Preparation of Thin Melanin-Type Films by Surface-Controlled

Apr 6, 2016 - The preparation of thin melanin films suitable for applications is challenging. In this work, we present a new alternative approach to t...
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Preparation of Thin Melanin-Type Films by Surface-Controlled Oxidation Mikko Salomak̈ i,*,†,§ Matti Tupala,† Timo Parviainen,† Jarkko Leiro,‡,§ Maarit Karonen,† and Jukka Lukkari*,†,§ †

Department of Chemistry and ‡Department of Physics and Astronomy, University of Turku, FI-20014 Turku, Finland Turku University Centre for Materials and Surfaces (MatSurf), Turku, Finland

§

S Supporting Information *

ABSTRACT: The preparation of thin melanin films suitable for applications is challenging. In this work, we present a new alternative approach to thin melanin-type films using oxidative multilayers prepared by the sequential layer-by-layer deposition of cerium(IV) and inorganic polyphosphate. The interfacial reaction between cerium(IV) in the multilayer and 5,6-dihydroxyindole (DHI) in the adjacent aqueous solution leads to the formation of a thin uniform film. The oxidation of DHI by cerium(IV) proceeds via known melanin intermediates. We have characterized the formed DHI−melanin films using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), UV−vis spectroscopy, and spectroelectrochemistry. When a five-bilayer oxidative multilayer is used, the film is uniform with a thickness of ca. 10 nm. Its chemical composition, as determined using XPS, is typical for melanin. It is also redox active, and its oxidation occurs in two steps, which can be assigned to semiquinone and quinone formation within the indole structural motif. Oxidative multilayers can also oxidize dopamine, but the reaction stops at the dopamine quinone stage because of the limited amount of the multilayer-based oxidizing agent. However, dopamine oxidation by Ce(IV) was studied also in solution by UV−vis spectroscopy and mass spectrometry in order to verify the reaction mechanism and the final product. In solution, the oxidation of dopamine by cerium shows that the indole ring formation takes place already at low pH and that the mass spectrum of the final product is practically identical with that of commercial melanin. Therefore, layer-by-layer formed oxidative multilayers can be used to deposit functional melanin-type thin films on arbitrary substrates by a surface-controlled reaction.

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INTRODUCTION Melanins are high molecular mass dark brown or black pigments which are formed by oxidative oligomerization of phenolic compounds bearing catechol moieties. Naturally occurring melanins, i.e., eumelanins, pheomelanins, neuromelanins in animals, and pyomelanins and allomelanins in plants, differ slightly in their chemical structure but share many similar biological functions. They are biological macromolecules with many identified and unidentified roles in the biosphere, serving as pigments,1 photoprotectors, and antioxidants.2 In addition, they have also other functions in living organisms, which are not thoroughly understood. Recently, eumelanins especially have become a subject of interest in materials chemistry due to their electrochemical,3 photochemical,4 and adhesion properties.5 In general, melanins possess several interesting physical and chemical properties,6 but the structure−property relationships are in many cases inadequately understood. Catechol reacts easily with amines and thiols and forms complexes with several metals and strong hydrogen bonds between surfaces and other molecules.7 In polymers, catecholcontaining groups are effective cross-linkers, and catechol groups help to attach several marine invertebrates tightly to © 2016 American Chemical Society

various surfaces. An extremely simple one-step coating process to form a catechol-based biomimetic melanin-type film was described by Lee et al.5 Their procedure uses a biological catechol derivative, dopamine, and utilizes the adhesion properties of catechols to synthesize a robust polydopamine film by simply putting the target material into a buffered, slightly basic solution of dopamine. Under these conditions, dopamine is slowly oxidized by atmospheric oxygen to a kind of synthetic eumelanin, both in the solution and on the surface of the immersed material. The initial reaction steps leading to the oxidative oligomerization of dopamine are well-known.8 Under successful conditions, the reaction proceeds further to dopamine− melanin, a material which has an as yet unresolved and debated structure.9 According to the generally accepted mechanism (Scheme 1), the reaction starts with the oxidation of dopamine to dopamine quinone (DQ), which then undergoes a cyclization reaction to form a heterocyclic indole, leukoaminochrome (LAC). Further oxidation of LAC leads to the Received: February 2, 2016 Revised: April 4, 2016 Published: April 6, 2016 4103

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dissolved in concentrated aqueous ammonia solutions can be spin-coated on silicon substrates to form thin high-quality films.13 However, ammonia may react with the catechol moieties in melanin and also destruct the structure of the material. In this work, we present an alternative method to obtain thin melanin-type films and study the surface-controlled oxidation of melanin precursors. We utilize the sequentially layer-by-layer (LbL) self-assembled oxidative films, which have been shown to be a general platform for the electrodeless polymerization of conducting polymers to form a conducting film on an inorganic material.23,24 The layer-by-layer technique is a general technique for a controlled and reproducible preparation of thin films, and the technique is practically insensitive to the nature, size, or form of the substrate.25 The thickness of the LbL prelayer can be tuned at nanometer precision, which provides an easy and universal way to control the amount of oxidant in the reaction. The goal is to generate a protocol for a controlled oxidative production of smooth melanin-type films on a substrate of choice.

Scheme 1

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formation of the corresponding quinone, aminochrome (AC). Dihydroxyindole (DHI) is formed by tautomerization of AC and is further oxidized to indole quinone (IQ). In the literature, the observed oxidative oligomerization products of DHI are mainly dimers, trimers, and tetramers.10 The oligomeric species are responsible for the further growth of the amorphous polydopamine particles. The molecular structure of eumelanin is not fully identified, and the relationship between the structure and biological role is being debated. Because melanin is an amorphous material, it is not possible to clearly distinguish between properties originating from the molecular structure and the supramolecular arrangement of the molecules. Melanins cannot be described as classic polymers since they do not possess welldefined repeat units, and the term polymer in connection with them is apparently misleading.11 The molecular dynamics simulations suggest that melanins consist of oligomeric protomolecules with intermolecular stacking interactions,12 which lead to higher secondary structures. Tertiary structures are then formed by noncovalent interactions between these secondary structures, and the final products are amorphous particles that are insoluble in most solvents. However, it is possible to generate stable dispersions in basic aqueous solutions because of the negative surface potential of the particles under these conditions.13 High-quality thin melanin films would be important for applications, especially in electronic devices.13 Relatively thin polydopamine films can be obtained by the spontaneous airinduced oxidation of dopamine. However, the method is timeconsuming,14,15 and the generated films show an uneven, granular morphology as they form by a three-dimensional island growth mechanism (Volmer−Weber mechanism).16 In addition, the process is not restricted to the surface but takes place also in the solution, thereby wasting a large part of the material. Many modifications of the original recipe have been studied in order to improve and control the film quality and properties. In the original method, the oxidant is dissolved oxygen, but several different oxidizing agents14,17−19 and variation of pH,20 temperature,21 and dopamine concentration20 have been tested, and in some cases, very thin melanintype films have been formed by controlled spontaneous oxidation of dopamine.15,22 On the other hand, melanin

EXPERIMENTAL SECTION

Preparation of Coating Solutions for the Layer-by-Layer Deposition. Potassium polyphosphate (potassium metaphosphate, ABCR) was solubilized (10 mM, referring to the monomer concentration) in an aqueous solution of 0.1 M NaCl. The solution was stirred for approximately 18 h before use. Cerium(IV) ammonium nitrate (Merck) was solubilized in water as 10 mM solutions. The pH of the cerium solution was unadjusted and was approximately 1.5. Oxidative Multilayer Deposition. At each deposition step the coating solution was allowed to stay in contact with the substrate for exactly 15 min and was then thoroughly rinsed with water three times. These steps were repeated sequentially with anionic (polyphosphate) and cationic (cerium) coating solutions until the required number of layers was obtained. Melanin Precursors. Dopamine hydrochloride (Sigma-Aldrich), was used as 1 mM aqueous solution adjusted to pH 1.5 (with sulfuric acid) or pH 4.5 (with 10 mM acetate buffer). 5,6-Diacetoxyindole (TCI Chemicals) was deacetylated under an oxygen-free atmosphere. Ca. 3 mg of 5,6-diacetoxyindole was dissolved in 1 mL of deoxygenated ethanol, and 2 equiv of NaOH in 1 mL of deoxygeneated water was added to the solution, which resulted in a solution of very light bronze color. Thereafter, 5 mL of 10 mM acetate buffer (deoxygenated, pH 4.5) was added to adjust the pH. The solution was used immediately for the DHI−melanin film synthesis. DHI−Melanin Film Formation. A quartz flow-through cuvette was used to follow dopamine and DHI oxidation by UV−vis spectroscopy. The cuvette was cleaned with piranha solution (a 3:1 (v/v) mixture of concentrated sulfuric acid and 30% hydrogen peroxide solution; warning: piranha solution is very corrosive and should not be stored in tightly closed vessels), rinsed with water, dried, and silanized using a 5% (v/v) solution of N-(trimethoxysilylpropyl)N,N,N-triethylammonium chloride (ABCR) in methanol for 5 min. The kinetic measurements of the film formation were carried out at 25 °C. Film and Product Characterization. Spectroelectrochemical experiments were performed in a cuvette using a DHI−melanin film covered ITO glass as a working electrode in a conventional singlecompartment cell with a three-electrode configuration. The multilayer film was deposited on ITO glass slide treated similarly as the quartz cuvette. The reference and auxiliary electrodes were a miniature Ag/ AgCl electrode and a platinum wire, respectively. Autolab PGSTAT101 potentiostat was used in the measurements. For the XPS, AFM, and SEM measurements the film was deposited on a silicon wafer treated similarly as the quartz cuvette. The XPS spectra were recorded on a PerkinElmer 5400 small-spot ESCA spectrometer using twin anode with Mg Kα radiation and the takeoff angle of 45°. The surface stripping analysis was conducted using argon 4104

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Langmuir sputtering of the film by a 1 mA current for 30 s. The spectra were analyzed using the Unifit 2013 software (Unifit Scientific Software GmbH, Leipzig, Germany). The morphology of the films was studied by scanning electron microscopy, SEM (LEO Gemini 1530), and atomic force microscopy, AFM (diCaliber AFM, Bruker), operated in the tapping mode. High-resolution MS analyses were performed on an electrospray quadrupole time-of-flight mass spectrometer (micrOTOFQ, Bruker Daltonics, Bremen, Germany). The mass spectrometer was controlled and the data were analyzed using Compass DataAnalysis Software (version 4.0 SP 5, Bruker Daltonics). The mass spectrometer was operated in both the negative and positive ion mode. The following settings were applied to the instrument: capillary voltage, 4000 V (negative), 4500 V (positive); end plate offset, −500 V; heated drying gas (N2) 8 L/min, 200 °C; the nebulizer gas (N2) 1.6 bar. For the MS analysis the melanin precipitate was solubilized in 25% ammonia (LCMS grade, Fluka) by using a tip sonicator (Hielscher UP100H, 125 W/cm) for 15 min. The sample was diluted to 5% (referring to ammonia) with distilled water before the analysis. Similar solution was prepared from commercial synthetic melanin (Sigma-Aldrich) for reference.

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RESULTS AND DISCUSSION Oxidation of Dopamine by Cerium(IV) in Film. The oxidative multilayer consisting of five bilayers of Ce(IV) and polyphosphate, (PP/Ce)5, was prepared on quartz or silicon substrates as described in our previous work.23,24 The thickness of a dry film was 12 nm, and these ultrathin films have been shown to possess sufficient oxidative capacity for the deposition of a thin conducting polymer film. The surface-controlled oxidation of dopamine, in dilute degassed aqueous solution and at controlled pH, by a (PP/Ce)5 multilayer, was followed spectroscopically. The spectra recorded during oxidation at two different pH’s (1.5 and 4.5) are shown in Figures 1a and 1b. In both cases, absorbance rapidly decreases around 300 nm, due to the reduction of Ce(IV) to Ce(III),23,24 and new bands appear at 400 or 480 nm at pH 1.5 or 4.5, respectively. On the basis of the absorption maxima, the oxidation products can be identified as dopamine quinone (DQ), at pH 1.5, and aminochrome (AC) at pH 4.5 (the second absorption maximum of AC at 300 nm is masked by the decrease of cerium absorption).26 Rinsing with water removes these features in the spectra, showing that the reaction products are not attached to the film. The reduction of cerium(IV), as indicated by the rapid drop of absorbance around 300 nm, is practically instantaneous in both cases. However, the growth of the new bands due to the oxidation products takes place much slower, requiring some half an hour to attain the final value. Therefore, the overall reaction can be divided into fast and slow steps, and the oxidation of dopamine is not a straightforward two-electron process. Cerium(IV), which strongly binds the catechol group in dopamine, is a very efficient single electron oxidant,27 but two electrons must be removed from dopamine for its complete oxidation. Although cerium(III) is not as good a complexing agent as Ce(IV), and the oxidation product may dissociate from the metal,, the product cannot react with another Ce(IV) atom because all oxidant is rapidly exhausted in the film due to the great excess of dopamine in solution. Therefore, we assume that cerium oxidizes dopamine first to a semiquinone form. The slow steps leading to dopamine quinone and aminochrome with absorbance at ca. 400 and 480 nm, respectively, result from the disproportionation reaction between two dopamine semiquinone molecules producing dopamine quinone and dopamine. At pH 1.5, these are the final products, but at pH 4.5

Figure 1. Spectra recorded during the reaction of a dopamine solution with an oxidative Ce/PP film at pH (a) 1.5 and (b) 4.5.

cyclization of dopamine quinone produces aminochrome. The cyclization by Michael addition requires an unprotonated amino group, but the pKa of the group in dopamine is 10.6, which should not allow the LAC or AC formation at this pH.28 However, indole formation has been observed at low pH also when using Cu(II), another catechol coordinating oxidant.17 We suggest that metal coordination may enhance the cyclization reaction by stabilizing the quinone imine resonance structure in the semiquinone intermediate with a closed nitrogen-containing five-membered ring. In addition, rapid formation of the LAC/AC redox pair has been observed during the electrochemical oxidation of dopamine at pH above 5.5.29,30 The redox potential of this couple is ca. 400 mV more cathodic than that of dopamine/dopamine quinone, and the latter can oxidize LAC to the observed AC. The proposed reaction scheme is presented in Scheme 2. The reaction products were rinsed from the cuvette and analyzed using electrospray mass spectrometry. The major constituent of the more acidic solution (pH 1.5, Figure S1) was dopamine (m/z = 154) because of its large excess in the solution, but a small peak at m/z 152 due to dopamine− quinone was observed, too. Although the redox potential of quinones is the more anodic the lower the pH, the (Ce/PP) multilayer can oxidize dopamine under these acidic conditions, but the intramolecular cyclization to leucoaminochrome is prevented at this low pH. At pH 4.5 (Figure S1), a new peak at m/z 150 was observed. The mass spectrum alone does not allow to differentiate between AC and DHI, but this peak can be assigned mainly to aminochrome, based on the UV−vis spectrum of the solution. DHI would have an absorption maximum at 295 nm but not at longer wavelengths (Figure 1b), and its formation cannot be completely ruled out at higher pH.26,31 At both pH, the reaction was terminated before the 4105

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Figure 2. Absorbance changes (normalized between 0 and 1) as a function of pH in a Ce(IV)−dopamine (10:1) aqueous solution at 300 (○), 395 (□), 480 (△), and 620 nm (▽). Lines are drawn only as a guide to the eye.

formation of any melanin-type oligomers or film formation because of the limited oxidation capacity of the ultrathin multilayer film. This is in striking contrast to the previous work, which has shown that conducting polymer films can be formed on similar thin oxidative multilayers.23,24 The one-electron oxidation of monomers of conducting polymers creates a reactive radicals, and the polymer is formed in an avalanche of radical coupling reactions. In this case, the oxidant has only to produce a high enough local concentration of monomer radicals, and the further redox processes can take place between reactive oligomers of different length. In the case of dopamine, the initial radical formed (semiquinone) can only produce the oxidized quinone, and the reactivity of this quinone or its cyclized product is not high enough to sustain the reaction without any external oxidant. The immersion of the Ce(IV)/PP multilayer in a dopamine containing solution destroys the film as evidenced by ellipsometric measurements. No multilayers can be grown using cerium(III) and polyphosphate, and without the rapid formation of a shielding polymer film, the reduced Ce/PP multilayer is shattered and lost into the solution. Some cerium may also be directly dissolved from the film as a result of complex formation with dopamine. Oxidation of Dopamine by Ce(IV) in Solution. The solution pH is clearly an important parameter in the polydopamine generation. In order to find out under which conditions cerium(IV) can produce melanin-type materials and to eliminate the problem of oxidant shortage, the oxidation reaction was studied in solution by UV−vis (Figure 2) and mass spectroscopy as a function of pH. The highly acidic (pH 1.5) dilute Ce(IV)−dopamine (10:1 mole ratio) solution has a slight yellow color and absorbance below 400 nm due to soluble Ce(IV) species. The MS analysis of this solution showed that the dominant species is dopamine quinone, in accordance with the results above (Figure S1), but in this case, no dopamine is left in solution because of a large excess of cerium(IV), which remains available for further oxidation reactions. As the pH was gradually increased to pH 5 by the addition of a strong base (NaOH), the solution turned slightly opaque due to clouding. This was seen as a rapid decrease of absorbance below 350 nm and increase of it at long wavelengths, caused by the formation of scattering Ce(IV) oxide particles. Simultaneously, there was a transient increase of absorbance at 395 nm, which signifies the formation and disappearance of dopamine quinone. In the range from ca. pH 5 to pH 8 the color of the solution changed from orange to blue, and there was an increase of absorbance at 300, 480, and

620 nm, attributed to the formation of aminochrome (300 and 480 nm) and oxidized dihydroxyindole. Finally, at ca. pH 8, there was a rapid increase in scattering accompanied by the decrease of aminochrome absorption at 300 nm. At the same time, visible particles were detected, indicating the formation of insoluble aggregates, evidenced by the rapid increase of scattering in the spectrum at all wavelengths. The aggregates, which slowly sediment to the bottom, become very dark, especially when higher concentrations of dopamine are used, and represent the final product of Ce-based dopamine oxidation. The observed formation of various intermediate species at different pH is well in accordance with the MS studies and the previous observations carried out at two different pH and temperature as a function of time.26 Product Analysis of the Cerium-Based Oxidation. The insoluble material formed at the end of the reaction of dopamine with excess Ce(IV) was treated with ammonia to produce a stable aqueous dispersion.13 Commercial synthetic melanin, similarly dispersed, was used as a reference material, and both were analyzed using electrospray mass spectrometry (Figure 3 and Figure S2). The major peaks found in the mass spectra are listed in the Table S1 and indicated also in Figure 3. The most important conclusion is that the same major peaks are found in both spectra. This is a clear indication that both

Figure 3. Positive ion electrospray MS spectra of commercial synthetic melanin and Ce(IV)-based melanin material dissolved in 5% ammonia solution. Only the major peaks are labeled. 4106

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dopamine as a reagent because of the limited amount of available oxidant in the film. With oxidative films, 5,6dihydroxyindole would be a better choice as it is a higher oxidation product of dopamine and is thought to represent the true monomeric unit of melanin. Unfortunately, because of the low stability of DHI toward spontaneous oxidation by dissolved oxygen, an acetoxy protected derivative must be used. The protecting groups were cleaved in an oxygen-free atmosphere, and the formed DHI was stabilized in deoxygenated acetate buffer at pH 4.5 immediately prior to exposure to an oxidative film. Upon contact of the DHI solution with the (PP/Ce)5 multilayer under oxygen-free conditions, a dark coating was formed on the surface of the substrate, and the process was completed in ca. 1 h. The color of the solution remained unchanged, which shows that the reaction was restricted on the film surface. The dark coating could not be removed from the surface by rinsing. In the following, we call this material DHI− melanin and characterize it by several techniques. Figure 4 shows the spectra of the formation of DHI melanin film on a PP/Ce multilayer. After an initial fast stage the

materials are basically similar although the commercial material is synthesized from tyrosine and therefore might contain carboxylic acid moieties in the dihydroxyindole units. This difference is probably reflected in the large amount of minor peaks with a higher m/z ratio than the major products. The identification of the major peaks is rather challenging, but a small DHI peak can be found near m/z 149. The experimental conditions might favor the detection of only the degradation products caused by the ammonia treatment, and the intact melanin material within the aggregates may be invisible in the electrospray analysis. In fact, we are unaware of any previous mass spectrometry analysis of ammonia solubilized melanin. The MS spectra of both the synthetic melanin and Ceoxidized melanin (Figure 3) seems to contain a series of major peaks at m/z 1037 (418 + 619), 836 (418 + 418), 619 (201 + 418), 418 and 201. The spectra were measured using positive ionization, and the observed m/z values should represent species of the form [M + H]+ or [M + M′ + H]+. Even though the integer m/z value sums coincide in the series of the peaks above, there is a mismatch in the decimal part, which implies that the elemental contents of the proposed constituent species must be different. However, there is still a possibility that the peaks are due to degradation of larger molecules. The peak m/z 418.09 is in excellent accordance with a trimer of two DHI and one pyrrole carboxylic acid (PCA), a structure that has been suggested by Ding et al.32 and a similar one for a DHI− carboxylic acid oligomer by Napolitano et al.33 The peaks at m/ z 201.10 and 108.04 can be attributed to the degradation products of the trimer (Scheme 3). The peaks with higher m/z Scheme 3

Figure 4. Absorbance changes during the formation of a DHI− melanin film on a (PP/Ce)5 multilayer in a deoxygenated pH 4.5 acetate buffer. The spectrum of (PP/Ce)5 is taken as reference. The first spectrum taken after 2 min followed by spectra at 5 min intervals. The inset shows the film spectrum (referenced to the spectrum before DHI addition) after water rinsing (the large dip at ca. 300 nm is due to the loss of Ce(IV) in the film).

values are more difficult to analyze. Speculatively, the peak at m/z 619 could represent a combination DHI2PCA2Pyrrole. The pyrroles in the chain were also suggested for DHI− carboxylic acid oligomer,33 but in this case, there would be only one carbon between the pyrrole units because of different bonding between the indoles. Consequently, the m/z 619 peak might represent a pentamer with three ring fissions. For the two peaks with the highest m/z values, we tentatively suggest that 836 represents a hexamer with two ring fissions and 1037 an octamer with four ring fissions. However, these peaks may well be various stacked combinations of smaller species. A model based on mass spectroscopic results of DHI−melanin degradation products suggests that the tetramers, pentamers, and hexamers are the major constituents of the material and the fraction of oligomers higher than octamers is insignificant.34 Formation of a Melanin-Type Film. The studies reported above suggest that reactive melanin precursor molecules and melanin-type material can be produced from dopamine by cerium oxidation. However, with oxidative multilayers, the controlled deposition of a melanin material is not possible using

reaction follows the (pseudo) first-order kinetics (Figure S21), in accordance with conducting polymer formation.23,24 The spectrum of the reaction product exhibits maxima at ca. 500− 550 and 620 nm, with a small maximum around 800 nm. With time, the absorbance grows above 600 nm relative to that below. Based on the melanin absorption coefficient in the literature, the ca. 0.1 AU increase at 620 nm implies a film thickness close to 11 nm.13 However, the spectrum differs from that generally reported for synthetic or natural melanin. The featureless UV−vis absorption spectrum typical of eumelanin is believed to result from the heterogeneous nature of the material.35 The DHI dimers exhibit maximum absorbance in the range 500−550 nm and above 650 nm36,37 On the other hand, oxidized poly(5,6-dimethoxyindole-2-carboxylic acid) films have a wide absorption band between 600 and 700 nm.38 This suggests that after the initial formation of DHI dimers the oxidation process prefers certain oligomeric species in their oxidized state, which then stack together in the film. 4107

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Langmuir The spectroelectrochemical studies of the film (vide inf ra) support this conclusion. Film Structure. Although the exposure of the PP/Ce multilayer to a DHI solution produces a visible dark stable coating on the surface, neither AFM nor SEM does not reveal marked surface changes in comparison to an unreacted (PP/ Ce)5 film. The morphology of the PP/Ce multilayer film consists of characteristic granular features with the size of ca. 30−40 nm (Figure 5a,c).23 The film formation does not

Figure 5. AFM (a, b) and SEM (c, d) images of a (PP/Ce)5 multilayer before (a, c) and after (b, d) the formation of a DHI−melanin film.

noticeably affect the feature size. In the AFM analysis, the rootmean-square roughness increases slightly from 1.1 to 1.8 nm as a result of the film deposition. The SEM images reveal some more details. Before the film deposition the granular structure of the PP/Ce multilayer is clearly visible. The most evident change after the DHI oxidation and film formation is the increased haziness in the image, which obscures the underlying features (a similar but weaker effect can be anticipated in the AFM images). However, the hazy surface film appears smooth and uniform without any characteristic features. Melanin normally appears in the form of nanoparticles, and such particles can be seen at high magnification also in smooth high quality melanin films reported in the literature.13 The basic difference between the reported films and the one formed on the oxidative multilayer is the film thickness. In this case, the thickness is of the order of 10 nm (vide inf ra), which is smaller than the characteristic size of melanin-based nanoparticles.39 Fundamentally, the difficulty of film imaging by SEM can be attributed to the much lower electron density of the deposited organic film compared to that of the underlying inorganic multilayer. The structural analysis of the DHI−melanin film is challenging, and for example, MALDI measurements of the thin film were completely unsuccessful. Instead, XPS was used to study the chemical composition of the films. Figure 6 shows the Ce 3d, N 1s, and the low binding energy (BE) regions in the survey spectra of the oxidative (PP/Ce)5 multilayer and the intact and argon-sputtered (PP/Ce)5(DHI−melanin) films. The deconvoluted high-resolution spectra of the O 1s, N 1s, C 1s, and other relevant regions are shown in the Supporting Information, and the results are collected in Table S2.

Figure 6. XPS spectra of the (PP/Ce)5 multilayer (black, top), the (PP/Ce)5(DHI−melanin) film (red, bottom), and the sputtered (PP/ Ce)5(DHI−melanin) film (green, center) in the (a) N 1s, (b) Ce 3d, and (c) low BE region.

The spectrum of the (PP/Ce)5 multilayer showed peaks for cerium, oxygen, and phosphor as assumed, together with an impurity carbon signal. In addition, signals originating from the underlying silicon and silicon oxide surface could be seen. However, no nitrogen signal was detected. The analysis of the Ce 3d, O 1s, and P 2p photoelectron peaks yielded results identical to those previously reported for such an oxidative multilayer.23 In a previous study, substrate signals were observed also after the deposition of a thin polydopamine film by successive dipping in fresh dopamine solutions, and they were attributed to structural changes of the film in the XPS chamber.15 On the other hand, the spectrum of the (PP/ 4108

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Langmuir Ce)5(DHI−melanin) film showed peaks only for oxygen, nitrogen, and carbon. All these signals remained after Ar sputtering, which also revealed peaks attributed to various cerium photoelectrons. The spectra show a relative loss of Ce(IV) species in the sputtered film, which has been used for the formation of DHI−melanin, a further indication of the redox nature of the film formation (see Figure S11). The absence of cerium or phosphor (or silicon) signals in the spectrum of the DHI−melanin covered oxidative multilayer shows that this film is uniform and thick enough to suppress the photoelectrons from the underlying layers. The attenuation of the photoelectron signal can be used to estimate the film thickness d according to the equation

oxidation of dihydroxyindole. In addition, the observation of only one nitrogen species, which can be assigned to pyrrolic nitrogen, indicates that the 5-membered ring of indole also stays intact in the oxidation. On the other hand, the N 1s spectrum of the argon-sputtered DHI−melanin film clearly displays two nitrogen species. One of them at 400.6 eV is the original pyrrolic nitrogen, but another and larger peak appears at 399.1 eV. This can be assigned to neutral primary amine group, and its appearance indicates the opening the of indole ring during the sputtering process.42 Sputtering does not affect the C 1s or O 1s regions markedly, except that a signal due to ceria appears as the underlying oxidative multilayer is exposed. Interestingly, all phosphate seems to be removed. This may be caused by sputtering or polyphosphate degradation and the decrease of cerium complexing power upon reduction (no multilayers can be formed using Ce(III) and polyphosphate). It should be noted that the interpretation of the XPS spectra is complicated by the significant variation of the reported binding energies in melanin and polydopamine samples.9,15,22,32,39,43−46 For example, the BE assigned to the aromatic C−CH carbons varies by ca. 1.5 eV, and similar variation can be found for other carbon (and oxygen) species, too. In most cases, the adventitious carbon signal provides the only method for the binding energy calibration. This signal is generally observed in the range 284.7−285.2 eV, and a value 284.8 or 285.0 eV is often used (the latter especially for polymer samples) for setting the BE scale. The C 1s peak observed at the lowest BE in this work (285.2 eV) can be assigned to a combination of adventitious and aromatic carbon. This shows that any charging effects in our spectra are within the instrumental error margins (ca. 0.3 eV), and therefore we have not applied any correction to the observed and deconvoluted BE values. On the other hand, the reported binding energy differences for various carbon species are more consistent. The signals assigned to C−O/C−N and CO are found at 1−1.5 and 3−3.8 eV higher BEs than the peak due to C−CH, respectively. These BE differences are well in accordance with our C 1s spectrum and its assignments. The XPS spectrum of the DHI−melanin film formed on the oxidative multilayer is in accordance with material consisting of intact (oxidized and reduced) dihydroxyindole moieties, but XPS cannot reveal the bonding pattern between the indole units. The absence of peaks due to cerium in the film spectrum rules out the possibility of a cerium-based dihydroxyindole complex. The mass spectra of solution-formed products showed that cerium oxidation produces various indole-based oligomers. In addition, the film is very robust toward rinsing, and attempts to use MALDI-TOF for structure determination were completely unsuccessful, the only fragments observed originating from the underlying multilayer. The color of the film was intense dark blue. All these facts imply that the structure of the DHI−melanin film consists of covalently bonded dihydroxyindole oligomers, which stack together due to noncovalent π−π interactions. These structural features are supposed to be typical for polydopamine and suggest that the DHI−melanin film formed on oxidative multilayers can be characterized as a polydopamine/melanin type material, too. Electroactivity of the DHI−Melanin Film. Melanins are known to be electronic−ionic hybrid conductors with conductivity resembling that of semiconductors.13,47−49 In humid environment, protons are the major contributor to the ionic conductivity whereas the charge carrier hopping effected

d = L cos θ ln(I0/I ) where I and I0 are the observed photoelectron intensities with and without the attenuating layer, θ is the takeoff angle, and L is the photoelectron attenuation length in the film material. Using the universal curve approach to the attenuation length in organic materials, we can estimate the value of L to be ca. 3.2 and 3.1 nm for the Ce 4d and P 2p photoelectrons, respectively.40 The melanin layer effectively blocks all these photoelectrons so that their intensity falls well below the noise level in the spectra. On the basis of the photoelectron signal and noise levels of the spectra, we can estimate that the minimum thickness of the DHI−melanin layer is in the range of 7−8 nm, which is in accordance with the value obtained from the UV−vis spectrum, especially considering that the UV−vis result refers to the total amount of DHI−melanin on the substrate surface, including material penetrating into the oxidative underlayer (vide inf ra), whereas the XPS measures only the thickness above the underlying film. The thickness below the generally observed aggregate size for melanin-type materials implies a smooth film, in accordance with the very small change in the rms roughness upon film deposition. The atomic ratio C:O:N calculated from the XPS spectra of the (PP/Ce)5(DHI−melanin) film was 9:3:1, close to the ratio 8:2:1 expected for polyDHI. The excess of carbon and oxygen over nitrogen can be attributed to impurities, which are commonly observed in XPS and are seen also in the (PP/Ce)5 spectra. Importantly, no nitrogen signal was observed in multilayers not exposed to DHI. The high-resolution N 1s spectrum of the (PP/Ce)5(DHI−melanin) film could be explained using only a single-component peak with the BE of 400.6 eV, which can be attributed to a pyrrolic nitrogen in the indole ring.41,42 On the other hand, two components were required to fit the O 1s spectrum. The one at lower BE at 531.7 eV was assigned to CO and the other at 532.9 eV to C− O.32,39 The ratio of the two types of oxygen was CO/C−O = 0.23, which suggests that the DHI−melanin exists in a partly oxidized form. Further support for this was obtained from the C 1s spectra, the deconvolution of which required three components at 285.2, 286.0, and 288.9 eV, assigned to aromatic C−CH (and some adventitious carbon), C−O/C−N, and C O, respectively.32,39 Two different oxygen species, assigned to quinone and catechol forms, have been reported in many studies of melanin-type materials. Although the cerium oxidation of 5,6-dihydroxyindole in this work initially produces the semiquinone form, its contribution to the XPS spectrum should be insignificant because of the favorable disproportionation reaction of quinonoid compounds in aqueous media. The simple C 1s spectrum with three clearly assignable components shows that Ce(IV) does not cause any over4109

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with various types of melanins, either spectrally or as two peaks in the voltammogram. This is in striking contrast with the quinone electrochemistry, which shows only one two-electron redox process in buffered aqueous media because of the instability of the semiquinone form.53 Generally, two oneelectron processes are seen only in nonaqueous media and in unbuffered aqueous solutions.54 With melanin films, the sluggishness of the redox reactions and their insensitivity to pH suggest that the reactions mostly take place within the film and shielded from the ambient environment, which can explain the two-step process observed. The conductivity of melanin is assumed to result from a mixed electronic−ionic charge transfer mechanism. The electronic conductivity depends on charge hopping between available sites (mixed-valence type mechanism) and the ionic contribution on the protons produced by the comproportionation reaction. Therefore, in the highly oxidized sample, which is overwhelmingly in the quinone form, neither of the mechanisms is operative and the material is no longer conducting. As a result, the film was passivated after complete oxidation. In addition, the second oxidation step is generally not completely reversible with o-quinones because of chemical side reactions.55 The spectroelectrochemical data can also explain the UV−vis spectrum of the film, which is atypical for melanins (Figure 4). The as-produced film is in a partially oxidized state and the spectrum corresponds to that measured at potentials close to +0.1 V in Figure 7.

by the comproportionation equilibrium and the redox reactions of the constituent moieties results in electronic conductivity. However, the redox properties of melanins are not very well characterized. The redox-active species in melanins should consist of the quinone, semiquinone, and hydroquinone forms of the dihydroxyindole moieties. Surprisingly, films formed by enzymatic oxidation of 3,4-dihydroxyphenylalanine exhibited no pH dependence in their redox behavior.50 In this case, two oxidation peaks were observed, whereas the electropolymerized film of DHI−melanin exhibited only a single peak and a synthetic eumelanin film one or several peaks.49,51,52 In most cases, the charge transfer rate in the films was low and the films were passivated upon oxidation. Very few spectroelectrochemical studies of melanins exist, and therefore a thin DHI−melanin film was formed on a glass/ ITO/(PP/Ce)5 substrate in order to study the redox processes. The DHI−melanin film exists in a partially oxidized form after deposition, and it was electrochemically reduced (at −0.5 V vs Ag/AgCl) before the spectroelectrochemical measurements. All spectra are referenced to this reduced state of the film and represent steady-state conditions (Figure 7). Upon oxidation a

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CONCLUSIONS Oxidation of dopamine or 5,6-dihydroxyindole in neutral or alkaline aqueous media by dissolved oxygen leads to the formation of melanin-type materials, which are in most aspects indistinguishable from the natural pigment. Solution studies presented here show that similar material can be obtained from dopamine by using Ce(IV) as an oxidizing agent already at relatively acidic pH, probably because of the stabilizing effect of the coordinated oxidant. Optical and mass spectrometry observations show that the reaction proceeds via the known intermediates dopamine quinone and aminochrome, and the final product has a mass spectrum practically identical to a commercial synthetic melanin. Thin oxidative multilayers, formed using the sequential layerby-layer procedure with cerium(IV) and polyphosphate, are a general platform for the electrodeless production of conducting polymer films. These oxidative films rapidly oxidize dopamine to the semiquinone form. It slowly disproportionates to form dopamine quinone, which cyclizes already in weakly acidic (pH 4.5) solutions. Because of the small amount of oxidant in the film, the reaction does not proceed further. However, when 5,6dihydroxyindole (DHI), a known intermediate in melanin synthesis, is used, a thin but uniform, dark, insoluble, and robust film is formed on the oxidative multilayer. The film formation is self-limiting and consumes practically all oxidant in the film. The thickness of the formed DHI−melanin film is ca. 10 nm, which is much smaller than the typical particle size of melanin aggregates. XPS characterization of the film shows that it completely covers the underlying multilayer, and its composition is typical for melanin-type materials. The DHI− melanin film is also electroactive, which proves that it penetrates into the underlying cerium/polyphosphate layer, and spectroelectrochemical measurements reveal a two-step oxidation process involving the semiquinone and quinone forms, of which only the first step is reversible. Although no

Figure 7. Spectral changes during oxidation of a (PP/Ce)5/DHI− melanin film (in aqueous 0.1 M Na2SO4) at different potentials (vs Ag/AgCl reference electrode). Spectra are referenced to a spectrum obtained at −0.5 V vs Ag/AgCl.

band starts to grow near 620 nm. This band can be attributed to the semiquinone radical species, in which the conjugation can extend through the molecule from the oxygen to the nitrogen atom. This band reaches its maximum at a potential around +0.1 V, at the same potential that a broad oxidation peak has been observed for electropolymerized DHI−melanin in neutral aqueous solutions.51 It starts to decrease at more anodic potentials, and another band, seen as a small shoulder at more cathodic potentials, grows at ca. 480 nm and becomes the dominant feature in the spectra at potentials above +0.3 V. Qualitatively similar behavior has been observed with electropolymerized DHI−melanin although the reported absorbance maxima are at lower wavelength, which could be explained by the different choice of the reference spectrum.51 The maximum wavelength of the band is the same as that of the quinonoid aminochrome (475 nm), and this band is accredited to the quinone form. Therefore, the spectral behavior shows two consecutive oxidation processes, which is in accordance with the hydroquinone−semiquinone−quinone redox system. Two consecutive oxidation processes have generally been observed 4110

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(8) Liu, Y.; Ai, K.; Lu, L. Polydopamine and its Derivative Materials: Synthesis and Promising Applications in Energy, Environmental, and Biomedical Fields. Chem. Rev. 2014, 114, 5057−5115. (9) Liebscher, J.; Mrowczynski, R.; Scheidt, H. A.; Filip, C.; Hadade, N. D.; Turcu, R.; Bende, A.; Beck, S. Structure of Polydopamine: A Never-Ending Story? Langmuir 2013, 29, 10539−10548. (10) Pezzella, A.; Panzella, L.; Natangelo, A.; Arzillo, M.; Napolitano, A.; d’Ischia, M. 5,6-Dihydroxyindole Tetramers with “Anomalous” Interunit Bonding Patterns by Oxidative Coupling of 5,5′,6,6′Tetrahydroxy-2,7′-Biindolyl: Emerging Complexities on the Way Toward an Improved Model of Eumelanin Buildup. J. Org. Chem. 2007, 72, 9225−9230. (11) d’Ischia, M.; Napolitano, A.; Ball, V.; Chen, C.; Buehler, M. J. Polydopamine and Eumelanin: From Structure-Property Relationships to a Unified Tailoring Strategy. Acc. Chem. Res. 2014, 47, 3541−3550. (12) Chen, C.; Ball, V.; de Almeida Gracio, J. J.; Singh, M. K.; Toniazzo, V.; Ruch, D.; Buehler, M. J. Self-Assembly of Tetramers of 5,6-Dihydroxyindole Explains the Primary Physical Properties of Eumelanin: Experiment, Simulation, and Design. ACS Nano 2013, 7, 1524−1532. (13) Bothma, J. P.; de Boor, J.; Divakar, U.; Schwenn, P. E.; Meredith, P. Device-Quality Electrically Conducting Melanin Thin Films. Adv. Mater. 2008, 20, 3539−3542. (14) Ball, V.; Gracio, J.; Vila, M.; Singh, M. K.; Metz-Boutigue, M.; Michel, M.; Bour, J.; Toniazzo, V.; Ruch, D.; Buehler, M. J. Comparison of Synthetic Dopamine-Eumelanin Formed in the Presence of Oxygen and Cu2+ Cations as Oxidants. Langmuir 2013, 29, 12754−12761. (15) Bernsmann, F.; Ponche, A.; Ringwald, C.; Hemmerle, J.; Raya, J.; Bechinger, B.; Voegel, J.; Schaaf, P.; Ball, V. Characterization of Dopamine-Melanin Growth on Silicon Oxide. J. Phys. Chem. C 2009, 113, 8234−8242. (16) Klosterman, L.; Riley, J. K.; Bettinger, C. J. Control of Heterogeneous Nucleation and Growth Kinetics of DopamineMelanin by Altering Substrate Chemistry. Langmuir 2015, 31, 3451−3458. (17) Bernsmann, F.; Ball, V.; Addiego, F.; Ponche, A.; Michel, M.; de Almeida Gracio, J. J.; Toniazzo, V.; Ruch, D. Dopamine-Melanin Film Deposition Depends on the used Oxidant and Buffer Solution. Langmuir 2011, 27, 2819−2825. (18) Wei, Q.; Zhang, F.; Li, J.; Li, B.; Zhao, C. Oxidant-Induced Dopamine Polymerization for Multifunctional Coatings. Polym. Chem. 2010, 1, 1430−1433. (19) Kim, H. W.; McCloskey, B. D.; Choi, T. H.; Lee, C.; Kim, M.; Freeman, B. D.; Park, H. B. Oxygen Concentration Control of Dopamine-Induced High Uniformity Surface Coating Chemistry. ACS Appl. Mater. Interfaces 2013, 5, 233−238. (20) Ball, V.; Del Frari, D.; Toniazzo, V.; Ruch, D. Kinetics of Polydopamine Film Deposition as a Function of pH and Dopamine Concentration: Insights in the Polydopamine Deposition Mechanism. J. Colloid Interface Sci. 2012, 386, 366−372. (21) Jiang, J.; Zhu, L.; Zhu, L.; Zhu, B.; Xu, Y. Surface Characteristics of a Self-Polymerized Dopamine Coating Deposited on Hydrophobic Polymer Films. Langmuir 2011, 27, 14180−14187. (22) Li, B.; Liu, W.; Jiang, Z.; Dong, X.; Wang, B.; Zhong, Y. Ultrathin and Stable Active Layer of Dense Composite Membrane Enabled by Poly(Dopamine). Langmuir 2009, 25, 7368−7374. (23) Salomäki, M.; Räsänen, M.; Leiro, J.; Huti, T.; Tenho, M.; Lukkari, J.; Kankare, J. Oxidative Inorganic Multi Layers for Polypyrrole Film Generation. Adv. Funct. Mater. 2010, 20, 2140−2147. (24) Salomäki, M.; Myllymäki, O.; Hätönen, M.; Savolainen, J.; Lukkari, J. Layer-by-Layer Assembled Oxidative Films as General Platform for Electrodeless Formation of Conducting Polymers. ACS Appl. Mater. Interfaces 2014, 6, 2325−2334. (25) Decher, G.; Schlenoff, J. B. In Multilayer Thin Films: Sequential Assembly of Nanocomposite Materials, 2nd ed.; Wiley: 2012; Vol. 1−2. (26) Bisaglia, M.; Mammi, S.; Bubacco, L. Kinetic and Structural Analysis of the Early Oxidation Products of Dopamine - Analysis of the

oxidant (cerium) is found in the DHI−melanin material, the nascent film is in an oxidized state and, therefore its UV−vis spectrum differs from the typical featureless melanin spectrum. The deposition of the DHI−melanin film takes place faster than that of the polydopamine films formed using spontaneous oxidation. In addition, the use of oxidative multilayers gives control of the film formation at nanolevel and can allow spatial control of the formation of redox active melanin-type thin films on practically any surface, irrespective of size, form, or chemical nature. This suggests application possibilities in e.g. bioelectronics and biocompatible energy storage. We are currently investigating the use of other oxidant metals, biopolymer-based oxidative films, and much thicker and stratified multilayers for the surface-controlled synthesis of melanin-type functional films.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b00402. Mass spectra of synthetic melanin and cerium oxidized dopamine solutions, UV−vis spectra of oxidation products, XPS spectra and binding energies for intact and sputtered DHI−melanin film and oxidative multilayer (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail mikko.salomaki@utu.fi (M.S.). *E-mail jukka.lukkari@utu.fi (J.L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Qi Wang for the help with MALDI-TOF measurements.

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REFERENCES

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