Biomedical applications of Surface Enhanced Raman Spectroscopy - a step forward to clinical practice

Lo scopo di questo progetto di dottorato \ue8 quello di utilizzare delle superfici metalliche nanostrutturate come substrati per la spettroscopia Raman amplificata da superfici (SERS) per l\u2019analisi di biofluidi. Questa tecnica analitica restituisce l\u2019impronta digitale vibrazionale del campione grazie alla presenza della nanostruttura metallica. Queste caratteristiche anticipano le potenzialit\ue0 della spettroscopia SERS in campo bioanalitico che ha visto un aumento esponenziale delle sue applicazioni nell\u2019ultimo decennio. In particolare, la SERS richiede la fabbricazione di substrati metallici nanostrutturati che possano funzionare da sensori. Questo progetto si basa sullo sviluppo di un approccio privo di marcatura (label-free:): nessuna funzionalizzazione \ue8 presente sulla superficie metallica al fine di rilevare in modo aspecifico gli analiti presenti della matrice di interesse biologico. Il risultato del segnale SERS sar\ue0 un\u2019istantanea della soluzione in analisi depositata sulla superficie metallica, cio\ue8 l\u2019impronta specifica del campione. Per esempio, l\u2019analisi label-free dei biofluidi riflette il suo contenuto metabolico. Nell\u2019era \u201comica\u201d, il SERS pu\uf2 essere integrato nella metabolomica non funzionalizzata in quanto fornisce il profilo metabolico del soggetto in esame e di conseguenza distinguere campioni diversi basandosi sulle differenze di ogni profilo analizzato. I colloidi stabilizzati elettrostaticamente sono stati scelti per la loro nota compatibilit\ue0 con i biofluidi. Verranno usati sia in forma colloidale in sospensione acquosa, sia fissati su un supporto di carta, definiti supporti solidi e sviluppati grazie a un protocollo validato nel nostro laboratorio. Il vantaggio portato dai supporti in carta risiede nella stabilit\ue0 della risposta spettroscopica: sono di lunga durata, facili da fabbricare e da maneggiare, economici e veloci, potenzialmente fabbricabili su ampia scala. Queste sono le caratteristiche che nell\u2019ambito delle applicazioni del SERS possono promuovere la costruzione di un dispositivo Point of Care. Basandosi sulle competenze acquisite dal nostro gruppo di ricerca, lo scopo di questa tesi di dottorato \ue8 duplice: aumentare le nostre conoscenze sull\u2019interazione biofluidi-nanostrutture e utilizzare il metodo SERS per lo studio di specifici problemi clinici. Al fine di soddisfare tali richieste questo lavoro \ue8 diviso in tre parti: 1. Sviluppare protocolli per l\u2019analisi label-free delle frazioni di sangue (siero, plasma, eritrociti, cellule mononucleate del sangue periferico, e sangue intero) con il SERS, sfruttando le loro caratteristiche in base alla diversa preparazione dei campioni e ai substrati SERS utilizzati; 2. Caratterizzare il comportamento delle biomolecole sulla superficie di nanoparticelle metalliche su sistemi modello, cio\ue8 capire il ruolo delle corone di proteine e non proteine nell\u2019interazione metabolita-nanoparticelle. Il sistema modello usato si basa su un insieme di albumina di siero umano (la pi\uf9 abbondante proteina del siero) e molecole che sono comunemente osservate nei biofluidi: adenina, ipoxantina e acido urico; 3. Applicare le nozioni di cui sopra per la diagnosi precoce di diverse malattie (tumore al seno, fegato grasso non alcolico, cirrosi e carcinoma epatocellulare) tramite campioni di sangue e plasma e l\u2019uso di analisi dati multivariata per spettri SERS. Lo scopo dell\u2019utilizzo del SERS in ambito medico \ue8 di proporre nuovi approcci diagnostici complementari alle tecniche gi\ue0 in uso in clinica come ad esempio i metodi di immunochimica e istopatologia. Il vantaggio del SERS risiede nella rapida risposta e in un approccio non invasivo tramite l\u2019utilizzo di biopsia liquida. Lo scopo futuro \ue8 lo sviluppo di una piattaforma SERS label-free come dispositivo point of care integrato allo strumento RamanThis PhD project aims to apply nanostructured metal surfaces as substrates for Surface Enhanced Raman Spectroscopy for the study of biofluids. This analytical technique provides the vibrational fingerprint of a sample assisted by nanostructured metal surfaces, which can enhance the scattering signal of analytes adsorbed on them: this allows detection of analytes in very low concentrations. These features tell a lot about the potential of SERS in the bioanalytics, and indeed, in this field, the use of SERS has increased over the past decade taking advantage of both sensitive detection and fingerprinting features. Above all, SERS requires the manufacturing of metal nanostructured substrates as sensors. In particular, this project is based on the development of a label-free approach: no functionalization is present on the nanoparticles surface, and, hence, no preferential affinity for a given analyte in the biological matrix is sought. Briefly, once nanoparticles are in contact with the specimen, the analytes may adsorb on them without any specific interaction other than their affinity for the metal. The outcoming SERS signal will be a snapshot of what actually reached the metal surface, namely a fingerprint of the sample. For instance, the label-free analysis of biofluids reflect the metabolic content of the fluid itself. In the \u201comic\u201d era, SERS can integrate with untargeted metabolomics and provide the metabolic profile of a specimen and distinguish different samples accordingly, based on differences in such profiles. Silver colloids have been chosen, given that their performances with biofluids are known. They have been used both as colloidal suspension in water, and fixed on a paper support, according to an in-house developed protocol for the fabrication of solid substrates. The coupling of metal nanostructures substrates with SERS acts as actual sensors, able to interact with aqueous environment and detect dissolved analytes. The real advantage of the paper supports lay in the stability of the spectroscopic response: they are long lasting, easy to fabricate and to handle, cost and time effective, prone to scale up. These reasons make them potential Point of Care tool in the frame of SERS applications. The aim of this PhD thesis is twofold: to push forward our fundamental knowledge of the nanostructure-biofluid interaction and to test the feasibility of the application of SERS for specific clinical problems. These goals were pursued in three steps: 1. to develop protocols for the label-free analysis of blood fractions (serum, plasma, erythrocytes, periphereal blood mononuclear cells, and whole blood) with SERS, exploiting their features according to several treatments and SERS substrates; 2. to characterize the behaviour of biomolecules at the interface with metal nanoparticles on model systems, namely to understand the role of the protein and non-protein corona in the metabolites-nanoparticle interaction. The model system used is based on mixture of human serum albumin (i.e. the most abundant serum protein) and molecules which are commonly detected in SERS of biofluids: adenine, hypoxanthine and uric acid; 3. to apply the aforementioned knowledge to the early diagnosis of several diseases (breast cancer, non-alcoholic fatty liver diseases, cirrhosis and hepatocellular carcinoma) through serum and plasma samples by means of multivariate data analysis of SERS spectra. Considering the latter application of SERS in the field of disease diagnosis, the aim is to provide new diagnostic methods complementary to the accepted gold standards such as immunochemistry and histopathology methods. The advantages of SERS lay on the rapid response and on the non-invasiveness of the liquid biopsy approach. As a future goal, the development of SERS platforms as label-free point of care tools integrated to portable Raman instruments could bring the diagnosi

Long-term stability of an injection-molded zirconia bone-level implant: A testing protocol considering aging kinetics and dynamic fatigue

Abstract Objective Separately addressing the fatigue resistance (ISO 14801, evaluation of final product) and aging behavior (ISO 13356, standardized sample) of oral implants made from yttria-stabilized zirconia proved to be insufficient in verifying their long-term stability, since (1) implant processing is known to significantly influence transformation kinetics and (2) aging, up from a certain level, is liable to decrease fatigue resistance. Therefore, the aim of this investigation was to apply a new testing protocol considering environmental conditions adequately inducing aging during dynamic fatigue. Methods Zirconia implants were dynamically loaded (107 cycles), hydrothermally aged (85\ub0, 60 days) or subjected to both treatments simultaneously. Subsequent, monoclinic intensity ratios (Xm) were obtained by locally resolved X-ray microdiffraction (\u3bc-XRD2). Transformation propagation was monitored at cross-sections by \u3bc-Raman spectroscopy and scanning electron microscopy (SEM). Finally, implants were statically loaded to fracture. Linear regression models (fracture load) and mixed models (Xm) were used for statistical analyses. Results All treatments resulted in increased fracture load (p 64 0.005), indicating the formation of transformation induced compressive stresses around surface defects during all treatment modalities. However, only hydrothermal and combinational treatment were found to increase Xm (p < 0.001). No change in Xm was observed for solely dynamically loaded samples (p 65 0.524). Depending on the variable observed, a monoclinic layer thickness of 1\u20132 \u3bcm (SEM) or 6\u20138 \u3bcm (Raman spectroscopy) was measured at surfaces exposed to water during treatments. Significance Hydrothermal aging was successfully induced during dynamic fatigue. Therefore, the presented setup might serve as reference protocol for ensuring pre-clinically long-term reliability of zirconia oral implants

Ergothioneine, a dietary amino acid with a high relevance for the interpretation of label-free surface enhanced Raman scattering (SERS) spectra of many biological samples

Intense SERS spectra of the natural amino acid ergothioneine (ERG) are obtained on different substrates upon 785 nm excitation. A characteristic spectral pattern with a distinctive intense band at 480\u2013486 cm 121 is conserved when substrates of different type and characteristics are used. On the basis of available literature, we propose ERG is adsorbed on the metal surface in its thiolate form via the sulphur and heterocyclic nitrogen. The same spectral pattern is obtained in SERS spectra of filtered erythrocytes lysates, confirming the presence of ERG in those cells. The occurrence of ERG bands in label-free SERS spectra of serum and plasma reported in literature by different authors is discussed, highlighting the importance of this amino acid for the interpretation of SERS spectra of these biofluids

Núcleos de Ensino da Unesp: artigos 2008

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq