NANOSTRUCTURED ANTI-BIOFOULING SURFACES: PHYSICAL DEPOSITION OF PS-b-PAA LANGMUIR-BLODGETT FILMS

Biofouling, the accumulation and proliferation of microorganisms, plants, and fouling animals on surfaces in an aqueous environment, poses a significant challenge. For example, the effects of fouling of ship hauls include hydrodynamic drag, increase in fuel consumption by ships whose hulls have been fouled, and increase in frequency of dry-dock cleaning. In the history of marine navigation, varieties of anti-biofouling control measures have been suggested but tributyltin self-polishing copolymer (TBT-SPC) paints have been the most effective and commercially viable option in curbing biofouling. However, leaching of tri-organotin biocides from TBT-SPC paints through self-polishing activity constitute pollution which led to the ban of biocidebase paints. We explored bio-inspired nature of lubricin and fabricated polyelectrolyte polymer brushes from commercially available polymer materials by Langmuir-Blodgett deposition technique (LB fabrication) in order to control grafting density and by ATRP. Interfacial tension results indicate that PS60-b-PAA29, based on steric and electrostatic interaction within the block copolymer, is very stable over ranges of pHand temperatures similar to that of the marine ecosystem. Fluorescence microscope and atomic force microscope imaging, as well as, advancing contact angle measurements on the physically fabricated samples shows that there was successful fabrication of PS60-b-PAA29brushes on glass surfacevia LangmuirBlodgett deposition. While biofouling test is underway on the brushes fabricated by LB deposition technique, preliminary biofouling testing by M. Callow‘s laboratory at the University of Birmingham on ATRP samples indicates that grafting duration (hence, thickness) of polyelectrolyte polymer brush has a direct impact on the film efficiency against biofouling.


LIST OF TABLES
. A main result of biofouling is hydrodynamic drag due to increase in surface roughness of ship hull which causes increase in fuel consumption. Table 1.1 shows the foul rating system used by the US Navy to classify degree of fouling. was estimated to be (US) $400 million -$540 million (Schultz et al, 2010).
In order to combat bio-fouling, biocides are used in surface coatings.
However, biocides themselves are toxic and constitute pollution when leached from the surface. Hence, there is a need to develop non-toxic antifouling surfaces that are economical and scalable. Nanostructured polymer brush coatings may provide such a surface by engaging steric and electrostatic repulsive forces in order to prevent biofouling.

Research objectives
This work is driven by the hypothesis that polyelectrolyte brushes can be fabricated from inexpensive commodity polymers and exhibit antifouling properties through engineered steric and electrostatic interactions combined with nanoscale topography. It has been derived from ongoing collaboration and support through the Naval Undersea Warfare Center (NUWC). To test this hypothesis, we have examined the fabrication of self-assembled amphiphilic block co-polymer brushes composed of poly(styrene)-block-poly(acrylic acid) (PS-b-PAA). The brushes were formed via physical Langmuir-Blodgett deposition on prepared glass slides as model surfaces.
This method (among others such as thermal evaporation, electrodeposition, and sputtering) was chosen because it enables us to control the monolayer thickness, as well as, surface coverage.
The main objectives of this M.S. project are stated below: 1. Examine the surface activity of PS-b-PAA at air/water interfaces under relevant system conditions. The premise behind this objective was to examine a priori the surface pressure of the brushes before depositing them on the substrates. Previous work has shown that film structure at an air/water interface can be transferred onto solid substrates (Currie et al, 2000).
2. Examine the effect of substrate treatment and preparation on PS-b-PAA film deposition. The premise behind this objective was to identify the best surface treatment and preparation method to achieve physisorption of PS-b-PAA film onto the substrate.
3. Fabricate and characterize physically-deposited PS-b-PAA brush coatings on prepared substrates as a function of surface pressure, which sets film morphology.
Characterization was conducted using atomic force microscopy AFM), surface pressure-area isotherm studies, and Uv-vis fluorescence spectroscopy.
4. Fabricate covalently grafted PAA and PS-b-PAA by atom transfer radical polymerization (ATRP), consistent with previous work by Qian Ni, and test the bio-fouling properties of these coatings in Professor Callow's laboratory at The University of Birmingham, UK. This work was intended to 1) test the performance of previously developed coatings and 2) provide a comparison between covalently and physically deposited brushes.
Chapter 2 presents the background of this project. It highlights the evolution of antifouling paints and focuses on the classifications of antifouling coatings such as non-biocide based, biocide-based, and non-toxic technologies.
Chapter 3 itemizes the materials used in this research and explains the methods used. It highlights the surface pre-treatment steps, hydroxylation, application of primer to the surface of the substrate, preparation of the copolymer solution, and explains how to physically deposit PS-b-PAA.
In Chapter 4, results and discussion are presented in detail. This includes an interpretation of the results from surface pressure-area isotherms, contact angle measurements, UV-vis measurements, and fluorescence microscope images.
Conclusions drawn will be presented in chapter 5.
Finally, historical development in monolayer science, information about monolayer characterization, calculation of the volume of the block copolymer required, and detailed description of instrumentations are provided as Appendices.

Targeted biofouling organisms
Marine or freshwater structures such as oilrig platform supports, ship hulls, cooling systems for power plants, culture rafts, and ocean thermal energy conversion systems are usually protected against fouling by coatings with compounds that deter settlement of fouling species (Stupak et all, 2003).  (Almeida et al, 2007).
The microorganisms that cause fouling are small in size when viewed individually, frail in nature, and well adapted to aqueous environments. However, the effects of their activities are very profound economically and environmentally.
Examples of fouling organisms are presented in Table 2.1. The scale of sizes of fouling organisms has been developed by (Magin et al, 2010) as shown below. It has been estimated that the weight of fouling organisms could be about 150 kg/m 2 when they completely cover a surface. This is equivalent to approximately 6000 tons of fouling materials. Typically, for large commercial vessels, the hull has an approximate surface area of about 40,000 m 2 (Howell et al, 2009). Consequently, the effective weight of the ship will be increased causing hull roughness, loss of velocity, reductions in fuel efficiency, and pollution due to greenhouse gas emission.

Classification of antifouling mitigation coatings
In order to control fouling, various methods have been used over the past centuries. Antifouling coatings can be classified into three major categories: biocidebased antifouling coating, biocide-free antifouling coating, and non-toxic technology.
The most successful method is the incorporation of additives with biocidal effects into antifouling paints.  systems are electrical current antifouling system, electrochemical reaction antifouling system, and radioactive antifouling system.

Electrical current antifouling system (ECAS)
Electrical antifouling alternative involves the use of electricity to produce toxic chemicals such as chlorine on ship hulls (Iselin, 1952;Swain, 1998;Huang, 1999). This results in large voltage drop and corrosion of the surface of the ship hull.
In addition, this method causes release of chlorine and organic chlorine derivatives into the ocean leading to localized pollution. Another disadvantage of this method is that uniform dispersion is not feasible leading to inefficient antifouling control (Bertram, 2000).

Electrochemical reaction antifouling system (ECRAS)
ECAS is environmentally unsafe and inefficient. This was one of the main reasons for exploring alternative means of controlling biofouling using the principle of electrochemical reaction to attack fouling organisms. This system uses electron transfer between an electrode and microbial cells resulting in electrochemical oxidation of the intracellular substances (Yebra et al, 2004). Other electrochemical systems used involve the development of conductive paint electrodes that were used to create an electrical potential (Okochi et al, 1995). The effect of the electrical potential is that it killed bacteria and fluctuation of the electrical potential to negative value causes the bacteria to be removed from the electrodes because most bacterial are negatively charged. ECRAS has some limitations. For instance, it is restricted to small scale applications such as control of bacteria fouling (among all fouling organisms) in pipes.

Radiation-based antifouling system (RBAS)
Due to the limitation of ECRAS mentioned above, investigators experimented with radiation-based antifouling system. An example of RBAS includes acoustic radiation (applied by vibration of piezoelectric coatings). Ultra-violet radiation has also been used for sea water sterilization (Swain, 1998). However, the power requirement of this technology is enormous (Swain, 1998); therefore it is not commercially feasible for large scale application.

Biocide based antifouling systems
Early biocide-based antifouling paints contain biocides such as copper, arsenic or mercury oxide. For example, copper is commonly used in antifouling paints as a metal, oxides, sulfides, and thiocyanates (Ranke et al, 1999). Another component of antifouling paint is zinc pyrithione. It is used as the active ingredient in anti-dandruff shampoo and certain antifouling pigments (Ranke et al, 1999). Other biocide-based antifouling paints contain naphtha or benzene (Iselin, 1952). In 1958, it was discovered that tributyltin acrylate ester can be used as an antifouling coating (Gitlitz et all, 1981). For instance, tributyltin acrylate and tributyltin methacrylate were known to be very potent against marine biofouling (Yebra et al, 2004). However, control of marine fouling through antifouling paint application was revolutionized by discovery of tributyltin-self polishing copolymer (TBT-SPC). TBT-SPC antifouling paints contain polymer backbones linked to tributyltin by an ester linkage (Figure 2.3) (Anderson, 1995). The hydrophobic nature of tributyltin prevents water from penetrating through the coating.
In sum, all biocide-based antifouling paints contain at least one or more of the following active ingredients: zinc pyrithione, naphtha, benzene, tributyltin acrylate ester, tributyltin acrylate, tributyltin methacrylate, tributyltin self-polishing copolymers, and combination of copper with metals, oxides, sulfides, and thiocyanates. These ingredients confer toxicity on the antifouling paints that contain them. Therefore, due to the leaching of these toxic ingredients into the marine environment, regulations were enacted to ban their use. The ban has motivated researchers to look for environmentally benign alternatives to biocide-based antifouling coatings. Non-toxic technology antifouling system The antifouling alternatives described thus far either have environmentally negative impacts or economic limitations that cause regulators or the ship industry to restrict their use. A good antifouling alternative must not be toxic, should not be expensive, should not be chemically unstable, and finally, it must be able to prevent fouling from any organism regardless of the species (Chambers et al, 2006). Some non-biocidal alternatives meet the requirement stated above. Non-toxic coatings can be divided into three broad categories, namely (a) foul release coatings, (b) smart coatings, and (c) hard marine coatings (Howell et al, 2009).

Foul-release coatings
Foul-release coatings are coatings that render a surface non-stick and extremely smooth; they confer low friction and low-surface energy characteristics on a surface, thus arresting the formation of biofilm on surface structures that are in contact with water by marine fouling species (CEPE Antifouling Working Group, 1999;Chapman, 2003;Howell et al, 2009).

Smart coatings
Smart coatings are materials that provide specific response to certain external stimuli. In other words, smart coatings can sense their environment and respond appropriately to the stimulus (Baghdachi, 2009). Such stimuli or environmental conditions could be temperature, stress/strain, pH, and ionic strength. Examples of smart coatings are antifouling applications, antimicrobial (in the medical field), stimuli response coatings, self-healing surfaces, self-cleaning, and super hydrophobic/hydrophilic switching coatings (Baghdachi, 2009;Yebra et al, 2004).
A much broader categorization of smart coatings are bioactive coatings (antimicrobial polymers, antifouling coatings, and photocatalytic coatings), nanotechnology-based coatings (self-assembling polymers and coatings, photonics, and molecular electronics), stimulus and response coatings (coatings functioning as sensors, color shifting coatings, and light sensing coatings) and self-assembled intelligent layers (self-repair and healing coatings, super hydrophobic coatings, and molecular brushes) (Tanner, 2005).

Polymer Brushes
Polymer brush describes an arrangement of bulky, polymer macromolecules (consisting of repeated units) that are physically or chemically anchored to a surface on one end. Not all polymer chains immobilized to a surface are polymer brushes. In a polymer brush arrangement, the grafting density is high enough such that the polymer chains are forced to stretch (Zhao et al, 2000).
Polymer brush arrangement confers special characteristics that can be explored in a number of applications such as adhesive materials (De Gennes et al, 1992;De Gennes et al, 1993), surface coatings that controls depositions of biocolloids like protein adhesion to a surface (Amiji et al, 1993;Currie et al, 2002), and as lubricants (Joanny, 1992). They can also be used as chemical gatekeepers and nanomaterial triggers that initiate drug delivery under certain conditions.   Polymer brushes can either be prepared by physisorption or by chemical grafting. There are two types of chemical grafting methods, namely, -grafting to‖ and -grafting from‖. In the chemical grafting method, the polymers are attached to the surface and to each other via covalent bonding.
The -grafting to‖ approach of brush synthesis can be referred to as the topdown method in which the polymerization is performed first to form the polymer chains and then attached to the surface afterwards as shown in Figure 2.6.
Specifically, monomers with reactive terminals are used in making the polymer chains. In order for the reaction to be successful, the surface on which the brush is to be grafted must be -activated‖ in order to accept the incoming polymer chains. One difficulty that is associated with this method of polymer brush fabrication is that after addition of few polymer chains, steric interaction may hinder available sites on the substrate surface from accepting other incoming chains.  Advantages and disadvantages associated with different methods of polymer brush fabrications have been summarized and they are presented in Table 2.2. Physisorption enables one to control the grafting density and surface coverage. In order to make the monolayer, a Langmuir-Blodgett trough (shown in Figure 2.8) can be used. First, a monolayer is prepared by spreading the surfactant, from which the polymer brush is to be made, on the surface of the subphase in the Langmuir-Blodgett trough. The spreading solvent is then allowed to evaporate from the subphase surface.  A substrate that has been pre-treated is then dipped vertically (Langmuir-Blodgett technique) or horizontally (Langmuir-Schaeffer technique) into the monolayer. Upon withdrawing the substrate, polymer brush at the air/water interface is deposited on the surface of the substrate (Figure 2.9).
The block copolymer used in this research is a polyelectrolyte polymer brush (poly(styrene) 60 -block-poly(acrylic acid) 29 (PS 60 -b-PAA 29 )). The major forces acting within the chosen polyelectrolyte brush are long-ranged electrostatic interactions and short-ranged steric repulsion. When PS 60 -b-PAA 29 brush is immersed in water, the PAA chains become negatively charged. These charges are surrounded by cations and the spatial organizations of the charges introduce the Debye screening length (1/k).
The Debye screening length determines the excluded volume between the polymer chains and the conformational behavior of the chain.
PS 60 -b-PAA 29 is a good candidate for non-toxic coating because of several qualities that it possesses: it has been shown to be an affective bioactive implant specifically for use in the oral cavity . It is non-toxic in nature, its tunable charge density stemming from PAA being a weak polyelectrolyte, and it is inexpensive. The polystyrene block in PS-b-PAA is hydrophobic and it provides mechanical support when the block copolymer is attached to substrate's surface.

CHAPTER 3 METHODOLOGY
Deposition of the PS 60 -b-PAA 29 was accomplished by physical deposition using the Langmuir-Blodgett (LB) technique. Chemical deposition using atomic transfer rapid polymerization (ATRP) -grafting from‖ approach was also used to deposit PS-b-PAA. First, I will itemize the materials used followed by methodologies for the physical deposition and chemical deposition respectively.
The solution was heated at 100 o C for 2 hours and then rinsed with deionized water, followed by methanol.

Hydroxylation
In order to maximize the surface of the glass slide, the concentration of hydroxide ion on the surface was increased by soaking the cleaned glass slides in 30:70 mixture of hydrogen peroxide/deionized water (30% H 2 O 2 )  for 45 min at 70 o C. After 45 min, 5mL of ammonium hydroxide was added.
Once cooled, the substrates were rinsed with deionized water and then dried in methanol.
Application of -primer‖ to the surface of glass slides Since we are interested in physically depositing the block copolymer on the surface of the glass slide, the LB film has to stick as soon as it touches the surface of the pre-treated glass slide. In order to promote -stickiness‖, polystyrene was deposited on the surface of the glass slide as follows: 11g/L of polystyrene was prepared by dissolving 275 mg of polystyrene in 25mL of chloroform. The solution was poured on the surface of glass slides in a beaker. The beaker was placed under the hood for 3 hours to 12 hours in order to allow complete evaporation of the chloroform. The polystyrene coated glass produced is then placed in a vacuum oven (using pressure ≥ 25mmHg) and heated at 150 o C for 3 days. This process will anneal polystyrene to the glass slides.
Once the annealing step was completed, the slides were washed with chloroform to remove excess polystyrene. The slides were allowed to sit under the hood for as long as necessary so the chloroform could evaporate.
Preparation of PS 60 -b-PAA 29 The block copolymer was prepared by dissolving 25 mg of PS 60 -b-PAA 29 in 15mL of 1,4-dioxane. The solution was heated at 60 o C for 2 days. Heating the solution allows all the PS 60 -b-PAA 29 powders to dissolve. 10 mL of toluene was added. The resulting solution was shaken to facilitate proper mixing.
Physical deposition of PS 60 -b-PAA 29 Prior to the deposition of PS 29 -b-PAA 60, the self-assembly monolayer (SAM) of PS 60 -b-PAA 29 was prepared by spreading 90µL of PS 60 -b-PAA 29 at the air/water (A/W) interface. The monolayer was allowed to settle for 15-30 min before transferring the block copolymer to the surface of the polystyrene-modified glass. The deposition pressure was determined by looking at the Baier curve ( Figure   3.2); a SAM deposited at a surface tension of 22-24mN/m will give a brush-modified surface with the lowest fouling (Magin, 2010).

Chemical deposition methodology
Surface pre-treatment The solution was subjected to heat at 100 o C for 2 hours and then rinsed with deionized water followed by methanol.

Hydroxylation
In order to maximize the surface of the glass slide, the concentration of hydroxide ion on the surface was increased by soaking the cleaned glass slides in 30:70 mixture of hydrogen peroxide/deionized water (30% H 2 O 2 )  for 45 minutes at 70 o C. After 45min, 5mL of Ammonium Hydroxide was added.
Once cooled, the substrates were rinsed with deionized water and then dried in methanol.

Silane modification
Place the freshly cleaned and hydroxylated glass slides into a 3-neck round bottom flask, add 270 mL of anhydrous toluene and install the reflux condenser.
Close the openings with rubber septa. Flush with nitrogen for 30 minutes. Add 2.7 mL of 3-(Trimethoxysilylpropyl)-2-bromo-2-methylpropionate (TMSPBMB) and heat under reflux at 60 o C for 4 hours. After 4 hours, stop the reaction and remove the silane modified glass slides. Wash with toluene, ethanol, and dry in a stream of nitrogen.
PAA modification of glass (Two 3-neck flasks were used for this reaction) Flask 1: Silane modified glass slides were placed in the 3-neck flask and sealed with airtight rubber septa on the outer openings while the middle opening is fitted with a condenser that connects to a running tap water and the sink for discharge.
Flask 2: Acetone, Cu(I)Br, tert-butyl acrylate, and stir bar in the amount specified in Table 3.2 was added to the 3-neck flask and sealed with rubber septa.
Place the flasks on two separate hot plates. Connect the flasks with cannula and insert needles into each flask to allow the escape of gas.  Hydrolysis of poly(tert-butyl acrylate) to poly(acrylic acid) Poly(tert-butyl acrylate) modified glass slides were placed in a round bottom flask, 20 mL of dioxane and 3 mL of concentrated hydrochloric acid were added to the flask. The mixture was then heated under reflux at 100 o C for 4 hours. Upon completion, the solution was allowed to cool, the glass slides were removed and cleaned with deionized water followed by methanol and dried in a stream of nitrogen.
PS ATRP modification of glass (Two 3-neck flasks were used for this reaction) Surface pre-treatment and silane modification steps are the same as in the production of poly(acrylic acid) brushes. There is no hydrolysis step for the fabrication of polystyrene brushes.
Simply replace the reagents in Table 3.1 with the reagents in Table 3.2 for the PS ATRP modification of glass slides and follow the same steps under PAA ATRP modification of glass.

PS-b-PAA ATRP modification of glass
Follow the steps for the each block as outlined above.

RESULTS AND DISCUSSIONS
Physical deposition of PS 60 -b-PAA 29 Langmuir-Blodgett film

Surface treatment
In hours. Polystyrene film formed on the surface of the glass but it was weakly bounded.
In order for the polystyrene film to bind tightly, it was heated in vacuum oven for 72 hours at about 25 mmHg. Excess polystyrene was then washed off with chloroform.
This caused thin film coating of polystyrene to be thermally bounded to the glass surface.
Upon deposition of PS 60 -b-PAA 29 on top of this modified glass at a preset surface pressure, the polystyrene block of the PS 60 -b-PAA 29 formed bond with polystyrene on the glass substrate; hence, polymer brush was formed.  In salt-free deionized water, the hydrophilic block of the copolymer, PAA, became solvated and the protons in water complement the anions present on the PAA chains as much as possible. In essence, the chains of PAA ‗diffuse' into water due to solvation.

Surface Activity of PS
In Figure 4.1, at 0 o C and 15 o C (deionized water), it was possible to pack the molecules very close to one another (as indicated by negligible pressure increase) as the barriers of the LB trough are closed. This is because the thermal (kinetic) energies of the molecules within the self-assembly monolayer (SAM) are low. However, as an area/molecule of 42A 2 -40A 2 was reached, a -phase change‖ (change from high state of disorderliness of polymer molecules (chains) to a more ordered state, that is, from ‗gaseous state' to expanded monolayer phase) was observed indicating that the short-ranged steric force, long-ranged electrostatic repulsive force, and hydrophobic interactions among the polymer chains acted in concert as the molecules resisted packing too close to each other. So the pressure began to increase until the maximum pressure was observed.
The short-ranged steric repulsion can be expressed mathematically as follows (Evans et al, 1999): where F is force, A represents the area, KT represents the thermal energy (1.38 x 10 -23 J/K * temperature), L represents polymer brush thickness, and h is the distance of separation. While the long-ranged electrostatic force can be expressed according to the following mathematical representation (Evans et al, 1999): This phenomenon explains why the surface pressure started to rise at 60A 2 when the temperature of the deionized water subphase was increased to 20 o C and 25 o C.
Equations (1) and (2) predict that surface pressure is directly proportional to temperature (thermal energy); however, in Figure 4.1, the surface pressure-area isotherm indicates that the thermal energy at 15 o C is lower than the thermal energy at 0 o C leading to higher packing density at 15 o C contrary to expectation. This unusual behavior presents a phenomenon that needs to be investigated further in order explain what is happening within the surfactant molecules at air/water interface at 15 o C.
In Figure 4.2, simulated sea water was used as the subphase while temperature and surface pressure were maintained at 20 o C and 40mN/m respectively. The pH of the sea water was varied: 4.01 (acidic pH), 7.06 (neutral pH), and 9.96 (basic pH).
The compositions of sea water are NaCl (58%), MgCl 2 .6H 2 O (26%), Na 2 SO 4 (9.75%),  When sea water with concentration of 300 mM (high concentration) was used as the subphase, the cations in the sea water such as Na + , Mg 2+ , Ca 2+ , K + , and Sr 2+ gathered around the negatively charged PAA chain in the salt solution and bind to those negative charges present on the surface of the chain. This phenomenon is called salt screening.
In a 300 mM -500 mM sea water, the entire surface of a polymer brush becomes homogeneous (Witte et al, 2010). When the pH of the 300 mM sea water was maintained at 4.01, the PAA block of PS 60 -b-PAA 29 became neutral because the pH is less than the pKa of PAA (4.5), this means that in acidic pH up to 4.5, the -COOH groups of the PAA macromolecule exist in non-dissociated form (Chibowski et al, 2006). Consequently, at pH below the pKa of PAA, we have NB regime. Also, there were minimal long-ranged electrostatic repulsive forces present within the molecules of the PAA group at pH 4.01 in the sea water; only the steric repulsive and hydrophobic forces were at play.  Hence, there was mass transfer (deposition) from the air/water interface to the polystyrene modified glass slides.    Table 4.1 presents the transfer ratio of PS 60 -b-PAA 29 from air/water and air/sea water interface. The transfer ratio was calculated as follows: It can be seen that as temperature increased from 15 o C to 25 o C, the transfer ratio decreased. The same trend was observed when the pH was increased from 4.01 through 9.96 while holding the temperature and pressure constant at 20 o C and 44mN/m respectively. Figures 4.1-4.4 and most importantly, the understanding that grafting density can be controlled by adjusting the pH of the subphase before deposition of any hydrophobic-block-hydrophilic block copolymer on to a hydrophobic surface, is the main advantage that physical deposition technique has over chemical deposition technique.

Contact angle measurements
Contact angle results from surface free energy between liquid and solid surfaces when surrounded by air or gases in general. Contact angle measurement can help one to understand wettability, affinity, adhesiveness, and repelling tendency of a surface. The mathematical expression for calculating contact angle is known as       Although, it has been indicated on the Baier curve that foul-release coating can be achieved when a surface has a surface energy of 22 mN/m-24 mN/m, additional factors such as nanoscale or microscale roughness can affect fouling (Carman et al, 2006).
In this nanoscale fabrication work, we suspect that contact angle variations can be attributed to nanoscale roughness of the polymer brush modified surface.
Although at this moment, we cannot directly determine how nanoscale roughness affected the contact angles but it is safe to assume that the contact angle measurement did not only relate to the degree of wettability of the surfaces but it also revealed the presence of nanoscale roughness via grafting density variation that is absent from ATRP deposition technique.

UV-vis transmittance
UV-vis spectroscopy is a technique in which the ability of electron to be excited and move between energy levels is utilized. These energy levels have direct correlation to the molecular orbital of the systems. Specifically, UV-vis spectroscopy takes advantage of electronic transitions involving π orbitals and lone pair electrons to identify conjugated systems which have stronger absorptions. The wavelength of ultraviolet light is 200 nm -400 nm while that of visible light is 400 nm -800 nm.
Detail discussion of this technique has been carried out in Appendix B.
It can be seen in Figure 4.9 that there are three regions of transmittance:

Fluorescence imaging studies
Fluorescence occurs when a material emits light within nanoseconds or femtoseconds upon absorption of light with short wavelength (Lichtman et al, 2005).
Not all the absorbed lights are emitted but the emitted light (known as fluorescence) by the material has longer wavelength than the incident light. Emitted fluorescence is then collected by the objective of the microscope and sent to the detector. In order to observe fluorescence, fluorophore is needed to -label‖ the sample molecules.
Fluorophores are molecules or compounds that possess fluorescence properties.
Details of the operation and principles of fluorescence microscopy have been discussed by Muller, 2006 andLichtman et al, 2005.
The fluorophore used in this work is acridine orange. It binds to the carboxyl group of the PAA in PS-b-PAA. The fluorophore solution was prepared by dissolving acridine orange in deionized water to make 1 mg/mL solution (probably too concentrated as shown by the fluorescence images below). The solution was poured on the polymer brush and allowed to stain the sample for approximately 30 minutes.
The samples were washed with deionized water after staining. Upon the completion of fluorophore rinsing, the samples were placed on the fluorescence microscope and images were obtained using confocal microscope at magnification of 200X -400X.    However, when the brushes were hydrated as shown in Figure 4.12, it reveals that the PAA blocks of the copolymer became extended and swollen. It can be seen that the density of the brushes increased due to wetting (evidence of hydrophilic nature of PAA).
In Figure 4.13, the surface pressure, subphase, and pH were changed in order to study the behaviors of the polymer brush. It can be seen that as the deposition pressure was changed from 22 mN/m, the surface energy of the resulting brush and surface morphology of the brush produced also changed. This dot-like appearance proved that the PAA blocks have stretched. This may have resulted in increase brush thickness. Also, Xu et al, 2006 provided evidence that at pH above the pKa of PAA (4.5), the PAA chains stretch as the pH increases. So, when the pH was raised to values above the pKa, brush density thickness increased.

Atomic force microscopic (AFM) analysis
AFM is a good analytical tool for characterization of polymer brush surfaces.
It can be used to study surface morphological and topographical features, measure thickness, and investigate mechanical properties of polymer brush modified surfaces.
AFM takes surface measurement by scanning the tip of the cantilever on a surface resulting in an attractive or a repulsive interplay with the surface. As a result of these interactions, the tip attached to a cantilever experiences a force that causes the cantilever to bend. A laser beam off the cantilever detects the deflection of the cantilever causing the degree of the laser beam to be translated to image in terms of height or topography (Kolasinski, 2008). More details on AFM can be found in  Therefore, the estimated thickness of the PS 60 -b-PAA 29 brush is 23.88 nm.

Chemical Deposition of PS 60 -b-PAA 29 Langmuir-Blodgett film
In an effort to compare the anti-biofouling efficiency of chemically and physically deposited PS-b-PAA films, ATRP reaction was conducted. Surface characterizations are hereby discussed first.

Uv-vis transmittance
Brushes fabricated by ATRP were tested for transparency by UV-vis spectroscopy and Figure 4.18 shows that chemically modified surface by ATRP also transmitted all the lights that passed through them as was the case with physical deposition surfaces in Although the regional grouping observed in Figure 4.9 was not observed in taken and discussed below.

Fluorescence imaging studies
Samples listed in Table 4.2 were deposited on hydroxylated, pre-treated glass slides by ATRP in order to study and compare the surface morphology of both covalently linked brushes and physisorbed brushes. Poly(acrylic acid) brush (6hrs) (PAA) 5 Poly(acrylic acid) brush (12hrs) 6 Polystyrene brush (6hrs) 7 Polystyrene-block-poly(acrylic acid) (PS-b-PAA) Only PS-b-PAA fluorescence micrograph is presented here because all the samples essentially look similar to PS-b-PAA micrograph under the fluorescence microscope.
Therefore, it is difficult to observe any pattern formation or deduce any grafting density variation information from the fluorescence micrographs of the chemically deposited brush shown Figure 4.19. Inability to visibly see what is going on the chemically deposited brushes could also be due to high brush thickness, thus forming an opaque carpet. In order to study the morphology, we need a more powerful tool such as atomic force microscope imaging.

Biofouling studies of covalently linked polymer brushes (ATRP)
Bio-adhesion studies were conducted by the Callow laboratory at the University of Birmingham, UK. The results are hereby presented below.
The bar labeled ‗Glass' in Figure 4.22 represents the control in the biofouling studies. The densities of attached spores varied with chemical composition of the modified surface and grafting duration. The lower the spore density of a modified surface compare to the glass spore density, the more effective the biofouling coating on that particular modified surface.
Hydroxylated and silane modified glass slides show lower spore settlement density. In the case of the poly(acrylic acid) modified surface, grafting duration of the brush has a direct impact on the film efficiency, that is, the PAA brush grafted for 6 hours shows higher settlement density than the unmodified glass slide while the 12 hours modified glass show almost half the settlement density of the unmodified glass slide. Polystyrene surface, a hydrophobic surface, is notorious for allowing settlement of spores (Finlay et al, 2011;Newey et al, 2007;Young et al, 1984). So, the 6 hour PS and 6h-6h of PS-b-PAA grafts in However, when the modified slides were hydrated as shown in Figure 4.9, stretching of the PAA block of the copolymer was observed. It can be seen that the thickness of the brush increase due to wetting (evidence of hydrophilic nature of PAA).
The pressure, subphase, and pH were changed in order to study the behaviors of the polymer brush. At basic pH (pH 9.97), the PAA block of the PS 60 -b-PAA 29 become ionized (the carboxylic groups of the PAA chain ionized) and acquire negative charges, these result in steric and electrostatic repulsions, as well as, charge screening. It can also be seen in Figure 4.13 that the brush look like dots instead of strand-like appearance that was observed when the film was dry. This dot-like appearance proved that the PAA blocks have stretched and they are standing upright.
Hence, brush density has decreased.
It is impossible to observe any pattern formation or deduce any grafting density variation information from the fluorescence micrographs of the chemically deposited brush shown Figure 4.19. Inability to visibly see what is going on the chemically deposited brushes could also be due to high brush thickness, thus forming an opaque carpet. In order to study the morphology, we need a more powerful tool such as atomic force microscope imaging.
The existence of these three different regions on the UV-vis of the physically deposited brushes, which was not observed in Figure 4.18, the UV-vis transmittance measurements of PS 60 -b-PAA 29 brush surfaces fabricated via LB and ATRP method suggest that the surfaces are semi-transparent, which is important for applications in lenses or windows.
Preliminary biofouling studies of surface modified with ATRP deposition shows that grafting duration (hence, thickness) of polyelectrolyte brush has a direct impact on the film efficiency against biofouling, that is, the PAA brush grafted for 6 hours shows higher settlement density than the unmodified glass slide while the 12 hours modified glass show almost half the settlement density of the unmodified glass slide. The attachment of the spores to the PS brushes shows that PS surfaces are not effective anti-biofouling brushes but adhesion of the spores to the PS-b-PAA modified surfaces suggests that the thickness of the brushes needs to be increased by increasing the grafting time. It will also be necessary to increase the hydrolysis time of poly(tert-butyl acrylate) to 8 hours or more. Therefore, for PS-b-PAA brush surface to be effective against biofouling, it may need to be grafted for at least for 24 hours if ATRP is to be used.

FUTURE WORKS
Physical Deposition: Biofouling studies on physically fabricated polyelectrolyte brushes are underway at URI aquarium where the samples are immersed in sea water pumped directly from the ocean. Future samples will be deposited on full size microscope slides via Langmuir-Schaeffer technique.
Quantitative AFM work will also be done to determine brush thickness, grafting density, and adhesive strength of the polymer brush transferred to the substrate's surface. one should spread oil on the surface of water at sea every time ships travel on the ocean but in the periods between seventeenth and eighteenth century, the commonly held believe was that oil on the surface of the water causes the sea to be calm.
John Aitken devised an instrument that was capable of detecting movement in water that has been subjected to air current after oil has been spread on the surface of such water and found out that oil did not calm water. Irvin Langmuir was a Metallurgical Engineer by training whose works led to gas-filled lamps (gas-filled lamps are known for their higher efficiencies and durabilities). He also worked extensively in the area of surface chemistry for which he won the Nobel Prize in Chemistry. Most importantly, Langmuir unified already known but scattered and neglected scientific theories such as the surface nature of adsorption, the kinetic theory of gases, and the range of intermolecular attractive forces by showing their relative relationships.
Langmuir measured the spreading pressures of thin films, developed the surface film balance, shed light on the molecular orientation at the surface of water on which a monolayer of organic substance has been spread, confirmed the existence of short-range forces, and finally, he explained why some molecules did not form monolayer films.
Katharine Blodgett (1898Blodgett ( -1979 Katharine Blodgett was first in many things such as first woman to work on the research staff at General Electric, the first person to obtain a doctorate degree from the Cavendish Laboratory in Cambridge, England in UK. She was also the first person to transfer fatty acid monolayer film on to a solid substrate such as glass slides. In her honor, any monolayer(s) transferred to solid substrate is/are known as Langmuir-Blodgett film(s). Finally, Katharine's attention was later directed to studying the optical properties of multilayer films.

MONOLAYER CHARACTERIZATION
Surface Pressure-Area Isotherm (Roberts, 1990;Petty, 1996) Molecules within a liquid have certain extent of attractions for each other.
This extent of attraction is referred to as cohesion. By comparison, molecules within a liquid have equal attraction from all directions compared to molecules at the surface of the liquid, which experience disproportionate attractions because of interaction with air on one side and interaction with molecules within the liquid on the other side.
Essentially, the molecules at the surface of a liquid experience much greater attractive forces towards the liquid than toward the air molecule. As a result, there is effective, prevailing attraction towards the liquid aggregate such that the air-water boundary automatically lowers its area and shrinks as a result.
The activities, as well as, the forces that are in play on the surface of the liquid and within the bulk of the liquid lead to a situation where the liquid often has excess free energy. The excess free energy is called surface tension which can be expressed thermodynamically according to the following mathematical expression: Where G represents the free energy, S is the surface area. The temperature, pressure, and composition (n i ) are held constant.
Furthermore, hydrogen bondingnotorious for its strength -forms loose networks especially in aqueous environment. The networks formed often undergo manipulation on the surface of the liquid bulk by actions such as compression of barriers of the Langmuir-Blodgett trough to reduce area and addition of surfactant to the surface of the subphase. Other intermolecular forces also exit in the aqueous subphase because of the polar nature of water. The overall effect of these intermolecular interactions is high surface tension.
The strength of the surface tension is reduced when temperature is increased and surfactants or contaminants are spread on the surface of the subphase. Hence surface pressure is observed because of the difference in environment between the molecules on the surface and those within the bulk of the subphase. It is therefore possible to quantify the surface pressure according to the following mathematical expression:

= −
where is the surface pressure, o  is the surface tension of pure deionized water, and  is the surface tension of water after the spreading of the surfactant. It should be noted that the maximum obtainable surface pressure on water surface is 73mN/m at 20 o C, however, it is could be lower in practice.
In monolayer science, surface pressure-area isotherm was the fundamental tool used in understanding the surface activities of surfactants at air/water interface.
Agnes Pockels was the first person to use π-area isotherm in 1893 in analyzing oil on water surface (Roberts, 1990).
Surface pressure-area isotherm is a 2-dimentional graph ( Figure B1) that shows the relationship between surface pressure on the vertical axis and the area/molecule (A 2 ) on the horizontal axis. It can be divided into the sections called ‗phases' named synonymously according to the three phases of matter's existence. Figure B1: Typical surface pressure-area isotherm of Langmuir Monolayer.
In the gas phase, the molecules of the surfactant have enough space between them such that intermolecular interactions takes place without one molecule interfering with the other, thus they exert very small or negligible force on one another. In addition, the molecules align themselves in a random manner on the surface of the subphase. However, as the area occupied by the monolayer is reduced (barrier compression), the hydrophobic tails start to interact with each other. As the hydrophobic tails are brought even closer, then the interaction will become significant resulting in rise in surface pressure until a constant pressure is observed. This constant pressure ushers in the extended phase and it signifies co-existence of two phases, that is, gas phase and expanded phase. This is a first order thermodynamic transition.
In reality, not all surfactants have all the three phases. Figure B2 is the pressure-area isotherm of PS 60 -b-PAA 29 in which the three phases are not observed whereas Figure B3 shows the surface-pressure area isotherm of DPPC with all the three phase. Figure B2: Surface pressure-area Isotherm of PS 60 -b-PAA 29 at 20 o C after 1 hr of spreading on the surface of the monolayer.
The next phase observed is the expanded phase. This phase corresponds to the liquid phase. In order to explain the expanded phase, it will be necessary to refer to the surface pressure-area isotherm of specific surfactants such as PS 60 -b-PAA 29 and Dipalmitoylphosphatidylcholine.
In Figure B2, the plateau occurs at about 1mN/m afterwards, the expanded phase appears. The case is the same in Figure B3; the constant pressure region  Figure B3: Surface pressure-area isotherm of dipalmitoylphosphatidylcholine (DPPC) at room temperature.
(steadily increasing), then the plateau and finally the expanded phase. These slight differences between the ideal surface pressure-area isotherm and the isotherms for actual surface active agent, as well as, the differences between the isotherms among various surfactants may be due to difference in the length of chain composition of the hydrophobic tails, higher order thermodynamic transition, and effect of residual solvent molecules at the interface of the subphase.
After the expanded phase, another first or higher order thermodynamic transition signified by a constant pressure region or lack thereof ushers in the condensed phase. However, as with the gas and expanded phases, the condensed phase is not always observed in all the monolayer materials.
The factors that contribute to the variations in the expanded to condensed phase transition include the length of the hydrocarbon chain in the hydrophobic tail DPPC, 20 o C DPPC (1mg/mL, 330uL) @ RT and temperature. Generally speaking, decreasing the chain length of hydrocarbon tail leads to an increase in the surface pressure of the phase transition. Also increase in temperature has the same effect (Petty, 1996). At the molecular level, a decrease in chain length leads to diminished intermolecular Van der Waals' forces. Moreover, if the temperature is reduced, the result is a decrease in thermal motion of the molecules within the film. The combined effect of the changes mentioned above result in the formation of the condensed phase.
Sometimes, there may be direct transition between the gas phase and the condensed phase because of extremely long hydrocarbon tail length.

Atomic force microscopy
Atomic force microscopy was developed in 1986 by Binnig, Quate, and Gerber. It is a microscopic method that allows researchers to see and quantify surface structures with extraordinary resolution and accuracy. Surfaces whose structures can be investigated by AFM range from solid materials to microorganisms to macromolecules. One great advantage of using AFM for surface characterization is that it is non-destructive and that is why it is suitable in measuring soft surfaces and biological molecules. It can measure samples between 5 nm to 250 µm (or more) in size. The figure below shows the linear scale of different microscopes used in material science. AFM takes surface measurement by scanning the tip of the cantilever on a surface resulting in an attractive or a repulsive interplay with the surface. As a result of these interactions, the tip attached to a cantilever experiences a force which causes the cantilever to bend. A laser beam off the cantilever detects the deflection of the cantilever causing the degree of the laser beam to be translated to image in terms of height or topography (Kolasinski, 2008).

Ultraviolet-Visible Spectroscopy
This is a technique in which the ability of electron to be excited and move between energy levels is utilized. These energy levels have direct correlation to the molecular orbital of the systems. Specifically, UV-vis spectroscopy takes advantage of electronic transitions involving π orbitals and lone pair electrons to identify conjugated systems which have stronger absorptions. The wavelength of ultraviolet light is 200-400nm while that of visible light is 400-800nm. Figure  B6: The electromagnetic spectrum adapted from http://sciencejunkies.com/page/3/. Ultraviolet-Visible Spectroscopy operates within the ultraviolet-visible light region of the electromagnetic spectrum.
Thus for a substance to be qualified for testing using UV-vis technique, it must have uninterrupted conjugated double, triple or a mixture of both bonds along a stretch of the molecule. Therefore, the smallest number of molecule of a material that can absorb electromagnetic radiation is called the chromophore.
In principle, the lowest transition of energy occurs between highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) in the ground state. For electrons to move from HOMO to LUMO, electromagnetic radiation must be absorbed, this event causes electrons to be excited to the LUMO. It should be noted that the more unsaturated the substance under test is the smaller the HOMO-LUMO spacing and the change in energy required and consequently, the lower the frequency which means the longer the wavelength. Figure B7: The molecular orbital energy representation of ground state and excited state of two electrons in a molecule.
The general outline of a UV-visible spectrometer can be seen below. An attempt is hereby made to briefly describe the optical principle of UV-vis spectrophotometer. Light from sources are filtered are they enter the monochromator.
Hence, as the light exits the monochromator, it becomes monochromatic light. The monochromatic light then illuminates the sample. A detector then measures the amount of light that passes through the sample.