Self-assembling peptides have emerged as new nanobiomaterials and received considerable attention in the areas of nanoscience and biomedical engineering. One important type is the ionic-complementary peptide, which contains special patterns of positive and negative charge distribution. This thesis explores the application of this special type of peptides for the modification of electrode surfaces. The ionic-complementary peptide modified electrode was then further used to immobilize biologically active molecules, glucose oxidase in the present case, to construct a biosensor. There are two major parts in this thesis. In the first part, an ionic-complementary peptide, EFK16-II, was used to modify a highly ordered pyrolytic graphite (HOPG) electrode surface. The nanofibre structure of the self-assembling peptide on the electrode surface was characterized by atomic force microscopy (AFM). Attenuated total reflection fourier transform infrared sectroscopy (ATR-FTIR) spectra showed that upon addition of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), EFK16-II molecules tend to be cross-linked among themselves. Cross-linking of the peptide diminishes the number of carboxyl groups available for immobilizing a sensing enzyme, i.e., glucose oxidase (GOx). A simple method based on pre-mixing the carbodiimide and GOx was developed; it inhibited peptide cross-linking and significantly improved enzyme immobilization. Biosensors constructed in this way showed increased overall signal intensity and a much higher sensitivity at 4.94mA M-1 cm-2, a six-fold increase compared to the previously-reported peptide-modified electrodes. In the second part, another ionic-complementary peptide, EAK16-II, was used to modify the HOPG electrode. AFM images showed that EAK16-II formed well-ordered nanofibre patterns on the electrode surface. The redox couple Fe(CN)63-/4- was used as a probe to detect the electrochemical properties of the EAK16-II modified electrode. The results showed that the electron transfer at the electrode surface does not change much before and after modification. GOx was immobilized onto the EAK16-II modified HOPG and showed a good response to the concentration change of glucose. Similar to the EFK16-II, inter- or intro-peptide cross-linking also occurs when the solution containing EDC and sulfo-NHS was injected onto EAK16-II modified electrode. The same method as in the first part was applied here to prevent peptide cross-linking. The sensitivity was improved from 0.53mA M-1cm-2 to 2.4mA M-1cm-2. A proposal for constructing a reagentless biosensor by immobilizing both enzyme and mediator onto the electrode was made. However, the results indicated that the mediator, ferrocene carboxylic acid (FCA), was not stable on the surface after being immobilized. A redox protein, cytochrome c (Cyt c), was also immobilized onto an EAK16-II modified electrode. Direct electron transfer (DET) between the redox center of Cyt c and the electrode was observed. However, cyclic voltammetry results indicated that the peptide did not help improve the DET of modified Cyt c. The results presented here demonstrate significant potential for ionic-complementary peptides for constructing electrochemical biosensors.

Self-assembling peptides have emerged as new nanobiomaterials and received considerable attention in the areas of nanoscience and biomedical engineering. One important type is the ionic-complementary peptide, which contains special patterns of positive and negative charge distribution. This thesis explores the application of this special type of peptides for the modification of electrode surfaces. The ionic-complementary peptide modified electrode was then further used to immobilize biologically active molecules, glucose oxidase in the present case, to construct a biosensor. There are two major parts in this thesis. In the first part, an ionic-complementary peptide, EFK16-II, was used to modify a highly ordered pyrolytic graphite (HOPG) electrode surface. The nanofibre structure of the self-assembling peptide on the electrode surface was characterized by atomic force microscopy (AFM). Attenuated total reflection fourier transform infrared sectroscopy (ATR-FTIR) spectra showed that upon addition of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), EFK16-II molecules tend to be cross-linked among themselves. Cross-linking of the peptide diminishes the number of carboxyl groups available for immobilizing a sensing enzyme, i.e., glucose oxidase (GOx). A simple method based on pre-mixing the carbodiimide and GOx was developed; it inhibited peptide cross-linking and significantly improved enzyme immobilization. Biosensors constructed in this way showed increased overall signal intensity and a much higher sensitivity at 4.94mA M-1 cm-2, a six-fold increase compared to the previously-reported peptide-modified electrodes. In the second part, another ionic-complementary peptide, EAK16-II, was used to modify the HOPG electrode. AFM images showed that EAK16-II formed well-ordered nanofibre patterns on the electrode surface. The redox couple Fe(CN)63-/4- was used as a probe to detect the electrochemical properties of the EAK16-II modified electrode. The results showed that the electron transfer at the electrode surface does not change much before and after modification. GOx was immobilized onto the EAK16-II modified HOPG and showed a good response to the concentration change of glucose. Similar to the EFK16-II, inter- or intro-peptide cross-linking also occurs when the solution containing EDC and sulfo-NHS was injected onto EAK16-II modified electrode. The same method as in the first part was applied here to prevent peptide cross-linking. The sensitivity was improved from 0.53mA M-1cm-2 to 2.4mA M-1cm-2. A proposal for constructing a reagentless biosensor by immobilizing both enzyme and mediator onto the electrode was made. However, the results indicated that the mediator, ferrocene carboxylic acid (FCA), was not stable on the surface after being immobilized. A redox protein, cytochrome c (Cyt c), was also immobilized onto an EAK16-II modified electrode. Direct electron transfer (DET) between the redox center of Cyt c and the electrode was observed. However, cyclic voltammetry results indicated that the peptide did not help improve the DET of modified Cyt c. The results presented here demonstrate significant potential for ionic-complementary peptides for constructing electrochemical biosensors.

Prolonged exposure of humans and experimental animals to microgravity is known to be associated with a variety of physiological and cellular disturbances. With advancements in aerospace technology and prolonged space flights, both organism and cellular level understanding of the effects of microgravity on cells will become increasingly important in order to ensure the safety of prolonged space travel. To understand these effects at the cellular level, on-line sensor technology for the measurement and control of cell culture processes is required. To do this measurement, multiple sensors must be implemented to monitor various parameters of the cell culture medium. The model analytes used in this study were pH and dissolved oxygen which have physiological importance in a bioreactor environment. In most bioprocesses, pH and dissolved oxygen need to be monitored and controlled to maintain ionic strength and avoid hypoxia or hyperoxia. Current techniques used to monitor the value of these parameters within cell culture media are invasive and cannot be used to make on-line measurements in a closed-loop system. In this research, a microfabricated hydrogel microarray sensor was developed to monitor each anlyte. Either a pH or an oxygen sensitive fluorescent agent was immobilized into a hydrogel structure via a soft lithography technique and the intensity image of the sensor varied from the target analyte concentration. A compact detection system was developed to quantify concentration of each analyte based on the fluorescence image of the sensor. The system included a blue LED as an illumination source, coupling optics, interference filters and a compact moisture resistant CCD camera. Various tests were performed for the sensor (sensitivity, reversibility, and temporal/spatial uniformity) and the detection system (temporal/spatial stability for the light source and the detector). The detection system and the sensor were tested with a buffer solution and cell culture media off-line. The standard error of prediction for oxygen and pH detection was 0.7% and 0.1, respectively, and comparable to that of commercial probes, well within the range necessary for cell culture monitoring. Lastly, the system was coupled to a bioreactor and tested over 2 weeks. The sensitivity and stability of the system was affordable to monitor pH and dissolved oxygen and shows potential to be used for monitoring those analytes in cell culture media noninvasively.

Nanofabrication has received substantial interest from scientists and engineers because of its potential applications in many fields. This was because nanoscale structures have unique properties that cannot be observed or utilized at other size scales. Our living environment and many of our daily necessities had been strongly influenced by these techniques. Computers, electronics, housewares, vehicles, and medical care are now all affected by this explosive nanotechnology. However, traditional methods in controlling nanoscale features and their properties were often time-consuming and expensive. The objective of my research was to design, fabricate, and test nanostructure platforms using a unique toolbox of bottom-up lithographic techniques recently developed in our laboratory. These novel methods can be utilized for the rapid prototyping of nanoscale patterns in a much easier and more economical way. Specifically, we also focused on applying these nanoscale patterns as sensor platforms. These platforms were easily produced with our unique methods, and provide ultra sensitive capability to detect diverse chemical or biological species. The demonstration of capabilities and applications of our unique technologies includes the following projects. Chapters II and III describe a simple, inexpensive, and rapid method for making metal nanoparticles ranging between 10 nm and 100 nm in size through metal photoreduction with templates. The process can be completed in approximately 11 minutes without the use of a clean room environment or vacuum techniques. A simple label-free biosensor fabrication method based on transmission localized surface plasmon resonance (T-LSPR) of this platform is also demonstrated. Chapters IV and V present a nanoscale patterning technique for creating diverse features in polymers and metals. The process works by combining evaporative ring staining with a colloidal templating process. Well-ordered hexagonally arrayed nanorings, double rings, triple rings, targets, and holes were all easily prepared. A line width as thin as ~15 nm can repeatably be performed with this technology. Finally, Chapter VI demonstrates an ultra-sensitive plasmonic optical device based on hexagonal periodic nanohole metal films produced through our evaporative templating technique. The optical properties of these sub-wavelength periodic hole array metal films are discussed.

Understanding protein stability and folding is of central importance in chemistry, biology, and medicine. Despite its importance, a molecular level understanding of protein stability still remains illusive due to the complexity of the system. In this study, we employed protein-like polypeptides to study several aspects of protein stability in different aqueous environments. The model system employed here is elastin-like polypeptides (ELPs). First, the modulation of the lower critical solution temperature (LCST) of neutral ELPs was investigated in the presence of 11 sodium salts that span the Hofmeister series for anions. It was found that the hydrophobic collapse/aggregation of these ELPs generally followed the series. Specifically, kosmotropic anions decreased the LCST by polarizing interfacial water molecules involved in hydrating amide groups on the ELPs. By contrast, chaotropic anions lowered the LCST through a surface tension effect. Additionally, chaotropic anions showed salting-in properties at low salt concentrations that were related to the saturation binding of anions with the biopolymers. These overall mechanistic effects were also compared to the results previously found for the hydrophobic collapse and aggregation of poly(N-isoproplyacrylamide). A positively charged ELP, ELP KV6-112, was used as a next model system. We observed both inverse and direct Hofmeister effects on LCST with five chaotropic salts. Next, the solvent isotope effects on the LCST of ELPs were investigated as a function of ELP chain length and guest residue chemistry using D2O and H2O. Differences in the LCST values with heavy and light water were correlated with secondary structure formation of the polypeptide chains which was quantified by circular dichroism, FTIR, and differential scanning calorimetry measurements. It was found that there is a great change in the LCST values between H2O and D2O for those polypeptides which form the greatest amount of b-spiral structure. This study suggests that hydrogen bonding rather than hydrophobicity is the key factor in the stabilization of ELPs in D2O over H2O. The phase transition property of ELPs can also be applied to development of stimuli responsive biosensor system. In this study, we employed ELP-conjugate solid supported lipid bilayer as a size selective binding sensor.

An affinity based bio-sensing technique was developed using an anti-transmissible spongiform encephalopathy monoclonal antibody as a bio-recognition molecule. Fluorescein iso-thio-cynate (FITC), labeled with a prion epitope (QYQRES), was used as a decoy for prions. Experiments done in 0.1M phosphate buffer revealed that the dye fluorescence increased with the pH of the buffer and was influenced by solvent polarity. Binding studies conducted at pH 6, 7, and 8 showed that the optimum pH for the antibody-decoy binding was 7. Maximum differences between control and antibody samples were observed at pH 7. The optimum incubation time was found to be less than 4 hours for the control, antibody, and the prion samples at room temperature. Prion detection curves were established at 4 and 10 nM antibody decoy concentrations. The lowest detectable prion concentration in phosphate buffer was 8 nM. Experimental conditions determined in the phosphate buffer were used to implement the technique in gelatin and baby formula. Prion detection curves were generated in 0.01, 0.4, 1.0 and 2.0 mg/ml of gelatin solution. The gelatin interfered with the binding and the displacement reaction of antibody, decoy and prion. Addition of an anionic surfactant, sodium dodecyl sulfate (SDS) at 0.3 mg/ml to gelatin samples facilitated prion detection in gelatin. The lowest detectable concentration of prion in gelatin was 0.5 nM at 0.4mg/ml gelatin. The baby formula samples produced light scattering and the intrinsic peak of baby formula at 526nm interfered with the dye peak at 514nm. Serial dilutions of baby formula were done to reduce the interference. Prion detection curves were then obtained at 1.31 and 5.34 mg/ml baby formula and 0.454 mg/ml of Triton-X-100 was added to the baby formula samples. The lowest detectable concentration of prion was 2 nM for baby formula. This developed bio-sensing technique can be used to detect prion in gelatin and baby formula solutions. Addition of surfactants assisted prion detection in foods, while high concentrations of gelatin and baby formula had an adverse effect on the detection system.

Zugl.: Dresden, Techn. Univ., Diss., 2009

The objective of this work is to develop mathematical models for predicting the thermal stability of commercial diagnostic assays. These assays are a product of the Point of Care division of Abbott laboratories, and are used for analyzing patient blood samples for specific substances. The accuracy of the results from these diagnostic tests relies on the activity of specific biological and/or chemical components of the sensors. Mathematical models that describe the stability of these active components are useful for supporting product shelf-life claims and for the design and implementation of accelerated testing protocols. In the thesis, the stability of two diagnostic assay systems of interest to Abbott Point of Care is investigated using mathematical modeling. For the first assay system investigated, the biosensor associated with the assay is identified as an important factor for product stability. A second-order dynamic model is developed to describe the thermal stability of this biosensor. The model corresponds to a reversible reaction followed by an irreversible reaction, with rate coefficients having Arrhenius temperature dependencies. The second-order dynamic model provides improved predictions relative to a first-order dynamic model, based on a comparison between model fits for two experimental datasets, and a comparison of predictive ability for a validation dataset. The second-order dynamic model is used to extend the concept of Mean Kinetic Temperature concept from the pharmaceutical industry to systems with higher-order dynamics. For the second assay system investigated, the calibration fluid is identified as a key factor in assay stability. A first-order model is developed to describe the stability of the analyte within the calibration fluid. The first-order model captures most of the trend present in the data from calibration fluid incubation experiments. Finally, model predictions are used to investigate the amount of change in assay response that can be attributed to changes in concentration of analyte in the calibration fluid (after storage at elevated temperatures). The results show that the changes observed in assay responses are consistent with the magnitude of changes in calibrant analyte concentrations predicted by the model. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-02-02 00:09:23.758

No description available.

This thesis describes the development and testing of a range of electrodes designed to be able to measure electrical current produced by the respiration of bacteria in direct contact with the electrode surface. The electrodes are designed to directly wire into redox processes in the cytoskeleton of the bacteria so that electron transfer can be measured in real time without the need for solution based mediator molecules. The rate of electron transfer from the bacteria is enhanced by nanostructuring the surface of graphite electrodes with vertically aligned single and multiwalled carbon nanotubes (CNTs) and covalently coupling mediator molecules to the CNT tips. A selection of the prepared electrodes are tested with the non-electrogenic bacteria Proteus vulgaris and Bacillus subtilis to demonstrate the potential of the electrode designs to be used with a wide range of microbial species.