Functional Silane Based Co-Polymers for Biofunctionalization Studies, Chemical Sensing and Separations

The focus of the research reported in this dissertation has focused on two areas: (1) Synthesis of silane-containing polymers and (2) the application of these polymers to surface modification of substrates such as PDMS (for microfluidic applications), glass, quartz, silicon wafers and various nanoparticles (magnetic iron oxide, silica) for use in chemical and biological sensing and separation applications as well as in biofunctionalization of magnetic nanoparticles. Chapter 1 of this dissertation described the preparation of a library of silane containing coand terpolymer prepared with various susbtitued maleimides along with their characterization by FTIR, UV-Visible, Hand C-NMR, TGA and DSC. The ability to control the reactive functionalities incorporated into the polymer structure affords direct control over the resulting properties of the materials. Chapter 2 describes the conversion of the polymers prepared in chapter 1 into various functional forms that can be used in the design of different sensing and separation platforms. More specifically, this chapter lays out the conversion of these polymers into polymer thinfilms, polymer-silica composites, polymer-coated nanoparticles and polymer facilitated nanoparticle arrays. In all cases the conversion or assembly of these polymers into these structures is made possible through the reaction of the alkoxysilane side groups of the polymers forming very robust siloxane bonds. These structures are being explored for their use in chemical sensing and separations of explosive and ammes and other species as described in other chapters of this dissertation. The conversion of the polymer into thin-films is accomplished through either dip-coating using a layer-by-layer deposition scheme or through a spin-coating scheme. The layer-by-layer scheme allowed for a controlled deposition of the polymers into films of desired dimension where a constant loading density is observed as a function of the deposition cycle (i.e. number of layers). A facile biofunctionalization of magnetic and silica nanoparticles is also describe in this dissertation. The ability to biofunctionalize these materials allows for their potenital application in the biomedical field where they can be used in detection and in targeted drug delivery. Also described herein is the functionalization of microfluidics systems for bioanalysis. Surface modification with silica nanoparticles would be a promising technique to provide an excellent interface on the conventional polymer surface. A sensitive biofunctionalization is carried in microfluidic chips. The antigens attached to the surface modified silanes via urea linkage and successful binding of antigens and antibodies is observed and can preserve the bioactivity of the antigens and antibodies and resist non specific adsorption. This study can be extended for a high-throughput system for bio-marker proteins. Finally, we demonstrated the applicability of these polymers for sensmg amines and nitro compounds. Substitutions on the phenyl ring help in tuning the sensitivity of these polymers to various amines including very weak aromatic amines. Increase in the intensity of fluorescence and a very noticeable change in color of the polymer solutions in presence of amines helps in their detection. The color of the solution depended on two factors: identity of the polymer and identity of the base. For different polymers, the color varied with the electron withdrawing/donating power of the substitutions on the phenyl ring and also the variation in the side chain allyl vs. vinyl and ethoxy vs. methoxy. Also the nature of the amines 1° vs. 2° vs. 3° affected the rate at which the polymers reacted with those amines and formed the final yellow product. The colored polymer solutions with high fluorescence intensity have been used to detect the presence of nitro compounds as their presence brought the fluorescence down and was directly proportional to the amount of nitro compound present. This work represents our ability to design, control and utilize novel materials for various applications.


INTRODUCTION
The recent advances in simulation, chemistry, processmg techniques, and analytical instrumentation allow a whole host of new types of polymer particles and polymer nanotechnology applications to be realized. Particles include; hollow, multilobed, magnetic, functionalized with reactive groups on the surface, conductive, etc.
Our ability to devise new process control strategies have led to the ability to control the shape, chemical composition, internal structure, and morphology of the nanoparticles so as to develop new levels of product performance and application 1 • Nanotechnology enables us to create functional materials, devices, and systems by controlling matter at the atomic and molecular scales, and to exploit novel properties and phenomena 2 · Nanosensors and nano-enabled sensors have applications in many industries, among them transportation, communications, building and facilities, medicine, safety, and national security, including both homeland defense and military operations. Consider nanowire sensors that detect chemicals and biologics 3 , nanosensors placed in blood cells to detect early radiation damage in astronauts4, and nanoshells that detect and destroy tumors 5 . Nanomaterials and nanostructures are other promising application areas.
Two functions often separated in many sensors, especially those for chemicals and biological substances, are recognition of the molecule or other object of interest and transduction of that recognition event into a useful signal. Nanotechnology will 1 enable us to design sensors that are much smaller, less power hungry, and more sensitive than current micro-or macrosensors. Few sensors today are based on pure nanoscience, and the development of nano-enabled sensors is in the early stages; yet we can already foresee some of the possible devices and applications. Sensors for physical properties were the focus of some early development efforts, but nanotechnology will contribute most heavily to realizing the potential of chemical and biosensors for safety, medical, and other purposes 8 . Nanotechnology will also enable the very selective, sensitive detection of a broad range of biomolecules 9 . Other areas we expect to benefit from nanotechnologybased sensors include transportation (land, sea, air, and space); communications (wired and wireless, optical, and RF) ; buildings and facilities (homes, offices, factories); humans (especially for health and medical monitoring); and robotics of all types 11 • We'll also see nano-enabled sensors increasingly integrated into commercial and military products. Many new companies will make nano materials and some will make sensors based on them. Nanotechnology is certain to improve existing sensors and be a strong force in developing new ones. The field is progressing, but considerable work must be done before we see its full impact. The second step in characterization of material after its design is its assembly.
The importance of self-assembly as a synthetic tool in the fabrication of polymeric materials has increased dramatically due to the complex chemical nature of current The main goal of this research work is to design and develop novel materials (polymers) using simpler, convenient techniques and which could be easily prepared.
Theses materials have unique properties and multiple applications which allow their use as thin films, be modified into polymer nanocomposites and used as multifunctional sensors all made of the same materials 33 -37 . The preparation of polymers, their characterization and its conversion into various forms for various applications has been mentioned below concisely and dealt with in detail in the following chapters 3746 .
In chapter two of this dissertation, we report the facile synthesis of a wide range of silane based copolymers. The work described in this chapter focuses on our ability to design materials which allows us to introduce various functional moieties into the matrix of the bulk materials which is finally used to tune the macroscopic material selectivity. Synthesis of these polymers has been carried out in three steps, In the first step, the maleic anhydride is reacted with various functionalized anilines to synthesize the maleamic acid and followed by the ring closure by dehydration to give the respective substitututed N-Phenylmaleimide. In the final step, the phenylmaleimide is copolymerized with a variety of allyl/vinyl alkoxy silanes to result in the desired polymers. The polymerization is achieved by radical initiator AIBN, the amount of initiator used can control the chain lengths and also the molecular weights of the polymers. A variety of methods have been used to characterize these materials.
FTIR, 1 H NMR and 13 C NMR have been used to determine the structure of these polymers. DSC (Differential Scanning Calorimetry) was used to study the thermal properties in order to understand the thermal behavior and robustness of these polymers. GPC (Gel Permeation Chromatography) to determine the molecular weights of the polymers.
In Chapter 3 we report how the prepared silane based copolymers are converted into various functional forms such as polymer-silica composite nanoparticles and modification of various substrates by deposition and by a facile layer-by-layer approach. These nanocomposite particles and thin-films were shown to be effective in the detection of amino and nitro containing compounds using fluorescence and fluorescence quenching as the detection scheme. UV-Visible Spectroscopy was used to confirm material deposition with each cycle when preparing polymer thin-films. We observe an increase in the intensity of the absorption band of the chromophore in the UV-Visible spectra with increase in the amount deposited with each deposition cycle in all the cases. Since the chemistries of the surfaces used such as silicon and glass is similar to those of capillaries (CE or CEC) the polymers deposited on these surfaces provided a two dimensional environment of how the polymer is effected under different conditions when these materials are made into columns. The modification of the surfaces has been accomplished by a facile spin-6 coating or dip-coating method and provides a very convenient alternative to the laborious lithographic techniques for preparation of arrayed interfaces.
In Chapter 4 we report facile attachment of proteins to the surface of magnetic iron oxide nanoparticles using our polymers as well as other simple silane compounds.
It was possible to demonstrate the retention of the biofunctionality of the proteins when a polymer underlayer is employed. This is most likely due to the polymer providing a cushion that protects the protein from the surface of the particles, where surface effects may adversely affect the protein. This work also demonstrated the successful attachment of antigens and antibody that can be used to design biosensors, as well as allowing for the design of targeted drug delivery and imagining systems.
In Chapter 5 we developed novel ways of functionalizing microfluidics systems for bioanalysis. Through the sensitive biofunctionalization carried in microfluidics chips, successful binding of antigens and antibodies is observed and can preserve the bioactivity of the antigens and antibodies and resist non-specific adsorption and prevents the surface fouling of proteins in the channels.
Chapter 6 describes the application of the polymers reported in chapter 2 for sensing organic molecules such as amines and nitro compounds. The polymers not the monomers or their precursors were found to be sensitive to the presence of amines.
These polymers were found to react with primary, secondary and tertiary amines at different rates giving rise to colored intermediates which finally turns to a yellow colored final product.
Both 1 H and 13 C NMR studies confirmed the formation of !mines when the polymers react with primary amines and enamine formation with secondary amines.
This work also describes how the properties of the bulk can be tuned by introducing different groups on the phenyl ring. More electronegative the group is more sensitive the polymer is to the presence of weak bases such as aromatic amines. Another advantage of these polymers as sensors is the dependence of the sensitivity on the polymer concentration rather than the amine. Hence even very low concentrations of amines can be detected by increased polymer concentration.
The distinct advantages of the materials reported in this dissertation are the ease of preparation and the reproducibility of method used for polymer preparation.
The simplicity of maleimides chemistry allows the introduction of a variety of functional groups which allows the materials to be used as sensors for a variety of organic molecules. The tunability of side chains also provides an advantage as they help in forming strong and stable Si-0-Si covalent bonds and also provides control on materials deposition.
These polymers are hence very easy to be modified into thin films which are very robust under harsh oxidizing conditions. The modification of these polymers to nanocomposites can be done by employing simple siloxane chemistry 33  have also been successfully entrapped. Because of their versatility of these materials have been used in both sensing and separation applications 6 . As in sensing, large number of materials has been studies to be used as a stationary phase in chromatographic separations 6 . In separation techniques silica based stationary phases are the most popular. The dominance of silica in the chromatographic industry reflects the many desirable properties that silica affords the chromatographer 7 . Silica can be prepared in a wide variety of pore size and particle size materials, and can be easily In this work we report the preparation and characterization of a group of stable polymers that have been designed to incorporate silane side-groups directly into the polymer backbone. These polymers allow the preparation of polymeric sol-gels, where the polymers are capable of participating directly in the sol-gel formation process, which we demonstrate in following chapter. We expect these materials to afford control over both the physical and chemical properties (selectivity, for example) of the resulting sol-gels by the judicious choice of the various moieties like allyl and vinylalkoxysilanes that can be incorporated into the polymer matrix 20 . We therefore intend to tailor the materials to specific sensing and separating applications.
Incorporation of the silane groups into the polymer backbone is expected to minimize or eliminate phase segregation, a common problem with polymer blending. When these alkoxysilane side groups are hydrolyzed, they can bond covalently within the sol-gel matrix, to give a more uniform sol-gel matrix with minimal or no phase separation. Phase separation would lead to micro heterogeneity in the sol-gel matrix, and would affect the properties of the material. If these materials were employed as a stationary phase in RP-LC, it would adversely affect the mass transfer of the analyte throughout the column and thus affect the separation. Similarly, if these non homogeneous materials were used in sensing application, they would not give uniform sensitivity in its microenvironment.
The ability to vary/control the functional moieties incorporated into the polymer backbone, as well as the molecular weights of the polymers through a simple synthetic scheme, has been described elsewhere in the literature 2 1 • The polymers prepared here were found to be insoluble in water and stable for extended periods in solution, with no evidence of cross-linking of the silane groups except in the presence of a catalyst (aqueous base, for example). This was confirmed with FTIR spectra which showed no evidence of -Si-0-Si band which would occur in the region of 1020-1090 cm-1 . Thus no special protocols are required to handle or store these polymers 22 . In the following chapter, we describe the conversion of these polymers into stable solgel materials, where we use tetraethoxysilane (TEOS) to promote conversion to the sol-gel. We also report in the chapter on the ability of these polymers to participate directly in sol-gel formation, even in the absence of any promoter (TEOS) through the cross-linking of the silane side groups which are an integral part of the polymer, describe the application of these polymers for various uses such as arrays, thin films their assembly and characterization, conversion to nano composites and their applications as sensors.

Experimental
The preparation of polymers in this study was accomplished by radical polymerization using 2, 2' -azobisisobutyronitrile (AIBN) as initiator of the various monomer units. The polymers were characterized using Gel Permeation Chromatography (GPC), solution phase 1 H-NMR, 13 C-NMR, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and Infrared spectroscopy (FTIR-ATR). The results obtained by the above studies are presented below along with a discussion of the results following.

Monomer Preparation:
The maleimide monomers were prepared by reacting maleic anhydride with a substituted aniline in a two step reaction as described in Scheme 1 in cases where the moiety was not commercially available. The reaction of a functionalized aniline with maleic anhydride leads to the formation of the corresponding maleamic acid (Scheme Reaction conditions and procedures were same as reported above. Typical yield for this polymerization was -70%.
Poly (2, 3, 4, 5, 6- By introduction of various electron donating and electron withdrawing groups on the phenyl ring of the above mentioned copolymers made the resulting materials amenable to a variety of sensing applications. All the functionalized monomers employed in the work are prepared in house and used after recrystallization with ethanol.

Terpolymer Preparation:
The terpolymer in this study to demonstrate a layer-by-layer deposition scheme was prepared by radical polymerization of n-phenylmaleimide, vinyltrimethoxysilane (VTMS) and pyrenemaleimide Scheme 2.4. In a fashion similar to the copolymers, this terpolymer is given the acronym n-Pyrenylmaleimide-VTMS. AIBN is used as initiator for preparation of this polymer and greater than 85% yield of this polymer was realized. Corporation. With the help of FT-IR analysis, we were able to characterize different groups. Monomer and polymer products were verified by IR spectra. We were also able to distinguish difference between allylalkoxy and vinylalkoxy polymers. It was also used to confirm that no hydrolysis has occurred by the absence of 0-H stretches.
An absence of hydrolysis proves the stability of the polymer in ambient conditions. 'H NMR and 13 C NMR analysis: 1 H NMR and 13 C NMR analysis was performed using Bruker 300 MHz NMR spectrometer. NMR analysis was used to confirm the structures of polymers. This study also helped to confum the stability of polymers against hydrolysis.

Molecular weight determination:
Gel permeation chromatography (Agilent 1100) was used in which THF was used as a solvent, with a flow rate of 0.5 ml per minute and a refractive index detector.

Thermal analysis:
Thermal stability of the polymers was studied using a DSC (Q 100 V8 build 261). At heating rate of 10°C/min, the polymers were analyzed in the temperature range of 30 to 450°C. Thermal properties were studied in order to understand the thermal behavior and robustness of these polymers as these materials will be used in separation and sensing applications, they should be stable and perform efficiently under various conditions such as high temperatures.

Results and Discussion:
The ultimate goal of the work was the preparation of stable polymeric materials and that can also be used in the preparation of polymeric sol-gels. Through

4-Chlorophenylmaleimide (4-CIPM), c) 4-Bromophenylmaleimide (4-BrPM).
These polymeric materials will find use in the design of various tailored materials for By incorporating the silane side groups diresctly into the copolymer backbone allowed for the direct participation of the polymers in the sol-gel formation process.
As expected the phase separation was very negligible, which can prove to be a significant problem using other methods to incorporate polymers into the sol-gel matrix. This finding was particularly important as these materials were prepared for predetermined applications such as chromatographic separation and sensing. These polymers can be incorporated in the tetraethoxysilane (TEOS) matrix to form sol-gel or the polymers itself can be converted to sol-gels. Eventually when preparing the polymer, maleimides tend to form alternating copolymers which ensures a regular structure to the polymers as well as having consistent properties. Having consistent properties in a polymer provides the stationary phase with uniform selectivity leading to a more reproducible separation. A non-uniform matrix, this will affect the interaction of the stationary and mobile phase with the analyte under investigation.
Hence the separation results will not be reproducible. Similar is true if these materials are used in sensing applications, the response of the materials will not be uniform if the materials is not homogeneous. Therefore, phase separation could pose a major threat to the performance of these materials, but owing to the presence of silane side groups in the polymer backbone they can covalently bind with the sol-gel matrix reducing the problem of phase separation to minimum. Additionally, the preparation of polymeric sol-gels affords us the ability to tailor the properties of the end product, by incorporating different polymers in TEOS matrix and by using different conditions for making materials. This allows us exquisite control over the design of materials with pre-determined functions.
To confirm the structures, the IR spectrum was taken of the monomer where However, when choosing the alkoxysilanes, the type of alkoxy and silane groups can give different properties. An allyl or vinyl group can be used to vary the chain length and influence the pore size within the copolymer. Depending on whether methoxy or ethoxy is utilized, will affect the reactivity of the polymers. These silane groups (SiOCH3 or Si0C 2 H 5 ) are the most important part of the copolymer by allowing the polymer to covalently link to a silica surface such as a silica particle or capillary wall. A methoxy group is more reactive than an ethoxy which determines how quickly the polymer can deposit or attach itself to silica. Silica particles and capillary walls contain silanol groups (Si-OH) which can react with the polymer when its silane groups are hydrolyzed into silanols. Combining all these properties provides a copolymer that can be tuned and attached to silica surface in order to prepare different forms of stationary phases for separation techniques.
The polymers prepared in this work were found to be very stable, showing no significant hydrolysis of the silane side groups as evidenced by the absence of the Si-OH stretching bands at 3200-3700 cm-1 in the FTIR spectrum. In the one-pot synthesis of polymer, a stable powdery precipitate was obtained. All polymers prepared in this study were solid powder-like materials that were found to be insoluble in water and in most organic solvents.  Br band lies at below 400 cm-1 is not seen. But the strong band at 800 cm-1 show that the benzene is para substituted.

39
In the 1 H NMR spectra, the resonance of the proton is influenced by other protons in its vicinity. In all 1 H NMR spectra no resonance of the proton around 8 = 5.0 was observed, which is the resonance value of the proton in the -Si-OH group.  between the maleimide and alkoxysilane. With this data, in combination with the IR spectra, it is proven that the copolymer was prepared successfully as well as stable under normal laboratory conditions with its methoxysilane side groups intact.
The polymers were characterized for their molecular weights using Gel Permeation Chromatography (GPC). The polymers in this study were synthesized using different concentrations of AIBN as a radical initiator. The purpose of GPC study was done to compare the number average molecular weight, weight average molecular weight, and polydispersity of different polymers with 5% and 10% radical initiator.  The important observation here was, an mcrease in polydispersity of each polymer, when we increased concentration of AIBN radical used for the polymerization process. When the initiator, used for polymerization process was increased, the polydispersity values increased. This suggest that as the initiator concentration increased, distribution range of molecular weights increases. This observation explained by the mechanism of radical polymerization process. The radical polymerization process occurs in three main steps. First step being the radical 45 initiation, radicals are generated and initiates the polymerization reaction. Second step is the chain propagation, the polymer chain increases by addition of monomers to the chain. The third step is chain termination, when two radicals in the reaction mixture react to neutralize each other thereby stopping the process of increasing chain length.
As the higher concentrations of AIBN are used, the number of radicals generated during polymerization are more and subsequently the probability of two radicals neutralizing each other in the polymerization mixture is higher. The GPC data of these polymers shows similar observations. As the AIBN concentration used for polymerization process increased, polydispersity values were found to be increased.
The polydispersity values for NPM-VTMS and for NPM-VTES were found to be higher than for NPM-ATMS and NPM-ATES. This observation can be explained by the fact that vinylalkoxysilanes are more reactive than allylalkoxysilanes. Therefore polymerization reaction of vinylalkoxysialnes will terminate faster than the allylalkoxysilanes, resulting in a wide distribution of molecular weights for vinylalkoxysilanes polymers.
Thermogravimetric analysis of the polymers show that the polymers were stable upto the temperature of 3 80°C .  This observation can be confirmed from differential thermal analysis (DTA).
In OT A change in mass with respect to temperature ( dm/d7) is plotted against temperature or time. DTA are derivative plots and any small changes in mass are clearly observed. 14 again clearly shows that change in mass for NPM-VTES begins at temperature of 150° C whereas for NPM-VTES, the mass change does not occur until after temperature of 400° C. Initial decomposition temperature is the temperature at which there is loss in weight of more than 10 % of the initial weight of the polymer.
Based on the initial decomposition temperature TGA revealed that the allyl polymers are thermally more stable than the vinyl polymers. The data suggests that the initial decomposition temperature is less in vinyl polymers as compared to allyl polymers.
Allyl polymers showed no sign of thermal degradation below temperatures of 420° to 48 at temperatures after 330° C there is significant change in mass (more than 10%). 70% change in mass (T 10%) and the temperature at which there is maximum rate of change in the mass of polymer (T max). For vinylmethoxy and vinylethoxy polymers the Tio% was 330° C and 340° C respectively and for allylmethoxy and allylethoxy polymers Tio% was 420° C and 430° C in that order. Tso% for all the polymers was observed to be similar which was 480° -490° C and T max for all the polymers was in the range of 480°-500° C. These data suggest that the only difference is in the initial This again suggests that the entropy of the allyl polymers is more than the entropy of vinyl polymers, which can attributed to the structural difference between the two polymers. Therefore the enthalpy required for melting vinyl polymers is more than the allyl polymers which is confirmed by differential scanning calorimetry.

CONCLUSION
Synthesis of phenylmaleimide monomers, silane-based copolymers and the materials that can be used in the efficient preparation of polymeric sol-gel materials have been achieved. The phenylmaleimides were created by reacting a functionalized aniline and maleic anhydride. Through IR spectra, the full conversion of the amine group from the aniline derivatives to the maleimide is verified by the absence of N-H stretches. With IR spectra, peaks from the carbonyl (maleimide) and silane (alkoxysilane) groups are visible. It also shows no silanol groups which would be present if any hydrolysis had taken place. This proves the stability of the copolymer in synthesis and ambient conditions. NMR reports further supports that there is no hydrolysis of the copolymer with the absence of resonance of the silanols. The resonance of the proton also confirms that the alkoxysilane is attached to the maleimide at the double bond between the two carbonyl groups. In the synthesis of the 51 polymers, molecular weights were controlled by the amount of radical initiator, AIBN.
The molecular weights were higher as the AIBN concentration increased.
Vinylalkoxysilane polymers were found to be more polydisperse than allylalkoxysilane polymers. The thermo gravimetric analysis suggests that the polymers are very much stable upto a temperature of 350°C. The allylalkoxysilane polymers were found to be thermally more stable than the vinylalkoxysilane polymers owing to difference in the structure of side chain. Due to the low polydispersity and

Applications ABSTRACT
In this work we prepared variety of copolymers that bear silane and alkoxysilane side groups converted into polymer-silica composite particles using a modified sol-gel protocol and their self-assembly at various interfaces such as silicon and glass. In this work we also employ the self-assembly of those polymers to prepare surfaces capable of templating/directing the assembly of nanoparticles and polymer composites into organized assemblies that will later be used in the preparation of arrays that are intended for sensing applications. In this work we also demonstrate the preparation and characterization of a group of pyrene-functionalized silane-based copolymers that have been converted into polymer particles and polymer-silica nanocomposites. Measurements employing these particles were shown to be an effective detection and quantification technique for amino-and nitro-containing compounds using fluorescence quenching as the detection scheme. In addition, we have demonstrated a facile method for the self-assembly of these particles onto various surfaces. This assembly protocol will be investigated for non-lithographic surface patterning for the development of array-based sensors.

Introduction
In recent years the materials chemistry and nanochemistry has gained significant attention due to the ability to design materials with unique properties on the scale and that can be assembled 1 • 2 in a controlled manner by providing materials that are both thermally 3 .4 and chemically robust 5 The drive has seen the development of a number of different types of materials and methods to assemble them into useful forms. Material science has witnessed major developments with the emergence of sol-gel chemistry. The popularity of the sol-gels stems from their ease of preparation, their robustness (chemical and thermal) and the ease with which they can be tailored for specific applications. The sol-gel method has been used extensively to prepare a variety of novel glasses 7 Many of the available methods are also time-consuming, taking upwards of several weeks in some cases, and long-range order is still often not achieved. The inclusion of a post-deposition ordering step may remedy this problem.
In this work we demonstrate a facile and robust method for the preparation and surface modification/patterning of interfaces able to facilitate nanoparticle self assembly via a non-lithographic technique. Nanoparticles have been modified to present various functionalities can be placed onto surfaces in an array pattern. The ability to assemble organized structures of nanoparticles, where these nanoparticles can be selectively modified will find use in the fields of chemical and biological sensing.
In this work the silane-based polymers were allowed to react directly with the surface silanol groups of glass and silica through the formation of siloxane (Si-0-Si) bonds as described elsewhere 47 ' 48 . Subsequent to polymer deposition the modified surfaces were exposed to a suspension of preformed silica nanoparticles or polymer silica nanocomposites. These particles were prepared via two major routes based on methods originally demonstrated by Stober et a1 49 and De et a1 50 . The modified Stober method is considered more efficient for the assembly of ordered arrays arising from the ability to form discrete particles of controllable size. Particle deposition was accomplished by one of the two facile techniques. The first was "dip-coating" method and the second a "spin-coating" method. Both were investigated to determine which of these simple methods would be more appropriate for ultimately producing ordered particle arrays. Again, ordered arrays are of great interest for several of the applications mentioned above. Ordered arrays produced by this self-assembly method would be desirable due to its facile and inexpensive nature.

EXPERIMENTAL Polymer Preparation:
The preparation of the polymers employed in this study was described in chapter 2 of this dissertation. For example a copolymer was prepared by radical copolymerization of n-phenylmaleimide and vinyltrimethoxysilane (NPM-VTMS) using AIBN as radical initiator with chloroform as solvent. A terpolymer of 1pyrenylmaleirnide with NPM and VTMS was also prepared as described in chapter 2 and used to prepare both particulate and thin-films in this work. Subsequent to polymerization the polymers were collected as powdered precipitates from hexane.
After purification the polymers were then used as described below. The alkoxysilane side groups were used as the functionality of attachment of the polymers to the various interfaces. Additionally, the side-groups facilitated the formation of the composite particles as the alkoxysilane groups were converted to siloxane cross-links.

Preparation of various functional forms:
Using the polymers prepared as above vanous functional forms can be prepared by simply modifying experimental conditions. Example structure include (1) polymer-silica nanocomposites, (2) Coated nanoparticles, (3) polymer thin-films and

a. Preparation of interfaces for thin-film deposition:
The glass and silicon interfaces used in this study were cleaned in piranha solution ( a mixture of 1 :3 30% H 2 0 2 and concentrated H 2 S0 4 -this mixture is very corrosive and can be explosive if exposed to organics, extreme care should be exercised) followed by extensive washing in distilled water. The interfaces were then used in the thin-film schemes as described below.

b. Polymer modified interfaces (dip-coating):
In this case of polymer-modified interfaces, the polymers prepared as reproducible. We have chosen this method to achieve two specific goals. The first was that the method should be time-efficient and uncomplicated. The second goal was that we desired to maintain a uniform deposition on the interface.

Form 4:
Nanoparticle Arrays -Nanoparticle Self-Assembly: The deposition of the various nanoparticles into organized arrays was accomplished in one of two schemes, either dip-coating or spin-coating similar to polymer thin-film preparation as described in Form 3 above.
a. Nanoparticle self-assembly -dip-coating: In the first scheme a simple "dip-coating" method is employed. Here, the polymer-silica composites or the polymer coated nanoparticles prepared as described b. Nanoparticle self-assembly -Spin-coating: Using the same spin-coating apparatus as described above, nanoparticles can be organized on the surface by spin-coating. In this scheme, the particles (polymer-

Ellipsometric Analysis:
The thickness of the polymer thin-films coated onto the silicon wafers was accomplished through Gaertner Ll 16C Ellipsometer using the surface characterization lab at University of Massachusetts -Amherst. It is based on the measurement of the variation of the polarization state of the light after reflection on a plane surface.

Fluorescence Analysis:
Fluorescence analysis of the pyrene labeled polymers was accomplish through steady-state analysis using a PTI-QuantaMaster system. Here we used an excitation wavelength of 340nm and scanned the emission monochromator from 350 to 650nm to collect the emission spectrum.

Microscopic Imaging
Scanning electron microscopic (SEM) images were collected on an FEI Quanta 200 instrument. Particle-deposited interfaces were sputtered with gold prior to analysis with SEM. Images were collected at voltages between 15 kV and 25 kV depending on particle composition.
Atomic force microscope (AFM) images were collected on a Park Instruments AFM/STM. AFM images of both the polymer-free and polymer-modified surfaces and particle-deposited surfaces were collected. Surface area and surface roughness of both polymer-free and polymer-modified interfaces were obtained from AFM also.

RESULTS and DISCUSSIONS
The aim of the work described in this chapter is the facile conversion of the polymer prepared in-house into various functional forms that can be used in the development of sensing and separation platforms. All of the polymer employed in this work were designed and synthesized within our research group as described in chapter 2 of this dissertation. Using these polymers that bear alkoxysilane side groups It should further be noted that the scope of this work was not to form well organized arrays based on deposition method alone, but rather how the polymers (both deposited on various surfaces and polymers within composite particles) influence the self-assembly. As previously mentioned, we intend to explore the self-assembly of nanoparticles at polymer modified interfaces in an attempt to develop patterned interfaces that can be tailored to specific sensing applications. The specifics of this work _ polymer preparation and surface modification and particle self-assembly are

b) two weeks after hydrolysis (c) and 3 weeks after hydrolysis (d) to form sol-gel.
There is also an appearance of a small band at 800 cm·' , which can be attributed to the formation oflong chain of siloxane bonds (-Si-0-Si).
Over a period of three weeks the intensity of the band at 800 cm- 1    We were also able to realize the deposition of polymer-silica composite nanoparticles at the polymer-modified interfaces as shown in Figure 3.7. where the sizes ranged from about 500 to 1,500 nm. Again, we observed the highest deposition of these particles from toluene as the deposition solvent. In future work we intend to attempt to better organize these assemblies by annealing the polymers at temperatures beyond the glass transition temperature (T g).

Trinitrophenol (Picric acid) (B). Trinitrotoluene (TNT) (C). Nitrobenzene {D). 2, -Nitrotoluene (2-NT).
Nitroaromatics and Nitramines classes of compounds are of interest due to their use in explosives. These compounds are ideally detected by fluorescence quenching due to their strong electron-withdrawing groups that are capable of forming charge-transfer complexes with pyrene leading to the quenching of its emission by either a static or dynamic quenching mechanism. In the studies performed in this 81 work a dynamic mechanism is evident due to the decreasing intensity as the concentration of the quencher is increased. Further confirmation was possible due to the decrease in the fluorescence lifetime, a finding that is consistent with a dynamic mechanism.
The collisional/dynamic fluorescenc quenching mechanism is defined by the     Nanoparticle are very small materials with dimension less than 1OOnm 1 which is particularly beneficial in that it allows for size-dependent properties that may not be properties, thereby resulting in loss of cell activity or even death of the cell or organism. Therefore, for the purposes of protein/organism identification, it is important for the protein not to denature during the assay.

Experimental Methods
The preparation of the functionalized magnetic nanoparticles proceeded in four distinct steps. The polymers were first prepared by the radical polymerization of the monomers. Next, the magnetic nanoparticles were prepared by the coprecipitation of Fe (II) and Fe (III) from basic solutions. The nanoparticles were then functionalized with the polymers or silanes, and finally the proteins were attached to the polymermodified nanoparticles. These steps are described in detail below.

Bromophenylmaleimide -vinyltrimethoxy silane (BrPM-VTMS) and ter-polymer
Chlorophenylmaleimide-viny 1 trimethoxysilane-Ally lisothiocyanate (CPM-VTMS-AITC) were synthesized according to the procedure described in Chapter 2 of this dissertation. All polymers were used as described below to functionalize the magnetic nanoparticles. In a second step, to illustrate a one-pot functionalization, the magnetite particles were mixed into a solution of 1,6-diisocyanatohexane to which the protein solution was added. Fluorescence spectra were collected to confirm the attachment of the protein to the magnetite.

Bio-Ferrograph Studies
In this experiment a Bio Ferrograph (Model # 2100, Guilfoyle Inc.) was employed for the analysis of the biofunctionalized magnetic iron oxide nanoparticles.
This particular instrument allows for the online analysis where both magnetic deposition and imaging of the nanoparticles is possible. Using a pump, a small amount of the ferro fluid is introduced into the system and flows over an electromagnet with a field strength of 1.2 Tesla. The electromagnet generates a high gradient magnetic field resulting in the deposition of the magnetic particles in a size gradient along the microscope slide attached to the instrument.

Preparation ofthe JgG solution:
The IgG solution was prepared by diluting a commercially available 11.0 mg/ml solution of human lgG in 10 ml of IX PBS buffer solution.

Preparation of the FTIC labeled anti-lgG solution:
The anti-IgG solution was prepared by diluting a commercially available 3.

Preparation of the egg albumin solution:
The egg albumin was prepared by dissolving 0.0132 g of egg albumin in 1 mL of 0.01 borate buffer at pH 8.4. The egg albumin was subsequently labeled with dansyl chloride according to a procedure described in reference 1. Briefly, the egg albumin was stirred with a 0.003M solution of dansyl chloride egg albumin for 1.5 hours at 25°C. During the labeling procedure the solution was wrapped in aluminum foil to avoid the photodegradation of the dansyl chloride. The resulting labeled egg albumin was stored at I 5°C until further studies.

Preparation of the isocyanatopropyltriethoxysilane functionalized nano particles:
The isocyanate nanoparticles were prepared by stirring a specific amount of the ferrofluid with the isocyanatopropyltrimethoxysilane. The resulting particles were rinsed, collected and resuspended in an appropriate solution depending upon the further needs of the method.

Preparation o(the JgG-functionalized magnetic nanoparticles:
The IgG-functionalized particles were prepared by suspending the isocyanatopropyltriethoxysilane functionalized nanoparticles prepared as described in the step above in a solution of IgG that had been prepared in IX PBS solution. This magnetic nanoparticle-IgG mixture was allowed to stir in an ice bath for 2 hours. The IgG-functionalized nanopartilces were collected, rinsed and stored at I 5 °c until needed for further studies.

Preparation of egg albumin-functionalized magnetic nanoparticles:
The egg albumin functionalized nanoparticles were prepared by suspending the isocyanatopropyltriethoxysilane functionalized magnetic nanoparticles prepared as described above in a solution of egg albumin that had been prepared as described above. This mixture was allowed to stir at 2s 0 c for 2 hours. The resulting nanoparticles were stored at I 5°C until further studies.

Preparation o[the ferrograph cassette:
In the first part of the ferrography experiment the five ports of the cartridge were prepared by introducing 0.3 mL of the various magnetic nanoparticles that were suspended in IX PBS solution.

Port 5). Plain (unfunctionalized) magnetic particles (Control 2)
After the cassette had been prepared as described above, the second step was to determine if there was any biorecognition between the IgG functionalized magnetic nanoparticles and the anti-IgG. Ports 4 and 5 as described above were used as control.
In port 4, the nano particles are only functionalized with isocyanatopropyltriethoxysilane to determine if there is any capture of anti-IgG by any unreacted isocyanato groups. Port 5 is plain magnetic nanoparticles.

Biorecognition experiment:
Next, the antibody (anti-IgG) and the control protein (egg albumin) were introduced into the ports as described below. The ferrography experiment was allowed to proceed and after drying, the magnetic particles were collected from the cassette and fluorescence spectra were collected in order to determine binding between the antibody and antigen. The control protein (egg albumin) was introduced to determine if there was any non-specific binding to either the IgG or unreacted isocyanato groups.
For these experiments, ram on Ferrograph is programmed to introduce the samples at a flow rate of 0.5 mL/min. Additional sample was introduced during sequential runs. 105

RESULTS and DISCUSSION
The aim of the work presented in this paper is the development of simple chemistries that can be used for the facile biofunctionalization of nanoparticle. In this case, we have focused our attention to the functionalization of magnetic iron oxide particles.
The functionalized magnetic nanoparticles can thus be used as multifunctional sensors in the capture and analysis of pathogens and particulates 10 Additionally, the ability to functionalize these materials with biological entities such as proteins and antibody can prove particularly attractive in the biomedical/bioimaging fields . For example, if these materials can be functionalized with a specific protein/antibody that can recognize a specific type of tumor, the it is conceivable that better images or medical decision can be made because only that specific tumor will be imaged since these "smart" nanoparticles can be directed to a specific target.
The preparation of the magnetic nanoparticles was accomplished by coprecipitation of Fe 2 + and Fe 3 + in the presence of ammonium hydroxide resulting in the formation of a blackish-brown precipitate, which was visibly attracted to an external magnet. Once the magnetic nanoparticles had been prepared they were then collected and functionalized in one of two schemes followed by attachment of a model protein (egg albumin). In the first scheme which is presented as Figure 4.3a, a polymer that had been synthesized to present terminal isothiocyanato groups was first attached to 110 the surface of the iron oxide nanoparticles by formation of bonds between the surface of the nanoparticle and the methoxysilane side groups of the polymer. In the second step presented as Figure 4.3b, the polymer-modified nanoparticle is then exposed to a solution of the egg albumin polymer that had been prepared in a buffered solution. In this scheme, the attachment of the protein to the nanoparticles is through the formation of a thiourea group between the amino terminus of the protein and the isothiocyanate group of the polymer.      This peak which is associated with fluorescence from the tryptophan moiety of the protein is also evidence of the fact that the proteins are not denatured under the modification conditions. In the even of protein denaturation this peak is typically redshifted. In this case we feel that this finding is due to the cushion-like layer of the polymer that protects the protein from the surface of the nanoparticle. It should be noted that we have control over the thickness of the polymer layer by controlling the modification chemistry as well as the time of exposure of the nanoparticle to the polymer solution.
In the Bioferrograph biofunctionalization studies it was necessary to determine an appropriate ratio of isocyantopropyltrimethoxysilane to the protein to obtain a satisfactory signal. To accomplish this study the model protein (egg albumin) labeled with dansyl chloride was employed. Here, 3 separate 10 mL batch of ferromagnetic fluid particles was stirred with volumes of 1, 2 and 3 mL of isocyantopropyltrimethoxysilane for 1 hour. These blends gave ratios of 10: 1, 10:2 and 10:3 respectively. Subsequently, each batch of particle was collected and stirred with lmL of dansyl-labeled egg albumin and stirred for 30 minutes. It was found in this study that the 10:3 ratio particles gave the most intense fluorescence signal. The increased signal scales with increasing ratio and is likely due to the increase in the number of reactive groups on the surface of the particles allowing for more efficient capture of the protein. Due to this finding all subsequent studies were performed using this 10:3 ratio. Figure 4. 7 is the spectra collected after the danysl chloridelabeled egg albumin was exposed to the magnetic particles prepared at different ratio. The experiment conducted in Port 2 of the ferrograph was IgG-functionalized magnetic nanoparticles that was subsequently exposed to dansyl chloride-labeled egg albumin. In this experiment when the magnetic particles are collected from the ferrograph and analyzed by fluorescence spectroscopy there was evidence of a binding event as indicated by the fluorescence peak at approximately 580 nm as seen in the spectrum below. In this particular case an excitation of 280 nm was employed which excited the protein, leading to the fluorescence and subsequent energy transfer to the dansyl chloride moiety which explains the 580nm peak. It should be noted that the observe peak at 580 nm is much higher than that expected for the dansyl chloride moiety, however, this is undoubtedly due to solvent effects.
by the fluorescence peak, it is unclear whether this binding is a result of non-specific binding to IgG (very unlikely) or due to any unreacted isocyanato groups still on the surface of the particles. The latter seems far more likely due to the fact that the isocyanato groups shows no preference for any particular protein to which it binds.
Further studies are underway to discern the nature of this binding. It is very likely that a blocking step using a small amine after exposure to the lgG will solve the problem of this unwanted binding as the amine group will bind to any unreacted isocyanato groups rendering them deactivated.
Magnetic particles +lsocyanato+lgG+Egg albumin   The experiment in Port 4 was the magnetic nanoparticles that had only been functionalized with isocyanatopropyltrimethoxysilane but with no lgG. This port was subsequently exposed to a mixture of anti-IgG and egg albumin. Aim was to determine if there is any binding between the proteins, particularly the anti-IgG. The spectrum of the sample collected from the ferrograph cassette showed no evidence of binding of either the egg albumin or anti-IgG. This finding is likely due to the short exposure time of the protein solution to the sample.  The control experiment conducted in Port 5 involved the use of the plain unfunctionalized magnetic nanoparticles. This port was then exposed to the mixture of anti-IgG and egg albumin. In this case the fluorescence spectrum revealed no fluorescence peak as expected. This finding is consistent with the fact that there is no group capable of binding the protein to the surface of the magnetic nanoparticles.
Below is the fluorescence spectra taken of the sample collected from the cassette of the ferrograph.  application. The use of less material is protecting our limited resources and the disposability of the microdevices helps to avoid contamination. It has also been reported that microchannels can improve the speed and accuracy of chemical reactions, as well as the speed, sensitivity, and repeatability of many assays 3 .
With a few exceptions, microfluidics is at an earlier stage of development for life science applications and, despite the potential, looks set to take a while to really invoke a paradigm shift 4 .

Microfluidic Lab-on-a-chip Devices
The purpose of these devices is to manipulate and process solution based samples and systems by carrying out typical procedures such as mixing, heating and separation. Processed samples may be delivered to some form of detector that subsequently transmits data. These devices typically consist of a monolithic material 133 that is patterned with microscale channels and features such as mixers, valves, injectors and separators that can assume the role of equivalent macroscopic laboratory equipment. Appropriate connection to the macroscale world for inputs and outputs (not trivial) is required and so the microfluidic device is likely to be part of a system 4 .
Typically, materials used are either glass or polymer although some hybrid silicon components may also be used. Microfluidic technology allows the reliable handling of smaller samples and provides greater control over a process with increased safety. True lab-on-a-chip devices would also incorporate some means of detection thus providing a 'reagents in -data out' apparatus although the material output of processed reagents may be together with a set of unique challenges which must be addressed to achieve optimal sealing results 5 .
While the use of glass or hard plastic is still employed in the design of microfluidic devices, many researchers have turned to poly dimethylsiloxane (PDMS), a rubbery material also used to make soft contact lenses as an alternative. According to Whitesides, who heads one of two groups that first worked with the polymer, "PDMS has become a popular choice for rnicrofluidic device fabrication for its ease of use" 6 .
In certain instances, glass is the most popular material employed for the fabrication of microchip such is the case when designing electrophoresis-based devices. This choice is primarily due to the similarity of the glass surface to that of fused-silica capillaries. In addition, glass has many other positive attributes for microchip electrophoresis applications, including good mechanical and optical properties, high electrical insulation, and low chemical reactivity. Glass microchips are usually fabricated using classical photolithography combined with wet chemical etching .Some disadvantages of glass chips are that they are expensive and relatively difficult to fabricate. Production of these chips requires access to a clean room and the use of corrosive etching solutions. In addition, the thermal bonding technique which is often used in the fabrication of glass microchips is time-consuming and often irreproducible 7 . Glass and hard plastics are easy to break and hard to etch and bond.
Glass, however, being susceptible to breakage, cracking, scratching and chipping, is a difficult material to process and handle. Bonding of glass plates can also be achieved at room temperature 20°c. This is based on hydrogen bonding at the glass interface, which has been achieved only after rigorous cleaning. And also during sealing of glass plates leads to thermal degradation of a chemically modified layer 6 .

137
The above outlined disadvantages related to glass microfabrication procedures have led scientists to investigate alternative materials for microchip fabrication. In particular, the production of microfluidic devices using polymeric substrates has generated significant interest due to their superior biocompatibility, greater flexibility, reduced cost, and ease of processing 8 . Additional advantages of polymers are that they are inexpensive, large numbers of microdevices can be fabricated from a single master, and the production of these devices does not require a clean room environment7. PDMS is the most popular polymer for microfluidic applications. It is elastomeric, inexpensive and possesses good optical clarity. Another significant advantage of PDMS is its ability to generate a tight seal to itself or other flat surfaces, reversibly or irreversibly, without distortion of the microchannels. An essential element of PDMS prototyping is the fabrication of a master template. This master is commonly fabricated in photoresist (such as SU-8), silicon, or nickel 7 . Disadvantages of PDMS include its hydrophobicity, which can lead to analyte adsorption. PDMS is just very easy to work with. There's a very simple procedure to go from design to master to mold, and you can seal the layers together almost effortlessly. A rubbery elastomer, PDMS won't break if dropped. It also breathes, so gases can exchange with the environment beyond the chip, while the material's springiness enables pneumatic controi3.

Applications of Microfluidic devices:
Microfluidic systems have diverse and widespread potential applications.
Some examples of systems and processes that might employ this technology include 138 inkjet printers, blood-cell-separation equipment, biochemical assays, chemical synthesis, genetic analysis, drug screening, electro chromatography, surface micromachining, laser ablation, and mechanical micro milling. Not surprisingly, the medical industry has shown keen interest in microfluidics technology 9 .
Lab-on-a-chip (LOC) technology has the potential to greatly simplify analytical analysis by providing a platform for chemical and biochemical reactions as The current use of robotic laboratory systems in conjunction with microtitre plates is well established and ongoing incremental developments allow this incumbent paradigm to remain competitive. Only when microfluidics offers a distinct and proven advantage over these solutions can they start to be replaced 15 • A glass surface is hydrophilic, meanmg it attracts water. This is often an advantage when working with glass microfluidic chips, but in certain applications you would need the surface to be hydrophobic or modified with a coating that prevents adsorption of small molecules 16 And the molds are heated at 60° C for 30 minutes. Then the second layer of PDMS poured on to the same mold and degassed and heated at 60° C for 2.5 hours. As the baking of PDMS is done using a scalpel, cut out the required shape and peeled off the silica wafer. A biopsy punch is used is used to cut out the holes for the ports.

Activated Surface Bonding:
Using a plasma asher create plasma activation on the surface of glass, silica wafer and PDMS. A plasma asher is intended for use with RIE (Reactive Ion Etching) as well as plasma cleaning. We were able to create oxygen free radicals on the surface of glass and PDMS then place the surfaces together for a permanent bond.
After plasma ashing gently press the PDMS layers against the wafer so there are no air bubbles between the PDMS. And bake them again with the PDMS facing up at 85°C for 80 minutes.
To ensure a better reversible seal between PDMS with glass, a mixing ratio of 20: 1 used, because this produces a less rigid polymer replica. More rigid PDMS chip, the sealing to another PDMS remained good. The PDMS channel slab bonded to a 144 cover substrate in order to create a sealed chip. This is achieved by irreversible bonding after oxidation of the PDMS layer to create the silanol groups. The PDMS surfaces are oxidized in an 0 2 plasma 6 . Zeiss Axioplan 2 light microscope: Zeiss Axioplan 2 Imaging system incorporates a research quality light microscope which enhances imaging capability with various filter sets. Image acquisition is achieved with the Zeiss AxioCam digital camera. Image Acquisition, processing and analysis are achieved using ZEISS Axio Vision software. This microscope is used for the examination of proteins, antigens and antibodies that are labeled with one or more fluorescent probes.

Fluorescence Spectrometer:
The fluorometry analysis was performed on Photon Technology International Spectrometer for measurements of fluorescence signals, excitation and em1ss10n spectra, and fluorescence suppression of fluorophores attached to proteins.

Typhoon Laser:
The typhoon imager offers multi-color fluorescence and provides better resolution. The machine's dual lasers and two detectors enable the investigator enables to choose from a wide range of fluorescent probes. This variable mode imager is used for automatic scanning of the fluorophores attached to proteins. The attachment of two or more fluorophores to a particular protein is confirmed.

Silica modified microchannels.
The immobilization of biological entities such as proteins, enzymes, cells is of great importance for the development of biosensors, immunoassays and biomicrofluidic devices. In this work, we study the in-situ bio-functionalization and cell adhesion in microfluidic channels. Here we are looking to develop a facile route to covalently anchor antibodies (bovine IgG) to the channel surface while retaining their biofunctionality 24 . In this scheme, a silica based monolith is grown directly within the channel. In order to prepare the monolith, a solution of ethanol, ammonium hydroxide 146 and water was prepared. A second solution of ethanol and and tetraethoxyorthosilane was added to the first solution above. These sol-gel precursors are mixed and stirred for 2 hours ex-situ and then injected into the microchannels and left overnight to cure.

Preparation of Bovine IgG (antigen):
Commercially available Bovine IgG of concentration 11.0mg/ml is used. The

Preparation of AntiBovine lgG labelled with Fluoro isothiocyanate (FITC):
Commercially available AntiBovine lgG of concentration 3.4 mg/ml is used. 0.080925g of dansyl chloride is dissolved in 100 mL ofDimethylformamide.

147
Labelling egg albumin solution with dansyl chloride: 1 mL of 0.003M dansyl chloride solution is mixed with 1 mL of 3.00e-6M egg albumin solution and stirred for one and half hours in an aluminum foil covered vial.
The mixture was stored in the refrigerator.

RESULTS and DISCUSSIONS:
As previously mentioned, the goal of this work is to develop a facile route to the modification of PDMS microfludic devices that allow for their biofunctionalization. The ability to biofunctionalize these devices opens up the possibility for the development of microdevices that can be used to rapidly and cheaply develop medically relevant devices where sample requirements can be kept at a minimum. Additionally, the ability to modify these devices in a facile manner also opens up the possibility of design coatings to prevent the biofoulding that is typically encountered in these devices.
The most reliable method to covalently functionalize PDMS is to expose it to an oxygen plasma, whereby surface Si-CH 3  Although PDMS is swollen in many organic solvents, it is unaffected by water, polar solvents (ethylene glycol) and perfluorinated compounds. It was found that non-    color which affords discrimination between the classes of amines, i.e. primary, secondary or tertiary. In the case of a primary amine, the originally non-fluorescence polymer changes to a deep pink/reddish color and eventually turns to an intense yellow color with a fluorescence signal shifted from the original 580 nm down approximately 520 nm. In the presence of secondary amines, the intense pink/reddish 163 color after exposure changes to an orange color over time. When exposed to tertiary amines, the solution remains the intense pink/reddish color over time. The focus of this work is to elucidate the species that are responsible for these observations and the mechanism by which they are formed.

Polymer preparation:
All polymers employed in this study were synthesized in-house through radical copolymerization of n-phenylmaleimide (NPM) with various comonomers, namely allyl-or vinyltrimethoxysilane (ATMS or VTMS), allyl-or vinyltriethoxysilane (A TES or VTES), and allylisothiocyanate (AITC). In all cases the syntheses were performed using chloroform as solvent and AIBN (2,2' azobisisobutyronitrile, 1 -10mol%) as radical initiator. Polymer syntheses and characterization has already been mentioned in Chapter 2. Figure 6.1 depicts a representative copolymer. Acronyms for the various polymers were developed by combining the various acronyms of each monomer as presented above. For example a copolymer prepared from nphenylmaleimide and vinyltrimethoxysilane is given the acronym (NPM-VTMS).
Similar acronyms will be employed throughout the remainder of the chapter.

Results and Discussion
The aim of the work presented in this paper is to explore the possible mechanism of the fluorogenic and sensing properties of the polymers that were The unique nature of these polymers to act as amines sensors lies in the fact that in addition to the fluorogenic response in the presence of amines, when the polymer-amine solution is allowed to sit, the solutions undergo further color changes as well as spectral changes that depend on the class of the amines. In the case of primary amines, the polymer solutions change from the pink/red/purple color to a bright yellow accompanied by a significant increase in fluorescence intensity (often saturating the detector) centered at -520nm. In the case of a secondary amine, the original pink/red/purple solution changes to orange with a peak centered at -560nm.
In the case of the tertiary amine, the solution typically retains its original pink/red/purple color. In this way, it is possible to distinguish between the different classes of amines.

Detection of primary amines:
When the polymer solution 1s exposed to a pnmary amme, the solution immediately turns to an intense deep pink/reddish color with an intense fluorescence peak centered about 580 nm. The broad nature of the fluorescence peak is suggestive of the formation of a complex of some kind. The proposed species leading to the 170 formation of this fluorescent species is the formation of an imine which results from the reaction of an amine with a carbonyl compound.
The proposed mechanism of the imine formation is due to a nucleophile attack on the carbonyl group by the lone pair electrons centered on the nitrogen of the amine group which leads to a dipolar tetrahedral intermediate. During this process, a proton transfer from the nitrogen of the amine to the oxygen yields a neutral carbinolamine.
The proposed mechanism is depicted below. Figure 6.4 depicts the initial attack on the carbonyl center by the amine. Figure 6.5 depicts the mechanism of the imine formation.
A n !mine 0

H-NMR of Polymer with Secondary amine (dipropyl amines ) forming
Enamines: The spectrum Figure 6.12 shows the successful formation of enamines between n-phenylmaleimide and vinyltrimethoxysilane and secondary amine (dipropylamine).
The terminal methyl protons of the secondary amine were observed as a triplet at 8 = 0.96 (peak h). The methylene protons adjacent to the methyl group are observed as a multiplet at 8 = 1.53 (peak i). The second set of methylene protons adjacent to amino group is seen as a triplet at 8 = 2.98 (peak j ). All these peaks of the secondary amine confirm that they are intact with the amino group and do not undergo any hydrolysis during the reaction. ..  polymer solution for detection. So the same polymer can be used for more than one sample to be detected for amines. The polymer-amine solution initially exhibits an emission in the red region in the visible spectrum and shows a marked blue shift spectroscopically after sitting. There is also a visual color change as well that allows for the discrimination between the classes of amines. These solutions were studied over time in an attempt to reveal the species likely responsible for the variation in the color of the polymer solution in the presence of the various base. Our initial assumption of imine and enamine formation between the amines and the carbonyl center of the maleimide is confirmed by NMR spectra. We rationalize that due to the distinctly different species formed with primary, secondary and tertiary amines, these differences gives rise to the variation in the observed color.

Conclusions
The research work presented in this dissertation is aimed at designing at new materials and facile assembly strategies for the design of materials that will find use in biofunctionalization studies, surface modification of microfluidic devices, chemical sensing and separations. We were able to manipulate the properties of the materials in a controlled way and also demonstrated various levels of control. First, we have successfully demonstrated a simple method to prepare a library of silane based polymers. We were able to tune the properties of the polymer by varying the identity of functional groups incorporated into the matrix of the materials by varying the substituted aniline used to prepare the maleimides. Secondly the conversion of polymers into various forms such as silica particles and silica-polymer composite particles to modify substrates by a simple dip-coating and spin-coating methods.
Modified various substrates like glass, silicon by covalent linkage adopting a facile layer-by-layer approach. Since the polymers were covalently linked by strong siloxane bonds Si-0-Si the layers built on the substrates as well as multilayers were very robust under very harsh oxidizing conditions. We have observed that in the case of polymer modified interface there is significantly higher amount of particle deposition relative to the polymer-free interfaces. This finding we have rationalized in the context of the increased surface area provided by the polymer chains on the surface. This ability to 201 control the loading density opens whole new avenues towards design of materials which can be used as sensors for detecting amines and nitro compounds.
In the other research work detailed the polymer facilitated protein functionalization of Magnetic Iron Nanoparticles as the polymer not only provides the skeletal support but also protect the protein from denaturing as a result of the surface polar functionalities. In this work, research goal is aimed at ability to attach proteins, antigens and antibodies to the functionalized magnetic nanoparticles in such a way that the protein retains its native properties and to prevent non-specific adsorption of proteins in a biological specific antigen-antibody interaction.
The other work employed in our research is to perform the surface modification in the microfluidic channels. Biofunctionalization is achieved in microfluidic chips using facile surface modification schemes by using in-house prepared polymers and C18 materials. This work is a promising avenue to provide an excellent interface to increase the surface roughness and area within the device, as well as serving as an inert site for attachment of the biological entities being employed in these systems. This study can be extended for a high-throughput system for biomarker proteins.
The last part of this research was application of the polymers prepared as sensors. The polymers are found to be sensitive to the presence of aliphatic amines, aromatic amines and weakly basic aromatic amines. The polymer solutions were intensely colored and fluorescence intensity increases in the presence of polymers.
And by changing the nature of groups on the phenyl ring of the polymers, the sensitivity can also be tuned. An important feature associated with these polymers was 202 their reusability as detectors for amines. The polymer solutions with base added can be made reverse and colorless by adding an acid and can reuse again to detect another base. Successfully we were able to synthesize and characterize a library of versatile polymers and studied their potential applications in this work.

Future work
The work we present in this dissertation serves as an opening to a variety of novel areas that will be further explored within the Major group in future.
With respect to conversion of polymers into various superstructures and developing a layer-by-layer approach our work currently underway to determine the influence on hydrophobic character and polymer distribution on the surface as a function of film thickness and exploring the possibility of organization of these particles at the polymer-modified interfaces by heating the interface to the T g of the polymer chains. It is expected that at the polymer T g when the polymer chains "melt" and begin to flow, this will facilitate particle rearrangement into a more favorable configuration leading to organized arrays that do not require labor-intensive lithographic techniques.
With respect to synthesis of polymers, by modifying the phenyl ring with various groups, these polymer can be made to sense other organic groups as well. For instance, by adding a boronic acid moiety to the phenyl ring, these polymers can be made to detect both amines and carbohydrates.
Another area where the polymers reported can be used is to detect explosives.
Since the polymers with base added have been found to be fluorescent, the presence of 203 any explosive can bring this fluorescence down and show a positive result. Not only as sensors, as these polymers are also being used to make columns (monolithic as well as HPLC) within the Major group, introduction of selective groups in the polymer can enhance the properties of these columns for selective as well as biological separations.
Magnetic Nanoparticles which are sub-micron size particles being smaller than typical cells, bacteria and viruses, they are ideal candidates for use in biological studies as their small size allows them to readily access the target species. The magnetic particles can be functionalized to attach antibodies labelled with fluorophores which can selectively attach to cancerous cells by recognizing the antigens via urea linkage. Upon magnetic resonance imaging, the magnetics can successfully help in the visualization of target cells. The magnetic particles modified biologically with drugs can be a promising treatment for cancer by Magnetic Resonance Imaging.
Simple maleimide chemistry used through out the research, combined with siloxane chemistry can also be used to prepare quantum dots whose surface is modified with known or unknown sequences of DNA oligonucleotides. These quantum dots can be linked to any of the surface used within the lab via Si-0-Si bonds. The size of the quantum dot can identify its position with the help of fluorescence.