ELEKTROKİMYASAL HİBRİT AFİNİTE ELEKTROTLARIN GELİŞTİRİLMESİ VE GENEL TARAMA KİTİ OLARAK KULLANILABİLİRLİKLERİNİN ARAŞTIRILMASI

Abstract

The emergence of the COVID-19 pandemic has once again highlighted the necessity for the development of rapid diagnostic kits. The detection of viral and microbial pathogens has become imperative. In addition to the pandemic, geographical hazards and public health concerns have also underscored the need for rapid, reliable, cost-effective, and easily accessible tests. In the presented thesis study, three different pathogens were detected using three different methods with four sensor systems. As part of the thesis, the first study focused on Pseudomonas aeruginosa, a significant threat in public areas such as swimming pools and saunas, which can be transmitted through contaminated water and contribute to increased mortality and morbidity rates in intensive care units. In this study, P. aeruginosa bacteria were imprinted onto a polydopamine polymer surface on a graphene-graphite hybrid substrate and detected electrochemically. Molecularly imprinted polydopamine films were prepared on graphene oxide-modified graphite electrodes by the chemical oxidation of dopamine in the presence of P. aeruginosa template molecules. The electrodes were electrochemically characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV). Additionally, the electrodes were chemically characterized using Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The results indicated that the films were successfully deposited onto the electrodes and exhibited enhanced electrical conductivity in the presence of graphene oxide. After template removal, selective cavities for P. aeruginosa were exposed. The analytical performance of the electrodes was tested using DPV within a concentration range of 10²–10⁸ CFU/mL. The sensor's selectivity was also evaluated against potential interfering competitors, including Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, demonstrating a high specificity for P. aeruginosa. The second study in the thesis involved the detection of the SARS-CoV-2 virus surface peptide, responsible for over 771 million reported cases and more than 7 million deaths since the first case was identified on November 17, 2019. The immunoaffinity sensor interface was developed using Anti-SARS-CoV-2 antibodies covalently immobilized onto poly(pyrrole-pyrrole-3-carboxylic acid) [p(Py-PyCOOH)] nanotube surfaces. The anti-SARS-CoV-2 immobilized nanotubes were drop-cast onto flexible screen-printed carbon electrodes. The electrodes were electrochemically characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Additionally, Raman spectroscopy, contact angle measurements, and scanning electron microscopy (SEM) were used for chemical characterization. The results demonstrated the successful deposition of nanotubes onto the electrodes. The sensor’s analytical performance was evaluated using EIS in a concentration range of 1–1000 pg/mL. Its selectivity was assessed against potential interfering agents, including immunoglobulin G (IgG), bovine serum albumin (BSA), hemoglobin (Hb), glucose, and uric acid. The final two sensors developed within the scope of the thesis were designed for the detection of the Crimean-Congo hemorrhagic fever acute virus, which is transmitted by ticks carried by migratory birds and poses a significant threat to our geographical region. The first sensor design for this virus incorporated DNA-based aptamers. Electrodes electrochemically coated with platinum nanoparticles were modified with p(Py- PyCOOH) polymer via cyclic voltammetry in two cycles to allow the binding of DNA aptamers. These electrodes, onto which DNA aptamers were covalently immobilized, were electrochemically characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and chronoamperometry (CA). Additionally, chemical characterization was performed using Raman spectroscopy (FTIR), contact angle measurements, and atomic force microscopy (AFM). The analytical performance of the electrodes was tested using EIS in a concentration range of 10²–10⁸ copies/mL. The sensor's selectivity was evaluated against potential interfering agents, including P. aeruginosa, E. coli, S. aureus, B. subtilis, and SARS-CoV-2, demonstrating high specificity. Finally, molecularly imprinted sensors were developed for the detection of the Crimean- Congo hemorrhagic fever acute virus. Electrodes electrochemically coated with PtNP were treated with a solution of 4-aminophenylboronic acid (4-APBA) containing the Crimean-Congo hemorrhagic fever acute virus and subjected to 10 cycles of cyclic voltammetry to form a virus-containing film. The viral particles within the structure were subsequently removed using phosphate-buffered saline (PBS) through 10 cycles of cyclic voltammetry. Non-imprinted electrodes were prepared by polymerizing a virus-free 4- APBA solution. These electrodes were electrochemically characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Chemical characterization was also conducted using Raman spectroscopy (FTIR), contact angle measurements, and atomic force microscopy (AFM). The analytical performance of the electrodes was tested using EIS in a concentration range of 10²–10⁸ copies/mL. The sensor's selectivity was evaluated against potential interfering agents, including P. aeruginosa, E. coli, S. aureus, B. subtilis, and SARS-CoV-2, demonstrating high specificity.