No description available.

Biofunctional interfaces capable of anchoring biomolecules and nanoparticles of interest onto a platform are the key components of many biomedical assays, clinical pathologies, as well as antibacterial and antiviral surfaces. In an ideal biofunctional surface, bio-entities and particles are covalently immobilized on a substrate in order to provide robustness and long-term stability. Nonetheless, most of the reported covalent immobilization strategies incorporate complex wet-chemical steps and long incubation times hindering their implementation for mass production and commercialization. Another essential factor in the biointerface preparation, specially with regard to biosensors and diagnostic applications, is utilization of an efficient and durable blocking agent that can inhibit non-specific adsorption of biomolecules thereby enhancing the sensitivity of sensors by diminishing the level of background noise. Many of the commonly used blocking agents lack proper prevention of non-specific adsorption in complex fluids. In addition, most of these agents are physically attached to surfaces making them unreliable for long-term usage in harsh environments (i.e. where shear stresses above 50 dyn/cm2 or strong washing buffers are involved). This thesis presents novel and versatile strategies to covalently immobilize nanoparticles and biomolecules on substrates. The new surface coating techniques are first implemented for robust attachment of TiO2 nanoparticles onto ceramic tiles providing self-cleaning properties. Further, we utilize similar strategies to covalently immobilize proteins and culture cells in microfluidic channels either as a full surface coating or as micropatterns of interest. The new strategies allow us to obtain adhesion of ~ 400 cells/mm2 in microfluidic channels after only 1-day incubation, which is not achievable by the conventional methods. Moreover, we show the possibility of covalently micropatterning of biomolecules on lubricant-infused surfaces (LISs) so as to attain a new class of biofunctional LISs. By integration of these surfaces into a biosensing platform, we are able to detect interleukin 6 (IL-6) in a complex biofluid of human whole plasma with a limit of detection (LOD) of 0.5 pg.mL-1. This LOD is significantly lower than the smallest reported IL-6 LOD in plasma, 23 pg mL-1, using a complex electrochemical system. The higher sensitivity of our developed biosensor can be attributed to the distinguish capability of biofunctional LISs in preventing non-specific adhesion of biomolecules compared to other blocking agents. / Thesis / Doctor of Philosophy (PhD) / The key goal of this thesis is to provide new strategies for preparation of robust and durable biointerfaces that could be employed for many biomedical devices such as self-cleaning coatings, microfluidics, point-of-care diagnostics, biomedical assays, and biosensors in order to enhance their efficiency, sensitivity, and precision. The introduced surface biofunctionalization methods are straightforward to use and do not require multiple wet-chemistry steps and incubation times, making them suitable for mass production and high throughput demands. Moreover, the introduced surface coating strategies allow for creation of antibody/protein micro-patterns covalently bound onto a biomolecule-repellent surface. The repellent property of the surfaces is resulted from infusion of an FDA-approved lubricant into the surface of a chemically modified substrate. While the surface repellency can effectively prevent non-specific adhesion of biomolecules, the patterned antibodies can locally capture the desired analyte, making them a great candidate for biosensing.

No description available.

In the characterization of many diseases, the tissues involved tend to have different chemical compositions when compared to those of normal tissues. For example, in certain types of cancer, metabolites or small peptides may be overproduced by the cancerous cells. If this difference can be measured, using bio-sensor technology, early on in disease progression and development, it may mean the difference between life and death for the patient suffering from the disease. Because of their substrate specificities and chemical recognition properties, Redox enzymes make ideal test subjects for studying the potential of enzymes for use in bio-sensing equipment. In this research, a target set of redox enzymes was cloned from the hyperthermophilic organism, Sulfolobus solfataricus. Many of the cloned enzymes were expressed and purified, so that biophysical characterizations, kinetic enzyme assays, and crystallization trials can be performed in the future.

Cholesterol and ethanol biosensors based on conducting polypyrrole (PPy), poly(3,4-ethylenedioxythiophene) (PEDOT) and poly(3,4-ethylenedioxypyrrole) (PEDOP) were constructed. Cholesterol oxidase (ChOx, from Pseudomonas fluorescens) and alcohol oxidase (AlcOx, from Pichia pastoris) were physically entrapped during electropolymerization of the monomers (Py, EDOT, EDOP) in phosphate buffer containing sodium dodecylsulfate (SDS) as the supporting electrolyte. The amperometric responses of the enzyme electrodes were measured monitoring oxidation current of H2O2 at +0.7 V in the absence of a mediator. Kinetic parameters, such as Km and Imax, operational and storage stabilities, effects of pH and temperature were determined for all entrapment supports. Based on Michaelis-Menten (Km) constants, it can be interpreted that both enzymes immobilized in PEDOT showed the highest affinities towards their substrates. Before testing the alcohol biosensors on alcoholic beverages, effects of interferents (glucose, acetic acid, citric acid, L-ascorbic acid) which might be present in beverages were determined. The alcohol content of the distilled beverages (vodka, dry cin, whisky, raki) was measured with these biosensors. A good match with the chromatography results (done by the companies) was observed.

In this thesis, six different biosensors based on conducting polymers of poly 4-(2,5-di(thiophen-2-yl)-1H-pyrrole-1-l) benzenamine [poly(SNSNH2)] and poly(1- (4-nitrophenyl)-2,5-di(2-thienyl)-1H-pyrrole [poly(SNSNO2)] were prepared. Electrochemical technique was used for polymerization of conducting polymers and two different immobilization techniques / crosslinking and adsorption were used for immobilizing enzyme or microbial in the conducting polymer matrices. The proposed biosensors were characterized and optimized. Optimum pH, thickness of conducting polymer and biological material amount were determined. Linearity, repeatability and operational stability experiments were performed. Carbon nanotubes and gold nanoparticles were also added to the biosensing system to see the effects of nanoparticles. The biosensors also used for ethanol and/or glucose biosensing in commercial samples. In the first part of thesis, a biosensor was designed by immobilizing Gluconobacter oxydans in poly(SNSNH2) matrix on graphite electrode. The biosensor preparation method was a two-step procedure where the cells were immobilized by adsorption on the surface after the electropolymerization step.Use of dialysis membrane to cover the surface after immobilization conserves the bioactive surface during the operation. The preparation is simple and not time consuming. Systems proposed showed good linearity and repeatability as well as high operational stability. Glucose amount in fruit juice, ethanol amount in vodka and whisky were determined. In the second part of thesis, a second biosensor was designed with electrochemical polymerization of 1-(4-nitrophenyl)-2,5-di(2-thienyl)- 1H-pyrrole via cyclic voltammetry on graphite electrode. Afterwards, Pseudomonas fluorescens and Gluconobacter oxydans were immobilized successfully on the conducting polymer matrix separately. The proposed biosensors showed good linear range, and repeatability as well as high operational stability. In the third and fourth parts, gold nanoparticle and carbon nanotube effects were studied on poly(SNSNH2)/glucose oxidase biosensor, respectively. Covalent binding of glucose oxidase was achieved to poly(SNSNH2) by the help of glutaraldehyde on the top of graphite and carbon paste electrodes. Nanoparticle amount and optimum pH were determined for both biosensors. After analytical characterization, glucose amount in two fruit juices were determined with poly(SNSNH2)/GOx/AuNP and poly(SNSNH2)/ GOx/CNT biosensors. In the last part, biosensor was designed with immobilizing alcohol oxidase in poly(SNSNH2) matrix via crosslinking with glutaraldehyde on platinum electrode. The proposed biosensor was characterized and optimized in terms of thickness, enzyme loading, pH, AuNPs, CNTs, linear range, repeatability and operational stability.

No description available.

published_or_final_version / abstract / Chemistry / Master / Master of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Suspended lipid bilayers, or black lipid membranes (BLMs), have been used to study the electrophysiological properties of ion channels (ICs); however, BLMs assembled from natural, non-polymerizable lipids are inherently unstable due to the non-covalent associations on which they are based. Lifetimes of several hours are commonly observed in BLMs until rupture due to mechanical, thermal, or chemical insults. One potential improvement is the use of polymerizable phospholipids (poly(lipids)). BLMs prepared using dienoyl functionalized poly(lipids) and binary mixtures of fluid, non-polymerizable lipids with poly(lipids) were investigated for IC recordings.poly(BLMs) exhibited enhanced lifetimes from several hours to upwards of 4 weeks while maintaining IC functionality for one week. Activity of ICs that require membrane fluidity was retained using binary phospholipid mixtures of fluid and polymeric phospholipids. IC activity was retained by inducing domain formation, wherein ICs incorporated into the fluid domains. The binary membranes exhibited marked enhancement in stability resulting from fractional poly(lipids) polymerization. Additionally, ICs can be reconstituted into the fluid domains following photopolymerization and subsequent domain formation, a key requirement when UV-sensitive ICs are utilized. Here, the electrical properties, stability, and incorporation of pore-forming ICs, including hemolysin, alamethicin, and gramicidin, into poly(lipid) membranes are reported. Potential applications developing ligand-gated IC based sensors for high throughput screening are being investigated.In parallel to the characterization of poly(lipids) for potential long-term IC membranes, a model ligand-gated IC was expressed, characterized, and reconstituted into non-polymerizable lipids. Mutant K_ATP channels were expressed in mammalian and yeast systems. The orientations of mutant K_ATP channels were studied using electrophysiological and immunohistochemical techniques. Large quantities were expressed and purified from <italic>Pichia pastoris</italic> and functionally reconstituted into BLMs. ATP and long-chaing coenzyme A ester sensitivity was maintained in reconstituted in BLMs. K_ATP channels will serve as a model system for testing the effect of poly(lipid) BLMs on IC function. Future utilization of poly(lipid) BLMs in combination with ligand-gated ICs offer major advancements to potential increased throughput for IC screening.