Master of Science / Department of Chemistry / Christopher T. Culbertson / Mixing is an essential process to many areas of science for example it is important in studying chemical reaction kinetics, chemical synthesis, DNA hybridization and PCR amplification. Mixing on the macroscale level is readily achieved through convection. Rapid mixing on microchips however, is problematic as the low Reynolds numbers and high Peclet numbers indicate that fluid flow is in the laminar regime and limits mixing on microchips to diffusion. Because of these limitations mixing on microchips is often relegated to diffusional mixing which requires long channels and long time periods. Several methods have been developed to increase the speed and efficiency of mixing on microfluidic devices. A variety of techniques have been employed to overcome these obstacles including for example 1) 3 dimensional channel designs to split up and recombine flows 2) employing sophisticated lithographic techniques to make grooves within a channel to generate transverse flows and 3) using lateral flow created by using spiral channels. Other groups have used outside energy sources to achieve mixing by changing of the zeta potential within the channel, using induced charge electroosmosis, and also by modifying the electrokinetic flow. We propose using dielectric elastomers (DEs) to modulate flow as a means to achieve rapid and active mixing on the microchip format. Electroactive polymers such as poly(dimethylsiloxane) function as DEs and are capable of converting electrical energy into mechanical energy. The application of an electrical potential across the PDMS results in a change in the dimensions of the PDMS dielectric layer between the two actuating electrodes creating an actuator. When employed in microfluidic devices this actuator can be used to change the volumes of the microfluidic channels on the PDMS. If the actuators are placed near a T-intersection where two components are entering the intersection the actuators can serve to improve mixing on microfluidic devices. Studies were conducted on how on the magnitude of the actuation, the frequency of actuation, the field strength, the electrode design and position relative to the T intersection, the channel dimensions and the overall channel design impacted mixing efficiency. Mixing results showed promise but further development of technology is necessary to achieve adequate mixing in microfluidic channels using DEs.

Electrochemical cycling of Ag surfaces under basic conditions has led to the discovery of a new, simple, rapid and cost effective method for the preparation of nano-structured Ag surfaces with features ranging from 30–150 nm in diameter. Our results indicated that during cyclic voltammetry, the surface was oxidized, resulting in the formation of soluble Ag complexes which were re-deposited as elemental Ag nanostructures (NSs) on the cathodic scan. The electrochemical properties of the Ag NSs were greatly affected by the presence of organophosphonates and other additives (vide infra), which also influenced the growth of nanostructures. The interaction of these Ag NSs with malathion and paraoxon were explored in more detail using Surface Enhanced Raman Spectroscopy. Results showed that generally smaller nanosized features resulted in high quality surface Raman enhancement. Next, Ag NSs’ properties in the presence of organophosphonates was investigated in tap water and apple juice in order to address issues related to matrix effects and potential interference from constituents in solution. Electrochemical and localized surface plasmon resonance results demonstrated the ability to detect organophosphonates in real samples, albeit at a lower limit of detections and without any selectivity to any particular organophosphonate. Next, the morphology and corrosion behaviour of Ag surfaces was explored in the absence and presence of surfactants and capping agents. Results demonstrated the protection of Ag surfaces against corrosion in the presence of Tween-20, while potassium citrate presence enhanceed corrosion of silver surfaces, resulting in the formation of a pitted surface with smaller Ag NSs. Lastly, the interactions of 2-cyano-3-(2′-(5′,10′,15′,20-tetraphenyl porphyrinato zinc-(II))yl) acrylic acid-modified Ag NSs with biomolecules were explored using spectro-electrochemical techniques. The photocurrent response of porphyrin-modified Ag NSs was quenched by the addition of adenosine-5’-monophosphate (AMP), guanosine-5’-monophosphate (GMP) and cytidine5’-monophosphate (CMP), with a quenching efficiency of 80%, 68% and 48% for AMP, CMP and GMP, respectively.

Electrochemical cycling of Ag surfaces under basic conditions has led to the discovery of a new, simple, rapid and cost effective method for the preparation of nano-structured Ag surfaces with features ranging from 30–150 nm in diameter. Our results indicated that during cyclic voltammetry, the surface was oxidized, resulting in the formation of soluble Ag complexes which were re-deposited as elemental Ag nanostructures (NSs) on the cathodic scan. The electrochemical properties of the Ag NSs were greatly affected by the presence of organophosphonates and other additives (vide infra), which also influenced the growth of nanostructures. The interaction of these Ag NSs with malathion and paraoxon were explored in more detail using Surface Enhanced Raman Spectroscopy. Results showed that generally smaller nanosized features resulted in high quality surface Raman enhancement. Next, Ag NSs’ properties in the presence of organophosphonates was investigated in tap water and apple juice in order to address issues related to matrix effects and potential interference from constituents in solution. Electrochemical and localized surface plasmon resonance results demonstrated the ability to detect organophosphonates in real samples, albeit at a lower limit of detections and without any selectivity to any particular organophosphonate. Next, the morphology and corrosion behaviour of Ag surfaces was explored in the absence and presence of surfactants and capping agents. Results demonstrated the protection of Ag surfaces against corrosion in the presence of Tween-20, while potassium citrate presence enhanceed corrosion of silver surfaces, resulting in the formation of a pitted surface with smaller Ag NSs. Lastly, the interactions of 2-cyano-3-(2′-(5′,10′,15′,20-tetraphenyl porphyrinato zinc-(II))yl) acrylic acid-modified Ag NSs with biomolecules were explored using spectro-electrochemical techniques. The photocurrent response of porphyrin-modified Ag NSs was quenched by the addition of adenosine-5’-monophosphate (AMP), guanosine-5’-monophosphate (GMP) and cytidine5’-monophosphate (CMP), with a quenching efficiency of 80%, 68% and 48% for AMP, CMP and GMP, respectively.

Proteome profiling is the identification and quantitation of all proteins in biological samples. An important application of proteome profiling that has received much attention is clinical proteomics, a field that promises the discovery of biomarkers that will be useful for early diagnosis and prognosis of diseases. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from complex biological fluids and (ii) extensive biochemical processing (reduction, alkylation and enzymatic digestion) prior to analysis. However, the lack of standardized sample handling and processing in proteomics is a major limitation for the field. The conventional macroscale manual sample handling requires multiple containers and transfers, which often leads to sample loss and contamination. For clinical proteomics to be adopted as a gold standard for clinical measures, the issue of irreproducibility needs to be addressed. A potential solution to this problem is to form integrated systems for sample handling and processing, and in this dissertation, I describe my work towards realizing this goal using digital microfluidics (DMF). DMF is a technique characterized by the manipulation of discrete droplets (100 nL – 10 L) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. This thesis demonstrates how DMF can be a powerful tool capable of automating several protein handling and processing steps used in proteomics.

The unique spectroscopic properties of quantum dots (QDs) are of interest for application in intracellular studies of gene expression. QDs derivatized with single-stranded probe oligonucleotides were used to detect complementary target sequences via hybridization and fluorescence resonance energy transfer (FRET). As nucleic acid targets are not labeled within cells, a displacement assay for nucleic acid detection featuring QDs as FRET donors was developed. QDs conjugated with oligonucleotide probes and then pre-hybridized with labeled target yielded efficient FRET in vitro. Studies in vitro confirmed that displacement kinetics of pre-hybridized target was a function of the stability of the initial hybridized complex. Displacement was observed as reduction in FRET intensity coupled with regeneration of QD fluorescence. By engineering the sequence of the labeled target, faster displacement was possible. The QDprobe+target system was successfully delivered into cells via transfection. Although QDs with their cargo remained sequestered in endosomal vesicles, fluorescent properties were retained.

Proteome profiling is the identification and quantitation of all proteins in biological samples. An important application of proteome profiling that has received much attention is clinical proteomics, a field that promises the discovery of biomarkers that will be useful for early diagnosis and prognosis of diseases. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from complex biological fluids and (ii) extensive biochemical processing (reduction, alkylation and enzymatic digestion) prior to analysis. However, the lack of standardized sample handling and processing in proteomics is a major limitation for the field. The conventional macroscale manual sample handling requires multiple containers and transfers, which often leads to sample loss and contamination. For clinical proteomics to be adopted as a gold standard for clinical measures, the issue of irreproducibility needs to be addressed. A potential solution to this problem is to form integrated systems for sample handling and processing, and in this dissertation, I describe my work towards realizing this goal using digital microfluidics (DMF). DMF is a technique characterized by the manipulation of discrete droplets (100 nL – 10 L) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. This thesis demonstrates how DMF can be a powerful tool capable of automating several protein handling and processing steps used in proteomics.

The unique spectroscopic properties of quantum dots (QDs) are of interest for application in intracellular studies of gene expression. QDs derivatized with single-stranded probe oligonucleotides were used to detect complementary target sequences via hybridization and fluorescence resonance energy transfer (FRET). As nucleic acid targets are not labeled within cells, a displacement assay for nucleic acid detection featuring QDs as FRET donors was developed. QDs conjugated with oligonucleotide probes and then pre-hybridized with labeled target yielded efficient FRET in vitro. Studies in vitro confirmed that displacement kinetics of pre-hybridized target was a function of the stability of the initial hybridized complex. Displacement was observed as reduction in FRET intensity coupled with regeneration of QD fluorescence. By engineering the sequence of the labeled target, faster displacement was possible. The QDprobe+target system was successfully delivered into cells via transfection. Although QDs with their cargo remained sequestered in endosomal vesicles, fluorescent properties were retained.

Doctor of Philosophy / Department of Chemistry / Takashi Ito / This research was focused on understanding how external stimuli affect the permeability of the chemically modified electrodes, and how the materials used in modifying the working electrodes respond to the changes in the surface charge. We adopted a voltammetric type electrochemical sensor to investigate the permeability effects induced by pH and organic solvents. The working electrodes used in this research were chemically modified with thioctic acid self assembled monolayer (TA SAM), track etched polycarbonate membranes (TEPCM) and PS-b-PMMA nanoporous films (polystyrene-block-polymethylmethacrylate). We studied the permeability behavior of each of the material upon application of external stimuli. In chapter 3, the permeability changes induced by change in surface charge of thioctic acid SAM was investigated. The surface charge of the monolayer was tuned by changing pH of the medium, which resulted in decrease of redox current of a negatively charged marker due to deprotonation of the surface –COOH groups of TA SAM. Decrease in redox current reflected a decrease in the reaction rate, and by using closed form equations the effective rate constants at several pKa values were extracted. In chapter 4, permeability changes induced by pH in TEPCM were investigated. We assessed the surface charge of these membranes via cyclic voltammetry generated for neutral and charged redox molecules. Limiting current of charged markers were affected by the surface charge induced by pH, where as the redox current for the neutral marker was not affected. Experimental redox currents were larger than the theoretical current, indicating that redox molecules preferentially distributed in a surface layer on the nanopore. Organic solvent induced permeability changes of PS-b-PMMA nanoporous films were investigated via electrochemical impedance spectroscopy and AFM. Higher response of pore resistance in the presence of organic solvents indicated either swelling of the nanoporous film or partitioning of organic solvents in the pores. However AFM data revealed that the permeability changes are due to partitioning of the solvents rather than swelling of the porous film, since there was no appreciable change if the pore diameter in the presence of solvents.

Doctor of Philosophy / Department of Chemistry / Daniel A. Higgins / In this dissertation, state-of-the-art fluorescence microscopy techniques are employed to probe unique nanoscale phenomena in poly(dimethylsiloxane) (PDMS) films and on single carbon nanofibers. In one study, the mobility and physical entrapment of single dye molecules in dry and solvent-loaded PDMS films is explored. Experiments are performed under dry nitrogen and at various levels of isopropyl alcohol (IPA) loading from the vapor phase, as monitored by a PDMS-coated quartz-crystal microbalance. Single molecules are shown to be predominantly immobile under dry conditions and mostly mobile under IPA-saturated conditions. FCS is used to measure the apparent diffusion coefficient, yielding a mean that is virtually independent of IPA loading and sample class. An increase in the population of mobile molecules under high IPA conditions is attributed to the filling of film micropores with solvent, rather than by incorporation of molecularly dispersed solvent into the PDMS. In a second study, the molecular mobility of both neutral and cationic molecules in cured PDMS films is studied as a function of oligomer extraction. Cross correlation and Bayesian burst analysis methods were used to quantify the populations of fixed and total molecules, respectively. The results show that the total concentration of dye increases with increased oligomer extraction, while the relative populations of fixed and mobile molecules decrease and increase, respectively. These results are relevant to the use of PDMS in microfluidics, nanofiltration and pervaporation membranes and solid phase microextraction fibers. In a final study, molecular beacons (MBs) were immobilized onto the ends of single, sol-gel encapsulated vertically-alligned carbon nanofibers (VACNFs) attached to a silicon electrode. MB fluorescence was monitored as a function of the potential applied to the VACNF in a three-electrode electrochemical cell. Application of positive potentials attracts the negatively charged backbone of the MB, causing hybridization of the stem and a reduction in beacon fluorescence. Negative potentials cause dehybridization of the stem, and an increase in MB fluorescence. This study presents the first measurement of potential-dependent dehybridization/rehybridization of MBs attached directly to the end of a single VACNF. These studies will help to characterize the mechanism by which future lab-on-a-chip devices will detect harmful bio-organisms.

Master of Science / Department of Chemistry / Christopher T. Culbertson / Chemists have been trying to relate the structure and composition of different cereal proteins to their physical properties to better inform their product use for more than 250 years now. Among these cereals, wheat is considered the most important due to its unique ability to form viscoelastic dough and retain gas during fermentation, the latter being important for bread making. This property is due to the endosperm part of wheat that contains proteins mostly gliadins and glutens. It is known that the composition and relative ratio of these proteins is determined by both the growing environment and genetics. Manipulation of the genetics allows one for control of only about 50% of the end use quality of the wheat and the rest is controlled by environment. Currently, the bread making quality of wheat is determined by baking test loaves of bread. This process is time consuming and wasteful. The main goal of this project was to create fingerprints of gliadin proteins for different wheat cultivars as a function of environmental conditions. This would then allow wheat kernels to be analyzed and assessed right after harvest to determine their appropriateness for making the various wheat products. Researchers have tried to create a catalogue of information for individual wheat cultivars by ‘fingerprinting’ the gliadins proteins in wheat using various analytical techniques including capillary electrophoresis (CE). CE offers advantages like high separation efficiency, and faster analysis. Further miniaturization of CE on microfluidic devices has enhanced the speed and efficiency of separation. Furthermore, it is possible to integrate multiple chemical analysis processes like sample preparation, separation and detection in a single microfluidics device. Microfluidic uses micron sized separation channels defined in a glass, quartz or polymer. This dissertation is focused on fabricating multilayer microfluidic devices from Poly(dimethylsiloxane) (PDMS) and using these devices to electrophoretically separate wheat gliadin proteins followed by detection using UV absorption in less than 5 min. PDMS is cheap, easy to fabricate and is optically transparent above ~230nm. Initial results of the UV absorbance detector developed for this device are presented.