Reverse Engineering To Characterize Redox Properties: Revealing

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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 Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.8b02428 • Publication Date (Web): 17 Aug 2018 Downloaded from http://pubs.acs.org on August 22, 2018

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Chemistry of Materials

Reverse Engineering to Characterize Redox Properties: Revealing Melanin’s Redox Activity through Mediated Electrochemical Probing

Mijeong Kang,a,b Eunkyoung Kim,a,b Zülfikar Temoçin,c Jinyang Li,a,b Ekaterina Dadachova,d Zheng Wang,e Lucia Panzella,f Alessandra Napolitano,f William E. Bentley,a,b Gregory F. Payne a,b*

a

Institute for Bioscience and Biotechnology Research, University of Maryland, 4291 Fieldhouse Drive , Plant Sciences Building, College Park, Maryland 20742, USA

b Fischell Department of Bioengineering, University of Maryland, 8228 Paint Branch Drive, 2330 Jeong H. Kim Engineering Building, College Park, Maryland 20742, USA c

Department of Chemistry, Science and Arts Faculty, Kırıkkale University, Yahȿihan, 71450 Kırıkkale, Turkey

d

College of Pharmacy & Nutrition, University of Saskatchewan, Canada

e

Center for Bio/Molecular Science and Engineering, US Naval Research Laboratory, Washington, DC, 20375, USA

f

Department of Chemical Sciences, University of Naples Federico II, Via Cintia 4, I-80126 Naples (Italy)

*Corresponding author Email: [email protected] Phone: 301-405-8389 FAX: 301-314-9075

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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 melanins’ proposed functions (e.g., antioxidant, pro-

oxidant and radical scavenging) involve an exchange of electrons, we developed an electrochemical reverse engineering methodology to probe melanin’s redoxproperties.

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

characteristics (e.g., currents) to reveal redox properties.

output

response

Here, we illustrate the

versatility of MEP and review results demonstrating 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: MELANIN


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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. Melanins are a vast class of black, brown or even yellowish and reddish natural biopolymers

of

biogenetically

diverse

from

the

nature

and

oxidation

of

chemical

composition

phenolic

metabolites

that (Figure

arise 1).

Traditionally the term melanins has been used to refer to two main groups of intracellular nitrogenous pigments, the dark eumelanins and the lighter, reddishyellow 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 fair complexioned

individuals

have

been

suggested

photosensitizing14 and pro-oxidant activity.15-18

to

have

an

opposite,

Yet the presence of melanins in

unexposed areas of the human body (e.g., the brain), suggests melanins



Page 4 of 41

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 officinalis,

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, due to its relatively high purity.

Unlike eumelanins, which are

relatively widespread in nature, pheomelanins are found only in mammalian skin, hair, 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

pathogenesis of Parkinson's disease.27,

29,

its 30

possible

implication

in

the

It is generally agreed that


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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 radiations.43, 44 Figure 1 illustrates that these fungal melanins are known to derive from oxidative coupling of 1,8-dihydroxynaphthalene (DHN), a naturally occurring polyketide. In summary, melanin is found in a variety 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 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 melanin’s ability 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



Page 6 of 41

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 last 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 (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 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 non-conducting hydrogel (typically ~ 0.5 mm wet film of the aminopolysaccharide chitosan) adjacent to an electrode surface (Figure S1 in 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 (Figure S2 in Supporting


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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 (Figure S3 in 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 the example in Figure 2c, the

schematic shows: 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 re-oxidized 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 redoxcycling 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.



Page 8 of 41

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. 20.20 (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 (timeinvarying) amplifications are signatures of reversible and repeated oxidation and reduction. Adapted with permission from ref. 54.54 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,


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the electrochemical currents associated with mediator oxidation/reduction is observed to be amplified and 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 at

the left in Figure 2c and two representations of the output curves are shown at the right.

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 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 redox-active (i.e., it can exchange electrons with the mediators). The conclusion from Figure 2c, is that melanin is redox active; 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,

Page 10 of 41

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 melanin-containing 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 hour 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-hour 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-invarying. 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. transfer

between

observations

have

two

While the results in Figure 2d show electron

electrochemical

been

extended

to

mediators other,


(Fc

more

and

Ru3+),

these

biologically-relevant,

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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 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



Figure 3.

Page 12 of 41

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. 64.64 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 3 mediators each with


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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 at the right 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 redox-probing 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 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 output response for probing of a film



containing the synthetic model pheomelanin. In this case, an amplification of Iroxidation (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 redox-cycling 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 near that of eumelanin.]64 From a biological perspective, the results from this reverse engineering study may suggest explanations for pheomelanin’s reported pro-oxidant activities. Specifically, if pheomelanin can act as an electron transfer catalyst, and its redox potential is comparatively oxidative (vs eumelanin), then it may exert prooxidant 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


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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 (N-acetyl-

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 More recent studies have suggested 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 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

neuromelanin.55,

71, 72

cysteinyldopamine−dopamine

core−shell

model

of

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. It is important to note however 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



Page 16 of 41

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 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-invarying) amplification of mediator currents requires a balance between the oxidative and reductive redox-cycling mechanisms. Adapted with permission from ref. 55.55 Figure 4b illustrates the importance of balancing oxidative and reductive redox-


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cycling mechanisms to generate steady (i.e., time-invarying) 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 period (first 190 min), the voltage was

cycled between +0.7 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 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 and 480 min, the input amplitude was again adjusted back to the initial amplitude (between +0.7 and −0.4 V) and the output curve in Figure 4b shows a return to time-invarying 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



Page 18 of 41

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 to be electrochemically “silent”). As expected, the output curve during this period of unbalanced probing (480 to 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 5 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 sideeffects.

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. versatility of MEP.

From a methods perspective, Figure 4 illustrates the

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


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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

approach.

cell

and

bimodal

measurement

(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. 54.54



Page 20 of 41

provided measures of the kinetics of these reactions.

Amplifications of

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. and

can

provide

Optical modalities are especially convenient

complementary

chemical

information.

Such

a

spectroelectrochemical approach is illustrated by studies with fungal 1,8dihydroxynaphthalene (DHN)-melanin (Figure S4 in 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 an incompletely-understood mechanism(s).

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


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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-sulphonic acid) (ABTS) can be electrochemically oxidized in a one-electron 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 non-radical 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 non-radical 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 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



Page 22 of 41

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 and 9 minutes when ABTS is oxidized, and this confirms the generation of the ABTS+• radical. +•

absorbance associated with this ABTS

The

radical is observed to decay after 9

minutes 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 a signature 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 and 20 min) and generate the PQ+• radical.

Analogous to 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

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perspective, the extension of output measurements to additional modalities enhances the richness of information that can be acquired by MEP. 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 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: 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.

Further, 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 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.

Further, 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



Page 24 of 41

redox-active but non-conducting organic materials useful?

We contend that

redox-activity (vs 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 redox-based antioxidant, pro-oxidant and radical scavenging activities)86-89, and to create redox-capacitor materials for bio-based energy conversion,90-93 and bio-electronics.94-97 Table 1. Contribution of mediated electrochemical probing (MEP) to understanding melanin Supporting evidence Melanin is redox active.

Extensions

20, 23, 45, 55, 64, 71, 97-

100

New information

Melanin’s redox activity is reversible and Melanin can redox-cycle with melanin can be repeatedly switched exogenous chemicals (e.g., toxins, between oxidized and reduced states.20, drugs).55, 71

54, 64

Melanin can scavenge free radicals (i.e., Melanin can repeatedly oxidative free radicals).12, 20, 54, 101-103 oxidative free radicals.20, 54 Eumelanin and pheomelanin different redox potentials.33, 64

scavenge Melanin can radicals.54

scavenge

reductive

have Pheomelanin’s redox potential is more oxidative than eumelanin’s.64

Melanin can serve as a redox buffer (e.g., to maintain redox homeostasis).15,

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 reductive free radical (e.g., PQ+•).54 • Melanin must be in a reduced state to generate ROS.20

20, 39, 52, 54, 64, 104

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


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groups have reported that melanins are redox-active23,

45, 97-100

with MEP provide supporting evidence for these claims.20,

and our results

55, 64, 71

Moreover,

the convenience of MEP enabled these claims to be extended through demonstrations that various melanins are reversibly redox-active and they can be repeatedly switched between oxidized and reduced states.20,

55, 64, 71

The

reversibility of melanins’ redox activity 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 as 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 vs 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



Page 26 of 41

The final row in Table 1 indicates that others have suggested that melanin may serve

as

a

homeostasis.39,

redox-buffer 52, 104

which

could

be

important

for

maintaining

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 mid-physiological range which means their functional activity depends 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).107109

Yet melanins often exist in complex redox contexts.

For instance, cell-wall

bound fungal melanin (Figure 5a) is localized at an interface between reducing intracellular conditions and more oxidative extracellular conditions. Their ability to exchange electrons with soluble mediators suggests 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.

And if it is possible for melanin to play a role in

the transduction and transmission of redox information, what about plant lignins which share some of melanin’s structural features and redox activities? And, if these possibilities are realities for melanin and lignin, what about humic


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acids that are prevalent in soil?

110-115

These possibilities suggest that our

biosphere may be 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 Supporting Information The supporting information is available free of charge on the ACS Publication Website at DOI: Supporting Information provides information for preparing the melaninchitosan films (Figure S1) and SEM images of these films (Figure S2), as well as schematics of the electrochemical system (Figure S3) and spectroelectrochemical system (Figure S4). AUTHOR INFORMATION Corresponding Author Gregory F. Payne e-mail: [email protected] Phone: 301-405-8389 FAX: 301-314-9075 ORCID Gregory F. Payne: 0000-0001-6638-9459



Eunkyoung Kim: 0000-0003-2566-4041 Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS The authors gratefully acknowledge financial support from the United States National Science Foundation (DMREF-1435957) and the Department of Defense (Defense Threat Reduction Agency; HDTRA1-13-1-0037 and HDTRA1-15-1-0058). REFERENCES (1) Ito, S.; Wakamatsu, K.; d'ischia, M.; Napolitano, A.; Pezzella, A., Structure of Melanins. In Melanins and Melanosomes, J. Borovansk, P.A. Riley ed.; Wiley-VCH: Weinheim, Germany, 2011; pp 167-185. (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 (1-13). (3) Napolitano, A.; Panzella, L.; Leone, L.; d’Ischia, M., Red Hair Benzothiazines and Benzothiazoles: Mutation-Inspired Chemistry in the Quest for Functionality.

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Page 34 of 41

Bentley, W. E.; Wang, Z.; Payne, G. F., Spectroelectrochemical Reverse Engineering

DemonstratesThat

Melanin’s

Redox

and

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