Functionalization and Characterization of Nanostructured Materials for Sensing Applications
Open Access
- Author:
- Dean, Stacey Lynne
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 10, 2010
- Committee Members:
- Christine Dolan Keating, Dissertation Advisor/Co-Advisor
Christine Dolan Keating, Committee Chair/Co-Chair
Mary Elizabeth Williams, Committee Member
Thomas E Mallouk, Committee Member
Carlo G Pantano, Committee Member - Keywords:
- surface chemistry
proteins
nucleic acids
colloid
nanoparticles
SERS
FETs
nanowires
sol-gel
silica - Abstract:
- The optimal functionalization of nanoparticle surfaces for sensing applications was investigated in this dissertation. Nanoparticles have emerged as major components in the field of biosensing. For example, metallic nanoparticles have been used as substrates for Surface Enhanced Raman Spectroscopy (SERS) and silicon nanowires have improved the sensitivity in the area of field effect transistors (FETs). Due to the applications of nanoparticles and nanomaterials in sensing, it is vital to have multiple routes to control the surface chemistry of the nanoparticles. Complete characterization of these surfaces is essential for the fabrication and production of reproducible, sensitive, and reliable biosensors. Silica has been used to modify the surfaces of nanoparticles offering advantages such as protection of the metallic core, prevention of aggregation, biocompatibility, and the ability to attach molecules to the surface of the nanowire through well-understood silane chemistry. Modification of these coatings has been accomplished by incorporation of different functional groups for different applications. Control of surface chemistry is crucial in the performance of nanomaterials and biofunctionalized nanomaterials in applications, such as sensing. In addition to control over the surface chemistry of the nanoparticles, themselves, different routes of biomolecule immobilization must be optimized. Biomolecules can be attached by direct adsorption, covalent attachment, or through cross-linking molecules, and the effect of the immobilization process on the biomolecule function must be characterized to produce reliable sensors. In the following chapters I will address the modification of silica coatings to control the surface chemistry of nanoparticles, and then I will address methods of biomolecule immobilization. Chapter 2 of this dissertation describes the coating of metallic nanowires with organically modified silica (ORMOSIL) coatings to incorporate different functionalities into a standard silica coating. The addition of different functional groups, e.g., dihydroimidazole or polyethylene oxide, enabled new chemistry to be incorporated into the coatings, such as metal ion binding or protein resistance, respectively, while still offering the same advantages as standard silane coatings. Metallic nanowires were coated with a variety of different modified silanes, and were then fully characterized with transmission electron microscopy (TEM) and infrared spectroscopy (IR) to confirm the presence of these modified silanes within the coating. Additionally, the metal nanowire cores were dissolved to leave behind silica nanotubes composed of different ORMOSILs. Nanotubes have applications in drug delivery, bioanalysis, and catalysis; the synthesis of nanotubes in this manner enabled control over the chemistry of the nanotubes. Proof-of-concept experiments for applications of these coatings demonstrated the protein resistance and ability to attach DNA using a cross-linking molecule to the polyethylene oxide-containing silica coating, and the metal ion binding of the dihydroimidazole-containing silica coating. Chapter 3 introduces silica coated, porous Au nanowires for use as Surface Enhanced Raman Spectroscopy (SERS) substrates for the detection of small molecules in biological samples. Au/Ag alloy nanowires were coated, as described in Chapter 2, with SiO2 or an organically-modified silica containing a protein-resistant polyethylene oxide functionality. Once coated, the sacrificial silver was etched from the alloy nanowires, leaving behind silica coated, porous Au nanowires, which were characterized using TEM. Due to the nanostructure of these nanowires, enhancement of the inherently weak Raman signal was achieved. A small molecule, 4-mercaptobenzoic acid, was detected, even after the nanowires were exposed to biologically relevant serum concentrations of protein. This demonstrated the potential usefulness of these coated nanowires for detection of small molecules, e.g., metabolites or metabolic products of drugs, in biological fluids using SERS. The immobilization of HRP onto spherical gold nanoparticles by direct adsorption was investigated in Chapter 4. Direct adsorption of enzymes onto nanoparticles is the most common method of protein immobilization, but it is often not fully characterized. It was determined that there was multilayer formation based on the quantification of the enzymes on the surface of the nanoparticles. Stability of the protein:nanoparticle conjugates was studied using both zeta potential and flocculation assays at different pH values. Additionally, the specific activity of the enzyme was determined. It is generally thought that fluorescent labels do not affect the structure and function of the enzyme. However, comparisons between HRP and fluorescein labeled HRP:Au conjugates determined that the stability of the conjugates differed, particularly at pH 8.0. Nanoparticle surface modifications for immobilization were further investigated in Chapter 5. The experiments presented in Chapter 5 explored the immobilization of enzymes onto nanoparticle surfaces using barcoded nanowires to detect small molecules in solution. Two different immobilization techniques were studied: encapsulation within a silica coating or within polyelectrolyte multilayers. These immobilization techniques did not require any covalent attachment to the surface of the nanowires, which may affect the activity of the enzymes, and also enabled encapsulation within a material, preventing reversibility of the immobilization. It was determined that the layer-by-layer (LbL) deposition of polyelectrolytes in the presence of enzymes, rather than encapsulation in silica, enabled the immobilization of a wide variety of enzymes of different molecular weights onto both nanowires and latex beads. Characterization of two different fluorescently labeled enzymes, malate dehydrogenase and citrate synthase, was completed by immobilization onto fluorescent beads. This enabled quantification of the enzyme molecules per bead and calculation of the specific activity, which provided information as to the stability of the immobilized enzymes and their ability to turnover product for biosensor fabrication. Progress towards an enzymatic sensor was accomplished by the enzymatic reaction of horseradish peroxidase (HRP), catalyzing the oxidation of the fluorescein derivative, 2,7′-dichlorodihydrofluorescein, by urea hydrogen peroxide. The production of the fluorescent product was concentrated around the nanowires, but diffusion away from the nanowires was problematic. The effect of lithographic processes on the ability of the probe DNA oligos to selectively hybridize target sequences was studied in Chapter 6. The use of lithography to fabricate more complex biosensor devices has become very prevalent, and therefore, it is necessary to understand the effect of lithographic processes on the nucleic acids. This was accomplished by exposing nanowires to different photoresists, i.e., Megaposit SP 3012, Shipley 1813, PMGI SF6, that require different pre-exposure bake temperatures (95°C, 100°C, 190°C, respectively). 3´-Thiolated single-stranded DNA was covalently attached to gold or silica coated wires through thiol-Au or Sulfo-SMCC attachment chemistry, respectively, and these wires were deposited onto a substrate prior to photoresist exposure. It was found that the exposure to standard photoresists, baked at lower temperatures (≤100°C), did not prevent hybridization of target sequences post photoresist removal. Post photoresist exposure, the DNA sequences were able to discriminate a single base mismatch. The fabrication of bioFETS for the detection of nucleic acids was studied in a collaborative project with Dr. Theresa Mayer’s group in the Department of Electrical Engineering. Previously it was shown that nanowires pre-functionalized with DNA probes were assembled onto a lithographically prepared chip using dielectrophoretic forces. By confirming the lithographic processes did not affect the biomolecules, this assembly method of silicon nanowires can be used for the fabrication of bioFETs. Currently, silicon nanowires are synthesized through vapor-liquid-solid growth off of a surface, but low yield and poor quality control and length homogeneity is often problematic. In collaboration with Xiahua Zhong in the Mayer group, progress towards lithographically prepared axially doped silicon nanowires was accomplished, as also shown in Chapter 6. The biofunctionalization of these particles and dielectric materials was studied. In conclusion, a variety of different sensing platforms, i.e., SERS substrates, suspension array enzymatic biosensors, and bioFETS, were studied for the development of biosensors to detect small molecules and DNA. The modification of the surface chemistry of nanoparticles was accomplished through the use of silica coatings and polyelectrolyte multilayers. The understanding of the interaction of biomolecules with nanoparticles, and the ability to control the surface chemistry of the nanoparticles enables the production and fabrication of reproducible and reliable biosensors. In the future, this knowledge can be used to fabricate sensitive bioFETs that will enable multiplexed detection of nucleic acid cancer markers and allow for statistical analysis of the results. Ultimately, this knowledge will permit the fabrication of inexpensive, point-of-care sensors for early detection of diseases based on an electrical readout mechanism.