Reverse Engineering To Characterize Redox Properties: Revealing

Methods/Protocols Cite This: Chem. Mater. 2018, 30, 5814−5826

pubs.acs.org/cm

Reverse Engineering To Characterize Redox Properties: Revealing Melanin’s Redox Activity through Mediated Electrochemical Probing Mijeong Kang,†,‡ Eunkyoung Kim,†,‡ Zülfikar Temoçin,§ Jinyang Li,†,‡ Ekaterina Dadachova,∇ Zheng Wang,⊥ Lucia Panzella,# Alessandra Napolitano,# William E. Bentley,†,‡ and Gregory F. Payne*,†,‡

Downloaded via UNIV OF SOUTH DAKOTA on September 11, 2018 at 12:26:43 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Institute for Bioscience and Biotechnology Research, University of Maryland, 4291 Fieldhouse Drive, Plant Sciences Building, College Park, Maryland 20742, United States ‡ Fischell Department of Bioengineering, University of Maryland, 8228 Paint Branch Drive, 2330 Jeong H. Kim Engineering Building, College Park, Maryland 20742, United States § Department of Chemistry, Science and Arts Faculty, Kırıkkale University, Yahşihan 71450 Kırıkkale, Turkey ∇ College of Pharmacy & Nutrition, University of Saskatchewan, Saskatoon SK, S7N 2Z4, Canada ⊥ Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory, Washington, District of Columbia 20375, United States # Department of Chemical Sciences, University of Naples Federico II, Via Cintia 4, I-80126 Naples, Italy S Supporting Information *

ABSTRACT: Melanins are ubiquitous in nature, yet their functions remain poorly understood, because their structures and properties elude characterization by conventional methods. Since many of the proposed functions of melanins (e.g., antioxidant, pro-oxidant, and radical scavenging) involve an exchange of electrons, we developed an electrochemical reverse engineering methodology to probe the redox properties of melanin. This mediated electrochemical probing (MEP) method (i) characterizes insoluble melanin particles that are localized adjacent to an electrode within a permeable hydrogel film, (ii) employs diffusible mediators to shuttle electrons between the electrode and melanin sample, and (iii) imposes complex sequences of input voltages and analyzes output response characteristics (e.g., currents) to reveal redox properties. Here, we illustrate the versatility of MEP and review results demonstrating that melanins have reversible redox activities, can exchange electrons with various reductants and oxidants, and can quench radicals either by donating or accepting electrons. These results suggest possible biological functionalities for melanin and motivate the use of MEP for characterizing additional (i.e., synthesized) materials whose functions rely on redox properties. More broadly, MEP is revealing a richness to redox activities that has previously been inaccessible to investigation.

■

INTRODUCTION

unexposed areas of the human body (e.g., the brain) suggests that melanins perform functions other than photoprotection.19 Typical eumelanins include the pigments that can be extracted from human and mammalian black-brown hair and irides, as well as those found in the inner ear and melanomas. The most remarkable example of eumelanin from lower animals is represented by the ink of cephalopods, including Sepia off icinalis, Octopus vulgaris, and Loligo vulgaris. Sepia melanin that exhibits a granular microstructure (100−200 nm)20−23 is traditionally regarded as a standard for eumelanins, because of its relatively high purity. Unlike eumelanins, which

Melanins are a vast class of black, brown or even yellowish and reddish natural biopolymers of diverse nature and chemical composition that arise biogenetically from the oxidation of phenolic metabolites (Figure 1). Traditionally, the term melanins has been used to refer to two main groups of intracellular nitrogenous pigments: the dark eumelanins and the lighter, reddish-yellow pheomelanins, which arise from a bifurcation of a common biosynthetic pathway.1−3 In humans, the dark eumelanins are believed to play a role in skin and eye photoprotection,4−8 and protection from free radicals and oxidative insults.9−13 In contrast, the lighter pheomelanins that are found in red-haired individuals with fair complexions have been suggested to have an opposite, photosensitizing14 and pro-oxidant activity.15−18 Yet, the presence of melanins in © 2018 American Chemical Society

Received: June 8, 2018 Revised: August 16, 2018 Published: August 17, 2018 5814

DOI: 10.1021/acs.chemmater.8b02428 Chem. Mater. 2018, 30, 5814−5826

Chemistry of Materials

Methods/Protocols

Figure 1. Melanins are ubiquitous in nature, have complex structures that are only partially understood, and have putative biological functionalities that often involve the exchange of electrons through redox reactions. [Every image is Public Domain or CC0 (Creative Commons Zero) and obtained from https://free-images.com.]

disease.27,29,30 It is generally agreed that neuromelanin arises by dopamine oxidation and polymerization driven by excess cytosolic catecholamines not accumulated into synaptic vesicles.31 Formation of cysteinyldopamine adducts and their oxidation is also an important contributory mechanism in neuromelanin formation. According to current views, neuromelanin exhibits a core−shell architecture resulting from a casing process in which an initially formed pheomelanin core is encapsulated into a eumelanin coating.32,33 Melanin synthesis in microbes is likely to provide a survival advantage in the environment. In several fungal species (e.g., Cryptococcus neoformans), black melanin polymers are implicated in virulence,34−38 host immune response,39−42 resistance to environmental stress, and protection against harmful radiation.43,44 Figure 1 illustrates that these fungal melanins are known to be derived from oxidative coupling of 1,8-dihydroxynaphthalene (DHN), a naturally occurring polyketide. In summary, melanin is found in a variety of living systems; it is proposed to perform various biological functions, and most of these proposed functions rely on redox activity. As a result, several methods have been used to characterize the redox activities of melanin.45−47 Conceptually, the simplest

are relatively widespread in nature, pheomelanins are found only in mammalian skin, hair, and eyes, and in hen feathers.24−26 Eumelanins and pheomelanins derive from the common precursor dopaquinone formed by oxidation of the amino acid L-tyrosine by tyrosinase. In the eumelanin-forming pathway, 5,6-dihydroxyindole intermediates are generated via intramolecular cyclization of dopaquinone and are converted to the final eumelanin pigments through an oxidative polymerization process. Regarding the production of pheomelanins, the intervention of sulfhydryl compounds (such as cysteine) gives rise exclusively to thiol adducts of dopa (cysteinyldopas) that, upon further oxidation, leads to the formation of the pheomelanin pigments via benzothiazine intermediates. The general structures of eumelanin and pheomelanin are shown in Figure 1. The presence of black/brown melanin granules has also been described in two mesencephalic areas of the human central nervous system: the substantia nigra and the locus coeruleus. The neuronal content of this pigment, termed neuromelanin, increases with age until 60 years and then decreases.27,28 Much interest in neuromelanin derives from its possible implication in the pathogenesis of Parkinson’s 5815



Methods/Protocols

Figure 2. Mediated electrochemical probing (MEP) to characterize redox properties. (a) Melanins have a complex hierarchical structure (Sepia melanin from cuttlefish). [Adapted with permission from ref 20. Copyright 2014, American Chemical Society, Washington, DC.] (b) Conventional approaches to understand materials focus on structure while MEP focuses on redox properties. (c) Schematic shows MEP with two mediators while electrical input (voltage) and output (current) curves reveal amplified mediator currents that are signatures of redox cycling and melanin’s redox activity. (d) Long-term cyclic experiments test for the reversibility of redox activity: steady (time-invariant) amplifications are signatures of reversible and repeated oxidation and reduction. [Adapted with permission from ref 54. Copyright 2017, American Chemical Society, Washington, DC.]

inputs and measuring the resulting response characteristics. As suggested in Figure 2b, the redox inputs are imposed through a combination of chemical inputs (e.g., mediator additions) and electrical inputs, while output responses can be measured through common sensing modalities (e.g., optical and electrical). Here, we review this electrochemical reverse engineering approach and show how it is revealing properties important to melanin’s putative biological functions. Figure 2c illustrates three important features of our mediated electrochemical probing (MEP) approach. First, we start by entrapping (i.e., embedding) the insoluble melanin particles within a nonconducting hydrogel (typically ∼0.5 mm wet film of the aminopolysaccharide chitosan) adjacent to an electrode surface (see Figure S1 in the Supporting Information shows the preparation processes of melanin−chitosan films on the electrode). This hydrogel film localizes the melanin near the electrode surface and entraps the melanin within a hydrogel matrix that allows the free diffusion of small molecules (see Figure S2 in the Supporting Information shows the SEM images of melanin−chitosan film). The second feature of MEP is the purposeful addition of soluble mediators (i.e., electron shuttles) to the surrounding solution (see Figure S3 in the Supporting Information shows the overview picture of an electrochemical system for the MEP experiment). These mediators can diffuse into and throughout the hydrogel matrix and serve to establish redox “communication” between the electrode and the embedded melanin. In

approach to study melanin’s electron accepting/donating activity is to contact a melanin sample with an oxidant/ reductant and then “titrate” the consumption of this oxidant/ reductant.48,49 Pulse radiolysis allows a free radical to be generated and enables study of the ability of melanin to exchange electrons to scavenge (i.e., quench) such free radicals.12 Electrochemical studies have shown that melanin can directly exchange electrons with an electrode50,51 and that melanin can be reduced by Fe(II).39,52 Recently, the oxidative potentials of eumelanosomes and pheomelanosomes isolated from black and red human hair were measured using photoelectron emission microscopy (PEEM).33,53 While these studies have contributed to our understanding of melanin’s redox activities, each of these methods has limitations, and no standard method has emerged to measure the redox properties of melanins.

■

MEDIATED ELECTROCHEMICAL PROBING Over the past few years, we have been developing an electrochemical reverse engineering approach to characterize the redox properties of materials and we have used melanin as our model. While most materials characterization approaches start by focusing on structure, the complexity of melanin’s hierarchical structure has made this approach less successful (see Figure 2a). Rather than focusing on structure, our reverse engineering approach treats melanin as a “black box” and characterizes its redox properties by imposing controlled redox 5816



Methods/Protocols

Figure 3. Multiple mediators can be used simultaneously to probe for redox potential. (a) Eumelanin and pheomelanin have different biosynthetic routes, structures, and proposed functional properties. (b) Redox probing with three mediators detected differences in redox potentials between eumelanin and pheomelanin. (c) Voltage input and current outputs for probing eumelanin and pheomelanin: Peak “D” is characteristic for pheomelanin and indicates pheomelanin has a more oxidative redox potential. [Adapted with permission from ref 64. Copyright 2015, Springer Nature, London, United Kingdom.]

amplified mediator currents are a signature pattern indicating redox activity of the film-entrapped material. This MEP approach is illustrated by an experiment in which a Sepia melanin was probed using two mediators: one oxidative mediator (50 μM ferrocene dimethanol, Fc, E0 = +0.25 V vs Ag/AgCl) and one reductive mediator (50 μM: Ru(NH3)6Cl3, Ru3+, E0 = −0.2 V vs Ag/AgCl). The input voltage curve is shown in the left-hand side of Figure 2c and two representations of the output curves are shown on the righthand side. The first output plot shows current as a function of time (i−t), while the second shows a cross-plot between output current and input voltage (i−V; time is not explicitly shown in this cross-plot). One control in this illustrative example is the response of a hydrogel film lacking melanin that is probed with the two mediators. The output current for this control shows small peaks associated with the electrochemical oxidation and reduction of the mediators at the electrode. A second control is a hydrogel film with embedded melanin probed in the absence of mediators (not shown).20,55 The output for such controls show very small currents under any condition, which indicates that there is minimal (if any) direct electron transfer between melanin and the electrode.20,55 When the experimental film containing embedded melanin was probed using both mediators, the output curve shows significant current amplification both in the oxidative region

the example in Figure 2c, the schematic shows the following: one mediator can donate an electron to the electrode under oxidative voltages; the oxidized form of this mediator can diffuse into the hydrogel matrix and accept an electron from melanin; and this re-reduced mediator can diffuse back to the electrode to be reoxidized by donating the electron to the electrode. This “oxidative redox−cycling” process serves to extract electrons from the melanin. The second mediator in Figure 2c is reduced when the electrode potential is changed to a reducing (more negative) voltage. This second mediator undergoes an analogous “reductive redox cycling” process that serves to transfer electrons from the electrode to reduce melanin. As indicated by the thermodynamic plot in Figure 2c, these redox cycling processes are controlled by the voltage input that is imposed at the electrode. and this voltage can be cycled to sequentially engage melanin in oxidative and reductive redox cycling. The third feature of our MEP approach is the imposition of controlled sequences of input voltages and interpretation of the output response characteristics (e.g., the observed current). The voltage inputs (along with the selection of mediators) control the redox cycling reactions that serve to probe the redox characteristics of the embedded melanin. When redox cycling occurs, the electrochemical currents associated with mediator oxidation/reduction is observed to be amplified and 5817



Methods/Protocols

pheomelanin is believed to be a pro-oxidant (under light or dark conditions) and has been suggested to confer sensitivity to radiation-induced melanomas for people with red hair and light skin.17,62,63 To discern differences, we prepared film-coated electrodes using synthetic models of eumelanin and pheomelanin, as well as human samples from eumelanin-rich black hair and pheomelanin-rich red hair. These film coated electrodes were then immersed in solutions containing three mediators each with different redox potentials (Ru2+ E0= −0.2 V; Fc E0= +0.25 V; Ir3+ E0 = +0.55 V). To initiate probing, we imposed various input voltage sequences and we discerned differences between these melanins by evaluating the response characteristics of the output current. The overall conclusion from this probing is illustrated in the schemes on the right-hand side in Figure 3b. Specifically, eumelanin was observed to be able to accept electrons from Ru2+ and donate electrons to both Fc+ and Ir4+. In contrast, pheomelanin was observed to accept electrons from both Ru2+ and Fc and also to donate electrons to Ir4+.64 The conclusions summarized in Figure 3b were supported by several independent measurements, using different redoxprobing sequences, and we highlight one such probing approach in Figure 3c. In this case, we imposed cyclic voltage inputs in the presence of the three mediators. Initially, the voltage is “swept” into the oxidative potential range, where first Fc can be oxidized and then Ir3+ can be oxidized. The output (i−V) curve for the case of eumelanin shows that oxidative current peaks are observed for both Fc (peak “B”) and Ir3+ (peak “C”), and these peaks are amplified for films containing the synthetic model eumelanin (compared to the melanin-free control film). This amplified current is a signature of oxidative redox cycling, which indicates that eumelanin is donating electrons to both Fc+ and Ir4+. When the voltage is swept into the reducing direction, then electrons can be transferred from the electrode to reduce Fc+ and Ru3+. The output for the film containing the synthetic model eumelanin shows a distinct amplification for Ru3+ reduction peak (peak “A”), while no amplification is observed for Fc+ reduction. This response characteristic indicates that eumelanin only engages in reductive redox cycling with Ru3+. This pattern suggests that eumelanin has a redox potential that is between those for Fc and Ru3+.64 The right-most plot in Figure 3c shows the output response for the probing of a film containing the synthetic model pheomelanin. In this case, an amplification of Ir-oxidation (peak “C”) is observed but the amplification of Fc oxidation (peak “B”) is considerably weaker (compared to the amplification in Fc oxidation observed for eumelanin). The most striking difference in output response for the pheomelanin-containing film is the observation of an amplified Fc+ reduction peak (peak “D”), which is completely absent from the eumelanin-containing film. This amplification in Fc+ reduction indicates that Fc is engaged in a reductive redoxcycling mechanism that mediates the transfer of electrons from the electrode to the pheomelanin. Such a redox cycling could only occur if pheomelanin has a redox potential that is more oxidative (more positive) than the E0 for Fc. This observation indicates that pheomelanin has a redox potential between those for Fc and Ir3+. [Note: the amplified Fc oxidation peak “D” suggests that pheomelanin may also have a second redox potential that is similar to that of eumelanin.]64 From a biological perspective, the results from this reverse engineering study may suggest explanations for pheomelanin’s

as well as the reductive region. Amplification of mediator currents is a characteristic feature of redox cycling and is a signature output response indicating that melanin is redoxactive (i.e., it can exchange electrons with the mediators). The conclusion from Figure 2c is that melanin is redoxactive; it can accept electrons from the reducing mediator, and it can donate electrons to the oxidizing mediator. While alternative experimental approaches have also indicated that melanin has redox activity,45,47−49,56,57 the advantage of MEP is that it provides a simple experimental approach that can be extended to reveal more-detailed features of melanin’s redox activity. Steady Input/Output Provides Evidence for Reversible Redox Activities. One important question of melanin’s redox activity is its reversibility: can melanin be repeatedly oxidized and reduced, or are the reactions irreversible? To answer this question, we performed the experiment illustrated in Figure 2d, in which the electrode with the melanincontaining film was immersed in a solution containing both the oxidative mediator (Fc) and reductive mediator (Ru3+), and the input potential (i.e., voltage) imposed at the electrode was repeatedly cycled over an 8-h period. The output current curves in Figure 2d show that when melanin was embedded in the film, the mediator currents were amplified during both oxidation and reduction (compared to the control film lacking melanin). Importantly, this amplification remained approximately constant over the entire 8-h period of the experiment, which indicates that melanin is reversibly redox-active and can be repeatedly oxidized and reduced (i.e., melanin is a “redox switch”). From a signal processing perspective, this output response is approximately steady or time-invariant. From a methods standpoint, MEP allows insoluble samples to be probed repeatedly to determine the reversibility of redox activities. The observation that melanin can be repeatedly oxidized and reduced could have significant biological consequences. Specifically, the reversibility of melanin’s redox activity indicates that melanin serves as a catalyst for the transfer of electrons from reductants to oxidants. While the results in Figure 2d show electron transfer between two electrochemical mediators (Fc and Ru3+), these observations have been extended to other, more biologically relevant, reductants and oxidants.58,59 If these in-vitro-observed properties also operate in vivo, then melanin may play an important role in redox biology. Specifically, various biological redox couples (e.g., NAD(P)/NAD(P)H, GSH/GSSG, ascorbate/ dehydroascorbate) have kinetic barriers that preclude them from equilibrating with each other [such barriers to electron transfer can be contrasted with the case of proton transfer where protonation−deprotonation reactions are generally believed to readily equilibrate]. A catalytic function for melanin that could equilibrate redox couples would indicate that melanin could be an important participant in redox biology (and not simply an inert “bystander”). Multiple Mediators Allow Differences To Be Detected. MEP allows subtle differences in redox characteristics to be detected, and this is illustrated for the case of eumelanin and pheomelanin. As illustrated in Figure 3a (also Figure 1), eumelanin is the black−brown melanin believed to be synthesized from tyrosine, while pheomelanin is believed to be synthesized from both tyrosine and cysteine.25,60,61 From a biological perspective, these melanins are hypothesized to have markedly different functional properties: eumelanin is believed to protect against radiation and free radical damage, while 5818



Methods/Protocols

Figure 4. Arbitrarily complex sequences of voltage inputs can be imposed to reveal more-detailed information. (a) Acetaminophen (APAP) is a redox-active drug and MEP shows that it can undergo oxidative redox cycling with melanin. (b) Long-term cyclic voltage inputs over differing voltage ranges illustrate that steady (time-invariant) amplification of mediator currents requires a balance between the oxidative and reductive redox-cycling mechanisms. [Adapted with permission from ref 55. Copyright 2017, American Chemical Society, Washington, DC.]

reported pro-oxidant activities. Specifically, if pheomelanin can act as an electron transfer catalyst, and its redox potential is comparatively oxidative (versus eumelanin), then it may exert pro-oxidant activities through a redox-buffering mechanism.64 From the perspective of the MEP experimental method, these results illustrate that materials can be probed in the presence of multiple mediators and these mediators exert their influences (i.e., engage the material in redox-cycling) only over a comparatively narrow range of imposed electrode voltages that can be tailored to probe for specific redox information (e.g., in this case for a melanin’s redox potential). Tailoring Voltage Inputs Enables Confirmation of Redox Interactions. In the above two examples, we used conventional electrochemical mediators, to probe for the intrinsic redox properties of melanin. However, it is also possible to use this method to probe for redox interactions with melanin. In particular, redox probing may reveal if a drug can undergo redox cycling with melanin (e.g., with neuromelanin). For instance, the analgesic acetaminophen (Nacetyl-para-aminophenol, APAP) is known to undergo redox cycling in the liver, which is believed to be responsible for the liver damage associated with APAP overdoses.65−67 Morerecent studies have suggested that APAP may exert redoxbased activities in the brain with low levels conferring protective antioxidant activities.68−70 To our knowledge, there are few simple in vitro methods available to test a material for such redox cycling interactions and, thus, we believe that such activities may be underappreciated in biology. Importantly, many biological contexts (e.g., the brain and gut)

are characterized by steep O2 gradients, which would provide the driving forces to favor redox cycling since only short diffusion distances separate oxidative and reductive conditions. To test APAP’s redox-cycling abilities, we probed the natural melanin from Sepia and a synthetic cysteinyldopamine− dopamine core−shell model of neuromelanin.55,71,72 Figure 4a indicates that APAP is redox-active with an oxidative redox potential (E0 = +0.3 V) that could potentially engage in oxidative redox cycling to accept electrons from melanin. In this in vitro experimental system, the electrode serves as the electron “sink” to oxidize APAP and potentially the electrode is mimicking oxidative physiological activities. However, note that melanin is not an infinite “source” of electrons and that continued oxidative redox cycling would deplete electrons from the melanin and preclude continued oxidative redox cycling. To replenish the melanin with electrons, we added the Ru3+ mediator and imposed reducing voltages that allowed the Ru3+ to engage melanin in the reductive redox-cycling mechanism that mediates the transfer of electrons from the electrode to melanin.55 While the biological questions and implications of these experiments are interesting, we primarily focus the following discussion on the experimental methodology. Figure 4b illustrates the importance of balancing oxidative and reductive redox-cycling mechanisms to generate steady (i.e., time-invariant) output currents. In this experiment, an electrode with a melanin-containing film was immersed in a solution containing both APAP (50 μM) and Ru3+ (50 μM), and cyclic voltage sequences were imposed. For the initial 5819



Methods/Protocols

Figure 5. The information accessed by MEP can be expanded by enlisting additional measurement modalities. (a) Cell-wall-bound DHN melanin has a unique chemical structure and is hypothesized to confer protection to fungal cells. (b) Simultaneous spectroelectrochemical measurements allow redox activities to be discerned from electrical outputs and radical-scavenging (quenching) activities to be detected from optical outputs. (c) Illustration of spectroelectrochemical cell and bimodal measurement approach. (d) Experimental results show the linkage between redox activities (evidenced from amplified electrical outputs) and radical-scavenging activities (evidenced from attenuated optical outputs): the oxidative free radical (ABTS+ •) is quenched by accepting electrons from melanin and the reductive free radical (PQ+ •) is quenched by donating an electron to melanin. [Adapted with permission from ref 54. Copyright 2017, American Chemical Society, Washington, DC.]

to be electrochemically “silent”). As expected, the output curve during this period of unbalanced probing (480−590 min) is observed to be unsteady, with an initial large amplification in oxidative currents decaying with each progressive cycle as the melanin becomes progressively more oxidized (depleted of electrons). Finally, at 590 min, the input voltage amplitude was adjusted back to the larger range and the output curves show a return to steady output currents. The bottom curves in Figure 4b show i−V representations of the same data for this sequence of five input voltage amplitude ranges.55 From a biological perspective, the results in Figure 4 indicate that the redox active analgesic APAP can undergo oxidative redox cycling with a material (i.e., melanin) that is present in the body (including the brain). These in vitro observations of redox interactions raise significant questions about whether a redox activity of a drug contributes to its mode of action, metabolism, or side effects. Obviously, in vitro electrochemical measurements cannot prove an in vivo activity, just as in vitro measurements of enzyme activity cannot prove a protein’s in vivo function. From a methods perspective, Figure 4 illustrates the versatility of MEP. Specifically, the imposed voltage sequence can be purposefully tailored to probe for specific redox information and the input sequence can be rapidly changed without requiring the addition or deletion of chemical reagents. This allows a single experiment to sequentially probe broad redox windows and also narrower, more focused, redox windows. Integrating Additional Measurement Modalities To Enrich Information. In the above examples, voltage inputs were used to provide the driving force for mediator-electrode electron transfer reactions, while the output currents provided measures of the kinetics of these reactions. Amplifications of

period (first 190 min), the voltage was cycled between +0.7 V and −0.4 V (vs Ag/AgCl). This large-amplitude input voltage sequentially provides the oxidative voltage needed to oxidize APAP and initiate its oxidative redox cycling, and also the reductive voltage needed to reduce Ru3+ and initiate its reductive redox cycling. Under these balanced conditions, the output response in Figure 4b shows near steady amplified currents.55 Between 190 min and 260 min, the amplitude of the input voltage was adjusted to cycle over a narrower voltage range between 0 and −0.4 V. This voltage range provides the reductive conditions for Ru3+ reductive redox cycling, but is insufficiently oxidative to allow APAP oxidative redox cycling. Under this narrower voltage amplitude, Ru3+ is expected to transfer electrons to melanin (i.e., reduce the melanin), APAP is expected to be “silent”, and the melanin is expected to become progressively reduced, since there is no oxidative mechanism available to remove electrons. During this period of unbalanced electron transfer (190−260 min in Figure 4b), the output currents were observed to be unsteady with a progressive decay in the amplification of Ru3+ reduction (note: a progressive increase in Ru2+ oxidation is also obvious in this output current curve).55 Between 260 min and 480 min, the input amplitude was again adjusted back to the initial amplitude (between +0.7 V and −0.4 V), and the output curve in Figure 4b shows a return to time-invariant steady output currents. At 480 min, the input voltage amplitude was again adjusted to a narrower range but, in this case, to an oxidative range between 0 and +0.7 V. This oxidative range is sufficient to allow the APAP oxidation that can initiate oxidative redox cycling, but is insufficient to allow Ru3+ reduction (i.e., under these conditions, Ru3+ is expected 5820



Methods/Protocols

and 9 min), ABTS was oxidized and a small oxidative output current was observed for the control film lacking melanin. The electrical outputs in Figure 5d show that films containing either Sepia melanin or fungal ghosts (fungal DHN melanin) had amplified oxidative currents, indicating that ABTS can undergo oxidative redox cycling with both types of melanin. The lower plot in Figure 5d shows the optical outputs generated during this redox probing. For the control (melanin-free film), a large increase in optical absorbance is observed between 4 min and 9 min when ABTS is oxidized, and this confirms the generation of the ABTS+ • radical. The absorbance associated with this ABTS+ • radical is observed to decay after 9 min, because this radical can diffuse out of the optical window and also because it can be electrochemically reduced back to ABTS under the increasingly reductive imposed voltage. Importantly, films containing either Sepia melanin or the fungal DHN melanin show attenuations in the optical outputs, which is an indication that these melanins are donating electrons that quench the ABTS+ • radical.54 The results in Figure 4d also show response characteristics when the imposed voltage was cycled to be reductive (between 17 min and 20 min) and generate the PQ+ • radical. Analogous to the results for ABTS oxidation, the incorporation of either type of melanin in the films resulted in amplified electrical output currents associated with PQ2+ reduction and attenuated optical outputs associated with PQ+ • quenching. Amplification of the output currents provides evidence for reductive redox cycling between PQ2+ and melanin, while the attenuated optical outputs indicates that melanin’s acceptance of an electron serves to quench the PQ+ • radical.54 The results in Figure 5d indicate that melanin can quench free radicals either by donating electrons to oxidative radicals (e.g., ABTS+ •) or by accepting electrons from reductive radicals (e.g., PQ+ •). Further studies demonstrated that these radical scavenging (i.e., quenching) activities are linked. Specifically, melanin must be reduced (i.e., have available electrons) to be able to donate an electron to quench an oxidative free radical, and melanin must be oxidized to be able to accept an electron to quench a reductive free radical. From a methods perspective, the extension of output measurements to additional modalities enhances the richness of information that can be acquired by MEP.

mediator currents are used as signatures of redox-cycling interactions that indicate the mediators are undergoing redox interactions with the embedded melanin. In some cases, it is possible to expand the information generated from mediated electrochemical probing by measuring additional outputs through orthogonal modalities. Optical modalities are especially convenient and can provide complementary chemical information. Such a spectroelectrochemical approach is illustrated by studies with fungal 1,8-dihydroxynaphthalene (DHN)-melanin (Figure S4 in the Supporting Information shows the overview picture of a spectroelectrochemical system for the MEP experiment). Fungal DHN-melanin has a different chemical structure and is generated through an entirely different biosynthetic pathway than eumelanin and pheomelanin.73,74 Interestingly, fungi that have been observed to survive the contaminated zones near the Chernobyl nuclear reactor have been observed to be enriched in DHN melanin, which is often preferentially localized in the cell wall.43,75 Cell-wall-bound DHN melanin is illustrated in Figure 5a, which shows SEM and TEM images of fungal “ghosts” prepared by treating the melanized fungus Wangiella dermatitidis with strong acid. It has been hypothesized that this cell wall melanin protects the fungi from radiation exposure through incompletely understood mechanisms. Possibly, fungal melanin-mediated radioprotection of the living cells results from a combination of Compton scattering by the high number of electrons in its macromolecules, energy attenuation of photons as a consequence of high angular scattering caused by the geometry of the spheres, and quenching of free electrons and free radicals generated by the radiolysis of water.6,43,49 To evaluate radical scavenging capabilities, we embedded fungal ghosts in films adjacent to an electrode surface and probed using two redox active chemical components (i.e., two diffusible mediators), as illustrated in Figure 5b. One component 2,2′-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) can be electrochemically oxidized in a oneelectron transfer reaction that generates the ABTS+ • free radical.76−78 If this ABTS+ • radical can diffuse into the film and accept an electron from the embedded melanin, then this redox interaction would serve to quench the ABTS+ • radical. It turns out that this ABTS+ • radical is green-colored while the nonradical ABTS is colorless and thus optical absorbance measurements (near 390 nm) can be used to observe the generation and subsequent quenching of this ABTS+ • radical.54 The second component used for redox-probing was paraquat (PQ2+) that can be electrochemically reduced in a one-electron transfer reaction that generates the PQ+ • radical.79−81 If this PQ+ • radical can diffuse into the film and donate an electron to melanin, then this redox interaction would serve to quench the PQ+ • radical. Similarly, the PQ+ • radical is blue-colored and the nonradical PQ2+ is colorless, and thus absorbance measurements (also near 390 nm) can be used to observe the generation and subsequent quenching of this PQ+ • radical.54 Figure 5c illustrates the spectroelectrochemical cell with a perforated gold electrode that allows the simultaneous measurement of electrochemical and optical outputs. In these experiments, we coated the perforated electrode with a transparent film containing embedded fungal ghosts. Figure 5d shows experiments with a single input voltage cycle. When the voltage was cycled to oxidative potentials (between 4 min

■

SUMMARY AND PERSPECTIVES Mediated electrochemical probing (MEP) allows the redox properties of insoluble materials to be revealed using convenient electrochemical methods. Conventional approaches to characterize the redox properties of insoluble materials use reagents (oxidants or reductants) that only allow probing in one direction. By using mediators and tailored voltage sequences, a single MEP experiment allows characterization of the following: redox activities in both oxidative and reductive directions, the reversibility of redox activities, and the voltage range (i.e., redox potential range) over which these activities are observed. Our examples illustrate that MEP enlists the typical advantages of electrochemistry for rapid, sensitive, and convenient analysis, while enabling analysis to be performed using arbitrarily complex input voltage sequences. Furthermore, the electronic format of the MEP input and output facilitates automation, allowing probing to be preprogrammed to run without further intervention (i.e., to perform the experiment overnight). Importantly, we envision the electronic format could also allow autonomous, adaptive 5821



Methods/Protocols

Table 1. Contribution of Mediated Electrochemical Probing (MEP) To Understanding Melanin supporting evidence

extensions

new information

melanin is redox-active20,23,45,55,64,71,97−100

the redox activity of melanin is reversible, and melanin can be melanin can redox-cycle with exogenous repeatedly switched between oxidized and reduced states20,54,64 chemicals (e.g., toxins, drugs)55,71

melanin can scavenge free radicals (i.e., oxidative free radicals)12,20,54,101−103

melanin can repeatedly scavenge oxidative free radicals20,54

eumelanin and pheomelanin have different redox potentials33,64

the redox potential of pheomelanin is more oxidative than that of eumelanin64

melanin can serve as a redox buffer (e.g., to maintain redox homeostasis)15,20,39,52,54,64,104

melanin can scavenge reductive radicals54

melanin’s redox properties are contextdependent: • melanin must be in a reduced state to scavenge an oxidative free radical (e.g., ABTS+ •)54 • melanin must be in an oxidized state to scavenge a reductive free radical (e.g., PQ+ •)54 • melanin must be in a reduced state to generate ROS20

probing.82,83 For instance, it should be possible for outputs to be analyzed in real time (e.g., using machine learning methods)84,85 and input sequences to be adapted to enhance the efficiency of materials characterization. Furthermore, the capability of integrating output measurements through multiple sensor modalities could enrich the information harvested through MEP. An obvious question is, why is a method that enables a characterization of redox properties valuable for materials science? While there are well-known applications for conducting polymers (e.g., for flexible electronics), why are redox-active but nonconducting organic materials useful? We contend that redox activity (versus electronic conductivity) is more relevant to biological applications, because the “flow” of electrons in the aqueous environments of biology occurs through redox reactions involving molecular intermediates (and not by a flowing “sea” of electrons typically envisioned in a metal). Thus, MEP could provide a valuable characterization tool to assist in the development of materials for emerging applications in the life sciences (e.g., materials with redoxbased antioxidants, pro-oxidants, and radical scavenging activities),86−89 and to create redox capacitor materials for biobased energy conversion,90−93 and bioelectronics.94−97 The development of MEP has been largely driven by the desire to characterize melanin, which is an abundant but poorly understood biological material. As illustrated in Table 1, we believe MEP has contributed to an understanding of melanin in several important ways. As indicated by the first row, previous groups have reported that melanins are redoxactive23,45,97−100 and our results with MEP provide supporting evidence for these claims.20,55,64,71 Moreover, the convenience of MEP enabled these claims to be extended through demonstrations that various melanins are reversibly redoxactive and they can be repeatedly switched between oxidized and reduced states.20,55,64,71 The reversibility of the redox activity of melanins suggests that melanin can serve as a redox catalyst for the transfer of electrons from a range of biologically relevant reductants to oxidants.49,57,105,106 This putative redox catalyst activity led to MEP studies that demonstrated (we believe for the first time) that melanin can redox-cycle with exogenous chemicals, such as the toxin paraquat71 and with drugs.55 Potentially, these new observations55,71 are important, because they suggest that redox cycling may be an under-

appreciated phenomenon that could contribute to chemical toxicities and therapeutic benefits. The second row in Table 1 indicates that others have reported that melanin has antioxidant activities that include free-radical scavenging abilities.12,101−103 Again, MEP provided supporting evidence that melanin can donate electrons to scavenge oxidative free radicals20,54 and MEP extended these observations by demonstrating that this radical scavenging activity is repeatable (i.e., melanin can serve as a catalyst and not simply as a “consumable” reagent for oxidative radical scavenging).54 In addition, we believe MEP led to a new observation, that melanin can also accept electrons to scavenge reductive free radicals.54 The third row in Table 1 indicates that previous researchers have suggested that the functional differences between eumelanin and pheomelanin (protective versus pro-oxidant) may result from differences in their redox potentials.15,33 MEP supported and extended these differences by showing that pheomelanin has a more-oxidative redox potential compared to eumelanin.64 The final row in Table 1 indicates that others have suggested that melanin may serve as a redox buffer, which could be important for maintaining homeostasis.39,52,104 We believe MEP has supported these suggestions and has provided new insights of the context dependence of melanin’s properties.20,54 Specifically, melanin must be in a reduced state to be able to donate electrons to quench oxidative free radicals, and melanin must be in an oxidized state to accept electrons to quench reductive free radicals.54 Further, the integration of our electrochemical methods with more conventional chemical analyses enabled us to show that melanin can generate reactive oxygen species (ROS) only if the melanin is in a reduced state and exposed to oxygen.20 Probably, the broader contribution of MEP to biology will be that it may yield deeper insights into possible redox-based activities in biology. Specifically, melanins have redox potentials in the midphysiological range which means that their functional activity is dependent on their local context. In reducing contexts, melanins can accept electrons, while in oxidizing contexts, melanins can donate electrons (even donate electrons to O2 to generate reactive oxygen species).107−109 Yet, melanins often exist in complex redox contexts. For instance, cell-wall-bound fungal melanin (Figure 5a) is 5822



Methods/Protocols

(2) Micillo, R.; Panzella, L.; Koike, K.; Monfrecola, G.; Napolitano, A.; d’Ischia, M. Fifty Shades” of Black and Red or How Carboxyl Groups Fine Tune Eumelanin and Pheomelanin Properties. Int. J. Mol. Sci. 2016, 17, 746. (3) Napolitano, A.; Panzella, L.; Leone, L.; d’Ischia, M. Red Hair Benzothiazines and Benzothiazoles: Mutation-Inspired Chemistry in the Quest for Functionality. Acc. Chem. Res. 2013, 46, 519−528. (4) Boulton, M.; Różanowska, M.; Różanowski, B. Retinal photodamage. J. Photochem. Photobiol., B 2001, 64, 144−161. (5) Brenner, M.; Hearing, V. J. The Protective Role of Melanin Against UV Damage in Human Skin†. Photochem. Photobiol. 2008, 84, 539−549. (6) Dadachova, E.; Bryan, R. A.; Howell, R. C.; Schweitzer, A. D.; Aisen, P.; Nosanchuk, J. D.; Casadevall, A. The radioprotective properties of fungal melanin are a function of its chemical composition, stable radical presence and spatial arrangement. Pigm. Cell Melanoma Res. 2008, 21, 192−199. (7) Halder, R. M.; Bridgeman-Shah, S. Skin cancer in African Americans. Cancer 1995, 75, 667−673. (8) Solano, F. Melanins: Skin Pigments and Much More-Types, Structural Models, Biological Functions, and Formation Routes. New J. Sci. 2014, 2014, 498276. (9) Panzella, L.; Gentile, G.; D’Errico, G.; Della Vecchia, N. F.; Errico, M. E.; Napolitano, A.; Carfagna, C.; d’Ischia, M. Atypical Structural and π-Electron Features of a Melanin Polymer That Lead to Superior Free-Radical-Scavenging Properties. Angew. Chem., Int. Ed. 2013, 52, 12684−12687. (10) Ju, K.-Y.; Lee, Y.; Lee, S.; Park, S. B.; Lee, J.-K. Bioinspired Polymerization of Dopamine to Generate Melanin-Like Nanoparticles Having an Excellent Free-Radical-Scavenging Property. Biomacromolecules 2011, 12, 625−632. (11) Sarna, T. New trends in photobiology: Properties and function of the ocular melanin  a photobiophysical view. J. Photochem. Photobiol., B 1992, 12, 215−258. (12) Rózȧ nowska, M.; Sarna, T.; Land, E. J.; Truscott, T. G. Free radical scavenging properties of melanin: Interaction of eu- and pheomelanin models with reducing and oxidising radicals. Free Radical Biol. Med. 1999, 26, 518−525. (13) Seagle, B.-L. L.; Rezai, K. A.; Gasyna, E. M.; Kobori, Y.; Rezaei, K. A.; Norris, J. R. Time-Resolved Detection of Melanin Free Radicals Quenching Reactive Oxygen Species. J. Am. Chem. Soc. 2005, 127, 11220−11221. (14) Panzella, L.; Szewczyk, G.; D’Ischia, M.; Napolitano, A.; Sarna, T. Zinc-induced Structural Effects Enhance Oxygen Consumption and Superoxide Generation in Synthetic Pheomelanins on UVA/ Visible Light Irradiation†. Photochem. Photobiol. 2010, 86, 757−764. (15) Panzella, L.; Leone, L.; Greco, G.; Vitiello, G.; D’Errico, G.; Napolitano, A.; d’Ischia, M. Red human hair pheomelanin is a potent pro-oxidant mediating UV-independent contributory mechanisms of melanomagenesis. Pigm. Cell Melanoma Res. 2014, 27, 244−252. (16) Mitra, D.; Luo, X.; Morgan, A.; Wang, J.; Hoang, M. P.; Lo, J.; Guerrero, C. R.; Lennerz, J. K.; Mihm, M. C.; Wargo, J. A.; Robinson, K. C.; Devi, S. P.; Vanover, J. C.; D'Orazio, J. A.; McMahon, M.; Bosenberg, M. W.; Haigis, K. M.; Haber, D. A.; Wang, Y.; Fisher, D. E. An ultraviolet-radiation-independent pathway to melanoma carcinogenesis in the red hair/fair skin background. Nature 2012, 491, 449−453. (17) Hill, H. Z.; Hill, G. J. UVA, Pheomelanin and the Carcinogenesis of Melanoma. Pigm. Cell Res. 2000, 13, 140−144. (18) Wenczl, E.; Smit, N. P. M.; Pavel, S.; Schothorst, A. A.; Van der Schans, G. P.; Timmerman, A. J.; Roza, L.; Kolb, R. M. (Pheo)Melanin Photosensitizes UVA-Induced DNA Damage in Cultured Human Melanocytes. J. Invest. Dermatol. 1998, 111, 678− 682. (19) Dubey, S.; Roulin, A. Evolutionary and biomedical consequences of internal melanins. Pigm. Cell Melanoma Res. 2014, 27, 327−338.

localized at an interface between reducing intracellular conditions and more-oxidative extracellular conditions. Their ability to exchange electrons with soluble mediators suggests that cell-wall melanins could allow intracellular energy resources (i.e., reducing equivalents) to be enlisted to perform functions outside the cell (e.g., to quench extracellular oxidative free radicals or transmit redox-based signals). This reasoning suggests that melanins are not simply inert waste products, but possibly may be active participants in redox biology. Moreover, if it is possible for melanin to play a role in the transduction and transmission of redox information, what about plant lignins that share some of melanin’s structural features and redox activities? And, if these possibilities are realities for melanin and lignin, what about humic acids that are prevalent in soil?110−115 These possibilities suggest that our biosphere may be a medium for redox-based communication and this activity has gone unnoticed, because of measurement limitations. In summary, we believe that mediated electrochemical probing provides a new and simple way to probe the redox properties of materials. We anticipate that the use of this new characterization method could enable observations of previously unknown phenomena of importance in nature and technology.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.8b02428. Information for preparing the melanin−chitosan films (Figure S1); SEM images of these films (Figure S2); schematics of the electrochemical system (Figure S3) and the spectroelectrochemical system (Figure S4) (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Tel.: 301-405-8389. Fax: 301-314-9075. E-mail: gpayne@ umd.edu. ORCID

Eunkyoung Kim: 0000-0003-2566-4041 Lucia Panzella: 0000-0002-2662-8205 Alessandra Napolitano: 0000-0003-0507-5370 William E. Bentley: 0000-0002-4855-7866 Gregory F. Payne: 0000-0001-6638-9459 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from the United States National Science Foundation (No. DMREF1435957) and the Department of Defense (Defense Threat Reduction Agency; Nos. HDTRA1-13-1-0037 and HDTRA115-1-0058).

■

REFERENCES

(1) Ito, S.; Wakamatsu, K.; d’Ischia, M.; Napolitano, A.; Pezzella, A. Structure of Melanins. In Melanins and Melanosomes; Borovansk, J., Riley, P. A., Eds.; Wiley−VCH: Weinheim, Germany, 2011; pp 167− 185. 5823



Methods/Protocols

(20) Kim, E.; Liu, Y.; Leverage, W. T.; Yin, J.-J.; White, I. M.; Bentley, W. E.; Payne, G. F. Context-Dependent Redox Properties of Natural Phenolic Materials. Biomacromolecules 2014, 15, 1653−1662. (21) Simon, J. D.; Hong, L.; Peles, D. N. Insights into Melanosomes and Melanin from Some Interesting Spatial and Temporal Properties. J. Phys. Chem. B 2008, 112, 13201−13217. (22) Nofsinger, J. B.; Forest, S. E.; Eibest, L. M.; Gold, K. A.; Simon, J. D. Probing the Building Blocks of Eumelanins Using Scanning Electron Microscopy. Pigm. Cell Res. 2000, 13, 179−184. (23) Watt, A. A. R.; Bothma, J. P.; Meredith, P. The supramolecular structure of melanin. Soft Matter 2009, 5, 3754−3760. (24) Del Bino, S.; Ito, S.; Sok, J.; Nakanishi, Y.; Bastien, P.; Wakamatsu, K.; Bernerd, F. Chemical analysis of constitutive pigmentation of human epidermis reveals constant eumelanin to pheomelanin ratio. Pigm. Cell Melanoma Res. 2015, 28, 707−717. (25) Ito, S.; Wakamatsu, K. Quantitative Analysis of Eumelanin and Pheomelanin in Humans, Mice, and Other Animals: a Comparative Review. Pigm. Cell Res. 2003, 16, 523−531. (26) Ito, S.; Wakamatsu, K.; Sarna, T. Photodegradation of Eumelanin and Pheomelanin and Its Pathophysiological Implications. Photochem. Photobiol. 2018, 94, 409−420. (27) Zucca, F. A.; Basso, E.; Cupaioli, F. A.; Ferrari, E.; Sulzer, D.; Casella, L.; Zecca, L. Neuromelanin of the Human Substantia Nigra: An Update. Neurotoxic. Res. 2014, 25, 13−23. (28) Mann, D. M. A.; Yates, P. O. Lipoprotein pigments-Their relationship to ageing in the human nervious systemII. The melanin content of pigmented nerve cells. Brain 1974, 97, 489−498. (29) Zucca, F. A.; Segura-Aguilar, J.; Ferrari, E.; Muñoz, P.; Paris, I.; Sulzer, D.; Sarna, T.; Casella, L.; Zecca, L. Interactions of iron, dopamine and neuromelanin pathways in brain aging and Parkinson’s disease. Prog. Neurobiol. 2017, 155, 96−119. (30) Segura-Aguilar, J.; Paris, I.; Muñoz, P.; Ferrari, E.; Zecca, L.; Zucca, F. A. Protective and toxic roles of dopamine in Parkinson’s disease. J. Neurochem. 2014, 129, 898−915. (31) Zucca, F. A.; Vanna, R.; Cupaioli, F. A.; Bellei, C.; De Palma, A.; Di Silvestre, D.; Mauri, P.; Grassi, S.; Prinetti, A.; Casella, L.; Sulzer, D.; Zecca, L. Neuromelanin organelles are specialized autolysosomes that accumulate undegraded proteins and lipids in aging human brain and are likely involved in Parkinson’s disease. npj Parkinson’s Disease 2018, 4, 17. (32) Ito, S. Encapsulation of a reactive core in neuromelanin. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 14647−14648. (33) Bush, W. D.; Garguilo, J.; Zucca, F. A.; Albertini, A.; Zecca, L.; Edwards, G. S.; Nemanich, R. J.; Simon, J. D. The surface oxidation potential of human neuromelanin reveals a spherical architecture with a pheomelanin core and a eumelanin surface. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 14785−14789. (34) Nappi, A. J.; Christensen, B. M. Melanogenesis and associated cytotoxic reactions: Applications to insect innate immunity. Insect Biochem. Mol. Biol. 2005, 35, 443−459. (35) Andersen, S. O. Insect cuticular sclerotization: A review. Insect Biochem. Mol. Biol. 2010, 40, 166−178. (36) Nakhleh, J.; El Moussawi, L.; Osta, M. A. The Melanization Response in Insect Immunity. Adv. Insect Physiol. 2017, 52, 83−109. (37) Cordero, R. J. B.; Casadevall, A. Functions of fungal melanin beyond virulence. Fungal Biol. Rev. 2017, 31, 99−112. (38) Butler, M. J.; Day, A. W. Fungal melanins: a review. Can. J. Microbiol. 1998, 44, 1115−1136. (39) Jacobson, E. S. Pathogenic Roles for Fungal Melanins. Clin. Microbiol. Rev. 2000, 13, 708−717. (40) Casadevall, A.; Rosas, A. L.; Nosanchuk, J. D. Melanin and virulence in Cryptococcus neoformans. Curr. Opin. Microbiol. 2000, 3, 354−358. (41) Liu, G. Y.; Nizet, V. Color me bad: microbial pigments as virulence factors. Trends Microbiol. 2009, 17, 406−413. (42) Nosanchuk, J. D.; Casadevall, A. The contribution of melanin to microbial pathogenesis. Cell. Microbiol. 2003, 5, 203−223.

(43) Dadachova, E.; Casadevall, A. Ionizing radiation: how fungi cope, adapt, and exploit with the help of melanin. Curr. Opin. Microbiol. 2008, 11, 525−531. (44) Khajo, A.; Bryan, R. A.; Friedman, M.; Burger, R. M.; Levitsky, Y.; Casadevall, A.; Magliozzo, R. S.; Dadachova, E. Protection of Melanized Cryptococcus neoformans from Lethal Dose Gamma Irradiation Involves Changes in Melanin’s Chemical Structure and Paramagnetism. PLoS One 2011, 6, e25092. (45) Meredith, P.; Sarna, T. The physical and chemical properties of eumelanin. Pigm. Cell Res. 2006, 19, 572−594. (46) d’Ischia, M.; Napolitano, A.; Pezzella, A.; Meredith, P.; Sarna, T. Chemical and structural diversity in eumelanins: unexplored biooptoelectronic materials. Angew. Chem., Int. Ed. 2009, 48, 3914−3921. (47) d’Ischia, M.; Wakamatsu, K.; Cicoira, F.; Di Mauro, E.; GarciaBorron, J. C.; Commo, S.; Galván, I.; Ghanem, G.; Kenzo, K.; Meredith, P.; Pezzella, A.; Santato, C.; Sarna, T.; Simon, J. D.; Zecca, L.; Zucca, F. A.; Napolitano, A.; Ito, S. Melanins and melanogenesis: from pigment cells to human health and technological applications. Pigm. Cell Melanoma Res. 2015, 28, 520−544. (48) Gan, E. V.; Haberman, H. F.; Menon, I. A. Electron transfer properties of melanin. Arch. Biochem. Biophys. 1976, 173, 666−672. (49) Dadachova, E.; Bryan, R. A.; Huang, X.; Moadel, T.; Schweitzer, A. D.; Aisen, P.; Nosanchuk, J. D.; Casadevall, A. Ionizing Radiation Changes the Electronic Properties of Melanin and Enhances the Growth of Melanized Fungi. PLoS One 2007, 2, e457. (50) Serpentini, C.-L.; Gauchet, C.; de Montauzon, D.; Comtat, M.; Ginestar, J.; Paillous, N. First electrochemical investigation of the redox properties of DOPA−melanins by means of a carbon paste electrode. Electrochim. Acta 2000, 45, 1663−1668. (51) Gidanian, S.; Farmer, P. J. Redox behavior of melanins: direct electrochemistry of dihydroxyindole-melanin and its Cu and Zn adducts. J. Inorg. Biochem. 2002, 89, 54−60. (52) Jacobson, E. S.; Hong, J. D. Redox buffering by melanin and Fe(II) in Cryptococcus neoformans. J. Bacteriol. 1997, 179, 5340− 5346. (53) Ye, T.; Hong, L.; Garguilo, J.; Pawlak, A.; Edwards, G. S.; Nemanich, R. J.; Sarna, T.; Simon, J. D. Photoionization Thresholds of Melanins Obtained from Free Electron Laser-Photoelectron Emission Microscopy, Femtosecond Transient Absorption Spectroscopy and Electron Paramagnetic Resonance Measurements of Oxygen Photoconsumption. Photochem. Photobiol. 2006, 82, 733−737. (54) Kim, E.; Kang, M.; Tschirhart, T.; Malo, M.; Dadachova, E.; Cao, G.; Yin, J.-J.; Bentley, W. E.; Wang, Z.; Payne, G. F. Spectroelectrochemical Reverse Engineering DemonstratesThat Melanin’s Redox and Radical Scavenging Activities Are Linked. Biomacromolecules 2017, 18, 4084−4098. (55) Temoçin, Z.; Kim, E.; Li, J.; Panzella, L.; Alfieri, M. L.; Napolitano, A.; Kelly, D. L.; Bentley, W. E.; Payne, G. F. The Analgesic Acetaminophen and the Antipsychotic Clozapine Can Each Redox-Cycle with Melanin. ACS Chem. Neurosci. 2017, 8, 2766−2777. (56) Nyhus, K. J.; Wilborn, A. T.; Jacobson, E. S. Ferric iron reduction by Cryptococcus neoformans. Infect. Immun. 1997, 65, 434− 438. (57) Turick, C. E.; Tisa, L. S.; Caccavo, J. F. Melanin Production and Use as a Soluble Electron Shuttle for Fe(III) Oxide Reduction and as a Terminal Electron Acceptor by Shewanella algae BrY. Appl. Environ. Microbiol. 2002, 68, 2436−2444. (58) Kim, E.; Liu, Y.; Bentley, W. E.; Payne, G. F. Redox Capacitor to Establish Bio-Device Redox-Connectivity. Adv. Funct. Mater. 2012, 22, 1409−1416. (59) Kim, E.; Leverage, W. T.; Liu, Y.; White, I. M.; Bentley, W. E.; Payne, G. F. Redox-capacitor to connect electrochemistry to redoxbiology. Analyst 2014, 139, 32−43. (60) Donato, P. D.; Napolitano, A. 1,4-Benzothiazines as Key Intermediates in the Biosynthesis of Red Hair Pigment Pheomelanins. Pigm. Cell Res. 2003, 16, 532−539. (61) Ito, S. A Chemist’s View of Melanogenesis. Pigm. Cell Res. 2003, 16, 230−236. 5824



Methods/Protocols

hydrogen-evolving gold and nickel electrodes. J. Electroanal. Chem. Interfacial Electrochem. 1981, 125, 367−386. (81) Bird, C. L.; Kuhn, A. T. Electrochemistry of the viologens. Chem. Soc. Rev. 1981, 10, 49−82. (82) Schmidt, M.; Lipson, H. Distilling Free-Form Natural Laws from Experimental Data. Science 2009, 324, 81−85. (83) Nikolaev, P.; Hooper, D.; Webber, F.; Rao, R.; Decker, K.; Krein, M.; Poleski, J.; Barto, R.; Maruyama, B. Autonomy in materials research: a case study in carbon nanotube growth. npj Comput. Mater. 2016, 2, 16031. (84) Jordan, M. I.; Mitchell, T. M. Machine learning: Trends, perspectives, and prospects. Science 2015, 349, 255−260. (85) Ghahramani, Z. Probabilistic machine learning and artificial intelligence. Nature 2015, 521, 452−459. (86) Liu, H.; Qu, X.; Kim, E.; Lei, M.; Dai, K.; Tan, X.; Xu, M.; Li, J.; Liu, Y.; Shi, X.; Li, P.; Payne, G. F.; Liu, C. Bio-inspired redoxcycling antimicrobial film for sustained generation of reactive oxygen species. Biomaterials 2018, 162, 109−122. (87) Liu, Y.; Ai, K.; Ji, X.; Askhatova, D.; Du, R.; Lu, L.; Shi, J. Comprehensive Insights into the Multi-Antioxidative Mechanisms of Melanin Nanoparticles and Their Application To Protect Brain from Injury in Ischemic Stroke. J. Am. Chem. Soc. 2017, 139, 856−862. (88) Valgimigli, L.; Baschieri, A.; Amorati, R. Antioxidant activity of nanomaterials. J. Mater. Chem. B 2018, 6, 2036−2051. (89) Solano, F. Melanin and Melanin-Related Polymers as Materials with Biomedical and Biotechnological ApplicationsCuttlefish Ink and Mussel Foot Proteins as Inspired Biomolecules. Int. J. Mol. Sci. 2017, 18, 1561. (90) Kim, Y. J.; Wu, W.; Chun, S.-E.; Whitacre, J. F.; Bettinger, C. J. Biologically derived melanin electrodes in aqueous sodium-ion energy storage devices. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 20912− 20917. (91) Kim, Y. J.; Khetan, A.; Wu, W.; Chun, S.-E.; Viswanathan, V.; Whitacre, J. F.; Bettinger, C. J. Evidence of Porphyrin-Like Structures in Natural Melanin Pigments Using Electrochemical Fingerprinting. Adv. Mater. 2016, 28, 3173−3180. (92) Kim, Y. J.; Wu, W.; Chun, S.-E.; Whitacre, J. F.; Bettinger, C. J. Catechol-Mediated Reversible Binding of Multivalent Cations in Eumelanin Half-Cells. Adv. Mater. 2014, 26, 6572−6579. (93) Lampel, A.; McPhee, S. A.; Park, H.-A.; Scott, G. G.; Humagain, S.; Hekstra, D. R.; Yoo, B.; Frederix, P. W. J. M.; Li, T.D.; Abzalimov, R. R.; Greenbaum, S. G.; Tuttle, T.; Hu, C.; Bettinger, C. J.; Ulijn, R. V. Polymeric peptide pigments with sequence-encoded properties. Science 2017, 356, 1064−1068. (94) Liu, Y.; Tsao, C.-Y.; Kim, E.; Tschirhart, T.; Terrell, J. L.; Bentley, W. E.; Payne, G. F. Using a Redox Modality to Connect Synthetic Biology to Electronics: Hydrogel-Based Chemo-Electro Signal Transduction for Molecular Communication. Adv. Healthcare Mater. 2017, 6, 1600908. (95) Liu, Y.; Kim, E.; Li, J.; Kang, M.; Bentley, W. E.; Payne, G. F. Electrochemistry for bio-device molecular communication: The potential to characterize, analyze and actuate biological systems. Nano Commun. Netw. 2017, 11, 76−89. (96) Kim, E.; Liu, Y.; Ben-Yoav, H.; Winkler, T. E.; Yan, K.; Shi, X.; Shen, J.; Kelly, D. L.; Ghodssi, R.; Bentley, W. E.; Payne, G. F. Fusing Sensor Paradigms to Acquire Chemical Information: An Integrative Role for Smart Biopolymeric Hydrogels. Adv. Healthcare Mater. 2016, 5, 2595−2616. (97) Sheliakina, M.; Mostert, A. B.; Meredith, P. An all-solid-state biocompatible ion-to-electron transducer for bioelectronics. Mater. Horiz. 2018, 5, 256−263. (98) Mostert, A. B.; Powell, B. J.; Pratt, F. L.; Hanson, G. R.; Sarna, T.; Gentle, I. R.; Meredith, P. Role of semiconductivity and ion transport in the electrical conduction of melanin. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 8943−8947. (99) Meredith, P.; Bettinger, C. J.; Irimia-Vladu, M.; Mostert, A. B.; Schwenn, P. E. Electronic and optoelectronic materials and devices inspired by nature. Rep. Prog. Phys. 2013, 76, 034501.

(62) Mitra, D.; Luo, X.; Morgan, A.; Wang, J.; Hoang, M. P.; Lo, J.; Guerrero, C. R.; Lennerz, J. K.; Mihm, M. C.; Wargo, J. A.; Robinson, K. C.; Devi, S. P.; Vanover, J. C.; D’Orazio, J. A.; McMahon, M.; Bosenberg, M. W.; Haigis, K. M.; Haber, D. A.; Wang, Y.; Fisher, D. E. An ultraviolet-radiation-independent pathway to melanoma carcinogenesis in the red hair/fair skin background. Nature 2012, 491, 449−454. (63) Napolitano, A.; Panzella, L.; Monfrecola, G.; d’Ischia, M. Pheomelanin-induced oxidative stress: bright and dark chemistry bridging red hair phenotype and melanoma. Pigm. Cell Melanoma Res. 2014, 27, 721−733. (64) Kim, E.; Panzella, L.; Micillo, R.; Bentley, W. E.; Napolitano, A.; Payne, G. F. Reverse Engineering Applied to Red Human Hair Pheomelanin Reveals Redox-Buffering as a Pro-Oxidant Mechanism. Sci. Rep. 2016, 5, 18447. (65) Lancaster, E. M.; Hiatt, J. R.; Zarrinpar, A. Acetaminophen hepatotoxicity: an updated review. Arch. Toxicol. 2015, 89, 193−199. (66) Yoon, E.; Babar, A.; Choudhary, M.; Kutner, M.; Pyrsopoulos, N. Acetaminophen-Induced Hepatotoxicity: A Comprehensive Update. J. Clin. Transl. Hepatol. 2016, 4, 131−142. (67) Du, K.; Ramachandran, A.; Jaeschke, H. Oxidative stress during acetaminophen hepatotoxicity: Sources, pathophysiological role and therapeutic potential. Redox Biol. 2016, 10, 148−156. (68) Nazıroğlu, M.; Cihangir Uğuz, A.; Koçak, A.; Bal, R. Acetaminophen at Different Doses Protects Brain Microsomal Ca2+ATPase and the Antioxidant Redox System in Rats. J. Membr. Biol. 2009, 231, 57−64. (69) Zhao, W.-X.; Zhang, J.-H.; Cao, J.-B.; Wang, W.; Wang, D.-X.; Zhang, X.-Y.; Yu, J.; Zhang, Y.-Y.; Zhang, Y.-Z.; Mi, W.-D. Acetaminophen attenuates lipopolysaccharide-induced cognitive impairment through antioxidant activity. J. Neuroinflammation 2017, 14, 17. (70) Ghanem, C. I.; Pérez, M. J.; Manautou, J. E.; Mottino, A. D. Acetaminophen from liver to brain: New insights into drug pharmacological action and toxicity. Pharmacol. Res. 2016, 109, 119−131. (71) Kim, E.; Leverage, W. T.; Liu, Y.; Panzella, L.; Alfieri, M. L.; Napolitano, A.; Bentley, W. E.; Payne, G. F. Paraquat−Melanin Redox-Cycling: Evidence from Electrochemical Reverse Engineering. ACS Chem. Neurosci. 2016, 7, 1057−1067. (72) Double, K. L.; Zecca, L.; Costi, P.; Mauer, M.; Griesinger, C.; Ito, S.; Ben-Shachar, D.; Bringmann, G.; Fariello, R. G.; Riederer, P.; Gerlach, M. Structural Characteristics of Human Substantia Nigra Neuromelanin and Synthetic Dopamine Melanins. J. Neurochem. 2000, 75, 2583−2589. (73) Eisenman, H. C.; Casadevall, A. Synthesis and assembly of fungal melanin. Appl. Microbiol. Biotechnol. 2012, 93, 931−940. (74) Bell, A. A.; Wheeler, M. H. Biosynthesis and Functions of Fungal Melanins. Annu. Rev. Phytopathol. 1986, 24, 411−451. (75) Gessler, N. N.; Egorova, A. S.; Belozerskaya, T. A. Melanin pigments of fungi under extreme environmental conditions (Review). Appl. Biochem. Microbiol. 2014, 50, 105−113. (76) Ivekovic, D.; Milardovic, S.; Roboz, M.; Grabaric, B. S. Evaluation of the antioxidant activity by flow injection analysis method with electrochemically generated ABTS radical cation. Analyst 2005, 130, 708−714. (77) Scott, S. L.; Chen, W. J.; Bakac, A.; Espenson, J. H. Spectroscopic parameters, electrode potentials, acid ionization constants, and electron exchange rates of the 2,2′-azinobis(3ethylbenzothiazoline-6-sulfonate) radicals and ions. J. Phys. Chem. 1993, 97, 6710−6714. (78) Konan, K. V.; Le Tien, C.; Mateescu, M. A. Electrolysisinduced fast activation of the ABTS reagent for an antioxidant capacity assay. Anal. Methods 2016, 8, 5638−5644. (79) Zak, J. K.; Butler, J. E.; Swain, G. M. Diamond Optically Transparent Electrodes: Demonstration of Concept with Ferri/ Ferrocyanide and Methyl Viologen. Anal. Chem. 2001, 73, 908−914. (80) Bowden, E. F.; Hawkridge, F. M. Thin-layer spectroelectrochemical kinetic study of viologen cation radicals reacting at 5825



Methods/Protocols

(100) d’Ischia, M.; Napolitano, A.; Pezzella, A.; Meredith, P.; Sarna, T. Chemical and Structural Diversity in Eumelanins: Unexplored BioOptoelectronic Materials. Angew. Chem., Int. Ed. 2009, 48, 3914− 3921. (101) Sarna, T.; Plonka, P. M. Biophysical Studies of Melanin. In Biomedical EPR, Part A: Free Radicals, Metals, Medicine, and Physiology; Eaton, S. R., Eaton, G. R., Berliner, L. J., Eds. Springer: Boston, MA, 2005; pp 125−146. (102) Panzella, L.; Gentile, G.; D'Errico, G.; Della Vecchia, N. F.; Errico, M. E.; Napolitano, A.; Carfagna, C.; d’Ischia, M. Atypical Structural and π-Electron Features of a Melanin Polymer That Lead to Superior Free-Radical-Scavenging Properties. Angew. Chem., Int. Ed. 2013, 52, 12684−12687. (103) Jacobson, E. S.; Hove, E.; Emery, H. S. Antioxidant function of melanin in black fungi. Infect. Immun. 1995, 63, 4944−4945. (104) Jacobson, E. S.; Goodner, A. P.; Nyhus, K. J. Ferrous Iron Uptake in Cryptococcus neoformans. Infect. Immun. 1998, 66, 4169− 4175. (105) Turick, C. E.; Caccavo, F.; Tisa, L. S. Electron transfer from Shewanella algae BrY to hydrous ferric oxide is mediated by cellassociated melanin. FEMS Microbiol. Lett. 2003, 220, 99−104. (106) Bryan, R.; Jiang, Z.; Friedman, M.; Dadachova, E. The effects of gamma radiation, UV and visible light on ATP levels in yeast cells depend on cellular melanization. Fungal Biol. 2011, 115, 945−949. (107) Liu-Smith, F.; Poe, C.; Farmer, P. J.; Meyskens, F. L. Amyloids, Melanins and Oxidative Stress in Melanomagenesis. Exp. Dermatol. 2015, 24, 171−174. (108) Munoz-Munoz, J. L.; García-Molina, F.; Varón, R.; Tudela, J.; García-Cánovas, F.; Rodríguez-López, J. N. Generation of hydrogen peroxide in the melanin biosynthesis pathway. Biochim. Biophys. Acta, Proteins Proteomics 2009, 1794, 1017−1029. (109) Premi, S.; Wallisch, S.; Mano, C. M.; Weiner, A. B.; Bacchiocchi, A.; Wakamatsu, K.; Bechara, E. J. H.; Halaban, R.; Douki, T.; Brash, D. E. Chemiexcitation of Melanin Derivatives Induces DNA Photoproducts Long after UV Exposure. Science 2015, 347, 842−847. (110) Newman, D. K.; Kolter, R. A role for excreted quinones in extracellular electron transfer. Nature 2000, 405, 94−97. (111) Aeschbacher, M.; Sander, M.; Schwarzenbach, R. P. Novel Electrochemical Approach to Assess the Redox Properties of Humic Substances. Environ. Sci. Technol. 2010, 44, 87−93. (112) Sun, T.; Levin, B. D. A.; Guzman, J. J. L.; Enders, A.; Muller, D. A.; Angenent, L. T.; Lehmann, J. Rapid electron transfer by the carbon matrix in natural pyrogenic carbon. Nat. Commun. 2017, 8, 14873. (113) Aeschbacher, M.; Graf, C.; Schwarzenbach, R. P.; Sander, M. Antioxidant Properties of Humic Substances. Environ. Sci. Technol. 2012, 46, 4916−4925. (114) Sander, M.; Hofstetter, T. B.; Gorski, C. A. Electrochemical Analyses of Redox-Active Iron Minerals: A Review of Nonmediated and Mediated Approaches. Environ. Sci. Technol. 2015, 49, 5862− 5878. (115) Aeschbacher, M.; Brunner, S. H.; Schwarzenbach, R. P.; Sander, M. Assessing the Effect of Humic Acid Redox State on Organic Pollutant Sorption by Combined Electrochemical Reduction and Sorption Experiments. Environ. Sci. Technol. 2012, 46, 3882− 3890.

5826


Reverse Engineering To Characterize Redox Properties: Revealing

Recommend Documents