In this study, we report the chemical synthesis and functionalization of magnetic and gold-coated magnetic nanoparticles and the immobilization of single-stranded biotinylated oligonucleotides onto these particles. Selected sequences specific to the BRCA1 gene were used as a test platform. The binding of oligonucleotides to these particles was achieved through a streptavidin-biotin bridge via a carbodiimide activation protocol. Particle size and oligonucleotide attachment were confirmed by transmission electron microscopy; oligonucleotide binding was characterized by Fourier transform infrared spectroscopy and hybridization confirmed by fluorescence emission from the fluorophore attached to the target oligonucleotide strand. The rate of hybridization was measured using a spectrofluorometer and a microarray scanner. The rate of hybridization of oligonucleotides bound to the synthesized particles depends on the inorganic support material and its surface chemistry. The rate of hybridization increased concomitantly with the concentration of the probe and the target in the reaction medium. Furthermore, exposure of probe and target oligonucleotide to a combination of target and noncomplementary DNA strand reduced the rate of hybridization, possibly because of steric crowding in the reaction medium and cross-linking between reacting oligonucleotides and the noncomplementary strands. The study undertaken opens several possibilities in bioconjugate attachment to functionalized iron and iron nanocomposite structures for controlled manipulation and handling using magnetic fields.

Magnetic nanoparticles (Fe 3 O 4 ) were synthesized by thermal co-precipitation of ferric and ferrous chlorides. The sizes and structure of the particles were characterized using transmission electron microscopy (TEM). The size of the particles was in the range between 9.7 and 56.4 nm. Cholesterol oxidase (CHO) was successfully bound to the particles via carbodiimide activation. FTIR spectroscopy was used to confirm the binding of CHO to the particles. The binding efficiency was between 98 and 100% irrespective of the amount of particles used. Kinetic studies of the free and bound CHO revealed that the stability and activity of the enzyme were significantly improved upon binding to the nanoparticles. Furthermore, the bound enzyme exhibited a better tolerance to pH, temperature and substrate concentration. The activation energy for free and bound CHO was 13.6 and 9.3 kJ/mol, respectively. This indicated that the energy barrier of CHO activity was reduced upon binding onto Fe 3 O 4 nanoparticles. The improvements observed in activity, stability, and functionality of CHO resulted from structural and conformational changes of the bound enzyme. The study indicates that the stability and activity of CHO could be enhanced via attachment to magnetic nanoparticles and subsequently will contribute to better uses of this enzyme in various biological and clinical applications.

BackgroundMagnetic nanoparticles have been significantly used for coupling with biomolecules, due to their unique properties.MethodsMagnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chloride using two different base solutions. Glucose oxidase was bound to the particles by direct attachment via carbodiimide activation or by thiophene acetylation of magnetic nanoparticles. Transmission electron microscopy was used to characterize the size and structure of the particles while the binding of glucose oxidase to the particles was confirmed using Fourier transform infrared spectroscopy.ResultsThe direct binding of glucose oxidase via carbodiimide activity was found to be more effective, resulting in bound enzyme efficiencies between 94–100% while thiophene acetylation was 66–72% efficient. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when subjected to thermal and various pH conditions. The overall activity of glucose oxidase was improved when bound to magnetic nanoparticlesConclusionBinding of enzyme onto magnetic nanoparticles via carbodiimide activation is a very efficient method for developing bioconjugates for biological applications

This article reports the chemical synthesis and functionalization of magnetic and gold-coated magnetic nanoparticles. The binding characteristics of streptavidin-conjugated nanoparticles were studied using prion protein as a target to a specific biotinylated aptamer. The size and structure of the particles were determined by transmission electron microscopy, and the binding of the prion to the particle was confirmed by Fourier transform infrared spectroscopy. The rate of prion binding to the aptamer was dose-dependent, and prion immobilization was more effective on L-aspartic acid-functionalized magnetic nanoparticles compared to the carboxyl-functionalized gold-coated magnetic nanoparticles. This study sets the stage for the development of prion detection platforms as well as our long-term goals in structure elucidation at the binding interface.

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