Article pubs.acs.org/Langmuir
Comparison of Synthetic Dopamine−Eumelanin Formed in the Presence of Oxygen and Cu2+ Cations as Oxidants Vincent Ball,†,‡,§ José Gracio,∥,⊥ Mercedes Vila,#,∇ Manoj Kumar Singh,∥,⊥ Marie-Hélène Metz-Boutigue,‡ Marc Michel,○ Jérôme Bour,○ Valérie Toniazzo,○ David Ruch,○ and Markus J. Buehler*,◆,¶ †
Faculté de Chirurgie Dentaire, Université de Strasbourg, 1 Place de l’Hôpital, 67000 Strasbourg, France Institut National de la Santé et de la Recherche Médicale, unité 1121, 11 rue Humann, 67085 Strasbourg Cedex, France § Fédération de Médecine Translationelle de Strasbourg, 1 Place de l’Hôpital, 67000 Strasbourg, France ∥ Center for Mechanical Technology and Automation (TEMA), Department of Mechanical Engineering, University of Aveiro, 3810-193, Portugal ⊥ Aveiro Institute of Nanotechnology, University of Aveiro, 3810-193, Portugal # Departamento Quimica Inorganica y Bioinorgánica, Facultad de Farmacia, Universtad Complutense de Madrid, Plaza Ramon y Cajal s/n, 28040 Madrid, Spain ∇ Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 28040 Madrid, Spain ○ Department for Advanced Materials and Structures, Centre de Recherche Public Henri Tudor, 5 rue Bommel, L-4940 Hautcharage, Luxembourg ◆ Laboratory for Atomistic and Molecular Mechanics, Department of Civil and Environmental Engineering, and ¶Center for Materials Science and Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States ‡
S Supporting Information *
ABSTRACT: Eumelanin is not only a ubiquitous pigment among living organisms with photoprotective and antioxidant functions, but is also the subject of intense interest in materials science due to its photoconductivity and as a possible universal coating platform, known as “polydopamine films”. The structure of eumelanin remains largely elusive, relying either on a polymeric model or on a heterogeneous aggregate structure. The structure of eumelanin as well as that of the closely related “polydopamine films” can be modified by playing on the nature of the oxidant used to oxidize dopamine or related compounds. In this investigation, we show that dopamine−eumelanins produced from dopamine in the presence of either air (O2 being the oxidant) or Cu2+ cations display drastically different optical and colloidal properties in relation with a different supramolecular assembly of the oligomers of 5,6 dihydroxyindole, the final oxidation product of dopamine. The possible origin of these differences is discussed on the basis of Cu2+ incorporation in Cu dopamine−eumelanin.
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INTRODUCTION Eumelanin is the brown-black ubiquitous pigment in vertebrates, where it plays an important role as a photoprotectant1,2 and as an antioxidant. It is also implied in pathologies like melanoma and Parkinson’s disease.1 In addition to its diverse functions in living organisms, eumelanin displays fascinating physical and chemical properties,4 among which its photoconductivity2 makes it a potential “biomimetic” material for optoelectronics.3 Its strong absorption over the entire UV− visible part of the electromagnetic spectrum and its extremely low fluorescence quantum yield4 will allow for the conversion of light into heat.5 Natural eumelanins are synthesized from the amino acid tyrosine, which undergoes an ortho hydroxylation, catalyzed through the enzyme tyrosine oxidase, to yield LDOPA. In the presence of an oxidant, among which is oxygen but also metal cations, this precursor undergoes an oxidation to © XXXX American Chemical Society
yield dopaquinone. After an intramolecular Michael addition and further oxidation steps, 5,6-dihydroxyindole (DHI) (Scheme 1) or 5,6-dihydroxyindole-2-carboxylic (DHICA) is obtained. These two molecules are extremely reactive and are the building blocks of eumelanins. The chemical pathway to the particulate eumelanin is however extremely complicated, because DHI (Scheme 1) can dimerize in many different manners yielding either 4,7′, 2,2′, or other dimers. The production of trimers and higher level oligomers of DHI or DHICA is even more complicated. Some DFT calculations highlight that among all of the isomers of the tetramers, the one having the configuration of a Received: April 13, 2013 Revised: September 1, 2013
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eumelanin in the rest of this Article, for convenience. Yet we recognize that it may be misleading to precede “eumelanin” with an indication of the used oxidant. Even if it may create some controversy, we prefer not to call these compounds “polydopamine” due to recent findings.8−10 It has been known for a long time that eumelanin interacts strongly with metallic cations,17 among which is Cu2+, and that iron(III) can lead to the oxidation of dopamine in anaerobic conditions.18 Herein, we investigate the Cu2+ triggered formation of Cueumelanin from three points of view: (i) The first is the kinetics of formation in the presence of Cu2+ and in the presence of O2. (ii) The second is the properties of the obtained Cu dopamine−eumelanin aggregates from a colloid science perspective, the size and the zeta potential of the aggregates versus the solution pH. It has already been shown that the nature of the used oxidant strongly influences the size distribution as well as the fractal dimension of the obtained eumelanin,19 but to our knowledge the influence of the used oxidant on the zeta potential of the eumelanin aggregates has not yet been investigated. (iii) The third is the structure of the Cu dopamine−eumelanin aggregates obtained by highresolution TEM, which will be compared to the onion-like structure of O2 dopamine−eumelanin.20 This investigation is hence aimed to highlight that eumelanins displaying different structures and properties can be obtained using different oxidants in a single one-step reaction. Note that Cu2+ is an oxidant strong enough (0.34 V vs the normal hydrogen electrode) to oxidize dopamine into dopaquinone, the standard redox potential of the dopaquinone/dopamine couple being close to 0.12 V versus the normal hydrogen electrode.
Scheme 1. Chemical Structure of DHI and Numerotation of Its Carbon Atoms
porphyrin is the most stable.6 Hence, the pathway to dopamine−eumelanin from such small oligomers leads to a heterogeneous7 but nevertheless robust material. More experimental and theoretical evidence points to the fact that eumelanin is not a linear polymer of DHI molecules. Using chemical degradation, d’Ischia et al. showed recently that the average degree of oligomerization of “polydopamine”, closely related to dopamine−eumelanin, is not higher than about 4−5.8 13 C NMR spectroscopy also showed the absence of 4−7′ covalent bridges in synthetic eumelanin prepared from dopamine solution in the presence of tris(hydroxymethyl) aminomethane buffer at pH 8.5 and in the presence of oxygen as an oxidant.9 In addition, in the so-called “polydopamine” compound, some of the products of dopamine oxidation are covalently bound, whereas others are associated through noncovalent selfassembly.10 This later study is particularly interesting to understand the structure of the so-called “polydopamine” films, which form spontaneously on almost all kinds of substrates when put in the presence of slightly alkaline dopamine,11 or the structurally related norepinephrine,12 solutions in the presence of dissolved oxygen. It has also been shown that similar eumelanin like coatings can be obtained on substrates using periodate13 or Cu2+ cations14 as oxidants. These coatings present exciting perspectives for applications in surface science, even if the detailed mechanism of their deposition is not yet understood.15 In the case of eumelanin like films produced from dopamine solutions in the presence of Cu2+ at 30 mM, it was found that their growth was much slower than the corresponding films obtained in the presence of oxygen in an open atmosphere.14 In addition, the final film thickness on silicon can be higher than 70 nm (after 80 h of reaction), whereas the film thickness saturates at about 45 nm in the presence of O2 and after 16−24 h of reaction,14 with all experiments being performed in the presence of a dopamine solution at 2 g L −1 . As a complementary finding, the dopamine−eumelanin films produced in the presence of Cu2+ cations exhibit a peak at 360− 370 nm, which is not present in eumelanin films produced in the presence of O2. These findings lead to the assumption that the synthetic eumelanin-like films produced in the presence of Cu2+ cations have a structure different from those produced in the presence of O2. This is highly probable because it has been demonstrated that the presence of Cu2+ favors the formation of 2,2′ dimers of DHI.16 It is the aim of this Article to show that synthetic dopamine− eumelanin produced in solution in the presence of Cu2+ at different concentrations displays different properties (optical and colloidal) as well as a structure different from that of eumelanin produced in the presence of O2. Both compounds will be called Cu dopamine−eumelanin and O2 dopamine−
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MATERIALS AND METHODS
Synthesis of Cu and O2 Dopamine−Melanin. The used chemicals, tris(hydroxymethyl)aminomethane (Euromedex, reference 26-1286-3094B), dopamine (Sigma, reference H. 8502, lot BCBG8676 V), and anhydrous copper sulfate (Aldrich, reference 62230, lot BCBH8060 V), were all purchased from Sigma Aldrich and used without further purification. All of the solutions were prepared from double distilled and deionized water (ρ = 18.2 MΩ cm, Milli QPlus, Millipore, Billerica, MA). The pH of the 50 mM Tris buffer was adjusted to 8.50 ± 0.02 before the dissolution of any other kind of solute. Dopamine was dissolved at a concentration of 2 g L−1 (i.e., 10.6 × −3 10 M) in the 50 mM Tris buffer. In the case where Cu2+ cations were used as the oxidant, the copper sulfate was dissolved at 30 or 60 mM before the dissolution of dopamine, which corresponds to t = 0 for the kinetics of Cu dopamine−eumelanin formation. In this case, the pH of the solution fell spontaneously to 4.5 ± 0.2, which is expected due to the Lewis acid character of Cu2+. Even if Tris does not play the role of a buffer any more in the presence of copper sulfate, we nevertheless worked in the presence of this molecule for the synthesis of Cu-eumelanin. Indeed, it has been shown that Tris can be covalently incorporated in synthetic eumelanin (produced from oxygenated dopamine solutions).8 The reaction vessel in which Cueumelanin was synthesized was kept closed and under an argon atmosphere. The reaction medium (100 mL) was shaken with a magnetic stirrer (300 rpm) for up to 288 h, and regularly small aliquots of the solution were removed for spectroscopic characterization. Before measurement of the absorption spectrum between 200 and 800 nm with a double beam Lambda 35 spectrophotometer (Perkin-Elmer), the solutions containing eumelanin and unreacted dopamine were diluted 20 times with distilled water. The reference quartz cuvette (Thuet, Blodelsheim, France) contained distilled water. Note that the reaction conditions of dopamine−melanin in the presence of Cu2+ cations correspond to a closed system from a B
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Structural Characterizations. A conventional high-resolution transmission electron microscopy (HRTEM) imaging and energy dispersive X-ray spectroscopy (EDS) were conducted using a JEOL 2100F TEM at 100 kV acceleration voltages of the electrons to study the molecular organization of synthetic dopamine−eumelanins. The composition of the dopamine−eumelanin grains synthesized in the presence of CuS04 30 mM was investigated by EDS. All of the samples were deposited on a Ni TEM grid. Degradation in the Presence of Sodium Hypochlorite. Both of the solutions containing O2 dopamine−eumelanin and Cu dopamine−eumelanin were diluted by a factor of 20 in a 0.1 M NaOH solution containing 1 g L−1 of sodium hypochlorite (NaClO), which is able to degrade the so-called “polydopamine” films.22 The used dopamine−eumelanins were synthesized as previously described, but the reaction time was 2 weeks at ambient temperature to ensure almost complete conversion of dopamine in dopamine−eumelanin (see the Results and Discussion). The dilution of the dopamine− eumelanin-containing solutions in the strongly oxidant NaOH− NaClO solution defines the time t = 0 for the kinetics of dopamine−eumelanin degradation. This degradation was followed by means of spectrophotometry at a constant wavelength of 300 nm during 2 h. One measurement was made every 30 s against a reference cuvette containing the NaOH−NaClO solution. To determine the difference in composition of both kinds of dopamine−eumelanins, both solutions underwent 24 h of oxidation in the presence of a 0.1 M NaOH solution containing 5 g L−1 of NaClO. The solutions were then filtered on Acrodisc membranes having a pore size of 200 nm before performing high performance liquid chromatography (HPLC) experiments. To that aim, both solutions were chromatographied by using the Ultimate 3000 Dionex system (Thermo Scientific Dionex, Villebon sur Yvette, France) with a Grace protein-peptide C-18 reverse phase (10 × 250 mm; particle size 3−20 μm and pore size 300 Angstrom) from Vydac (Deerfield, Ireland). The Chromoleon chromatography data system was used for the analytic process and the data collection. Elution was performed with a gradient of solvent B (0.1% trifluoroacetic acid in 70% acetonitrile−Milli-Q water) in solvent A (0.1% trifluoroacetic acid in Milli-Q water) as indicated on the chromatogram. The elution rate was 1 mL min−1, and the different peaks were detected at a wavelength of 340 nm at which both solutions issued from the degradation of dopamine−eumelanins displayed some absorbance.
thermodynamic point of view, because the initial ratio between the oxidant and dopamine was equal to 3 and 6 when the reactions were performed in the presence of 30 and 60 mM CuSO4, respectively. These ratios should nevertheless allow for a quantitative oxidation of dopamine because in the classical reaction mechanism leading to 5,6dihydroxyindole, dopamine undergoes three oxidation steps, each one implying 2 electrons, whereas each Cu2+ cation can be reduced to Cu gaining 2 electrons in this reduction step. In the case of O2 dopamine−eumelanin, the experimental conditions were the same with one major exception: the reaction vessel was left open to allow for a permanent supply of O2. For the synthesis of both Cu and O2 dopamine−eumelanins, some distilled water was regularly added to the reaction medium to keep the reaction volume constant and equal to (100 ± 1) mL. The reactions leading to Cu dopamine−eumelanin and to O2 dopamine−eumelanin were performed at 25 ± 2 °C. Dynamic Light Scattering and Zeta Potential Measurements. At the end of the reaction kinetics, when the UV−vis spectra of the Cu dopamine−eumelanin and O2 dopamine−eumelanins did not undergo significant changes with time, the solutions were removed from the reaction vessels and dialyzed against 1 L of Tris buffer at pH 8.5 during 24 h. The dialysis membrane had a molecular weight cutoff of 10 000 g mol−1 (Spectra Por) to allow for the small oligomers of dopamine or 5,6-dihydroxyindole to be removed from the reaction medium. In the case of Cu dopamine−eumelanin, the dialysis should also allow one to remove nonreduced Cu2+ cations as well as small Cu nanoparticules formed upon reduction of Cu2+ in Cu. We can however not exclude that larger Cu clusters remain in the reaction medium. After dialysis, the O2 dopamine−eumelanin containing solutions were titrated with diluted NaOH up to pH 10 and then titrated back to pH 3.5 using diluted HCl (0.1 M). At some particular pH values, the zeta potential was calculated from the measured value of the electrophoretic mobility using the Schmolukowski equation. Using the same device, a Nano ZS from Malvern, the hydrodynamic size distribution of the dopamine−eumelanin particles was determined by means of dynamic light scattering experiments. The hydrodynamic diameter of the particles was calculated from the diffusion coefficient obtained by fitting the intensity autocorrelation function by means of an inverse Laplace transform. For each sample, at least 10 successive zeta potential and dynamic light scattering experiments were performed. The given results correspond to the average value ± one standard deviation. Film Deposition on Quartz Slides. Quartz slides (4 cm × 1 cm × 0.1 cm) (Thuet, Blodelsheim, France) were cleaned by successive immersion in a 2% (v/v) Hellmanex solution (Hellma Gmbh, Müllheim, Germany) in the presence of ultrasonic agitation during 30 min, rinsed with distilled water, immersed in 0.1 M HCl solutions, and intensively rinsed with distilled water. This cleaning was performed just before the beginning of the dopamine−eumelanin film deposition. The quartz slides coated with O2 or with Cu dopamine−eumelanin were removed from their respective reaction medium after the given reaction times, rinsed with distilled water, and blown dry under a nitrogen stream. They were used for characterization by UV−vis spectroscopy and for X-ray photoelectron spectroscopy (XPS). The zeta potential of the O2 dopamine− eumelanin films versus pH curves were obtained using a ZetaCad device (Cad Instrumentation, Les Essarts le Roi, France) as detailed elsewhere.21 X-ray Photoelectron Spectroscopy (XPS). XPS analysis was performed with a Hemispherical Energy Analyzer SPECS (PHOIBOS 150) employing a monochromatic Al Kα radiation (1486.74 eV) operating at 200 W with an anode voltage of 12 kV. The pressure in the analysis chamber was fixed at 10−9 mbar. The pass energies were set to 80 and 20 eV for survey and higher resolution scans, respectively. The binding energy scale was calibrated from the carbon contamination using the C1s peak at 284.6 eV. Core peaks were analyzed using a nonlinear Shirley-type background. For the analysis of the high-resolution spectra, the peak positions and areas were optimized by a weighted least-squares fitting method using 70% Gaussian and 30% Lorentzian line shapes.
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RESULTS AND DISCUSSION As a function of the reaction time in the presence of either O2 from air or dissolved Cu2+ (in a deoxygenated solution), the spectral properties of the aerated dopamine solutions (Figure 1 of the Supporting Information) or of the degazed dopamine solution in the presence of 30 mM Cu2+ cations (Figure 2 of the Supporting Information) change with a progressive reduction in the absorbance of the peak at λ = 280 nm and an increase of the absorbance at longer wavelengths. Such an evolution is characteristic for the formation of eumelanins.23 However, in the presence of Cu2+ as an oxidant, the kinetics of the absorbance reduction at 280 nm is much slower (Figure 1) than that in the presence of air (with dissolved oxygen acting as an oxidant). Figure 1 displays the spectra of both solutions, after a dilution by a factor of 20 with distilled water, at the end of the dopamine−eumelanin formation kinetics, when no further spectral changes are observed (see Figures 1 and 2 of the Supporting Information). Additionally, one has to note that the presence of a residual peak at λ = 280 nm even after prolonged reaction time in the presence of oxygen may be due to the presence of either nonoxidized dopamine, small aggregates of this molecule, (dopamine)2-DHI non covalent clusters whose presence in eumelanin has been proven by Lee et al.10 Because of its reactivity in presence of oxygen and in basic conditions, the C
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Figure 2. Absorption spectra of dopamine−eumelanins deposited on quartz slides from dopamine solutions (2 g L−1 in the presence of 50 mM Tris buffer) using O2 from air (blue −) or Cu2+ (− at 30 mM) as the oxidant. The reaction time was of 23 h in both cases. The inset shows digital pictures of the coated quartz slides.
Figure 1. UV−visible absorption spectra of a dopamine solution (− · − · −), O2 dopamine−eumelanin solution (after 212 h of agitation, blue −), and Cu dopamine−eumelanin solution (−) after 240 h of agitation in a closed bottle. Initial concentration in copper sulfate: 30 mM.
absorbance of the O2 counterpart, whereas the absorbance of the oxygenated solutions is always higher than the absorbance of the solution containing Cu2+ cations (Figure 1 and Figures 1 and 2 of the Supporting Information). This may be due to different formation kinetics in solution and on the substrates. The particles issued from the oxidation of dopamine in solution were dialyzed against Tris buffer during 24 h to remove species of small molecular weight as well as small copper particles that may be obtained in the case of Cu dopamine−eumelanin. Note that after 260 h of reaction, some sediment was present at the bottom of the reaction vessel. This trend was particularly pronounced in the case of O2-eumelanin. This observation points for the formation of large sized aggregates that phase separate out from the solution. This is in line with the known insolubility of eumelanins. These large sedimented particles were discarded from further analysis by carefully removing the black-brownish solutions. The particles obtained at the end of the dialysis step were then titrated with diluted HCl or NaOH solutions, and their zeta potential as well as their size distribution were measured as a function of the pH. The characterization of the particles was only performed when the pH of the solution reached a constant value after titrant addition. This equilibration time was surprisingly long, up to 30 min in most experiments. The particles issued from the dialysis were also used for structural investigations using highresolution TEM. It appears that O2 dopamine−eumelanin displays the same zeta potential versus pH curve as dopamine− eumelanin deposited on glass slides from aerated dopamine solutions as long as the pH is lower than about 7 (Figure 3 of the Supporting Information). For higher pH values, the zeta potential of the film continues to decrease, whereas the zeta potential of the O2 dopamine−eumelanin particles plateaus at −25 mV above pH 8. The zeta potential versus pH curves are however markedly different for O2 and Cu dopamine− eumelanins (Figure 3). The point of zero charge shifts from 4 for O2 dopamine−eumelanin to 6.2 for Cu dopamine− eumelanin produced in the presence of 30 mM CuSO4. When the copper sulfate concentration is further increased to 60 mM, the point of zero charge shifts to an even higher pH, and the eumelanin particles barely acquire a negative zeta potential even upon a further increase in pH above pH 8.
presence of unreacted dopamine, free in solution, is highly unlikely. The residual peak is also broader than the peak of unreacted dopamine and slightly red-shifted (Figure 1). The absorption spectrum of O2 dopamine−melanin also displays a shoulder at around 420 nm, which may be attributed to the presence of DHI or other species resulting from the oxidation of dopamine. On the other hand, the spectrum of the Cu dopamine−eumelanin solution displays a shoulder at λ = 360 nm as for the spectra of Cu-eumelanin deposited on quartz slides.14 The spectra of Cu dopamine−eumelanin displayed in Figure 1 and in Figure 2 of the Supporting Information are nevertheless different from the spectrum of eumelanin produced in the presence of air and KOH from a solution of 5,6-diacetoxyindole and to which Cu2+ was subsequently bound.17 In this case, the spectrum of eumelanin displayed a peak at λ ≈ 420 nm, which was attributed to copper bound to quinone-imine groups of eumelanin as well as a peak at λ ≈ 240 nm attributed to Cu−catechol complexes. We also investigated the spectra of the compounds deposited on quartz slides put in the presence of the dopamine solutions in the presence of both air or Cu2+ cations (30 mM in copper sulfate). The substrates were intensively rinsed with distilled water and blown dry before characterization. It appears that both for O2 dopamine−eumelanin and for Cu dopamine− eumelanin, the peak close to λ = 280 nm attributed to (dopamine)2−DHI clusters is considerably reduced with respect to the peaks observed in solution (Figure 1). This means that those species are either free in solution or weakly bound to the dopamine−eumelanin clusters and can be easily removed by washing with distilled water. In addition, the peak observed at λ = 360 nm in the solution containing the Cu dopamine−eumelanin remains present in the spectrum of the deposited film. The same holds true for the shoulder observed at λ = 420 nm for O2 dopamine−eumelanin (Figure 2). It is also apparent from the inset in Figure 2 that the color of both compounds deposited on the quartz slides is markedly different: the deposited O2 dopamine−eumelanin appears black, whereas the Cu dopamine−eumelanin is brownish. It has to be noted that the absorbance of the Cu dopamine− eumelanin films is higher, after 23 h of deposition, than the D
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the O2 dopamine−eumelanin investigated in the present study.25 The higher is the concentration in Cu2+ used to oxidize melanin, the smaller are the particles. This may be related to the fact that the dopamine−eumelanin particles formed at a higher concentration in Cu2+ are stabilized against flocculation and aggregation due to the presence of an excess of Cu2+ cations. As another major difference, the Cu2+ dopamine−eumelanin is stable in the acidic pH range, whereas O2 dopamine− eumelanin is stable only above pH 6. To explain both spectroscopic differences (Figures 1 and 2), as well as the differences in the colloidal properties of dopamine−eumelanins (Figures 3 and 4) produced using O2 or Cu2+ as the oxidant, we make the assumption that the complexation of Cu2+ by catechol or quinone groups changes not only the self-assembly pathway of DHI but also its acidobasic properties. The catechols being bound to copper undergo a drastic pKa change and do not deprotonate up to pH 6 in the presence of 30 mM Cu2+ or do not deprotonate at all when the synthesis is performed at 60 mM in Cu2+. This assumption holds only true if Cu is detected in the Cu-eumelanin. Indeed, XPS spectroscopy detected Cu, mostly in the form of Cu2+ on the extreme surface of Cu dopamine−eumelanin films. The presence of CuO (Cu2+) rather than metallic Cu and Cu2O (Cu+) is confirmed26,27 on the high-resolution Cu2p XPS spectrum (Figure 5) by (i) a characteristic value of binding
Figure 3. Zeta potential versus pH titration for dopamine−eumelanin particles synthesized in the presence of air (○, O2 being the oxidant), in the presence of Cu2+ at 30 mM (blue ■) and 60 mM (red ⬢). The dopamine concentration was the same in all experiments (2 g L−1 in the presence of 50 mM Tris buffer at pH = 8.5), and the reaction time was 250 h before dialysis and pH titration. The error bars correspond to one standard deviation over at least 10 measurements on each solution.
The hydrodynamic diameter of the dopamine−eumelanin particles is also significantly affected by the reaction conditions as shown in Figure 4.
Figure 5. High-resolution XPS spectrum of a Cu dopamine− eumelanin film in the Cu2p region.
Figure 4. Hydrodynamic diameter versus pH for dopamine− eumelanin particles synthesized in the presence of air (○, O2 being the oxidant), in the presence of Cu2+ at 30 mM (blue ■) and 60 mM (red ⬢). The dopamine concentration was the same in all experiments (2 g L−1 in the presence of 50 mM Tris buffer at pH = 8.5), and the reaction time was 250 h before dialysis and pH titration. The error bars correspond to one standard deviation over at least 10 measurements on each solution. One measurement corresponds to the acquisition of one autocorrelation function.
energy for Cu2p3/2 at 934.5 eV (Cuo and Cu+ are visible at around 932−933 eV), (ii) a large full-width at half-maximum (fwhm) value of the Cu2p peaks (fwhm = 3.7 eV for Cu2p3/2) only observed for the CuO form, and (iii) the presence of a broad satellite (shake up) in the binding-energy range of 939− 946 eV, in addition to the main line, which only appears for CuO. The presence of copper in Cu dopamine−eumelanin was also confirmed by EDS analysis, which showed that the weight percentage in copper was of 0.49% (Figure 4 of the Supporting Information). The presence of Cu2+ in the Cu dopamine− eumelanin means that metallic copper, which is necessarily formed in solution during the oxidation of dopamine, is not significantly incorporated in the bulk of the material. The
It is found that the O2 dopamine−eumelanin particles, up to 5.5 μm in hydrodynamic diameter, are much bigger than the Cu2+ dopamine−eumelanin particles, which never exceed 3 μm in size. Note that the particle size determined in this study is consistent with the measurements reported in the literature24 even if the DHI-eumelanin characterized by D’Ischia et al. by means of DLS is somewhat smaller (1.1−1.2 μm in radius) than E
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Figure 6. High-resolution TEM images of O2 dopamine−eumelanin and Cu2+ dopamine−eumelanin (produced in the presence of 30 mM Cu2+ cations) represented in (a) and (b), respectively. In both cases, the reaction was performed in the presence of dopamine at 2 g L−1 and 50 mM Tris buffer.
presence of Cu2+ reflects the strong affinity of eumelanins for these cations.17 In addition, the structures of O2 and Cu dopamine− eumelanins are markedly different as shown in the highresolution TEM micrographs of Figure 6. When the synthesis of dopamine−eumelanin is performed in the presence of Cu2+ at 30 mM, the secondary structures present in O2 dopamine− eumelanin in the form of platelets and onion like organization are totally lost. Note that a similar secondary structure has been described by Meredith et al.28 We make the assumption that the particle-like organization in Cu dopamine−eumelanin originates from a different mode of organization of DHI and its oligomers in both types of dopamine−eumelanins. It has been demonstrated that DHI dimerizes favorably in the 2,2′ dimer in the presence of Cu2+,16 which could well explain the absence of platelet-like and onion structures in the Cu dopamine−eumelanin. Indeed, the presence of such secondary structures in O2-eumelanin was explained by the π stacking of tetramers of DHI having a porphyrin like configuration.29 The fact that Cu2+ changes the structure of dopamine−eumelanin goes in the same direction as the results obtained by Gallas et al. from small-angle X-ray reflectivity experiments.30 Of course, coordination of Cu2+ cations present in the Cu dopamine−eumelanin films through different coordination modes17 plays certainly a major role in the supramolecular organization of the corresponding particles and films. It is also possible that the lack of secondary structures in Cu dopamine−eumelanin is due to a lower degree of oligomerization of DHI units with respect to O2 dopamine− eumelanin. To investigate the mode of association of the Cu dopamine−eumelanin, we thought to use structural methods like NMR spectroscopy. Unfortunately, the use of 13C solidstate NMR spectroscopy on the Cu dopamine−eumelanin colloids is not possible due to the presence of Cu2+ cations, as revealed by means of XPS spectroscopy (Figure 5). If the structures of both dopamine−eumelanins are different, we make the assumption that their stability in the presence of a strong oxidant should also be different. To that aim, we investigated the degradation kinetics of O2 and Cu dopamine− eumelanins in the presence of 0.1 M NaOH solutions containing 1 g L−1 NaClO as an oxidizing agent. It appears that the decrease in absorbance at λ = 300 nm of the Cu dopamine−eumelanin solutions is much faster than that for the O2 dopamine−eumelanin solution (Figure 7). This clearly shows that O2 dopamine−eumelanin is much more stable in the presence of strong oxidants than Cu dopamine−eumelanin. To show that both dopamine−eumela-
Figure 7. Degradation kinetics of O2 (−) and Cu (blue −) dopamine−eumelanins in the presence of 0.1 M NaOH and NaClO at 1 g L−1 as followed by means of UV−vis spectroscopy at λ = 300 nm. The concentration of both eumelanins was the same, and the absorbance was set at 0 at time t = 0 when the dopamine−eumelanin containing solution was diluted with the solution containing the oxidant.
nins also have different compositions, they were submitted to a longer degradation reaction, 24 h in the presence of a 0.1 NaOH solution containing 5 g L−1 of NaClO. These solutions were then filtered to remove residual colloids and were analyzed by HPLC. The chromatograms of the degradation products of O2 dopamine−eumelanin and of Cu dopamine− eumelanin are significantly different, as shown in Figure 5 of the Supporting Information. More precisely, there are three major differences between the two chromatograms: (i) Concerning the two peaks at retention times between 8 and 10 min, the Cu dopamine−eumelanin displays a first peak that is more intense than the second one, whereas the peak intensities of these two peaks are reversed for the degradation products of O2 dopamine−eumelanin. (ii) The chromatogram of the degradation products of Cu dopamine−eumelanin displays an important peak at a retention time close to 27 min; such a peak is absent in the solution issued from the degradation of O2 dopamine−eumelanin. The same holds true for some additional small peaks at higher retention times. (iii) The peak eluting at 37.5 min is much more intense in the case of Cu than in the case of O2 dopamine−eumelanin degradation products. F
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Overall, it appears that the degradation products of Cu dopamine−eumelanin are more hydrophobic, eluting at higher retention times, than those of the corresponding O 2 dopamine−eumelanin. Because those degradation products are expected to be pyrrole dicarboxylic and pyrrole tricarboxylic acids,31 we would expect that the O2 dopamine−eumelanin leads to a higher proportion of the more polar pyrrole tricarboxylic acids, which is not inconsistent with the presence of more highly branched structures than in Cu dopamine−eumelanins. Accordingly, this finding is not inconsistent with the different structures found by HRTEM images in Figure 6: the presence of platelet and onion-like structures in O2 dopamine−eumelanin is only possible when the oligomers of DHI are highly cross-linked, leading more probably to a higher fraction of pyrrole tricarboxylic acid upon degradation. Unfortunately, we were not able to perform electrospray ionization mass spectrometry on those solutions due to the presence of a too high concentration in NaClO, leading to a fast contamination of the focusing lenses in the mass spectrometer.
CONCLUSIONS In this investigation, we demonstrated that synthetic dopamine−eumelanins produced from dopamine solutions in the presence of oxygen or Cu2+ cations as oxidants display different optical and colloidal (size distribution and zeta potential titration curves) properties. In addition, their formation kinetics is markedly different. The colloids obtained at the steady state of the oxidation kinetics display also a markedly different structure: the O2 dopamine−eumelanin is essentially made of onion-like structures, which are not present in the Cu dopamine−eumelanin. We make the assumption that Cu2+ cations modify markedly the dimerization kinetics of 5,6dihydroxyindole to yield dopamine−eumelanins with different structures and properties. Indeed, the Cu dopamine− eumelanin contains copper in the form of Cu2+ cations as evidenced from XPS spectroscopy. In future investigations, we aim to simulate the self-assembly of Cu dopamine−eumelanin by means of molecular dynamics, making assumptions about the structure of small DHI oligomers. ASSOCIATED CONTENT
S Supporting Information *
Absorption spectra of dopamine/O2-eumelanin and dopamine/ Cu eumelanin containing solutions, after different reaction times, zeta potential versus pH titration curve for O2-eumelanin particles and for O2-eumelanin films deposited on glass, EDAX analysis of Cu-melanin produced from a 2 mg mL−1 dopamine solution in the presence of 50 mM Tris buffer and 30 mM copper sulfate, and chromatograms of the solutions issued from the degradation of O2 and Cu dopamine−eumelanins. This material is available free of charge via the Internet at http://pubs.acs.org.
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