University of Southern California Dissertations and Theses

I. Layered nano fabrication. II. Adhesion layers for hippocampal neurons

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I. Layered nano fabrication. II. Adhesion layers for hippocampal neurons

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Content I. LAYERED NANO FABRICATION II. ADHESION LAYERS FOR HIPPOCAMAL NEURONS Copyright 2003 by Diana Yvonne Lewis A Thesis Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTERS OF SCIENCE (CHEMISTRY) May 2003 Diana Yvonne Lewis R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES, CALIFORNIA 90089-1695 This thesis, written by \S L Y \< \. YvOfU \ e under the direction of htZjC_ thesis committee, and approved by all its members, has been presented to and accepted by the Director of Graduate and Professional Programs, in partial fulfillment of the requirements for the degree of Director Date May16. 2003 Thesis Committee Chair R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Acknowledgements I would like to thank Professors Mark Thompson, Ari Requicha, and Bruce Koel for giving me the opportunity to study such an interesting topic of Nanotechnology. I admire their creativity and intellect. I would also like to thank my family for their love and support throughout this process. Especially I would like to thank my daughter Kamila Reeder for being a source of great inspiration to me. I would also like to thank Dr. Michael Quinlan for many helpful conversations about surface science, basic research and the occasional bad joke. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Table of Contents Acknowledgements..........................................................................................................ii List of Tables....................................................................................................................v List of Figures................................................................................................................. vi List of Schemes............................................................................................................... ix A bstract............................................................................................................................. x Chapter 1: Layered Nano Fabrication -Analytical and Selection C riteria...........1 1.1 Introduction.................................................................................................... 1 1.2 The Selection Criterion for the Multilayer Film ........................................3 1.2.1 Repeatable height of layers.....................................................................3 1.2.2 Growing a Multilayer.............................................................................. 4 1.2.3 Remove layer without damage to the nanostructure.......................... 4 1.2.4 Well-ordered film allows for a second deposition of particles...........5 1.2.5 The deposition conditions of the film should be relatively mild and efficient of multilayer growth.................................................................................5 1.3 Characterization methods and equipment.......................................................6 1.4 Substrate Cleaning Preparation........................................................................7 1.5 Multilayer Targets- Molecule Candidates for LNF....................................... 8 1.5.1 Synthesis of 11-methyl-trichlorosilyl-undecanoate and 11-methyl- (dimethyl-monochlorosilyl) undecanoate............................................................. 8 1.5.2 Evaluation of 11-methyl-trichlorosilyl-undecanoate and 11-methyl- (dimethyl-monochlorosilyl) undecanoate for LNF.............................................11 1.6 Synthesis and Evaluation of Zinc-bis-phosphonate Multilayers..............15 1.6.1 Introduction.........................................................................................15 1.6.2 Preparation of the Anchoring Layer...................................................15 1.7 Multilayer Film Growth.............................................................................. 19 1.8 Conclusion.....................................................................................................25 Chapter 2: Usage of Zinc Bis-Phosphonate Multilayers in LNF...........................24 2.1 Introduction...................................................................................................26 2.2 Embedding 5nm Cl-capped Gold Colloids..................................................26 2.3 10 nm Amino Alkane and Cl-capped Colloids............................................ 29 2.4 15nm Cl-Capped Gold Colloids.................................................................... 31 2.5 Deposition of silver PVP capped particles on silicon and gold substrates. 33 2.6 Preparation of Anchoring Layer on Gold..................................................... 34 2.7 Deposition of Silver Colloids on anchoring layer........................................34 2.8 Embedding Silver Colloids......................................................................... 35 2.10 Conclusion and Outlook of Chapter 2 ........................................................ 36 Chapter 3 Selective Adhesion layers for a Tight Neural Contact....................... 39 3.1 Introduction.................................................................................................. 39 iii R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 3.1.1 Method and Setup.............................................................................. 41 3.2 SAM Characterization.................................................................................43 3.2.1 Contact Angle Measurements........................................................... 43 3.2.2 Atomic Force Microscopy.................................................................44 3.3 Detecting Antibody Activity at the Surface..............................................45 3.4 SAMs Preparation for Terminal Carboxylate groups...............................45 3.5 Experimental Results................................................................................... 48 3.6 Antibody Activity at the Surface............................................................... 51 3.7 Cell Attachment to SAM Surfaces............................................................ 53 3.7.1 Experimental Results.........................................................................56 3.8 Conclusion and Outlook............................................................................63 Bibliography...................................................................................................................65 iv R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. List of Tables Table 1 Stoichiometric determination between Zinc and Phosphorous Atoms 24 Table 2 Overall film comparison....................................................................................25 Table 3 Contact angles measured on gold pattern glass substrate............................... 44 Table 4 Various attachments on Surfaces..................................................................... 56 Table 5 Contact angles measure on gold and glass......................................................62 v R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. List of Figures Figure 1 (a) 11-methyl-trichlorosilyl-undecanoate (1). Figure 1 (b) 11-methyl- dimethylmono-........................................................................................................... 8 Figure 2 XPS spectra of cleaned Silicon wafer............................................................. 11 Figure 3 2x2 micron image of the terminal amine layer on silicon.............................15 Figure 4 lx l micron image of the terminal phosphorous layer on silicon.................. 15 Figure 5 2 x 2 micron image of the second method for deposition. No erosion to the pattern is detected......................................................................................................16 Figure 6 (a) XPS Survey Scan of the anchoring layer on silicon.................................18 Figure 7 2x2 micron image of one layer of Zinc Bis-Phosphonate film..................... 18 Figure 8 Monolayer growth over time (upper). Multilayer growth (lower)................19 Figure 9 XPS spectra of ZBP multilayers corresponding to 6 cycles......................... 20 Figure 10 The graph of the Inelastic Mean Free Path.................................................. 21 Figure 11 The Inelastic Mean Free Path as a function of Kinetic Energy................ 22 Figure 12 1 x 1 micron image of 5nm thiol-capped gold colloid on the anchoring layer...........................................................................................................................25 Figure 13 (2 micron) 5nm thiol-capped colloid after growth of 4 layers....................25 Figure 14 5nm thiol-capped gold colloids after removal of ZBP............................... 25 Figure 15 500nm image of Phosphorlation 5nm Chloride-capped gold colloids on APTS..........................................................................................................................26 Figure 16 lx l micron image of 10 nm Chloride-capped gold colloids on Phosphorlated Si02 surface.....................................................................................28 Figure 17 Lowering the surface pH increased coverage of 10 nm Chloride-capped gold colloids............................................................................................................. 28 Figure 18 10 nm chloride-capped gold colloids after growth of 5 layers lowered pH Phosphorlated surface...............................................................................................29 vi R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 19 10-15 nm chloride-capped colloids after removal of ZBP......................... 30 Figure 20 lx l micron image of the anchoring layer on a gold substrate.................... 31 Figure 21 3x3 micron image of 10 nm Ag nano-particles deposited on the anchoring layer............................................................................................................................32 Figure 22 500 x 500 nm image of silver nanoparticles after 5 layers film growth.... 32 Figure 23 Neuron bound to substrate via SAM.............................................................36 Figure 24 Antibody from the Immunoglobin Superfamily..........................................39 Figure 25 Contact Angles. Hydrophilic surface (left); hydrophobic surface (right).. 44 Figure 26 4x4 micron image of Antibodies on gold- without SAM........................... 49 Figure 27 Methyl-terminated SAM following exposure to Antibody solution......... 45 Figure 28 COOH-terminated SAM exposed to Antibody solution............................. 50 Figure 29 Activated SAM exposed to Antibody solution............................................51 Figure 30 Afm images of AB1204 following exposure to the enzyme Alkaline Phosphatase............................................................................................................... 52 Figure 31 Bound Antibodies with AP after exposure to Tween 20............................ 49 Figure 32 (a) Dead cells, (b) Healthy cells, (c) Debris................................................ 54 Figure 33 Zoomed area of fixed cell bodies.................................................................. 55 Figure 34 Cells 3days old: non-specific Antibody from rabbit deposited in 0.135 M NaCl {thin film: thiotic acid/butyl mercaptan followed by EDC......................... 57 Figure 35 Cells 3days old: Sodium Channel-specific Antibody from rabbit deposited in 0.135 M NaCl {thin film: thiotic acid/butyl mercaptan followed by EDC}... 58 Figure 36 Cell Culture 11 days old and before fixing with 4% aldehyde solution. Strong attachment 1. Dead cells 2. Thicker branching -live cells clumped together with some dead cells on top. 3. Thinner branching-cells less clumped......................................................................................................................59 vii R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 37 Presence of EDC on glass confirmed by antibody attachment................... 60 Figure 38 Afin images of 4-Methyl Thiol Phenol.........................................................59 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. List of Schemes Scheme 1 The deposition and growth process of LNF...................................................2 Scheme 2 General synthesis of tri-chlorosilanes.............................................................9 Scheme 3 Multilayer growth on Silicon.........................................................................19 Scheme 4 Synthesis of di-substituted 1, 10-phenantroline...........................................37 Scheme 5 Growth on Gold Substrate............................................................................ 46 Scheme 6 Synthetic routes improve selective modification to a gold substrate 61 ix R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Abstract In chapter 1, the analytical and selection criteria for Layered Nano Fabrication (LNF) are presented. It was determined that multilayer of Zinc Bis-Phosphonate films (ZBP's) grow in about 10 minutes per layer under ambient conditions. Atomic Force Microscopy (AFM), X-Ray Photoelectron Spectroscopy (XPS), and Ellispometry characterized multilayer growth. Alkyl silanes were grown on Silicon substrates. ZBP’s were grown on silicon and gold substrates. Chapter 2 describes the application of ZBP’s as a scaffolding layer as it embeds nano-sized particles (NSP’s). AFM was used to analyze and monitor the embedding of the nsp and the removal of the scaffolding film. These results represent a novel demonstration to Layered Nano Fabrication (LNF). Chapter 3 describes the formulation of antibody-adhesion layers and their effect they have on the attachment of hippocampal neurons. The initial results determined that about 50% of the bound antibodies have the appropriate geometry for active binding. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Chapter 1: Layered Nano Fabrication -Analytical and Selection Criteria 1.1 Introduction Size limitations of photolithography are due to optical diffraction1,35. In that process, there is a need for shorter wavelengths to obtain smaller features. However, that would require complex technology and expensive budgets. Micron Contact Printing (pCP) avoids diffraction limits22,23, a n d 3 8. In principle, it involves making a master, a mold and that imprints the pattern. However, high levels of defects in the mask and non-compliance with manufacture requirements are the major obstacles to incorporating its use towards sub-micro patterning. The defects of the mask are due to the elastomer materials used to make the masks. These problems include shrinking, sagging, and swelling. There is also a need to find a material(s) that demonstrate reproducibility of printing35. There have been considerable contributions in patterning at the submicron scale, but these still have to integrate well with modem manufacturing technology. For example, they need to improve reproducibility, quality of products, masks, and materials that do not leave a residue or produce defects. There is also an issue of incorporating SAM technology into the manufacturing level of production. This thesis in part, introduces Layered Nano Fabrication (LNF) as a novel fabrication approach to sub-micron patterning. This approach fulfills some requirements of industry including requirements include reproducibility and simplicity. LNF is also to section off and transfer to production environments and standards. Its process has 1 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. simple discrete steps, using manufacture compatible solvents. The master is formulated using nano-sized colloids that have a low size dispersion, which would give reproducible control over the resolution of the master and printing defects. The type of Thin Film Architecture would combine with the successful demonstration of Multiple Tip AFM for Particle Manipulation (MTAPM) 4 ’ 2 6 '2 8. That is a fully automated process and thus would ease incorporating with manufacturing. AFM can perform in situ; therefore it is possible to conduct process validation. The problem areas that confront LNF include validating successful linkage of nano structures. For example, optimizing detection of the bonds between the metal and the linker atoms Overall idea Deposit Particles Arrange Particles Embed the first layer by multi-layered film scaffold Embed the second layer by multi-layered film scaffold does not seem straightforward or easy. Arrange second deposition of Particles Remove scaffold Scheme 1 The deposition and growth process o f LNF. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Layered Nano Fabrication (LNF) is a process that uses multilayers to build a nanoscale structures in a vertical (3-dimentional) direction. In a typical process (scheme 1) an anchoring layer forms at the substrate and after the deposition and manipulation of the nanoscale particles, multilayers are grown to embed the particles. When the first deposition of particles is nearly embedded, a second layer of nanoparticles are deposited and manipulated accordingly, followed by the removal of the multilayer leaving the constructed 3-D structure. The main questions addressed in chapter one focus on the selection requirements for the multilayer film, followed by and introduction of the available analytical techniques used to evaluate the film. 1.2 The Selection Criterion for the Multilayer Film Important functions of the film are to embed particles with minimal movement, be inert to the particle surface and will not damage to structure to the particles after its removal. The following sections comment further about the selection process. 1.2.1 Repeatable Height of Layers The first requirement of a sacrificial layer to be effective in LNF is that the layer(s) embed the nano-particles and this indicated by measuring the decrease in the particle's height as the film embeds the particle; therefore it is important to characterize the film and determine is this is a suitable choice for the LNF process. 3 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Ideally, this film should behave similar to a self-assembly monolayer (SAM): reproducible, consistent increases in thickness that correlate to the heights of the molecular monolayer. The LNF process forms multilayers form covalent bonds at both ends. The self-assembly process consists of a chemically active part of the molecule that forms a covalent bond with an atom at the substrate. As the chemical active sites are being filled, the inactive part of the molecule begins to pack together using attractive forces like Van der Waals forces in hydrophobic chains. 1.2.2 Growing a Multilayer Homogeneity of the film after each growth cycles will limit the number of multilayers that are possible, and also determines the ability to decipher the film from the particles. Defects in the multilayer film after several cycles occur and this could prohibit particle embedding and their detection because after the deposition of several layers, defects in the film (holes) deteriorate how well the film packs. 1.2.3 Remove Layer without Damage to the Nanostructure This criterion produces two important issues about the solvent selection for SAM growth. Firstly, the dissolving solution should not solvate the nanoparticles to the extent that it competes with the linking agents, nor should it cause particle aggregation. For example, tight ion pairs between the surface and solvent could 4 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. move the particle around. Secondly, the solvent used to suspend the nanoparticles should not dissolve the multilayer. 1.2.4 Well-Ordered Film Allows For a Second Deposition of Particles The packing of the film should allow for a second deposition of particles. Although the molecule used in the formation of the SAM is the primary dictate in the packing of the film, optimizing deposition conditions, molarities, solvent, and the deposition time can have an influence. 1.2.5 The Deposition Conditions The film should be relatively mild and efficient of multilayer growth because it is a compilation of many simple steps, which greatly affect the overall LNF process. The molecules used to cap the nano particles should not have a strong interaction with the multilayer; the solvent of the particles must not destroy the growth of the multilayers. Particle manipulation and linking particles involve several steps that are potentially points for damage. For example, dithiolalkanes are used as a linking agent for gold nanoparticles. In a procedure described elsewhere, a drop of the thiol solution is placed on the sample surface. Although this resulted in linking the particles, it often leaves the surface congested with a thiol residue. Carefully rinsing the sample with 5 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. the appropriate solvent or immersing it in an ultrasound bath can easily destroy the sample. Eventually, the particle manipulation will be preformed by automated multi- AFM-tip arrays. Although in relatively stable environments, there is still a problem with thermal drift. Having a short growth time would minimize the problem of the drift. In addition, the using the SAMs that require inert deposition conditions would impede the imaging and manipulation part because of the difficulty to image insitu. There are many opportunities where the sample preparation, the structure formulation, or the film growths are vulnerable to damage the overall process. 1.3 Characterization Methods and Equipment This LNF approach to nano-engineering uses analytical methods to characterize the film: Atomic Force Microscopy (AFM), ellipsometry, and X-ray Photoelectron Spectroscopy (XPS), and Contact Angle measurements. The AFM images were taken with a Nanoscope III in tapping mode. The tips used were TSR-7 (silicon nitride) the approximate radius tips were between 40nm-70nm. Additional imaging was taken with the Park Instruments AFM, in non-contact mode. Particle manipulation was preformed using this AFM through a modification to the AFM controller. It enables the user to turn off the feedback loop and to manipulate nanosized particle using the scanning tip. (A detailed description can be found in a later section). The images were preformed in air with a relative humidity between 30-60%. The image processing consisted of line flatting program, and sectional analysis. The ellipsometry measurements were preformed on a Rudolph ER 2300. 6 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. The optical constants for Silicon were ns = 3.8 and k = 0.123. The optical constants for gold were ns = 0.702 and k = 3.825. The Angle of incidence was 70 degrees incidence to the surface. The calculation of PSI and Delta, were generated using DAFIBM software3 1 ’3 6 ’34. XPS experiments were recoded on single channel detector using A1 K alpha non- monochromatic source (1486.6 eV) used for all samples, and 300 watts total power (15kV and 20mA) at a 0.01% detection limit. The analyzer width is 30 mm. The emission angle is 53.8 degrees. Peak fitting data analysis (multilayer stoichometry) was performed using ESCA tools software. The sensitivity factors used were C ls = 0.25, N ls = 0.42, P2p3 = 0.36, Si = 0.25, Zn = 0.21, C ls = 0.25, and O ls = 0.66. The Contact Angle measurements were taken in dynamic mode. The volume of the liquid droplet was lpL. The hydrosilation additions used a UV light source and a custom built reaction chamber. 1.4 Substrate Cleaning Preparation The protocol development and optimization are an important part of reproducibility and are as follows: silicon wafers (n-p doped) were cleaned in a 20-minute ultrasonic bath of Acetone, followed by 20minutes in Isopropanol, 20 minutes in Methanol, blown with N2 gas, and 3 minutes in the Ozone chamber. This procedure resulted in a hydrophilic surface with an oxide thickness of approximately 25 to 35 nm. Another 7 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. method for preparing a hydrophilic layer is to use Piranha solution for 30 minutes (3:1 96% H2SO4, 30% H2O2), followed by rinsing with copious amounts of DI water, and blew dry with N2 gas. This procedure resulted in a hydrophilic surface with an oxide thickness of approximately 18 to 22 nm. Although both cleaning methods produced a thin oxide layer that allowed the growth of an anchoring layer and multilayers, the second method was too corrosive for silicon substrates with e- beam lithography patterns30,15'1 7 . 1.5 Multilayer Targets- Molecule Candidates for LNF Two types of film were considered for the LNF process. The first SAMs studied in LNF were from the trichlorosilane family, ll-Methyl-(mono) and (tri) chlorosilyundecanoate (MoSUD) and (MtSUD) respectively, and the second was zinc-bis-phosphonates. This section describes the synthesis, analysis and evaluation of the two types of film as a Multilayer for LNF, followed by a brief summary of unanswered questions and suggestions for future experiments. 1.5.1 Synthesis of 11-methyl-trichlorosilyl-undecanoate and 11-methyl- (dimethyl-monochlorosilyl) undecanoate a) B) o 8 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 1 (a) 11-methyl-trichlorosilyl-undecanoate (1). Figure 1 (b) 11-methyl-dimethylmono- chlorosilyl-und ecanoate (2). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Since previous studies1 5 '1 7 , 3 3 have shown that Octyldecyltrichlorosilane, (OTS) (CH3-(CH2)18-SiCl3) can embed nano particles. 11-methyl- HSiC13 O ' UV 15 hrs Cl Scheme 2 General synthesis of tri-chlorosilanes. diimethylmonochlorosilyl-undecanoate and 11-methyl-trichlorosilyl-undecanoate were considered for LNF because of a recent report by Hoffman et al. They formed multilayers o f 1 and used UV light to form thin silicon layers. In that experiment, the Methyl ester groups are at the surface followed by reduction to an alcohol via LiAlH4. Repeating this cycle produced a well-packed multilayer as the new hydroxyl surface forms another silicon oxide matrix. Ozone oxidation of the organic layer results in a silicon layer whose thickness is equal the number of multilayer cycles. Compound 2 was commercially available (Gelest) and 1 was synthesized by hydrosilation of the alkene group of 11-Methyl-undecenoate. All reactions must be preformed under dry conditions. In a typical reaction, excess amounts of distilled HSiCl3 were added to a quartz test tube containing a solution of 11-methyl- undecenoate. The mixture stirred for several hours under UV - light. The product 10 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. was isolated under vacuum distillation (30 mtorr, 179 °C). The 1 H NMR confirmed the product. 54% yield. 1.5.2 Evaluation of 11-methyl-trichlorosilyl-undecanoate and 11-methyl- (dimethyl-monochlorosilyl) undecanoate for LNF The films must meet requirements mentioned in the previous section. They include flatness and repeatable thickness of multilayer growth. Typical film growth commences as small islands at the surface. Several samples were prepared and their deposition times varied from 20 seconds until 4 hrs. Unexpectedly the islands were intermixed with small aggregates (4-5 nm in height). Since alkyl silane chemistry is widely studied, it was considered appropriate to further investigate the aggregation problem. It was perceived suspicious that the aggregates were relatively uniform in size because moisture contamination form aggregates in the film usually have wide size dispersion. However, trichlorosilanes are very moisture sensitive. The first strategy was to optimize the deposition conditions. Using a monochloride SAM was expected to minimize the aggregates. Two chlorine atoms are replace by methyl groups thus removing the silicon oxide matrix at the surface. However, replacement with 2 did not eliminate the presence of the aggregates. Next, it was then though that the aggregates were due to moisture contamination in the deposition environment, which was in an organic chemistry laboratory under ambient conditions. Repeating this experiment inside an inorganic glove box did not remove the presence of aggregates at the surface. (At the time, 5nm thiol-capped gold colloids were used and 11 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. they were difficult to decipher.) AFM images of the silicon wafer before and after ozone oxidation indicated that the aggregates were surface damage of silicon and not water contamination in the SAM. Using Piranha cleaning method, and decreasing the ozone chamber cleaning time from 20 to 3 minutes removed the presence of the aggregates at the surface. In order to meet the requirements for LNF, there must be some way to detect the growth of these multilayers. The XPS is a simple technique to determine such growth. Specifically, is it possible to detect the presence of alcohol groups following the reduction of surface ester groups, and detect the silicon from 1. Min: 351 Max: 1819 N(E) 120 117 114 111 108 105 102 99 96 93 90 Binding Energy (eV) Figure 2 XPS spectra o f cleaned Silicon wafer In figure 2, the Si 2p region of the spectrum is shown. The silicon 2p region has two peaks of interest. The SiOx peak (found at (102.5 eV)) is equal height as the Si' peak (found at 99 eV) 2. The ellipsometry thickness was measured at about 22 A. After 12 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. one monolayer, the peaks have equal height. After the reduction and growth of a second layer, the Si0 peak is twice the size at the SiOx peak. Si-C peaks are found at 100 eV and this would explain the increase to the peak area found at 99-100 eY. Comparing the Cls region of spectra before and after deposition would also serve as an evaluation method. For example, the ejected electrons from the aliphatic carbon region are typically shown at 284.5 eV. The presence of CO, CO2 shift this peak down field to 287-289 eV because of the higher oxidation state of the carbon (it increases the required binding energy to eject the core electrons). After the deposition of a monolayer, two smaller peaks are consistent with the ranges found in literature for C=0, 0-C =0 (289.4eV), and H-C-0 (287.0 eV )1 2 ’ 1 5 '1 7 . The sample is treated with LiAH4 to reduce the ester to an alcohol, where an increase to the peak area at 287eV (H-C-O) and the disappearance of the carbonyl peak at 289.4 eV is found. For the ester carbon 0-C =0 peak, the height decreased, and the HC-0 peak increased. C ls peaks of Si-C are typically found at 282.4 eV. There is also the presence of Li and A1 at the surface is possible and mostly likely from the careful rinsing of the sample because too harsh rinsing would destroy any nanostructures6,1. Two alternative surface techniques that would be better suited to evaluate multilayer growth are surface IR and ellipsometry1 517 ’ 3 0 ~ 3 3. IR would easily decipher the disappearance of the carbonyl carbon and the appearance of the OH. Ellipsometry could be used as an alternative to determine the thickness of the multilayer. Several 1 3 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. papers have used ellipsometry and IR to study the growth of multilayer. One example used a 22-carbon length chain tri-cholosilyl methyl ester. Even though they reported successful growth of up to 25 layers, at the 4th layer it was also reported that the film began to display major defects such that it was determined to actually have 52% coverage after the 4th and 5th layers1 5 . These molecules did not meet the usability and efficiency requirements for LNF. One reason is the trace amounts of Li salts at the surface would be a major obstruction to AFM imaging of nanostructures. Another disqualifying reason would be its experimental setup for the monolayer synthesis. It would require inert conditions for the hydrosilation and distillation, and multilayer growth. The deposition time and conditions was also another factor since one monolayer took about 2.5 hours. Finally, removing the multilayer via ozone oxidation would also remove any organic molecules used to link the nanostructure. Overall, these results were helpful in the selection process for the multilayer. Some of these include shorter deposition time, ambient growth conditions, and distinct atom detection with XPS. The next section discusses a successful multilayer for LNF. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 1.6 Synthesis and Evaluation of Zinc-bis-phosphonate Multilayers 1.6.1 Introduction Michaelis-Arbuzov reaction is employed to form the bis-phosphonates . Starting with a di-alkyl bromide, tri-phosphoethylesters react via an unstable trialkoxyphosphonium intermediate. Multi-layers using Zinc -bis-phosphonates (ZBPs) are formed in two parts: the anchoring layer and the mulitlayer, which consists of a divalent metal ion, and the organic bis-phosphonate. The immediate advantages for using ZBPs are they have simple experimental setups, the deposition time of a monolayer is 20 minutes, and the film is dissolved in water, and would not remove any organic linkers between nanoparticles. These advantageous features will be evaluated along with answering questions like the quality and reproducibility with embedding particles. These following sections describe their film's synthesis, characterization and evaluation. 1.6.2 Preparation of the Anchoring Layer The preparation of the anchoring layer was completed in two parts: modify the surface with free amines, followed by phosphorylation. In one method, the sample was refluxed in dry Toluene with 3% 3-aminopropyl dimethylmonoethoxysilane (APDMES) (or 3-aminopropyltriethoxysilane APTES) for 16 hrs. After the reaction cooled to room temperature, the sample was rinsed with Toluene, MeOH, and blown dry with N2 gas. Next, the sample was placed in lOmM solution of phosphorous 1 5 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. oxylchloride (POC13) and 2, 4-Lutidine in dry Acetonitrile overnight. This worked well on Silicon wafers. However, this method removed the lithography pattern. Therefore, a second method was necessary. Although the problem could be the reaction time for phophosnation, the first strategy was to eliminate the toluene/reflux step. A second method consisted of using 2% APTES in MeOH for 5 minutes, rinsed with MeOH, blew dry with N2 gas, and baked in an oven at 110 °C for 1.5 hours. XPS and ellipsometry data (not shown) was comparable to the first method. Phosphorylation was performed at lOmM solution of phosphorous oxylchloride (POC13) and 2, 4-Lutidine in dry Acetonitrile for 5 hours. A H sigjit P ro file [A ] Figure 3 2x2 micron image of the terminal amine layer on silicon Figure 4 lx l micron image o f the terminal phosphorous layer on silicon 16 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 40. The AFM measurements in figure 3 and 4 indicated that the anchor layer is a flat film with complete monolayer coverage. The deposition of the monolayer film should have a similar roughness to the substrate. Silicon roughness was measured between 0.1 - 0.2 nm, and the resulting amine-terminated layer was (RMS = 0.3nm), which are within expected range. Contact angle measurements were 90 -110°’ degrees. The contact angle of the Phosphorous surface was 68-80° since the presence of phosphoric acid groups improved the hydrophilic surface. Figure 5 2 x 2 micron image of the second method for deposition. No erosion to the pattern is detected. V fin : 2863 Max: 98102 „ , Zn 2p3 Si 2p3 990 880 770 660 550 440 330 220 110 0 Binding Energy (eV) 17 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Min: 18491 M ax: 28731 N Is 418 413.6 409.2 404.8 400.4 396 391,6 387.2 382.8 378.4 374 Binding Energy (eV) Figure 6 (a) XPS Survey Scan of the anchoring layer on silicon. Min: 18491 Max: 28731 Min: 9388 Max: 20766 N(E) N(E) P 2p3 418 413.6 409,2 404.8 400.4 396 391.6 387.2 382.8 378.4 374 Binding Energy (eV) Figure 6 (b) Nitrogen Region. 143 140,8 138.6 136.4 134.2 132 129.8 127.6 125.4 123.2 121 Binding Energy (eV) Figure 6 (c) Phosphorous Region. In figures 6 a, b and c, the survey scan of the anchor layer shows the presence of a Nitrogen peak at 440 eV and Phosphorous atoms at 182 eY. The sensitivity factors for these atoms are very similar and results show equal percentages of both atoms. Based on XPS and AFM, ellipsometry and contact angles, each method produced a reasonably flat, monolayer with a thickness of 5-7A. XPS showed a 1: 1 ratio of N (Is) and P (2p). 18 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 1.7 Multilayer Film Growth 0)0 or o )o or X 0 o(o ^ 0 o(0 Zn Zn Zn Zn Zn Zn Zn ,0H OH ,0H ,0H 0 0 'O 0 O 0 ' 0 0 ' 0=P0H 0 :P 0 H 0 :P 0 H 0 :P0H Q W w w / NH ^NH ^NH NH Jmmol Zn(0Ac)2 NH 'NH HN HN \ Ethand, lOmin < --------------- - S i — - S i — —S i— - s i — - S i — - S i — - S i — 0 0 0 0 0 0 0 1 I I I I I I SiOx SiOx Ethanol, lOmin SiOx Scheme 3 Multilayer growth on Silicon Scheme 3 depicts the multilayer growth using 1, 8-BPA. Multilayers using 1, 12- 9 1 EBP is also formed using this method . After the deposition of the anchoring layer, the substrate is placed in 5mM Zinc Acetate in Ethanol for 10 minutes, followed by 10 minutes in 5 mm 1, 8-bis phosphonate. The substrate was placed in a few milliliters of Ethanol and gently agitated, the sample left to air-dry. 19 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 7 2x2 micron image o f one layer of Zinc Bis-Phosphonate film. In figure 7, the resulting monolayer is typically flat with some artifacts. It was thought that the artifacts could be from the zinc layer, however further studies determined that crystal aggregates could form when there is a presence of sodium or ammonium. The surface of the film should provide a flat background to the particles. The film’s roughness after several multilayers is about 1.4 nm. This information is also helpful to determine the appropriate particle size in the second deposition. It was determined that 10< nm size particles would have the best resolution on the multilayers. Several modifications where used to optimize growth8,12, 1 3 and the optimal films seem to depend on whether the phosphate molecule had an aliphatic or aryl chain, zinc purity, and whether methanol or ethanol was used. Mallouk et. al. reported2 1 that with respect to aliphatic chains, ZnC12 was used for >C8, and Zn (OAc)2 was used for <C8. However, using a C12 length chain with ZnC12 did not show any film growth. Using 96% Zn (OAc)2 to grow 1, 8-bis phosphonate multilayers worked half 20 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. of the time, and never with 1, 12-diethyl, di-phenyl bis-phosphante (1,12-EBP). Adding a few drops of MeOH did not increase solubility of 96% Zn. Using 99+% purity of Zinc improved the deposition for both 1, 8-bis phosphonate and 1, 12-EBP. M u Itilaye r Film t h i c k n e s s 12 0 10 0 - £ 8 0 « 6 0 ^ 40 2 0 0 2 4 6 N u m b e r of la ye rs + S e ri e s 1 Figure 8 Monolayer growth over time (upper). Multilayer growth (lower). In figure 8, the upper graph depicts monolayer growth over time. Starting with the thickness of the oxide and anchor layer (25 A), the thickness was measured at 1, 5, 10, 15, 20, and 25 minutes. After about 10 minutes, the total thickness levels off to 21 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. about 41 A, and the actual film thickness is 16 A. The lower graph measures the resulting thickness per the number of cycles (layers). Ellipsometry measures 4 layers of 1, 8-bis phosphonate, and starting with an oxide/anchor layer thickness of 21 A, the thickness increases 12-14 A after each cycle. These measurements gave confidence to the optimized depositions conditions for multilayer growth. The next analysis was used to determine the stoichiometry of the film. Min: 2863 Max: 98102 Zn 2p3 N(E) Si 2p3 1100 880 990 770 660 550 440 330 220 0 110 Binding Energy (eV) Figure 9 XPS spectra o f ZBP multilayers corresponding to 6 cycles XPS was used to follow the multilayer process as is provides both quantitative and stoichometric information. There is a Zn 2p3 peak at 1040 eV, N Is at 440 eV, and P 2p3 at 13 eV. Beside elemental analysis, XPS can provide stoichiometeric information (figure 9). The assumption is that the detection peak is from those core electrons throughout the sample and not only at the surface. The area underneath each detection peak is the sum contribution o f the element core electron from all the layers. In order to determine stoichometry1 8 , 2 0 '3 0 , the signal must be corrected for 22 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. the attenuated contributions from each layer underneath attenuated and follows a Beer's law. Filled core states 100 so « A u # c so too ELECTRON ENERGY $00 10 0 0 2000 Figure 10 The graph o f the Inelastic Mean Free Path The Inelastic Mean Free Path (IMFP) (figure 10) should be thought about in terms of as “bands” for groups of materials, with the metallic band at the bottom, followed by semi conductors above metals and then thin organic films like Zinc bis- phosphonates. For example, as a multilayer film, the sensitivity values of Zinc = 60 and Phosphorous = 136 are 8 times higher than would be expected from this curve. This is due to the packing of the film because it affects the tilt angle of the packed film seems to focus the ejected electrons towards the detector (figure 11). 23 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 100.00 \ £ £ CL U . E 10.00 -■ 1.00 ■ \ \ \ 0.10 10 100 Energy (eV] 1000 10000 Figure 11 The Inelastic Mean Free Path (IMFP) as a function o f Kinetic Energy. This means that IMFP of low energy electrons corresponds to only a few atomic layers and therefore is a surface sensitive technique. This knowledge is used to calculate the attenuated contributions of atoms (Zn or P) from the underlying layers, and from that, one can determine the stoichiometry of the multilayers. Phosphate terminated surface model; Zn:P =1:2 Layer# zinc phosphorous 0 0 1.00 1 0.79 0.90 repeat distance = 14.5 2 0.62 0.81 mean free path (Zn) = 60 3 0.48 0.73 mean free path (P) “ 136 4 0.38 0.65 5 0.30 0.59 ASF Zn/P = 15.08 6 0.23 0.53 Stoichiometric corrected sum 2.80 10.40 Intensity*ASF ratio 42.22 10.40 Calculated Zn/P ratio M easured Zn/P ratio 0 .2 7 02 -2 Calculated raw peak area ratio 4.06 Table 1 Stoichiometric determination o f 6 layers o f Zinc and Phosphorous Atoms. 24 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. For example, consider the silicon substrate with anchor layer and 6 layers of growth as shown in Table 1. The XPS signal from the underlying layers will be attenuated (i.e. reduced in intensity) due to inelastic scattering of some of the photoelectrons as they traverse through the layer. This reduces the overall intensity of a XPS signal (Zn and P). For Zn/P ratio there is a very good correlation between the calculated value 0.27 and the measured value 0.27 thus confirming that the 6 cycles of film growth of ZBP has multilayers structure. 1.8 Conclusion Monolayer Deposition Time (monolayer) Reaction Conditions Embed Particles 2n d Deposition o f Particles Manipulation on Film Removal of Layers Trichlorosilanes 2.5 hours Dry, inert atmosphere No* No* No* Ozone Zinc Bis- Phosphonates 20 minutes Ambient, In Ethanol Yes Yes Yes Water * = unknown Table 2 Overall film comparison. Table 2 summarizes the results of both films. ZBPs are preferred films to grow. These films grown are flat, well ordered and packed multilayers, have ambient conditions and short deposition times, relative to trichloro-silanes, and are easy to detect and characterize. The next chapter discusses the embedding process. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Chapter 2: Usage of Zinc Bis-Phosphonate Multilayers in LNF. 2.1 Introduction Chapter two compiles the results of initial experiments concerning nanoparticle embedment. These initial experiments planned the sequence of steps and optimized the conditions essential to successful embedment using the LNF process. These results should also avail themselves to future experiments that attempt to address questions including particle linkage, manipulation14,26'2 8 . These results are organized into sections that discuss preparation, deposition conditions, and embedment. The requirements that must meet are that no strong bonding between the surface and particle shell; and that the solution that suspends the particles must not destroy the film. 2.2 Embedding 5nm Cl-capped Gold Colloids Thus far, 5 nm thiol capped particles9 , 1 3 , 3 8 can be difficult to manipulate and distinguish from the surface roughness in comparison. The particles are transferred and suspended in toluene, which must not be water saturated.1 4 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. d i s "t a. rii o « ? Figure 12 1 x 1 micron image o f 5nm thiol-capped gold colloid on the anchoring layer * -*>■' #*• * >*- * * -r • ♦ ■ •% " * . l * : - • ' - ’ »-<*« ^ v » * « * * -. ^ •* * •* * > » *' **, ~ j* . .-*'1 * - V: »j* - * " ’“ »*“** * * < " £ * w J » , J*. . . . J?.;„ * ...,> & ,.,. Uert distance Oert distance llert distance 2.002 nM 2.428 m 1.707 n M Figure 13 (2 micron) 5nm thiol-capped colloid after growth o f 4 layers 27 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Uert distance Uert distance Figure 14 5nm thiol-capped gold colloids after removal of ZBP Figures 12, 13, and 14 demonstrate that the multilayer growth of ZBPs embeds 5nm alkyl-thiol-capped particles. The particles' height decreases during film growth, and resume after dissolving the film. Line H e i g h t 0: 4 9 .7 A [ A ] 1: 55.1 A H s ig h t P rofile [A] 0.2 Figure 15 500nm image of Phosphorlation 5nm Chloride-capped gold colloids on APTS. Figure 15 shows the heights and homogeneity of 5 NM Chloride-capped particles that were added to the anchoring layer on Silicon, followed by phosphorylating the 28 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. amine surface. No significant changes to the heights of these particles were made and so it can be concluded that there is no damage by that reaction. Further experiments should include repeating this with various sizes and with metal colloids. These initial results show a potential use to fabricate high-resolution masks combined with a multilayer film as mentioned in section l.D. Further experiments should include removing the particles, growing a multilayer and analyzing the step edges. This was an important alternative to explore because the particle manipulation conditions on an amine surface have been optimized. 2.3 10 nm Amino Alkane and Cl-capped Colloids lOnm-amine capped particles are better resolved and manipulated by AFM. The organic capped particles depend on some water content for dispersion. Molecular sieves were added and left in the suspension for 3 days. The solution was clear with a small aggregation of particles. It was possible to deposit on the (P03)2- surface. Flowever, foreseen problems about particle linkage, water stabilization, and manipulation necessitated replacing them with Cl-capped particles. For example, the alkyl amine coverage at the gold colloid surface is not known. Therefore it is not clear how much water is needed to uniformly disperse in toluene. A careful water titration experiment may resolve this. Starting with the aggregated particles in the very dry toluene, one may titrate water into the solution and monitor any change to colloid aggregates. Additionally, if water was directly at the colloid surface, could a linker molecule replace it? It would be interesting to determine if this amount of water would have any affect to the multilayers. Initially, the manipulation of these 29 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. particles was not straightforward and further experiments need to optimize those conditions. 30 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 2.4 15nm Cl-Capped Gold Colloids o o Line H eight • 80.0 [A ] 0.8 Figure 16 lx l micron image o f 10 nm Chloride-capped gold colloids on Phosphorlated Si02 surface. In ethanol, the anchor layer surface exits as phosphate groups that have a 2 charge per group, which is expected to repel like-charged particles such as Cl-capped gold particles (figure 16). As a result, there is a low coverage of particles at the silicon surface. Lowering the pH at the surface by rinsing with 12 mM HCL significantly increased the coverage of the particles (figure 17). 31 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 17 Lowering the surface pH increased coverage o f 10 nm Chloride-capped gold colloids There is noticeable increase in coverage after treating the sample with 12 mMol HC1. At a lower pH, overall positive surface charge attracts the Cl-capped particles. A drop is left on the substrate for 25 minutes followed by a drop of 15 nm Cl-capped particles for 25 minutes. The particle heights are between 12 -15 nm. [A ] 0: 66.1 A i y j U : Jb.ifA - 2 0.0 A J A .v -M----- 0.2 0.4 0.6 0.8 Qm Figure 18 10 nm chloride-capped gold colloids after growth o f 5 layers lowered pH Phosphorlated surface. Figure 18 shows that a majority of these sized particles (green line with blue numbering) have a height of 35 A on average. Assuming that the original heights were between 7-10nm, the difference correlates to the thickness of 5 cycles of ZBP film. In figure 19, after removing the layers, the line scan shows that most of heights of these particles increase back to the original height range of the particles before the growth of the multilayer film. This shows that removing the film does not damage any arrangement or structure. 32 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 19 10-15 nm chloride-capped colloids after removal of ZBP 2.5 Deposition of silver PVP capped particles on silicon and gold substrates. Selecting nano-particles that were stabile in ethanol, would simply the LNF process because that the solvent is also used to grow zinc Bis Phosphonate multilayers. EtOH is the solvent used to disperse PVP-capped Ag particles and the synthesis of Silver nano-scaled particles is reasonably straightforward. AgNo3 (8.5 mg, 500 mmole) was dissolved in 50 mL of Ethanol. To the solution was added polyvinylpyrrolidone, (PVP-K30, 220 mg, 2 mmol as monomer unit) and the solution refluxed overnight (100 °C) under inert atmosphere. The resulting intense yellow solution (X max = 400nm) was characterized as monodisperse silver colloids of average size (aprox. 12 nm). It is valuable to deposit Silver particles and perform LNF on silicon substrates because of the lithograph map that allows for manipulation of the same particles. The preparation for the anchor layer on Silicon has been discussed previously. Silver particles are well resolved on the anchor layer surface. 33 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 2.6 Preparation of Anchoring Layer on Gold Preparing the anchor layer (figure 20) on a gold surface is a two-step process. Sonicating in Acetone, Isopropanol, and Methanol for 20 minutes each, followed by 10 minutes in the ozone chamber cleans gold substrates. The resulting oxide layer was hydrophilic with a contact angle of 11 degrees. The substrate was placed in 2- Mercapto-ethyl phosphoric acid (HS(CH2)2P03H2) 5 mmol in Ethanol solution for 5 hours. The contact angle was 68 degrees, which is a consistent value for P03H2 surfaces on silicon. Figure 20 lxl micron image of the anchoring layer on a gold substrate. 2.7 Deposition of Silver Colloids on anchoring layer The substrate was immersed in a few milliliters of the colloid solution. The sample was removed after 5-10 minutes and left to air dry. Silver nano-particles on gold substrates are clearly resolved at the surface (figure 21). U ert d is ta n c e 34 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. U ert d is ta n c e 9.41B nw Uert distance 10.959 ret Uert d is ta n c e 10.214 r < M o 1 Figure 21 3x3 micron image o f 10 nm Ag nano-particles deposited on the anchoring layer 2.8 Embedding Silver Colloids After imaging several different areas, the multilayer of ZBP embeds these particles in the same manner as gold colloids. The thickness of the films, after 5 layers growth is about 7 nm, which is the approximate decrease to the particles in the line scan of figure 21. Figure 22 shows embedded particles with an expected height of 3nm. U ert d is t a n c e Figure 22 500 x 500 run image o f silver nanoparticles after 5 layers film growth 35 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 2.9 Conclusion and Outlook of Chapter 2 The sections presented in chapter 2 compile the initial results of nano-particle deposition, embedding, and manipulation using the multilayer film Zinc Bis- Phosphonate. These initial results meet the selected criterion for Layered Nano Fabrication. Therefore, the contents within this chapter should be included as direction toward future experiments that optimize these results, or begin to address new issues. For example, there is the question of how to link particles. Recently, alkyl di-thiols were used to link gold sized-sized particles into simple structures. The structures were maintained after several manipulation steps. However there was some difficulty in removing excess di-thiols from the substrate, and those efforts could easily damage the structure. In conclusion here are some brief suggestions for future experiments to solve this problem. One experience is to link the particles after embedding them with a few multilayers. Since the di-thiols are suspended in Ethanol, damage to the film is not anticipated. Dissolving the film away should remove the excess di-thiol because the excess dithiol will settle onto the surface of the multilayer film rather than the substrate. Another experiment is to chance the geometry of the linker molecule. Currently, aliphatic, linear di-thiols are used. It is suspected that at room temperature, these molecules are is rapid much motion, and to allow for one molecule to link with two colloid particles molecules in considerable yield. This rationale follows the successful stabilizing of gold colloids using a thiolalkane. Restricting the degrees of 36 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. freedom by using 1, 4-di-thiol phenyl, and di-phenyl showed no initial results, however this could be largely to a limitation of the angle between the thiol atoms, which was only at 180 degrees. (Scheme 4) Synthesis of a di-thiol linker molecule using 1, 10-phenantroline has the potential for various angles between the thiol atoms, and extra alignment for second thiol bonding provided by the nitrogen groups. H H H H B r2 Reflux. 2hrs B r MeC12 Pd(PPh3)4 B (R)(OH)2 MeC12 H Soni cation '' H H R R= alkyl or aryl thiol, phosphate, or amine Scheme 4 Synthesis o f di-substituted 1, 10-phenantroline. 1, 10-Phenantroline is rigid and planar, containing two nitrogen atoms at the 1 and 10 positions, which may provide some binding to metallic colloids. The 8 protons located at the 2 thru 9 positions. The nitrogen atoms could bind to the colloids. It has 37 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. been reported that Nitrogen chelate well to metals including Gold, Silver, and Ruthenium. Starting with a bromide precursor, Suzuki coupling can modify the 2 thru 9 positions of these compounds. For example a one-pot reaction for a di-aryl- substitution at the 3, 8 positions in about 40% yield, via Suzuki coupling. Modifying compounds with alkyl thiol chains containing one or two degrees of freedom may improve di-thiol linkage. Introducing thiol groups at the various positions of 1, 10- phenantroline may provide the appropriate geometry for linking coinage colloid metals. Additionally the nitrogen atoms could secure the molecule such that the second thiol group is in a better position to form a bond to the colloid. Another potentially interesting application of LNF would be to fabricate high- resolution masks out of a Multilayer. This could be achieved by using the nanoparticles as photo-resistant shadow masks. For example, a typical procedure would include deposition of colloids, followed by manipulation, linkage. The next step would be to remove un-shadowed areas via hu exposure, removal of particles (magnetic or electric field), growing a multi-layer SAM and analyzing the step edges via AFM, TEM, or SEM. The rationale for this experiment can be inferred from figure 14, which showed that phosphorylating the surface after depositing 5 nm Cl- capped particles did not erode them. There was no change to their size before and after phosphorylation. Since manipulation conditions for these particles are optimized, on amine surface. Linking them into a desired shape would be feasible. 38 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Chapter 3 Selective Adhesion layers for a Tight Neural Contact 3.1 Introduction Chapter 3 presents initial results that focus on the experimental set up conditions to deposit adhesion layers used in the culture of neural networks. This chapter’s results fit into a larger scheme, which is to develop chemistry methods that promote selective adhesion and attachment of neurons to electrodes through ligand-receptor binding (figure 23). A self-assembled monolayer (SAM) is viewed as the ligand (on a gold or titanium substrate) and the receptors are either the binding domains of the axon or the neural cell body. Neurons need biocompatible surfaces to improve the neuron-electrode interface through using biologically active adhesion layers. A sub goal is the optimization of an adhesion layer that shows activity towards the promotion of cell adhesion proteins, such that it supports cell life and propagates neural network2 4 ,2 9 ,3 4 . Neuron elect Ligand-receptor binding Figure 233 Neuron bound to substrate via SAM. The development of a reliable mode to study the firing patterns of neural networks is highly recognized and desirable. There are many advantages to understanding these 39 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. patterns and their relationships to brain function. Neuron cells operate on discrete on/off signal. This is similar to the 1 and 0 bits that comprise a computer’s binary code. The sequence of a binary code, the ASCII code, correlates to symbols of our alphanumeric system for communication. Having the ability to relate the firing patterns to a similar type of ASCII code would allow researchers to formulate a catalogue where brain functions are related to the brains firing pattern. The current methods are designed to accommodate the activity associated with the firing capacity. However the current experimental set up has many problems that impede the amount of information that is obtainable. Some of these problems include the size of the electrodes. Being large and bulky, they inhibit the social/ physical movement of the host and therefore can affect the data. Another way to get information is to study a slice of the brain. The relative position of the cells would affect any information about firing patterns. The cells are subject to migration from the address electrode. The initial results outlined in the following sections attempt to address the need to produce reproducible, secured cell attachment within a micro array of electrodes the application of SAM technology12,34,36, Therefore, it is import to find an adhesion layer that will sustain the attachment of healthy cell cultures without any compromise to the signal output, it is also desirable to modify the glass and electrode (gold or titanium) surfaces such that the neurons will selectively attach to electrodes. This chapter discusses two approaches. The first approach incorporates active antibodies that are specific to the neural axons. Both 40 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. combine surface selective SAMs with either the antibody or cell specific bio-mimic peptides. Synthetic SAMs provide longevity of the monolayer through covalent bond linkage to the substrate. For example, to achieve en vivo reproducibility en vitro, it is important to find an adhesion layer that will sustain the attachment of healthy cell culture. SAMs form well ordered monolayers spontaneously on various substrates. For example, alkylsilanes form on silicon oxide, alkanethiols on coinage metals (gold), and alkyl phosphates on Titanium oxide and Titanium nitride surfaces. The shape of the cells and the branching are greatly affected by the surface it attaches to, therefore using SAM technology is a good approach because it gives control of topography and flatness. The hydrophobic component of SAMs contributes to flat, homogenous surfaces through Van Der Waals forces. Derivatization of the terminal functional groups provides further control of cell attachment, which affects the neuron’s shape and branching. The following sections in chapter 3 present initial conditions to bind voltage-dependant Calcium channel to the SAM surface on an electrode using AFM. 3.1.1 Antibody Background The antibodies used are from the Immunoglobulin G family of glycoproteins. The advantages of IgG are they are large, widely available and have well characterized sequences this allows for constructing good models to determine activity. They 41 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. contain two pairs of polypeptide chains and each pair contains a heavy and a light chain. Fab arm waving Fab elbow bend Fab rotation Fc wagging t MkeCkxk 1994 Figure 24 Antibody from the Immunoglobin Superfamily In figure 24, the light chain (smaller inner section) is responsible for binding specificity. Both chains are linked together via disulfide bonds. Immobilization to the surface occurs through amide bond formation between the antibody’s lysine groups and the activated carboxyl groups at the surface. Various orientations of the Antibody are reported at the surface because of approximately 60 lysine groups found at the surface of the antibody (AB) and therefore seem to be a random process regardless of the conditions. What is the most probable attachment of antibodies at a gold substrate? Should one think about them approaching the surface as would an open umbrella sinking in a pond? The rationale behind this is that the lysine groups 42 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. at the base of the heavy chains would be expected to make contact with the surface due to it’s gravity. Control may be d by the incorporation of a SAM. These experiments employ a SAM with terminal COOH groups (activated by EDC), which would protrude away from the surface. Future surface experiments should explore where does each of the SAM components (COOH or CH3 terminus) prefer to deposit on the gold surface? Would modifying an AFM tip with free amine groups provide topography information? Following the deposition of antibodies, would staining with flurosceinamine (a dye that tags free amine groups) identify antibodies at the surface?3 ’1 1 ’1 7 ’ 2 4 ’ 2 9 ’ 3 7 3.2 SAM Characterization The available analytic tools were contact angle meter and Atomic Force Microscopy. Here is a brief description of how they are implemented to analyze the experiments. 3.2.1 Contact Angle Measurements The contact angle meter (figure 25) is a simple, non-destructive method for monitoring growth progress of surface modification. Other non-destructive methods are important to surface characterization like ellipsometry, however the roughness of the gold samples prevent its use the increasing thickness of the film would be hidden within the roughness of the metal substrate. 43 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 25 Contact Angles. Hydrophilic surface (left); hydrophobic surface (right) Contact angles are a useful method to follow the surface modification. Using a small sessile droplet of water, the angle formed tangent to the surface can be related to the hydrophillicity and hydrophobicity of the surface of the film. For example, starting with a clean hydroxylated gold surface, the expected angles are less than 20 degrees. That angle increases as the hydrophobic SAM coverage increases. The measurements were done in ambient laboratory conditions (aprox. 50% humidity). Deionized water was used and the angles were measured in dynamic mode. 3.2.2 Atomic Force Microscopy Measurements were preformed in air using Nanoscope III (Digital Instruments). AFM is becoming a widely used surface technique in the study of biological materials. This type of scanning probe microscopy provides topological surface information under quick and relatively simple conditions. AFM uses optical beam reflection to monitor the displacement of a silicon nitride cantilever, (k = 80 N/m) as it scans over a specified surface area. It is used to follow the deposition of the antibodies by comparing surface scans looking before and after antibody deposition. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 3.3 Detecting Antibody Activity at the Surface It was important to gain a qualitative, and eventually, quantitative assessment about the Antibodies that attached to the surface. They need to have the appropriate geometry where the antibody remains biologically active after attachment to the SAM. The method to analyze this at the surface was to compare the volume verses surface areas. Adding either a specific secondary antibody or the enzyme alkaline Phosphatase, would identify any active antibodies bound to the surface. This is detected by the height increase of any primary antibodies bound to the surface. 1 1 ’ 2425, 3.4 SAMs Preparation for Terminal Carboxylate groups The growth of the SAM is as follows: R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Thiotic Acid C.Shannon et al Annal. Chem. 2000, 72,2371 C O O H COOH Im M soln. (1:1) 24 hrs Butyl Mercaptan Au 3HC2HC- - N = C = N (CH2)3N(CH3)2 l-ethyl-3-(3-diniethylam inopropyl)carbodiim ide HC1 '(C H 2 )3 N (C H 3 )2 5ug/mL (1mm diameter) 24 hrs Scheme 5 Growth on Gold Substrate R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. The sample was glass microscope slide that was patterned via vacuum deposition with an array of gold dots (2-10 mm diameter). It was cleaned in a piranha solution (3 H2S04: 1 H202) for 15-25 minutes, rinsed with copious amounts of water and ethanol and blew dry with N2 gas. Next, the sample (scheme 5) was transferred to a ImM solution of thiotic acid and butyl mercaptan in ethanol for 24 hrs; afterwards, the un-bound molecules were removed by rinsing with Ethanol, acetonitrile and blowing dry with a stream of N2 gas. Next the sample was placed in 1% EDC (1, 3- ethyl (di-methylpropyl carboimide) in acetonitrile for 5 hours, rinsed with Acetonitrile, and blown dry with N2 gas17,40. The immobilization of non-specific antibodies was preformed under non-sterile conditions. Prior to antibody immobilization, the sample was rinsed with PBS buffer and transferred to a 5 -12 pM solution of antibody in PBS buffer (pH = 7.4) for 24 hours at 4 °C. The sample was transferred to 5% ethylamine in ImM Borate Buffer for 5 hours. This was to cap the un-reacted carboxyl groups at the surface. The final SAM was carefully rinsed with DI water and left to air-dry before taking AFM measurements. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. ■ clean ■ SAM ■ EDC Methyl COOH Glass Table 3 Contact angles measured on gold pattern glass substrate 3.5 Experimental Results Contact Angles were measured after each step of the film growth (Table 3). Immediately following the cleaning procedure, the contact angles of the gold and glass surfaces (green) were <20 degrees, which is characteristic of hydrophilic surfaces. The angles increased to 60 degrees after treatment with the SAM solution containing thiotic acid and butylmercaptan (COOH), and after exposure to 1% EDC solution, increased to 80 degrees (powder blue). After treatment with 100% butymercaptan (Methyl), the contact angle increased to 84 degrees. After exposure to 1% EDC solution, this angle remained the same, which was expected since this surface does not have carboxyl groups. The contact angle of the glass area (glass) was not expected to increase. However, after exposure to EDC, this angle increased 48 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. sharply to 70 degrees, which implied that EDC had bonded to the glass surface. The issue of non-selective EDC modification to glass is addressed later. This following results show that the use of a SAM controls antibody immobilization to a gold surface. The first look was how antibodies bind to the bare gold surface without the use of a SAM. Figure 26 4x4 micron image of Antibodies on gold- without SAM 5 pg/mL solutions of antibodies were deposited on a clean, bare gold surface following the protocol described previously. Figure 26 shows uncontrolled deposition. Without an adhesion layer, the deposition of antibodies appears to form large aggregates at the surface. Figure 27 Methyl-terminated SAM following exposure to Antibody solution 49 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 27 depicts a second negative control where the monolayer surface is methyl terminated. As expected, antibody attachment is not detected. The large protrusion (noted in white) is most likely debris and the smaller ones could be antibody attachment due to holes in the SAM. Figure 28 COOH-terminated SAM exposed to Antibody solution Figure 28 shows a third negative control where the COOH terminated SAM is exposed to antibody solution. As expected, in the absence of EDC, the carboxyl carbons are not activated to form amide bonds with the antibody’s lysine groups and shows a low coverage of antibody at the surface. U e rt distance Uert distance Uert distance R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 29 Activated SAM exposed to Antibody solution In figure 29, the image depicts antibodies deposited on the activated SAM. The antibodies appear as small discrete spheres (white) at the surface. AFM images depict them with a height dispersion is between 6.5nm -13 nm the most populated heights are 6.5-7 nm and 10-13 nm. 3.6 Antibody Activity at the Surface This section addresses how to detect the antibodies' activity at the surface. Initially, alkaline phosphatase was used for visual colorimetric read on the activity. Unfortunately, the highly reflective surface of gold and the low concentration of antibodies that are attached to the surface did not allow for visual detection. An alternate method was to deposit antibodies specific for Alkaline Phosphatase (AP), an enzyme used in colorimetric detection, on the gold surface. These enzymes have {several} binding sites that are specific to Fab regions of antibodies with the appropriate geometry. From AFM, one would expect to see height increases from their binding event, and help estimate their activity at the surface. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 30 Afm images of AB1204 following exposure to the enzyme Alkaline Phosphatase. Figure 30 shows an a typical process of estimation of the number of active antibodies. On average, 52 % of the antibodies at the surface are active. This was done by a visual tally of antibody that increased in size following treatment of the enzyme alkaline phosphatase. The first column is for the number of antibodies counted. Following the standard image processing, the line scan analysis bar was carefully moved along the image to depict any antibodies from the surface. The second column is the z-scale that was used to set the appropriate color bar, which is the height minimum. Any item at the surface above the height minimum is shown in white. The last column on the right is the percent of antibodies that increased in height, which could be viewed as having the appropriate geometry. Following the deposition of primary antibodies, 140 were observed to have a minimum height of 7.5 nm. Following the addition of the enzyme alkaline phosphatase, 52 showed a minimum height of 15nm (52%), of which 20 showed a minimum height of 25 nm (14%). 52 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. To eliminate any antibodies surface that were not covalently bound to the SAM, the sample was submerged in a solution of Tween 20, a detergent used to minimize non specific binding (typically Tween 80 is widely used, but was not readily available at the time). The expectation was to remove any abound antibodies not covalently bonded to the SAM. Figure 31 Bound Antibodies with AP after exposure to Tween 20 Figure 31 shows a similar trend as in figure 30. After treatment with Tween 20, the same tally procedure was preformed. There is a consistent trend in the number of antibodies attached to the surface with respective heights. This suggests that the antibodies attached to the surface are covalently bound to the SAM and approximately 50% show some activity towards the enzyme alkaline phosphatase. The next section shows how the cells bound to the SAM. 3.7 Cell Attachment to SAM Surfaces The following protocol was used to for cell adhesion. The hippocampi of E-18 rats are dissected and mechanically dissociated after treatment with 1% trypsin. Cells are count z-Bcale ht, roin h it rang* % 84 15 nm trim 6 4 2 nm 30 nm 16 nm 15-20 nm 67% 60 nm 23 nm 35-36 nm 28% 53 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. cultured in Neuro Basal Medium (Gibco) with 0.5 mM glutamine, B-27 Supplement, pen-strep, and for the first few days, 25 mM glutamate. The cells are cultured at about 125,000 cells/cm2 , in a 10% C02 incubator and fed twice a week. To examine the cell attachment and branching on the gold surface the cells must be fixed to the surface and the sample is turned over for viewing. The cell culture is treated with a solution of 4% formaldehyde solution for 15 minutes. The skeletal structure of the cell is examined via a microscope. Before comparing the attachment that these cells have on variations of the SAM, some visual guidelines (figure 32) have been noted for healthy cells, dead cells, and non-cellular debris. Figure 32(a) Dead cells, (b) Healthy cells, (c) Debris 54 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. In figure 32b, dark grey spheres depict the healthy cells with white halos surrounding them; dead cells 32a are small white dots and debris appear in various shapes 32c. In a few days, healthy cells will show the beginnings of axon and dendrite branching (thin dark lines). The focus of incorporating the antibodies is to determine if they increase the amount of branching by the cells. Comparing the neuronal branching at the gold-glass interface does this. Cell Fixation Figure 33 Zoomed area o f fixed cell bodies The spheres represent the fixed-dead cell bodies and the neural branching is also apparent by the thinner dark lines. Fixing the cell cultures permits direct evaluation of the neural branching at the gold-glass interface. 55 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 3.7.1 Experimental Results Summary of Cell Attachment to Modified Surfaces Surface group A ttachm ent cm G lass A ttachm ent on Gold CO O H Expected: n Expected: y O bserved: n O bserved: n EDC Expected: n Expected: y O bserved: y O bserved: y Blank Expected: n Expected: n O bserved: n O bserved: n Glass with EDC only Expected: y Expected: N/A O bserved: y Observed:N/A Specific antibody Expected: y Expected: y O bserved: y O bserved: y Non-specific antibody Expected: y E xpected: y O bserved: y O bserved: y Non-specific antibody-biotin Expected: y Expected: y label O bserved: y O bserved: y CO O H with Strepavidin -FTIC Expected: n Expected: y O bserved: y weak O bserved: y Table 4 Various attachments on Surfaces Table 4 summarizes the cell attachment results-expected and observed respectively. As mentioned earlier the substrate is gold patterned on glass and the goal is to only attach neurons to gold through selective SAM modification to gold. It is known that the cell surface has an electro-negative charge and prefers to attach to surfaces that are positive, and rough. Before using the specific antibodies, it was important to examine how the cells would attach to the SAM itself. In the cell medium, the carboxyl group is mostly de-protonated and so the surface would have a net electro negative charge. A little attachment was expected in this case, mostly due to proton transfer from the cell medium (pH 7). The possibility of some protonated carboxyl groups had no observed effect. The EDC activated SAM before antibody exposure 56 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. unexpectedly showed strong cell attachment. This was repeated (Glass with EDC only) and gave similar cell attachment (not shown). Figure 34 Cells 3days old: non-specific Antibody from rabbit deposited in 0.135 M NaCl {thin film: thiotic acid/butyl mercaptan followed by EDC. The cells that are cultured on a SAM with non-specific antibody (figure 34) have good attachment to the glass (blue area) and gold regions (dark spot). The coverage and axon growth will be compared to a cell cultured with axion-specific antibodies. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 35 Cells 3days old: Sodium Channel-specific Antibody from rabbit deposited in 0.135 M NaCl {thin film: thiotic acid/butyl mercaptan followed by EDC}. The cells that are cultured on a SAM with specific antibody (figure 35) also have good attachment. Monitoring the culture of both over time will show any affects to neural branching. Observing the gold glass interface for a difference in attachment would give some indication of the antibody affects, since the assumption is that only the antibody is bound to gold because that was where the SAM-EDC was expected to be. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 36 Cell Culture 11 days old and before fixing with 4% aldehyde solution. Strong attachment 1. Dead cells 2. Thicker branching -live cells clumped together with some dead cells on top. 3. Thinner branching -cells less clumped. In figure 36 that there is strong attachment to the glass region. Since the cells do not bind to the bare glass, or inactive COOH terminated SAM surfaces, it is concluded that the presence of cells in the glass region indicates that there are EDC and Antibody bound to the surface. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 37 Presence of EDC on glass confirmed by antibody attachment. Subsequent AFM imaging shown in figure 37 confirms that antibodies attach to the glass due to the presence of EDC. This was further confirmed by the Addition of Alkaline Phosphatase, rinsing with Tween 20 and AFM analysis. This sample has seen the SAM solution, been activated with EDC, seen antibody (AB1204), been capped with ethylamine, and then seen the enzyme alkaline Phosphatase. The height range of AB1204 is 7-10 nm. After seeing Alkane Phosphatase, the heights consistently increase to 15-17nm and 25-28nm. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Route 1 Current experiments). Selective modification of Gold with an active surface towards antibody deposition. HS . 0 4-M ethylthiolbenzaIdehyde (MTBA Route 2 o o HS- + H' 'H 4-Aminothiolphenol glutaric dialdehyde Scheme 6 Synthetic routes improve selective modification to a gold substrate Since EDC in non-selective to the OH surfaces of glass and gold, it use should be eliminated. Two alternative methods (scheme 6) were identified for the selective modification of the gold surface. The first route is one step SAM deposition that incorporates 4-Methylthiobenaldehyde (MTBA) under the same deposition conditions as were used in the previous method. It has contains the thiol group and a reactive aldehyde group-both required to link the antibody to the gold. The aldehyde group has been shown to react with antibodies without any coupling agents, and the thiol group has been a well documented to form strong bonds to the gold surface. Another advantage of this molecule is that it shortens the distance between the electrode and neuron. Research has shown that shorter linker distances improve the contact. Assuming a tightly packed SAM with full coverage, the approximate thickness of MTBA would be 8.5 Angstroms. Compared to the original SAM 61 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. thickness of approximately 16 Angstroms (thiotic acid and butyl mercaptan 1:1 ratio) the contact is improved by 10 angstroms. Increasing Contact angles implies selective modification ■ C l e a n g la s s U G l a s s U G o l d B C I e a n g o ld M T B A A T P - G D A T A - B M - E D C Table 5 Contact angles measured on gold and glass. The contact angle increased from 10 to about 80 degrees following treatment with MTBA solution. The glass area showed very little change in the contact angles, which was expected. The contact angle increased from 13 to about 80 degrees following the treatment of 4-amino thiophenol and glutaricdialdyde, with very little change to the glass area (table 5). Figure 38 shows AFM images of the 4 Methyl thiol benzaldhyde followed by treatment of the antibody show attachment. The antibody heights are comparable to the previously observed heights and therefore selective modification was successful. 62 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. S e c tio n A nalysis Section Analysis M TBA-SAM before antibody M TBA -SA M w ith Antibody (specific) Figure 38 Afm images of 4-Methyl Thiol Phenol. (a) Before antibody (b) After antibody 3.8 Conclusion and Outlook The overall goal of this project was to deposit a specific antibody to the surface of an electrode that would support neural attachment to a gold surface. The interim milestones included identifying the appropriate SAM conditions to attach antibodies and analytical tools to characterize coverage and activity. Contact Angle measurements and Atomic Force Microscopy were implemented and these initial results support that antibodies have covalent attachment to the SAM deposited onto gold, with about 50% activity. Cells initially show good attachment to the SAM and to the antibody. Future experiments should include follow up cell cultures on the alternate SAM molecules. Would the terminal aldehyde groups be adequate for cell 63 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. attachment? There is also the possibility that the channel-specific antibodies would block the axon's firing ability. Recent experiments are currently being performed using integrand-peptide mimics, such as RGS may be a straightforward alternative. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Bibliography 1. Andres. R. et. al. Science 1996, 273, Sept. 20. 2. Applied Surface Science (141) 1999, 237-243. 3. Bamess, Y. et. al. Langmuir 2000 (16): 247-251. 4. Baur, C.; Bugacov, A.; Koel, B.E.; Madhukar, A.; Montoya, N.; Ramachandran, T. R.; Requicha, A. A. G.; Resch, R.; Will, P.; Nanotechnology, 1998, (9): 360- 364. 5. Bizios, R.; Kam, L.; Shain, W. Turner, J.N.; Biomaterials 2002; (23): 511-515. 6. Burning, P.A.; Humbel, B.M.; Philipse, A. P.; Verkleij, A.J.; Langmuir 1997, 13- 3921-3926. 7. Cacciafesta, P.; Humphris, A.; Jandt, K.; Miles, M.; Langmuir 2000; (16): 8167- 8175. 8. Clearfield, A. et. al. Inorg. Chem. 1996, (35):5254-5263. 9. Dressick, W.J. et.al. Langmuir, 1999, (15): 5429-5432. 10. Eastman, J.A.; Choi, S.U. S.; Li, S.; Yu, W.; Thompson, L.J. Appl. Phys. Lett. (78)6: 718-720. 11. Effenberger, F.; Grunze, M.; et. al.; Adv. Mater. 2000 (12): 901-905. 12. Ferguson, G.; Freund, M..S.; Angew. Chem. Int. Ed. 2000 (39) 7: 1227-1230. 13. Friedbacher, G.; Hoffman, H.; J. Phys. Chem. B. 1998, (102): 7190-7197. 14. Heath, J.; Knobler, C.; Leff, D.; J. Phys. Chem. B. 1997, (101): 189-197. 15. Hoffman, H.; Mayer, U.; Krischanitz, A.; Langmuir 1995 (11): 1304 -1312. 16. Hoffman, H.; Mayer, U.; Valiant, H.B.; Langmuir, (1996): 4614-4617. 65 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 17. Karyakin, A.; Presnova, G.; Yu, M. et. al. Anal. Chem. 2000 (72): 3805-3811. 18. Langmuir, 1999, (15): 7244-7251. 19. Leff, D., Ohara, P., Heath, J.; J. Phys. Chem. B 1995 (99) 18: 7036-7041. 20. Leff, D.; Brandt, L.; Heath, J.R. Langmuir 1996 (12): 4723-4730. 21. Michel, B. et. al. Langmuir 1996 (12): 1997-2006. 22. Mirsky V.M. et. al. Mikrochim. Acta. 1999, (131): 29-34. 23. Negishi, N.; Takeuchi, K.; Thin Solid Films 2001; (392): 249-253. 24. O'Brien, J.O.; Jones, V. W.; Porter, M. D. Anal. Chem. 2000 (72): 703-710. 25. Offenhaousser, A. et. al.; J. Biomater. Sci. Polymer Edn. 1996; (8) 1: 19-39. 26. Resch, R. Baur, C.; Baugacov, A.; et. al. J. Phys. Chem. B 1999, (103): 3647- 3650. 27. Resch, R.; Baur, C.; Bugacov, A.; Koel, B.E.; Madhukar, A.; Requicha, A. A. G.; Will, P.; Langmuir, 1998, (14) 23: 6613-6616. 28. Resch. R.; D. Lewis, Meltzer, S.; Ultramicroscopy; 2000; (82): 135-139. 29. Sorribas, H.; Braun, D. et. al.: J. Neuro. Sci. Meth. 2001; (104): 133-141. 30. Thompson, M.; McGovern, M.; Can. J. Chem. 1999, (77) 1678-1689. 31. Thompson, M.E. et. al. Chem. Mater. 1996, (8): 1490-1499. 32. Ulman, A.; Tillman, N.; Langmuir 1999, (5): 101-111. 33. Valiant, T.; Kattner, J.; Brunner, H.; Mayer, U.; Hoffman, H. Langmuir 1999 (15): 5339-5346. 34. White, R.M. IEEE IFCS, 1998, 587-594. 35. Whitesides, G.; Xia, Y.; Angew. Chem. Int'l. Ed. 1998, 37; 550-575. 66 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 36. Whitesides, G.M.; Self-Assembling Material, A web-posting of his lecuture; http://www.nanothinc.com/nanoscie/... des/selfassembling materials.htm. 37. Willner, I . ; Katz, E. Angew. Chem. Int. Ed. 2000 (39): 1180-1218. 38. Yang H.C. Aoti, K., Hong, H.G., Sackett, D.D., Arendt, M. F., Bell, C., Mallouk, T.E.; JACS 1993 (115):11855-11862. 39. Yang, X.; Shi, J.; Johnson, S.; Swanson, B.; Langmuir 1998 (14) 17: 1505. 40. Yang, W., Shannon, C. Annal. Chem. 2000, (72):2371. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. UMI Number: 1416564 Copyright 2003 by Lewis, Diana Yvonne All rights reserved. ® UMI UMI Microform 1416564 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.

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Asset Metadata

Creator Lewis, Diana Yvonne (author)

Core Title I. Layered nano fabrication. II. Adhesion layers for hippocampal neurons

School Graduate School

Degree Master of Science

Degree Program Chemistry

Degree Conferral Date 2003-05

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, neuroscience,chemistry (physical chemistry),engineering, biomedical,engineering, materials science,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Thompson, Mark E. (committee chair), Koel, Bruce (committee member), Requicha, Aristides (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-305477

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Legacy Identifier 1416564.pdf

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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