Dissertation committee:
1. Γαρδέλης Σπυρίδων, Αναπληρωτής Καθηγητής Τμήματος Φυσικής Ε.Κ.Π.Α.
2. Λυκοδήμος Βλάσιος, Αναπληρωτής Καθηγητής Τμήματος Φυσικής Ε.Κ.Π.Α.
3. Παπανικολάου Νικόλαος, Διευθυντής Ερευνών, Ε.Κ.Ε.Φ.Ε. Δημόκριτος
4. Πέτρου Παναγιώτα, Διευθύντρια Ερευνών, Ε.Κ.Ε.Φ.Ε., Δημόκριτος
5. Στεφάνου Νικόλαος, Καθηγητής Τμήματος Φυσικής, Ε.Κ.Π.Α.
6. Σιμσερίδης Κωνσταντίνος , Αναπληρωτής Καθηγητής Τμήματος Φυσικής, Ε.Κ.Π.Α.
7. Κόντος Αθανάσιος, Αναπληρωτής Καθηγητής Σ.Ε.Μ.Φ.Ε., Ε.Μ.Π.
Summary:
Raman spectroscopy and fluorescence imaging are considered reliable methods for evaluating the molecular structure of substances, with a wide range of applications. In recent years, there has been a strong research interest in establishing non-invasive analytical techniques for detecting substances of biological interest in a variety of biomedical applications based on these two methods. However, due to the weak signals provided by these two techniques and the particularly low concentrations limits for these substances in the samples, these methods are very difficult to fulfill the requirements of modern research. The observation of signal enhancement with the use of metal na-noparticles and especially noble metals, such as silver and gold, has spurred further development of spectroscopic methods. The discovery of Surface Enhanced Raman Spectroscopy-SERS as well as Surface Enhanced Fluorescence-SEF, satisfied the need for high signals and allowed detection of very low analyte concentrations, even down to a single molecule (single molecule detection). Concurrently with the discovery of these phenomena, a significant and systematic effort has begun in order to fabricate suitable substrates through a multitude of methods and structures to maximize signal amplifications and achieve the detection of very low concentrations with high repeatability.
The primary goal of this doctoral thesis is the development of sensitive optical biosensors for the immunochemical determination of cancer markers and the direct and indirect determination of oxidative stress markers. The determination of these markers is based on the SERS spectroscopy as well as the fluorescence enhancement through the interaction with the plasmons excited in metallic nanostructures adjacent to the substances under investigation. The key core in this investigation is the fabrication and development of suitable nanostructured silicon/silver active substrates that can provide the plasmonic enhancement increasing the detection sensitivity compared to other detection techniques. The substrate growth method is based on Metal-Assisted Chemical Etching (MACE) technique. Specifically, these substrates consist of silicon nanowires, where silver nanoparticles are grown on their surface in the form of aggregates or dendrites. These active substrates were evaluated initially for standard substances such as rhodamine, crystal violet and finally for the detection of cancer markers and in particular three markers related to gynecological cancers, and especially of ovarian cancer: Cancer antigen 125 (CA-125), Human epididymis secretory protein 4 (HE4), and Survivin or BIRC5. In addition, the same substrates were evaluated for the detection of markers of oxidative stress, specifically the substances glutathione (GSH), malondialdehyde (MDA), catalase (CAT), uric acid (UA), Manganese superoxide dismutase (MnSOD) as well as the protein carbonyls.
From the study with the standard substance rhodamine (R6G), it was observed that both types of active substrates developed showed high detection sensitivity, however, silver aggregates exhibited lower detection limit and significantly better homogeneity than silver dendrites. Therefore, these specific substrates were selected as the most suitable for the detection of biomolecules through SERS experiments. Oxidative stress markers were detected in buffer solutions and artifi-cial saliva. Specifically, for GSH and MDA, measurements were also carried out in natural saliva. Similarly, the detection of cancer markers detection was performed in standard solution prepared in buffer. From the results of the Raman measurements, it emerged that the surfaces with the silver aggregates present high detection sensitivity (Limit of Detection-LOD) and their dynamic range covers the concentration ranges determined in both healthy individuals and patients.
A similar study with the standard substance R6G was carried out on both types of substrates for detection through Photoluminescence (PL). The study revealed that the substrates with the silver dendrites are more suitable for PL experiments for the detection of the above biomolecules compared to those with silver aggregates. Measurements of the cancer markers and the oxidative stress markers GSH, MDA, MnSOD and protein carbonyls, showed that the substrates with the silver dendrites offer a satisfactory sensitivity and dynamic range that covers the concentrations determined in healthy individuals as well as in patients.
Thus, the substrates developed in this thesis are suitable for the development of sensitive optical sensors for the determination of biomarkers through Raman or PL measurements.
Keywords:
Silicon nanowires, metal-assisted chemical etching, silver dendrites, silver aggregates, surface-enhanced Raman spectroscopy, surface-enhanced fluorescence,optical sensors
