Development of electrochemical biosensors for detection of some inflammatory biomarkers related to cardiovascular diseases

Cardiovascular diseases (CVDs) are the leading cause of mortality and morbidity worldwide. Early detection of CVDs is of vital importance, as timely diagnosis can significantly improve clinical outcomes. A growing body of evidence indicates that inflammation plays a significant role in the pathogenesis of many CVDs, including atherosclerosis, hearth failure, myocardial infarction, and pericardial diseases. The most widely accepted strategy for detecting inflammation in CVDs involves measuring circulating biomarkers of inflammation. Growth Differentiation Factor-15 (GDF-15) and Galectin-3 (Gal-3) are new inflammation-related biomarkers that are increasingly valuable in the early diagnosis and follow-up of CVDs. In this thesis, two electrochemical immunobiosensors were developed for the detection of GDF-15 and Gal-3. A solid gold electrode (SGE) was used as the working electrode for the GDF-15 biosensor, while disposable indium-tin oxide-coated polyethylene terephthalate (ITO/PET) sheets were utilised for the Gal-3 biosensor. In the design of biosensors, a similar and step-efficient surface modification strategy was employed. For this purpose, surfaces of the working electrodes in both biosensors were hydroxylated using different methods; self-assembled monolayers (SAMs) were formed on the SGE surface with 6-mercaptohexanol molecules, while active hydroxyl groups were formed on the surface of ITO/PET electrodes by chemical hydroxylation. Epoxy silane SAMs were formed on the hydroxylated electrode surfaces using the same silanization agent, trimethoxy[2-(7-oxabicyclo[4.1.0]hept-3-yl)ethyl]silane (TMOHES), but at different temperatures. Then, antibodies specific to GDF-15 and Gal-3 biomarkers were immobilized onto the modified KAE and ITO/PET electrode surfaces, respectively. Electrochemical impedance spectroscopy and cyclic voltammetry techniques were used to evaluate the analytical performance of the developed biosensors, and to monitor the design, optimization and characterization studies. The surface modifications were verified by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX) and Fourier-transform infrared spectroscopy (FTIR). Under optimised conditions, the GDF-15 biosensor exhibited a linear range of 125–1500 pg mL⁻¹ (R² = 0.998) with a LOD of 83.8 pg mL⁻¹ and a LOQ of 254.2 pg mL⁻¹. The Gal-3 biosensor showed a linear range of 2.5–40 ng mL⁻¹ (R² = 0.996) a LOD of 0.73 ng mL⁻¹ and a LOQ.of 2.43ng pg mL⁻¹, demonstrating high linearity and low detection limits. Repeatability (n=10) and reproducibility studies were conducted for both of the sensors and interference effects of various proteins were evaluated. Gal-3 and GDF-15 analyses were performed in commercial human serum to assess matrix effects and the potential applicability of the sensors to real clinical samples. For the Gal-3 biosensor regeneration, inter-user reproducibility and storage stabilityanalyses were carried out. Single-frequency impedance (SFI) measurements were performed for the GDF-15 biosensor, and the electro-active surface area of the sensor was calculated using CV. The findings demonstrate that these two label‑free impedimetric immunosensors, developed through a simple yet effective surface modification strategy, are suitable for the sensitive and rapid detection of GDF‑15 and Gal‑3 with the advantages such as low sample volume requirement, high repeatability, reproducibility, sensitivity, and selectivity. The Gal‑3 biosensor, in particular, stands out as a cost‑effective, disposable platform with reusability potential due to its regeneration capability. Consequently, the developed biosensors hold promise for contributing to inflammation‑focused biomarker assessment and to the risk stratification and management of CVD in research and clinical practice.