Understanding the Role of Aggregation in the Broad Absorption Bands

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Understanding The Role Of Aggregation In The Broad Absorption Bands Of Eumelanin Kuk-Youn Ju, Martin C Fischer, and Warren S. Warren ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b04905 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on December 4, 2018

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Understanding The Role Of Aggregation In The Broad Absorption Bands Of Eumelanin Kuk-Youn Ju†, Martin C. Fischer†,‡, Warren S. Warren†,‡,§,* †

Department of Chemistry, Duke University, Durham, NC 27708, USA

‡

Department of Physics, Duke University, Durham, NC 27708, USA

§

Department of Radiology, Duke University, Durham, NC 27710, USA

* Email: [email protected] Present address for Kuk-Youn Ju: Department of Radiology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, Korea.

Abstract: In this work, we investigate the relationship between the complex hierarchical assembly structure of eumelanin, its characteristic broad absorption band, and the highly unusual nonlinear dynamics revealed by pump-probe or transient absorption microscopy. Melanin-like nanoparticles (MelNPs), generated by spontaneous oxidation of dopamine, were created with uniform but adjustable size distributions, and kinetically controlled oxidation was probed with a wide range of characterization methods. This lets us explore the broad absorption bands of eumelanin models at different assembly levels, such as small subunit fractions (single monomeric and oligomeric units and small oligomer stacks), stacked oligomer fractions (protomolecules) and large-scale aggregates of protomolecules (parental particles). Both the absorption and pump-probe dynamics are very sensitive to these structural differences, or to the size of intact particles (a surprising result for an organic polymer). We show that the geometric packing order of protomolecules in long-range aggregation are key secondary interactions to extend the absorption band of eumelanin to the low energy spectrum, and produce drastic changes in the transient absorption spectrum.

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Keywords: Eumelanin; hierarchical assembly structure; aggregation; geometric packing order; broad absorption band

Eumelanin is a black pigment found in most living organisms, where it plays antioxidant and photoprotection roles in addition to coloration. In the past decade, eumelanin has also been an attractive biomolecular target for material science, because of intriguing physico-chemical properties not found in common natural organic chromophores. This includes the broad UV-vis absorption band increasing toward high energy,1 strong non-radiative relaxation of photoexcited electronic states,2 high chemical stability compared to its free monomeric units,3 ion-exchange behavior,4 permanent free radical character,5 and hydration-dependent ionic-electronic hybrid conductor behavior.6 Based on these properties, there have been numerous experimental attempts to exploit eumelanin-inspired materials as functional platforms for technological and biomedical applications, for example, in batteries,7 organic electronics,8,98-9biomimetic interfaces,10 drug delivery systems11, and bioimaging.12 However, the fundamental relationships connecting eumelanin’s structure to its peculiar physicochemical properties have been poorly understood due to its intrinsic structural complexity. It is widely accepted that eumelanin is derived from oxidation of two key fundamental units, 5,6dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA).13 Recent structural models of eumelanin describe a hierarchical self-assembled system, where various oligomers derived from DHI and DHICA are stacked and the stacked oligomers (protomolecules) forms aggregated substructures about 10 ~ 20 nm in dimension and growth to particulate structure in a


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size about hundreds of nanometers through further aggregation of the substructures.14-18 Structural complexity originates from both chemical diversity at the level of fundamental oligomers and the hierarchical assembly architecture, which has made it difficult to explore. Among the intriguing properties of eumelanin, the origin of the black color presented by monotonically increasing absorption spectrum from near IR to UV region has been the most fundamental question. Eumelanin is the only biomolecule exhibiting such a broad absorption band in the natural organic chromophore world; it is more similar to an amorphous semiconductor than organic chromophore. In addition, electronically excited eumelanin exhibits extremely low fluorescence emission.2 Considering that intense UV light irradiation leads to toxic and/or mutagenic effect in mammalian cells, the combination of a broad absorption band increasing toward high energies and extremely low fluorescence quantum yield allows most of the absorbed UV-visible light to be efficiently transferred to heat, which is beneficial for photoprotection. The structure of eumelanin may be an optimized and efficient organic system for photoprotection that nature developed through evolution. Another motivation for understanding the melanin structure arises from near-IR pump-probe (or transient absorption) microscopy of melanin in tissue19. There are many different molecular processes which can contribute to a pump-probe signal, so the signal as a function of inter-pulse delay can be quite complex. The signal is heterogeneous in skin20-22, conjunctival,23,2423-24and vulvar25 tissue, and this heterogeneity correlates directly with clinical concern.26,27 It has been accepted that the broad absorption band of eumelanin originates from a chemical diversity at the level of the oligomers, which is the so-called chemical disorder model.28 According to this model, the broad absorption of eumelanin is from ensembles of numerous chromophore bands arising from various chemically distinct oligomers; configurational and conformational


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disorder, and redox state of oligomers are primary factors that change the π electron density of eumelanin oligomers and determine the absorption spectrum of eumelanin. Recent theoretical studies have widened this picture by including non-covalent interactions between fundamental oligomers in the hierarchical assembly structure, such as π-π stacking between oligomers29-31 and aggregation of stacked oligomers.32 Proximity between oligomers in the hierarchical assembly structure leads to further change of their π electron delocalization aspect and consequent variation of the absorption spectrum. However, detailed experimental studies supporting this picture have been restricted because of the lack of a practical approach to link complex structure and material properties. Because of the high reactivity of intermediate species during oxidation of eumelanin precursors, precise control of eumelanin assembly structure has not been achieved in most synthetic procedures. In addition, lack of an experimental strategy to characterize the absorption spectrum of eumelanin at different assembly levels have made it hard to study structure-optical property relationship. In this work, we use melanin-like nanoparticles (MelNPs), generated by spontaneous oxidation of dopamine or chemical oxidation of DOPA into very uniform but adjustable size distributions, to study the relationship between the complex hierarchical assembly structure of eumelanin, its characteristic broad absorption band, and the highly unusual nonlinear dynamics revealed by pump-probe or transient absorption microscopy. Oxidation of dopamine primarily leads to generation of DHI as the monomer unit, but oxidation of DOPA leads to generation of oligomers with both DHI and DHICA.13, 33 This synthetic approach avoids complications from impurities, such as chelated metal ions, present in the commonly used biological melanins such as sepia eumelanin. By controlled disintegration into subunits, the broad absorption bands of stacked oligomers (protomolecules) and their parent particles formed by large aggregates of


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protomolecules were explored. We find a remarkably large particle size dependence for an organic polymer. Characterization of the absorption and transient absorption spectra at different assembly levels shows that stacking between fundamental oligomers and geometric packing order of protomolecules in long-range aggregation are key secondary interactions that extend the absorption band of eumelanin to lower energies. Results and Discussion A. Size-dependent absorption spectra of MelNPs Oxidation of dopamine has been employed to generate a versatile coating of polydopamine (PDA) on a variety of substrates.34 In the absence of a substrate, neutralization of dopamine hydrochloride with proper amount of NaOH in oxygen-dissolved water leads to spontaneous oxidation of dopamine followed by generation of MelNPs (spherical particles of PDA).35 Several papers have shown that the structure of PDA is very similar to the hierarchical assembly of natural eumelanin.36-38 In addition to the assembly structure, characteristic physical properties of MelNPs are similar to natural eumelanin, such as a broad absorption band and free radical character. These features make MelNPs a promising model to study the relationships between eumelanin’s supramolecular structure and its physico-chemical properties.35 It was previously found that the size of MelNPs can be controlled by tuning synthetic conditions influencing the reaction rate for spontaneous oxidation of dopamine, such as temperature, pH, and concentration of dopamine.35 By controlling the reaction conditions (see experimental section), three narrow size distributions of MelNPs were prepared, centered around 60 nm, 100 nm and 250 nm (Figure 1b, c, and d, respectively); these will be denoted as 60-MelNPs, 100-MelNPs and 250MelNPs. Intriguingly, prepared MelNPs showed size-dependent absorption spectra (Figure 2a). Larger MelNPs showed higher attenuation at longer wavelengths compared to smaller particles.


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To evaluate if the attenuation difference arises from absorption or scattering (which also changes with particle size), we measured the sample’s photoacoustic response, which is proportional to their absorption at the excitation wavelength (minus the emission, but differently sized MelNPs did not exhibit any appreciable emission upon excitation at any visible or IR wavelength). Therefore, the photoacoustic signal of MelNPs can be used as parameter to estimate intrinsic absorption. MelNPs exhibited different photoacoustic signal depending on particle size and excitation wavelength (Figure 2-b), which is well matched with the size-dependent attenuation spectra of MelNPs. Thus, this result indicates that the attenuation spectrum of MelNPs reflects the absorption spectrum and is not significantly altered by scattering.


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Figure 1. Structural disassembly of synthetic MelNPs under de-oxygenated alkaline condition. (a) Schematic illustration of hierarchical assembly structure of MelNPs and their structural disintegration under de-oxygenated alkaline condition. Particle character of synthetic MelNPs originates from hierarchical self-assembly driven by π-π stacking between fundamental oligomers and aggregation of stacked oligomers. This hierarchical structure is reminiscent of natural eumelanin structure. Exposure to de-oxygenated condition leads to de-aggregation of assembling subunits, which is possibly associated with attenuation of hydrogen bonding responsible for edge-to-edge joining between stacked oligomers. Note that deoxygenation is important to prevent chemical oxidation of the eumelanin structure by generated reactive oxygen species (ROS) because catechol units of eumelanin could activate dissolved oxygen to be ROS at high pH.39 TEM images of (b) 60-MelNPs, (c) 100-MelNPs and (d) 250-MelNPs. TEM images of (e) 60-MelNPs, (f) 100-MelNPs and (g) 250-MelNPs after exposure to de-oxygenated alkaline condition for 1h. Arrows indicate parental MelNPs and released subunits. AFM images of the highMW subunit fractions (MW > 2000) released from (h) 60-MelNPs, (i) 100-MelNPs and (j) 250MelNPs. Subunits were deposited on mica substrate before AFM analysis. (k) Height distribution of the high-MW subunit fractions (MW > 2000) deposited on mica substrate.

B. pH-controlled disassembly of MelNPs Recently, a practical approach to induce disintegration of the eumelanin structure was developed40 based on elevating the pH level of the solution under deoxygenated conditions. This condition leads to disintegration of eumelanin structure into self-assembling subunits (stacked oligomers). Solid state C13 NMR analysis showed negligible signal changes for eumelanin particle after the disassembly process, which indicates that the process only induces changes in the physical interactions.40 In light of the supramolecular structure of eumelanin, an increase in pH would lead to disruption of hydrogen bonding responsible for edge-to-edge joining between adjacent subunits in the hierarchical assembly structure (Figure 1-a). Synthetic MelNPs can also be disassembled into subunits under this condition,40 so MelNPs are not a single component of high molecular weight, but rather aggregates of assembling units as elucidated in the supramolecular assembly structure of PDA.


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As shown in Figure 1e, f, and g, the structural disintegration of MelNPs was observed by transmission electron microscopy (TEM) after deoxygenated alkaline exposure. TEM images showed that amorphous subunits are released from particles. Subunits released from parental particles were isolated by eliminating dissolved salts and some portion of residual particles by sequential dialysis (molecular weight cutoff at 2000) and centrifugation. In the following we term the fraction retained by the dialysis membrane the “high-MW subunit fraction” and the fraction passing through the membrane the “low-MW subunit fraction.” Even though the dialysis causes a loss of subunits capable of passing through the dialysis membrane, the fraction is very small compared to the de-aggregated subunits released from MelNPs (Figure S1). The thickness of the isolated high-MW subunit fractions was measured by atomic force microscopy (AFM) after depositing them on a mica substrate. AFM height images showed that subunits are dispersed on the substrate (Figure 1-h, i, and j). The thickness of the subunits released from differently sized MelNPs ranged between 0.5- 2.8 nm (Figure 1-k). Given that pH-controlled disassembly process is possibly associated with perturbation of hydrogen bonding between stacked oligomers in eumelanin,40 the high-MW subunit fraction (the major subunit fraction resulting from disassembly of MelNPs), would be expected to be stacked oligomers. Recent studies based on experimental observations and theoretical calculations have suggested that the eumelanin model generated by oxidation of dopamine is constructed by aggregation of stacked oligomers41,4241-42 and the products generated by oxidation of dopamine are very similar to natural eumelanin in terms of their self-assembled structure. In addition, the thickness distribution of the subunits resulting from disassembly of MelNPs is well matched with the height dimension observed in the eumelanin protomolecules (stacked oligomers) by STM, AFM and small angle x-ray scattering.43,4443-44 In this regard, considering the inter-sheet distance of about 0.34 nm in natural eumelanin


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protomolecules,14 we assigned the high-MW subunit fraction as stacked oligomers composed of 2 ~ 8 oligomer sheets. An interesting feature of the de-aggregated subunits is that their thickness distribution depends on the parent particle size. As indicated in Figure 1k, the thickness distribution increases with size of the parent MelNPs. 60-MelNPs are primarily composed of stacked layers of 2~5 oligomers, while the major stacking number of subunits released from 100-MelNPs and 250-MelNPs are about 3~5 and 4~8, respectively. In a previous report, it was found that the size of MelNPs can be controlled by tuning synthetic parameters influencing the reaction rate for spontaneous oxidation of dopamine.35 For example, synthetic conditions that allow for an increase in reaction rate for oxidation of dopamine lead to small MelNPs. As shown in Figure 1k, the stacking level of deaggregated subunits was decreased as their parental particle size is decreased. Given that MelNPs would be formed by a multistep process including generation of fundamental oligomers, stacking between generated oligomers and aggregation of stacked oligomers, a kinetically-controlled assembly process of the fundamental oligomers would be a reasonable explanation for the different stacking degree. For example, a low degree of stacking between fundamental oligomers and their aggregation to form particle structure may be kinetically more favorable during the assembly process, whereas a higher degree of stacking and the particle growth might be thermodynamically favorable. C. Absorption spectra of MelNPs and subunit fractions The low-MW subunit fractions were selectively isolated by collecting the portion that passed through the dialysis membrane (molecular weight cutoff 2000 Daltons). Mass spectroscopic analysis showed that they contain various monomeric and oligomeric units with molecular weight ranging from 150 to 922 (Figure S2). In addition to the monomeric and oligomer units, some


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portion of the stacked oligomers that are not detected in MS due to its low ionization efficiency would be in the subunit fraction. Interestingly, the mass data indicated that the subunits fractions show almost identical molecular weight distribution despite the different parental particle size. This implies that the chemical diversity generated from differently size MelNPs at the single monomeric and oligomeric unit level is very similar and not influenced by the oxidative reaction rate of dopamine. The low-MW subunit fractions showed broad and featureless absorption increasing toward higher energies (Figure 2-c), and high UV optical density compared to the larger fragments. In addition, they exhibited excitation-dependent emission in the visible range and their corresponding excitation spectra showed different band edges (Figure S3), which is very similar to natural eumelanin.45 This indicates that they are composed of chemically distinct species. The broad absorption band of eumelanin has been interpreted as a superposition of various chromophore bands with different HOMO-LUMO gap energy,28 in which case chemical diversity at the fundamental oligomer level is a key factor to achieve broad absorption band. Considering that they contain numerous monomeric and oligomers units with different molecular weight, the chemical diversity arising from various units would contribute to the broad absorption spectrum. The low-MW subunit fractions showed absorption spectra that were not very sensitive to the original particle size. Absorption spectra were normalized at 320 nm to identify absorption difference clearly (Figure 2-c). The normalized absorption spectra indicate that their absorption in the UV and near-IR region is almost equivalent and absorption in the visible region (320 ~ 500 nm) is slightly different. Subunits derived from larger MelNPs exhibit slightly higher absorption around 320 ~ 500 nm than those from smaller MelNPs. Considering that they are composed of qualitatively identical monomeric and oligomeric units as determined in MS analysis (Figure S2),


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a quantitative difference in constituting units (monomeric units, oligomeric units, and small oligomer stacks) could lead to the difference, but the absorption features of the subunit fractions are nearly identical. This indicates that the chemical diversity at the constituting unit level is not the structural factor to cause the size-dependent absorption of MelNPs.

Figure 2. Linear absorption spectra of differently sized MelNPs at the different assembly level. (a) UV-vis absorption spectra of differently sized MelNPs. Weight concentration of samples was tuned to be identical. (b) Photoacoustic response of differently sized MelNPs with excitation at 723 and 821 nm (c) UV-vis absorption spectra of the low-MW subunit fractions released from differently sized MelNPs. (d) UV-vis absorption spectra of the high-MW subunit fractions released from differently sized MelNPs. UV-vis absorption spectra shown in (c) and (d) were normalized at 320 nm.


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The high-MW subunit fractions also showed monotonic broad absorption, but the absorption feature was different from the low-MW subunit fractions with enhanced absorption in the visible and near IR compared to the UV region. (Figure 2-d) The increased absorption contribution at low energies could be attributed to several possible factors. Generation of charge transfer complexes between oligomers in a stacked structure would contribute to enhancement of absorption at low energies. Because quinhydrone formation between catechol units is a well-known charge transfer complex,46 the possible charge transfer band would lead to increased absorption at low energy. With the formation of quinhydrone complexes, the redox equilibrium of oligomers would be also changed. Because it has been accepted that the oxidized form of eumelanin oligomer has lower HOMO-LUMO gap energy,47 a change of redox state toward the oxidized form would be another possibility. However, reductive treatment of the stacked oligomer fraction with NaBH4 did not show any appreciable absorption change (Figure S4), suggesting that charge transfer complexes and redox state are not structural factors contributing to the broad absorption band of stacked oligomer fractions. Chemical heterogeneity of the fundamental oligomers (in terms of molecular weight and planarity of molecular structure) could be generated at the early stage of assembly process. In this regards, two structural factors may contribute to the relatively high absorption of the high-MW subunit fraction at low energy compared to the small-MW subunit fraction. For example, the highMW subunit fraction may be composed of oligomers with higher molecular weight compared to the low-MW subunit fraction. Thus, the more extended pi-electron delocalization of oligomers in the high-MW subunit fraction would make their HOMO-LUMO gap energy lower compared to that of the low-MW subunit fraction. The lack of single oligomers in the high-MW subunit fraction as shown in AFM analysis suggests that they would be comprised of oligomers capable of efficient


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pi-pi stacking, suggesting a planar structure of oligomers. A planar oligomer structure can enhance low energy absorption as compared to non-planar structures by exhibiting more extended pielectron delocalization within oligomers48 (planar geometry also favors a higher stacking order, as discussed further below).

Figure 3. (a) Emission spectra of de-aggregated subunits released form differently sized MelNPs. Emission spectra were taken at 320 nm excitation. For comparison of emission intensity, absorbance of all samples was made equivalent at 320 nm by adjusting the relative concentration. For minimization of re-absorption, highly diluted samples were used, resulting in a < 0.1 around 425 nm of all samples. The high-MW subunit fractions showed significantly lower emission intensity compared to the low-MW subunit fractions. (b) Normalized emission spectra of deaggregated subunits. It appears that emission spectra of the high-MW subunit fractions are redshifted and broadened compared to the low-MW subunit fractions.

In contrast to the low-MW subunit fractions containing single monomeric and oligomeric units, the high-MW subunit fractions are primarily composed of stacked oligomers (Figure 1k), which suggests that secondary interactions supporting the stacking structure would cause the enhancement of absorption at low energies. Since previous theoretical studies have consistently concluded that the electronic structure of eumelanin oligomers can be changed when eumelanin oligomers are in proximity through π- π stacking,29-31 electronic coupling between oligomers in a


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stacked structure would be a likely explanation for enhanced absorption intensity at low energies. Most computational studies predicted that stacking structure causes a red-shift and broadening of eumelanin oligomers, leading to an extension of the spectrum to lower energies. The emission spectra of stacked oligomer fractions provide insight into the electronic coupling between oligomers in stacked structures. The high-MW (stacked oligomer) subunit fractions exhibit redshifted and significantly lower emission compared to the low-MW subunit fractions (Figure 3). Strong fluorescence quenching of aromatic chromophores is a characteristic feature found in selfassembled organic chromophore systems, where electronic coupling occurs by combination of locally excited states on different chromophores.49,5049-50 In this regard, increased absorption contribution at low energy would be the result of electronic interaction between oligomers with different HOMO-LUMO gap energies in the stacked structure. As indicated in Figure 1k, the stacked oligomer fractions released from differently sized MelNPs show different thickness distributions. However, it appears that absorption spectra of the stacked oligomer fractions from differently sized MelNPs are superimposable. Hence, even though the absorption band of the stacked oligomer fraction differs from the low-MW subunit fractions, it appears that their absorption spectra is not dependent on the stacking degree. In addition, the emission and excitation spectra were shown to be very similar (Figure 3). These results indicate that electronic coupling between oligomers in the stacked structure leads to enhancement of the absorption band at low energy as suggested in previous computational studies,29-31 and 2~5 oligomer layers observed in the stacked oligomer fraction released from 60-MelNPs may be a maximum number to cause stacking-mediated absorption change. Thus, stacked oligomers derived from 100-MelNPs and 250-MelNPs may not induce additional absorption increase in the low energy region of the spectrum compared to those from 60-MelNPs. From this observation, we


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concluded that the stacking structure of oligomer units is not a factor contributing to sizedependent absorption of MelNPs.

D. Aggregation-induced absorption change of subunits The very similar absorption spectra between de-aggregated subunits released from differently sized MelNPs suggest that another structural factor (beyond the chemical diversity at the level of free monomeric and oligomeric unit and the stacking structure) is associated with the sizedependent absorption of MelNPs. Aggregation of eumelanin protomolecules has been considered as a key structural factor to changes in the absorption band. Experimental observations revealed that aggregation leads to extension of the absorption spectrum to lower energies.40, 47 In general, aggregation of organic chromophores causes changes of their absorption band, which has been elucidated by exciton coupling theory,51 but the absorption changes depend on the orientation of transition dipoles. The edge-to-edge joining between eumelanin protomolecules by quinhydrone formation has been suggested as a possible secondary interaction responsible for their aggregation. Thus, edge-to-edge joining between protomolecules would lead to J-type aggregation that leads to broadening and red-shift of absorption bands. The generation of charge transfer bands would be another possibility; charge transfer complex by quinhydrone forms between protomolecules would lead to enhancement of absorption at low energy. Upon formation of a quinhydrone complex, the redox state of eumelanin oligomers can be also changed. The changed redox state of eumelanin oligomers would contribute to change of absorption band. E. Transient absorption spectroscopy of different sized MelNPs Transient absorption spectroscopy provides insight into the effect of aggregation on the broad absorption band of MelNPs. Femtosecond transient absorption spectroscopy has been shown to be


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a sensitive method to discriminate melanin pigment as a function of iron binding, photo-damage, chemical oxidation, and the molecular weight of melanin constituents.52 One of the many possible non-instantaneous processes observed in transient absorption of melanin pigment is ground state bleach (GSB) signal. When the pump pulse excites the chromophore, its ground state is depleted and the absorption by a second (probe) pulse will be decreased. This decrease in absorption (increase in probe transmission) as the pump is turned on is recorded as a negative GSB signal. A GSB signal is generated only if both pump and probe pulses are in resonance with a chromophore band and if both share a common ground state. For a fixed pump wavelength, the GSB signal as a function of probe wavelength can reveal features of individual absorption bands within the MelNP’s broad absorption spectrum. In some cases stimulated Raman (overlapped with the pulses) can be observed as well.

Figure 4. Transient absorption spectra of differently sized MelNPs and subunits (a-c) Transient absorption signal of (a) 60-MelNPs, (b) 100-MelNPs, and (c) 250-MelNPs obtained with 720 nm pump and variable probe wavelength (770 ~ 830 nm). (d) Time-delay signal at 1 ps as a function of probe wavelength for 60-MelNPs, 100-MelNPs, and 250-MelNPs. Signal was normalized at 770 nm. (e-g) Transient absorption signal of the high-MW subunit fractions released from (e) 60-MelNPs, (f) 100-MelNPs, and (c) 250-MelNPs. Transient absorption signal was obtained with 720 nm pump and variable probe wavelength (770 ~ 830 nm). (h) Time-delay signal


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at 1 ps as a function of probe wavelength for the high-MW subunit fractions. Signal was normalized at 770 nm.

As shown in Figure 4a, b, and c, a non-instantaneous negative GSB signal in different sized MelNPs was observed when applying 720 nm pump and 770 nm probe, which indicates that individual chromophore bands of MelNPs in the near-IR region are in resonance with both pump and probe pulses. When changing the probe wavelength from 770 nm towards 830 nm, the GSB signal decreased and finally changed to a positive signal indicative of excited state absorption (ESA). Interestingly, MelNPs exhibited strongly size-dependent GSB signals. In contrast to the very similar transient absorption signal between subunits with different stacking degree, MelNPs showed broader GSB band as their size increased (Figure 4d). Hence, large-sized MelNPs have broader chromophore bands than small-sized MelNPs. The low-MW subunit fractions have almost no pump-probe signal at this wavelength combination. The high-MW subunit fractions did not show any time-delayed GSB at this pump and probe combination (Figure 4e, f, and g) and the subunit fractions showed very similar timedelayed ESA signal regardless of different stacking degree (Figure 4e, f, g, and h). This observation indicates that the electronic structures are very similar between the high-MW subunit fractions, but clearly different from the parent particles. The different transient absorption behavior between high-MW subunit fractions and parent particles implies that electronic structure of the subunit fraction is changed when they aggregate to form the particle structure. From transient absorption spectroscopy, we concluded that aggregation of subunits is the crucial structural factor that changes individual chromophore bands and consequently results in sizedependent absorption behavior. As described above, quinhydrone formation responsible for aggregation of eumelanin protomolecules is an important factor causing red-shifted and broadened


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chromophore bands, which can be explained by exciton coupling or the formation of charge transfer bands and a change of redox state. Geometric packing of the eumelanin protomolecules in the large-scale aggregation system may be the key determinant for efficient quinhydrone formation. Aggregates of highly stacked oligomers would have geometric preference forcing their lateral faces to interact with each other efficiently and form numerous quinhydrone complexes. The geometric preference would be lowered with a decrease in the number of stacked oligomer layer. In this regard, thickness of de-aggregated subunits would be indicative for their geometric packing order when they are confined in large-scale aggregation systems. MelNPs derived from highly stacked oligomer would have high geometric packing order giving rise to numerous quinhydrone complex and consequent strong red-shifted and broadened chromophore bands. F. Effect of geometric packing order observed in DOPA-MelNPs A

eumelanin

particle

model

derived

from

oxidation

of

racemic

DOPA

(3,4-

dihydroxyphenylalanine, a biological precursor to dopamine) was further examined as another eumelanin model system. DOPA-MelNPs with size of approximately 100 nm were prepared by chemical oxidation of rDOPA as previously reported53 (Figure 5a). The particle structure was disintegrated by exposure to deoxygenated alkaline condition and characterized with same method described above. AFM analysis showed that thickness distribution of the stacked oligomer fraction is lower than that of 100-MelNPs (Figure 5b and c). It has been suggested that the packing aspect of eumelanin protomolecules in the hierarchical assembly structure is closely related to the chemical structure of the fundamental oligomers.48 Fundamental oligomers derived from DHI are prone to form stacked layers because of their planar structure. Thus, DHI eumelanin particles would be formed by aggregation of highly stacked oligomers. On the other hand, oxidation of DHICA leads to oligomers with non-planar structure


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because of the inter-unit dihedral angles of about 47°.48 Therefore, DHICA eumelanin would be formed by aggregation of oligomers with low stacking degree. Given this structural picture of eumelanin assembly, it would be expected that the chemical structure of the MelNP precursor acts as a key factor determining their assembled structure because DOPA-MelNPs is composed of a relatively high portion of DHICA monomeric unit compared to MelNPs derived from dopamine. (Figure S5) Of course, it cannot be ruled out that the structure difference arises from different oxidation condition. Note that DOPA-MelNPs is generated by chemical oxidation of DOPA while spontaneous oxidation of dopamine leads to generation MelNPs.


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Figure 5. Structural-optical property relationship of DOPA-MelNPs. (a) TEM image of DOPAMelNPs. (b) AFM image of the high-MW subunit fractions (MW > 2000) deposited on a mica substrate. (c) Height distribution of the high-MW subunit fractions released from 100-MelNPs and DOPA-MelNPs. (d-f) Comparison of UV-vis absorption spectra between (d) 100-MelNPs and DOPA-MelNPs at oligomer, (e) stacked oligomer, and (f) large-scale aggregates level. All absorption spectra were normalized at 320 nm. (g) Transient absorption spectra of DOPA-MelNPs


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and (h) their high-MW subunit fractions. Spectra were obtained with 720 nm pump and variable probe (770 ~ 830 nm). (i) Time-delayed transient signal at 1 ps as a function of probe wavelength. Signal was normalized at 830 nm. (j) Proposed structure-property relationship of eumelanin emphasizing the hierarchical assembly structure. The hierarchical assembly would play a role to extend the broad absorption band of eumelanin to low energies.

The thinner thickness distribution of DOPA-MelNPs than 100-MelNPs implies that DOPAMelNPs should have a lower geometric packing order than 100-MelNPs and thus should have lower absorption contribution at low energies. Comparison of absorption spectra between two eumelanin models supported this expectation: The UV-vis absorption spectra show that DOPAMelNPs exhibit less absorption at low energies than 100-MelNPs (Figure 5f). At the de-aggregated subunit level, DOPA-MelNPs exhibits slightly higher absorption at low energies compared to 100MelNPs (Figure 5d and e), indicating a more extended π electron delocalization at the free oligomer and stacked oligomer level. However, electronic coupling between protomolecules with low geometric packing order in large-scale aggregation level seem to be very weak compared to the systems with higher geometric packing order. The transient absorption signals of DOPA-MelNPs further support this expectation. Figure 5g shows transient absorption spectra of DOPA-MelNPs taken with 720 nm pump and variable probe wavelength (770 ~ 830 nm). In contrast to 100-MelNPs, the DOPA-MelNPs did not exhibit any GSB signal, suggesting chromophore bands of DOPA-MelNPs that are very narrow compared to 100-MelNPs. De-aggregated subunits released from DOPA-MelNPs showed very similar transient absorption signal (Figure 5h and i) to their parent particles, indicating that the aggregation-induced change of their electronic structure is very weak in this system. From these observations, thus, it is plausible to conclude that aggregation of eumelanin protomolecules is the key structural factor to determine its broad absorption bands (Figure 5j). In particular, the geometric packing order of


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protomolecules in the large-scale aggregation system is crucial to cause substantial extension of the broad absorption band to low energies. Conclusions Synthetic MelNPs generated by spontaneous oxidation of dopamine and chemical oxidation of DOPA were utilized as eumelanin model systems, to link between hierarchical assembly structure of eumelanin and its characteristic broad absorption band. A top-down approach to control the disintegration of eumelanin models into subunits allowed us to observe the absorption spectra at different assembly levels. We investigated subunit fractions composed of single monomeric and oligomeric units and small oligomer stacks, stacked oligomer fractions (protomolecules) and largescale aggregates of protomolecules (parental particles). This experimental finding supports the hypothesis that broad absorption bands governed by intrinsic π electron delocalization within fundamental eumelanin oligomers can be further changed by secondary interactions such as π-π stacking and aggregation in the hierarchical assembly system of eumelanin. Eumelanin models generated by kinetically controlled oxidation of dopamine and chemical oxidation of DOPA further indicated that the geometric packing order of eumelanin protomolecules in the large-scale aggregation system plays a key role to extend broad absorption band significantly to low energies. We believe that the experimentally observed relationship between complex hierarchical assembly structure of the eumelanin model and its optical properties provides insights into the way nature developed these self-assembled, efficient, photo-protective systems. This study also provides clues for designing eumelanin-based functional materials.

Methods Synthesis of MelNPs


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MelNPs were prepared as described in a previous report.35 For 250-MelNPs, 180 mg of dopamine hydrochloride was dissolved in 90 mL deionized water. A NaOH solution (630 L, 1 N) was added to the solution of dopamine hydrochloride under vigorous stirring at room temperature. After 5 h, 250-MelNPs were retrieved by centrifugation (5000 rpm for 10 min) and dispersed in deionized water. MelNPs were purified with sequential centrifugation (5000 rpm for 10 min) and re-dispersion in deionized water for 3 times. For 100-MelNPs, 180 mg of dopamine hydrochloride was dissolved in 90 mL deionized water. A NaOH solution (770 L, 1 N) was added to the solution of dopamine hydrochloride under vigorous stirring at 50 °C. After 5 h, 100-MelNPs were retrieved by centrifugation (20000 rpm for 10 min) and dispersed in de-ionized water. 100MelNPs were purified with sequential centrifugation (20000 rpm for 10 min) and re-dispersion in deionized water for 3 times. For 60-MelNPs, 180 mg of dopamine hydrochloride was dissolved in 135 mL deionized water. A NaOH solution (790 L, 1 N) was added to the solution of dopamine hydrochloride under vigorous stirring at 50 °C. After 5 h, 60-MelNPs were retrieved by centrifugation (20000 rpm for 10 min) and dispersed in de-ionized water. 60-MelNPs were purified with sequential centrifugation (20000 rpm for 10 min) and re-dispersion in deionized water for 3 times. Synthesis of DOPA-MelNPs DOPA-MelNPs were prepared as described in a previous report.53 70 mg of L-DOPA was dissolved in deionized water (50 mL) by heating. KMnO4 solution (360 μL, 1 N) was added to the solution of L-DOPA under vigorous stirring at room temperature. After 7 h, DOPA-MelNPs were retrieved by centrifugation (20000 rpm for 10 min) and dispersed in de-ionized water. DOPAMelNPs were purified with sequential centrifugation (20000 rpm for 10 min) and re-dispersion in deionized water for 3 times. To remove Mn2+ ions present in DOPA-MelNPs, HCl solution (10 mL


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of 10 N) was slowly added to DOPA-MelNPs suspension. After 30 min, DOPA-MelNPs were retrieved by centrifugation and re-dispersed in deionized water. DOPA-MelNPs were further purified with sequential centrifugation (20000 rpm for 10 min) and re-dispersion in deionized water for 3 times. pH-controlled disassembly process Differently sized MelNPs and DOPA-MelNPs were disassembled by exposure to de-oxygenated alkaline solution as described in a previous report.40 MelNPs and DOPA-MelNPs were dispersed in deionized water with weight concentration of 2 mg/mL. The solution was purged with N2 for 20 min to eliminate dissolved oxygen. Under purging with N2, 3.5 mL of NaOH solution was added to 10 mL of solution of dispersed synthetic melanin particles. NaOH solution (1N) was prepurged for 20 min before addition. After 5 h, 5 mL of deoxygenated KH2PO4 solution (1M) was added to the solution for neutralization. The resulting solution was dialyzed using a dialysis kit (Thermo Scientific Slide-A-Lyzer Dialysis, molecular weight cutoff at 2000) for 1 day. After dialysis, the fraction entrapped in the dialysis membrane was centrifuged to eliminate residual particles (19000 rpm, 10 min). The high-MW subunit fractions (MW > 2000) were obtained by collecting the supernatant after centrifugation. The low-MW subunit fractions (MW < 2000) were obtained by collection the fraction that passed through the dialysis membrane. Characterization of MelNPs, DOPA-MelNPs and their de-aggregated subunits. The morphology of synthetic melanin particle models before and after pH-controlled disassembly was examined by TEM (FEI Tecnai G2 Twin). The thickness of the high-MW subunit fractions was characterized by AFM. For AFM analysis, the subunit fraction was deposited onto a mica substrate. Even though the subunits could be linked in the lateral dimension during the deposit process, the subunits’ height distribution observed by AFM provides structural information such


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as degree of stacking.43 For the deposition, diluted subunit solution was dropped onto the spinning mica substrate (4000 rpm, 30 sec). The height distribution of subunits deposited on the mica substrate was analyzed by tapping mode AFM (Digital Instruments Dimension 3100 AFM). UVVis absorption spectra of synthetic melanin models and their de-aggregated subunits were acquired with a Shimadzu UV-1700 spectrophotometry system. Emission and excitation spectra of deaggregated subunits released from eumelanin particle models were obtained using a spectrofluorometer (Fluorolog-3, HORIBA). The photoacoustic (PA) response of MelNPs was obtained in the same manner as described in a previous report.40 To measure photoacoustic signals, the transparent plastic tubes were positioned in a custom-made container filled with distilled water. Water temperature was stably maintained to be 24 °C. Differently sized MelNPs were injected into the tubes. Their weight concentration was tuned to be equivalent. Radio-frequency (RF) PA data from the samples were captured with a commercial ultrasonic scanner equipped with a SonixTouch research package (Ultrasonix Corp.,Vancouver, BC, Canada) and a linear array transducer (L14− 5/38) connected to a 128channel SonixDAQ parallel system. The RF channel data acquisition sequence was initialized for every laser pulse excitation event of an Nd:YAG OPO laser system (Surelite III-10 and Surelite OPO Plus, Continuum Inc., Santa Clara, CA, U.S.A.) at the rate of 10 Hz. The laser energy was delivered to the samples by a custom-made bifurcated optical fiber bundle (Fiberoptic Systems, Inc., Simi Valley, CA, U.S.A.) integrated into the linear array transducer. The pulse length of the excitation laser was 7 ns. Photoacoustic response of the samples was measured at 723 nm and 821 nm. The distance between the integrated PA probe and targets was fixed at 30 mm and the energy density on the samples was maintained at 6.7 mJ/cm2.


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Transient absorption signal were obtained as presented in previous reports with some modifications.52 A titanium-sapphire laser (80 MHz, Chameleon, Coherent, Inc.), tuned between 770 nm and 830 nm, pumped an optical parametric oscillator (Mira-OPO, Coherent, Inc.), providing a 720 nm output. The output pulses had a cross-correlation of approximately 210 fs FWHM, when measured by two-photon absorption in rhodamine-6G. The pump beam (720 nm) was amplitude-modulated at 2 MHz with an acousto-optic modulator. Part of the titanium-sapphire laser’s output (tuned from 770 nm to 830 nm) was used as the probe beam and remained unmodulated. Through a mechanical delay stage, the interpulse delay was controlled and the two beams were combined on a dichroic mirror. The two beams were sent to a home-built scanning microscope fitted with an objective (40X, 0.75 NA, Olympus). Each beam power was adjusted to 0.6 mW at the sample. After transmission through the sample, both beams were collected with a 1.1 NA condenser (Olympus). The pump beam was rejected by a stack of chromatic filters and the probe was detected by an amplified photodiode. All samples were homogeneously dispersed in agarose gel with thickness of about 100 m on a glass substrate. High-Resolution Liquid Chromatography-Mass Spectrometry. The subunit fraction which passed through a dialysis membrane (molecular weight cutoff at 2000) was collected and concentrated by evaporating the solvent (water). For the high-resolution mass measurement of the subunit fraction, they were analyzed by high-performance liquid chromatography, followed by mass spectrometry. 10 L of the subunit fraction (MW < 2000, water) was injected into an Agilent 1200 Series high-performance liquid chromatography system (HPLC; Agilent Technologies Inc.) and separated through an Agilent Bonus RP column (2 x 100 mm, 1.8 m, ultrahigh performance column). The HPLC was connected to a standard Agilent ESI interface. The mobile phase was 0.29% formic acid, 96.8% water and 2.9% methanol (A) and


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0.29% formic acid, 96.8% acetonitrile and 2.9% water (B) at flow rate of 160 L/min with elution gradient. The gradient program started with 0% B at 0 min and increased to 40% B during the 13 min program. The MS used an electrospray ionization (ESI) source in the positive-ion mode. Preparation of PDCA and PTCA For synthesis of pyrrole-2,3,5-tricarboxylic acid (PTCA), 100 mg of 5-hydroxyindole-2carboxylic acid was dissolved in 100 ml of 1 M K2CO3 and then oxidized with 4 mL of 30% H2O2 by heating under reflux for 20 min. After cooling, 5 mL of 10 % Na2SO3 was added and the mixture was acidified with 40 mL of 6 M HCl. The acidified solution was extracted twice with 200 mL of ether. The ether extract was washed with 50 ml of water, dried over anhydrous Na2SO4, and concentrated to dryness in vacuo. Pale brown PTCA crystals formed on the walls of the flask and were collected for HPLC-MS analysis. Pyrrole-2,3-dicarboxylic acid (PDCA) was synthesized using 5-hydroxyindole as a same manner described in the case of the procedure for PTCA. Peroxide oxidation of melanin models Chemical oxidation of melanin models was performed as described in a previous study 54 with slight modifications. 860 l of 1 M K2CO3 and 40 l of 3 % H2O2 were added into 100 l of melanin suspension (1 mg/mL) and the suspension was heated in a boiling water bath for 20 min. After cooling, the residual H2O2 was decomposed by adding 20 ml of 10 % Na2SO3. The mixture was acidified with 500 l of 6 M HCl. The resulting solution was extracted twice with 7 mL of peroxide-free ether. The ether extract was evaporated to dryness and the residue was dissolved in 200 l of water. The resulting solution was centrifuged with centrifugal filter equipped with semipermeable membrane (Amicon Ultra centrifugal filter MWCO 3 kDa) and the fraction that passed through the filter was injected into the chromatograph. High-Resolution Liquid Chromatography-Mass Spectrometry.


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The oxidation products were analyzed by HPLC using an Ultimate 3000 RS system (Thermo fisher scientific Inc.), U-VDSpher PUR C18-E column (1.8 m, 50 x 2.0 mm, VDS optilab). The mobile phase was 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). At 1.0 mL/min, the elution gradient was (min, %B): 0, 0; 1, 0; 12, 25; 14, 25; 16, 0. The UV detector was set at a 255 nm absorbance. The standards for peak identification were pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA).

ASSOCIATED CONTENT This work was funded by NIH under grant R01 CA166555 and by NSF under grant CHE-1610975. We thank Jeeun Kang, Jin Ho Chang at Sogang University for their help in measurements of the photoacoustic response for MelNPs. Supporting Information. Supporting information includes HPLC chromatograms, ESI-MS spectra, and emission/excitation spectra. The Supporting Information is available free of charge on the ACS Publications website at DOI: AUTHOR INFORMATION Corresponding Author: Warren S. Warren (email: [email protected])


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REFERENCES 1. Crippa, P. R.; Cristofoletti, V.; Romeo, N., A Band Model for Melanin Deducted from Optical Absorption and Photoconductivity Experiments. Biochim. Biophys. Acta 1978, 538, 164170. 2. Meredith, P.; Riesz, J., Radiative Relaxation Quantum Yields for Synthetic Eumelanin. Photochem. Photobiol. 2004, 79, 211-216. 3. Meredith, P.; Sarna, T., The Physical and Chemical Properties of Eumelanin. Pigm. Cell Res. 2006, 19, 572-594. 4. Larsson, B.; Tjalve, H., Studies on the Melanin-Affinity of Metal Ions. Acta Physiol. Scand. 1978, 104, 479-484. 5. Commoner, B.; Townsend, J.; Pake, G. E., Free Radicals in Biological Materials. Nature 1954, 174, 689-691. 6. 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. 7. Kim, B. G.; Kim, S.; Lee, H.; Choi, J. W., Wisdom from the Human Eye: A Synthetic Melanin Radical Scavenger for Improved Cycle Life of Li-O-2 Battery. Chem. Mater. 2014, 26, 4757-4764. 8. Tarabella, G.; Pezzella, A.; Romeo, A.; D'Angelo, P.; Coppede, N.; Calicchio, M.; d'Ischia, M.; Mosca, R.; Iannotta, S., Irreversible Evolution of Eumelanin Redox States Detected by an Organic Electrochemical Transistor: En Route to Bioelectronics and Biosensing. J. Mater. Chem. B 2013, 1, 3843-3849. 9. 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. 10. Bettinger, C. J.; Bruggeman, J. P.; Misra, A.; Borenstein, J. T.; Langer, R., Biocompatibility of Biodegradable Semiconducting Melanin Films for Nerve Tissue Engineering. Biomaterials 2009, 30, 3050-3057. 11. Zhang, R.; Fan, Q.; Yang, M.; Cheng, K.; Lu, X.; Zhang, L.; Huang, W.; Cheng, Z., Engineering Melanin Nanoparticles as an Efficient Drug-Delivery System for Imaging-Guided Chemotherapy. Adv. Mater. 2015, 27, 5063-5069. 12. Ju, K. Y.; Lee, J. W.; Im, G. H.; Lee, S.; Pyo, J.; Park, S. B.; Lee, J. H.; Lee, J. K., BioInspired, Melanin-Like Nanoparticles as a Highly Efficient Contrast Agent for T1-Weighted Magnetic Resonance Imaging. Biomacromolecules 2013, 14, 3491-3497. 13. Ito, S., The Ifpcs Presidential Lecture: A Chemist's View of Melanogenesis. Pigm. Cell Res 2003, 16, 230-236. 14. Cheng, J.; Moss, S. C.; Eisner, M.; Zschack, P., X-Ray Characterization of Melanins .1. Pigm. Cell Res. 1994, 7, 255-262. 15. Cheng, J.; Moss, S. C.; Eisner, M., X-Ray Characterization of Melanins .2. Pigm. Cell Res. 1994, 7, 263-273. 16. Zajac, G. W.; Gallas, J. M.; Alvaradoswaisgood, A. E., Tunneling Microscopy Verification of an X-Ray Scattering-Derived Molecular-Model of Tyrosine-Based Melanin. J. Vac. Sci. Technol., B: Nanotechnol. Microelectron.: Mater., Process., Meas., Phenom. 1994, 12, 1512-1516.


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17. Zajac, G. W.; Gallas, J. M.; Cheng, J.; Eisner, M.; Moss, S. C.; Alvarado-Swaisgood, A. E., The Fundamental Unit of Synthetic Melanin: A Verification by Tunneling Microscopy of XRay Scattering Results. Biochim. Biophys. Acta. 1994, 1199, 271-278. 18. Clancy, C. M.; Simon, J. D., Ultrastructural Organization of Eumelanin from Sepia Officinalis Measured by Atomic Force Microscopy. Biochemistry 2001, 40, 13353-13360. 19. Fischer, M. C.; Wilson, J. W.; Robles, F. E.; Warren, W. S., Invited Review Article: Pump-Probe Microscopy. Rev. Sci. Instrum. 2016, 87, 031101. 20. Wilson, J. W.; Degan, S.; Gainey, C. S.; Mitropoulos, T.; Simpson, M. J.; Zhang, J. Y.; Warren, W. S., Comparing in Vivo Pump-Probe and Multiphoton Fluorescence Microscopy of Melanoma and Pigmented Lesions. J. Biomed. Opt. 2015, 20, 051012. 21. Simpson, M. J.; Wilson, J. W.; Robles, F. E.; Dall, C. P.; Glass, K.; Simon, J. D.; Warren, W. S., Near-Infrared Excited State Dynamics of Melanins: The Effects of Iron Content, PhotoDamage, Chemical Oxidation, and Aggregate Size. J. Phys. Chem. A 2014, 118, 993-1003. 22. Matthews, T. E.; Wilson, J. W.; Degan, S.; Simpson, M. J.; Jin, J. Y.; Zhang, J. Y.; Warren, W. S., In Vivo and Ex Vivo Epi-Mode Pump-Probe Imaging of Melanin and Microvasculature. Biomed. Opt. Express 2011, 2, 1576-1583. 23. Wilson, J. W.; Vajzovic, L.; Robles, F. E.; Cummings, T. J.; Mruthyunjaya, P.; Warren, W. S., Imaging Microscopic Pigment Chemistry in Conjunctival Melanocytic Lesions Using Pump-Probe Laser Microscopy. Invest. Ophthalmol. Vis. Sci. 2013, 54, 6867-6876. 24. Robles, F. E.; Wilson, J. W.; Warren, W. S., Quantifying Melanin Spatial Distribution Using Pump-Probe Microscopy and a 2-D Morphological Autocorrelation Transformation for Melanoma Diagnosis. J. Biomed. Opt. 2013, 18, 120502. 25. Robles, F. E.; Deb, S.; Fischer, M. C.; Warren, W. S.; Selim, M. A., Label-Free Imaging of Female Genital Tract Melanocytic Lesions with Pump-Probe Microscopy: A Promising Diagnostic Tool. J. Low. Genit. Tract. Dis. 2017, 21, 137-144. 26. Matthews, T. E.; Piletic, I. R.; Selim, M. A.; Simpson, M. J.; Warren, W. S., Pump-Probe Imaging Differentiates Melanoma from Melanocytic Nevi. Sci. Transl. Med. 2011, 3, 71ra15. 27. Robles, F. E.; Deb, S.; Wilson, J. W.; Gainey, C. S.; Selim, M. A.; Mosca, P. J.; Tyler, D. S.; Fischer, M. C.; Warren, W. S., Pump-Probe Imaging of Pigmented Cutaneous Melanoma Primary Lesions Gives Insight into Metastatic Potential. Biomed. Opt. Express 2015, 6, 36313645. 28. Tran, M. L.; Powell, B. J.; Meredith, P., Chemical and Structural Disorder in Eumelanins: A Possible Explanation for Broadband Absorbance. Biophys. J. 2006, 90, 743-752. 29. Stark, K. B.; Gallas, J. M.; Zajac, G. W.; Golab, J. T.; Gidanian, S.; McIntire, T.; Farmer, P. J., Effect of Stacking and Redox State on Optical Absorption Spectra of Melanins -Comparison of Theoretical and Experimental Results. J. Phys. Chem. B 2005, 109, 1970-1977. 30. Tuna, D.; Udvarhelyi, A.; Sobolewski, A. L.; Domcke, W.; Domratcheva, T., Onset of the Electronic Absorption Spectra of Isolated and Pi-Stacked Oligomers of 5,6-Dihydroxyindole: An Ab Initio Study of the Building Blocks of Eumelanin. J. Phys. Chem. B 2016, 120, 34933502. 31. Prampolini, G.; Cacelli, I.; Ferretti, A., Intermolecular Interactions in Eumelanins: A Computational Bottom-up Approach. I. Small Building Blocks. RSC Advances 2015, 5, 3851338526. 32. Chen, C. T.; Chuang, C.; Cao, J.; Ball, V.; Ruch, D.; Buehler, M. J., Excitonic Effects from Geometric Order and Disorder Explain Broadband Optical Absorption in Eumelanin. Nat. Commun. 2014, 5, 3859.


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Understanding the Role of Aggregation in the Broad Absorption Bands

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