Farklı Nanopartiküller Kullanılarak Bakteri Tayinine Yönelik Gradient Yöntemi Geliştirilmesi

Abstract

Nowadays, development of label-free, easy handling, cost-effective and fast responsive analysis methods have gained great importance. In this study, it was aimed to develop an easy handling and cost-effective nanoparticle based density gradient method intended for the pathogenic bacteria detection. To this end, unbound bacteria, bacteria captured nanoparticles and unbound nanoparticle-antibody conjugates after immunological interaction were collected depending on their density and size through the sucrose density gradient. A simple and low-cost optical system was used to measure optic density variation of the phases, and the data was processed graphically on the computer with MatLab software. Gold nanoparticles, magnetic nanoparticles, semiconductor quantum dots and CNP, which are commonly used nanoparticles in bioassays, were utilized in the preliminary studies of density gradient centrifugation to understand the formation and shift of bacteria-particle bands in detail. Utilizing the principle of rate-zonal centrifugation was found eligible for the investigation of nanoparticle and bacteria-nanoparticle phases. The gradient parameters affecting the bacteria-particle band formation such as volume of particle solution, effect of single phase and two phase gradient, concentrations and volumes of sucrose solutions, effect of buffer solutions were investigated respectively. TEM and UV-Vis spectroscopy were used for the characterization of the nanoparticles. Fluorescence spectroscopy was used for characterization of quantum dots and FTIR-ATR was used for surface characterization of CNP. In solid media, single colonies were counted from the samples taken from gradient bands, and bacterial binding efficiencies were calculated for particle-specific and non-target interactions. It was seen that the migration distance of quantum dot bands and dispersion of carbon sphere phases displayed differences with respect to the change in bacteria amount in the sucrose gradient. This system was calibrated with CNPs to be prepared for real sample analysis. The detection and quantification of Escherichia coli in skimmed milk were examined by the developed method. The limit of determination and limit of quantitation was determined as 8 and 28 cfu/ml, respectively. The selectivity of the method has been proven with Enterobacter aerogenes studies. As a result, it was demostrated that rapid and sensitive bacteriological diagnosis can be performed with an easy set-up using a centrifuge and a mobile phone camera, which are easily found in almost every laboratory. The developed method that based on bacteria-NP phases for the pathogenic bacteria detection has also provided an innovative approach to centrifugation studies in the literature. In addition to this, due to cost-effective, easy handling, and having shortest response time by reducing the steps of the process; this method has become an alternative to the analysis methods in the literature.