ion implantation (PIII) provides an efficient method for achieving covalent protein attachment onto polymer surfaces by a simple one-step incubation in protein solution 26-27. Ion implantation in this approach is achieved by accelerating


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ions from plasma through the high-voltage sheath formed around an electrode that is negatively biased with respect to the plasma. The ions are accelerated to energies up to a maximum given by the product of the ion charge and the bias voltage applied. When these energetic ions impact the polymer they implant below the surface. As they move deeper into the polymer, they produce collision cascades which result in the breaking of chemical bonds and ionization and excitation of macromolecules 28. The bias voltages typically used in PIII limit the effects of the collision cascades to a surface layer of no more than a few hundred nanometers, leaving the bulk polymer unmodified 29-31. The surface-treated region is rich in radicals containing reactive unpaired electrons in dangling bonds 32. It is these embedded radicals that allow for covalent coupling of protein molecules 26, 33-34. This method has been demonstrated to provide robust bioactive attachment of enzymes such as horseradish peroxidase, soybean peroxidase and soybean catalase to polyethylene, polystyrene, polyamide and polytetrafluorethylene (PTFE or Teflon) 35-38. Biomolecules have been shown to retain a native functional conformation while being tethered to the surface by covalent bonds, thereby producing a bioactive surface 26, 33, 39-42 that can prevent the foreign body response whilst promoting optimal integration with the body. Previous work has shown that polypyrrole can be patterned using PIII with shadow masking to control cellular interaction through protein binding43.

In this study, we assess the use of ion implantation to modify polypyrrole in order to allow direct covalent functionalization with active biomolecules. The ion-implanted polymer surface layer is analyzed for chemical, electrical and structural changes. The extracellular matrix protein tropoelastin is used to assess the potential for direct covalent immobilization of biomolecules to the ion-implanted surface. The emphasis



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here is the investigation of changes in the structure and properties of polypyrrole to give a better understanding of the mechanism of protein binding.

2 Experimental Section

The pyrrole monomer was obtained from Merck and distilled prior to use. Paratoluenesulfonic acid (pTS) from Merck was used as the dopant. The solution was prepared with deionized Milli-Q water (18.2 MU). Gold-coated Mylar was cut into 1.5 cm x 2 cm strips before being cleaned with methanol and Milli-Q water. An aqueous monomer solution of 0.2 M pyrrole and 2 mg/mL of the pTS dopant was degassed in N2 for 10 min prior to pyrrole electropolymerization. Polypyrrole (PPy) films were grown galvanostatically at a current density of 0.25 mA/cm2 for 10 min in the aqueous monomer solution using an eDAQ EA161 potentiostat (eDAQ, NSW, Australia). Polymer growth was performed in a standard 3-electrode electrochemical cell with the gold-coated Mylar as the working electrode, a platinum mesh counterelectrode and Ag/AgCl reference electrode. After growth, the films were washed with Milli-Q water, gently dried with N2 gas and placed in petri dishes. The total number of samples used was 35.

Plasma immersion ion implantation was conducted in an inductively coupled radiofrequency (13.56 MHz) nitrogen plasma (99.99% nitrogen gas purity). The base pressure in the plasma vessel was 1 x 10-6 Torr (~10-4 Pa) and the nitrogen pressure during ion implantation was 2 x 10-3 Torr (0.3 Pa). The rf plasma power was 100 W and the reverse power was 12 W when matched. The plasma density during treatment (1.2 x 1015 ions/m3) was determined using a Langmuir probe with an rf block (Hiden


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Analytical Ltd). Acceleration of ions from the plasma was achieved by the application of high voltage (20 kV) negative bias pulses of 20 µs duration to the sample holder at a frequency of 50 Hz. The samples were treated for durations of 20 - 1600 seconds, corresponding to implanted ion fluences of 0.5 - 20 x 1015 ions/cm2.

After PIII treatment, the samples were incubated for 24 hours in tropoelastin solution (50 µg/ml in 10 mM sodium phosphate buffer, pH 7) at 23 ºC. After incubation, the samples were washed six times (20 minutes each wash) in buffer solution (10 mM sodium phosphate buffer, pH 7). Samples for FTIR and XPS spectral analysis were further washed in deionized water for 10 seconds to remove buffer salts from the surface before being dried. Washing to assess covalent immobilization was done in 2% SDS at 70 ºC for 1 hour.

The wettability of the polymer samples was measured using the sessile drop method with a Kruss DS10 contact angle instrument. Deionized water and diiodomethane were the test fluids. Measurements have been carried out on dried samples. The drop volume was 0.5 µl. Five drops of each liquid for every experimental point were tested. Only drops with symmetrical shape were selected for measurement. The central part of the sample was used for measurement. The drops were always placed at a new position to exclude contamination from previous drops. The samples used for the contact angle measurements had not been in contact with other materials and had not been used for other methods of measurement. The samples have been stored in closed Petri dishes and fixed on the bottom of the dish with double-sided adhesive tape to prevent any contact with the measured top surface of the sample. Total surface energy



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and its polar and dispersive components were calculated using the Owens, Wendt, Rabel and Kaelble model and a regression method 44.

AFM imaging was performed using a JPK Biowizard II (JPK Instruments, Germany). AFM images were obtained in air using a 40 N/m silicon nitride cantilever in intermittent-contact mode with a scan rate of 1 Hz. Root-mean-square (rms) roughness values were calculated from the AFM height images using the JPK AFM analysis software.

ATR-FTIR spectra were recorded using a Digilab FTS7000 FTIR spectrometer fitted with an ATR accessory (Harrick, USA) and a horizontal multiple reflection germanium crystal with a 45º incidence angle. The samples were clamped onto the ATR crystal with a standard holder. Generation of spectra with sufficiently high spectral resolution and signal-to-noise ratio required 500 scans at a resolution of 1 cm-1. Difference spectra, obtained by subtraction of spectra of samples before treatment from those of samples after treatment, were used to characterize the effect of the treatments. Base line correction with a polynomial function has been applied to all spectra. All spectral analyses were undertaken using GRAMS software.

The micro-Raman spectra were recorded on a Horiba Raman spectrometer with excitation at 632 nm. Spectra were taken over a 20 minute period using a 1 minute collection time repeated 20 times to reduce any fluorescence effects while maintaining an appropriate signal-to-noise ratio. Raman spectra were recorded with low energy flux of the laser beam to minimize structural changes due to heating. For the spectra presented here the laser power was not more than 0.8 mW as regulated by


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the objective and diaphragm of the spectrometer. During the measurements, the spectra were recorded from different locations on the surface. The surfaces were checked after laser irradiation and no signs of burning or heat induced changes were observed. Measurements were also repeated at the same location to confirm that the spectra did not change with the number of measurements. Therefore, we conclude that the observed Raman spectra are related to the structure of the polypyrrole and not to effects of laser beam heating. The micro-Raman penetration depth was estimated to be at least 10 µm.

The resonance micro-Raman spectra were collected over a range of acquisition times. Excitation was with 0.1 mW laser irradiation at 514 nm using a 50X lens creating a spot of diameter 1 µm at the sample. Prior to acquisition, the area was exposed to the laser to reduce the fluorescence yield. However, significant fluorescence was still observed, especially for the PIII-treated samples.

X-ray Photoelectron Spectra were collected using a spectrometer (SPECS-XPS from SPECS, Germany) equipped with a PHOIBOS 150-9 MCD energy analyzer, capable of detecting medium sensitivity elements to a trace level of 100 ppm, and a Al Kα X-ray source (1486.74 eV). Survey spectra were taken prior to five repeats of high-resolution spectra taken for the C1s, N1s, O1s and S2p peaks. The XPS spectra of PIII treated samples were recorded 3 weeks after PIII treatment. Elemental compositions for untreated and treated polypyrrole were determined. The atomic concentrations were calculated using the commercial code CasaXPS (Version 2.3).



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Cyclic voltammetry (CV) measurements were carried out under ambient conditions using an eDAQ EA161 potentiostat and e-corder (eDAQ, NSW, Australia) with EChem software for controlling the voltage and current signals. CV measurements were performed in phosphate buffer saline (PBS) solution with a scan rate of 100 mV/s for 10 cycles. Measurements were taken in a cell consisting of a polypyrrole working electrode, a platinum mesh auxiliary electrode and an Ag/AgCl (3.0 M NaCl) reference electrode operating cyclically between -0.5 and 0.5 V. The measurements were performed on untreated films and then the same films after PIII treatment, for treatment times of 80 s, 400 s, and 1600 s.

3 Results

3.1 Color The as-electrodeposited polypyrrole films are black with smooth surfaces. Short treatment times (up to 400 s) result in minimal color change, while those longer than 800 s result in a greying which can be attributed to carbonization occurring at the surface of the film.

3.2 Wettability and Surface Energy The water contact angle of untreated polypyrrole was found to be between 66 and 87 degrees. PIII treatment significantly decreases the water contact angle (figure1). 22 minutes after PIII treatment, the contact angle is in the range 23-25 degrees. With storage in air, the contact angle increases and returns to 70-85 degrees within 2 weeks. The similarity of water contact angle measurements for all treated samples in


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the range 80-1600 s at each measurement time point indicates that the wettability of the treated polypyrrole does not depend on PIII treatment time (fluence) in the range 80-1600 s (1015-2x1016 ions/cm2). Similar kinetics were observed for the diiodomethane contact angle (not shown), which was 48 degrees for untreated polypyrrole, decreasing to 22 degrees 22 minutes after PIII treatment and then rising to 53 degrees after two weeks’ storage.

Using the contact angles of water and diiodomethane, the polar and dispersive components of the surface energy were calculated (figure 2). The total surface energy of untreated polypyrrole is 40 mN/m, consisting of a polar component of 5 mN/m and dispersive component of 35 mN/m. At the first measurement after PIII treatment, the total surface energy was recorded as 70 mN/m with a 24 mN/m polar component and 46 mN/m dispersive component. After 2 weeks of storage in air, the total surface energy decreases and stabilizes at 35 mN/m with the polar and dispersive components decreasing to 3 mN/m and 32 mN/m respectively. The evolution in surface energy observed for polypyrrole shows that PIII treatment causes a shift towards a hydrophilic structure that subsequently undergoes a hydrophobic recovery.

Surface energy kinetics can be fitted using exponential functions assuming first-order reactions of free radicals 34, 45-46. The experimentally-determined kinetics of the dispersive surface energy were well-fitted with an exponential decay function (1) as follows (figure 2): −

σ D = AD ⋅ e

t tD 0

+ CD

(1)



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where σD is the dispersive component of the surface energy; CD=32 mN/m is the dispersive surface energy after infinite storage time since PIII treatment. The sum of AD=14 mN/m and CD is the dispersive surface energy immediately after PIII treatment as estimated by the fitting procedure, and tD0=4000 min is the characteristic time for the dispersive surface energy decay.

The experimentally determined kinetics of the polar component of the surface energy were well fitted with the sum of two exponential decay functions (figure 2): −

σ P = AP ⋅ e

t tP10

−

+ BP ⋅ e

t tP 20

+ CP

(2)

where σP is the polar component of the surface energy and CP=3 mN/m is the polar surface energy after infinite storage time since PIII treatment, the sum of AP=30 mN/m, BP=17 mN/m and CP is the polar surface energy immediately after PIII treatment as estimated by the fitting procedure, and tP10=15 min and tP20=10,000 min are characteristic times associated with polar surface energy decay.

The total surface energy of polypyrrole 22 minutes after PIII treatment was measured to be 70 mN/m. Such a high surface energy cannot be attributed to oxygen-containing functional groups alone. Total surface energies for organic polymers are usually in the range of 10-35 mN/m. Polymers with a high concentration of polar oxygen-containing groups show the highest values of the surface energy: poly(methyl methacrylate) with C=O groups has 49 mN/m, polycarbonate with aromatic rings and carbonyl groups has 46.7 mN/m, while poly(ether ether ketone) PEEK has 46 mN/m 47. High surface energies are observed for materials with extended state conduction electrons (such as the free electron gas found in metals). Metals have surface energies between 100 and


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1000 mN/m 48. Graphite sheets with unpaired electrons at the edges have surface energies in the range of 1750-3338 mN/m 49. In the case of PIII-treated polypyrrole, the increase in surface energy can be explained by the presence of unpaired electrons in radicals generated when high energy ions, implanted beneath the surface, break chemical bonds along their paths. With storage time, the surface energy decays, corresponding to a decrease in the radical concentration in the treated surface layer50-51. The radical decay process involves the radicals reacting with other radicals, environmental species and other components of the polymer matrix.

The main contribution to the increase in the total surface energy caused by PIII treatment is the increase in the polar component. At our earliest time point of 22 minutes after the PIII treatment, the polar energy component is 24 mN/m. This decreases with storage time to 3 mN/m. The polar part of the surface energy characterizes polar interactions that are due to the unpaired electrons associated with radicals and polar chemical groups at the surface. The variation of the dispersive component is not as large: it decreases from 46 mN/m shortly after PIII to 32 mN/m after long term storage. The dispersive part is associated with the appearance of stable chemical groups on the surface. The radical kinetics and the formation of new chemical groups is inferred by the characteristic times for the kinetics of the polar and dispersive components of total energy (as seen in figure 2): 15 and 10,000 min for the polar component and 4000 min for the dispersive component of surface energy.

The total surface energy of PIII-treated polypyrrole at very long storage time is 35 mN/m, lower than the surface energy of untreated polypyrrole (40 mN/m). The dispersive (32 mN/m) and polar (3 mN/m) components of the surface energy at a very



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long storage time are both lower than the untreated polypyrrole values (35 mN/m and 5 mN/m accordingly). This reduction in surface energy corresponds to the production of a highly carbonized structure formed by radical reactions within the treated layer. Such a shift towards a disordered carbonized structure is also observed for other ion beam implanted polymers after long storage times 34, 45.

3.3 Surface Morphology: AFM imaging Figure 3 shows AFM images of untreated and PIII-treated (for periods of 800 s) polypyrrole films. AFM height images show no significant difference in the morphology and surface roughness between the untreated and treated polymer films for all PIII treatment times examined. All of the films display the typical nodular morphology of PPy 52 and have an average surface roughness (Ra) value in the range 4-6 nm. In contrast, AFM phase images reveal a difference in the phase signal correlated with the film morphology. The phase image shows darker and lighter areas corresponding to the nodule structures and peripheries of the nodules, respectively. This phase difference is greatest for untreated films and decreases with PIII treatment time. The phase image post PIII treatment shows significantly less granulation, indicating a homogenizing of the chemical structure consistent with the surface becoming carbonized and more chemically uniform with increased PIII treatment.

3.4 Surface Chemistry: Raman, FTIR and XPS spectra The ATR-FTIR and Raman spectra of untreated polypyrrole we measured are in agreement with the literature 53-54. The interpretation of the vibrational modes is presented in Table 1. Changes due to PIII treatment were detected by subtracting the


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untreated spectra from the PIII-treated spectra (figure 4). In the ATR-FTIR spectra, the low intensity line at 2210 cm-1 appearing after PIII is attributed to stretching vibrations of C≡N groups. New lines in the regions 1647-1620 cm-1 and 1708-1711 cm-1 correspond to vibrations in C=C and C=O groups respectively. These lines are attributed to carbonization and oxidation processes in polypyrrole occurring with ion bombardment and subsequent exposure to oxygen-containing atmosphere respectively. After the longest treatment time (1600 s), new lines at 1467-1440 cm-1 and 1418 cm-1 corresponding to the δ(CH2) vibrational group appear. These groups are attributed to the transformation of pyrrole ring structures into saturated hydrocarbon groups. New lines in the 1277-1215 cm-1 region attributed to ν(C-O) vibrations are observed and interpreted as the result of oxidation during aging in atmosphere.

The Raman spectra of PIII-treated polypyrrole were also analyzed as subtraction spectra (figure 5). The differential spectra show new lines at 1620 and 1501 cm-1 attributed to aromatic ring vibrations, at 1420 cm-1 attributed to δ(CH2) vibrations, and at 1300-1337 and 1261 cm-1 attributed to ν(C-C) vibrations in newly saturated structures. The new lines at 989 and 942 cm-1 are attributed to aromatic ring vibrations.

The irradiation of polypyrrole with a green laser generates fluorescence (figure 6). The fluorescence spectrum of untreated polypyrrole is weak and has three broad peaks at wavelengths of 610, 660 and 720 nm. After PIII treatment with a fluence of 1016 ions/cm2, the positions of the fluorescence peaks do not change, but the peak at 660 nm becomes stronger and broader. This shows that the electron energy levels 15 ACS Paragon Plus Environment


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excited by the laser beam in untreated and PIII-treated polypyrrole are similar, with broadening of the treated samples’ peaks due to the creation of a disordered carbonized surface.

The resonant Raman spectra and characteristic D- and G-peaks show the presence of graphitic structures in untreated and PIII-treated polypyrrole (figure 7). The resonance Raman spectrum for untreated polypyrrole excited with the green laser shows two strong peaks at 1583 and 1379 cm-1. The 1583 cm-1 peak corresponds to the E2g vibrational mode, and the 1379 cm-1 peak corresponds to the Ag vibrational mode of a graphitic ring. Following the model of Ferrari for Raman spectra of carbon structures, we can conclude that the samples contain nanoclusters of graphite 55. The position of the G-peak at 1583 cm-1 and the ID/IG ratio of 0.61 suggest that the structure contains nanocrystalline graphitic clusters of about 1.9 nm in size. The Raman spectrum changes only subtly with PIII treatment. Fitting with Gaussian functions indicates that the G-peak has shifted to 1574 cm-1 and the D-peak to 1390 cm-1 after a treatment with a fluence of 1016 ions/cm2. According to the position of the G-peak and the ID/IG ratio, the carbonized structure contains a mixture of nanocrystalline graphitic and glassy carbon clusters of about 1.7 nm in size. The intensity of these spectra was high, indicating resonance effects during excitation. This observation is supported by the presence of a fluorescence spectrum that indicates excitation of electrons in untreated and PIII-treated polypyrrole.

Table.1. Assignments used in the interpretation of vibrational spectra (infrared and Raman) of polypyrrole before and after PIII treatment. Numerical values are wavenumbers in cm-1.


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FTIR Untreated PIII-treated 2210 2210 1708-1711 1684 1647-1620 1551 1532 1475

Raman Untreated PIII-treated

1620 1581 1495 1501

1452 1467-1440 1418

1420 1380 1300-1337

1302 1277

1261 1246

1215 1167 1108-1121 1034

1052/1078 1055 989, 942 930/965 693 680 615

Vibrations assigned C≡N C=O C-C inter-ring, asym C=C C-N,CNH C-N,CNH C-N, C=C sym C=C C-N, C=C asym CH2, CH3 CH2 C-C C-C, C=C C-C C-C CCH, CNH C-O Ring breathing C-O CCH, CNH C-O CCH Ring deformation, sym Ring deformation, sym Ring deformation, sym

The extent of structural transformation of the polypyrrole depends on the fluence of implanted ions. An observation of the chemical structure transformation was done by an analysis of the nitrile groups. The nitrile group, observed by the absorbance of the ν(C≡N)=2210 cm-1 line in the ATR-FTIR spectra, appears with PIII treatment and increases with increasing PIII treatment time (figure 8). The presence of nitrile group absorbance is also observed in some untreated samples. The figure 4 shows embedded spectra of 4 untreated samples for illustration of this effect. The concentration of nitrile groups is stable with storage time after PIII treatment and the absorbance of the nitrile group vibrations can be fitted with the following function of ion fluence:



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Abs(C ≡ N ) = A ⋅ (1− e−kf ) + B

(3)

where B is the absorbance of nitrile group vibrations before PIII and (A+B) is the absorbance of nitrile group vibrations after a long treatment time. The experimental points taken from the ATR-FTIR spectra for the C≡N group absorbance are wellfitted with this function when (1/k) = 2x1015 ions/cm2, which is the fluence obtained after a PIII treatment of 150 seconds.

The oxidation of polypyrrole occurs with exposure to air after PIII treatment (figure 9). The kinetics of oxidation with time after PIII were observed by tracking the changes in the ν(C=O) line absorbance at 1708 cm-1 in the ATR-FTIR spectra. The intensity of this line is low in the spectra of polypyrrole immediately after treatment for all PIII fluences, and it increases to a saturation level after two weeks of storage in air. The saturation level of the oxidation depends on the PIII treatment time. The strongest oxidation is observed for 400 and 800 second PIII treatment times (5x1015 and 1x1016 ions/cm2 fluences, respectively). The oxidation processes in PIII polymers usually follow first-order reactions27, when a concentration of oxygen-containing groups is described by exponential functions. We therefore fitted the experimental results of oxygen-containing groups in PIII-treated polypyrrole with exponential functions.

The experimental data of the carbonyl group concentration can be fitted with two exponents (figure 9): −

t k1

[C = O] = A1 ⋅ (1− e ) + A2 ⋅ (1− e

−

t k2

) + A0

(4) 18

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where A0 is the initial concentration of carbonyl groups, A0+A1+A2 is the final concentration of carbonyl groups after infinite storage time, and k1 and k2 are reaction constants. This model corresponds to a sum of two kinds of reactions: quick (A1, k1) and slow (A2, k2) reactions. The products of the slow reaction result in a higher contribution to the carbonyl group concentration than the products of the quick reaction (figure 10). The contribution of the quick reaction (k1) reaches a maximum at an ion fluence of 5x1015 ions/cm2 (400 s), while the contribution of the slow reaction (k2) reaches a maximum at an ion fluence of 1016 ions/cm2 (800 s). The quick reaction constant is stable up to a fluence of 1016 ions/cm2 and then decreases, while the slow reaction constant grows with fluence and saturates after 1016 ions/cm2 (figure 10). The range of the slow reaction constant (5000-15,000 min) is similar to the higher characteristic time constant of the polar part of the surface energy decay (10,000 min). The range of the quick reaction constant (from 20 to 200 min) is similar to the lower characteristic time constant of the polar part of surface energy decay (15 min). This suggests a correlation between the decay of the polar part of the surface energy and oxidation of the surface after exposure to air, suggesting that these effects are due to the same processes.

XPS spectra taken after PIII treatment show small changes in elemental concentrations with PIII ion fluence (figure 11). All observed elements (carbon, nitrogen, oxygen and sulfur) are presented for untreated and PIII-treated polypyrrole. The concentration of nitrogen increases from 11.5% for untreated polypyrrole to 13.5% for PIII-treated polypyrrole after a fluence of 2x1016 ions/cm2. The oxygen concentration increases from 12.8 to 14% after a short PIII treatment (40 s or 5x1014



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ions/cm2 fluence) and decreases with further PIII treatment time to 11.5%, whereas the sulfur content increases from 1.5 to 2%.

3.5 Conductivity Cyclic voltammetry curves (Figure 12) were measured before and after PIII treatments carried out with 20 kV bias to accelerate the implanting ions for various treatment times. A decrease in the size of the hysteresis loop after treatment is observed. After 40s of treatment, the shape of the hysteresis curve is maintained but the magnitude decreases significantly. At longer treatments, the symmetry of the loops disappears.

The changes in the observed hysteresis loops give insight into the changes in the redox properties and the conductive mechanism for polypyrrole with PIII treatment. Untreated polypyrrole exhibits its polaron transition associated with the doping process, observed in the range of 0-0.1V in the anodic cycle. The 80s treated sample still shows evidence of this transition, indicating that some of the original redox behaviour of the untreated polypyrrole is still present. At longer treatment times, these peaks have diminished and a reversible hysteresis curve associated with a symmetry between charging and discharging cycles is not observed. This result aligns with the observed spectral analysis indicating that there is an increase in the degree of surface carbonization within the polypyrrole structure after treatment. The increase in carbonization correlates with a reduction in conductivity caused by a change in molecular structure that is observed in the CV data in the form of a decrease in current amplitude from the untreated to treated samples. The underlying mechanism of charge transfer for the virgin samples is reliant on the conjugated bonding structure


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coupled with the dopant molecules present in polypyrrole giving rise to polarons and bipolarons along the polymer chain when the polymer is oxidized56. Plasma treatment of the samples causes atomic rearrangement of the polymer, altering its electrical properties by removing the electroactive conjugated structure and increasing the amount of crosslinking between the chains as the polymer becomes increasingly graphitic. Shorter treatment times retain significantly more of the original structure, resulting in a CV curve that is similar to the untreated in shape, but charge transfer is significantly reduced. The depth of graphitization could be reduced by using lower energy ions (Figure 13). Shallower treatments would further reduce the impact of the treatment on the CV behavior whilst preserving covalent protein attachment.

3.6 Protein attachment on treated and untreated polypyrrole Following the production of radical species during PIII treatment, polypyrrole is able to attach protein covalently to its surface. Tropoelastin is observed on PIII treated samples by ATR-FTIR analysis (Figure 14a) as seen by the presence of amide I and II peaks 57. The same samples soaked in buffer solution under the same conditions (PIII treatment time, temperature, concentration, storage time after PIII) without protein have been used for subtracting spectra to prevent the intrusion of the spectral bands of polypyrrole into the spectra of the protein. Untreated polypyrrole does not covalently bind the tropoelastin, as the protein is removed easily with gentle washing in buffer solution. Directly after PIII treatment, polypyrrole becomes significantly more hydrophilic, yet tropoelastin is strongly attached to the surface and resistant to washing in fresh buffer. Even after washing in SDS detergent, the amount of tropoelastin on the treated films shows only a slight decrease as non covalently bound



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molecules are removed (Figure 14b). We therefore conclude that covalent attachment of tropoelastin on polypyrrole can be achieved through PIII activation of the surface layer.

4 Discussion

The electropolymerization of pyrrole onto gold electrodes creates a crosslinked, nodular polypyrrole film with pTS dopant. In accordance with literature data and the Raman spectra obtained from untreated samples, we conclude that the structure of the films is characterized by conjugated π orbitals55, which are delocalized and provide high-mobility electrons. During PIII treatment, high-energy nitrogen ions bombard the polypyrrole film, causing collisions and recoiling of atoms from the polypyrrole macromolecules, which produce radicals with unpaired electrons throughout the depth of the treated surface regions. These radicals are able to react with a range of chemical groups and molecules, giving rise to surface oxidation, crosslinks, double bonds and condensed aromatic structures, as observed in the AFM and spectroscopy studies. The effect of these reactions with storage time after PIII treatment is to increase the carbonyl group absorbance and cause a decrease in surface energy. Following this trend, the surface of polypyrrole after PIII treatment remains reactive with molecules even at long times after treatment.

At short treatment times, the conduction mechanism in polypyrrole is modified as a result of structural distortion of the dopants, the polypyrrole molecules, and the


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disruption of polaron excitations. At high fluences, new graphitic structures are formed that provide a new mechanism of conduction based on the mobility of electrons in the π orbitals of graphitic islands. These results show that the use of polypyrrole films as conductive electrodes should not be compromised by the PIII treatment. The thickness of the modified layer is directly related to the ion stopping depth, which is calculated to be 65 nm using SRIM calculations as shown in Figure 1358. The energy of the ion treatment can be selected to minimize the effects on the contact resistance between the polypyrrole film and the solution, by minimizing the thickness of the treated layer whilst allowing for sufficient covalent immobilization of biological macromolecules. The longevity of the covalent immobilization has been shown to scale with the depth of the ion-modified layer, which in turn scales with the ion energy used in the PIII treatment 33. Therefore, the application-specific need for long-term covalent coupling activity will determine the minimum ion energy required.

Unpaired electrons are stabilized on the edges of graphitic islands due to the delocalization of π-electrons in the condensed aromatic structures. These unpaired electrons provide strong intermolecular interactions at the air-film interface. This is indicated by the high surface energy, and in particular, by its polar component. The chemical groups with unpaired electrons provide sites of chemical activity in the modified surface layer. It is these sites that allow for the chemical reactions with molecules in the atmosphere and covalent binding of protein molecules on incubation in protein solution. This same mechanism of radical reactions providing covalent attachment of proteins has been observed for PIII-treated polyethylene, polystyrene and polytetrafluoroethylene 26, 35-39, 42, 59-64. However, in the case of polypyrrole, the attachment of protein is observed even after short PIII treatment times, something that



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is not observed in other polymers 26, 35-39, 42, 59-64. This may be due to untreated polypyrrole having condensed aromatic unsaturated groups, which stabilize the unpaired electrons of radicals. Poor protein attachment of untreated polypyrrole arises from a combination of factors. First, the untreated polypyrrole surface is hydrophilic, and hence shows less physical adsorption of protein than hydrophobic polymers. Second, polymerization has reached completion so that the surface has no remaining active radicals. As a result, the protein can be easily washed away from the untreated polypyrrole by buffer solution. Since condensed aromatic structures already exist in polypyrrole, whereas these structures need to be created by the ion modification in the case of other polymers, much shorter PIII treatments generate the required concentration of radicals stabilized by aromatic structures to immobilize protein covalently on the polypyrrole surface layer. The advantage of the PIII method is that is does not require the additional wet chemical steps that are needed to add linker molecules. These additional steps add complexity and cost as well as introducing the possibility that traces of toxic reactants may remain that could impact the subsequent use of the surfaces for cell attachment.

Conclusions

Plasma immersion ion implantation has been shown to provide the surface of electrosynthesized polypyrrole with covalent binding sites for immobilizing protein molecules. Spectral analysis has shown that PIII treatment of polypyrrole films creates a nanometer-scale carbonized, radical-rich surface that enables immobilization of bioactive molecules, while the film remains electrically conductive. The surface morphology becomes more uniform after PIII treatment. Covalent protein


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immobilization has been achieved on the PIII-treated surfaces and shown to be resistant to SDS elution, while the untreated surfaces released adsorbed protein when washed gently with fresh buffer. Sufficient surface activation for covalent coupling of biomolecules was achieved after treatment times and ion fluences much shorter than those required to similarly activate most other polymers. This work has shown that electrosynthesized PIII-treated polypyrrole may potentially be employed as a biologically active, conductive electrode for interfacing with living systems. The ability to covalently bind biological molecules on the surface allows the study of the interactions of the bound molecules with other molecules and cells near the interface. This study suggests PIII treatment of polypyrrole as a promising platform technology to provide a biologically active interface for neurological stimulation and electrical biosensing.

Acknowledgments

We gratefully acknowledge the Australian Research Council (ARC) our industry partners Cochlear Ltd, LfC and SpineCell for financial support through grant LP110200316.

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For Table of Contents Use Only

Structural Analysis and Protein Functionalization of Electroconductive Polypyrrole Films Modified by Plasma Immersion Ion Implantation Alexey Kondyurin, Kostadinos Tsoutas, Quentin-Xavier Latour, Michael J. Higgins, Simon E. Moulton, David R. McKenzie, Marcela M.M. Bilek


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10000

100000

Time after PIII treatment, min

Fig.2. Evolution of the dispersive (full symbols) and polar (empty symbols) components of surface energy of PIII treated polypyrrole as a function of storage time after treatment. The PIII treatment times were 80 s (blue rhombi), 400 s (green squares) and 1600 s (red triangles). The circles indicate dispersive and polar components of the surface energy for untreated polypyrrole. The lines show the fitted functions (1) and (2) discussed in the text.


Page 32 of 45

Page 33 of 45

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


Figure 3. AFM images (topography and phase) of polypyrrole surfaces before and after PIII treatment (800 s treatment time). Image size is 2 x 2 μm



2210 1551

1167 1302 1452

.02 1684

Untreated .015

.01

.005

1215

1630 1708

40

a.u.

Absorbance, a.u.

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

Page 34 of 45

80 400 800 1600

0

1441, 1277 3500

3000

2500

Wavenumber, cm

2000

1500

-1

Figure 4. ATR-FTIR spectra of polypyrrole. The top spectrum (blue) is from untreated polypyrrole. The other spectra are from polypyrrole after treatment, for a range of PIII treatment times. These spectra have had the untreated polypyrrole spectrum subtracted from them to show the differences that appear due to treatment, and their scales have been expanded (x10) to make the new features more clearly visible. Base line correction with a polynomial function has been applied to all spectra and the region containing noisy peaks from carbon dioxide contamination has been removed. The embedded spectra are zoomed to 100 times to show a weak CN peak. The spectra of 4 untreated samples are presented to show deviations between the untreated samples.


Page 35 of 45

1380 930 600

Raman intensity, a.u.

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


615 Untreated

400

80

1246 1052

680 1420

989 1055 693

942

1108

1261

1620

1501

200 200

0

400 800

400

600

800

1000

1200

Wavenumber, cm

1400

1600

1800

-1

Fig.5. Micro-Raman spectra of polypyrrole. The top spectrum (blue) is from untreated polypyrrole. The other spectra are from polypyrrole after treatment for a range of PIII treatment times. These spectra have had the untreated polypyrrole spectrum subtracted from them to show the differences that appear due to treatment, and their scales have been expanded (x10) to make the new features more clearly visible.



3500

PIII-treated

3000

Intensity, counts

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

Page 36 of 45

2500 2000 1500 1000

Untreated

500

743

692

647

608

573

542

Wavelength, nm Fig.6. Fluorescence of untreated and PIII-treated (1016 ions/cm2) polypyrrole. The spectra were excited with a laser beam with a wavelength of 514 nm.


6000

(a)

1379

4000

1633 1687

1505

1385 1420

1246

1000

1189

2000

1050 1070

937 979

3000

921


5000

0 800

1000

1200

1400

1600

1800

2000

1800

2000

Wavenumber, cm- 1

1574

14000

(b) 12000

1390

10000 8000

1620 1686

1510

1400 1419

1200 1245

2000

1070

4000

1051

6000

948 985


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


1583

Page 37 of 45

0 800

1000

1200

1400

Wavenumber, cm

1600 -

1

Figure 7. Resonance micro-Raman spectra of (a) untreated polypyrrole and (b) PIII- treated polypyrrole, treated with a fluence of 1016 ions/cm-2. The spectra also show the individual Gaussian components that have been fitted. The fluorescence signal has been subtracted.



0.01

0.008

Absorbance, a.u.

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

Page 38 of 45

0.006

0.004

0.002

0 0

500

1000

1500

2000

PIII time, s

Fig.8. Absorbance of the C≡N line at 2210 cm-1 in ATR-FTIR spectra of polypyrrole as a function of PIII treatment time. Experimental data points are shown overlaid with a fitted exponential function, A = 0.007 exp(-t/150) + 0.0011.


Page 39 of 45

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


Fig.9. Absorbance of the 1708 cm-1 line from C=O group vibrations as a function of storage time after PIII for a range of treatment times. Symbols indicate experimental data points, whilst the lines are solutions of equation 8. The inset shows the same data on a linear scale.



0.16

0.025

0.02

0.12

A2

0.1

0.015

0.08 0.01

0.06 0.04

0.005

A1

0.02 0 0

500

1000

A1 constant, a.u. A1 constant, a.u.

1500

PIII treatment s time,time, s time, PIII PIII treatment s

25000

0 2000

250

20000

200

k2 15000

150

10000

100

k1

5000

0 0

500

1000

1500

k1 constant, 1/min

A22 constant, A constant,a.u. a.u.

0.14

k2 constant, 1/min

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

Page 40 of 45

50

0 2000

PIII treatment time, s PIII treatment time, s Fig.10. Fitting coefficients of equation 8, used to fit C=O absorbance data as a function of PIII treatment time.


Page 41 of 45

2.5

2.0

S2p

75

1.5 74 1.0 73

0.5

C1s

72 0

500

1000

Sulfur content, %

Carbon content, %

76

1500

0.0 2000

PIII treatment time, s

15 Nitrogen and oxygen content, %

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


14

N1s

13

12

O1s

11

10 0

500

1000

1500

2000

PIII treatment time, s

Fig.11. Element content of polypyrrole surface determined using XPS as a function of PIII treatment time.



Untreated 0.006

0.004

Current, mA

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

Page 42 of 45

0.002

80 s 1600 s

0

400 s

-0.002

-0.004 -0.6

-0.4

-0.2

0

0.2

0.4

0.6

Voltage, V

Figure 12. Cyclic voltammograms of untreated and 80s, 400s and 1600s PIII treated polypyrrole in buffer.


Page 43 of 45

3.5 5 keV 10 keV

3.0

15 keV

Vacancies, ion-1 nm-1

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


2.5

20 keV

2.0 1.5 1.0 0.5 0.0 0

20

40

60 80 Depth, nm

100

120

Fig.13. Vacancy distribution in polypyrrole with pTS dopant after bombarding by nitrogen ions of 20, 15, 10 and 5 keV kinetic energy as calculated by SRIM.



0.01 0.009

Amide II Amide I Amide A

0.008

1600

0.007

Absorbance, a.u.

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

Page 44 of 45

800 0.006 380 0.005 200 0.004 80 0.003 40 0.002 20 0.001 Untreated 0 1400

1900

2400 2900 -1 Wavenumber, cm

a


3400

Page 45 of 45

0.03 Amide II Amide I 0.025

Amide A 1600

Absorbance, a.u.

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


0.02

800

0.015 380 0.01

200

80 0.005 40 20 0 1400

1900

2400

2900

3400

-1

Wavenumber, cm

b Fig.14. FTIR ATR spectra of protein attached on PIII treated polypyrrole before (a) and after washing in SDS detergent (b). The bands of Amide A, I and II are observed for all PIII treated samples. The spectrum of untreated polypyrrole does not show Amide bands. The spectra of polypyrrole was subtracted.


Structural Analysis and Protein Functionalization of

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