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Mechanism of the biomolecular synthesis of PEDOT:PSS: importance of heme degradation by hydrogen peroxide† J. D. Morris,a,b,c K. M. Wong,a,b C. D. Peñaherreraa,b,c and C. K. Payne*a,b The use of biomolecules as oxidants for the synthesis of conducting polymers provides an important tool for the control of polymer properties. Using PEDOT:PSS as a representative conducting polymer, we compare a set of heme proteins (soybean peroxidase, cytochrome c, and horseradish peroxidase) used as oxidants. The resulting PEDOT:PSS was characterized with visible and near IR spectroscopy, Fourier trans-
Received 19th September 2015, Accepted 16th November 2015
form infrared spectroscopy, electron spin resonance spectroscopy, and four point probe conductivity
DOI: 10.1039/c5bm00399g
measurements. We find that the relative concentrations of bipolarons and polarons vary as a function of the protein used for polymerization. We then show that heme degradation by hydrogen peroxide plays a
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critical role in determining polymer properties.
Introduction The conducting polymer poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS)1–3 is transparent in the visible region of the spectrum,4,5 dispersible in water,6 and biocompatible7,8 allowing its use in a diverse range of fields including light emitting diodes,9–11 photovoltaics,12–14 regenerative medicine,15,16 and chemical and biological sensing.17–20 The oxidative polymerization of ethylenedioxythiophene (EDOT) to PEDOT:PSS is typically performed using iron compounds, combined with peroxodisulfates or hydrogen peroxide, as oxidants.21 Alternatively, proteins such as catalase (CAT), hemoglobin (Hb), soybean peroxidase (SBP), horseradish peroxidase (HRP), and laccase can be used as oxidants.22–30 This biomolecular approach takes advantage of the naturally occurring structural diversity of proteins to control polymer conductivity or provide more environmentally friendly reaction conditions.22,23,26,27,30 In addition to PEDOT: PSS, other conducting polymers, including polyaniline and
a School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA. E-mail: [email protected] b Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia 30332, USA c School of Science and Technology, Georgia Gwinnett College, Lawrenceville, Georgia 30043, USA † Electronic supplementary information (ESI) available: Graphical representations of protein structures, X-ray photoelectron spectra of polymer films, visible and near IR of PEDOT:PSS after the addition of protein, visible and near IR of PEDOT:PSS polymerized with degraded protein in the presence of iron(III) chloride, four point probe conductivity of PEDOT:PSS after the addition of protein. See DOI: 10.1039/c5bm00399g
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polyphenols, have been synthesized using biological oxidants.22,23,31–37 Initially the biomolecular synthesis of PEDOT:PSS was assumed to be enzymatic.26,28,37 However, recent work in our lab demonstrated that enzymatic activity is not required for the polymerization of PEDOT:PSS.29 Both CAT and denatured, inactive, CAT can be used to synthesize PEDOT:PSS. Additional experiments showed that transferrin, a non-enzymatic, ironbinding protein, and Hb, a classic heme protein, can also be used as oxidants for the polymerization of PEDOT:PSS.29,30 Being able to use any iron-containing protein, rather than just enzymes, dramatically expands the range of biomolecules that can be used for polymerization. The heme protein database, for example, includes over 800 different heme proteins.38 Most importantly, different proteins result in different PEDOT:PSS properties. Hb-polymerized PEDOT:PSS possesses primarily bipolarons and has high conductivity, while CAT-polymerized PEDOT:PSS possesses primarily polarons and low conductivity.30 This difference is ultimately due to a difference in the quantity of free iron released during the polymerization.30,39 Free iron (FeCl3) results in PEDOT:PSS with polarons and low conductivity.39 In contrast, heme-bound iron (hemin) results in PEDOT:PSS with bipolarons and high conductivity.39 PEDOT:PSS often requires post-processing to enhance conductivity.40–43 Methods to enhance conductivity include secondary doping with organic solvents,42,44–46 heating PEDOT:PSS films,47 and drop casting inorganic salts and acids over PEDOT:PSS films.41 Using the oxidant to control conductivity is advantageous as it removes the need for post-processing, allowing a simple one-pot synthesis. Proteins serve as a natural library of structurally diverse oxidants for this purpose.
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To fully use biomolecular oxidants to control polymer properties, it is necessary to gain a mechanistic understanding of the relationship between iron release and final polymer properties. In this work, we use a small library of proteins to polymerize PEDOT:PSS. We use two classic enzymes for conducting polymer synthesis, SBP and HRP, and a new protein for PEDOT:PSS polymerization, cytochrome c (cyt c). These proteins, in addition to the CAT and Hb used previously,30 are all heme proteins (Fig. S1, ESI†). In the literature, the conditions used for PEDOT:PSS polymerization, including concentrations, pH, and reaction time, often vary making direct comparison of final polymer properties difficult.25–30 Here, we carry out each reaction under identical conditions allowing for a direct comparison of polymer properties as a function of protein oxidant. We characterize the spectral properties of the PEDOT:PSS with visible and near IR spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and electron spin resonance (ESR) spectroscopy. We characterize the electronic properties with four point probe. We find that cyt c- and HRP-polymerized PEDOT:PSS possess low conductivity and polarons, similar to CAT-polymerized PEDOT: PSS. SBP-polymerized PEDOT:PSS possesses high conductivity and bipolarons, similar to Hb-polymerized PEDOT:PSS. To understand how the protein affects polymerization under these reaction conditions, we investigated the reaction of hydrogen peroxide with CAT, Hb, SBP, cyt c, and HRP. We find that the reaction of hydrogen peroxide with these proteins leads to the degradation of the heme group.48,49 When the heme subunit degrades, it releases free iron into solution.50 Any PEDOT:PSS polymerized by this free iron will possess polarons.39 We show that this degradation process leads to an increase of free iron during PEDOT:PSS polymerization and hence increased polaron content at longer reaction times. These findings show the central importance of heme degradation in the biomolecular synthesis of PEDOT:PSS and highlight key variables in the synthesis of conducting polymers that can be controlled to enhance conductivity.
Experimental Materials 3,4-Ethylenedioxythiophene (EDOT, #483028), poly(styrenesulfonate) (MW = 70 kDa, #243051), CAT (#C40), Hb (#H2500), cyt c (#C2506), HRP (#77332), high conductivity grade PEDOT: PSS (#739332) and 30% hydrogen peroxide (#16911) were purchased from Sigma-Aldrich. SBP (#512) was purchased from Bio-Research Products. All solutions were prepared in 18 MΩ cm water generated by an EASYpure II water purification system (Thermo Fisher Scientific). Polymerization and purification of PEDOT:PSS PEDOT:PSS was polymerized by combining the appropriate biomolecule (4.8 μM by heme concentration, quantified by UV-Vis), EDOT (50 mM), and PSS (MW = 70 kDa, 25 mM) in an
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HCl–KCl buffer ( pH = 1) while stirring. The polymerization was initiated by adding hydrogen peroxide (50 mM). The final reaction volume was 2.5 mL. Unless otherwise noted, the reaction was allowed to proceed for 6 hours at room temperature. The polymer mixtures were then dialyzed (cutoff MW = 1000 Da, #132636, Spectrolabs) against 400 ml of deionized water to remove the HCl–KCl buffer and any unreacted monomer. Water was exchanged at 1, 2, and 3 hours and again after dialysis proceeded for 17 hours. Solutions were then centrifuged at 700g for 5 minutes to remove insoluble material. This protocol was adapted from previous research using CAT as an oxidant.29 For 24 hour polymerizations, the reactions were run under identical conditions except the EDOT concentration was increased to 100 mM and the polymerization was allowed to continue for 24 hours before dialysis. Degraded proteins used in PEDOT:PSS polymerization were incubated at reaction conditions (without EDOT) for 12 hours before the polymerization was initiated. For polymerizations with an iron chelator, the reactions were run in the presence of 7 μM ethylenediaminetetraacetic acid (EDTA). For heme degradation experiments, protein solutions (4.8 μM by heme concentration) were prepared in HCl–KCl ( pH = 1) and combined with hydrogen peroxide (50 mM). Solutions were stirred continuously. UV-visible spectra (DU 800 Spectrophotometer, Beckman Coulter) were collected every 60 minutes. Prior to characterization, each sample was purified by dialysis and centrifugation. However, these purification steps do not necessarily remove the protein used as an oxidant. XPS shows a nitrogen peak indicative of protein in the final PEDOT:PSS films (Fig. S2, ESI†). To ensure that the presence of the protein in the polymer did not give rise to the shift in dominant charge carrier, three control experiments were carried out. First, commercial PEDOT:PSS (Sigma-Aldrich) was characterized by visible and near IR spectroscopy after the addition of SBP (Fig. S3, ESI†). The presence of protein does not alter the visible and near IR absorption of PEDOT:PSS. This is consistent with our prior findings that the addition of Hb did not affect the properties of commercial PEDOT:PSS.30 Second, to demonstrate that the presence of protein during polymerization does not alter polymer properties, we used proteins with degraded heme groups in the presence of FeCl3 to polymerize PEDOT:PSS. The resulting polymer shows visible and near IR absorption similar to FeCl3 alone (Fig. S4, ESI†). Third, we show that the presence of protein in the PEDOT:PSS film does not alter conductivity. Polymer films were prepared with commercial PEDOT:PSS with and without SBP (4.8 μM by heme concentration). The two sets of films showed the same conductivity (Fig. S5, ESI†). Characterization of PEDOT:PSS Visible and near IR (DU 800 Spectrophotometer, Beckman Coulter) spectra were recorded of PEDOT:PSS solutions in deionized water. FTIR (Alpha FTIR, Bruker) spectra were taken on films drop casted on germanium wafers (#160-1191, Pike Technologies, Madison, WI). Films were dried in ambient
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atmosphere for 30 minutes at 80 °C. The FTIR data in Fig. 1a and 2b are scaled (multiplied by a constant value) with an offset maintained to allow comparison of the broad absorption feature. X-band ESR (Bruker) spectra were collected using PEDOT:PSS powder freeze dried (FreeZone, Lanconco) at −50 °C overnight. ESR spectra were collected at a fixed frequency of 9.878 GHz at a power of 1 mW. Signal intensity was integrated for 5 scans of 40 seconds each and the total signal intensities were normalized by PEDOT:PSS weight. All spectroscopic measurements were collected in triplicate on a minimum of 2 separate polymerizations. Conductivity was recorded using a four point probe (SYS-301 Probe Station, Signatone) on films of PEDOT:PSS drop cast on glass. High conductivity grade PEDOT:PSS was used as a standard to calibrate the four point probe measurements. Films were allowed to dry at room temperature overnight and then heated at 60 °C for 30 minutes. Film thickness was measured from the average of three scans with a profilometer (P15, Tencor). Conductivity
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was measured for a minimum of three separate polymerizations. In each case, a minimum of four spots on four films were measured. X-ray photoelectron spectroscopy (XPS, k-alpha X-ray photoelectron spectrometer, Thermo Scientific) spectra were collected on PEDOT:PSS films drop cast on glass wafers and heated at 60 °C for 30 minutes.
Results and discussion PEDOT:PSS characterization PEDOT:PSS synthesized using SBP, cyt c, and HRP as oxidants was characterized spectroscopically and electrically. The visible and near IR spectra of SBP-, cyt c-, and HRP-polymerized PEDOT:PSS show a polaron absorption peak at ∼815 nm, characteristic of PEDOT:PSS (Fig. 1a).21 These polymers also show enhanced absorption at longer wavelengths, indicative of bipolarons.30,39,51 This long wavelength feature can be more
Fig. 1 Spectroscopic and electrical characterization of SBP-, cyt c-, and HRP-polymerized PEDOT:PSS. (a) Representative visible and near IR spectra of PEDOT:PSS polymerized by SBP (blue), cyt c (green), and HRP (yellow). (b) Representative FTIR spectra of PEDOT:PSS polymerized with SBP (blue), cyt c (green), and HRP (yellow). (c) Representative ESR spectra of PEDOT:PSS polymerized by SBP (blue), cyt c (green), and HRP (yellow). (d) Conductivity of PEDOT:PSS polymerized by SBP, cyt c, and HRP as determined by four point probe. Error bars represent the standard deviation of a minimum of 18 measurements across 6 films.
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Fig. 2 Synthesis of PEDOT:PSS in the presence (dashed line) and absence (solid line) of EDTA, an iron chelator. (a) Visible and near IR spectra. (b) FTIR spectra.
clearly seen in the FTIR spectrum of SBP-polymerized PEDOT: PSS as a broad absorption peak centered at 3400 cm−1 that is associated with bipolarons (Fig. 1b).52 Cyt c- and HRP-polymerized PEDOT:PSS show a weaker absorption band in the mid-IR indicative of a reduced bipolaron population (Fig. 1b). ESR spectroscopy (Fig. 1c) is a useful tool for observing unpaired charges, such as polarons, while paired charges, such as bipolarons, show no ESR absorption.53–55 SBP-polymerized PEDOT: PSS shows a relatively weak absorption at a field strength of 3512 G due to residual polarons. Cyt c- and HRP-polymerized PEDOT:PSS show a stronger absorption feature at 3511 G and 3508 G, respectively. This enhanced absorption indicates an increased density of polarons in cyt c- and HRP-polymerized PEDOT:PSS compared to SBP-polymerized PEDOT:PSS. Shifts in the peak position in ESR are due to variation in the local magnetic field, suggesting the polarons in SBP-, cyt c-, and HRP-polymerized PEDOT:PSS are in slightly different local environments.56 The conductivity of each polymer was measured by four point probe (Fig. 1d). SBP-polymerized PEDOT:PSS is significantly more conductive (0.10 S cm−1) compared to cyt c- and HRP-polymerized PEDOT:PSS (2.1 × 10−2 S cm−1 and 2.3 × 10−2 S cm−1, respectively). This increased conductivity is consistent with the higher charge carrier mobility of bipolarons compared to polarons.55 Overall, spectroscopic characterization shows that PEDOT:PSS synthesized using SBP, cyt c, and HRP as oxidants leads to a polymer with both polarons and bipolarons, in agreement with our previous work using CAT and Hb as oxidants.30 For the proteins examined here, SBP is unique in showing a relatively greater concentration of bipolarons, observed in the FTIR (Fig. 1b) and supported by the ESR (Fig. 1c) spectra. This difference in charge carrier is also observed in the conductivity of the polymers (Fig. 1d) with SBP-polymerized PEDOT:PSS having a 5× higher conductivity. Similar to our findings, previous polymerization of PEDOT: PSS using HRP performed by Rumbau, et al. lack any strong absorption in the mid IR and show no enhanced absorption due to bipolarons in the near IR.26 Prior polymerizations of PEDOT:PSS with SBP have either been copolymer systems
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(with polypyrrole)27 or have used terthiophene as a radical initiator.28 In contrast to our results, the pyrrole/PEDOT copolymers show no absorption in the near IR or mid IR indicative of bipolaron formation. The data shown for SBP-polymerized PEDOT:PSS using terthiophene do not include near IR absorption beyond 800 nm or absorption in the mid IR beyond 1600 cm−1, making a clear determination of the dominant charge carrier difficult. The previous reaction was also carried out at much higher pHs (3.5 to 5.5) in the presence of 20% by volume dimethyl sulfoxide. These differences limit comparisons to the SBP-polymerized PEDOT:PSS described here. Iron coordination during polymerization The proteins we have used as oxidants are all heme proteins, yet result in PEDOT:PSS with different spectral properties and conductivities. SBP leads to a relatively high conductivity polymer with high bipolaron absorption (Fig. 1b) and low polaron absorption (Fig. 1c). Cyt c and HRP lead to polymers which are less conductive and possess higher polaron to bipolaron ratios. We hypothesized that the coordination of the iron, which serves as the actual oxidant in the polymerization reaction,29 is critical in determining polymer properties. Our previous polymerizations of PEDOT:PSS in the presence of EDTA, which chelates free iron, but not heme-bound iron, show that when heme-bound iron is the active oxidant, a bipolaron rich polymer results. By contrast, when free iron is the active oxidant, a polaron rich polymer results.30 This same shift to bipolarons is also observed with PEDOT:PSS polymerized by SBP, cyt c, and HRP in the presence of EDTA. EDTA results in a 2× decrease in PEDOT:PSS yield (Fig. 2a), indicating that roughly half of the PEDOT:PSS is polymerized with free iron as the oxidant. Increased absorption in the mid IR indicates an increased concentration of bipolarons (Fig. 2b). Similar results were obtained for cyt c and HRP (data not shown). Heme degradation by hydrogen peroxide The most likely mechanism of free iron release under our reaction conditions ( pH = 1, 50 mM hydrogen peroxide) is the
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degradation of the heme subunit of the protein and subsequent release of iron. Hydrogen peroxide is known to degrade the heme group of Hb at physiological pHs.49 It is expected that a similar degradation occurs for the other heme proteins used for polymerization of PEDOT:PSS and that this degradation releases iron into solution. To test this hypothesis, we monitored the UV-visible absorption of the heme group of each protein after exposure to hydrogen peroxide (Fig. 3). In addition to HRP, cyt c, and SBP, we examined the reaction of hydrogen peroxide with CAT and Hb, which we have previously used to polymerize PEDOT:PSS.29,30 After exposure to hydrogen peroxide for 6 hours, the absorbance of the heme group of Hb, SBP, and HRP was significantly decreased (Fig. 3b, c, and e). CAT and cyt c show nearly complete loss of the heme peak
Fig. 3 UV-visible spectroscopic characterization of (a) CAT, (b) Hb, (c) SBP, (d) cyt c, and (e) HRP proteins, at identical heme concentrations, before (solid) and after (dashed) incubation with hydrogen peroxide.
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(Fig. 3a and d). This indicates that in each case the heme group of the protein was degraded during the course of the reaction, likely releasing iron into solution. It should be noted that the extent of degradation in these experiments may differ from the extent of degradation during polymerization since hydrogen peroxide will be consumed by both the polymerization and the degradation reaction. Additionally, these rates may differ in the presence of EDOT or PEDOT:PSS. Heme degradation leads to a shift in the active oxidant in the biomolecular polymerization of PEDOT:PSS To test our hypothesis that hydrogen peroxide-degraded protein leads to greater concentrations of polarons relative to bipolarons, we polymerized PEDOT:PSS after pre-treating each protein with hydrogen peroxide for 12 hours, thereby degrading it prior to the start of the reaction. Visible and near IR spectra of PEDOT:PSS polymerized with degraded protein show a strong enhancement in polaron absorption at ∼815 nm (Fig. 4). In the case of each protein, the absorption at 815 nm (due to polarons) is stronger than the absorption at 1100 nm (due to bipolarons). This is in stark contrast to the polymerizations with proteins prior to degradation (Fig. 1a), which show lower absorption at 815 nm compared to 1100 nm. This indicates that the degradation of the heme subunit shifts the dominant charge carrier of PEDOT:PSS from bipolarons to polarons. Degradation of the heme subunit by hydrogen peroxide (Fig. 3) highlights the dynamic nature of the active oxidant. Initially, especially for Hb and SBP, heme-bound iron is present in solution. As the reaction proceeds and the heme degrades, the oxidant is expected to shift from heme-bound iron to free iron. This suggests that at long reaction times the polymerization should be increasingly dominated by free iron resulting in an increase in polarons relative to bipolarons. To test this hypothesis, we polymerized PEDOT:PSS over 24 hours, instead of the 6 hours used in previous experiments. If the
Fig. 4 Visible and near IR spectra of PEDOT:PSS polymerized with CAT (black), Hb (red), SBP (blue), cyt c (green), and HRP (yellow) after each protein was incubated with hydrogen peroxide for 12 hours. PEDOT:PSS polymerized with Hb before degradation (dashed red) is replotted from Fig. 1a for comparison.
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Fig. 5 Longer (24 h) reactions result in a reduction in bipolarons, observed by decreased absorption at longer wavelengths. (a) Representative visible and near IR spectra of PEDOT:PSS polymerized using CAT (black) and Hb (red) for 6 (solid) and 24 (dashed) hours. (b) Representative visible and near IR spectra of SBP (blue), cyt c (green), and HRP (yellow) polymerized for 6 (solid) and 24 (dashed) hours.
degradation of heme yields more free iron, then longer polymerizations should result in increased relative polaron concentration. After 24 h, CAT-polymerized PEDOT:PSS shows a dramatic reduction in bipolaron absorption at longer wavelengths (Fig. 5a). Hb-polymerized PEDOT:PSS, in contrast, shows minimal change in bipolaron absorption (Fig. 5a). PEDOT:PSS polymerized by SBP and cyt c show significant reductions in bipolaron absorption, while HRP shows virtually no change (Fig. 5b). The extent of this shift towards decreasing bipolaron content indicates a difference in the rate of protein degradation. Complete degradation leads to a dramatic reduction in bipolaron content (Fig. 4). The lack of such a reduction in the case of Hb and HRP indicates that under these reaction conditions these proteins do not significantly degrade. In contrast, the reduction of bipolaron absorption in CAT, SBP, and cyt c indicates these proteins are degraded during the reaction. During the reaction hydrogen peroxide is consumed by the polymerization process and protein degradation. Differing rates of degradation may be due to a competition between these two processes. These results show that using CAT, SBP, and cyt c as biomolecular oxidants leads to a shift in the active species during polymerization due to heme degradation. For these proteins, the dominant charge carrier can be altered by careful control of the reaction duration. In comparison, Hb and HRP show little change in the dominant charge carrier as a function of reaction duration.
PEDOT:PSS with a mixture of polarons and bipolarons. The relative concentrations of polarons and bipolarons can be controlled by the amount of free iron, compared to hemebound iron, present during the course of the reaction, demonstrated by polymerization in the presence of a free iron chelator, EDTA (Fig. 2). Free iron in the reaction mixture results from the degradation of the heme group by hydrogen peroxide (Fig. 3). When completely degraded proteins are used for polymerization, a polaron-dominated polymer results (Fig. 4). Additionally, when the time allowed for polymerization is increased, the extent of degradation of each heme group is increased for CAT-, SBP-, and cyt c-polymerized PEDOT:PSS leading to decreased bipolaron absorption in the resulting PEDOT:PSS (Fig. 5). This level of understanding enables the rational design of PEDOT: PSS with pre-selected properties through choice of protein used as an oxidant and reaction time.
Acknowledgements The authors would like to thank Prof. Amit Reddi for helpful discussion, Prof. Jake Soper for the use of the FTIR, and Dr. Robert Braga for help with the ESR. This work was supported by a Vasser Woolley Faculty Fellowship to CKP.
Notes and references Conclusions We have polymerized PEDOT:PSS using a library of heme proteins. We show that different protein oxidants result in PEDOT:PSS with different spectral and electrical properties (Fig. 1). SBP-polymerized PEDOT:PSS possesses high conductivity and a lower polaron to bipolaron ratio, while cyt c-, and HRP-polymerized PEDOT:PSS possess low conductivity and an increased polaron to bipolaron ratio. All proteins lead to
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1 B. L. Groenendaal, F. Jonas, D. Freitag, H. Pielartzik and J. R. Reynolds, Adv. Mater., 2000, 12, 481–494. 2 S. Kirchmeyer and K. Reuter, J. Mater. Chem., 2005, 15, 2077–2088. 3 K. Sun, S. Zhang, P. Li, Y. Xia, X. Zhang, D. Du, F. Isikgor and J. Ouyang, J. Mater. Sci.: Mater. Electron., 2015, 26, 4438–4462. 4 M. Dietrich, J. Heinze, G. Heywang and F. Jonas, J. Electroanal. Chem., 1994, 369, 87–92.
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5 G. Heywang and F. Jonas, Adv. Mater., 1992, 4, 116–118. 6 F. Jonas, W. Krafft and B. Muys, Macromol. Symp. Symposia, 1995, 100, 169–173. 7 X. Cui and D. C. Martin, Sens. Actuators, B, 2003, 89, 92– 102. 8 K. A. Ludwig, J. D. Uram, J. Yang, D. C. Martin and D. R. Kipke, J. Neural Eng., 2006, 3, 59–70. 9 W. H. Kim, A. J. Makinen, N. Nikolov, R. Shashidhar, H. Kim and Z. H. Kafafi, Appl. Phys. Lett., 2002, 80, 3844– 3846. 10 C. Zhong, C. Duan, F. Huang, H. Wu and Y. Cao, Chem. Mater., 2010, 23, 326–340. 11 C. A. Zuniga, S. Barlow and S. R. Marder, Chem. Mater., 2011, 23, 658–681. 12 W. J. Hong, Y. X. Xu, G. W. Lu, C. Li and G. Q. Shi, Electrochem. Commun., 2008, 10, 1555–1558. 13 G. Li, V. Shrotriya, J. Huang, Y. Yao, T. Moriarty, K. Emery and Y. Yang, Nat. Mater., 2005, 4, 864–868. 14 F. L. Zhang, A. Gadisa, O. Inganäs, M. Svensson and M. R. Andersson, Appl. Phys. Lett., 2004, 84, 3906–3908. 15 S. M. Richardson-Burns, J. L. Hendricks, B. Foster, L. K. Povlich, D. H. Kim and D. C. Martin, Biomaterials, 2007, 28, 1539–1552. 16 S. M. Richardson-Burns, J. L. Hendricks and D. C. Martin, J. Neural. Eng., 2007, 4, L6–L13. 17 J. A. Arter, D. K. Taggart, T. M. McIntire, R. M. Penner and G. A. Weiss, Nano Lett., 2010, 10, 4858–4862. 18 K. C. Donavan, J. A. Arter, R. Pilolli, N. Cioffi, G. A. Weiss and R. M. Penner, Anal. Chem., 2011, 83, 2420–2424. 19 Y. Cao, A. E. Kovalev, R. Xiao, J. Kim, T. S. Mayer and T. E. Mallouk, Nano Lett., 2008, 8, 4653–4658. 20 N. K. Guimard, N. Gomez and C. E. Schmidt, Prog. Polym. Sci., 2007, 32, 876–921. 21 A. K. Elschner, S. Lovenich, W. Merker, U. Reuter and K. Reuter, PEDOT: Principles and Applications of an Intrinsically Conductive Polymer, CRC Press, Boca Raton, Fl, 2010. 22 S. Kobayashi, J. Polym. Sci., Part A: Polym. Chem., 1999, 37, 3041–3056. 23 S. Kobayashi, H. Uyama and S. Kimura, Chem. Rev., 2001, 101, 3793–3818. 24 G. V. Otrokhov, O. V. Morozova, I. S. Vasil’eva, G. P. Shumakovich, E. A. Zaitseva, M. E. Khlupova and A. I. Yaropolov, Biochemistry, 2013, 78, 1539–1553. 25 G. Shumakovich, G. Otrokhov, I. Vasil’eva, D. Pankratov, O. Morozova and A. Yaropolov, J. Mol. Catal. B: Enzym., 2012, 81, 66–68. 26 V. Rumbau, J. A. Pomposo, A. Eleta, J. Rodriguez, H. Grande, D. Mecerreyes and E. Ochoteco, Biomacromolecules, 2007, 8, 315–317. 27 A. Tewari, A. Kokil, S. Ravichandran, S. Nagarajan, R. Bouldin, L. A. Samuelson, R. Nagarajan and J. Kumar, Macromol. Chem. Phys., 2010, 211, 1610–1617. 28 S. Nagarajan, J. Kumar, F. F. Bruno, L. A. Samuelson and R. Nagarajan, Macromolecules, 2008, 41, 3049–3052. 29 S. M. Hira and C. K. Payne, Synth. Met., 2013, 176, 104– 107.
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30 J. D. Morris, D. Khanal, J. A. Richey and C. K. Payne, Biomater. Sci., 2015, 3, 442–445. 31 W. Liu, S. Bian, L. Li, L. Samuelson, J. Kumar and S. Tripathy, Chem. Mater., 2000, 12, 1577–1584. 32 R. A. Gross, A. Kumar and B. Kalra, Chem. Rev., 2001, 101, 2097–2124. 33 W. Liu, J. Kumar, S. Tripathy, K. J. Senecal and L. Samuelson, J. Am. Chem. Soc., 1999, 121, 71–78. 34 K. S. Alva, J. Kumar, K. A. Marx and S. K. Tripathy, Macromolecules, 1997, 30, 4024–4029. 35 J. S. Dordick, M. A. Marletta and A. M. Klibanov, Biotechnol. Bioeng., 1987, 30, 31–36. 36 H. Uyama, H. Kurioka, I. Kaneko and S. Kobayashi, Chem. Lett., 1994, 23, 423–426. 37 B. Ryan, K. Akshay, R. Sethumadhavan, N. Subhalakshmi, K. Jayant, A. S. Lynne, F. B. Ferdinando and N. Ramaswamy, in Green Polymer Chemistry: Biocatalysis and Biomaterials, American Chemical Society, 2010, ch. 23, vol. 1043, pp. 315–341. 38 C. J. Reedy, M. M. Elvekrog and B. R. Gibney, Nucleic Acids Res., 2008, 36, D307–D313. 39 J. D. Morris and C. K. Payne, Org. Electron., 2014, 15, 1707– 1710. 40 Y. Xia and J. Ouyang, J. Mater. Chem., 2011, 21, 4927–4936. 41 Y. Xia, K. Sun and J. Ouyang, Adv. Mater., 2012, 24, 2436– 2440. 42 J. Ouyang, Displays, 2013, 34, 423–436. 43 H. Shi, C. Liu, Q. Jiang and J. Xu, Adv. Electron. Mater., 2015, 1, DOI: 10.1002/aelm.201500017. 44 R. Po, C. Carbonera, A. Bernardi, F. Tinti and N. Camaioni, Sol. Energy Mater. Sol. Cells, 2012, 100, 97–114. 45 J. Y. Kim, J. H. Jung, D. E. Lee and J. Joo, Synth. Met., 2002, 126, 311–316. 46 J. Ouyang, Q. F. Xu, C. W. Chu, Y. Yang, G. Li and J. Shinar, Polymer, 2004, 45, 8443–8450. 47 J. Huang, P. F. Miller, J. C. de Mello, A. J. de Mello and D. D. C. Bradley, Synth. Met., 2003, 139, 569–572. 48 T. M. Florence, J. Inorg. Biochem., 1985, 23, 131–141. 49 E. Nagababu and J. M. Rifkind, Biochem. Biophys. Res. Commun., 1998, 247, 592–596. 50 J. M. Gutteridge, FEBS Lett., 1986, 201, 291–295. 51 M. Cho, S. Kim, I. Kim, B. Kim, Y. Lee and J. Nam, Macromol. Res., 2010, 18, 1070–1075. 52 F. L. E. Jakobsson, X. Crispin, L. Lindell, A. Kanciurzewska, M. Fahlman, W. R. Salaneck and M. Berggren, Chem. Phys. Lett., 2006, 433, 110–114. 53 D. A. Svistunenko, Biochim. Biophys. Acta, 2001, 1546, 365– 378. 54 A. Zykwinska, W. Domagala, A. Czardybon, B. Pilawa and M. Lapkowski, Chem. Phys., 2003, 292, 31–45. 55 J. K. Lee, S. You, S. Jeon, N. H. Ryu, K. H. Park, K. MyungHoon, D. H. Kim, S. H. Kim and E. A. Schiff, J. Appl. Phys., 2015, 118, 015501. 56 J. A. B. Weil and J. R. Bolton, Electron Paramagnetic Resonance: Elementary Theory and Practical Applications, John Wiley & Sons, Hoboken, New Jersey, 2007.
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Cite this: DOI: 10.1039/c5bm00399g
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Mechanism of the biomolecular synthesis of PEDOT:PSS: importance of heme degradation by hydrogen peroxide† J. D. Morris,a,b,c K. M. Wong,a,b C. D. Peñaherreraa,b,c and C. K. Payne*a,b The use of biomolecules as oxidants for the synthesis of conducting polymers provides an important tool for the control of polymer properties. Using PEDOT:PSS as a representative conducting polymer, we compare a set of heme proteins (soybean peroxidase, cytochrome c, and horseradish peroxidase) used as oxidants. The resulting PEDOT:PSS was characterized with visible and near IR spectroscopy, Fourier trans-
Received 19th September 2015, Accepted 16th November 2015
form infrared spectroscopy, electron spin resonance spectroscopy, and four point probe conductivity
DOI: 10.1039/c5bm00399g
measurements. We find that the relative concentrations of bipolarons and polarons vary as a function of the protein used for polymerization. We then show that heme degradation by hydrogen peroxide plays a
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critical role in determining polymer properties.
Introduction The conducting polymer poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS)1–3 is transparent in the visible region of the spectrum,4,5 dispersible in water,6 and biocompatible7,8 allowing its use in a diverse range of fields including light emitting diodes,9–11 photovoltaics,12–14 regenerative medicine,15,16 and chemical and biological sensing.17–20 The oxidative polymerization of ethylenedioxythiophene (EDOT) to PEDOT:PSS is typically performed using iron compounds, combined with peroxodisulfates or hydrogen peroxide, as oxidants.21 Alternatively, proteins such as catalase (CAT), hemoglobin (Hb), soybean peroxidase (SBP), horseradish peroxidase (HRP), and laccase can be used as oxidants.22–30 This biomolecular approach takes advantage of the naturally occurring structural diversity of proteins to control polymer conductivity or provide more environmentally friendly reaction conditions.22,23,26,27,30 In addition to PEDOT: PSS, other conducting polymers, including polyaniline and
a School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA. E-mail: [email protected] b Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia 30332, USA c School of Science and Technology, Georgia Gwinnett College, Lawrenceville, Georgia 30043, USA † Electronic supplementary information (ESI) available: Graphical representations of protein structures, X-ray photoelectron spectra of polymer films, visible and near IR of PEDOT:PSS after the addition of protein, visible and near IR of PEDOT:PSS polymerized with degraded protein in the presence of iron(III) chloride, four point probe conductivity of PEDOT:PSS after the addition of protein. See DOI: 10.1039/c5bm00399g
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polyphenols, have been synthesized using biological oxidants.22,23,31–37 Initially the biomolecular synthesis of PEDOT:PSS was assumed to be enzymatic.26,28,37 However, recent work in our lab demonstrated that enzymatic activity is not required for the polymerization of PEDOT:PSS.29 Both CAT and denatured, inactive, CAT can be used to synthesize PEDOT:PSS. Additional experiments showed that transferrin, a non-enzymatic, ironbinding protein, and Hb, a classic heme protein, can also be used as oxidants for the polymerization of PEDOT:PSS.29,30 Being able to use any iron-containing protein, rather than just enzymes, dramatically expands the range of biomolecules that can be used for polymerization. The heme protein database, for example, includes over 800 different heme proteins.38 Most importantly, different proteins result in different PEDOT:PSS properties. Hb-polymerized PEDOT:PSS possesses primarily bipolarons and has high conductivity, while CAT-polymerized PEDOT:PSS possesses primarily polarons and low conductivity.30 This difference is ultimately due to a difference in the quantity of free iron released during the polymerization.30,39 Free iron (FeCl3) results in PEDOT:PSS with polarons and low conductivity.39 In contrast, heme-bound iron (hemin) results in PEDOT:PSS with bipolarons and high conductivity.39 PEDOT:PSS often requires post-processing to enhance conductivity.40–43 Methods to enhance conductivity include secondary doping with organic solvents,42,44–46 heating PEDOT:PSS films,47 and drop casting inorganic salts and acids over PEDOT:PSS films.41 Using the oxidant to control conductivity is advantageous as it removes the need for post-processing, allowing a simple one-pot synthesis. Proteins serve as a natural library of structurally diverse oxidants for this purpose.
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To fully use biomolecular oxidants to control polymer properties, it is necessary to gain a mechanistic understanding of the relationship between iron release and final polymer properties. In this work, we use a small library of proteins to polymerize PEDOT:PSS. We use two classic enzymes for conducting polymer synthesis, SBP and HRP, and a new protein for PEDOT:PSS polymerization, cytochrome c (cyt c). These proteins, in addition to the CAT and Hb used previously,30 are all heme proteins (Fig. S1, ESI†). In the literature, the conditions used for PEDOT:PSS polymerization, including concentrations, pH, and reaction time, often vary making direct comparison of final polymer properties difficult.25–30 Here, we carry out each reaction under identical conditions allowing for a direct comparison of polymer properties as a function of protein oxidant. We characterize the spectral properties of the PEDOT:PSS with visible and near IR spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and electron spin resonance (ESR) spectroscopy. We characterize the electronic properties with four point probe. We find that cyt c- and HRP-polymerized PEDOT:PSS possess low conductivity and polarons, similar to CAT-polymerized PEDOT: PSS. SBP-polymerized PEDOT:PSS possesses high conductivity and bipolarons, similar to Hb-polymerized PEDOT:PSS. To understand how the protein affects polymerization under these reaction conditions, we investigated the reaction of hydrogen peroxide with CAT, Hb, SBP, cyt c, and HRP. We find that the reaction of hydrogen peroxide with these proteins leads to the degradation of the heme group.48,49 When the heme subunit degrades, it releases free iron into solution.50 Any PEDOT:PSS polymerized by this free iron will possess polarons.39 We show that this degradation process leads to an increase of free iron during PEDOT:PSS polymerization and hence increased polaron content at longer reaction times. These findings show the central importance of heme degradation in the biomolecular synthesis of PEDOT:PSS and highlight key variables in the synthesis of conducting polymers that can be controlled to enhance conductivity.
Experimental Materials 3,4-Ethylenedioxythiophene (EDOT, #483028), poly(styrenesulfonate) (MW = 70 kDa, #243051), CAT (#C40), Hb (#H2500), cyt c (#C2506), HRP (#77332), high conductivity grade PEDOT: PSS (#739332) and 30% hydrogen peroxide (#16911) were purchased from Sigma-Aldrich. SBP (#512) was purchased from Bio-Research Products. All solutions were prepared in 18 MΩ cm water generated by an EASYpure II water purification system (Thermo Fisher Scientific). Polymerization and purification of PEDOT:PSS PEDOT:PSS was polymerized by combining the appropriate biomolecule (4.8 μM by heme concentration, quantified by UV-Vis), EDOT (50 mM), and PSS (MW = 70 kDa, 25 mM) in an
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HCl–KCl buffer ( pH = 1) while stirring. The polymerization was initiated by adding hydrogen peroxide (50 mM). The final reaction volume was 2.5 mL. Unless otherwise noted, the reaction was allowed to proceed for 6 hours at room temperature. The polymer mixtures were then dialyzed (cutoff MW = 1000 Da, #132636, Spectrolabs) against 400 ml of deionized water to remove the HCl–KCl buffer and any unreacted monomer. Water was exchanged at 1, 2, and 3 hours and again after dialysis proceeded for 17 hours. Solutions were then centrifuged at 700g for 5 minutes to remove insoluble material. This protocol was adapted from previous research using CAT as an oxidant.29 For 24 hour polymerizations, the reactions were run under identical conditions except the EDOT concentration was increased to 100 mM and the polymerization was allowed to continue for 24 hours before dialysis. Degraded proteins used in PEDOT:PSS polymerization were incubated at reaction conditions (without EDOT) for 12 hours before the polymerization was initiated. For polymerizations with an iron chelator, the reactions were run in the presence of 7 μM ethylenediaminetetraacetic acid (EDTA). For heme degradation experiments, protein solutions (4.8 μM by heme concentration) were prepared in HCl–KCl ( pH = 1) and combined with hydrogen peroxide (50 mM). Solutions were stirred continuously. UV-visible spectra (DU 800 Spectrophotometer, Beckman Coulter) were collected every 60 minutes. Prior to characterization, each sample was purified by dialysis and centrifugation. However, these purification steps do not necessarily remove the protein used as an oxidant. XPS shows a nitrogen peak indicative of protein in the final PEDOT:PSS films (Fig. S2, ESI†). To ensure that the presence of the protein in the polymer did not give rise to the shift in dominant charge carrier, three control experiments were carried out. First, commercial PEDOT:PSS (Sigma-Aldrich) was characterized by visible and near IR spectroscopy after the addition of SBP (Fig. S3, ESI†). The presence of protein does not alter the visible and near IR absorption of PEDOT:PSS. This is consistent with our prior findings that the addition of Hb did not affect the properties of commercial PEDOT:PSS.30 Second, to demonstrate that the presence of protein during polymerization does not alter polymer properties, we used proteins with degraded heme groups in the presence of FeCl3 to polymerize PEDOT:PSS. The resulting polymer shows visible and near IR absorption similar to FeCl3 alone (Fig. S4, ESI†). Third, we show that the presence of protein in the PEDOT:PSS film does not alter conductivity. Polymer films were prepared with commercial PEDOT:PSS with and without SBP (4.8 μM by heme concentration). The two sets of films showed the same conductivity (Fig. S5, ESI†). Characterization of PEDOT:PSS Visible and near IR (DU 800 Spectrophotometer, Beckman Coulter) spectra were recorded of PEDOT:PSS solutions in deionized water. FTIR (Alpha FTIR, Bruker) spectra were taken on films drop casted on germanium wafers (#160-1191, Pike Technologies, Madison, WI). Films were dried in ambient
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atmosphere for 30 minutes at 80 °C. The FTIR data in Fig. 1a and 2b are scaled (multiplied by a constant value) with an offset maintained to allow comparison of the broad absorption feature. X-band ESR (Bruker) spectra were collected using PEDOT:PSS powder freeze dried (FreeZone, Lanconco) at −50 °C overnight. ESR spectra were collected at a fixed frequency of 9.878 GHz at a power of 1 mW. Signal intensity was integrated for 5 scans of 40 seconds each and the total signal intensities were normalized by PEDOT:PSS weight. All spectroscopic measurements were collected in triplicate on a minimum of 2 separate polymerizations. Conductivity was recorded using a four point probe (SYS-301 Probe Station, Signatone) on films of PEDOT:PSS drop cast on glass. High conductivity grade PEDOT:PSS was used as a standard to calibrate the four point probe measurements. Films were allowed to dry at room temperature overnight and then heated at 60 °C for 30 minutes. Film thickness was measured from the average of three scans with a profilometer (P15, Tencor). Conductivity
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was measured for a minimum of three separate polymerizations. In each case, a minimum of four spots on four films were measured. X-ray photoelectron spectroscopy (XPS, k-alpha X-ray photoelectron spectrometer, Thermo Scientific) spectra were collected on PEDOT:PSS films drop cast on glass wafers and heated at 60 °C for 30 minutes.
Results and discussion PEDOT:PSS characterization PEDOT:PSS synthesized using SBP, cyt c, and HRP as oxidants was characterized spectroscopically and electrically. The visible and near IR spectra of SBP-, cyt c-, and HRP-polymerized PEDOT:PSS show a polaron absorption peak at ∼815 nm, characteristic of PEDOT:PSS (Fig. 1a).21 These polymers also show enhanced absorption at longer wavelengths, indicative of bipolarons.30,39,51 This long wavelength feature can be more
Fig. 1 Spectroscopic and electrical characterization of SBP-, cyt c-, and HRP-polymerized PEDOT:PSS. (a) Representative visible and near IR spectra of PEDOT:PSS polymerized by SBP (blue), cyt c (green), and HRP (yellow). (b) Representative FTIR spectra of PEDOT:PSS polymerized with SBP (blue), cyt c (green), and HRP (yellow). (c) Representative ESR spectra of PEDOT:PSS polymerized by SBP (blue), cyt c (green), and HRP (yellow). (d) Conductivity of PEDOT:PSS polymerized by SBP, cyt c, and HRP as determined by four point probe. Error bars represent the standard deviation of a minimum of 18 measurements across 6 films.
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Fig. 2 Synthesis of PEDOT:PSS in the presence (dashed line) and absence (solid line) of EDTA, an iron chelator. (a) Visible and near IR spectra. (b) FTIR spectra.
clearly seen in the FTIR spectrum of SBP-polymerized PEDOT: PSS as a broad absorption peak centered at 3400 cm−1 that is associated with bipolarons (Fig. 1b).52 Cyt c- and HRP-polymerized PEDOT:PSS show a weaker absorption band in the mid-IR indicative of a reduced bipolaron population (Fig. 1b). ESR spectroscopy (Fig. 1c) is a useful tool for observing unpaired charges, such as polarons, while paired charges, such as bipolarons, show no ESR absorption.53–55 SBP-polymerized PEDOT: PSS shows a relatively weak absorption at a field strength of 3512 G due to residual polarons. Cyt c- and HRP-polymerized PEDOT:PSS show a stronger absorption feature at 3511 G and 3508 G, respectively. This enhanced absorption indicates an increased density of polarons in cyt c- and HRP-polymerized PEDOT:PSS compared to SBP-polymerized PEDOT:PSS. Shifts in the peak position in ESR are due to variation in the local magnetic field, suggesting the polarons in SBP-, cyt c-, and HRP-polymerized PEDOT:PSS are in slightly different local environments.56 The conductivity of each polymer was measured by four point probe (Fig. 1d). SBP-polymerized PEDOT:PSS is significantly more conductive (0.10 S cm−1) compared to cyt c- and HRP-polymerized PEDOT:PSS (2.1 × 10−2 S cm−1 and 2.3 × 10−2 S cm−1, respectively). This increased conductivity is consistent with the higher charge carrier mobility of bipolarons compared to polarons.55 Overall, spectroscopic characterization shows that PEDOT:PSS synthesized using SBP, cyt c, and HRP as oxidants leads to a polymer with both polarons and bipolarons, in agreement with our previous work using CAT and Hb as oxidants.30 For the proteins examined here, SBP is unique in showing a relatively greater concentration of bipolarons, observed in the FTIR (Fig. 1b) and supported by the ESR (Fig. 1c) spectra. This difference in charge carrier is also observed in the conductivity of the polymers (Fig. 1d) with SBP-polymerized PEDOT:PSS having a 5× higher conductivity. Similar to our findings, previous polymerization of PEDOT: PSS using HRP performed by Rumbau, et al. lack any strong absorption in the mid IR and show no enhanced absorption due to bipolarons in the near IR.26 Prior polymerizations of PEDOT:PSS with SBP have either been copolymer systems
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(with polypyrrole)27 or have used terthiophene as a radical initiator.28 In contrast to our results, the pyrrole/PEDOT copolymers show no absorption in the near IR or mid IR indicative of bipolaron formation. The data shown for SBP-polymerized PEDOT:PSS using terthiophene do not include near IR absorption beyond 800 nm or absorption in the mid IR beyond 1600 cm−1, making a clear determination of the dominant charge carrier difficult. The previous reaction was also carried out at much higher pHs (3.5 to 5.5) in the presence of 20% by volume dimethyl sulfoxide. These differences limit comparisons to the SBP-polymerized PEDOT:PSS described here. Iron coordination during polymerization The proteins we have used as oxidants are all heme proteins, yet result in PEDOT:PSS with different spectral properties and conductivities. SBP leads to a relatively high conductivity polymer with high bipolaron absorption (Fig. 1b) and low polaron absorption (Fig. 1c). Cyt c and HRP lead to polymers which are less conductive and possess higher polaron to bipolaron ratios. We hypothesized that the coordination of the iron, which serves as the actual oxidant in the polymerization reaction,29 is critical in determining polymer properties. Our previous polymerizations of PEDOT:PSS in the presence of EDTA, which chelates free iron, but not heme-bound iron, show that when heme-bound iron is the active oxidant, a bipolaron rich polymer results. By contrast, when free iron is the active oxidant, a polaron rich polymer results.30 This same shift to bipolarons is also observed with PEDOT:PSS polymerized by SBP, cyt c, and HRP in the presence of EDTA. EDTA results in a 2× decrease in PEDOT:PSS yield (Fig. 2a), indicating that roughly half of the PEDOT:PSS is polymerized with free iron as the oxidant. Increased absorption in the mid IR indicates an increased concentration of bipolarons (Fig. 2b). Similar results were obtained for cyt c and HRP (data not shown). Heme degradation by hydrogen peroxide The most likely mechanism of free iron release under our reaction conditions ( pH = 1, 50 mM hydrogen peroxide) is the
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degradation of the heme subunit of the protein and subsequent release of iron. Hydrogen peroxide is known to degrade the heme group of Hb at physiological pHs.49 It is expected that a similar degradation occurs for the other heme proteins used for polymerization of PEDOT:PSS and that this degradation releases iron into solution. To test this hypothesis, we monitored the UV-visible absorption of the heme group of each protein after exposure to hydrogen peroxide (Fig. 3). In addition to HRP, cyt c, and SBP, we examined the reaction of hydrogen peroxide with CAT and Hb, which we have previously used to polymerize PEDOT:PSS.29,30 After exposure to hydrogen peroxide for 6 hours, the absorbance of the heme group of Hb, SBP, and HRP was significantly decreased (Fig. 3b, c, and e). CAT and cyt c show nearly complete loss of the heme peak
Fig. 3 UV-visible spectroscopic characterization of (a) CAT, (b) Hb, (c) SBP, (d) cyt c, and (e) HRP proteins, at identical heme concentrations, before (solid) and after (dashed) incubation with hydrogen peroxide.
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(Fig. 3a and d). This indicates that in each case the heme group of the protein was degraded during the course of the reaction, likely releasing iron into solution. It should be noted that the extent of degradation in these experiments may differ from the extent of degradation during polymerization since hydrogen peroxide will be consumed by both the polymerization and the degradation reaction. Additionally, these rates may differ in the presence of EDOT or PEDOT:PSS. Heme degradation leads to a shift in the active oxidant in the biomolecular polymerization of PEDOT:PSS To test our hypothesis that hydrogen peroxide-degraded protein leads to greater concentrations of polarons relative to bipolarons, we polymerized PEDOT:PSS after pre-treating each protein with hydrogen peroxide for 12 hours, thereby degrading it prior to the start of the reaction. Visible and near IR spectra of PEDOT:PSS polymerized with degraded protein show a strong enhancement in polaron absorption at ∼815 nm (Fig. 4). In the case of each protein, the absorption at 815 nm (due to polarons) is stronger than the absorption at 1100 nm (due to bipolarons). This is in stark contrast to the polymerizations with proteins prior to degradation (Fig. 1a), which show lower absorption at 815 nm compared to 1100 nm. This indicates that the degradation of the heme subunit shifts the dominant charge carrier of PEDOT:PSS from bipolarons to polarons. Degradation of the heme subunit by hydrogen peroxide (Fig. 3) highlights the dynamic nature of the active oxidant. Initially, especially for Hb and SBP, heme-bound iron is present in solution. As the reaction proceeds and the heme degrades, the oxidant is expected to shift from heme-bound iron to free iron. This suggests that at long reaction times the polymerization should be increasingly dominated by free iron resulting in an increase in polarons relative to bipolarons. To test this hypothesis, we polymerized PEDOT:PSS over 24 hours, instead of the 6 hours used in previous experiments. If the
Fig. 4 Visible and near IR spectra of PEDOT:PSS polymerized with CAT (black), Hb (red), SBP (blue), cyt c (green), and HRP (yellow) after each protein was incubated with hydrogen peroxide for 12 hours. PEDOT:PSS polymerized with Hb before degradation (dashed red) is replotted from Fig. 1a for comparison.
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Fig. 5 Longer (24 h) reactions result in a reduction in bipolarons, observed by decreased absorption at longer wavelengths. (a) Representative visible and near IR spectra of PEDOT:PSS polymerized using CAT (black) and Hb (red) for 6 (solid) and 24 (dashed) hours. (b) Representative visible and near IR spectra of SBP (blue), cyt c (green), and HRP (yellow) polymerized for 6 (solid) and 24 (dashed) hours.
degradation of heme yields more free iron, then longer polymerizations should result in increased relative polaron concentration. After 24 h, CAT-polymerized PEDOT:PSS shows a dramatic reduction in bipolaron absorption at longer wavelengths (Fig. 5a). Hb-polymerized PEDOT:PSS, in contrast, shows minimal change in bipolaron absorption (Fig. 5a). PEDOT:PSS polymerized by SBP and cyt c show significant reductions in bipolaron absorption, while HRP shows virtually no change (Fig. 5b). The extent of this shift towards decreasing bipolaron content indicates a difference in the rate of protein degradation. Complete degradation leads to a dramatic reduction in bipolaron content (Fig. 4). The lack of such a reduction in the case of Hb and HRP indicates that under these reaction conditions these proteins do not significantly degrade. In contrast, the reduction of bipolaron absorption in CAT, SBP, and cyt c indicates these proteins are degraded during the reaction. During the reaction hydrogen peroxide is consumed by the polymerization process and protein degradation. Differing rates of degradation may be due to a competition between these two processes. These results show that using CAT, SBP, and cyt c as biomolecular oxidants leads to a shift in the active species during polymerization due to heme degradation. For these proteins, the dominant charge carrier can be altered by careful control of the reaction duration. In comparison, Hb and HRP show little change in the dominant charge carrier as a function of reaction duration.
PEDOT:PSS with a mixture of polarons and bipolarons. The relative concentrations of polarons and bipolarons can be controlled by the amount of free iron, compared to hemebound iron, present during the course of the reaction, demonstrated by polymerization in the presence of a free iron chelator, EDTA (Fig. 2). Free iron in the reaction mixture results from the degradation of the heme group by hydrogen peroxide (Fig. 3). When completely degraded proteins are used for polymerization, a polaron-dominated polymer results (Fig. 4). Additionally, when the time allowed for polymerization is increased, the extent of degradation of each heme group is increased for CAT-, SBP-, and cyt c-polymerized PEDOT:PSS leading to decreased bipolaron absorption in the resulting PEDOT:PSS (Fig. 5). This level of understanding enables the rational design of PEDOT: PSS with pre-selected properties through choice of protein used as an oxidant and reaction time.
Acknowledgements The authors would like to thank Prof. Amit Reddi for helpful discussion, Prof. Jake Soper for the use of the FTIR, and Dr. Robert Braga for help with the ESR. This work was supported by a Vasser Woolley Faculty Fellowship to CKP.
Notes and references Conclusions We have polymerized PEDOT:PSS using a library of heme proteins. We show that different protein oxidants result in PEDOT:PSS with different spectral and electrical properties (Fig. 1). SBP-polymerized PEDOT:PSS possesses high conductivity and a lower polaron to bipolaron ratio, while cyt c-, and HRP-polymerized PEDOT:PSS possess low conductivity and an increased polaron to bipolaron ratio. All proteins lead to
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1 B. L. Groenendaal, F. Jonas, D. Freitag, H. Pielartzik and J. R. Reynolds, Adv. Mater., 2000, 12, 481–494. 2 S. Kirchmeyer and K. Reuter, J. Mater. Chem., 2005, 15, 2077–2088. 3 K. Sun, S. Zhang, P. Li, Y. Xia, X. Zhang, D. Du, F. Isikgor and J. Ouyang, J. Mater. Sci.: Mater. Electron., 2015, 26, 4438–4462. 4 M. Dietrich, J. Heinze, G. Heywang and F. Jonas, J. Electroanal. Chem., 1994, 369, 87–92.
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5 G. Heywang and F. Jonas, Adv. Mater., 1992, 4, 116–118. 6 F. Jonas, W. Krafft and B. Muys, Macromol. Symp. Symposia, 1995, 100, 169–173. 7 X. Cui and D. C. Martin, Sens. Actuators, B, 2003, 89, 92– 102. 8 K. A. Ludwig, J. D. Uram, J. Yang, D. C. Martin and D. R. Kipke, J. Neural Eng., 2006, 3, 59–70. 9 W. H. Kim, A. J. Makinen, N. Nikolov, R. Shashidhar, H. Kim and Z. H. Kafafi, Appl. Phys. Lett., 2002, 80, 3844– 3846. 10 C. Zhong, C. Duan, F. Huang, H. Wu and Y. Cao, Chem. Mater., 2010, 23, 326–340. 11 C. A. Zuniga, S. Barlow and S. R. Marder, Chem. Mater., 2011, 23, 658–681. 12 W. J. Hong, Y. X. Xu, G. W. Lu, C. Li and G. Q. Shi, Electrochem. Commun., 2008, 10, 1555–1558. 13 G. Li, V. Shrotriya, J. Huang, Y. Yao, T. Moriarty, K. Emery and Y. Yang, Nat. Mater., 2005, 4, 864–868. 14 F. L. Zhang, A. Gadisa, O. Inganäs, M. Svensson and M. R. Andersson, Appl. Phys. Lett., 2004, 84, 3906–3908. 15 S. M. Richardson-Burns, J. L. Hendricks, B. Foster, L. K. Povlich, D. H. Kim and D. C. Martin, Biomaterials, 2007, 28, 1539–1552. 16 S. M. Richardson-Burns, J. L. Hendricks and D. C. Martin, J. Neural. Eng., 2007, 4, L6–L13. 17 J. A. Arter, D. K. Taggart, T. M. McIntire, R. M. Penner and G. A. Weiss, Nano Lett., 2010, 10, 4858–4862. 18 K. C. Donavan, J. A. Arter, R. Pilolli, N. Cioffi, G. A. Weiss and R. M. Penner, Anal. Chem., 2011, 83, 2420–2424. 19 Y. Cao, A. E. Kovalev, R. Xiao, J. Kim, T. S. Mayer and T. E. Mallouk, Nano Lett., 2008, 8, 4653–4658. 20 N. K. Guimard, N. Gomez and C. E. Schmidt, Prog. Polym. Sci., 2007, 32, 876–921. 21 A. K. Elschner, S. Lovenich, W. Merker, U. Reuter and K. Reuter, PEDOT: Principles and Applications of an Intrinsically Conductive Polymer, CRC Press, Boca Raton, Fl, 2010. 22 S. Kobayashi, J. Polym. Sci., Part A: Polym. Chem., 1999, 37, 3041–3056. 23 S. Kobayashi, H. Uyama and S. Kimura, Chem. Rev., 2001, 101, 3793–3818. 24 G. V. Otrokhov, O. V. Morozova, I. S. Vasil’eva, G. P. Shumakovich, E. A. Zaitseva, M. E. Khlupova and A. I. Yaropolov, Biochemistry, 2013, 78, 1539–1553. 25 G. Shumakovich, G. Otrokhov, I. Vasil’eva, D. Pankratov, O. Morozova and A. Yaropolov, J. Mol. Catal. B: Enzym., 2012, 81, 66–68. 26 V. Rumbau, J. A. Pomposo, A. Eleta, J. Rodriguez, H. Grande, D. Mecerreyes and E. Ochoteco, Biomacromolecules, 2007, 8, 315–317. 27 A. Tewari, A. Kokil, S. Ravichandran, S. Nagarajan, R. Bouldin, L. A. Samuelson, R. Nagarajan and J. Kumar, Macromol. Chem. Phys., 2010, 211, 1610–1617. 28 S. Nagarajan, J. Kumar, F. F. Bruno, L. A. Samuelson and R. Nagarajan, Macromolecules, 2008, 41, 3049–3052. 29 S. M. Hira and C. K. Payne, Synth. Met., 2013, 176, 104– 107.
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30 J. D. Morris, D. Khanal, J. A. Richey and C. K. Payne, Biomater. Sci., 2015, 3, 442–445. 31 W. Liu, S. Bian, L. Li, L. Samuelson, J. Kumar and S. Tripathy, Chem. Mater., 2000, 12, 1577–1584. 32 R. A. Gross, A. Kumar and B. Kalra, Chem. Rev., 2001, 101, 2097–2124. 33 W. Liu, J. Kumar, S. Tripathy, K. J. Senecal and L. Samuelson, J. Am. Chem. Soc., 1999, 121, 71–78. 34 K. S. Alva, J. Kumar, K. A. Marx and S. K. Tripathy, Macromolecules, 1997, 30, 4024–4029. 35 J. S. Dordick, M. A. Marletta and A. M. Klibanov, Biotechnol. Bioeng., 1987, 30, 31–36. 36 H. Uyama, H. Kurioka, I. Kaneko and S. Kobayashi, Chem. Lett., 1994, 23, 423–426. 37 B. Ryan, K. Akshay, R. Sethumadhavan, N. Subhalakshmi, K. Jayant, A. S. Lynne, F. B. Ferdinando and N. Ramaswamy, in Green Polymer Chemistry: Biocatalysis and Biomaterials, American Chemical Society, 2010, ch. 23, vol. 1043, pp. 315–341. 38 C. J. Reedy, M. M. Elvekrog and B. R. Gibney, Nucleic Acids Res., 2008, 36, D307–D313. 39 J. D. Morris and C. K. Payne, Org. Electron., 2014, 15, 1707– 1710. 40 Y. Xia and J. Ouyang, J. Mater. Chem., 2011, 21, 4927–4936. 41 Y. Xia, K. Sun and J. Ouyang, Adv. Mater., 2012, 24, 2436– 2440. 42 J. Ouyang, Displays, 2013, 34, 423–436. 43 H. Shi, C. Liu, Q. Jiang and J. Xu, Adv. Electron. Mater., 2015, 1, DOI: 10.1002/aelm.201500017. 44 R. Po, C. Carbonera, A. Bernardi, F. Tinti and N. Camaioni, Sol. Energy Mater. Sol. Cells, 2012, 100, 97–114. 45 J. Y. Kim, J. H. Jung, D. E. Lee and J. Joo, Synth. Met., 2002, 126, 311–316. 46 J. Ouyang, Q. F. Xu, C. W. Chu, Y. Yang, G. Li and J. Shinar, Polymer, 2004, 45, 8443–8450. 47 J. Huang, P. F. Miller, J. C. de Mello, A. J. de Mello and D. D. C. Bradley, Synth. Met., 2003, 139, 569–572. 48 T. M. Florence, J. Inorg. Biochem., 1985, 23, 131–141. 49 E. Nagababu and J. M. Rifkind, Biochem. Biophys. Res. Commun., 1998, 247, 592–596. 50 J. M. Gutteridge, FEBS Lett., 1986, 201, 291–295. 51 M. Cho, S. Kim, I. Kim, B. Kim, Y. Lee and J. Nam, Macromol. Res., 2010, 18, 1070–1075. 52 F. L. E. Jakobsson, X. Crispin, L. Lindell, A. Kanciurzewska, M. Fahlman, W. R. Salaneck and M. Berggren, Chem. Phys. Lett., 2006, 433, 110–114. 53 D. A. Svistunenko, Biochim. Biophys. Acta, 2001, 1546, 365– 378. 54 A. Zykwinska, W. Domagala, A. Czardybon, B. Pilawa and M. Lapkowski, Chem. Phys., 2003, 292, 31–45. 55 J. K. Lee, S. You, S. Jeon, N. H. Ryu, K. H. Park, K. MyungHoon, D. H. Kim, S. H. Kim and E. A. Schiff, J. Appl. Phys., 2015, 118, 015501. 56 J. A. B. Weil and J. R. Bolton, Electron Paramagnetic Resonance: Elementary Theory and Practical Applications, John Wiley & Sons, Hoboken, New Jersey, 2007.
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