Yüzeyde Güçlendirilmiş Raman Spektroskopisi (SERS) Temelli Platformların Geliştirilmesi ve Biyoanalizlerde Kullanımı

Abstract

Surface enhanced Raman spectroscopy (SERS) is a powerful technique that enables the determination of analytes at low concentrations. Using the high plasmon resonance of the metallic nanostructures, the signals of the vibrational energies of the chemical bonds belonging to the targeted molecule are enhanced. Colloidal nanoparticles and plasmonic SERS-based platforms formed on solid surfaces were frequently utilized for the application of SERS technique. In this thesis, platfroms that can be used in SERS measurements for the determination of biomolecules in different food samples and biological environments were developed. Enhancement factors (EF), limit of detection (LOD) and limit of quantification (LOQ) values were calculated for the developed detection platforms of each targeted analyte. Gold nanorod particles (Au-NRs) and spherically shaped silver nanoparticles (AgNPs) were synthesized to form SERS-based platforms. The characteristics of the synthesized nanoparticles were determined and the plasmon bands, shapes and sizes were determined. Using two different techniques in bioassays; suspension of nanoparticles in colloidal state and immobilization of nanoparticles on gold-coated glass surfaces by chemical and physical bonds have resulted with three different SERS-based platforms. These platforms were named as Au-NRs, AgNPs and Au-AgNPs and were chosen to be used in bioassays developed for the targeted biomolecules. In the context of the present thesis, an indirect method was developed by using SERS active reporter molecules for the quantitative determination of ascorbic acid. LOD and LOQ values were found to be 1,37 μg ml-1 and 4,56 μg ml-1, respectively. The EF value calculated for bromocresol green, that was used as the reporter molecule, was found to be 1,33x109. SERS measurements were taken and obtained EF values of three different SERS-based platforms were compared in terms of their performance for caffeine analysis. Au-AgNPs surfaces which were constructed by the formation of self assmbled monolayer of silver nanoparticles on gold surface showed the best performance for caffeine analysis. The EF value for this platform was calculated as 6,7x109. SERS measurements were taken by using 20X and 50X objectives. LOD and LOQ values calculated from the obtained spectra were 0,904 and 0,6346 mg ml-1 for 20X objective and 0,0678 and 0,2260 mg ml-1 for 50X objective, respectively. Spectra of asesulfame K, cyclamate, sucralose, aspartame and saccharin were taken and EF values were calculated for each platform so that artificial sweeteners could be determined using SERS-based platforms. Based on the results obtained, a SERS-based method was developed using AgNPs nanoparticles to determine the amount of aspartame in artificial sweeteners in mineral waters. LOD values in plain and fruit flavored mineral waters were respectively; 0,13, 0,14 mg ml-1. With the developed method, aspartame has been found to be easily detectable at the upper and lower levels of the legal limit of 0,6 mg ml-1 in beverages. Alkaline phosphatase enzyme (ALP) is an important enzyme that plays a significant role in clinical trials and metabolic activities. SERS measurements were taken to determine ALP activity with the developed SERS-based platforms and it was determined that the best performance was obtained using Au-NRs nanoparticles in measurements between 10-11-10-15 M concentration range. The SERS active property of the 5-bromo-4-chloro-3-indole (BCI) product resulting from the enzymatic reaction of the ALP was exploited in this platform. The sandwich immunoassay was developed and LOD and LOQ values were obtained as 10 and 30 cfu ml-1 Escherichia coli.

Keywords: Surface Enhanced Raman Spectroscopy (SERS), gold nanoparticles, silver nanoparticles, ascorbic acid, caffeine, artificial sweeteners, alkaline phosphatase enzyme, Escherichia coli