CONVECTIVE ASSEMBLY OF ROD-SHAPED MELANOSOME IN DILUTE
POLYMER SOLUTION
A Thesis
Presented to
The Graduate Faculty of The University of Akron
In Partial Fulfillment
of the Requirements of the Degree
Master of Science
Jiuzhou Zhao
May, 2016 CONVECTIVE ASSEMBLY OF ROD-SHAPED MELANOSOME IN DILUTE
POLYMER SOLUTION
Jiuzhou Zhao
Thesis
Approved: Accepted:
______Advisor Dean of the College Dr. Ali Dhinojwala Dr. Eric J. Amis
______Co-Advisor Dean of the Graduate School Dr. Matthew Shawkey Dr. Chand Midha
______Department Chair Date Dr. Coleen Pugh
ii ABSTRACT
Bird feathers have developed most diverse structural colorations. Many of them are produced by orderly packing melanosomes (submicron melanin-filled organelles) structures. These melanosomes come in different morphologies such as spherical, rod-like, and disk-like shapes and they may be solid or hollow. But it is still unclear how such ordered structures of anisotropic melanosomes are formed and it is also challenging to mimic those structures using in-vitro assembly technique. In this work, we extracted rod-shaped melanosomes from crow feathers and investigated their assembly behavior in the evaporating sessile droplets containing polyvinyl pyrrolidone (PVP) or polyethylene oxide (PEO). We used zeta potential to monitor the interaction between polymer and melanosomes, high-speed video to track melanosome movement during evaporation, and optical/electron microscopy to study the packing behavior of melanosomes. We have found melanosomes packed densely at the deposition edge, forming a “coffee ring” pattern after drying PEO-melanosome droplet. While at the same position of PVP-melanosome solution the coffee ring effect was suppressed and melanosome did not form densely packing structure. This is mostly due to the adsorption of PVP on melanosome surface that can bring the particle to the air-water interface instead of arriving at contact line. This work helps
iii our better understanding of anisotropic particles assembly in polymer solution and also makes it possible towards mimicry of the structures of bird feathers.
iv ACKNOWLEDGEMENTS
I would like to thank my advisor, Dr. Ali Dhinojwala, and my committee member, Dr.
Matthew Shawkey, for their encouragement and guidance on my work. I really enjoy the process of scientific research. Also, I want to express my gratitude to my mentor,
Ming Xiao for teaching me a lot of basic knowledge and the operation method of many machines. I cannot learn so many things without his help at the beginning of my experiment. I appreciate the help from all the group members in Dr. Dhinojwala’s lab and Dr. Shawkey’s lab. We had a great time working together in the past two years.
I am deeply grateful to my parents for their support. They give me everything without preconditions so that I can finish this work with all my efforts.
v TABLE OF CONTENTS Page
LIST OF FIGURES…………………………………………………………………viii CHAPTER I. INTRODUCTION………………………………………………………………..1 1.1 Structure Color……………………………………………………………………1 1.1.1 Melanosome and Melanin…………………………………………………1 1.1.2 Structure Color on Avian Plumage ……………………………………..3 1.2 Coffee Ring Effect………………………………………………………………..6 1.2.1 Capillary Flow……………………………………………………………...7 1.2.2 Marangoni Flow……………………………………………………………8 1.3 Description of This Work ……………………………………………………….10 II. EXPERIMENT……………………………………………………………………12 2.1 Material…………………………………………………………………………..12 2.1.1 Extraction of Natural Melanosome……………………………………….12 2.1.2 Clean Silicon Wafer………………………………………………………14 2.1.3 Water-Soluble Polymer…………………………………………………...14 2.2 Experiments………………………………………………………………………15 2.2.1 Characterization Method………………………………………………….15 2.2.2 Experimental Detail……………………………………………………….15 2.2.3 Detection on Experiment Solution………………………………………..16 2.2.4 High-Speed Video and Scanning Electron Microscopy…………………..17 III. RESULTS AND DISCUSSION…………………………………………………18 3.1 Morphology and Size of Melanosomes…………………………………………..18 3.2 Assembled Patterns after Drop Casting………………………………………….19 3.3 High Speed Video………………………………………………………………..24
vi 3.4 Influence Factors Analysis ……………………………………………………….28 3.4.1 Effect of Particle Charge………………………………………………….28 3.4.2 Effect of Viscosity………………………………………………………...30
3.4.3 Effect of Surface Tension…………………………………………………31 IV. CONCLUSION………………………………………………………………...... 32 V. FUTURE WORK…………………………………………………………………34 REFERENCES……………………………………………………………………….35
vii LIST OF FIGURES
Figure Page 1.1-1. Biosynthetic way of eumelanin and pheomelanin……………………………..2
1.1-2. Generation process of melanosomes……………………………………………3
1.1-3. The breast feathers of male bird change color from (A) yellow-orange to (B) blue-green by changing the observe angle of barbules. (C) Transverse section gives the angle of the bent is nearly 120 degree. The span of right hand slope of 15 layers is 8 micrometers. (D) Cross section model of Parotia Lawesii barbules…………………………………………………………………………4
1.1-4. SEM images of cortex cross section in barbules for green (a) and brown (b)….5
1.1-5. (a) Pica Pica with a long tail; (b) Cross section of a barbule; (c) Structure model……………………………………………………………………………6
1.2-1. Coffee Ring Effect…………………………………………………………...... 7
1.2-2. Cross Section of Capillary Flow Regime……………………………………….7
1.2-3. Tears of wine……………………………………………………………………9
1.3-1. Radially arrangement due to the phase separation…………………………….10
1.3-2. Slow evaporation for high aspect ratio silica rods…………………………….11
2.2-1. Drop Casting Method………………………………………………………….16
3.1-1. SEM image of extracted melanin particles……………………………………18
3.2-1. Optical and SEM images for melanosome solution …………………………..19
3.2-2. Optical & SEM image for PEO-Melanosome solution. Form top to bottom the PEO concentration is 0.002 mg/mL, 0.004 mg/mL, 0.02 mg/mL. The evaporation condition is 21°C, 15% relative humidity………………………..20
viii 3.2-3. Optical & SEM image for PVP-Melanosome solution. Form top to bottom the PVP concentration is 0.002 mg/mL, 0.004mg/mL, 0.02 mg/mL. The evaporation condition is 21°C, 15% relative humidity………………………..21
3.2-4. Order parameter for PEO-melanosome solution under 15% RH, 21℃……….23
3.2-5. Particle-packing density under 15% RH, 21℃………………………………..24
3.3-1. Particle speed under 15% RH, 21°C condition………………………………..25
3.3-2. Snapshot of melanosome solution at start……………………………………..26
3.3-3. Meniscus thickness of different solutions……………………………………..27
3.4-1. Zeta potential for different solutions…………………………………………..29
3.4-2. Surface tension measure for different solutions……………………………….31
ix CHAPTER I
INTRODUCTION
1.1 Structure Color
Structural coloration is the production of color by microscopically structured surfaces
fine enough to interfere with visible light, sometimes in combination with pigments.
In bird feathers, the colors have close relationship with melanin.
1.1.1 Melanosome and melanin
Melanin is a general term for a series of pigments that widely existed in organisms.
The mainly two types of melanin are eumelanin and pheomelanin.1 They are generated by different precursors in biosynthetic pathway. Figure 1.1-1 shows the current understanding of the very beginning step of melanin biosynthetic pathways.1
Because of the chemical structure of eumelanin and pheomelanin are different, their
synthesis way also different.
1 Figure 1.1-1 Biosynthetic way of eumelanin and pheomelanin1
Melanosomes are organelles found in living body tissues throughout the kingdom of animals. And melanosomes also have different shapes: diameter until 1µm sphere particles, and ellipsoid particles with scale up to nearly 2 µm, but varies in aspect ratios.1 Melanosomes also have many different functions. It has strong adsorption of
UV-visible light, low quantum yield of radioactive, good property of anti-oxidize, and
2 Figure 1.1-2 Generation process of Melanosomes2 it can get rid of the free radicals.3 Melanosomes contain varieties of biomolecules such as lipids and proteins. But the main constituent is melanin. The functions that melanosomes have mainly attribute to the chemical properties that melanin have.
Recent research found melanosomes generate from vesicles.2 The whole process is shown in Figure 1.1-2. This kind of vesicles created by the trans-Golgi network. High degrees of dendritic melanocytes under through a series of morphology change stages due to the network contain tyrosinase. The whole procession begins with pigment precursors and ends with the membrane-enclosed bladder of melanin.
1.1.2 Structure color on avian plumage
In avian feather barbules, the nanostructure of air, keratin, and melanosomes are well organized, which generates iridescent colors.4 Here is the picture of breastplate feathers of male bird of paradise (Parotia lawesii). The feathers display color changes from yellow-orange to blue-green when viewed from different angle of barbules on
3 feather tips.4 And the color showing on this kind of birds is really beautiful so that it’s
attractive people to explore why it can generates this color.
Figure 1.1-3 The breast feathers of male bird change color from (A) yellow-orange to (B) blue-green by changing the observe angle of barbules. (C) Transverse section gives the angle of the bent is nearly 120 degree. The span of right hand slope of 15 layers is 8 micrometers. (D) Cross section model of Parotia Lawesii barbules.5
These kinds of feathers have unique thin-film and multi-film interference combination
in single feather barbules. Melanin multi-layers in the barbules can generate
yellow-orange reflection and the cortex play an important role as an outside thin-layer
that can reflect blue color side beams. This special property is mainly due to its bent-shape cross section, which is shown in Figure 1.1-3 (C). And this structure can work as a mirror that can create three kinds of colors: one orange-yellow reflector within the feather plane, two symmetrically bluish reflectors at the angles respectively about 30 degree. This kind of plumage can let the bird of paradise to get more suddenly color change when they keep flying than they are in static condition.4
4 Another mechanism of showing iridescent feathers is 2-D photonic crystals, which
incorporated on the barbules, for example, the peacock feathers. The study shows that cortex contain 2-D photonic crystal in peacock feather barbules should take charge for the coloration.6 In the green, blue, and yellow barbules, the shape of lattice is approximately square (Figure 1.1-4 a). While in brown barbules, it shows the rectangular lattice (Figure 1.1-4 b). The differences between them are lattice constant and period’s number toward the direction of cortex surface. Next part is the 2-D photonic crystal to creat structure color.
Figure 1.1-4 SEM images of cortex cross section in barbules for green (a) and brown (b)6
Figure 1.1-5 shows the specific picture of 2-D photonic crystal structure in nature.
The black-billed magpie bird has iridescent yellow-green color tail and blue color
reflector on the wings dark areas. The picture of cross section shows the central core
are surrounded by the cortex. And cortex is the thin-film about melanin and keratin
containing cylindrical holes of air. According to the picture, we can see parallel
5 Figure 1.1-5. (a) Pica Pica with a long tail; (b) Cross section of a barbule; (c) Structure model7
channels contains an lattice of hexagonal. Relatively, the jungle crow give a very
simple structure about melanin granules.4
1.2 Coffee ring effect
If a drop of coffee drips on the table, after all the solvent evaporated, there is a ring-shape pattern leaving on the table instead of a homogeneous circle shape (Figure
1.2-1). The phenomenon is not limited to coffee droplet. The ring-shape pattern forms as long as the droplets contain colloids dispersed in the solution. Two of flow scheme are widely used to explain this phenomenon: Capillary flow (driving force is continuity) and Marangoni flow (driving force is gradient of surface tension).8
6 Figure 1.2-1 Coffee Ring Effect9
1.2.1 Capillary flow
Figure 1.2-2 Cross Section of Capillary Flow Regime9
When a droplet drips on the solid substrate, the edge of circle will get pinned at the three phase contact line (Three phases are gas, liquid and solid). The evaporation happens faster at the edge of the droplet, leading to radially flow from the center bulk liquid to the edge of the droplet to make compensation of evaporated liquid. When the contact line gets pinned, the radius of the ring will not change. The outward flow will bring the particle to the contact line and finally deposit at the edge of ring, forming a ring-shape pattern. So this is the coffee ring effect. It can be observed as long as 1) contact line get pinned at the first location, 2) the contact angle of droplet and surface
7 is not zero, 3) solvent can evaporate.9 Figure 1.2-2 shows the evaporate condition in this kind of flow regime. Picture (a) indicates that if solvents evaporate without compensation flow, the droplet will shrink and the radius will decrease. Picture (b) shows that with the compensation flow, the radius of droplet can keep constant. That is why particle get deposit at the contact line.9
1.2.2 Maragoni flow
When liquid film affected by external disturbances, such as temperature, concentration change. A part of liquid film will attenuate. There will be a flow from thick film to thin film due to surface tension gradient. This is Marangoni flow. It was first observed as “tears of wine” (Figure 1.2-3). Because usually there is some wine attach to the glass above the liquid level. Due to the alcohol has faster evaporation rate than water, so the surface tension difference generate between bulk wine and the wine film on the glass. The fluid tends to flow away from the lower surface tension region. So the wine will flow from the regions that have higher concentration of alcohol because water has larger surface tension. But due to the effect of gravity, the flow will return to the bulk liquid, forming the tear of wine. The Maragoni flow is caused mainly by surface tension.
8 Figure 1.2-3 Tears of wine20
The effect of Marangoni flow is just opposite to the effect of capillary flow.8 Due to
previous analysis, in the capillary flow regime, outward flow can keep a constant
radius of droplet. But Marangoni flow can cause the fluid inward. As we know, with
the temperature increase, the surface tension will decrease. In a whole droplet, the
bottom of liquid has lower temperature while the top of it has higher temperature. The
temperature difference causes the surface tension difference. So the surface tension
gradient is generated due to the temperature gradient. Such gradient can create shear
stress that can drags the fluid along droplet interface.10 Also it can set up the recirculating flows. Temperature will change in the process of evaporation, but it is non-uniform change along the droplet surface due to the non-uniform of evaporate speed and non-uniform of the heat transfer speed from inside droplet to the surface of droplet. All of the non-uniformity is the influenced factor of the droplet shape. The very important factor is the ratio of radius to height of the droplet. Smooth droplet has the faster evaporation at the edge than the center. The thermal conduct between the droplet and solid substrate can also affect temperature gradient on the droplet surface.
9 Depend on the two kinds of speed, Marangoni flow can cause outward flow that can
bring particle to the contact line, and also can cause inward flow that can bring
particles to the center of the droplet.10
1.3 Description of this work
In material science, coffee ring effect often used to arrange nanoparticles into ordered structure at the edge of ring. Here is an example that the author disperse polystyrene sphere particle into the PVP solution and during the evaporation, the stripe pattern shows at the periphery11. The pattern was shown in Figure 1.3-1.
Figure 1.3-1. Radially arrangement due to the phase separation11
Here is another example to certify coffee ring effect that could help particle form
order structure by increasing humidity using high aspect ratio (nearly 12) silica
particle. They first disperse silica rods into water and evaporation process last for
nearly 3 hours.12 The order structure report by them shows in Figure 1.3-2.
10 Figure 1.3-2. Slow evaporation for high aspect ratio silica rods12
Based on this effect, melanosomes could also form ordered structure in polymer
solution. In this project, drop-casting method would be used to assemble melanin
particles. As far as we know, no one has tried to order anisotropic melanosomes in
polymer solution before. Because the formation of order structure of melanosomes in keratin matrix for bird feather barbules is still an open question. We hope this work could provide some insights to this open question. The effect of different polymer on assemble behavior of melanosomes is the purpose of this work. It will help us to learn more about the ordered structure of melanosomes in bird feather barbules.
11 CHAPTER II
EXPERIMENT
2.1 Materials
As described in abstract part of this thesis, the material we used in this experiment are
melanosomes extracted from crow feathers and 2 kinds of water soluble polymer.
2.1.1 Extraction of natural melanosome
We extracted rod-like shaped melanosomes from crow feathers based on a modified
protocol from the previous study.13 The whole process took seven days.
Day 1: The feathers were soaked into acetone 2 times, each time for 5 minutes. Then
immerse the feathers into the dichloromethane and ether one after another, each
solvent for 5 minutes. After that, wash the feathers according to the following
sequences: acetone, water, and acetone. The feather was dried at room temperature in the vacuum. Cut feathers into small pieces (nearly 2 mm) and put them into glass tube.
In this work, total 2.376g feathers were used. Then added 47.52 mL 0.1M phosphate
12 buffer (pH is 7.4) and 0.4752 g DTT into the tube. After degassing the solution by nitrogen, put the tube into the shaker. The reacted condition is 37 centigrade and rotor speed is 150 r/min.
Day 2: We added 1.188 mL (20mg/mL) Proteinase-K and 0.2376 g DTT into the tube.
After degassed by nitrogen, reacted for 20 h under the same condition as previous described.
Day 3: The product were rinsed by water for 6 times and centrifuged at 4000 g for 15 minutes after each rinse. And finally suspended pellet in 40 mL phosphate buffer and
19 mg Papain and 95 mg DTT. After degassed by nitrogen, reacted for 20 h.
Day 4: We rinsed the production by water for 6 times and centrifuged at 4000 g for 15 minutes after each rinse. Finally suspended the pellet in 19 mL phosphate buffer and
0.4752 mL (20 mg/mL) Proteinase-K and 38.016 mg DTT. After nitrogen degassed, reacted for 20 h.
Day 5: The product was washed by water and centrifuged at 4000 g for 15 min. Then added 7.128 mL phosphate buffer and 2% (volume fraction of buffer) Triton X-100.
Degassed by nitrogen and reacted at same condition for 4h. After reacted, centrifuged at 18000 g for 15 minutes. And washed the product by methanol 3 times and water 3 times. After each wash, product was centrifuged at 4000 g for 15 minutes. Then added
19 mL phosphate buffer and 0.4752 mL (20 mg/mL) Proteinase-K and 38.016 mg
DTT. After degassed by nitrogen, reacted for 20 h.
Day 6: We put the outcome tube into Branson 1510 sonicator for 5 minutes. Filtered with filter paper Grade 2. Centrifuged the filtrate at 4000 g for 15 minutes. Washed
13 the product 3 times by water and centrifuged at 4000 g for 15 minutes after each
washing. Then added 9.504 mL buffer and 0.2376 mL (20 mg/mL) Proteinase-K and
19 mg DTT. Degassed by nitrogen, and reacted for 20 h.
Day 7: The product were washed 3 times by water and centrifuged at 4000 g for 15
minutes after each wash. Stock the solution into the fridge and measure the
concentration.
2.1.2 Clean silicon wafer
Cut the silicon wafer into nearly 0.8×0.8 cm2 pieces. Use Piranha solution (70% volume fraction concentrated sulfuric acid and 30% hydrogen peroxide mixture) treatment to clean the silicon wafer. After that the wafers were rinsed in DI water for several times and sonicated in the sonicator with DI water. Use nitrogen to blow dry.
2.1.3 Water-soluble polymer
The principle of choosing polymer is: 1) should dissolve in water; 2) contain oxygen or hydroxyl group and some other atom such as nitrogen. These polymers have good solubility in water and contain some group that may have some interaction with melanosomes. That’s why they were used in this experiment. The following polymers were all purchased from GAF Chemicals Corporation and Aldrich Chemical
Company and were used directly.
14 1. PVP: polyvinyl pyrrolidone. The molecular weight of PVP used in experiment was
40,000.
2. PEO: polyethylene oxide. The molecular weight of PEO used in experiment was
20,000.
2.2 Experiments
In this project, the mainly method is drop casting of melanosome-polymer solution.
And we use SEM and optical to observe the deposition pattern. Also some supplementary experiment had been done to support our theory.
2.2.1 Characterization method
The melanin particles were characterized by SEM (scanning electron microscope)
JSM-7401F. And the deposition pattern was characterized by SEM and optical microscope (Olympus DP70).
2.2.2 Experimental detail
The drop casting method was used here to assemble melanin particles (Figure 2.2-1).
It is a simple way to help particle form order packing.
15 Figure 2.2-1 Drop Casting Method14
Firstly, the melanosome solution and polymer solution were mixed together and ultrasonic agitated for 30 seconds to form homogeneous dispersion. After that, certain volume of mixed solution was draw by a pipette, usually 2µL, and dripped onto the silicon substrate that had been cleaned according to the procedures described before.
Then let the water evaporated under dry and humidity conditions.
2.2.3 Detection on experiment solution
To investigate the effect of adding polymer into evaporating liquid drop containing crow melanosomes, we chose polyethylene oxide (PEO, 20k) and polyvinyl pyrrolidone (PVP, 40k) as model water-soluble polymer. The concentration of melanosomes was kept to be 0.02mg/mL and vary the concentration of polymer from
0.002 mg/mL to 0.02 mg/mL. The zeta potential value were measured by the
Zetasizer (Malvern Instrument, ZEN3690) from electrophoretic mobility at 25°C using the Smoluchowski model approximation. All measurements repeated 3 times on
separately prepared solutions. We also measured the surface tension using DroImage
software by pendent drop model. Each solution was measured three droplets and each
16 droplet was measured ten times every 5 seconds. We also measured the polymer concentration at 0.1 mg/mL and 0.2 mg/mL. The water evaporated at relative humidity of 15% and 23°C.
2.2.4 High-speed video and scanning electron microscopy
To explore the motion of anisotropic melanosomes during drying and how the deposition patterns formed, we used high-speed camera (Photron 120K-M2) to take videos for evaporating sessile droplet of all solutions. The video was taken under 60 frame/s and the resolution was 512*512 pixels. The images of final deposition pattern were recorded by optical microscope and scanning electron microscope. ImageJ
(http://imagej.nih.gov/ij/) was used to measure the orientations of particles relative to the edge of the deposit. The order parameter of deposited particles at the edge was calculated using the equation s=!(3
17 CHAPTER III
RESULTS AND DISCUSSION
3.1 Morphology and size of melanosomes
Figure 3.1-1. SEM image of extracted melanin particles
From SEM image (Figure 3.1-1), we can see individual rod-like shaped melanosomes with removal of keratins. We measured size of at least 50 melanosomes using software Image J: the average length was 1.56 ± 0.23 µm and the average width was
0.24 ± 0.07 µm (average aspect ratio is 6.5).
18
3.2 Assembled patterns after drop casting
Firstly, we did drop casting for melanosome solution (0.02 mg/mL) under room condition (15% RH and 21℃). Figure 3.2-1 showed it formed ring deposition pattern.
In SEM images rod shaped melanosomes formed densely packing structure at the edge of the ring.
Figure 3.2-1 Optical and SEM images for melanosome solution
Peter J. Yunker et al. have reported that whena sessile droplet containing ellipsoids
(aspect ratio of 3.5) dries, coffee ring effect was suppressed and a uniform disk shaped pattern left on solid substrate.16 This is because the attraction force between anisotropic particles is stronger than the sphere shape particles and it is easily to form cluster. The energy that needs to break this cluster is large so it makes particles harder to move. They also provide video that shows that the particles cluster resist the outward capillary flow.
But in our experiment, the high aspect ratio rod-shaped particles can form coffee ring structure. There are two possible reasons. Firstly, the particle shape difference leads to different deposition pattern. Because the ellipsoids are not easy to form densely
19 packing structure (side by side) and rod-shaped particles like melanosomes can form
this kind of packing easily. Secondly, it may due to the concentration difference of
particles cause the pattern difference. In our experiment, the particle concentration
was controlled at 0.02 mg/mL for the convenience of tracking single particle. Maybe
this concentration is not enough to form clusters resisting the outward flow.
Figure 3.2-2 Optical & SEM image for PEO-Melanosome solution. Form top to bottom the PEO concentration is 0.002 mg/mL, 0.004 mg/mL, 0.02 mg/mL. The evaporation condition is 21°C, 15% relative humidity.
To detect the effect of dilute polymer solution on particle convective assemble
process, we added different concentration of polymer into melanosome solution
20
according to certain proportion and performed drop casting in same condition (15%
RH and 21℃). As shown in Figure 3.2-2 d-f, PEO-melanosome solution can form condensed packing structure at the ring edge.
Figure 3.2-3 Optical & SEM image for PVP-Melanosome solution. Form top to bottom the PVP concentration is 0.002 mg/mL, 0.004mg/mL, 0.02 mg/mL. The evaporation condition is 21°C, 15% relative humidity.
Although the evaporation condition is as same as PEO-melanosome solution, the deposition pattern of PVP-melanosome solution is totally different from
PEO-melanosome solution. With low concentration of PVP (Figure 3.2-3 d and e), melanosomes dispersed on the substrate instead of forming densely packing structure
21
which appeared in PEO solution (Figure 3.2-2 d-f). And for the highest concentration of PVP (Figure 3.2-3 f), the number of particles deposited at the ring edge is smaller than same concentration of PEO (Figure 3.2-2 f).
From optical images we can see when polymer concentration is higher (Figure3.2-2 b, c and Figure 3.2-3 c), the deposition pattern shows multi-ring structure. The droplet containing particles pinned on surface because of the wetting hysteresis. With the evaporation of liquid, the contact angle decreases, which leads to extra free energy.
When this energy is large enough to overcome hysteretic energy barrier, the contact line will retract and get pinned again in the form of same contact angle but smaller droplet radius. This process repeats several times and the multi-ring structure leaves on the substrate.
To characterize the regularity of the particles at the ring edge, we used a concept called order parameter. It was used to describe the rod molecule in nematic liquid crystal. In this type of liquid crystal, almost all the molecules align along same direction. The mean value of this direction can be used as unit vector. The degree of orientation can be described by order parameter, as shown in the equation s=!(3
22
angle difference between each individual particle and the united vector was calculated, we use the equation above to obtain the order parameter. Each condition we took 10
SEM images to calculate order parameter separately and get the average value and the standard deviation. We only calculated the order parameter for PEO systems, due to non-close packing of melanosomes in PVP systems. (Figure 3.2-4) The information shows here is under low humidity condition (15%RH), the order parameter is almost constant (0.55 ± 0.05) when we add PEO into melanosome solution.
0.8
0.7
0.6
0.5
0.4 Order ParameterOrder
0.3
0.2 0.000 0.005 0.010 0.015 0.020 PEO Concentration (mg/mL)
Figure 3.2-4 Order parameter for PEO-melanosome solution under 15% RH, 21℃
But for PVP-melanosome system, where melanosomes cannot form densely packing structure, we used particle packing density to describe the influence of PVP on coffee ring effect, which is defined as the number of melanosomes per unit area. In each
SEM image, we selected a rectangular area (30 µm × 9 µm) that starts from the outmost ring edge of the deposition pattern by ImageJ. We also calculated the particle packing density of PEO-melanosome solution for comparison. The result shows in
23
Figure 3.2-5. We know pure melanosome solution deposition pattern is coffee ring and particles packing densely at the edge, and the particle density of melanosome solution is (0.74 ± 0.1) num/µm. When we added PVP into the solution, the particle density decreased obviously at all the PVP concentration. The particle packing density for PEO-melanosome solution only decreased at the concentration of 0.002 mg/mL and all of the value are larger than PVP. This result demonstrated that PVP help in reducing coffee ring effect. And the video can used as the directed evidence to illustrate the deposition formation process.
0.9 PVP PEO 0.8 ) 2 0.7
0.6
0.5
0.4 Pckingdensity(num/um 0.3
0.2 0.000 0.005 0.010 0.015 0.020 Polymer Conc. (mg/mL)
Figure 3.2-5 Particle-packing density under 15% RH, 21℃
3.3 High speed video
To understand what happened during the droplet evaporated that can lead to those different packing, we used high-speed camera to record the drying process. We took videos for all kinds of solutions and by tracking melanosomes, the coordinate of particles in each frame would be recorded. After plotting the coordinate that is along
24
the particle moving direction with time, we can use linear fitting to get the slope, which is the particle velocity.
PVP 30 PEO
25
20
15 ParticleSpeed (um/s)
10
0.000 0.005 0.010 0.015 0.020 Polymer Conc. (mg/mL)
Figure 3.3-1 Particle speed under 15% RH, 21°C condition
Without polymer, the average velocity of particles is around 25µm/s. When we started to add polymer, the velocity of particle in PVP solution increase while in PEO solution it decrease. And when we continue to add polymer, the particle speeds in both solutions start to decrease. In the highest concentration of polymer solution, the behaviors of two kinds of polymer are different again. Particles velocity in highest concentration of PEO increased while for PVP it decreased. When we analyzed the videos, we found that for particles in two lower concentrations PVP solution, they cannot even reach the contact line of droplet. We first hypothesized it is because the difference of water thickness near the contact line that will lead to different packing in the deposition pattern. Due to the interference of light, it will form bright and dark stripe patterns near the contact line area. See Figure 3.3-2, this is the snapshot of
25
melanosome solution without polymer at start of video. We measured the distance between center of the first dark stripe and the center of third dark stripe. Imagine that the contact angle is θ, which can represent the water thickness, also we know that water thickness t at darkest point can be calculated by the following equations:
2t=(x+!)!.17 Here λ is the wavelength of the light and n is the refractive index of ! ! water, and x=0,1,2… While t3-t1=λ/n, and tanθ=(t3-t1)/d, d is the horizontal distance between the dark stripe. So the distance between first dark line and third dark line is inverse proportional to contact angle. Here we plot time with 1/d and results shows in
Figure 3.3-2.
Figure 3.3-2. Snapshot of melanosome solution at start
26
0.40 MN PEO0.02 0.35 PEO0.004 PEO0.002 0.30 PVP0.02 PVP0.004 0.25 PVP0.002
0.20
1/d (1/um) 1/d 0.15
0.10
0.05
0.00 -20 0 20 40 60 80 100 120 140 160 180 Time (s)
Figure 3.3-3. Meniscus thickness of different solutions
From the water thickness calculate results we could find that melanosome solution and 0.004 mg/mL PVP solution nearly have the same meniscus thickness, but melanosome solution can form closely packing structure while PVP-Melanosome solution cannot. Therefore, the meniscus thickness may not be the primary reason causing the packing difference.
Through this graph we can also get some more information. As pink curve (0.002 mg/mL PEO) shows, the contact line recedes at nearly 120s, while the blue curve
(0.004 mg/mL) and the red curve (0.02 mg/mL) show their contact line recede at nearly 180s. The particle packing density at the outmost part of the deposition pattern should be determined by two factors: particle speed and the time that contact line first recede. We suppose the second one is the dominant reason cause the particle packing
27
density decreased at 0.002 mg/mL of PEO but the velocity of particles in this solution is larger than the other two concentration of PEO.
3.4 Influence factors analysis
There are many influence factors can affect the melanosome movement during the evaporation process. We chose particle surface charge, solution viscosity and solution surface tension to analysis how these factors influence the particle moving and packing.
3.4.1 Effect of particle charge
We measured the particle charge by measuring the zeta (ξ) potential value of the melanosomes in different polymer solution. We could also use this technique to explore the interaction between melanosomes and polymer. As Figure 3.4-1 shows, the ξ-potential for pure melanosome particles was -46mV. And the absolute value of zeta potential decreased with adding PVP into the solution while with the increasing concentration of PEO, the zeta value did not change significantly. We attributed this difference to the adsorption of PVP chains on melanosome surface. And because the adsorption of PVP on melanosomes, the velocity of particles in highest concentration of PVP is also lower than in the highest concentration of PEO. Here we provide a suppose that PVP have strong interaction with melanosomes than PEO, and
28
melanosomes adsorb PVP polymer chains cannot move to the contact line and get stuck in the air-liquid interface and finally deposited at the area near the contact line.
-25 PVP PEO
-30
-35
-40
Zeta Potential (mV) Zeta -45
-50
0.00 0.05 0.10 0.15 0.20 Polymer Conc. (mg/mL)
Figure 3.4-1. Zeta potential for different solutions
Manos Anyfantakis and co-authors have reported that when negative charged particles disperse in positive charge surfactant solution, surfactant would attach to the particle. The deposition pattern of the mixed solution would not shown coffee ring due to the absorbance of particles at the liquid-gas interface.18 In the
PVP-melanosome systems, PVP similarly was absorbed onto melanosomes surface, causing the non-close packing of melanosomes (reduced coffee ring effect). Because the water soluble polymer are seldom. So we still trying to find anther kind of polymer can reduce melanosome surface charge to perform drop casting again to directly approve this.
29 3.4.2 Effect of viscosity
Assuming that the effect of viscosity were all owing to the polymer, we use following
2 2 equation to calculate viscosity: η=η0(1+[η]C+kH[η] C +...) η is the viscosity of
solution, η0 is the viscosity of solvent, [η] is the intrinsic viscosity of polymer, C is
the concentration of polymer, kH is Huggins parameter. The intrinsic viscosity of polymer could be obtained by Mark-Houwink equation: [η]=KMα, K and α are
constant value for certain polymer and M is viscosity average molecular weight,
which is similar to weight average molecular weight. Through calculation we can see
that at this low concentration of polymer, the increasing of viscosity is limited. So the
effect is not significant in bulk liquid but at the edge of the droplet, with evaporation
of water, the viscosity of liquid here is increasing. This can help particle get stable
when it arrive at the contact line. From the high-speed video we can see when no
polymer added into solution, particle would move left and right after it arrive at the
contact line. But with the increasing of polymer concentration, melanosomes become
more stable after it arrives at the contact line. Therefore, viscosity is only minor in the
particles assemble process. But if the concentration of polymer is very high, the
deposition will become a disk according to other researchers report.19
3.4.3 Effect of surface tension
The surface tension of different solution was measured by a pendent droplet model.
We can see from Figure 3.4-2 that with the increasing concentration of PVP, the
30 3.4.3 Effect of surface tension
The surface tension of different solution was measured by a pendent droplet model.
We can see from Figure 3.4-2 that with the increasing concentration of PVP, the
surface tension not decreases a lot. But for PEO, it decreased obviously.
66 PVP PEO 64
62
60
58 Surfacetension(mN/m) 56
54 0.000 0.005 0.010 0.015 0.020 Polymer Conc. (mg/ml)
Figure 3.4-2. Surface tension measure for different solutions
As far as we know, adding polymer reduces surface tension, so at this point, the
surface tension may play a role in leading to different packing of particles. But how
this factor affects the process of melanosome assembly is still unclear.
31 CHAPTER IV
CONCLUSION
In this work, PVP and PEO were used as two standard water-soluble polymers to investigate their effects on the assembly behavior of rod-shaped melanosomes during convective process under low humidity condition. Pure rod-shaped melanosomes deposited at the edge of the contact line during water evaporation process. Adding dilute PEO or PVP solution into the melanosomes solution affected the deposition patterns in a different way. In PEO-melanosome solution, melanosomes can still form condensed packing structure at the outmost part of the whole deposition pattern. The particle-packing density of PEO-melanosome solution is similar value at the concentration of 0.004 mg/mL and 0.02 mg/mL as that of pure melanosomes. The order parameter is almost constant with adding PEO. For PVP-melanosome solution, melanosomes formed more disordered structure instead of densely packing when lower concentration of PVP (0.002 mg/mL and 0.004 mg/mL) and the particle-packing density shows that adding PVP could suppress coffee ring effect.
Through combination of high-speed video and zeta potential measurements, we proposed that the adsorption of PVP chains on rod-shaped melanosomes, bringing
32 melanosomes onto the air-liquid interface, resulted in the loose packing structure in PVP-melanosomes system.
33 CHAPTER V
FUTURE WORK
Understanding the effect of high humidity on the melanosomes movement will help in improving the order packing, which paves the way for design of biomimic structural colors. So we need to take video of evaporation process under high humidity and analysis the particle movement as well as particle packing.
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