This work has explored the capability of a mixed film composed of oligonucleotides and oligomers to improve the selectivity for the detection of fully complementary oligonucleotide targets in comparison to partially complementary targets which have one and three base-pair mismatched sites. The intention was to introduce a “matrix isolation” effect on oligonucleotide probe molecules by surrounding the probes with oligomers, thereby reducing oligonucleotide-to-oligonucleotide and/or oligonucleotide-to-surface interactions. This resulted in a more homogeneous environment for probes, thereby minimizing the dispersity of energetics associated with formation of double-stranded hybrids. The mixed film was constructed by immobilizing pre-synthesized oligonucleotides onto a mixed aminosilane layer and then growing the oligomer portion by surface-initiated atom transfer radical polymerization (ATRP) of 2-hydroxy methacrylate (PHEMA). The performance of the mixed film was compared to films composed of only oligonucleotides in a series of hybridization and melt curve experiments. Surface characterization techniques were used to confirm the growth of the oligomer portion as well as the presence of both oligonucleotides and oligomer components. Polyatomic bismuth cluster ions as sources for time-of-flight secondary ion mass spectrometry experiments could detect both components of the mixed film at a high sensitivity even though the oligomer portion was at least 200-fold in excess. At the various ionic strengths investigated, the mixed films were found to increase the selectivity for fully complementary targets over mismatched targets by increasing the sharpness of melt curves and melting temperature differences (delta Tm) by 2- to 3-fold, and by reducing non-specific adsorption. This resulted in improved resolution between the melt curves of fully and partially complementary targets. A fluorescence lifetime investigation of the Cy3 emission demonstrated that Cy3-labeled oligonucleotide probes experienced a more rigid microenvironment in the mixed films. These experiments demonstrated that a mixed film composed of oligonucleotides and PHEMA can be prepared on silica-based substrates, and that they can improve the selectivity for SNP discrimination compared to conventional oligonucleotide films.

ABSRACT: This research focuses on two hydrophilic polymers that form hydrogels when they sorb water: Poly(2-hydroxyethyl methacrylate) (PHEMA) and Poly(2,3-dihydroxypropyl methacrylate) (PDHPMA). Present work in the field obviated the need to properly characterize the thermal and dielectric properties of these materials.The dielectric permittivity, e', and the loss factor, e", of dry poly(2-hydroxyethyl methacrylate) and poly(2,3-dihydroxypropyl methacrylate) were measured using a dielectric analyzer in the frequency range of 0.1Hz to 100 kHz and between the temperature range of -150 °C to 275°C. The dielectric response of the sub-Tg gamma transition of PHEMA has been widely studied before but little to no DEA data above 50°C is present in the literature. This study is the first to present the full range dielectric spectrum of PHEMA, PDHPMA and their random copolymers up to and above the glass transition region. The electric modulus formalism and several mathematical proofs were used to reveal the gamma, beta, alpha and conductivity relaxations. Dielectric analysis gives insight into the network structure of the polymer; it has been shown through thermal analyses that as the DHPMA content increased in HEMA-DHPMA copolymers the polymer matrix increased in available free volume and facilitated the movement of ions in its matrix. This is of significance as we then investigated the feasibility of using PHEMA, PDHPMA and their random copolymers as materials for a biocompatible coating for an implantable glucose sensor. The biocompatibility of hydrogels can be attributed to the low interfacial tension with biological fluids, high gas permeability, high diffusion of low molecular weight compounds, and reduced mechanical and frictional irritation to surrounding tissue. Once the biocompatibility of the hydrogels was established, the task to coat the polyurethane (PU)/epoxy coated metal glucose sensor was addressed. Plasma polymerization was found to be the most feasible technique for the application of the biocompatible hydrogel as a coating on the implantable glucose sensor. It has also been shown that thermal analysis techniques provide a mode of investigation that can be used to investigate the interfacial interactions of a novel hydroxylated, self-assembled nanoparticle with two functionally different polymers, poly(2-dihydroxyethyl methacrylate) and poly(methyl methacrylate).

This work has explored the capability of a mixed film composed of oligonucleotides and oligomers to improve the selectivity for the detection of fully complementary oligonucleotide targets in comparison to partially complementary targets which have one and three base-pair mismatched sites. The intention was to introduce a “matrix isolation” effect on oligonucleotide probe molecules by surrounding the probes with oligomers, thereby reducing oligonucleotide-to-oligonucleotide and/or oligonucleotide-to-surface interactions. This resulted in a more homogeneous environment for probes, thereby minimizing the dispersity of energetics associated with formation of double-stranded hybrids. The mixed film was constructed by immobilizing pre-synthesized oligonucleotides onto a mixed aminosilane layer and then growing the oligomer portion by surface-initiated atom transfer radical polymerization (ATRP) of 2-hydroxy methacrylate (PHEMA). The performance of the mixed film was compared to films composed of only oligonucleotides in a series of hybridization and melt curve experiments. Surface characterization techniques were used to confirm the growth of the oligomer portion as well as the presence of both oligonucleotides and oligomer components. Polyatomic bismuth cluster ions as sources for time-of-flight secondary ion mass spectrometry experiments could detect both components of the mixed film at a high sensitivity even though the oligomer portion was at least 200-fold in excess. At the various ionic strengths investigated, the mixed films were found to increase the selectivity for fully complementary targets over mismatched targets by increasing the sharpness of melt curves and melting temperature differences (delta Tm) by 2- to 3-fold, and by reducing non-specific adsorption. This resulted in improved resolution between the melt curves of fully and partially complementary targets. A fluorescence lifetime investigation of the Cy3 emission demonstrated that Cy3-labeled oligonucleotide probes experienced a more rigid microenvironment in the mixed films. These experiments demonstrated that a mixed film composed of oligonucleotides and PHEMA can be prepared on silica-based substrates, and that they can improve the selectivity for SNP discrimination compared to conventional oligonucleotide films.

ABSTRACT Covalent Immobilization of Glucose Isomerase on Poly (2-hydroxyethyl methacrylate) Particles Yildiz, Hakan &Uuml / mit M.S., Department of Chemistry Supervisor: Prof. Dr. Nesrin Hasirci July 2004, 54 pages In this study, poly (2-hydroxyethyl methacrylate), P(HEMA), particles were prepared by suspension polymerization of the monomer 2-hydroxyethyl methacrylate with addition of ethylene glycol dimethyacrylate, EGDMA, as cross linker. Glucose isomerase, GI, enzyme was covalently immobilized on the prepared P(HEMA) particles after activation of the particles with cyanuric chloride. The activities of the free and immobilized enzymes were measured with Ethanol-Carbazole method. The immobilization of GI on P(HEMA) particles promoted enzyme stability and as a result, the enzyme became more stable to temperature, storage, and reuse. For maximum substrate conversion, optimum temperature was determined as 70 oC for free GI and this value shifted to 60 oC for immobilized enzyme. Optimum pH for maximum substrate conversion was found to be 7.0 for free GI and 8.0 for immobilized GI. The change of enzyme activity with substrate concentration were determined to calculate Km and Vmax values of the free and immobilized enzymes. Km values were found to be 1.7x10-2 mol/L and 3.1x10-1 mol/L while Vmax values were 1.01x10-4 mol/L.min, 1.65x10-3 mol/L.min for free and immobilized GI, respectively. Reuse capability of immobilized GI on P(HEMA) particles was measured and compared with commercial GI. Both systems retained 80 % of their original activities after 40th use, within 6 days. The change of enzyme activities upon storage were detected at certain time intervals for the samples stored in buffer solution at 4 oC. Immobilized enzyme was retained 60% of its original activitiy in 60 days of storage at 4 oC. Immobilized GI and commercial GI both retained 90% of their activities under continuous flow after 180 mL of substrate solution passed through the column.

Due to sensor -tissue interactions, currently none of the commercially available glucose sensors are capable of continuous, reliable monitoring of glucose levels during long-term implantation. In order to improve the lifetime of implanted glucose sensors, two series of biocompatible novel hydrogel coatings were designed, synthesized and the physical properties were measured. Different hydrogels with various 2,3-dihydroxypropyl methacrylate (DHPMA) compositions were coated onto glucose sensors. Results show that the higher freezable water content, swelling rate and uniform porosity that resulted from high DHPMA content increased the sensitivity and shortened the response time of glucose sensors. The linear range of a glucose sensor coated only with hydrogel is short, however, the range can be improved by coating the epoxy- polyurethane (PU) with a layer of hydrogel. Since the hydrogel minimizes the fibrosis and inflammation, it shows promise for use in implantable glucose sensors. However, the in vivo experiment shows only 25% of sensors still worked after 4 weeks. In order to overcome problems present in the first series of experiments, another series of novel hydrogels with various N-vinyl pyrolidone (VP) contents was developed. This study has provided a feasible approach to design and select the properties of the copolymer for coating implantable biosensors. The in vivo experiments demonstrate that a hydrogel coating significantly improved the performance of implanted glucose sensors. In order to suppress the acute inflammation caused by the surgery, dexamethasone-21 phosphate disodium salt (DX-21) was incorporated to a series of poly (HEMA-DHPMA-VP) hydrogels to investigate the drug delivery in vitro. All hydrogels showed a high initial release, followed by slow, long term release during the next hours to days. This release pattern is believed to be optimum for implanted glucose sensors suppressing the acute and chronic inflammation. Water structures in hydrogels swollen in different media water, PBS and DX-21 solution were also investigated. 1HEMA:1DHPMA copolymer and VP-HEMA-DHPMA copolymers imbibed higher freezable water fractions in DX-21 solution. The ratio of transporting water mass to DX-21 mass is 9.6 which is independent of the hydrogel composition.

<p>In the presented work, immunosensors for detection of Aflatoxin B1 based on different immobilization platforms were studied. Synthesis of an electroactive hydrogel was also carried out. Aflatoxins are a group of mycotoxins that have deleterious effects on humans and are produced during fungal infection of plants or plant products. Electrochemical immunosensor for the determination of Aflatoxin B1 (AFB1) was developed with anti-aflatoxin B1 antibody immobilized on Pt electrodes modified with polyaniline (PANi) and polystyrene sulphonic acid (PSSA). Impedimetric analysis shows that the electron transfer resistances of Pt/PANi-PSSA electrode, Pt/PANi-PSSA/AFB1-Ab immunosensor and Pt/PANi-PSSA/AFB1-Ab incubated in BSA were 0.458, 720 and 1066 k&Omega / , respectively. These results indicate that electrochemical impedance spectroscopy (EIS) is a suitable method for monitoring the change in electron-transfer resistance associated with the immobilization of the antibody. Modelling of EIS data gave equivalent circuits which showed that the electron transfer resistance increased from 0.458 k&Omega / for Pt/PANi-PSSA electrode to 1066 k&Omega / for Pt/PANi-PSSA/AFB1-Ab immunosensor, indicating that immobilization of the antibody and incubation in BSA introduced an electron transfer barrier. The AFB1 immunosensor had a detection limit of 0.1 mg/L and a sensitivity of 869.6 k &Omega / L/mg.</p>

<p>In the presented work, immunosensors for detection of Aflatoxin B1 based on different immobilization platforms were studied. Synthesis of an electroactive hydrogel was also carried out. Aflatoxins are a group of mycotoxins that have deleterious effects on humans and are produced during fungal infection of plants or plant products. Electrochemical immunosensor for the determination of Aflatoxin B1 (AFB1) was developed with anti-aflatoxin B1 antibody immobilized on Pt electrodes modified with polyaniline (PANi) and polystyrene sulphonic acid (PSSA). Impedimetric analysis shows that the electron transfer resistances of Pt/PANi-PSSA electrode, Pt/PANi-PSSA/AFB1-Ab immunosensor and Pt/PANi-PSSA/AFB1-Ab incubated in BSA were 0.458, 720 and 1066 k&Omega / , respectively. These results indicate that electrochemical impedance spectroscopy (EIS) is a suitable method for monitoring the change in electron-transfer resistance associated with the immobilization of the antibody. Modelling of EIS data gave equivalent circuits which showed that the electron transfer resistance increased from 0.458 k&Omega / for Pt/PANi-PSSA electrode to 1066 k&Omega / for Pt/PANi-PSSA/AFB1-Ab immunosensor, indicating that immobilization of the antibody and incubation in BSA introduced an electron transfer barrier. The AFB1 immunosensor had a detection limit of 0.1 mg/L and a sensitivity of 869.6 k &Omega / L/mg.</p>

Philosophiae Doctor - PhD / In the presented work, immunosensors for detection of Aflatoxin B1 based on different immobilization platforms were studied. Synthesis of an electroactive hydrogel was also carried out. Aflatoxins are a group of mycotoxins that have deleterious effects on humans and are produced during fungal infection of plants or plant products. Electrochemical immunosensor for the determination of Aflatoxin B1 (AFB1) was developed with anti-aflatoxin B1 antibody immobilized on Pt electrodes modified with polyaniline (PANi) and polystyrene sulphonic acid (PSSA). Impedimetric analysis shows that the electron transfer resistances of Pt/PANi-PSSA electrode, Pt/PANi-PSSA/AFB1-Ab immunosensor and Pt/PANi-PSSA/AFB1-Ab incubated in BSA were 0.458, 720 and 1066 kΩ, respectively. These results indicate that electrochemical impedance spectroscopy (EIS) is a suitable method for monitoring the change in electron-transfer resistance associated with the immobilization of the antibody. Modelling of EIS data gave equivalent circuits which showed that the electron transfer resistance increased from 0.458 kΩ for Pt/PANi-PSSA electrode to 1066 kΩ for Pt/PANi-PSSA/AFB1-Ab immunosensor, indicating that immobilization of the antibody and incubation in BSA introduced an electron transfer barrier. The AFB1 immunosensor had a detection limit of 0.1 mg/L and a sensitivity of 869.6 kΩL/mg. / South Africa

Philosophiae Doctor - PhD / In the presented work, immunosensors for detection of Aflatoxin B1 based on different immobilization platforms were studied. Synthesis of an electroactive hydrogel was also carried out. Aflatoxins are a group of mycotoxins that have deleterious effects on humans and are produced during fungal infection of plants or plant products. Electrochemical immunosensor for the determination of Aflatoxin B1 (AFB1) was developed with anti-aflatoxin B1 antibody immobilized on Pt electrodes modified with polyaniline (PANi) and polystyrene sulphonic acid (PSSA). Impedimetric analysis shows that the electron transfer resistances of Pt/PANi-PSSA electrode, Pt/PANi-PSSA/AFB1-Ab immunosensor and Pt/PANi- PSSA/AFB1-Ab incubated in BSA were 0.458, 720 and 1066 kΩ, respectively. These results indicate that electrochemical impedance spectroscopy (EIS) is a suitable method for monitoring the change in electron-transfer resistance associated with the immobilization of the antibody. Modelling of EIS data gave equivalent circuits which showed that the electron transfer resistance increased from 0.458 kΩ for Pt/PANi-PSSA electrode to 1066 kΩ for Pt/PANi- PSSA/AFB1-Ab immunosensor, indicating that immobilization of the antibody and incubation in BSA introduced an electron transfer barrier. The AFB1 immunosensor had a detection limit of 0.1 mg/L and a sensitivity of 869.6 k ΩL/mg. In the second platform an immunosensor based on gold nanoparticles (AuNP) and polythionine-modified glassy carbon electrode (GCE) for the determination of aflatoxin B1 (AFB1) was developed. Aflatoxin B1-BSA conjugate was immobilised on the modified GCE. Horseradish peroxidase (HRP) or Bovine serum albumin (BSA) were used to block sites against non-specific binding of the AFB1- conjugate with other compounds such as the salts used in preparing the buffer when the antibody interacts with the AFB1 conjugate and free AFB1. Competition reaction was allowed to take place between the free AFB1 and AFB1-conjugate for the binding sites of the anti-aflatoxin B1 antibody. Cyclic voltammetry (CV) was employed to characterize the electrochemical properties of the modified process. The peak separation of the immunosensor (ΔEp) was 62 mV indicating a quasi reversible process. Differential pulse voltammetry (DPV) was used to monitor the analytical signal. The response decreased with an increase in AFB1 concentration in the range of 0.6-2.4 ng/mL with a limit of detection of 0.07 and 0.16 ng/mL for HRP and BSA blocked immunosensors respectively. Significantly the low detection limit of 0.07 ng/mL is within the limits set by worl health organization (WHO) for AFB1 and its derivatives which is 2 ng/mL The proposed method eliminates the use of secondary antibody enzymatic labels. Synthesis and characterization of (p-(HEMA)-polyaniline hydrogels were investigated. The hydrogels were synthesized using: 2-Hydroxyeththyl methacrylate (HEMA), N-Tris (hydroxymethyl) methyl] acrylamide, 3- Sulfopropyl methacrylate potassium salt, Tetraethylene glycol diacrylate, Poly-(2- hydroxyethyl methacrylate), 2, 2-Dimethoxy-2-phenylacetophenone and aniline by UV irradiation. Two sets of the hydrogels were prepared using water / 1, 3, 3, 3-(tetramethyl butyl phenyl polyethylene glycol [Triton X-100] and water / ethylene glycol as the solvent. Scanning electron microscopy (SEM) revealed a more uniform pore size when Triton X 100 (TX-100 HG) was used as compared to ethylene glycol (EG-HG). Thermogravimetric analysis (TGA) showed that both hydrogels were stable up to 270 oC. Fourier transform-Infra red (FTIR) spectrum confirmed the incorporation of polyaniline (PANi) and HEMA in the composite. Electrochemical properties of the hydrogels evaluated using Cyclic Voltammetry and Electrochemical Impedance Spectroscopy (EIS) demonstrated the electroactivity and conductivity.