Bio-Inspired Melanin-Based Structural Colors Through Self

BIO-INSPIRED MELANIN-BASED STRUCTURAL COLORS THROUGH SELF-

ASSEMBLY

A Dissertation

Presented to

The Graduate School of the University of Akron

In Partial Fulfillment

of the Requirements for the Degree

Doctor of Philosophy

Ming Xiao

August, 2017

BIO-INSPIRED MELANIN-BASED STRUCTURAL COLORS THROUGH

SELF-ASSEMBLY

Ming Xiao

Dissertation

Approved: Accepted:

______Advisor Department Chair Dr. Ali Dhinojwala Dr. Coleen Pugh

______Co-advisor Dean of the College Dr. Matthew D. Shawkey Dr. Eric J. Amis

______Committee Member Dean of the Graduate School Dr. Mesfin Tsige Dr. Chand Midha

______Committee Member Date Dr. Toshikazu Miyoshi

______Committee Member Dr. Hunter King

______Committee Member Dr. Thein Kyu

ABSTRACT

Structural colors enable creation of a spectrum of non-fading visible colors without using pigments, potentially reducing the use of toxic metal oxides and conjugated organic pigments. Although many top-down and bottom-up methods have been used to produce structurally colored materials, significant challenges remain to achieve the contrast needed for producing a complete gamut of colors and a scalable process for industrial applications.

Nature provides some intriguing palettes of structural colors in avian feathers using three main ingredients, melanin, keratin, and air. Recently, we have demonstrated that the rainbow-like iridescent colors in a single feather of Australia pigeon (common bronzewing) are caused by a slight variation of the layer thickness of multilayered melanosome (organelles filled with melanin) nanostructures. Learning from these color production mechanisms, we have synthesized melanin nanoparticle (SMNPs) ranging from 100-200 nm in diameter. Using an evaporation-based process we have assembled these nanoparticles to produce a wide spectrum of visible colors. We have shown the high absorption of SMNPs leads to more saturated colors than those produced using polystyrene or glass nanospheres. In addition, SMNP films show rapid, large, reversible color changes responsive to variation in ambient humidity, due to swelling/shrinking of

SMNPs.

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To produce brighter colors in a scalable process, we have designed core-shell nanoparticles with melanin core (RI ~1.74) and silica shell (RI ~ 1.45). A one-pot reverse emulsion process has been used to assemble these core-shell nanoparticles to produce a full-spectrum of colorful supraballs. Similar to mixing pigmentary colors, we can also blend these core-shell SMNPs with different shell thicknesses to control the spacing and continuously tune colors. The new bio-inspired melanin-based structurally materials pave the way for producing novel photonic inks, suitable for applications like painting, textiles, and displays.

DEDICATION

This dissertation is dedicated to my family. A deep gratitude to my father

Renping Xiao, my mother Zixia Wang, my wife Hong Chen, and my sister Xian Xiao.

Their constant love and support encourage me all the time.

ACKNOWLEDGEMENTS

My PhD has been a really fruitful journal filled with struggle with what to start, curiosity for new knowledge, excitement of examining on new hypothesis, and personal mental maturity. During this process, I own my gratitude to lots of people.

First and foremost, I would like to acknowledge my advisors, Prof. Ali Dhinojwala and Prof. Matthew Shawkey for insightful mentorships and continuous supports. They are both best supervisors and are so nice, tolerant to allow me to try all types of new things. Ali has taught me a lot on the way of thinking and solving problems. I have also learnt a lot from Matt, ranging from TEM imaging, scientific writing, to keeping broad curiosities. I really feel lucky that Ali started the collaboration with Matt for my project and then Matt provided the chance to collaborate with Prof. Nathan Gianneschi group.

Secondly, I want to thank all my collaborators: Dr. Yiwen Li, Ziying Hu, Zhao

Wang, and Prof. Gianneschi at UCSD for continuously providing different types of melanin samples; Dr. Wei Chen, Dr. Youlee Hong and Prof. Toshikazu Miyoshi for the help with solid-state NMR experiments; Alejandro Diaz Tormo, Prof. Nicolas Thomas for teaching me how to use finite-difference time-domain simulations; Dr. Min Gao for the help with environmental SEM experiment; and Boxiang Wang for performing the scattering calculation.

I should also extend my thanks to three master students, Jiuzhou Zhao, Weiyao Li, and Xiaozhou Yang, I worked and have been working with for their hardworking and

great help on several of my projects. I also want to thank Dr. He Zhu, Dr. Yu Zhang,

Mena Klittich, Michael Wilson, Siddhesh Dalvi and all other Dhinojwala lab members for lots of discussion and different types of help.

I am grateful to Dr. Chad Eliason, Dr. Rafael Maia, Dr. Liliana D’Alba, Dr.

Branislav Igic, Asritha Nallapaneni and other Shawkey lab members for their instrument tutoring and helpful discussions on all my manuscripts.

Finally, I want to thank my family and friends for their love and support.

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TABLE OF CONTENTS

Page

TABLE OF CONTENTS ...... viii

LIST OF TABLES ...... xiii

LIST OF FIGURES ...... xiv

CHAPTER I RESEARCH OVERVIEW

...... 1

1.1 Introduction ...... 1

1.2 Melanin structure and functions ...... 4

1.3. Melanin-based colors in nature ...... 8

1.3.1 An absorbing layer...... 9

1.3.2 Arrays of solid melanosomes ...... 11

1.3.3 Hollow melanosomes ...... 14

1.4. Optical benefits of melanins for structural colors ...... 15

1.5 Melanin in synthetic structural colors ...... 17

1.5.1 Absorber to increase saturation ...... 18 viii

1.5.2 Solid thin film ...... 19

1.5.3 Solid melanin nanoparticles...... 20

1.5.4 Core-shell melanin nanoparticles ...... 22

1.5.6 Conclusions ...... 23

CHAPTER II NANOSTRUCTURAL BASIS OF RAINBOW-LIKE IRIDESCENCE IN

COMMON BRONZEWING PHAPS CHALCOPTERA FEATHERS ...... 24

2.1 Introduction ...... 24

2.2 Materials and Methods ...... 25

2.2.1 Barbule macrostructure ...... 25

2.2.2 Reflectance measurement ...... 25

2.3 Barbule nanostructure ...... 28

2.3 Results and Discussion ...... 29

2.3.1 Rainbow-like iridescent reflectance ...... 29

2.3.2 Nanostructure of iridescent barbules ...... 31

2.3.3 Multilayer interference modeling ...... 33

2.4. Conclusions ...... 42

CHAPTER III BIO-INSPIRED STRUCTURAL COLORS PRODUCED VIA SELF-

ASSEMBLY OF SYNTHETIC MELANIN NANOPARTICLES...... 45

3.1 Introduction ...... 45

3.2 Materials and Methods ...... 47

3.2.1 Synthesis and characterization of SMNPs ...... 47

3.2.2 Evaporation-based assembly ...... 48

3.2.3 Characterization of the SMNP film ...... 48

3.2.4 Measurements of the complex RI of SMNPs ...... 50

3.2.5 Optical Modeling ...... 52

3.3 Results and Discussion ...... 52

3.4. Conclusions ...... 60

CHAPTER IV STIMULI-RESPONSIVE STRUCTURALLY COLORED FILMS FROM

BIOINSPIRED SYNTHETIC MELANIN NANOPARTICLES ...... 62

4.1 Introduction ...... 62

4.2 Materials and Methods ...... 63

4.2.1. Preparation of SMNP films...... 63

4.2.2. Characterization of dynamic colors ...... 64

4.2.3. Water absorption measurement ...... 65

4.2.4. In-situ investigation of SMNP film thickness ...... 67

4.3 Results and Discussion ...... 67

4.4. Conclusions ...... 80

CHAPTER V BIO-INSPIRED BRIGHT NON-IRIDESCENT PHOTONIC MELANIN

SUPRABALLS ...... 81

5.1 Introduction ...... 81

5.2 Materials and Methods ...... 83

5.2.1 Characterization of nanostructures in bird feathers ...... 83

5.2.2 Synthesis and characterizations of CS-SMNPs ...... 83

5.2.2 Supraball preparation ...... 85

5.2.3 Supraball characterization ...... 86

5.3 Optical Model ...... 87

5.3.1 FDTD simulation ...... 87

5.3.2 Scattering theory ...... 88

5.4 Results and Discussion ...... 93

5.4. Conclusions ...... 103 xi

CHAPTER VI SUMMARY

...... 105

REFERENCES ...... 108

APPENDIX A: MATRIX TRANSFER METHOD ...... 134

APPENDIX B: COPYRIGHT NOTICE ...... 137

APPENDIX C: AUTHOR PROFILE ...... 139

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LIST OF TABLES

Table Page

Table 2-1. Spacing and diameter of melanosomes in the barbule nanostructure measured

...... 33

Table 5-1 Synthesis conditions for different sized CS-SMNPs ...... 84

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LIST OF FIGURES

Figure Page

Figure 1-1. Five mechanisms to generate structural colors: (A) Dispersion causes a white light beam to split into different colors and a photo of a rainbow; (B) A cartoon showing lighter scattered by particles and a photo of sunset (photo credit, Xiaozhou Yang); (C) A scheme illustrating the thin-film interference and a photo a colorful oil film; (D) A scheme showing how diffraction gratings split white light and a photo of a CD disc; (E)

A cartoon representing 1D, 2D, and 3D photonic crystals and a photo of an opal (photo credit, Dave Ault)...... 3

Figure 1-2. Biosynthetic pathways leading to eumelanin and pheomelanin production.

Note that the activities of tyrosinase, Tyrp1 and Tyrp2 are involved in the production of eumelanin, while only tyrosinase (and the amino acid cysteine) is necessary for the production of pheomelanin.10 Reproduced with permission from the American Society for Photobiology, copyright 2008...... 5

Figure 1-3. Proposed hierarchical structure of natural melanosomes.12 ...... 6

Figure 1-4. Melanin in natural colorations. (A) Randomly disordered packed melanosomes in feathers of California quail.24 Reproduced with permission from the

American Physical Society, copyright 2006. (C) Multilayer of melanosomes in wing feathers of common bronzewing and breast feathers of bird of paradise.28,29 Reproduced with permission from The Optical Society, copyright 2014; Reproduced with permission from the Royal Society, copyright 2011. (D) Two-dimensional ordered packing of melanosomes in mallard and peacock feathers.30,31 Reproduced with permission from the

Royal Society, copyright 2012; Reproduced with permission from the National Academy of Sciences, copyright 2003. (E) Different organizations of hollow melanosomes in the feathers of Himalayan monal,32 hummingbird,33 and wild turkey.30 Reproduced with permission from the American Philosophical Society, copyright 1960; Reproduced with permission from the Royal Society, copyright 2013; Reproduced with permission from the Komm. Gebr. Fretz, copyright 1977...... 10

Figure 1-5. Light absorbers to increase the saturation of structural colors. (A) Carbon black was added into binary sizes of polystyrene nanoparticles to make green color more saturated but darker when increasing the concentration of carbon black.55 Reproduced with permission from the John Wiley & Sons, copyright 2010. (B) Black polypyrole was coated onto silica nanoparticles to enhance color visibility.57 Reproduced with permission from the American Chemical Society, copyright 2016. (C) Addition of carbon black into spray coated amorphous silica nanoparticles decreases reflectance intensity, but increases color saturation.54 Reproduced with permission from the John Wiley & Sons, copyright

Figure 1-6. Melanin for synthetic structural colors. (A) Sepia melanin is used a light absorber to decrease incoherent scattering.64 Reproduced with permission from the John

Structural colors from the interference of a layer of melanin nanoparticles.50 Reproduced with permission from the American Chemical Society, copyright 2015. (D) Structural colors from scattering of melanin nanoparticles.67 Reproduced with permission from the

Advancement of Science, copyright, 2016. (F) Photonic supraball inks made of melanin core and silica shell nanoparticles...... 20

Figure 2-1. (a) Stereo light microscope image. Panels (b-d) show higher magnification images of barbules in distal region, middle region, and proximal regions, respectively.

Scale bars, (a) 2 mm and (b-d) 200 μm...... 25

Figure 2-2. Schematic for the geometry of spectrometer for specular reflectance measurement. is the angle between the incident beam and the surface normal; similarly,

is the angle between the detector and the surface normal; and is the angle between feather rachis (bold black line with an arrow) and the plane-of-incidence (yellow plane).

The values of and can be read directly from the spectrometer. The arrow of the feather stands for the distal end of the feather. is the angle between the stage plane on which the feather lies and the horizontal plane...... 27

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Figure 2-3. Specular reflectance spectra for the green region of barbules at incident angles from 10 to 45 ...... 30

Figure 2-4. (a-c) Microscopic images of blue, green and red barbules from MSP, respectively and small black squares in the middle indicate the sampled spot (the length of the black square is 4 μm). (d) Color variation of the barbules measured by MSP, the vertical axis is the normalized reflectance intensity with arbitrary unit. The color for each curve is based on the human visual perception according to the standard CIE1931.88 .... 31

Figure 2-5. TEM images of (a) cross section of a red barbule, (b) longitudinal section of a green barbule, and (c) cross section of a non-iridescent brown barbule. Scale bars, 500 nm...... 32

Figure 2-6. TEM images of cross-sections of barbules with different colors under the same magnification: (a) red, (b) green, and (c) blue. Scale bars, 100 nm ...... 33

Figure 2-7. Schematic of multilayer structure in barbules. is the keratin cortex thickness; , the diameter of melanosomes; , the spacing between melanosomes layers and , the spacing between neighboring melanosomes ...... 34

Figure 2-8. (a) Measured (green line) and modeled spectra (black line) on the average layer thickness for barbules in green region. (b-c) The modeled bluest and reddest spectra

(black lines) based on largest and smallest thickness of melanosome and keratin layers in blue and red barbules; and the blue and red curves are the bluest and reddest spectra measured by MSP, respectively...... 36

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Figure 2-9. Modeled results for angle-resolved reflectance spectrum for middle barbules

(green solid line). The black square data points are obtained from experimental spectra in

Figure 2-3...... 38

Figure 2-10. The modeled color range for barbules in the blue zone and the experimentally measured green...... 39

Figure 2-11. Reflectance of green barbule measured by MSP using differently polarized input beams. The blue curve is the unpolarized reflectance, and the red, green are the s- and p- polarization, respectively...... 40

Figure 2-12. The hue dependence on the outmost keratin layer (cortex layer) thickness based on multilayer modeling result...... 41

Figure 2-13. Green solid line is the decrement rate of full width at half maximum

(FWHM) , which is the derivative of FWHM with respect to layer number and blue dash line is the increment rate of intensity which is the derivative of intensity with respect to layer number...... 42

Figure 3-1. Characterizations of SMNPs. (a) Size distribution of SMNPs, where y axis is the contribution of scattered light intensity from different sizes of particles to the total light intensity. Inset: TEM image of SMNPs with a scale bar of 100 nm. (b) Square of effective RI of SMNP solution changes against the volume fraction. The slope of the

22 linear fitting is nnmw . The coefficient of determination for the linear fitting is 0.998.

(c) Transmission spectra for SMNPs solutions at different concentrations (blue, 10 mg/L; red, 25 mg/L; black, 50 mg/L). (d) Imaginary part of the RI as function of the wavelength.

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Different colored curves are for each concentration (blue, 10 mg/L; red, 25 mg/L; black,

50 mg/L) and green curve is the averaged value of all three concentrations...... 53

Figure 3-2. Optical images of films deposited at 60 C with an evaporation rate of 0.50-

0.55 mm/h and different concentrations: (a) 0.17 mg/mL, (b) 0.6 mg/mL (c) 1.0 mg/mL, and (d) 3 mg/mL. Scale bars: 0.5 mm...... 54

Figure 3-3. Optical characterizations of SMNP films. (a) Optical images of colored films.

The red and orange colors are from different regions of the film in Figure 3-2b; and the yellow and green colors are from different locations of the film in Figure 3-2c. Scale bars:

100 μm. (b) Measured (red curve) and modeled (black curve) reflectance spectra of red film in (a). (c) Measured (green curve) and modeled (black curve) reflectance spectra of green film in (a)...... 55

Figure 3-4. Specular reflectance of the silicon wafer measured using the microspectrophotometer...... 56

Figure 3-5. Hyperspectral analysis of (a) red and (b) green SMNP films. In each case, two distinct spectra (shown with different color codes) contributed to the color measured in specular reflectance. Each spectrum corresponded to a different pixel percent of the scanned area, and the low-occurrence spectrum always appeared randomly scattered across the scanned area (see inserts)...... 57

Figure 3-6. SEM images of structure of SMNP films. (a) and (b) are SEM cross sectional images of the red film and green film, respectively. (c) and (d) are top view SEM images of the red and green films, respectively. Scale bars: 500 nm...... 58

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Figure 3-7. Modeled reflectance spectra for films of PS nanoparticle and SMNPs. The colors of the curves are colored with RGB standards using “rgb2spec” in Pavo package of

444 nm, peak area ratio between the shorter wavelength peak (390 nm) and the longer wavelength peak (510 nm) is 0.64 for PS; while peak area ratio between the shorter wavelength peak (405 nm) and the longer wavelength peak (550 nm) is 0.22 for SMNPs.

...... 59

Figure 4-1. Dynamic light scattering measurement of SMNPs in an aqueous solution. .. 64

Figure 4-2. A scheme of custom-build humidity control setup with real-time monitor of the relative humidity...... 65

Figure 4-3. Water uptake of a standard aluminum DSC pan with the increase of humidity.

...... 67

Figure 4-4. (a) A TEM image of SMNPs, scale bar, 100 nm. (b) A scheme of film formation via evaporation induced self-assembly process. Optical images for blue (c) and red (g) SMNP films, scale bar, 100 μm. (d) and (e) Top view and side view SEM images for the blue film, where insets are 2D Fourier transform power spectra. (f) and (h) Top view and side view SEM images for the red film. Scale bars in (d-h), 1 μm...... 68

Figure 4-5. Specular reflectance spectra of a SMNP film at various incident angles with respect to the film normal. Black curve is for 10 , red for 20 , green for 30 , and blue for

40 ...... 69

Figure 4-6. Dynamic colors for blue (a) and red (b) films at various RH. (c) The color changes during humidity change from 10% to 30%, 50%, 70%, and 90% for blue (black dots) and red (white dots) films, as presented in the CIE 1931 color space...... 70

Figure 4-7. Reflectance spectra of a bare silicon wafer under relative humidity of 10%

(black curve) and 90% (red curve)...... 71

Figure 4-8. Maximum peak positions in the spectra of blue (a) and red (b) films shift during eight times of cycling RH. In (b), black curve is for the primary peak position and red is the secondary peak position...... 72

Figure 4-9. Reflectance spectra of blue (a) and red (b) SMNP films before and after eight times cycles at RH of 10% and 90%...... 72

Figure 4-10. Water uptake in mass of SMNPs changes with RH. Solid line is the linear fitting (%  1.81  0.156RH , R2 0.96 )...... 73

Figure 4-11. The scheme of the structure of SMNP films...... 74

Figure 4-12. (a) Measured (solid blue) and model spectra (solid black) for blue film at

RH of 10%. (b) Measured (solid red) and model spectra (solid black) for red film at RH of 10%. (c) Measured (dash blue) and model spectra (dash black) for blue film at RH of

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90%. (d) Measured (dash red) and model spectra (dash black) for red film at RH of 90%.

...... 75

Figure 4-13. (a) Scheme of model 1, where SMNP film thickness swells by 17% from

RH 10% to 90%. (b) Scheme of model 2, assuming water condenses into pores of SMNP films...... 76

Figure 4-14. Theoretical reflectance for both blue (a) and red (b) SMNP films at RH of

90% based on model 2. Black spectra are the simulated spectra for both blue and red film.

Blue curve represents experimental reflectance of the blue film and red curve stands for experimental reflectance of the red film...... 77

Figure 4-15. Cross-sectional ESEM images of a SMNP film at different water vapour pressures. Scale bar, 500 nm...... 78

Figure 4-16. Side view ESEM images on cross section of a SMNP film under dry nitrogen conditions at various pressures (1 Torr, 5 Torr, and 10 Torr). Scale bar, 500 nm.

...... 78

Figure 4-17. Top view ESEM images on a SMNP film at different water pressures, (a) high vacuum, (b) 5 Torr, (c) 15 Torr, and (d) back to high vacuum. Scale bars, 5 μm. ... 79

Figure 5-1. Natural inspirations and the optical model. (A) Two biological examples to enhance color brightness from left to right: an iridescent turkey wing feather and a cross- sectional TEM image of a single barbule, and the duck wing feather and cross-sectional

TEM image of a single barbule. Scale bars, 500 nm. Image credits, Ferran Pestana/Teddy

Llovet. (B) Normal reflectance spectra from the (111) plane of FCC lattices made of xxii

core-shell nanoparticles and homogenous nanoparticles with similar sizes and equivalent refractive indices: high RI core/low RI shell nanoparticles (core: RI = 1.74, diameter =

200nm; shell: RI = 1.45, thickness = 50 nm), equivalent homogenous nanoparticles (RI =

1.54, diameter = 300 nm), and low RI core/high RI shell nanoparticles (core: RI = 1.45, diameter = 267 nm; shell: RI = 1.74, thickness = 16.5 nm). (C) The reflectance intensity ratio between core-shell and homogenous structure changes as we vary the ratio of core radius to total core-shell nanoparticle radius...... 82

Figure 5-2. UV-vis absorption of pure SMNPs, CS-SMNPs, and pure silica nanoparticle solution at the concentration of 20 mg/L...... 85

Figure 5-3. The complex refractive index for synthetic melanin cores used in the FDTD calculations, which is the best fit with the measurements from our previous paper.50 ..... 88

Figure 5-4. Core-shell SMNPs synthesis and self-assembly. (A) A scheme of the method of synthesizing silica coated melanin nanoparticles. (B) TEM images of core-shell

SMNPs: 160/0 nm, 160/36 nm, and 160/66 nm, respectively. Scale bar, 100 nm. (C) A scheme showing the self-assembly of supraball structures via a reverse emulsion process.

(D) A photo of rainbow-like flowers, painted with supraball inks made of five different sizes of CS-SMNPs: navy, 123/36 nm; green, 123/43 nm; olive, 160/36 nm; orange,

160/50 nm; and red, 160/66 nm...... 94

Figure 5-5. Characterization of supraballs. (A) Optical images of supraballs made of four types of nanoparticles: 224 nm pure silica nanoparticles, 160/0 nm, 160/36 nm, and

160/66 nm CS-SMNPs. Scale bars, 0.5 mm. (B) Reflectance spectra and optical images

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for individual supraball consisting of 224 nm pure silica nanoparticles (black curve, cyan supraball), 160/0 nm CS-SMNPs (blue curve, purple supraball), 160/36 nm CS-SMNPs

(green curve, olive supraball) and 160/66 nm CS-SMNPs (red curve, red supraball). The shaded area indicates the standard deviation from 12 samples, plotted using Pavo package

88 in R . Black box in insets is 44 μm. (C) Angle-resolve spectra for olive inks as shown in Fig. 2D. The inset scheme shows setup for angle-resolved backscattering measurements where we fixed 휶=15° and varied angle 휽 between source and sample from 40° to 90°. (D) FDTD simulation of normal reflectance spectra from supraballs composed of three different sizes of CS-SMNPs...... 97

Figure 5-6 Microstructures of supraballs made of pure CS-SMNPs with three different sizes. (A) SEM images of whole supraball morphologies. (B) High resolution SEM images of top surfaces of supraballs. (C) Cross-sectional TEM images of inner structure of supraballs. Scale bars, (A) 2 μm, (B) 500 nm, and (C) 500 nm...... 98

Figure 5-7. Supraballs from binary CS-SMNPs. Optical images, SEM images of top surface of supraballs, and cross sectional TEM images for supraballs consisting of (A)

160/0 nm & 160/36 nm CS-SMNPs (B) 160/0 nm & 160/66 nm CS-SMNPs, and (C)

160/36 nm & 160/66 nm CS-SMNPs. The mixing ratio was 1:1 by mass. Scale bars, 500 nm. (D) Optical images of supraballs mixing different mass ratios of 160/36 nm and

160/66 nm CS-SMNPs. Black box is 4 4 μm...... 100

Figure 5-8. (A) Reflectance of single supraballs of pure 160/0 nm, mixed 160/0 nm &

160/36 nm CS-SMNPs, and mixed 160/0 nm & 160/66 nm CS-SMNPs. (B) Reflectance

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of single supraballs of pure 160/36 nm CS-SMNPs, pure 160/66 nm CS-SMNPs, and mixed 160/36 nm & 160/66 nm CS-SMNPs...... 101

Figure 5-9. TEM images of the inner structure of supraballs of binary CS-SMNPs: (A)

160/0 nm & 160/36 nm CS-SMNPs, (B) 160/0 nm & 160/66 nm CS-SMNPs, and (C)

160/36 nm & 160/66 nm CS-SMNPs. Scale bars, 500 nm...... 102

Figure 5-10. Normal reflectance spectra of supraballs making of binary CS-SMNPs

(160/66 nm and 160/36 nm) with different mixing ratios...... 102

Figure 5-11. Inverse of normalized transport mean free path changes with the different mixing ratios of 160/36 nm and 160/66 nm CS-SMNPs based on the scattering theory calculations. (A) Short-range order was taken into account. (B) We used independent scattering approximation without considerations of short-range order. The legend represents the mixing mass ratios of two sizes of CS-SMNPs...... 103

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CHAPTER I

RESEARCH OVERVIEW

1.1 Introduction

Structural colors, caused by physical interaction of light with periodic or aperiodic nanostructures have lots of advantages over conventional pigmentary colors that are produced by light absorption of conjugated organic molecules or metal-containing dyes.

These advantages include iridescent color effects, wide tunability of full-spectrum colors, resistance to photo/chemical bleaching, flexibility to use nontoxic materials, and capability to design responsive dynamic colors.

There are mainly five ways to produce structural colors, including dispersion, scattering, interference, diffraction, and photonic crystals (Figure 1-1). (1) As shown first by Isaac

Newton in 1672,1 a simple prism could disperse a beam of white light into rainbow colors because electromagnetic waves have wavelength-dependent refractive indices (called dispersion) and shorter wavelength light will bend more than longer wavelength light when passing through the prism. The most common example of this mechanism in nature is the production of rainbows, where water droplets act as prims to disperse the white light into seven different colors. (2) Scattering is a process of excitation and reradiation of electromagnetic waves when they meet an obstacle.2 A single particle having similar size to the wavelength of light leads to Mie scattering and a system filled with multiple particles will cause more complex scattering phenomenon depending the particles

concentration and organization. The blue color of sky and the red color of sunset are caused by the scattering of light. (3) Constructive and destructive interference at different wavelength is another way to structural color production. The simplest example is the rainbow-like colors of the oil film on a road, which are caused by interference of two reflected light beams at air/oil interface and oil/substrate interface. Multiple alternating dielectric layers can be used as interference filter to create bright colors. A larger layer number leads to an increase in the reflection intensity. (4) Diffraction gratings can also split the white light into colorful rays and the grating size is often larger than the visible light wavelength. The colors of CD discs belong to this category. The diffraction gratings are also widely used in the spectrophotometers. (5) Photonic crystals (PhCs) represent structures that have dielectric constant (namely refractive index) periodicity in one, two, or three dimensions. Such periodicity can prohibit the propagation of light at certain wavelength, leading to photonic band gaps and thus structural colors at the reflection mode.

With the development of self-assembly and photolithography as well as the concept of photonic crystals, research on structural colors have been explosive during last two decades. However, there remain challenges to achieve high visible structural colors in a scalable process. It is always difficult to obtain enough refractive index contrast without using inorganic materials, to reduce incoherent background scattering without adding absorbing materials, and to scale up lab fabrication methods for mass productions. In addition, it is always desired that other functionalities can be incorporated with structural 3

colors. Nature has developed millions of striking structural colors across species. Studies on structural colors in bird feathers in the last decade have revealed that the melanin contributes to colorations in feathers in different ways. Inspired by this, a few examples

(including some of my work) on melanin-based synthetic structural colors have been demonstrated since I started my research in 2012. In this chapter, we will review the melanin-based structural colors in nature and synthetic melanin-based structural colors.

Most of my PhD work will be referred or introduced within the context in this chapter and the details will be further covered in following chapters.

1.2 Melanin structure and functions

Melanin is an omnipresent pigment in bacteria, fungi, plants, animals, and even prehistoric organisms.3-5 Melanin is mainly divided into black to brown eumelanin and yellow to reddish pheomelanin based on the different biosynthetic paths (Figure 1-2).

Eumelanin is made from two major precursors, 5,6-dihydroxyl indole (DHI) and 5,6- dihydroxyindole-2-carboxylic acid (DHICA). Pheomelanin consists of two isomers of benzothiazine intermediates, which are synthesized in melanocytes when there is enough amount of cysteine.6 Pheomelanin is often combined with eumelanin and no pure pheomelanin in nature has been reported up to now. The ratio of these two types of melanin is related to photoreactivity and likely associated with the skin cancer susceptibility.7,8 Based on the fact that DHICA-eumelanin degrades to pyrrole-2,3,5- tricarboxylic acid (PTCA) by oxidation of KMnO4 and pheomelanin turns into 4-amino-

3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP), the degradation products have been used to estimate the ratio of two melanins (Details see

the review by Ito9). Currently, eumelanin is better understood than pheomelanin and no study has related the pheomelanin with structural coloration, therefore we will mainly focus on eumelanin here. For simplification, “melanin” we will mention in the following sections means “eumelanin”.

Figure 1-2. Biosynthetic pathways leading to eumelanin and pheomelanin production. Note that the activities of tyrosinase, Tyrp1 and Tyrp2 are involved in the production of eumelanin, while only tyrosinase (and the amino acid cysteine) is necessary for the production of pheomelanin.10 Reproduced with permission from the American Society for Photobiology, copyright 2008.

Melanin is synthesized from tyrosine in the specialized cells called melanocytes. Melanin is in a form of amorphous granules in some lower-level species like fungi, while it is synthesized in melanocytes to well-defined submicron particles for more advanced vertebrate species.11 It is common to term those melanin particles from epidermis melanosomes. Recently, we have proposed that different shaped melanosomes are all made of 30-50 nm nanoparticles, where melanin precursors (DHI, DHICA, and their oxidized forms) cross-link and stack together by - interactions (Figure 1-3).12

sphere 30-50nm

Solid rod ~4 Å

Hollow rod

Self-assembled π-π stacking nanoparticle

Figure 1-3. Proposed hierarchical structure of natural melanosomes.12

Despite the variation in the morphology, melanin in nature shares special biological functions and physiochemical properties. Melanin absorbs light broadly across UV-vis range, and it generates pigmentation in many organisms. In some animals like cephalopods, controlling the melanin density and distribution in the integuments using nerve stimulus result in dynamic colors.13 For human, exposure to UV enhances melanogenesis and it creates more melanin to protect the skin from UV damage. Melanin has been reported to dissipate almost 90% of the UV radiation to heat within a

nanosecond or even faster.14,15 Cuttlefish can release melanin inks as a defending weapon to escape from predators, due to the high light absorption of melanin. Despite of multiple biological functions based on from broadband absorption of melanin, the relationship between the molecular structure and its optical properties are not well understood.

Meredith et al. have demonstrated that melanin is chemically heterogeneous, consisting of numbers of different chemical species and the superposition of absorption of all species at different wavelength leads to a monotonic broadband absorption curve.16,17

Recently, Chen et al. have found the excitonic couplings within melanin also affect the absorption and proposed that geometric disorder of melanin aggregates can also broaden the absorption spectrum in addition to the chemical disorder.18

Melanin has a combination of different oxidized forms of monomers, like DHI, DHICA, and semioxidized (semiquinone) and fully oxidized (quinone) states. The transformation between those different oxidized forms provides the opportunities for electron exchange.19 Melanin contains intrinsic free radicals, and can quench poison reactive oxygen species (ROS) generated by UV radiation or some biological reactions within the tissues. Due to the catechol groups, melanin can bind with a variety of metal ions, like

Cu2+, Fe3+, Mg2+ through metal coordination bonds, which helps to reduce metal toxicity in cells or form cross-linking networks to enhance mechanical properties. In addition, melanin has unique electronic properties. In 1970s, McGinness et al. have demonstrated that melanin has properties of an amorphous seminconductor.20,21 Later, melanin has been discovered that electronic conductivity is affected by to humidity or light.22,23

Despite of many years of research on melanin, there remain open questions: (1) is melanin a chemically cross-linked heterogeneous polymer or assembled oligomers bound by the physical interaction like hydrogen bonding and charge transfer; (2) how the hierarchical chemical structure affects the optical properties, free radical quenching ability, and other functions. Many research articles and review papers have discussed melanin’s structure and biological functions, and current open questions; however, few have highlighted the role of melanin in the structural color productions and there was no study on the use of melanin for creation of artificial structural colors before 2014.

Therefore, in the following sections of this chapter we will put the mysteries on melanin structure behind and focus on the optical performance of melanin, including both natural and synthetic structural colors.

1.3. Melanin-based colors in nature

Nature has developed diverse colors across species during millions of years of evolution.

Colors can be originated from pigmentary or structural colors based on the coloration mechanisms. Melanin, as a pigment, can absorb light, leading to dark colors like brown and black in the feathers of California quail (Callipepla californica) (Figure 1-4A).24 We will not go into deep discussions on pigmentary colors rising from melanin and mainly concentrate on the significant roles that melanin plays in the production of structural colorations in nature. Melanin is synthesized in melanocytes and these particles (termed melanosomes) have well-defined sizes and diverse morphologies such as spheres, cylinders, or hollow shapes in bird feathers.11 Those melanosomes are transferred into barbules, where they assemble to different structures leading to structural colors.

1.3.1 An absorbing layer

Melanin has broadband absorption of light and sometimes melanosomes form a disordered background layer incoherent scattering light to enhance the color saturation.

Many non-iridescent colors in feathers are caused by coherent scattering of three- dimensional amorphous or quasi-ordered porous spongy -keratin layer (channel-type or spherical-type structure) in medullary cells of barbs.25 This spongy layer lies above a layer of melanosomes. Shawkey and Hill have investigated the color and nanostructure differences between a white amelanotic Steller’s jay (Cyanocitta stelleri Gmelin) feather and a blue Steller’s jay feather (Figure 1-4B).26 Based on the observation that both types of jay feathers have the similar spongy keratin layer and the amelanotic jay does not have a melanosome layer underneath, they have proposed the melanosome layer absorbs incoherently backscattered white light and increases the saturation of the color generated by the spongy-like structure.

Figure 1-4. Melanin in natural colorations. (A) Randomly disordered packed melanosomes in feathers of California quail.24 Reproduced with permission from the Royal Society, copyright 2010. (B) Absorbing layers of melanosomes in the barbs of blue jays and barbules in domestic neck feathers.26,27 Reproduced with permission from Company of Biologists Ltd, copyright 2006; Reproduced with permission from the American Physical Society, copyright 2006. (C) Multilayer of melanosomes in wing feathers of common bronzewing and breast feathers of bird of paradise.28,29 Reproduced with permission from The Optical Society, copyright 2014; Reproduced with permission from the Royal Society, copyright 2011. (D) Two-dimensional ordered packing of melanosomes in mallard and peacock feathers.30,31 Reproduced with permission from the Royal Society, copyright 2012; Reproduced with permission from the National Academy of Sciences, copyright 2003. (E) Different organizations of hollow melanosomes in the feathers of Himalayan monal,32 hummingbird,33 and wild turkey.30 Reproduced with permission from the American Philosophical Society, copyright 1960; Reproduced with permission from the Royal Society, copyright 2013; Reproduced with permission from the Komm. Gebr. Fretz, copyright 1977.

In addition to enhancing the purity of non-iridescent colors in some bird feathers, a basal layer of melanosomes have also been found in iridescent feathers. In those cases, a thin -

keratin cortex layer is on top of thick layer of melanin granules. Due to thin-film interference effect (Figure 1-1C) , the thickness variation of cortex can contribute to different colors, as purple and green colors seen in domestic pigeon (Columba livia domestica) neck feathers (Figure 1-4B).27 Another example is the iridescent blue color in satin bowerbird (Ptilonorhynchus violaceus minor) plumages where the top smooth keratin layer (160~170 nm thick) sits on a basal layer of densely packed melanosomes.34

In fact, no one has pointed out the role of basal melanin layer to the iridescent colors. We here propose that a basal melanin layer not only absorbs incoherent scattering light (small amount due to the ordered structure producing iridescent colors), but create a new keratin-melanin interface with enough refractive index contrast (melanin 1.65~2.0, keratin 1.54) to reflect more light, which is significant for thin-film production (More discussions will be covered in section 1.4).

1.3.2 Arrays of solid melanosomes

Most iridescent feathers contain arrays of solid melanosomes. Based on the organization patterns of melanosomes, they can be divided into the multilayer structure and the two- dimensional photonic crystal structure.

Multilayer structure

The simplest case for multilayer interference is a layer of keratin cortex and a layer of melanosomes. Maia et al. have demonstrated that the color of blue-black grassquits

(Volatinia jacarina) is caused by the light interference of a keratin layer (thickness, 127 nm) and a melanosome layer (thickness, 422 nm).35 As another example, tree swallow

(Tachycineta bicolor) mantle feathers show iridescent blue-green color because of the interference from a 148 nm keratin layer and a 173 nm melanosome layer.36 Interestingly, the keratin layer swells in high humidity condition, causing a 25 nm of red shift in the reflectance spectrum of the feather.

Some bird feathers contain multilayers of melanosomes with ~100 nm spacing between each layer. We have recently found the rainbow colors (blue to red, reflectance peak at

462 nm to 647 nm) in a single piece feather of common bronzewing (Phaps chalcoptera) feathers are caused by 6~7 layers of melanosome with equal spacing in keratin matrix

(Figure 1-4C).28 The slight variation in the thickness and spacing of melanosome layer leads to the color variation from blue up to red across the single feather. The details of this work will be further covered in Chapter II. The male bird of paradise (Parotia lawesii)29 has developed the boomerang-shaped barbules at a respective angle of 30 in the breast-plate plumages (Figure 1-4C). In the barbules, two reflectors making of multilayer melanosomes are crossed at a respective angle of 30 . This geometry allows the feather colors to change dramatically between yellow, blue, and black when the bird moves during their courtship.

2D Photonic crystals

Bird melanosomes mostly are rod-like and they tend to align together along the barbule surface, forming different types of lattice-like structure. On the plane of cross-section of a barbule, various two-dimensional photonic crystal structures have been identified, like the hexagonal and square lattice. The colors are determined by the melanosomes size, spacing, and the packing pattern. In the barbules of some ducks, melanosomes organize

to relatively close-packed hexagonal lattice. A study of six species of dabbling ducks has showed that the two-dimensional hexagonal lattice of melanosomes and a thin keratin cortex both contribute to the iridescent colors (Figure 1-4D).37 The optical modeling suggests that increasing the spacing between melanosomes from close to non-close packing leads to broader and larger reflectance peak, and thus brighter colors. When the diameter of melanosomes is around 60% of melanosome spacing, the reflectance peak intensity reaches the maximum value. Even though some ducks have non-close hexagonal packed melanosomes, but the spacing is still less than the optimal limit. That is probably because relatively close-packed hexagonal lattice is more energetically preferred.

The iridescent colors of peacock feathers have drawn the attention since 1704 when

Newton suggested the possible optical effect similar to thin-film structure caused the iridescence. Until last century (1962), Durrer first found the square lattice of melanosomes in peacock feather using the electron microscope and he simply used a multilayer reflector to explain how the color was produced.38 In 2003, Zi et al. demonstrated the different color in the feather eye pattern was due to square lattice spacing using photonic band gap calculation (Figure 1-4D).31 This unusual non-close square lattice is not energetically favorable and how such structure is produced in the barbules remains elusive.

Melanosomes can in some instances be well-preserved in the fossils. Vinther et al. have recently reported a feather fossil (found in the Messel Pit in Germany) showing vivid structural colors.39 The closed packed melanosomes are believed to produce structural

colors. However, it is not clear what is the exact packing of these melanosomes in these fossils that generates the colors.

1.3.3 Hollow melanosomes

No other animals like birds have developed hollow melanosomes, which is thought to be key ornamental innovations that help to produce higher phenotypic trait variability.11

Examples can be seen in feathers of African starlings (Sturnidae),11,40 magpie (Pica pica sericea),41 red junglefowl, Himalayan monal (Lophophorus impejanus),32 hadeda ibis

(Bostrychia hagedash)42, and wild turkey (Meleagris gallopavo)30. The packing pattern of hollow melanosomes varies from species to species (Figure 1-4E). Fischer's starling

(Spreo fischeri) contains circular, hollow melanosomes randomly scattered within the barbules,40 while magpies have almost closed hexagonal-packed melanosomes. In the

Himalayan monal feather, hollow melanosomes form equally spaced multilayer structures.32 Hummingbirds have close-stacked hollow melanosome plates, which are large filled with many air bubbles.33,43 Their colors have been demonstrated to be caused by the multilayer interference, however, the brightness and sharp iridescence cannot be explained by these current models

The air pockets inside the melanosome increase the refractive index contrast from melanosome/keratin to melanosome/air interface. To illustrate the importance of hollow structure generating colors, Eliason et al have recently studied wild turkey and violet- backed starlings (Cinnyricinclus leucogaster) iridescent feathers to investigated how the hollowness affected the structural colorations.30 They have showed that hollow melanosomes produced larger variations in colors than solid melanosomes in close-

packed hexagonal lattice and increasing hollowness leads to the increase in brightness of the colors.

1.4. Optical benefits of melanins for structural colors

Melanin forms well-defined particle morphology in bird feathers, serving an important self-assembly building block for creating a wide variety of structures for structural colors.

We will discuss what optical benefits melanin/melanosomes have in terms of coloration in this part.

Melanin is believed to have a high refractive index; however the exact value of RI is not well characterized. Conventionally a value of 2.0 for the real part of the RI has been used for lots of optical simulations to predict color productions.31,37 It has always been a challenge to directly measure the RI of melanin due to difficulty in extracting melanins from living organisms and insolubility of melanin in most solvents. A RI value of 1.7-1.8 for melanin has been reported in the visible range and this value has been obtained from fitting several unknown parameters to match theoretical spectra with experimental measurements based on some optical models.44,45 Some direct measurements on natural melanin were based on the multilayer structure (electron-dense and electron-light layers) in insects, assuming the electron-dense material of the multilayer structure to be melanin without any chemical analysis.46-48 Kurtz et al. have reported that isolated melanin free acid from Sepia ink has a RI of 1.66 at the wavelength of 633 nm,49 but it is still unclear about the difference between the modified melanin and natural melanin found in tissues.

Despite of difficulty to characterize the RI of natural melanin, we have directly measured the real part of RI synthetic melanin to be 1.74 at wavelength of 589 nm.50 (See details in

Section 3.2.4). Despite that we still do not know the exact RI value of melanin, melanin has higher RI than keratin (RI of 1.54)51 and probably has a value lower than 2.0. Its high

RI offers enough RI contrast when melanin particles embedded in keratin matrix in bird feathers, which cannot be achieved from common polymers.

Melanin has broad band absorption of light across the whole UV-vis region. This could result in a large absorption of light. Such absorption can help increase the saturation of structural colors. As mentioned in section 1.3.1, having a melanin layer to absorb incoherent scattering light can enhance the color saturation, making the blue color in the blue Steller’s jay feather in comparison to the white amelanotic Steller’s jay feather that does not contain melanin. In mimicking structurally colored materials, light absorbing components have often been doped to increase the color purity. Carbon black is a common absorber that has been added into the quasi-order packing of polystyrene or silica nanoparticles to enhance the colors (Figure 1-5A, C);52-55 gold nanoparticles

(absorbing due to its plasmonic effect) have been used in photonic crystal supraballs

(Figure 1-5D).56 Recently, Yang et al have coated polystyrene nanoparticles with black polypyrrole to increase the purity of color in their quasi-order packed structures (Figure

1-5B).57

High refractive index and broadband absorption in melanin has inspired researchers to create shining structural colors similar to bird feathers using melanin; however, natural melanin is insoluble in most solvents, strongly associated with tissues, and lacking tunability in sizes. It is challenging to extract or isolate natural melanin from animal integuments. In the last decade, efforts have been spent in developing various approaches to synthesize melanin-like nanoparticles (synthetic melanin), which has similar properties as the natural melanin.58 Synthetic melanin can not only offer the optical advantages of natural melanin, also allows a better control in the nanoparticle size, surface chemistry, and packing of these melanin nanoparticles.

1.5 Melanin in synthetic structural colors

Inspired by the use of melanin in structural colorations in bird feathers, both natural and synthetic melanin have been recently used for fabrication of man-made structural colors.

Natural melanin most used is extracted from sepia inks due to the relative easy purification process than other types of melanin isolated from animal integuments.

Synthetic melanin can be made through enzymatic oxidation of tyrosine and L-DOPA (L-

3,4-dihydroxyphenylalanine), or chemical oxidation of dopamine.59 When using tyrosine or L-DOPA as precursors, it is difficult to control the particle formation and dispersion stability in solvents. Synthetic melanin made from dopamine is easy to prepare with great control in size. In addition, polydopamine-melanin nanoparticles are well dispersed in solution without aggregation issues, which is nontrivial for different self-assembly approaches. In a typical synthesis process, dopamine hydrochloride is oxidized and polymerized to black nanoparticles in the NaOH aqueous solution.58,60 Later, it has been reported using ammonium as the base source and a mixture of water and short-chain alcohol as the reaction medium offers a better control to produce monodispersed synthetic melanin nanoparticles.61-63 Synthetic melanin can be either used as a solid thin film or fabricated as nanoparticles that can assemble into different structures to produce colors.

1.5.1 Absorber to increase saturation

Incoherent scattering often impedes the visibility of structural colors, especially for non- iridescent colors from short-range ordered structures. That is the reason a second component is needed to absorb light (Figure 1-5). In the section 1.3, we have discussed the significance of natural melanin to absorb a wide spectrum of visible light. Zhang et al.

have used natural melanin nanoparticle directly isolated from cuttlefish ink into the amorphous polystyrene nanoparticles (Figure 1-6A).64 Adding sepia melanin nanoparticles (diameter, ~110 nm) into mondodispersed polystyrene nanoparticles breaks the crystalline structures and leads to the formation of short-range ordered packing of polystyrene nanoparticles, which is important to create angle-independent colors. Those colors results in much brighter contrast.

1.5.2 Solid thin film

Wu and Hong have reported structurally colored dopamine-melanin thin films which are created by oxidation and self-polymerization of dopamine at surfaces of the Tris buffer solution (Figure 1-6B).65 In the bulk phase, amorphous melanin granules are formed and settle down to the bottom, serving as a light absorbing layer when the water is taken out.

The color is caused by the dopamine-melanin thin film interference and they have controlled the dopamine concentration from 0.01 M to 0.04 M to tune the film thickness from 105 nm to 179 nm to get various colors. The colorful solid melanin thin film can be transferred to substrate, however, the final color depends the substrate. Recently, a mixture of dopamine monomer and isopropylacrylamide has been polymerized together and deposited together onto a large silicon wafer (up to 10 cm) triggered by

66 CuSO4/H2O2. Interestingly, the co-deposited thin films show angle-independent colors and the colors can be tuned either by changing the film thickness or temperature. In this research, it is unclear that if dopamine is polymerized to nanoparticles or forms co- polymers with isopropylacrylamide.

Figure 1-6. Melanin for synthetic structural colors. (A) Sepia melanin is used a light absorber to decrease incoherent scattering.64 Reproduced with permission from the John Wiley & Sons, copyright 2015. (B) Structural colors of dopamine-melanin solid film.65 Reproduced with permission from the American Chemical Society, copyright 2015. (C) Structural colors from the interference of a layer of melanin nanoparticles.50 Reproduced with permission from the American Chemical Society, copyright 2015. (D) Structural colors from scattering of melanin nanoparticles.67 Reproduced with permission from the Royal Society of Chemistry, copyright 2015. (E) Polystyrene core and melanin shell nanoparticles.68 Reproduced with permission from the American Association for the Advancement of Science, copyright, 2016. (F) Photonic supraball inks made of melanin core and silica shell nanoparticles.

1.5.3 Solid melanin nanoparticles

To achieve a better control in the color production, synthetic melanin nanoparticles with tunable sizes need to be synthesized so that the packing patterns of nanoparticle can be controlled to create structural colors. We have recently demonstrated that using a simple evaporation process we assemble synthetic melanin nanoparticles into structurally colored films (Figure 1-6C, see more in Chapter III).50 The colors are iridescent and can be controlled by changing the layer thickness of packed nanoparticles through tuning the

concentration of synthetic melanin nanoparticles solution before evaporation. A thin-film optical model with measured optical parameters shows that the colors are due to thin-film interference. Using melanin nanoparticles can offer higher color purity than using common polymer nanoparticles due to their high RI and broad band absorption of light across the whole visible wavelength. More interestingly, these films can show dynamic color changes within seconds under different humidity conditions (See details in Chapter

IV).69 This color change is faster and larger than that in chameleons. The dynamic color results from reversible swelling/shrinking of melanin nanoparticles during drying and wetting cycles. Use of nanoparticles to make a thin film can offer the air gaps between nanoparticles and thus larger surface areas between melanin and air so that water vapor more easily diffuses into/out the nanoparticles. In addition, the air pockets maintain the stability of these films in repeating swelling/shrinking cycles.

Kohrit et al. have reported that centrifugation of the solution of synthetic melanin nanoparticles (130 nm to 256 nm) results in precipitates that shows deep blue to red non- iridescent structural colors (Figure 1-6D).67 Powders from drying those nanoparticle solutions in silicon rubber mold also show non-iridescent colors. They have contributed this angle-independent colors to the polydispersity which results in disordering the the crystalline packing of nanoparticles. Recently, Cho et al. have demonstrated synthetic melanin nanoparticles aqueous solution show colors under a strong white light.70 The high absorption of synthetic melanin attenuates the multiple scattering and the colors are caused by resonant Mie scattering. The concentration of the solution varies from 0.025 to

0.4 wt% and the average interparticle distance in solution ranges from 1.2 to 1.9 μm.

Within such concentration range, the colors of the solution depend on the size of melanin particle.

1.5.4 Core-shell melanin nanoparticles

Core-shell melanin nanoparticles have recently been used to create structural colors so that decreased light absorption makes the color brighter. One can use melanin either as the core or the shell. We have recently used finite-difference time-domain methods to predict that a conventional FCC lattice made of nanoparticles with melanin core and lower refractive index shell reflects more light than homogenous nanoparticles, while the core-shell nanoparticles with melanin shell and lower refractive index core reflects less light (Details in Figure 5-1B-C).71 Based on those calculations, we have designed silica core-melanin shell nanoparticles and used a simple reverse emulsion process to create non-iridescent supraball inks. Adjusting the melanin layer thickness and core size, we can obtain full-spectrum colors (Figure 1-6F). In addition, we can tune the colors just by simply mixing two sizes of core-shell nanoparticles, similar to color blending by mixing two pigments. The detail of this work will be fully covered in Chapter V.

Kawamura et al. have coated synthetic melanin onto polystyrene nanoparticles of 221 nm to 285 nm and the melanin shell thickness is controlled from 2.5 nm to 22 nm by changing the dopamine monomer concentration from 0.3 to 2.0 mg/mL. If the melanin shell thickness is thin, the pellets from drop casting appears angle-dependent colors; while thick shell produces non-iridescent colors (Figure 1-6E). This is because the rougher surface of core-shell nanoparticles with thick melanin layers disrupted the large

domains of crystalline structure forming in comparison to the smooth surface of core- shell nanoparticle with think shells.

1.5.6 Conclusions

Melanin, as a ubiquitous biomaterial across species in nature, has been extensively investigated to have a variety of physiochemical properties, but its role in structural colors has been understudied. In this chapter, we have briefly discussed structural coloration mechanisms, the structure and functions of melanin; and we have systematically summarized different roles of melanin in structural colors in bird feathers due to its high refractive index, broadband absorption, and diverse morphologies. We have also discussed all recent researches using melanin or melanin-based materials to make bio-inspired synthetic structural colors, including most of my PhD researches. In the following chapters, we will systematically cover what I have contributed to this field.

CHAPTER II

NANOSTRUCTURAL BASIS OF RAINBOW-LIKE IRIDESCENCE IN

COMMON BRONZEWING PHAPS CHALCOPTERA FEATHERS

2.1 Introduction

Natural structural colors, arising from light scattering by tissues that vary periodically in refractive index at the nanometer scale, are ubiquitous in many diverse taxa, including plants,72,73 molluscs,74 fish,75 insects,45,48,76-79 birds,27,29-31,34,37,80 and mammals.81 Avian feathers exhibit varied and complex structural colors for aposematism, sexual display or camouflage.82 The mechanisms producing these colors have thus far been identified as: (1) thin-film/multilayer interference from alternating layers of melanosomes (melanin containing organelles) and keratin in barbules,27,29,34 (2) photonic band gaps within the wavelength of visible light formed by two dimensional packing of melanosomes in a keratin matrix in barbules,30,31,37 and (3) coherent scattering from three dimensional quasi-ordered spongy matrices of keratin and air in barbs.80

In most cases studied thus far, single feathers have only one structural color that may vary depending on the incident angle and viewing angle.83-85 However, the covert feathers of the common bronzewing (Phaps chalcoptera) contain a color gradient from blue to red over the proximo-distal length of individual barbs that is present even at a constant illumination and viewing angle (Figure 2-1). This continuous rainbow-like iridescence is distinct from the four discontinuous colors (blue, green, yellow, brown) distributed in

well-defined regions in male peacock (Pavo muticus) feathers,31 and as far as we are aware has not been described before. Moreover, it suggests a high degree of nanostructural control during feather development that may serve as inspiration for the design of multi-colored fibers or coatings. We therefore investigated the nanostructural basis of this color gradient using optical and electron microscopy, spectrophotometry and optical modeling.

2.2 Materials and Methods

2.2.1 Barbule macrostructure

An iridescent covert feather of a male common bronzewing (Phaps chalcoptera) was obtained from the National Museum of Natural History (Washington, D.C. USA). We used a Leica S8 APO stereomicroscope (Leica Microsystems GmbH, Wetzlar, Germany) to examine the color variation of barbules along the distal-proximal axis of individual barbs (Figure 2-1)

Figure 2-1. (a) Stereo light microscope image. Panels (b-d) show higher magnification images of barbules in distal region, middle region, and proximal regions, respectively. Scale bars, (a) 2 mm and (b-d) 200 μm.

2.2.2 Reflectance measurement 25

Angle-resolved specular reflectance

We visually observed slight change in hue with the observation angle in the feather

(suggesting interference effects), so we quantified its iridescent properties using an

AvaSpec spectrometer with a xenon light source (Avantes Inc., Broomfield, USA, beam size ~ 5 mm) attached to a custom-built goniometer. Before any spectral measurements were collected, two calibration steps were done to maximize reflectance and thereby ensure all measurements were done using the same protocol.86 First, we made the incident beam ( ) and detector angle ( ) equal by mounting the feather onto the stage and rotating the stage until the reflectance intensity reached a maximum. The rotation angle was , shown in Figure 2-2. Second, we tilted the stage on which the feather sat to the angle where reflectance was maximized. Through these two adjustments, the plane of the color-producing nanostructures in the barbule was made perpendicular to the bisector line of the angle between incidence and detection directions. To quantify the effect of incident angle, we measured the specular reflectance at incident angles varying from 10 to 45 by 5 increments. Because the red and blue regions were smaller than the beam size (5 mm), we only performed these measurements on the green portions (Figure 2-1a).

Figure 2-2. Schematic for the geometry of spectrometer for specular reflectance measurement. is the angle between the incident beam and the surface normal; similarly, is the angle between the detector and the surface normal; and is the angle between feather rachis (bold black line with an arrow) and the plane-of-incidence (yellow plane). The values of and can be read directly from the spectrometer. The arrow of the feather stands for the distal end of the feather. is the angle between the stage plane on which the feather lies and the horizontal plane.

Single barbule normal reflectance

To precisely quantify the color of differently colored sections of the feather, we used a

CRAIC AX10 UV-Visible-NIR microspectrophotometer (MSP) (CRAIC Technologies,

Inc, San Dimas, USA, beam size ~ 4 , range 300-800 nm) to measure (once for each) the normal reflectance of single barbules across the different color regions of the feather.

We then measured the reflectance of 10 green barbules in the same barb that was then examined with transmission electron microscopy. We then performed three measurements on the normal reflectance at p- and s-polarized illumination for barbules with different colors.

2.3 Barbule nanostructure

To investigate the nanostructures of barbules, we prepared ultra-thin cross sections following the procedure described by Shawkey et al.87 We separated one barb containing all colors from blue to red into three different color regions (blue, green and red) with the help of an optical microscope. We again cut each color region into two parts to increase the number of pieces for sampling. We then dehydrated barbs and barbules using 100% ethanol for 20 minutes twice and sequentially infiltrated them with 15%, 50%, 70% and

100% Embed 812 resin. Each infiltration step was performed on a Thermolyne Vari-Mix rocker (Thermo Scientific, Waltham, USA) for about 24 hours. Next, we placed embedded medium and samples into block molds and cured them at 60 overnight. We trimmed the blocks with a Leica S6 EM-Trim 2 (Leica Microsystems GmbH, Wetzlar,

Germany) and cut 80 nm thick ultrathin sections with an Ultra 45 diamond knife

(Diatome Ltd, Biel, Switzerland) on a Leica UC-6 ultramicrotome (Leica Microsystems

GmbH, Wetzlar, Germany). Sections were transferred onto the copper grids that were viewed under a JEM-1230 transmission electron microscope (JEOL Ltd, Tokyo, Japan).

From the obtained TEM images, we measured the following parameters for the three differently colored regions 100 times via the software Image J (http://fiji.sc/Fiji): 1) diameters of melanosomes, ; 2) spacing (edge-to-edge distance) between melanosome layers, ; and 3) spacing (center-to-center distance) between adjacent melanosomes in the same layer, . Because the end-to-end spacing (35 15 nm) between melanosome rods in each melanosome layer is small compared to the length of melanosome rods (870 112 nm), we can assume infinite melanosome cylinders are evenly spaced (spacing = ) in

each melanosome layer and the volume fraction of melanosomes in each layer can be

2 calculated as Vddmelmm d( m d/ 2) // d 400.

2.3 Results and Discussion

2.3.1 Rainbow-like iridescent reflectance

The iridescent color of the bronzewing is limited to the exposed side of the feathers, as illustrated in Figure 2-1a. The color varies from red to blue along the distal-proximal gradient. These color differences are shown more clearly in magnified images for barbules in distal, middle and proximal regions (Figure 2-1b-d). The barbules are oriented uniformly. Angle-resolved specular reflectance of the green region of the feather is shown in Figure 2-3, with the incident angle ranging from 10 to 45 by 5 increments.

As the incident angle increased, the reflectance was blue-shifted (from 531nm to 492 nm) and the intensity decreased. The range of hues (we define hue as the wavelength of peak reflectance) across the whole feather, as measured by microspectrophotometer (MSP), is

185 nm (462nm to 647 nm, Figure 2-4d).

 20 10 15 20 25 15 30 35 40 10 45

Reflectance / % Reflectance /

0 400 450 500 550 600 650 700 750

Wavelength / nm

Figure 2-3. Specular reflectance spectra for the green region of barbules at incident angles from 10 to 45 .

2.3.2 Nanostructure of iridescent barbules

We used TEM to examine the nanostructure of individual barbules. Barbule cross- sections revealed 6-7 layers of melanosomes arranged parallel to the periphery of the barbule cell membrane (Figure 2-5a). This multilayer structure is found in all iridescent barbules, but not in the non-iridescent brown barbules at the very tip of the same feather

(Figure 2-1b and Figure 2-5c). Longitudinal sections (Figure 2-5b) reveal that these melanosomes are cylindrical. Multilayer structures in red, green and blue barbules are similar (Figure 2-6) and the size and spacing of melanosomes are slightly different. The mean diameter of melanosomes ( in distal barbules is around 10 nm larger than that in middle and proximal barbules, which are similar to one another (Table 2-1). The melanosome layer spacing ( , the keratin layer thickness) is very similar in distal and middle barbules, but is about 10 nm smaller in proximal barbules. The spacing between neighboring melanosomes in the same layer ( ) is nearly identical, resulting in little variation in volume fraction (packing density, of melanosomes in the keratin matrix for each melanosome layer (Table 2-1).

Figure 2-5. TEM images of (a) cross section of a red barbule, (b) longitudinal section of a green barbule, and (c) cross section of a non-iridescent brown barbule. Scale bars, 500 nm.

Figure 2-6. TEM images of cross-sections of barbules with different colors under the same magnification: (a) red, (b) green, and (c) blue. Scale bars, 100 nm

Table 2-1. Spacing and diameter of melanosomes in the barbule nanostructure measured using TEM results (Errors are standard deviation from 100 measurements)

Melanosome Melanosome 2 Volume 1 Inner-layer spacing of 3 Group Diameter (풅풎) layer spacing fraction melanosomes (풅ퟎ) /nm /nm (풂) /nm (푽풎풆풍 Distal/Red 92.8 6.4 86.1 14.2 112.7 10.0 0.65 0.12 Middle/Green 83.9 7.4 87.2 15.2 104.7 10.6 0.63 0.12 Proximal/Blue 82.0 6.8 77.4 14.0 101.8 10.2 0.63 0.13 1 distance between edges of neighboring melanosome layers, which is also the thickness of keratin layer

2 distance between centers of neighboring melanosomes in the same layer

3 volume fraction of melanosomes in each melanosome layer

2.3.3 Multilayer interference modeling

We used a standard multilayer interference model34,89 to calculate the theoretical reflectance for barbules of different colors. In this planar multilayer system (Figure 2-7), the top layer is the thin layer of keratin with thickness 12 5 nm. Below it are layers of alternating melanosomes and keratin. Thickness for all melanosome layers and keratin layers in individual barbules is consistent (Table 2-1), therefore we assume that the

thickness of melanosome layer and keratin layer are constant. Complex refractive index

(RI) is described as ̃ , where the real component is related to the phase velocity and the imaginary part indicates the absorption of the medium. The refractive index is supposed to dependent on wavelength. The real component ( ) for keratin RI based on by Leertouwer et al., 90 is

2 nker 1.532 5890 /  (1.1)

Here is wavelength of light in the unit of nm. Keratin is almost transparent and has

91 negligible absorption, thus we assume 0.

The RI for melanin is less well characterized due to its strong absorption.92 In previous

93 studies, was assumed to be 2, and some modeling results have in some cases confirmed this value34,37 while others suggested that it is lower than 2.45,46 Taking dispersion effect of RI into consideration, we refer to the empirically measured values for a dense melanin-like substance. 46

Real part:

2 nmel 1.56 36000 /  (1.2)

Imaginary part:

  142 kemel 1.62 (1.3)

In Equations (1.2) and (1.3), is wavelength of light in nm.

Because the melanosome layer is a mixture of melanosomes and keratin (Figure 2-5a and

Table 2-1), the RI for this layer can be corrected by averaging the refractive index of two components based on various effective medium theories.94 In this case, the volume averaging theory, applicable for absorbing materials is used to calculate the average RI of melanosome layer,95

nnimel, avg effeff  (1.4)

22 2 nAeff  B[A]/  2 (1.5)

22 2 eff [  A]/  2AB (1.6)

The constants A and B can be determined using the following equations:

2 22 2 A Vmel() nV mel  (1  n melmel )() ker ker (1.7)

B22(1 Vmel nV mel melmel ) n ker ker (1.8)

In Equations (1.7-1.8), is the average melanosome volume fraction in each melanosome layer (Table 2-1).

Figure 2-8. (a) Measured (green line) and modeled spectra (black line) on the average layer thickness for barbules in green region. (b-c) The modeled bluest and reddest spectra (black lines) based on largest and smallest thickness of melanosome and keratin layers in blue and red barbules; and the blue and red curves are the bluest and reddest spectra measured by MSP, respectively.

The mean values for melanosome layer thickness, keratin layer thickness, and melanosome inner-layer volume fraction (Table 2-1) for green barbules were used in modeling multilayer interference. The model result was compared with average measured spectra of green barbules on the same barb for TEM imaging. The measured peak intensity is a relative parameter which is dependent on the white reference, so we standardize all the intensities in measured and modeled spectra to 1, as has been done in other literature.30 Figure 2-8a shows that the modeled peak wavelength, peak width and peak shape match closely with those in the experimental curve. Since there is still some color variation in the same color region (e.g. 620~650 nm in red region), the average dimensional values from TEM images include both (e.g. red) colors with shorter wavelength and longer wavelength. To further test the contribution of multilayer structure to the color gradient in the feather, we obtained the theoretical color range (hues for bluest and reddest barbules) by modeling the color resulting from the smallest layer thickness for blue barbules (75.2 nm for melanosome layer and 63.4 nm for keratin layer) and the largest layer thickness for red barbules (99.2 nm for melanosome layer and 100.3 nm for keratin layer). The predicted hues are 477 nm and 638 nm for the bluest and reddest barbules, respectively, which agree quite well with the color range (462-647 nm) experimentally identified by MSP (Figure 2-8b-c)

600 modeled curve measured green 550

500

450

0 10 20 30 40 50

Maximum Peak Wavelength / nm Angles / degrees

Figure 2-9. Modeled results for angle-resolved reflectance spectrum for middle barbules (green solid line). The black square data points are obtained from experimental spectra in Figure 2-3.

700 Modeled Green Range 650 Measured Green

600

550

500

450

400 10 20 30 40 50

Maximum Peak Wavelength / nm Angle /Degrees

Figure 2-10. The modeled color range for barbules in the blue zone and the experimentally measured green.

The modeling calculations for reflectance as a function of angle predict that hue should decrease with increasing incident angle, as is observed in our empirical results (Figure

2-9). Taking into consideration the standard deviation of thickness of melanin and keratin layers in green barbules, we obtain the color range where the measured points for green barbules fall (Figure 2-10). Additionally, s- and p-polarized specular reflectance spectra at normal incident illumination almost overlap each other and have an identical wavelength of peak reflectance with unpolarized incidence light for all colored barbules and we only show polarized spectra for the green barbule (Figure 2-11). These results are consistent with TEM images (Figure 2-5) where we have observed the periodicity perpendicular to the thickness direction and not within the melanosome layers (parallel to the thickness direction). This is also consistent with polarization dependent iridescence in

negative controls with two dimensional photonic crystal structure in green-winged teal

(Anas. carolinensis)37 and peacock (Pavo cristatus ).31

Based on this model, we explored how variation in the nanostructure could potentially affect color. First, we found that small changes ( 12 nm) in the thickness of keratin cortex (outmost layer) result in only 7 nm variation in the value of reflectance maximum (Figure 2-12). Thus, the outer cortex plays a relatively small role in the color production of the common bronzewing. Experimentally, it was difficult to precisely measure the thickness of outmost layer because of the possible shrinkage of the barbule boundaries during embedding. The result of our model, however, demonstrates that a

precise value for the thickness of the keratin cortex layer is not critical to accurate modeling.

Figure 2-12. The hue dependence on the outmost keratin layer (cortex layer) thickness based on multilayer modeling result.

By contrast, the number of melanosome/keratin layers can strongly affect reflected color.

After six layers, the reflectance hue changes little with increasing number of layers. The full width at half maximum of reflectance peak decreases sharply and its decrement rate

(negative value) with respect to increasing layers plateaus off at around 12 layers (Figure

2-13). It is well know that the peak reflectance intensity increases with the number of layers for a multilayer system,83 but the increment of intensity for bronzewing feathers declines after 5 layers. The fact that increment rate of intensity is less than 1% after 12 layers indicates that there is diminishing reward for increasing the number of layers beyond 12. Therefore a sharp peak with enough reflectance intensity can be produced

with about 12 layers of melanin and keratin (~ 6 melanosome layers), the number most frequently found in rainbow-like iridescent bronzewing feathers.

1.5 decrement rate of FWHM increment rate of intensity 0

1.0 -10

-20 0.5 -30

-40 0.0

Increment Rate of Intensity / % Intensity / Increment Rate of

Decrement Rate of FWHM / nm FWHM / Decrement Rate of 5 10 15 20 25 30 Number of Layers

Figure 2-13. Green solid line is the decrement rate of full width at half maximum (FWHM) , which is the derivative of FWHM with respect to layer number and blue dash line is the increment rate of intensity which is the derivative of intensity with respect to layer number.

2.4. Conclusions

We report here for the first time a continuous color span ranging from blue to red in a single feather. We explain by optical modeling and TEM results that this large span (462-

647 nm) is caused by subtle shifts in both spacing and diameter of melanosomes in a multilayer structure. Although spacing differences in multilayers are common, subtle variation in melanosome size has never before been reported. This is particularly intriguing because it necessitates the existence of a mechanism in the developing feather

cells to precisely control (within a few nanometers) the size of the melanosomes. The diameters of melanosomes increase by 10 nm from blue (proximal) to red (distal) barbules and we hypothesize that this spatial size control is regulated by decreasing size of melanosomes as feather development proceeds, since the distal barbules grow earlier than proximal ones.96 How such fine control over size and spacing along the barb is achieved is an intriguing question that has barely been addressed. Maia et al.97 proposed that depletion attraction of melanosomes drives arrangement of melanosomes to aggregate into a single one-layer film beneath the barbule surface during keratinization.

However, the more complex and precise structuring observed here suggests additional forces may also be important in self-assembly of melanosomes. Whether these forces are subject to perturbation by external stressors, for example, will suggest whether they may serve as honest indicators of quality, as has been suggested for other iridescent feathers.97

Such multilayers of alternating high and low refractive index layers have also been utilized by many others animals, ranging from nacres of mollusks,74 fish scales,75 beetle elytra,48 butterfly wing scales98 to damselfly wing veins.45,77 However, use of multilayer solid melanosome rods in a keratin matrix for color production has only been reported in the bird of paradise (Parotia lawesii)29,99 since seminal early studies.38,100 Therefore, this paper may provide new inspiration for the design of multi-colored coatings or fibers from multilayer of high refractive index nanoparticles in a low refractive index matrix, which may be used for antireflection or spectral filtering. For example, bio-inspired tunable colored fibers have been made via multilayer rolling,101 but these are only a single color. Our results suggest that color gradients can be produced using spaced layers of high refractive index particles (e.g. melanosome particles) whose size can be tuned to 43

control the thickness of these layers and whose volume fraction can be manipulated to change the refractive index of these layers, providing a novel strategy for the design of synthetic multilayer structures with optical properties.

CHAPTER III

BIO-INSPIRED STRUCTURAL COLORS PRODUCED VIA SELF-ASSEMBLY

OF SYNTHETIC MELANIN NANOPARTICLES

3.1 Introduction

Structural colors, remarkable for their color tunability and resistance to chemical and photo bleaching, have broad applicability in colorimetric sensors,102-104 full color displays,105 and photonic pigments.106 Avian feathers likely possess the highest diversity of structural colors found in nature, with spectral features arising from arrays of melanosomes (submicron sized melanin-containing organelles in spherical, rod-like, or disk-like shapes with solid or hollow morphologies),11,107 such as in the multilayer structures present in the feathers of birds of paradise (Parotia lawesii) and in common bronzewings (Phaps chalcoptera).28,99 Other structural color morphologies, including two-dimensional photonic crystal structures are found in species such as peacocks (Pavo muticus) and mallards (Anas platyrhynchos).31,37 The vast array of colors and the ubiquitous use of this approach to coloration and patterning make mimicry of such assemblies highly desirable for synthetic materials. This presents a challenge in nanoscale synthesis of well-defined particles, their self-assembly to generate films, and ultimately in understanding of the underlying principles governing the observed effects.

Melanins, produced in melanosomes, are ubiquitous pigments found in bacteria, fungi, plants, extant animals,5 and in prehistoric organisms including dinosaurs.3,4,108,109 They

are classified as black/brown eumelanins and yellow/reddish pheomelanins based on their precursors.110 Eumelanins are more intensively studied and have intriguing physicochemical properties, including a monotonic broadband UV-vis absorption, an intrinsic radical center, and electrical and photoconductive properties.111 Melanins in animal integuments (feathers, hair or skin) are thought to absorb UV radiation to protect living organisms.110 However, no organism other than birds uses melanosomes to form organized structures for producing colors.84 Although the physical principles behind structural colors have inspired numerous efforts to generate photonic crystals,112,113 few have tried to take advantage of melanins or melanin-like material to mimic structural colors.65,67

Polydopamine (PDA), the most common type of synthetic melanins, has been used extensively in fields as diverse as biology, energy science, sensor development and environmental science,59 since its first use as a multifunctional coating was inspired by the foot protein in mussels.114 Although the exact polymerization mechanism for PDA has not yet been clearly elucidated, recent advances have revealed that the physicochemical properties of PDA-based synthetic melanins are generally similar to those of natural melanins,59,111 giving them promise as photo-protectors,15 antioxidants,58 semiconductors,21 and biomedical materials.115

Recently, Kohri et al.67 used different sizes of PDA particles to spray coat films of different colors. In this case, colors were mostly generated due to scattering phenomena.

Wu et al.65 fabricated colored reflectors by placing a thin PDA film on top of a thick layer of amorphous PDA particles that served as a strong absorbing layer. However,

despite their prevalence in the natural world, and obvious advantages in developing diversity of colors produced by birds in particular, direct bio-inspired coloration from ordered structures of PDA particles has not yet been reported. In addition, what advantages PDA particles may offer relative to common polymeric particles in terms of structural coloration has not yet been elucidated. Here, inspired by structural coloration arising from assembled melanosomes in avian feathers, we have prepared and assembled synthetic melanin nanoparticles (SMNPs) to construct structurally colored films using an evaporative process. We have measured the complex refractive index (RI) of SMNPs and have established a thin-film interference model that explains the origin of observed colors.

The SMNPs and their self-assembly into films provide an approach for mimicking the vibrant colors found in avian feathers with a wide range of potential applications in the design of optical devices, functional coatings, and biocompatible products.

3.2 Materials and Methods

3.2.1 Synthesis and characterization of SMNPs

The SMNPs were synthesized through the oxidation and self-polymerization of dopamine molecules in a solution consisting of water, ethanol and ammonia at room temperature, which is modified from the previous literature.116 Although many parameters could affect the final size of the SMNPs, we optimized the required size by tuning the molar ratio of ammonia and dopamine hydrochloride. Typically, to synthesize SMNPs with average diameter of ~146 nm, 50 mL deionized water and 20 mL ethanol were fully mixed with

1.2 mL ammonia aqueous solution (28-30%) under stirring at room temperature for about

1 hour. A 5 mL dopamine hydrochloride aqueous solution (4 mg/mL) was quickly

injected into this solution. It was observed that the solution color turned to pale yellow immediately and then gradually changed to black after 1 hour. After 18 hours, the targeted SMNPs were separated by centrifugation and washing with deionized water for three times. All chemicals are purchased from Sigma-Aldrich. We characterized the size of SMNPs in solution by DLS measurements using a BI-HV Brookhaven instrument with a 633 nm solid-state laser (Brookhaven Instruments Corp.). DLS data were analyzed using CONTIN software to determine the hydrodynamic diameter. The size of the nanoparticles was also confirmed using a JEM-1230 transmission electron microscope

(TEM) (JEOL Ltd.) after drying.

3.2.2 Evaporation-based assembly

Silicon wafers (Silicon Inc.) were cut into 11 cm2 and ultrasonicated in 2 wt.% sodium dodecyl sulfate (Sigma) solution for 30 minutes, followed by washing with deionized water for 30 minutes, and a final rinse using acetone for 30 minutes. After drying, the silicon substrates were subjected to a 5 minute air plasma treatment (Harrick Plasma,

PDC-32G). 1 mL of SMNPs in water of a known concentration were ultrasonicated for

15 minutes and placed into a plastic cuvette (Brandtech). A clean silicon wafer was held vertically in the solution at 60 C until all the water evaporated. A separate cuvette filled with water was used to monitor the evaporation rate (expressed as change in height (mm) per unit hour). We also measured the thickness of the top thin silica layer of bare silicon wafer using a multiple wavelength mode ellipsometer (J.A. Woollam Corp.) before evaporative deposition of SMNPs.

3.2.3 Characterization of the SMNP film

Optical images of deposited films were taken using an Olympus BX51microscope

(Olympus Corp.). CRAIC AX10 UV-Visible-NIR microspectrophotometer (MSP)

(CRAIC Technologies, Inc., a 15X objective, range 400-800 nm) was used to measure the normal reflectance spectra of deposited SMNPs films at various locations that showed uniform colors. We normalized the reflectance with respect to a white standard of high density Teflon tape (TaegaTech).

To characterize the purity of the color and the spectral homogeneity of the film at a fine spatial scale, we performed measurements using a PARISS® hyperspectral imaging system (LightForm, Inc.) mounted on a Nikon 80i microscope outfitted with a monochrome Retiga 2000DC CCD camera (QImaging). The system was radiometrically calibrated with accuracy better than 2 nm. The film was analyzed under specular reflectance (with a tungsten halogen white light, neutrally color balanced using a Nikon

NCB11 filter) using a 100x air objective, capturing spectra (400-800nm) from all digital pixels of an area of 0.050.1 mm2. The reflectance spectra were then normalized with respect to a standard silver mirror (ThorLabs Inc.) and smoothed with a moving average of 3 as recommended by the vendor. For relative comparison of spectral intensity, all acquisition parameters were kept the same from one mapped area to the other. All spectra from one individual area showing >99% closeness of fit were identified by one single representative spectrum. For data presentation (Figure 3-5), we show the mapping of the different spectra using artificial color coding. We display the normalized spectra corresponding to the color coded map and show the ratio of the number of pixels of the mapped area that is associated with each spectrum.

For electron microscopy, the top-view and cross sectional structures were obtained using

SEM (JEOL-7401, JEOL Ltd.) after silver sputter coating using a K575X turbo sputter coater (Emitech). To measure the thickness of the deposited film, we cut the SMNPs deposited wafer into half and vertically aligned the cut edge for SEM imaging. We used

ImageJ (http://imagej.nih.gov/ij/) to measure the thickness of the melanin film from the

SEM images.

3.2.4 Measurements of the complex RI of SMNPs

The complex RI of SMNPs can be expressed as nnicompmm .We determined the real

part nm , and the imaginary partm , separately. We prepared solutions of SMNPs with concentrations of 0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, and 2.0 mg/mL. The real part of

the RI of the solution ( neff ) was measured using a digital refractometer J157 (Rudolph

Research Analytical, light wavelength 589 nm), which was first calibrated using ultra-

pure water ( nw =1.33251) and a standard solution ( =1.41796). Each sample was measured four times, and we took an average value of 30 scans for each time step. Both calibration and measurements were performed at 25 C . Several effective medium theories can be used to calculate the average refractive index of composite materials, which are based on aeraging dielectric constant ( n2 ).117 Here, we used the Drude model94 and the effective RI of the solution is given by the following equation:

222 nVeffm nV mm n w  (1 ) (3.1)

Where Vm and nm are the volume fraction and real part of the RI of SMNPs,

respectively; and nw is the real part of the RI of water. We converted mass concentration into the volume fraction using the reported density of melanin, 1.3 g cm-3.118,119

To measure m of SMNPs, we prepared solutions of concentrations of 10 mg/L, 25 mg/L, and 50 mg/L. We used such low concentrations because the Beer-Lambert equation is only valid for dilute solution. The UV-vis transmission spectra were obtained using a

Cary 100 Bio UV-visible spectrophotometer (Agilent Technologies). Based on the Beer-

Lambert law, the transmission is related to the extinction coefficient, u() and optical length, :

Te()  ud() (3.2)

The electromagnetic wave theory gives the relation between the imaginary part of the RI,

eff and extinction coefficient

u()  (3.3) eff 4

Combining Equations (3.2) and (3.3), we can calculate the imaginary part of the RI of melanin solution using the following equation:

lnT ( )   (3.4) eff 4d

Because the absorption of water is negligible in wavelength range from 400 nm to 800 nm 120, the measured absorption of SMNPs solution results solely from the SMNPs themselves. The imaginary part of the RI of SMNPs was determined using the equation

meffm(  )/ V (3.5)

where Vm is the volume fraction of the SMNP solution.

3.2.5 Optical Modeling

A matrix method89,121 was used to calculate reflectivity data for a four-layer thin film model, which consists of a layer of air, a randomly packed film of SMNPs, a silicon oxide layer, and a thick silicon substrate. The thickness of SMNPs film was measured by analyzing cross sectional SEM images (Figure 3-6a-b). The silicon oxide layer was 119 

3nm thick measured using ellipsometry before depositing the melanin films, which was consistent with the value of 121  6 nm measured using the SEM cross-section images.

The RI values of silicon oxide and silicon are 1.458 and 3.973, respectively.122,123

3.3 Results and Discussion

Inspired by the dimension of natural melanosomes in structurally colored feathers (e.g. the diameter of rod-like shaped melanosomes is 120-170 nm for ducks37 and 140-180 nm for peacocks31), we have fabricated SMNPs with an average diameter of 146  15 nm as measured via transmission electron microscopy (TEM) (Figure 3-1a). Dynamic light

scattering (DLS) measurements are consistent with TEM data, revealing that SMNPs in solution have a narrow distribution with an average diameter of 184 nm. Measurements

2 of effective RI of SMNPs aqueous solutions ( neff ) as a function of volume fractions of

SMNPs were performed showing a near perfect linear relationship (Figure 3-1b), which matches the Drude model (Equation 3.1),94. The real part of the RI of SMNPs was calculated to be 1.741  0.001 at 589 nm (Equation 3.1). These calculations reveal the value of RI for synthetic melanin is appreciably higher than most synthetic polymers

(~1.4-1.6 at 589 nm).51 The imaginary component of RI was calculated (Equation 3.5) using the transmittance (or absorption) data for the SMNP solution (Figure 3-1c).

0.6 (a) (b) 1.7775

0.4 1.7770

)



1.7765



0.2 neff^2 1.7760

0.0 1.7755 10 100 1000 0.00 0.05 0.10 0.15 R (nm) h Volume fraction (%) 100 0.40 (c) 50 mg L-1 (d) 50 mg L-1 80 25 mg L-1 0.35 25 mg L-1 10 mg L-1 10 mg L-1 0.30 average 60 0.25

40 0.20

20 Imaginary RI 0.15 Transmission (%) 0.10 0 0.05 400 500 600 700 800 400 500 600 700 800 Wavelength (nm) Wavelength (nm)

effective RI of SMNP solution changes against the volume fraction. The slope of the 22 linear fitting is nnmw . The coefficient of determination for the linear fitting is 0.998. (c) Transmission spectra for SMNPs solutions at different concentrations (blue, 10 mg/L; red, 25 mg/L; black, 50 mg/L). (d) Imaginary part of the RI as function of the wavelength. Different colored curves are for each concentration (blue, 10 mg/L; red, 25 mg/L; black, 50 mg/L) and green curve is the averaged value of all three concentrations.

As expected from theory, we obtained similar values for the imaginary part of the RI of

SMNPs for all the three concentrations (10 mg/L, 25 mg/L, and 50 mg/L), with a small discrepancy for the values below 400 nm, probably due to the measurement errors at short wavelength from low transmittance. Previous reports describe experimental and modeling data suggesting a range of 1.65 to 2.0 for RI of natural melanins, although much debate remains concerning the exact value.31,37,45 Certainly, our measurements show that both real and imaginary parts of RI for SMNPs are similar to those reported for natural melanins45 with the broad absorption of light by SMNPs in the UV-visible region being comparable to that of natural melanin.110

(a) (b) (c) (d)

Figure 3-2. Optical images of films deposited at 60C with an evaporation rate of 0.50- 0.55 mm/h and different concentrations: (a) 0.17 mg/mL, (b) 0.6 mg/mL (c) 1.0 mg/mL, and (d) 3 mg/mL. Scale bars: 0.5 mm.

We prepared colorful SMNP films using a vertical evaporation-based self-assembly approach.124 Subsequently, a wide range of colors (red, orange, yellow, and green) was obtained by evaporating 0.6 mg/mL and 1.0 mg mL-1 solutions of SMNPs at an evaporation rate of 0.50-0.55 mm/h (Figure 3-3a). The concentration of the solution was

important for controlling the uniformity of the films with separate strips of colors running perpendicular to the evaporation front formed at low concentration, and grey or black films formed at high concentrations (Figure 3-2). An inherent feature of this approach to the assembly of ordered films is that the concentration changes during evaporation, making it difficult to obtain one uniform color across the entire 1 1 cm2 area of the sample. However, the uniformity over 1 mm was sufficient for both optical characterization and cross-sectional scanning electron microscopy (SEM) analyses.

(a)

(b) red-fit (c) green-fit

red-measured green-measured

400 500 600 700 800

Normalized reflectance (A.U.) 400 500 600 700 800 Normalized reflectance (A.U.) Wavelength (nm) Wavelength (nm)

Figure 3-3. Optical characterizations of SMNP films. (a) Optical images of colored films. The red and orange colors are from different regions of the film in Figure 3-2b; and the yellow and green colors are from different locations of the film in Figure 3-2c. Scale bars: 100 μm. (b) Measured (red curve) and modeled (black curve) reflectance spectra of red film in (a). (c) Measured (green curve) and modeled (black curve) reflectance spectra of green film in (a).

The reflectance spectra for red and green films show two peaks in the visible region with primary peak positions at 675 nm and 550 nm corresponding to the red and green color,

respectively (Figure 3-3b-c). As a control, the reflectance spectra for blank silicon wafers show no discernable peak and very high reflectance at short wavelengths, demonstrating that the colors observed for SMNPs films are not caused by the silicon substrate (Figure

3-4). Furthermore, hyperspectral images (Figure 3-5) show that the green color has a purity of 84%, with red color purity as high as 95% for a scanning area of 0.050.1 mm2.

The reflectance spectra measured using hyperspectral imaging are very similar to those obtained using the microspectrophotometer, complementarily confirming the uniformity of the films in terms of colors.

Normalized reflectance (A.U.) 400 500 600 700 800

Wavelength (nm)

Figure 3-4. Specular reflectance of the silicon wafer measured using the microspectrophotometer.

(a) 95% (b) 84% 5% 16%

0.05 0.1mm2 0.05 0.1mm2

Relative intensity (A.U.) Relative intensity (A.U.) 400 500 600 700 800 400 500 600 700 800 Wavelength (nm) Wavelength (nm)

SEM cross sectional images of regions of the films associated with green and red colors revealed thicknesses measured to be approximately 338  9 nm and 444  15 nm respectively (Figure 3-6). Top view SEM images of the films showed close-packed

SMNPs lacking long-range crystalline order; an observation confirmed by analysis of the two-dimensional Fourier power spectra (Figure 3-6c-d). In each case the films have small and evenly distributed cracks, due to drying and shrinkage. These cracks do not affect the color measurements because the measured spot size (10  10 μm2 for microspectrophotometer measurements, 0.05  0.1 mm2 for hyperspectral imaging) is on a longer length-scale than crack widths. We further modeled the reflectivity data using a four- layer thin-film interference model (air plus SMNPs film plus silicon oxide plus silicon.

For details, see Experimental Section). We used an interactive method on the packing density of SMNPs in the film to obtain the best match with measured reflectance spectra, with the best fitting packing density of 54 % for red film and 56 % for green film (Figure

3-3b-c). The obtained optimal packing density is reasonable, and lower than the theoretically maximum random closed packing density of 63.4%.125 Small discrepancies at shorter wavelengths may be due to the dispersion of the real part of the RI of SMNPs in the visible range. The optical model of red and green colors (longest and shortest wavelengths we achieved) shows that the variation of colors is primarily due to differences in the thickness of the SMNPs layers. The color generated here is not directly related to the particle size. The particle size influences the packing density, indirectly affecting the refractive index and the color produced by thin-film interference, but this effect is beyond the scope of this study.

Although self-assembly of colloidal nanoparticles to generate color has been well documented,126,127 most established systems use polymeric particles such as polystyrene

(PS) and poly(methyl methacrylate) (PMMA), which usually display negligible absorption of light in the visible range. However, the broad absorption spectrum of

SMNPs is important for generating more saturated colors. In recent studies it has been 58

shown that adding carbon black nanoparticles reduces incoherent scattering and enhances color saturation.54,55 We simulated the reflectance spectra (Figure 3-7) for films consisting of PS nanoparticles with similar packing density and thickness as the red and green films, as showed in Figure 3-3a. We used the following dispersion equation for real part of the RI of PS.128

3108 3.478 108 n 1.573 (3.6) 24

In Equation (3.6),  is the wavelength (nm). The imaginary part of the RI of PS is ~ 0, due to negligible absorption between 400 to 800 nm.128 Both spectra for SMNPs and PS particles have two peaks in the visible range. Interestingly, the two peaks for PS films have similar intensities (peak area ratio = 0.64~0.70), while the peak at shorter wavelength is much attenuated, reaching only 11% of the area of the peak at longer wavelength for the red SMNP film and 22% for the green SMNP film.

(a) (b) PS PS

SMNPs SMNPs

Figure 3-7. Modeled reflectance spectra for films of PS nanoparticle and SMNPs. The colors of the curves are colored with RGB standards using “rgb2spec” in Pavo package of

R.88 (a) The thickness of nanoparticle layer is 338 nm, peak area ratio between the shorter wavelength peak (420 nm) and the longer wavelength peak (620 nm) is 0.70 for PS; while peak area ratio between the shorter wavelength peak (440 nm) and the longer wavelength peak (680 nm) is 0.11 for SMNPs; (b) The thickness of nanoparticle layer is 444 nm, peak area ratio between the shorter wavelength peak (390 nm) and the longer wavelength peak (510 nm) is 0.64 for PS; while peak area ratio between the shorter wavelength peak (405 nm) and the longer wavelength peak (550 nm) is 0.22 for SMNPs.

SMNPs have unique absorption patterns (high absorption at short wavelengths and low absorption at long wavelengths) that enhance color purity and provide UV-protection.

Additionally, RI of SMNPs is much higher than most polymers, providing a relatively high RI contrast necessary for the design of highly sensitive colorimetric sensor.129

Relative to some reported metal oxide nanoparticles with even higher RI used to create structural colors,130 bio-inspired SMNPs potentially are less toxic, more biodegradable, and are inherently biocompatible.116 Moreover, these biomimetic structural colors can be directly obtained via assembly of SMNPs in aqueous solution, potentially offering a route towards a biocompatible structural color palette.

3.4. Conclusions

We have demonstrated a biomimetic approach for generating structural colors via evaporation- induced self-assembly of well-defined SMNPs. We have shown that SMNPs possess a high RI and broad absorption spanning the UV-visible range, similar to natural melanin, providing the necessary contrast for structural colors. Colors ranging from green to red were produced by evaporation-based self-assembly of these nanoparticles.

Controlling the thickness of the assembled nanoparticles produced different colors, which were successfully predicted using a thin-film interference model. In addition, SMNPs can be manufactured in large quantities and are biocompatible. Our results show the unique

advantage of using melanosomes to generate colors found in the animal kingdom and also offer numerous new opportunities towards multi-functional photonic devices and biocompatible products. In the future, increased control of the self-assembly process will be investigated as a necessity in obtaining large-scale films capable of producing the full color spectrum. Ongoing studies will explore alternative assembly procedures and utilize analogous SMNPs with various morphologies and compositions.

CHAPTER IV

STIMULI-RESPONSIVE STRUCTURALLY COLORED FILMS FROM

BIOINSPIRED SYNTHETIC MELANIN NANOPARTICLES

4.1 Introduction

Colors in nature play a variety of roles ranging from warning coloration to interspecific communication and protection from ultraviolet radiation.82,131,132 One critical function of colored patterns is camouflage, prevalent in nature in both predator and prey.82 In some cases camouflage is active and dynamic with changeable colors enabling some animals to disguise themselves in variable environments. Cuttlefish are a noteworthy example, wherein changes to their skin color, pattern, and texture are made to closely mimic or match their environment.133 Such color changes are facilitated by the use of structural colors arising from nanostructures, as these can be easily tuned by varying their periodic spacings. Although cuttlefish skin is likely under direct neural control,13 extrinsic factors like humidity (a significant parameter not only to living organisms, but also in chemical reactions and industrial applications), also affect spacing and thus color in organisms.

Structural colors, for example, of Morpho butterfly wings,134 tree swallow feathers,36 and

Hercules beetle elytra135 change with humidity. In parallel with nature, researchers have developed responsive synthetic optical materials under various stimuli, such as temperature,136-138 chemistry,139 pH,140 light,141 and mechanical force.142 Humidity- responsive systems showing dynamic colors can be divided into one-dimensional,143-145 two-dimensional,146 and three-dimensional photonic crystals.147-149 Other geometries like

supraballs and etalons have also been used.150,151 To increase colorimetric sensitivity to humidity, most systems have employed inorganic materials to increase refractive index143-145 or water absorbing hydrogels to maximize moisture uptake.147-149 Replacing hydrogels with natural materials like silk-fibroin allows only limited color change (20 nm shift in wavelength) when relative humidity (RH) is increased from 30% to 80%.152 There is great demand for a more facile approach using a single biocompatible/biodegradable material for highly efficient dynamic colorimetric performance.

Synthetic melanin nanoparticles (SMNPs) have been used as a single component to create structural coloration through thin-film interference50 or scattering,67 improving on other structurally colored systems by enhancing color saturation through light absorption54,56,153,154. Here, we show for the first time that self-assembled thin films of

SMNPs produce sensitive and reversible color change in response to humidity changes.

We quantitatively demonstrate that water absorption by SMNPs leads to changes in film thickness and thus color.

4.2 Materials and Methods

4.2.1. Preparation of SMNP films.

We first synthesized SMNPs via oxidation polymerization of dopamine monomer (20 mg) in a mixture of 50 mL water, 20 mL ethanol and 1.0 mL ammonia aqueous solution (28-

30%) at 25 C .50 The size and distribution of SMNPs were characterized using transmission electron microscopy (JEM-1230, JEOL Ltd.) and dynamic light scattering measurement (BI-HV Brookhaven instrument with a 633 nm solid-state laser) (Figure

4-1). We then cleaned silicon wafers (University Wafer) using a piranha solution (a mixture of 98% H2SO4 and 30% H2O2 with a volume ratio of 7:3) at 80 C . Clean wafers were held vertically in a SMNP aqueous solution at 60 C and SMNPs were deposited via evaporative self-assembly onto the wafer as the water evaporated. The deposited films were cut in half and both top view and side view scanning electron microscopy (SEM) images were obtained using the scanning electron microscope (JEOL-7401, JEOL Ltd.) without sputter coating. All chemicals were purchased from Sigma-Aldrich.

Light intensity (A.U.) 100 1000 Hydrodynamic radius (nm)

Figure 4-1. Dynamic light scattering measurement of SMNPs in an aqueous solution.

4.2.2. Characterization of dynamic colors

A CRAIC AX10 UV visible-NIR microspectrophotometer (MSP) (CRAIC Technologies

Inc.) was used to measure the normal reflectance spectra of SMNP films in a custom- built humidity chamber (setup in Figure 4-2). We used Teflon tape as a white reference 64

standard. We controlled RH from 10% to 90% by adjusting the mixing ratio of dry and wet nitrogen gas, which was monitored using a traceable hygrometer with accuracy of 1.5%

RH (<10 second response time, Model 4080, Control Company). At five humidity points

(10%, 30%, 50%, 70%, and 90%) after one minute of equilibrium time, we measured reflectance of SMNP films three times with one minute interval. To investigate how fast the color changes, we recorded the real-time color change (both optical images and spectroscopic scans) during the wetting and drying process. We also tested the stability of dynamic colors by measuring the spectra when cycling the humidity eight times. As a control, we measured reflectance of a bare silicon wafer at RH of 10% and 90% RH. We also measured angle-resolved reflectance spectra of SMNP films using an AvaSpec spectrometer with a xenon light source (Avantes Inc.).

Figure 4-2. A scheme of custom-build humidity control setup with real-time monitor of the relative humidity.

4.2.3. Water absorption measurement

To quantify how much water SMNPs can absorb, we used a CAHN 21 automatic electrobalance (CAHN/Ventron) with an accuracy of  0.005% to measure the mass change of small amounts (2~3 mg) of SMNP powders as a function of the relative humidity. First, we measured the water uptake of three empty standard aluminum DSC pans (13.65  0.02 mg at RH 10%) separately at various RHs and found the changes in mass due to water absorption by the aluminum pans was small and consistent among individual pans (Figure 4-3). The net water uptake by the melanin particles can be calculated by subtracting the water uptake by the aluminum pan at certain RH. We mixed dry and wet nitrogen gas to obtain specific RH and then started to record the mass every

3 minutes until equilibrium when the variations among the last five continuous readings were smaller than 0.010 mg. We repeated the measurements of two separate SMNP samples for both drying and wetting processes.

0.15 0.12 0.09

0.06 0.03 0.00 Net weight gain (mg) -0.03 0 20 40 60 80 Relative humidity (%)

Figure 4-3. Water uptake of a standard aluminum DSC pan with the increase of humidity.

4.2.4. In-situ investigation of SMNP film thickness

A FEI Quanta FEG 450 environmental scanning electron microscopy (ESEM) was used to study the microstructural dependence of SMNP films on gas (water vapor and nitrogen) pressure at room temperature. We performed both cross-section and top view imaging of

SMNP films without sputter coating at different humid conditions (various water vapor pressures). The ESEM chamber was first pumped to ~10-5 Torr before water vapor was introduced. After each desired pressure was reached, 5 minutes stabilizing time was allowed before ESEM observation. The water vapor pressure used in this study ranged between 5 Torr and 18 Torr, corresponding to RH 4.5% and 81% at 24 C . Dry nitrogen

(1 Torr to 10 Torr) was also introduced for a comparative study, which was not possible for >10 Torr nitrogen environment due to a poor imaging quality.

4.3 Results and Discussion

We synthesized monodispersed SMNPs with a diameter of (192  10) nm (Figure 4-1 and

Figure 4-4a) and assembled them into structurally colored films with blue and red colors using an evaporation-based process following our previous protocol (Figure 4-4b, c, g).50

Both blue and red films without long range in-plane order were obtained, as shown by two dimensional Fast Fourier power spectra of the top view SEM images (Figure 4-4d,

55 f). Blue and red films are (274  11) nm and (602  17) nm in thickness, based on cross- sectional SEM images (Figure 4-4e, h). The color of the films is caused by interference,50 which showed angle-dependent reflectance (Figure 4-5). This coloration mechanism is

similar to that observed in feathers with single or multiple layers of melanin and keratin.28,36

Figure 4-5. Specular reflectance spectra of a SMNP film at various incident angles with respect to the film normal. Black curve is for 10 , red for 20 , green for 30 , and blue for 40 .

Using a custom-built humidity control system (Figure 4-2), we varied the relative humidity from 10% to 90%, where these SMNP films displayed clearly visible dynamic color changes (Figure 4-6). The color of the blue film turned green, with the maximum peak position shifting from 475 nm to 530 nm when RH increased from 10% to 90%. The red film showed two peaks in the reflectance spectrum, a major peak at 638 nm and the secondary at 483 nm when RH was 10%. After RH increased to 90%, the red film turned green with the major peak shifting to 714 nm and the secondary peak shifting to 551 nm, which makes the secondary peak become the dominant color based on human visual perception (400-700 nm155). The colors cover almost a full spectrum of visible light from blue to red in the color space (Figure 4-6c). Interestingly, the remarkable color change of the red film between RH 70% and 90% offers great benefit in sensing high humidity

(Figure 4-6b-c). The reflectance spectra of a control consisting of only a bare silicon

wafer stayed constant in the same humidity range (10%-90%) (Figure 4-7), demonstrating that changes in the SMNP layer explains dynamic colors of SMNP films.

Relative reflectance (A.U.) 400 500 600 700

Wavelength (nm) 70

Figure 4-7. Reflectance spectra of a bare silicon wafer under relative humidity of 10% (black curve) and 90% (red curve).

The color of SMNP films quickly changed back to their original colors when the humidity was reduced to 10%. The rate of the color changes is mainly determined by how fast the humidity changes. Our observations are on those times scales, since it took 20 seconds to decrease RH from 90% to 10% or increase RH from 10% to 80%. Increasing

RH from 10% to 90% took more than 120 seconds with the first 20 seconds shown herein.

This color change is faster than active color changes in chameleon skin (~350 seconds from red to green) and passive, humidity-induced color change in feathers (25 nm wavelength shift in ~80 seconds).36,156 We measured the reversible response by cycling the RH eight times (longer experimental time windows led to drift in the intensity of the light source of the microspectrophotometer). The positions of the peak maxima are reversible (Figure 4-8) and we also found no change in the color saturation or broadening of the reflectance spectra after eight cycles (Figure 4-9), which is advantageous in designing humidity sensors in comparison to other previously-used approaches.145,147

(a) 530 (b) 750

520 700 510 650 500

490 600 480 550 470

Max peak positon (nm) 500 Max peak position (nm) 460 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Cycle number Cycle number

(a) (b)

Before-10% Before-10% Before-90% 400 Before-90% After-10% After-10% After-90% 300 After-90%

200

100

0 400 500 600 700 400 500 600 700

Normalized reflectance (A.U.) Normalized reflectance (A.U.) Wavelength(nm) Wavelength(nm)

Figure 4-9. Reflectance spectra of blue (a) and red (b) SMNP films before and after eight times cycles at RH of 10% and 90%.

Many colorimetric humidity sensors show visible color changes and these results have been attributed to the swelling of photonic crystal structures. However, these mechanisms have been inferred from indirect evidence.143,145,149 Here, we quantitatively measured the mass of absorbed water as a function of the RH (Figure 4-10). The mass uptake of water increased linearly with increase in RH.

Water uptake (wt%)

0 20 40 60 80

Relative humidity (%)

Figure 4-10. Water uptake in mass of SMNPs changes with RH. Solid line is the linear fitting (%  1.81  0.156RH , R2 0.96 ).

From linear fitting, we extrapolated that SMNPs increased mass (  ) by 13% upon increasing the RH from 10% to 90%. This change in mass can be converted to a change in volume.

VVwater  smnp smnp /  water (4.1)

In Equation (4.1), smnp , water , and Vsmnp are the density of SMNPs, water, and volume of

SMNPs in the film, respectively. Based on these measurements, a SMNP film can absorb water of approximately 17% in volume with increasing RH from 10% to 90%.

SMNPs layer

SiO2 n=1.458

Thick Si layer, n=3.973

Figure 4-11. The scheme of the structure of SMNP films.

To investigate the mechanism behind the observed color change, we used a standard thin film interference model (Figure 4-11) and calculated the reflectance spectra for both blue and red SMNP films.28,89,157 By using the values of the refractive index and extinction coefficient of SMNPs reported in our previous work and assuming the volume fraction of

SMNP film to be 55%,50 we calculate the reflectance spectra of SMNP films. The thickness measured using SEM is the lower limit due to high vacuum conditions and for fitting the data we start with this minimum thickness and increase the thickness until we obtain a best fit (Figure 4-12a-b): the thickness for blue and red film is 328 nm and 654 nm at RH of 10%, respectively. Since SMNP films are confined onto silicon wafers without the freedom to swell laterally when exposed to humid conditions, we used a simplified model (Figure 4-13a) where SMNP films only swell vertically and thus the swelling ratio is equal to the volume expansion. The thickness of SMNP film has a 17% growth when RH rises from 10% to 90%. With this increase in thickness of SMNP films, we have calculated the theoretical spectra and compared them with those measured experimentally. After increasing the RH to 90%, absorption of water vapor will not only 74

swell the film but also reduce the effective refractive index of SMNPs. Therefore we allowed the refractive index to vary and the best fitting was obtained with a value of 1.64 at 90% RH (Figure 4-12c-d). Based on water absorption, we calculated that the RI value of wet SMNPs is 1.68 at RH of 90% using the following equation.94

222 nnVsmnp-wet n smnp(1 V  water ) water water (4.2)

50 Where the RI of dry SMNPs, nsmnp is 1.74. The slight difference in the RI values obtained from these two methods might be due to the minor swelling of SMNPs laterally.

blue_expt_10% red_expt_10% (a) blue_model_10% (b) red_model_10%

blue_model_90% red_model_90%

400 500 600 700 400 500 600 700

Normalized reflectance (A.U.) Wavelength (nm) Wavelength (nm)

Figure 4-12. (a) Measured (solid blue) and model spectra (solid black) for blue film at RH of 10%. (b) Measured (solid red) and model spectra (solid black) for red film at RH of 10%. (c) Measured (dash blue) and model spectra (dash black) for blue film at RH of 90%. (d) Measured (dash red) and model spectra (dash black) for red film at RH of 90%.

(a) Model 1: The thickness of the SMNP film increases

Low humidity 10% High humidity 90%

(b) Model 2: SMNPs don’t swell and water condenses into the gap

Low humidity 10% High humidity 90%

Figure 4-13. (a) Scheme of model 1, where SMNP film thickness swells by 17% from RH 10% to 90%. (b) Scheme of model 2, assuming water condenses into pores of SMNP films.

To further eliminate other possibility to cause the color change than swelling of SMNPs, we calculated that how much color shift is caused if we assume all observed water fills into the pores of SMNP films. In this model (Figure 4-13b), SMNPs do not swell and their refractive index keeps constant. As shown in Figure 4-14, simulated reflectance spectra based on this model is not enough to produce large color change as observed experimentally. Actually there is only 15 nm shift for the blue film (55 nm shift in experiment); and 19 nm shift for the red film (76 nm shift in experiment). Therefore, the thickness expansion model discussed in the manuscript is responsible for observed dynamic colors.

(a) (b)

1.5 blue_expt_90% 1.0 red_expt_90% model2-blue-90% model2-red-90% 0.8 1.0 0.6

0.5 0.4 0.2

0.0 0.0 Normalized reflectance (a.u.)

Normalized reflectance (a.u.) 400 500 600 700 400 500 600 700 Wavelength (nm) Wavelength(nm)

Figure 4-14. Theoretical reflectance for both blue (a) and red (b) SMNP films at RH of 90% based on model 2. Black spectra are the simulated spectra for both blue and red film. Blue curve represents experimental reflectance of the blue film and red curve stands for experimental reflectance of the red film.

We further used ESEM to directly investigate the structural response of SMNP films to humidity variation. The relative humidity was controlled by water vapor pressure that varied from high vacuum (10-5 Torr, ~ RH 0%) up to ~18 Torr (RH 81%). Our observations of cross-sectional samples showed a prompt and noticeable thickness increase (sometimes up to 20% at high humidity) with increasing humidity, which seemed to be caused mainly by the increasing particle size (Figure 4-15). In contrast, no distinguishable thickness increase was observed in our control experiment where the same SMNP film was exposed to dry nitrogen gas at the similar pressures (Figure 4-16).

This result confirmed that the change in film thickness is due solely to absorption of moisture by the particles rather than change in atmospheric pressure. Top view ESEM images demonstrated SMNPs swell, with cracks in the film disappearing when humidity increased and this process is reversible when decreasing humidity (Figure 4-17).

Compared with solid films, the cracks in the SMNP films help release strain from SMNP

swelling at high humidity. This possibly enhances the reversibility of the observed color change during wetting and drying cycles.

Figure 4-15. Cross-sectional ESEM images of a SMNP film at different water vapour pressures. Scale bar, 500 nm.

1 Torr, N2 5 Torr, N2 10 Torr, N2

Figure 4-16. Side view ESEM images on cross section of a SMNP film under dry nitrogen conditions at various pressures (1 Torr, 5 Torr, and 10 Torr). Scale bar, 500 nm.

Figure 4-17. Top view ESEM images on a SMNP film at different water pressures, (a) high vacuum, (b) 5 Torr, (c) 15 Torr, and (d) back to high vacuum. Scale bars, 5 μm.

In this work, we achieved rapid and substantial color responses with humidity without any additional hygroscopic materials for water absorption or inorganic fillers to increase refractive index. Here, we can define the sensitivity of the color change to the humidity as,

S peak shift ( nmwt ) / water uptake ( %) (4.3)

For SMNP films, only 13 wt% water absorption can lead to 77 nm color shift, therefore

S 5.9 ( nm ) / ( wt %) , which is a factor of two larger than that reported for polyelectrolyte films ( 2.2 (nm ) / ( wt %) , 195 nm shift is achieved with 90% water uptake).103 With lower water uptake, the SMNP film is more likely to avoid structural deterioration after multiple wetting and drying cycles. In contrast to conventional

multilayer structures,16, 17 the SMNP film can be facilely prepared via a one-step coating process. More importantly, the porosity of the films makes it easier to absorb/desorb water vapor, increasing the response speed of the dynamic colors. In addition, SMNP films show larger and faster dynamic colors than natural systems like butterfly scales, bird feathers, or beetle elytra.36,134,135

Water or moisture can dramatically increase the electric conductivity of melanins possibly through an electronic-ionic hybrid conductor mechanism.21-23,158 However, quantification of water absorption or taking advantage of this property to make dynamic color changes had never been explored. Our work combining hydroscopic and unique optical properties (high refractive index and high absorption) of melanins will further broaden the capabilities of SMNPs in photonics, in addition to their potential applications in bio-imaging, drug delivery, and cancer diagnosis.59,159

4.4. Conclusions

In summary, we have developed bioinspired SMNP films and have demonstrated that these films show rapid, reversible dynamic color changes in response to humidity. By measuring water absorption, imaging the changes in diameter using ESEM, and by employing optical modeling, we have shown that color change is caused by swelling of

SMNP particles, which leads to changes in the layer thickness. The fact that remarkable color change is achieved with a single biocompatible component and simple evaporative process will lead to intriguing applications of synthetic melanin into sensing and dynamic colors. This strategy is an example of biomimicry, where we have applied principles of structural coloration in natural systems to achieve dynamic coloration.

CHAPTER V

BIO-INSPIRED BRIGHT NON-IRIDESCENT PHOTONIC MELANIN

SUPRABALLS

5.1 Introduction

In the colorful world in which we live, colors not only bring aesthetics and pleasure, but are also significant for communication, signaling, and security. Colors are produced through either absorption of light by molecules (pigmentary colors) or scattering of light by nanostructures (structural colors).160 Structural colors are superior in many ways to pigmentary colors, due to their tunability, resistance to (photo- or chemical) bleaching, and reduced dependence on toxic materials. Many recent papers have demonstrated the use of self-assembly to produce photonic crystals that generate colors across the visible spectrum.127 However, we still face significant challenges. Many traditional structural colors are iridescent and are thus not useful for wide-angle displays. Recent examples of non-iridescent structural colors do not have sufficient color saturation without adding absorbing materials (carbon black, gold nanoparticles, or black polypyrrole) to reduce the incoherent scattering.52,54,56,57,64 Core-shell nanoparticles with a shell refractive index similar to water have been used to tune the spacing between cores to achieve optimal scattering for non-iridescent colors, but these colors only exist in solution.106,161 Even though both bottom up and top-down methods have been widely used,55,162,163 there is a demand for a scalable process for mass production of structural colors.

Nature provides many spectacular examples of structural colors such as duck wing feathers (Anas crecca) that use non-close hexagonal packed melanosomes to increase color brightness,37 and wild turkey (Meleagris gallopavo) feathers with hollow, high RI contrast melanosomes that brighten feather colors (Figure 5-1A).30,107 These examples inspire the design of design core-shell synthetic melanin nanoparticles (CS-SMNPs) described herein, for the production of bright structural colors. Further driven by the demand for scalable production of structural colors, we have developed a facile one-pot reverse emulsion process to assemble CS-SMNPs into bright and non-iridescent photonic supraballs. These melanin-based supraballs may find use as photonic inks and multifunctional (UV-protective and superhydrophobic) coatings.

Llovet. (B) Normal reflectance spectra from the (111) plane of FCC lattices made of core-shell nanoparticles and homogenous nanoparticles with similar sizes and equivalent refractive indices: high RI core/low RI shell nanoparticles (core: RI = 1.74, diameter = 200nm; shell: RI = 1.45, thickness = 50 nm), equivalent homogenous nanoparticles (RI = 1.54, diameter = 300 nm), and low RI core/high RI shell nanoparticles (core: RI = 1.45, diameter = 267 nm; shell: RI = 1.74, thickness = 16.5 nm). (C) The reflectance intensity ratio between core-shell and homogenous structure changes as we vary the ratio of core radius to total core-shell nanoparticle radius.

5.2 Materials and Methods

5.2.1 Characterization of nanostructures in bird feathers

We obtained the iridescent wild turkey (Meleagris gallopavo) breast feather and the duck

(Anas crecca) wing feather from The University of Akron collection. We followed our previous protocol to prepare barbule cross sections for transmission electron microscopy.28 Briefly, we dehydrated cut feathers using 100% ethanol and infiltrated them with 15%, 50%, 70% and 100% embed 812 resin (Electron Microscopy Science) every 24 hours. Next, we placed embed 812 resin and samples into block molds and cured them at 60 C overnight. We trimmed the blocks and cut 80 nm thick sections on a microtome (Leica UC-6, Leica Microsystems GmbH.). Sections were placed onto the copper grids for TEM imaging (JEM-1230, JEOL Ltd).

5.2.2 Synthesis and characterizations of CS-SMNPs

We first synthesized pure SMNPs by oxidative polymerization of dopamine molecules

(Sigma Aldrich) under base environment according to our previous work,50 and then

164 coated silica shell (SiO2) onto the surface of SMNPs via the modified Stober method.

Typically, SMNPs (3.25 mg) were firstly dispersed in a mixture of 5 mL 2-propanol and

0.88 mL deionized water under ultrasonic treatment and then magnetic stirring. Then,

125 μL ammonia solution (NH4OH, 28-30%) was added and stirred for 10 min. We control the amount of tetraethyl orthosilicate (TEOS) injected into the mixture and hydrolysis reaction time to regulate the coated SiO2 shell thickness (See details in Table

5-1). Finally, CS-SMNPs were collected by centrifuge, washed with deionized water three times and re-dispersed in deionized water for the use in supraball preparation.

Table 5-1 Synthesis conditions for different sized CS-SMNPs Supraball Core diameter Shell thickness TEOS (μL) Reaction Time Colors (nm) (nm) Red 160 ± 7 66 ± 8 64 18 h Orange 160 ± 7 50 ± 8 53 3.5 h Olive 160 ± 7 36 ± 7 40 75 min Green 123 ± 10 43 ± 9 82 3 h Navy 123 ± 10 36 ± 6 82 100 min To exam the core-shell morphology and particle distributions, we drop-cast CS-SMNPs onto carbon black coated copper grid for transmission electron microscopy (JSM1230,

JEOL Ltd.). We measured around 40 CS-SMNPs using Image J to obtain particle size and standard deviation. We also measured UV-vis absorption of aqueous solutions of CS-

SMNPs and solid silica particles using UV-1800 UV-vis spectrometer (Shimadzu

Corporation).

Figure 5-2. UV-vis absorption of pure SMNPs, CS-SMNPs, and pure silica nanoparticle solution at the concentration of 20 mg/L.

5.2.2 Supraball preparation

Typically, a solution of 30 μL aqueous CS-SMNPs with a concentration of 30 mg/mL was added to 1 mL anhydrous 1-octanol (Sigma Aldrich). The water-in-oil emulsion rapidly formed upon shaking on a digital vortex (Genie 2, Scientific Industries) at a shaking speed of 1600 rpm for 2 min. The shaking speed was then reduced to 1000 rpm for 3 min when supraballs were formed upon shrinking of the aqueous droplets with water dissolving into the oil phase.165 After supraball sediments settled down, we removed most of supernatant (0.9 mL) to concentrate the supraballs. Colorful supraballs were obtained by removing the 1-octanol at 60 C . In this method it was also important to make the glass vials hydrophobic so that aqueous droplets did not adhere to glass vial and break upon contact with the glass vial. We coated an OTS self-assembled monolayer

(SAM) onto glass vials following a modified protocol.166,167 Into dry and clean glass vials, added 2 vt% OTS toluene solution and degassed for 15 min before tightly closing the cap.

After 16 hours at room temperature, we rinsed the vials with toluene and ethanol for three times. Finally, the vials were annealed at 120 C under vacuum for 2 hours. To quantify if

OTS SAM was successfully grown onto glass vial, we put a clean glass slide inside to vial during OTS growth and measured the contact angle of the glass slide (water contact angle 112±0.6°).

5.2.3 Supraball characterization

The dried supraballs were imaged under Leica M80 stereo microscope (Leica

Microsystems) and we used high density Teflon tape (TaegaTech) as white balance. The microscope contained LED lights as the source and was connected to camera Leica DMC

4500. The reflectance spectrum of individual supraball was measured using a CRAIC

AX10 UV visible-NIR micro-spectrophotometer (MSP) (CRAIC Technologies Inc.) with a 75 Watt Xenon short arc lamp (Ushio UXL75XE) as the light source. We averaged spectra from twelve random supraballs and calculated the standard deviation using

PAVO package in R programming software.88 To investigate if the colors of supraballs are angle non-independent, we deposited a thick films of supraballs and measured the scattering spectra from different angle using an AvaSpec spectrometer with a xenon light source (Avantes Inc.) attached to a custom-built goniometer (inset in Figure 5-5C).

The nanostructure of supraball surfaces was characterized using a field-emission scanning electron microscope (JEOL-7401, JEOL Ltd.). To investigate the inner structure of supraballs, we dispersed powders of supraballs into embed 812 resin in block molds

and cured them at 60 C for 16 hour. The hard blocks were trimmed to a sharp trapezoidal tip using a Leica S6 EM-Trim 2 (Leica Microsystems) and we then cut 80 nm thick sections using a diamond knife (Diatome Ltd.) on a Leica UC-7 ultra-microtome for transmission electron microscopy.

5.3 Optical Model

5.3.1 FDTD simulation

We used Lumerical FDTD solutions 8.15 to run the optical modeling. In the simulation, we created a FCC colloidal lattice with its plane (111) as X-Y plane and a plane wave light was injected down from Z direction, perpendicular to plane (111). For the simulation in Figure 5-1B, we compared theoretical reflectance spectra from core-shell nanoparticles and homogenous nanoparticles. The homogenous nanoparticle is the same size as core-shell nanoparticles with a RI of averaged RI value of core-shell nanoparticles based on the equation,

2 2 2 nhomo () Vcore V shell  n core V core  n shell V shell (5.1)

We set the RI value of 1.74 for high RI material (same with synthetic melanin, but without any absorption) and 1.45 for low RI material (same with fused silica) in our calculations. The lattice has 30 periods along X and Y directions and 12 periods along Z directions. We chose auto non-uniform mesh type with accuracy of 6. Both core and shell materials contained no absorption so that we could decouple optical response from core- shell structures and absorption. When comparing simulation results with experimental reflectance of supraballs (Figure 5-5D), we considered absorption from SMNPs core.50 In

this calculation, the Lumerical software used the fitting RI values based on experimental

RI values (Figure 5-3). Based on our observation that only top layers of supraball contributes to the colors, we calculated only 6 layers along z directions. We chose auto non-uniform mesh type with accuracy of 5, due to computation memory limitations.

Figure 5-3. The complex refractive index for synthetic melanin cores used in the FDTD calculations, which is the best fit with the measurements from our previous paper.50

5.3.2 Scattering theory

Because the supraballs made of mixed type 1 (160/36 nm) and type 2 (160/66 nm) CS-

SMNPs are far from the ordered FCC packing (Figure 5-7C), we can not simply regard them as photonic crystals. To consider the disorder effect, we used the multiple scattering theory to explain the color shifting from blending two sizes of CS-SMNPs with different mass ratio.168,169 The multiple scattering theory of light can provide basic analytical methods to address light scattering problem in disordered photonic media. To predict the reflectance spectra profile analytically without resorting to time-consuming numerical 88

1 simulations, here we define the parameter Ak l 0 t  to present the diffuse reflectance of

the supraballs, where k0 lltt 2/ First, lt directly determines the diffuse reflectance of supraballs, which plays a key role in the production of structural colors. For the diffusive

light transport without absorption, transmission is proportional to lt as Tl~ t and thus

168,169 reflectance increases when lt is reduced. When the optical absorption of CS-

SMNPs is considered, the reflectance is still negatively related to lt , because smaller lt leads to a stronger multiple scattering effect (note l doesn’t rely on absorption), which is t the only source of backscattering and thus diffuse reflectance. .168,169 Secondly, there are

significant interference effects when lt is comparable with the wavelength  ( A ~1), which are also observed in the scenarios such as coherent backscatteringand Anderson localization.170,171 Constructive interference effects lead to a reduction in diffusion constant D (i.e. coherent backscattering gives rise to a correction as DDA/~ 2 )172 and thus an increase in reflectance, which can be also quantified by the parameter A .

Therefore, we can use the parameter A to estimate spectral profile of diffuse reflectance.

This parameter has also been used by F. Scheffold et al in predicting optical spectra of

173 densely packed TiO2 nanoparticles.

Since CS-SMNPs are densely packed in supraballs, the short-range order induced interference mechanism among binary CS-SMNPs in multiple scattering of light likely

173-175 plays a key role in lt as well as the observed reflectance spectra. To support this argument, here we consider the short-range order induced interference effect in the multiple scattering using a theoretical model where two-particle correlation is taken into

account (known as Born’s approximation).176 Then we compare this result with that from the independent scattering approximation (ISA) without consideration of the short-range order.168,169,172 The theoretical model predicts the transport mean free path of light in supraballs as l   1 where  is the number density of particles.  is calculated in tt  t the following expression, 176

   Bd(  )sin (1 cos ) (5.2) t k 2 0

where kn 2/eff and B() F11  ()()(1 SF 1122  SF 2212  ) S 12 () ()2  (1 )()() .

We calculate effective refractive index neffeff  using the Maxwell-Garnett formula for three-component medium (core, shell, and air) as,177

eff 1 core11 shell ffcore shell (5.3) eff2  core  22  shell 

33 33 rr rr1,core   2,core  where f f1,core f 2,core and f ff 11       . f and f are core 1233 shell 12rr33    1 2 rr1,total 2,total 1,total   2,total 

the volume fraction of type 1 and type 2 CS-SMNPs. core and  shell are permittivity of

core (synthetic melanin) and shell (silica). r1,core and r1,total are the core radius and total

radius of type 1 CS-SMNPs, while r2,core and r2,total are the core radius and total radius of type 2 CS-SMNPs.  NNN/() is the number fraction of type-1 CS-SMNPs. 1 1 2

S () , S () , and S () are partial structure factors of the binary-particle system 11 12 22 calculated based on Percus-Yevick hard sphere model.178 The Percus-Yevick model is a

sufficient approximation for calculating pair correlation function characterizing short- range order in packing hard-sphere systems. F () , F () , and F () are form factors 11 12 22 derived from the Mie theory for core-shell particles. They are calculated as follows,

** Ff111() ff 11 f 1s sp p (5.4)

** Ff222() ff 22 f 2s sp p (5.5)

** Ff121( f ) 2 Re f 1 f 2s s p p (5.6)

  where and fsjm( j ) m (2  m  1) j a m, (cos  )  b m, (cos ) fpjm( j ) m (2  m  1) j a m, (cos  )  b m, (cos ) m1 m1

with j 1,2 denoting different particle species. Here  m and  m are functions defined as

P1 (cos ) dP1 (cos ) (cos )  m and (cos )  m , where P1 (cos ) is the associated m sin m d m

2,179 Legendre function. amj, and bm, j are Mie coefficients calculated as,

Dm,1 j//()() n shell m y j m y j m y j amj,  (5.7) Dm,1 j//()() n shell m y j m y j m y j

nshell G m,1 j m/()() y j m y j m y j bmj,  (5.8) nshell G m,1 j m/()() y j m y j m y j

where yjj kr ,total is the size parameter for the total radius of type-j particle and

kn 2/eff is the wavenumber in the surrounding medium with effective refractive

index neff as calculated previously. We defined n nshell/ n core , where nshell  shell and

ncorecore  are complex refractive indices of shell and core materials. The parameters,

Dmj, , Gmj, are calculated as

Dm()()/() n shell y j A m, j m n shell y j m n shell y j Dmj,  (5.9) 1 Am, j m ( n shell y ) / m ( n shell y )

Dm()()/() n shell y j B m, j m n shell y j m n shell y j Gmj,  (5.10) 1 Bm, j m ( n shell y j ) / m ( n shell y j )

And Amj, , Bmj, are

nDm()() n core x j D m n shell x j Am, j m() n shell x j (5.11) nDm()()() n core x j m n shell x j m n shell x j

Dm()/() n core x j n D m n shell x j Bm, j m() n shell x j (5.12) Dm()()() n core x j m n shell x j m n shell x j

where xjj kr ,core is the size parameter for the core radius of type-j particle. In above

equations,

m (), m () and Dm () are special functions defined using argument  as

mm()()  j  ,

(1) mm()()  y  , mm()()  h  and Dm()()/()  m   m  . Here jm () , ym ()

(1) and hm () are spherical Bessel functions of the first kind and second kind, and spherical

2 Hankel function of the first kind, in the order of n respectively. m ()and m ()denote

the first-order derivative respect to argument . 92

Equations (5.7-5.12) are also applicable for homogeneous spheres by setting

rrjj,total,core .

We calculated the A parameter for supraballs consisting of binary CS-SMNPs with different mass ratios. Figure 5-11A showed the normalized A for different mass ratios for 160/36 nm and 160/66 nm CS-SMNPs. A clear trend of blue-shift was observed when increasing the amount of 160/36 nm CS-SMNPs, which is consistent with the experimental observation.

As a comparison, we also calculated without consideration of short-range order and interference effects (Figure 5-11B), where we set the partial structure factors

SS( ) ( ) 1 , S ( ) 0 and wavenumber kk2/, unlike using 1122 12 0 kn 2/in the short-range-order case because using the effective index also takes eff partial interference effects into account.180 This approximation for calculating transport mean free path is called independent scattering approximation (ISA).180 No significant shift of peaks was observed by changing the composition of binary CS-SMNPs, which was invalid according to experimental results. This finding supports our conclusion that short-range order plays the crucial role in producing the blended colors of supraballs when mixing binary sizes of CS-SMNPs.

5.4 Results and Discussion

To aid in designing an optimal core-shell morphology for producing colors, we first used the FDTD (finite-difference time-domain) method to calculate the theoretical reflectance spectra (normal incidence) from a (111) plane of the most common photonic crystal with

a FCC packing composed of core-shell nanoparticles and homogenous nanoparticles (See section 5.3.1 for details). Relative to the lattice of homogenous nanoparticles, the lattice of core-shell nanoparticles with high RI cores and low RI shells peaked in reflectance at a similar wavelength, but had higher intensity (Figure 5-1B). However, the reverse core- shell structure consisting of low RI cores and high RI shells had lower intensity than the lattice of homogenous nanoparticles. Varying the ratio of core-to-total radius, we found the highest reflectance (~130% relative to homogenous nanoparticles) for high RI core/low RI shell structure when the core radius was ~ 60% of the radius of whole core- shell nanoparticle. The lowest reflectance (~52% relative to homogenous nanoparticles) was for core-shell nanoparticles with low RI cores when the core radius was ~80% of total core-shell nanoparticle (Figure 5-1C). Therefore, we designed core-shell nanoparticles with high RI cores and low RI shells to obtain higher reflectance and brighter colors.

Figure 5-4. Core-shell SMNPs synthesis and self-assembly. (A) A scheme of the method of synthesizing silica coated melanin nanoparticles. (B) TEM images of core-shell SMNPs: 160/0 nm, 160/36 nm, and 160/66 nm, respectively. Scale bar, 100 nm. (C) A

scheme showing the self-assembly of supraball structures via a reverse emulsion process. (D) A photo of rainbow-like flowers, painted with supraball inks made of five different sizes of CS-SMNPs: navy, 123/36 nm; green, 123/43 nm; olive, 160/36 nm; orange, 160/50 nm; and red, 160/66 nm.

We chose synthetic melanin as the core material due to its high RI (~1.74) and broadband absorption in the visible spectral region that helps to reduce incoherent scattering and thereby enhance color purity.50 We used silica (RI ~ 1.45) as the low RI shell and employed a sol-gel reaction to coat silica onto synthetic melanin nanoparticles (SMNPs) to synthesize core-shell SMNPs (CS-SMNPs) (Figure 5-4A). We used the synthetic melanin cores from 120 to 160 nm and tuned the coated shell thickness from 36nm to 66 nm by adjusting the reaction time and sol-gel precursor concentration (Table 5-1). In

Figure 5-4B, the core diameter was fixed to 160  7 nm and the shell thickness was changed from 0 nm up to 66 nm (e.g. 160/0, 160/36 and 160/66 nm). The shell helped to control the spacing between melanin nanoparticles, improving on the duck’s non-close packing structure. We employed an easily deployed water-in-oil reverse emulsion template method to assemble CS-SMNPs into micro-size supraballs, mimicking the structure found in rainbow wracks (Figure 5-4C). No surfactant molecules were used to stabilize the emulsion and the transient stable emulsion droplets were formed upon shear mixing. The oil phase was 1-octanol, which absorbs small amounts of water,165 reducing the amount of water in the aqueous phase containing CS-SMNPs. This process shrank the droplets and aided in the assembly of CS-SMNPs to form supraballs with full spectrum colors depending on the sizes of CS-SMNPs (Figure 5-4D). This was a one-pot process that took place at room temperature without post-treatment for water removal, and the particles could be easily separated by centrifugation. This process has a clear advantage over other emulsion-like processes used to produce colorful supraballs that require 95

microwaves or heat to remove water.181-183 The reverse emulsion method is also easily scalable to produce larger quantities than microfluidic devices.56,106,184

We investigated the supraballs consisting of four types of nanoparticles. Under stereo microscopy (mostly collecting scattering light), supraballs made of CS-SMNPs (160/36 nm, and 160/66 nm) showed highly visible olive and red colors, while supraballs making of 160/0 nm CS-SMNPs appear almost black (Figure 5-5A). As a control, we prepared supraballs using pure silica nanoparticles (224 16 nm) and they displayed whitish cyan color. The reflectance spectra for individual supraballs contained single peaks located at

~430 nm, ~540 nm, and ~660 nm for supraballs composed of 160/0 nm, 160/36 nm, and

160/66 nm CS-SMNPs, respectively (Figure 5-5B). The reflectance intensity of a single supraball increased with the increase in thickness of the silica shell due to reduced absorption by CS-SMNPs in comparison to pure SMNPs (Figure 5-2). The reflectance spectrum for silica particles peaked at ~465 nm and was superimposed by a high intensity broad background signal that led to a whitish color. This was caused by intense incoherent scattering.80,183 The reduced light absorption and incoherent scattering of CS-

SMNPs produced more saturated colors that were visible to the naked eye. In addition, these colors are non-iridescent, offering them great potential in applications such as wide- angle photonic inks (Figure 5-5C).

Figure 5-5. Characterization of supraballs. (A) Optical images of supraballs made of four types of nanoparticles: 224 nm pure silica nanoparticles, 160/0 nm, 160/36 nm, and 160/66 nm CS-SMNPs. Scale bars, 0.5 mm. (B) Reflectance spectra and optical images for individual supraball consisting of 224 nm pure silica nanoparticles (black curve, cyan supraball), 160/0 nm CS-SMNPs (blue curve, purple supraball), 160/36 nm CS-SMNPs (green curve, olive supraball) and 160/66 nm CS-SMNPs (red curve, red supraball). The shaded area indicates the standard deviation from 12 samples, plotted using Pavo package 88 in R . Black box in insets is 44 μm. (C) Angle-resolve spectra for olive inks as shown in Fig. 2D. The inset scheme shows setup for angle-resolved backscattering measurements where we fixed 휶=15° and varied angle 휽 between source and sample from 40° to 90°. (D) FDTD simulation of normal reflectance spectra from supraballs composed of three different sizes of CS-SMNPs.

We used electron microcopy to investigate the nanostructure to reveal the color production mechanism of the supraballs. SEM results showed that supraballs were spherical and composed of close packed nanoparticles (Figure 5-6A). High-resolution

SEM images of supraball outer surfaces revealed that nanoparticles formed a quasi- ordered packing, as reflected in the 2D fast Fourier Transform power spectra (Figure

5-6B). The quasi-ordered packing can reduce the iridescence observed in well-

crystallized supraballs.185 The spherical geometry of supraballs also likely leads to non- iridescent colors.56 Cross-sectional TEM images of supraballs showed that the supraballs were solid and filled with close packed nanoparticles (Figure 5-6C). This solid morphology can increase the mechanical strength of these supraballs. To form dense supraballs, it is critical to reduce the initial amount of water phase to be lower than the total solubility of water in 1-octanol (65 μL/mL) during the self-assembly process.165

We compared our empirical results with theoretical predictions of colors of melanin- based supraballs using FDTD simulations. In the model, we created a flat FCC photonic crystal consisting of six layers and calculated the normal reflectance from the (1, 1, 1) crystal plane without considering the curvature of the supraball surface. We used the 98

refractive index and absorption of SMNPs based on our previous measurement (Figure

5-3).50 The absorption decreased the overall reflectance, but attenuated the non-primary peaks, thus increasing the color purity. This advantage of melanin to increase the contrast was also demonstrated on self-assembled structures using only melanin particles.50 The calculated spectra for the supraballs contained a max peak position at ~440 nm, ~550 nm, and ~670 nm for 160/0 nm, 160/36 nm, and 160/66 nm CS-SMNPs (Figure 5-5D), which was in close agreement with the experimental measurements shown in Figure 5-5B. The simulated spectra appeared sharper than the experimental measurements because we used a flat photonic crystal in our calculations, which was not a perfect example based on our experimental data. However, the agreement with the maximum peak position clearly indicated that this simple model was able to capture the origin of colors of these supraball structures.

Figure 5-7. Supraballs from binary CS-SMNPs. Optical images, SEM images of top surface of supraballs, and cross sectional TEM images for supraballs consisting of (A) 160/0 nm & 160/36 nm CS-SMNPs (B) 160/0 nm & 160/66 nm CS-SMNPs, and (C) 160/36 nm & 160/66 nm CS-SMNPs. The mixing ratio was 1:1 by mass. Scale bars, 500 nm. (D) Optical images of supraballs mixing different mass ratios of 160/36 nm and 160/66 nm CS-SMNPs. Black box is 4 4 μm.

Similar to how pigmentary colors can be tuned by mixing two types of pigments, we used the same reverse emulsion process to assemble CS-SMNPs with binary sizes (same core diameter but different shell thicknesses) into spherical supraballs. Mixing pure SMNPs and CS-SMNPs with different shell thickness at mass ratio of 1:1 resulted in a similar purple color to those produced by pure SMNP supraballs (Figure 5-7A-B and spectra in

Figure 5-8A). Both SEM images of supraball outer surfaces and cross sectional TEM of supraballs demonstrated that only pure SMNPs segregated to the surfaces after mixing with CS-SMNPs (Figure 5-7A-B). However, mixing two sizes of CS-SMNPs with 1:1

100

ratio by mass resulted in an orange color, and nanoparticles of both sizes of CS-SMNPs were randomly mixed at the surface and the bulk (Figure 5-7C and Figure 5-8B). The differences in blending and segregation of nanoparticles can be explained by the higher affinity of melanin than silica to the oil-water interface. When mixing SMNPs and CS-

SMNPs, SMNPs aggregate to the interface; while the two different CS-SMNPs have similar hydrophobicity and are mixed in the supraballs without any long range order

(Figure 5-9). Taking advantage of the segregation and the significance of the top layers for color generation, we varied the mixing ratio of CS-SMNPs, providing a simple knob to tune colors without synthesizing new nanoparticles (Figure 5-7D, spectra in Figure

5-10).

Figure 5-8. (A) Reflectance of single supraballs of pure 160/0 nm, mixed 160/0 nm & 160/36 nm CS-SMNPs, and mixed 160/0 nm & 160/66 nm CS-SMNPs. (B) Reflectance of single supraballs of pure 160/36 nm CS-SMNPs, pure 160/66 nm CS-SMNPs, and mixed 160/36 nm & 160/66 nm CS-SMNPs.

101

Figure 5-9. TEM images of the inner structure of supraballs of binary CS-SMNPs: (A) 160/0 nm & 160/36 nm CS-SMNPs, (B) 160/0 nm & 160/66 nm CS-SMNPs, and (C) 160/36 nm & 160/66 nm CS-SMNPs. Scale bars, 500 nm.

Figure 5-10. Normal reflectance spectra of supraballs making of binary CS-SMNPs (160/66 nm and 160/36 nm) with different mixing ratios.

To understand the color blending effect, we used the inverse of normalized transport mean free path to calculate the scattering intensity of supraballs made of 160/36 nm and

102

160/66 nm CS-SMNPs based on the multiple scattering theory (details provided in

Section 5.3.2).168,169 The scattering model based on short-range order captures the reflectance trend observed in the experiments compared to a model that assumes independent scattering in supraballs (Figure 5-11). Therefore, the interference effect from the short-range order is critical for the color production in supraballs made of mixed CS-

SMNPs.

In addition to the brightness and saturation of the color produced using melanin core- shell nanoparticles, melanin has a broad absorption spectra and can dissipate almost 90% of the UV radiation into heat within a nanosecond.14,186 Therefore, an additional advantage of melanin-based supraballs is in the production of UV-resistant inks. Their colors can be easily tuned by changing the thickness of the silica or by mixing CS-

SMNPs of different shell thickness.

5.4. Conclusions 103

Inspired synergistically by non-close packing of melanosomes in duck feathers, and hollow melanosomes in turkey feathers, as well as theoretical FDTD modeling, we have designed CS-SMNPs that can self-assemble into micron-sized colorful supraballs through a one-pot, scalable reverse emulsion process. This control of spacing leads to supraballs with tunable colors across the entire visible spectrum. The structure of high RI cores and low RI shells increases reflectance to produce brighter colors. The use of melanin is critical to the success of this strategy because it provides the required RI contrast between the cores and the shells and the broad absorption that helps to enhance the color saturation by absorbing incoherent scattering. In addition to all the optical merits of using

CS-SMNPs, the reverse emulsion method to fabricate supraballs is simple, fast, and easily scalable. Similar to mixing pigmentary colors, one can match a desired color by simply mixing binary CS-SMNPs. Therefore, this novel two component strategy, melanin and silica (sand), has the potential to revolutionize the use of structural colors in place of toxic organic- and metal-based pigments.

104

CHAPTER VI

SUMMARY

Structural colors have a variety of advantages over pigmentary colors, but there remain challenges in the fabrication of bright, pure structural colors at a large scale. In this dissertation, a comprehensive summarization of current knowledge of melanin in nature structural color and a deep investigation of a rainbow-like bird feather have inspired us to utilize synthetic melanin nanoparticles as a tool box for creating structural colors. This not only leads to a deeper understanding of relations of melanin and structural colors, but also offers a pathway for creating pure, bright structural colors for applications like coatings and paintings.

Generally single feathers or other integuments contain only one structural color, but those of the common bronzewing display a consistent color gradient from blue to red (462-647 nm) over the proximo-distal length of individual barbs. We used optical microscopy and macro- and micro-spectrophotometry to characterize this color gradient, and transmission electron microscopy to investigate the nanostructure. Combining optical modeling and experimental results, we demonstrate that the rainbow-like iridescence is caused by multilayer interference from organized arrays of melanosome rods in a keratin matrix and that the color gradient results from the subtle shifts in both diameter and spacing of melanosome rods. This result illustrates tight developmental control feathers and may provide inspiration for the design of multi-colored coatings or fibers.

105

Directly inspired by the extensive use of self-assembled melanosomes to produce colors in avian feathers, we set out to synthesize and assemble synthetic melanin nanoparticles in an effort to fabricate colored films. We have quantitatively demonstrated that synthetic melanin nanoparticles have a high refractive index and broad absorption spanning across the UV-visible range, similar to natural melanin. Utilizing a thin-film interference model, we demonstrated the coloration mechanism of deposited films and showed that the unique optical properties of synthetic melanin nanoparticles provide advantages for structural colors over other polymeric nanoparticles (i.e. polystyrene colloidal particles).

In addition, we show for the first time fast, significant, and reversible changes of structural coloration in self-assembled synthetic melanin nanoparticle films in response to changes in humidity. The mechanism was elucidated as a process driven by the hygroscopic nature of the particles, leading to changes in the thickness of the synthetic melanin nanoparticle layer that alter the interference color. This mechanistic explanation is supported by water absorption measurements, an optical model, and environmental scanning electron microscopy. The synthetic melanin nanoparticles and their assembly into films showing dynamic colors but only provide an approach to mimicking vibrant colors found in avian feathers and a wide range of potential applications in designing optical devices and functional coatings, but also offers possible routes for synthetic melanin as a material of importance in sensors and coatings.

At last, we show a simple yet practical approach to use melanin and silica core-shell nanoparticles to produce photonic inks that show bright, non-iridescent colors across the visible spectrum. These core-shell nanoparticles are assembled into micron-size supraballs using a simple, scalable, and one-pot reverse emulsion process to produce 106

colors that can be tuned by controlling the thickness of the silica layer. Similar to mixing pigmentary colors, we can tune the colors by blending melanin nanoparticles of different shell thickness to control spacing between melanin cores. The brightness and saturation of the color produced using a high refractive index melanin and an emulsion process has provided a practical process to manufacture photonic inks or coatings.

A variety of challenges and opportunities still exist in this field. The first big open question is that how melanosomes assemble into different structures (i.e. multilayer, square or hexagonal lattice) in bird feathers. Addressing this problem can help us to mimic the assembly process happening in nature to create similar bright structural colors like bird. Secondly, it is challenging to synthesize submicron rod-shaped melanin nanoparticles similar to bird melanosomes. Those anisotropic nanoparticles will display unique self-assembly behavior and they can assemble into photonic crystals or liquid crystal phases that probably show interesting optical phenomena. Thirdly, it is promising to use melanin into non-iridescent structural colors for creation of wide-angle photonic inks. There is demand for more scalable, cheaper methods of making such colors (like the reverse emulsion process in Chapter V) for applications at a large scale. In addition, it will be interesting to explore other properties of melanin-based structural colors, because melanin has diverse properties, like UV protection, free radical scavenger, and semiconductivity. Finally, it is also desired to use melanin-hybrid materials for not only creating bright, saturated structural colors, but also incorporating new functionalities from the other component. Therefore, biologists, chemists, and physicists should work together to design optimal melanin-based materials and advance the fabrication and application of edge-cutting structural colors. 107

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APPENDIX A: MATRIX TRANSFER METHOD

The matrix method for multilayer interference calculation we used was developed by

Azzam and Bashara187 and has been used in both material science121 and biological fields.34

First, we need to define three matrices as follows:

Interface matrix for j th interface

1 1 rj I  (Appx. 1) (jj 1) r 1 t j j

In equation (Appx. 1), rj and t j are Fresnel reflection and transmission coefficients.

For p-polarization

nnjcoscos j11 j j 2 rj  Rr (Appx. 2) nnjcoscos j11 j j

For s-polarization

134

nnjjjj11coscos rj  (Appx. 3) nnjjcoscos jj  11

Based on the Snell’s law, we can correlate all the incident angles with refractive indices by

n0sin 0 n 1 sin  1    njj sin  (Appx. 4)

Layer matrix for j th layer

eibj 0 L  (Appx. 5) j ib 0 e j

Where bj 2 d j n j cos  j /  .

135

The scattering matrix including the reflection and transmission properties of multilayer structure

SS11 12 SI L I L I01 L 1 I 12 2 (m 1) m m m ( m 1) (Appx. 6) SS21 22

What we need to obtain from modeling is the reflectivity R ,

Rr 2 (Appx. 7) where r is the reflection coefficient which can be calculated from the scattering matrix

by r S11/ S 21 .

At last, we average out p- and s- polarization reflectivity to get the modeled reflectance.

136

APPENDIX B: COPYRIGHT NOTICE

This dissertation is based on the following papers:

[1] Chapter I: Xiao, M.; Shawkey, M. D.; Dhinojwala, A., Bio-Inspired Melanin-Based

Structural Colors. In preparation.

[2] Chapter II: Xiao, M.; Dhinojwala, A.; Shawkey, M., Nanostructural Basis of

Rainbow-Like Iridescence in Common Bronzewing Phaps chalcoptera Feathers. Opt.

Express 2014, 22 (12), 14625-14636.

This paper is under the open access.

[3] Chapter III: Xiao, M.; Li, Y.; Allen, M. C.; Deheyn, D. D.; Yue, X.; Zhao, J.;

Gianneschi, N. C.; Shawkey, M. D.; Dhinojwala, A., Bio-Inspired Structural Colors

Produced via Self-Assembly of Synthetic Melanin Nanoparticles. ACS Nano 2015, 9 (5),

5454-5460.

This paper is an open access article published under an ACS Editors' Choice License

[4] Chapter IV: Xiao, M.; Li, Y.; Zhao, J.; Wang, Z.; Gao, M.; Gianneschi, N. C.;

Dhinojwala, A.; Shawkey, M. D., Stimuli-Responsive Structurally Colored Films from

Bioinspired Synthetic Melanin Nanoparticles. Chem. Mater. 2016, 28 (15), 5516-5521.

137

[5] Chapter V: Xiao, M.; Hu, Z.; Wang, Z.; Li, Y.; DiazTormo, A.; LeThomas, N.; Wang,

B.; Gianneschi, N. C.; Shawkey, M. D.; Dhinojwala, A., Bio-Inspired Bright Non- iridescent Photonic Melanin Supraballs submitted.

138

APPENDIX C: AUTHOR PROFILE

Ming Xiao is a PhD candidate at the Department of Polymer Science, The

University of Akron. He received his Bacholor of Engineering Degree in Polymer

Materials and Science from Sichuan University in Chengdu, China, in 2012. His research focuses on bio-inspired melanin-based structural colors through self-assembly. During the PhD, the author has published five papers and has three more manuscripts submitted.

This manuscript is typed by the author.

139