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Radiative Relaxation Quantum Yields for Synthetic Eumelanin

We report absolute values for the radiative relaxation quantum yield of synthetic eumelanin as a function of excitation energy. These values were determined by correcting for pump beam attenuation and emission re-absorption in both eumelanin samples and fluorescein standards over a large range of concentrations. Our results confirm that eumelanins are capable of dissipating>99.9% of absorbed UV and visible radiation through non-radiative means. Furthermore, we have found that the radiative quantum yield of synthetic eumelanin is excitation energy dependent. This observation is supported by corrected emission spectra, which also show a clear dependence of both peak position and peak width upon excitation energy. Our findings indicate that photoluminescence emission in eumelanins is derived from ensembles of small chemically distinct oligomeric units which can be selectively pumped. This hypothesis lends support to the theory that the basic structural unit of eumelanin is oligomeric rather than hetero-polymeric.


INTRODUCTION
The melanins are an important class of pigmentary macromolecules found throughout nature (1). Eumelanin is the predominant form in humans, and acts as the primary photoprotectant in our skin and eyes. Physiochemically, all melanins are broadband UV and visible light absorbers, and potent free radical scavengers and antioxidants (1,2). In direct contradiction with its photoprotective properties, eumelanin (along with pheomelanin -the less prevalent red-brown pigment also found in humans), is implicated in the cytotoxic chain of events that ultimately lead to melanoma skin cancer (3). For this reason, the photophysics, photochemistry and photobiology of melanins are subjects of intense scientific interest.
In a more general sense, the broader structure-property-function relationships that dictate the behavior of these important biological macromolecules are still poorly understood (4). In particular, major questions still remain concerning the basic structural unit (5). It is fairly well accepted that eumelanins are macromolecules of DHI and DHICA, and that pheomelanins are cysteinyl-dopa derivatives (6,1). However, it is still a matter of debate as to whether eumelanin (in particular) is actually a highly cross-linked extended heteropolymer, or composed of DHI/DHICA oligomers condensed into 4 or 5 oligomer nano-aggregates (7). This is an absolutely fundamental issue, and is the starting point for the construction of consistent structure-property-function relationships. The answer to this question also has profound implications for our understanding of the condensed phase properties of melanins. In 1974, McGinness, Corry and Proctor showed that a pellet of dopamelanin could be made to behave as an amorphous electrical switch (8). They postulated that these materials may be disordered organic semiconductors. Several studies since have claimed to show that melanins in the condensed solid-state are indeed semiconductors (9,10). However, it is by no means certain that the conductivity reported is electronic in nature. A clear idea of the basic structural unit is fundamental to developing a consistent model for condensed phase charge transport in such disordered organic systems.
Recently, steady state and time-resolved optical spectroscopies have shed some light on the photophysics and photochemistry of melanins. Additionally, such techniques have been used to probe electronic and molecular structure in order to address the vexing question of the basic structural unit. In particular, Nofsinger et al. have published several comprehensive steady state and dynamic studies (11,12). They report that sepia eumelanin (a good model for human eumelanin) has a low radiative relaxation quantum yield (φ r =3x10 -3 ). Additionally, they have found that the time decay of the emission is non-exponential, and postulate that this may arise from a number of chemically distinct species. This proposition is supported by measurements on isolated mass fractions, which clearly show that radiative relaxation in sepia eumelanin is affected by aggregation. Their work lends support to the theory of an oligomeric basic structural unit, and from a photophysical perspective, suggests that non-radiative relaxation modes are the dominant energy dissipating pathways (13,7).
In general, there are relatively few reports of quantitative emission and excitation studies on melanins. Notable works include an early study by Kozikowski et al. (14), a detailed wavelength and concentration dependent study by Gallas and Eisner (15), and steadystate measurements on opioimelanins by Mosca et al. (16). The lack of quantitative data (radiative relaxation quantum yields in particular) is related to the inherent difficulties of performing optical spectroscopy on broad band absorbing macromolecules.
Photoluminescence emission from melanins is heavily re-absorbed across a large range of wavelengths. This phenomenon, coupled with highly non-linear absorption and heavy pump beam attenuation, makes extracting quantitative information from emission or excitation data very difficult without appropriate re-normalisation. In general, the effects of probe beam attenuation and re-absorption in melanin studies are minimized by using low concentration samples. The aforementioned estimates of radiative relaxation quantum yield by Nofsinger et al. were made in this low concentration limit (11,12). Gallas and Eisner (15) report a correction method, but then fail to apply it to produce a meaningful quantum yield.
In this paper we report absolute values for radiative relaxation quantum yield for a synthetic eumelanin. These values were obtained by applying a re-normalisation procedure to account for probe beam attenuation and emission re-absorption in melanin samples and the fluorescein standards over a large range of concentrations. We have determined quantum yields as a function of excitation wavelength, and found a clear and systematic dependency. This work is motivated by a desire to gain a better understanding of melanin photophysics and photochemistry, and also, to address the fundamental question of the melanin basic structural unit. As such, it is part of a broader integrated program of quantum chemical, molecular and condensed phase (solid-state) studies aimed at elucidating the structure-property-function relationships of melanins.

MATERIALS AND METHODS
Sample Preparation. Synthetic eumelanin (dopamelanin) derived from the non-enzymatic oxidation of tyrosine was purchased from Sigma Aldrich (Sydney, Australia), and used without further purification. Eumelanin solutions were prepared at a range of concentrations (0.001% to 0.005%) by weight macromolecule in high purity 18.2 MΩ MilliQ de-ionised water. To aid solubility, the pH of the solutions was adjusted using 0.01 M NaOH to ~11.5, and the solutions gently heated with stirring. Under such conditions, pale brown, apparently continuous eumelanin dispersions were produced. Fluorescein (φ r =0.92 ± 0.02) was purchased from Sigma Aldrich (Sydney, Australia) and used without further purification to prepare standard solutions at ten different concentrations varying from 1.2 ×10 -4 % to 5 × 10 -6 % by weight in 0.1M NaOH solution Emission Re-normalisation. All emission spectra were re-normalised to account for probe beam attenuation and emission re-absorption. The following procedure was used: The measured PL emission intensity ( ( ) ) is the background contribution to the measured emission intensity (solvent and impurity emission, and Raman scattering of the excitation by the solvent), and k(λ) is a scaling factor defining the probe beam attenuation and emission re-absorption. If we assume that only emission arising from a small volume at the waist of the excitation beam is collected by the spectrometer detection system, then k(λ) can be written as: where, α 1 is the absorption coefficient (cm -1 ) at the excitation wavelength, d 1 (cm) is the effective path length responsible for attenuating the excitation beam, and α 2 (λ) (cm -1 ) and d 2 (cm) are respectively the wavelength dispersive absorption coefficient (over the emission range) and path length responsible for emission re-absorption. Eq. 1 also holds for PLE measurements, but in this case, the scaling factor k(λ) is given by: The only difference between Eq. 2 and Eq. 3 is that α 1 (λ) is now dispersive in wavelength and α 2 is the absorption coefficient at the detection wavelength. If the geometry of the measurement system is known, then the path lengths d 1 and d 2 can be found by inspection. For example, for a collimated excitation beam incident upon a square cross-section cuvette (sides of length x cm) with collection at 90˚with respect to excitation (and in the same horizontal plane), then: However, if the geometry of the system is ill-defined, an estimate for the scaling factor can be found by analyzing the relative attenuation of the Raman scattered probe in the sample with respect to the solvent. This technique is useful in the case of melanin emission and excitation studies, since the relatively weak photoluminescence is the same order of magnitude as the signal resulting from Raman scattering of the probe from water (the solvent). In this case, Eqs.
2 and 3 (PL emission and excitation respectively) can be re-written as: where, d is now an effective path length (reflecting both the pump beam attenuation and emission re-absorption), and α 1 (λ) and α 2 (λ) are as before. Additionally, I RB is the intensity of the Raman peak in the background, and I R is the attenuated Raman peak intensity in the sample. These values can be found by fitting Gaussian line shapes to the Raman and PL features in the emission spectra. The absorption coefficient at the Raman peak (α R ) can be found from the absorbance spectra (Fig. 1). This method is particularly useful in PL thin film experiments where pump beam attenuation may be relatively weak, but emission re-absorption significant. In the data presented in this paper, all eumelanin and fluorescein emission spectra were re-normalised using Eqs. 1, 2 and 4, although we have shown that approximately similar results can be obtained using Eq. 5 to calculate k(λ) (17). Absorption coefficients (α 1 and α 2 ) were determined from the absorbance spectra for each concentration. The errors associated with this re-normalisation procedure were estimated by considering the uncertainty in the scaling factor according to the equation: Quantum Yield Calculations. Radiative relaxation quantum yields at three excitation wavelengths (350 nm, 380 nm and 410 nm) were calculated for the synthetic eumelanin using a standard procedure (18). Plotting the integrated photoluminescence emission vs. absorbance for a range of concentrations allows the quantum yield (φ mel ) to be determined according to the equation: where, ∆g mel and ∆g s are the uncertainties associated with the gradients determined from the integrated emission vs. absorption plots. In this analysis, we have assumed ∆g mel , and ∆g s and ∆φ s are uncorrelated.

Synthetic eumelanin absorption
It is well known that eumelanins in solution have a broad, monotonic absorption profile. In the absence of Rayleigh and Mie scattering, or any significant residual monomer or other contamination, the absorbance increases exponentially towards shorter wavelengths. This behavior is demonstrated in Fig. 1, which shows a series of spectra corresponding to concentrations of 0.001%, 0.0025% and 0.005% by weight synthetic eumelanin in pH11.5 de-ionised water. All spectra show a simple first order exponential dependency of absorption coefficient (α) vs.
wavelength. Indeed, a plot of lnα vs. λ yields an approximate straight line (not shown). One could expect some contribution to attenuation from Rayleigh scattering. This would likely manifest itself at the low wavelength (high energy) end of the spectrum (below 350nm). Rayleigh scattering has a strong λ -4 dependency (2), and hence a plot of lnα vs. lnλ would give a gradient of approximately -4 in regions of the spectrum where the effect was significant. This was not observed in any of our samples. Mie scattering from un-dissolved aggregates is also a possible attenuation mechanism.
This type of scattering is relatively wavelength independent, and is clearly visible as a broad feature in aggregated solutions at the concentrations of interest in our studies. None of the spectra showed such behavior. Hence, we conclude that our synthetic eumelanin was well solublised at pH 11.5, and that the attenuation shown in Fig. 1 is derived mainly from absorption. Fig. 2 shows a plot of absorption coefficient at 380 nm vs. concentration for the same three solutions. The relationship is clearly linear, and once again confirms that we have insignificant Mie scattering (this process would impose a non-linearity as a function of concentration). Our findings with respect to the absorption of eumelanin aqueous solutions at high pH agree with those reported by Nofsinger et al. (11), who found that under these conditions, no more than 15% of the attenuation could be attributed to scattering.

Synthetic eumelanin photoluminescence emission
Raw photoluminescence emission spectra (380 nm excitation wavelength) for synthetic eumelanin solutions at concentrations of 0.001%, 0.0025% and 0.005% by weight are shown in Fig. 3. There are several points to note in these raw spectra: a prominent Raman peak  Fig. 1) corrects for these effects. Fig. 4 shows the re-normalised emission spectra resulting from this procedure.
We recover the expected intensity vs. concentration relationship, and the central maxima are now at the same wavelength. Fig. 5 confirms that the emission intensity is linearly dependent upon concentration. From this re-normalised data we can now see that the eumelanin emission is red shifted by ~100 nm relative to the excitation. The emission consists of a single, broad feature (not the double peaked feature reported by Gallas and Eisner (15)), similar to that seen by Nofsinger et al. (11,12) and Kozikowski et al. (14), with a full width at half maximum of ~ 130 nm.  Fig. 7a), b) and c), we are led to the conclusion that this behavior is an inherent property of the eumelanin solution. One might argue that the reduction in photoluminescence emission at longer pump wavelengths is a function of the reduced absorption cross-section ( Fig. 1). However, the quantum yield data (to follow) shows that the radiative emission efficiency is indeed excitation energy dependent.

Radiative relaxation quantum yield
The integrated photoluminescence emission vs. absorbance (at the excitation wavelength) plots for all fluorescein and synthetic eumelanin samples are shown in Figs. 8a) and b) respectively. Five concentrations (0.001% to 0.005%) were measured for the eumelanin, and ten (1.2 ×10 -4 % to 5 ×  (19). This picture fits with the broad band absorbance and the clear asymmetry between excitation and emission characteristics. Although our data is consistent with the oligomeric nanoaggregate theory for the basic structural unit, it does not completely preclude the co-existence of oligomers (the emissive species) and large heteropolymers (the non-emissive species) which serve to statically quench.
In conclusion, we have determined radiative relaxation quantum yields for synthetic eumelanin. A re-normalisation procedure was used to correct for pump beam attenuation and heavy re-absorption of the emission. These are particular issues with broad band absorbing materials such as melanins. By applying the procedure, we were able to obtain data over a range of concentrations and for several different pump wavelengths. Our results confirm that eumelanin is capable of dissipating >99.9% of the absorbed UV and visible radiation non-radiatively.        and re-normalised to account for pump attenuation and emission re-absorption (solid line).
Errors were derived from the re-normalisation procedure (Eq. 8).