J. Phys. Chem. B 2000, 104, 7871-7873
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A Hierarchical Self-Assembly of Eumelanin Christine M. R. Clancy,† J. Brian Nofsinger,† R. Kyle Hanks,† and John D. Simon*,†,‡ Department of Chemistry, Duke UniVersity, Durham, North Carolina 27708, and Department of Biochemistry, Duke UniVersity Medical Center, Durham, North Carolina 27710 ReceiVed: April 27, 2000; In Final Form: May 26, 2000
Atomic force microscopy is used to examine the structure of a natural eumelanin isolated from the ink sacs of cuttlefish (Sepia officinalis). The experimental data presented clearly show that the 100-200 nm spherical eumelanin particles imaged previously by SEM are not a fundamental structural unit. While these spherical particles are stable structures, as is evidenced by their cohesiveness under mechanical stress, the AFM images reveal that these particles are composed of smaller constituents. Taking recent scattering and mass spectrometry results into consideration, we conclude that the self-assembly of Sepia eumelanin is a hierarchical process with small units assembling into hundred-nanometer structures, which then aggregate to form the morphology of the macroscopic pigment.
Despite a significant experimental effort on a variety of natural and synthetic melanins, the molecular structure of this class of pigments remains unknown. There are two distinct types of melanin found in humans: black to brown eumelanin and yellow to red pheomelanin.1-3 Eumelanin is the more ubiquitous pigment, and a natural eumelanin that is used as a human model system can be isolated from the ink sacs of Sepia officinalis in quantities large enough to study using physical techniques.4 Previously reported scanning electron microscopy (SEM) images on Sepia melanin suggest that the pigment is an aggregated structure consisting of subunits that have a lateral dimension on the order of 150 nm.5 The existence of smaller particles (∼15 nm) adhering to these larger subunits was recently reported by our group.6 Matrix assisted laser desorption ionization (MALDI) mass spectrometry studies reveal molecular constituents that have molecular weights between 500 and 1500 amu.7-12 Wideangle X-ray diffraction measurements of dried eumelanin have led to the proposal of a “fundamental aggregate” that is a graphite-like layered structure 4-5 layers thick in which each layer is composed of a small, oligomeric structure.13 The existence of such a fundamental building block is supported by images recorded using scanning tunneling microscopy (STM)14,15 and by small-angle neutron scattering and small-angle X-ray scattering data on the solution structure of copper ion-induced aggregation of synthetic melanin.16 The proposed aggregation structure is generally viewed as a π stacked molecular system, building on the models proposed for the molecular structure of the melanin polymer.7-12 Melanin is a solid, and its underlying microscopic and overall macroscopic structures determine its biological activity. In this letter, we report an investigation on the structure of Sepia eumelanin as determined by atomic force microscopy (AFM). The three-dimensional spatial resolution of AFM provides new insights into the structure of the pigment. Specifically, the AFM and SEM images support both the existence of small particles (diameters on the order of 5-25 nm) and that the spherical subunits revealed in SEM (average diameter of ∼150 nm) are * Corresponding author. † Department of Chemistry. ‡ Department of Biochemistry.
a secondary aggregation structure. Figure 1 shows a tapping mode AFM image of Sepia eumelanin dried on freshly cleaved mica from a solution of D2O collected with a Digital Instruments Nanoscope IIIa (Bioscope). This image reveals that micronsize eumelanin deposits are composed of aggregated structures whose diameters are on the order of 1-200 nm. This structural morphology is characteristic of Sepia eumelanin and is consistent with previous SEM studies. The inset in the figure plots the height of the aggregate along the line indicated by the arrow. If the width of the silicon AFM tip is taken to be 10 nm, this plot indicates that these constituent particles are nearly spherical. Figure 2 shows two AFM images of regions of Figure 1 taken at higher resolution. These latter images clearly reveal smaller eumelanin particles that are approximately 15-25 nm in diameter. Figure 3 shows a set of contact-mode AFM images (height image on the left, deflection image on the right) of an aggregated structure of melanin. This region was chosen for study because the images reveal the presence of mostly subunits of diameters between 100 and 200 nm. The deflection image, which is sensitive to changes in the slope as a function of spatial coordinate, confirms this basic structure. However, in the upper left-hand regions of the image, it appears that the aggregate is coated by material that has not assembled into the spherical structures observed throughout the remainder of the image. After imaging this area, the AFM tip was repeatedly lowered into and raised out of one point in the sample. The AFM tip was then removed and imaged by SEM. The eumelanin that adhered to the AFM tip as a result of this process is shown in Figure 4. There are a number of significant features in this SEM image. First, the material extends approximately 1.5 microns up the tip, or 2.2 microns along the angled side of the tip. However, the eumelanin deposit on the mica surface was only 100-200 nm deep. This implies that the pigment pushes itself up the tip upon multiple exposures to the sample. In general, the material that is picked up does not coalesce into a single particle. Second, the material shows a variety of shapes. Several ∼150 nm spheres have been picked up by the tip and remain intact. This indicates that these aggregated structures are quite stable. There are also
10.1021/jp001630q CCC: $19.00 © 2000 American Chemical Society Published on Web 08/17/2000
7872 J. Phys. Chem. B, Vol. 104, No. 33, 2000
Letters
Figure 1. Tapping mode AFM image of dried Sepia eumelanin on mica. The structure resembles that previously reported by SEM. Inset: The graph is a plot of the height of the aggregate as a function of distance along the line indicated by the arrow. This plot emphasizes that the morphology results from the aggregation of nearly spherical particles of diameter on the order of 150 nm.
Figure 2. High resolution tapping mode AFM images of two regions of the aggregate shown in Figure 1. These images clearly reveal smaller eumelanin particles that are approximately 15-25 nm in diameter. The scale bars represent 165 nm (left image), 83 nm (right image).
smaller pieces of material visible (∼70 nm) which must be the result of the disassembly of medium particles. Finally, amorphous material is seen. This could result if the tip caused the pigment to de-aggregate, and reassembly of the “spherical” structure does not occur. It is also possible that this material is
the as yet unidentified coating material seen in the upper lefthand corner of Figure 3a. The images collected lead to two conclusions. First, the ∼150 nm spherical eumelanin particles imaged by AFM and previously by SEM are not a fundamental structural unit of Sepia
Letters
J. Phys. Chem. B, Vol. 104, No. 33, 2000 7873
Figure 3. Contact AFM images of dried Sepia eumelanin on mica: left, height; right, deflection. These images reveal that this region of the aggregate is essentially composed of subunits whose diameters are between 100 and 200 nm. The scale bar in both images represents 210 nm.
particles, which then self-assemble into ∼100 nm spherical structures. Finally, these medium particles aggregate to form the morphology of the macroscopic pigment. Further work is needed to identify the chemical structure of molecular building blocks, and thereby develop an understanding of the forces that govern the formation of the observed structure. Acknowledgment. This project is supported by a grant from the National Institute of General Medical Sciences, GM-56882. We gratefully acknowledge the Lord Foundation of North Carolina for purchasing the AFM used in this study. References and Notes
Figure 4. SEM images of the silicon nitride AFM tip that was pressed into the eumelanin aggregate shown in Figure 3. The scale bar represents 180 nm. The arrows indicate spherical subunits that appear to have remained intact upon adhering to the AFM tip.
eumelanin. AFM images show that the medium particles are composed of smaller constituents, and SEM images of AFM tips that are pushed into the eumelanin show different sized material than the medium particles. Second, the ∼150 nm spherical particles are stable structures, as is evidenced by their cohesiveness under mechanical stress. These imaging experiments also have important implications for the nature of melanin structure. The aggregated structure formed by evaporative drying of D2O solutions of the pigment (as revealed in Figure 1) is also observed in solid samples isolated from the ink sacs.6 These solutions do not contain micron size particles, and so this structure is the result of the self-assembly of the molecular constituents of the pigment as the solvent evaporates. Furthermore, it is apparent that there is a hierarchy in the aggregation process. Previously reported MALDI data suggest that the molecular constituents of eumelanin are small compared to the size particles revealed by imaging experiments.5-12 Thus it is reasonable to hypothesize that these molecular units aggregate to form the observed small eumelanin
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