La spettroscopia Raman Surface Enhanced è una potente tecnica analitica, in cui sono combinati l'elevata sensibilità e il riconoscimento delle specie chimiche, forniti rispettivamente dall'aumento del segnale di superficie e dal fingerprinting vibrazionale della spettroscopia Raman. Pertanto, lo scattering anelastico (o Raman scattering) della luce viene utilizzato per catturare le cosiddette vibrazioni attive Raman in presenza di una fonte di luce esterna. Sulla base di questo fenomeno, una vibrazione unica può consentire di identificare con elevata selettività un numero enorme di strutture chimiche. Ma la dispersione Raman è di per sé un evento raro, che si verifica solo quando si ottengono determinate circostanze. Nella spettroscopia SERS gli elettroni conduttivi oscillanti coerenti sulla superficie del metallo vengono utilizzati per amplificare l'energia vibrazionale mediante la produzione di un campo elettromagnetico molto confinato su scala nanometrica, fornendo un segnale di esito fino a 10 volte più forte di quello iniziale. Ci riferiamo a questo effetto come risonanza plasmonica di superficie localizzata LSPR, che consente a specifiche superfici nanostrutturate di raggiungere anche la risoluzione molecolare. SERS è anche una tecnica veloce e facile da usare, in cui queste caratteristiche, unite al progresso nell'ottica e nella fotonica (oggi è possibile acquistare strumentazione da banco, potabile e palmare) hanno creato un campo fertile per la diffusione delle applicazioni SERS. Tuttavia, superfici plasmoniche, o più propriamente dette substrati SERS, possono essere ottenute con una semplice procedura, ad esempio ricoprendo sulla carta nanoparticelle di argento, ottenute con riduzione di sale in acqua. Alla fine, la spettroscopia SERS è una tecnica non distruttiva che consente la misurazione in mezzi acquosi. Per tutte queste caratteristiche è stato introdotto SERS, come uno strumento promettente nella medicina di precisione e nell'analisi dei biofluidi. In questo contesto, lo scopo della mia tesi è stato quello di trovare un metodo semplice ed economico per estrarre informazioni biochimiche da matrici biologiche complesse.

Surface Enhanced Raman spectroscopy is a powerful analytical technique, in which are combined high sensitivity and chemical species recognition, which are provided by the surface signal enhancement and the vibrational fingerprinting of the Raman spectroscopy, respectively. Thus, the inelastic scattering (or Raman scattering) of the light is used to capture so-called Raman active vibrations in presence of an external source of light. On the base of this phenomenon, unique vibration can allow identifying with high selectivity a tremendous number of chemical structures. But, Raman scattering is di per se a rare event, that occurs only when certain circumstances are achieved. In SERS spectroscopy coherent oscillating conductive electrons on the metal surface are used to amplify the vibrational energy by the production of a very confined electromagnetic field at the nanoscale, providing an outcome signal up to 10 times stronger than the initial one. We refer to this effect as localized surface plasmon resonance LSPR, which allows specific nanostructured surfaces to reach molecular resolution too. SERS is also a fast, user-friendly technique, in which these features coupled with the advancement in optic and photonics (bench, potable and palmar instrumentation can be bought nowadays) have created a fertile field for the spreading of SERS applications. Nevertheless, plasmonic surfaces, or more properly called SERS substrates, can be obtained with a simple procedure, e.g by coating on the paper silver nanoparticles, obtained with salt reduction in water. Eventually, SERS spectroscopy is a non-destructive technique allowing measurement in aqueous media. For all these features SERS has been introduced, as a promising tool in precision medicine and biofluid analysis. In this frame, the scope of my thesis has been to find a simple and inexpensive method to extract biochemical information from complex biological matrixes. The simplest way to do SERS biosensing is to use the Label-free SERS approach. Thus, it is possible, accordingly to the sample compositions and affinity for the metal surface, to identify small molecules and more specifically metabolites. This provides a snapshot of a pool of metabolites of which the variation can be used to extract information on the state of illness of patients. For instance, many studies of label-free SERS ha reached interesting results such as in monitoring the therapeutic window and in the classification of patients' state of illness on the base of the relative concentration of small metabolites. In this frame I have developed for my PhD project, a protocol to extract biochemical information from stool samples that were adopted to study for the first time stool samples of the celiac patient (celiac disease can be defined as an uncontrolled immunogenic response to the gluten ingestion), with promising results in assessing the compliance of gluten-free diet patient. On the other hand, we have developed a fast SERS sampling protocol for human serum on the paper that allow achieving reproducible SERS spectra via the centrifugation of few µl of samples spotted on the paper in the presence of silver colloidal Nanoparticles. Thus, it was possible to obtain a fast and reliable SERS protocol to be adopted for sampling and SERS bioanalytic.

Sviluppo e caratterizzazione di substrati SERS label-free per analisi di biofluidi e applicazioni biomediche

ESPOSITO, ALESSANDRO
2022

Abstract

Surface Enhanced Raman spectroscopy is a powerful analytical technique, in which are combined high sensitivity and chemical species recognition, which are provided by the surface signal enhancement and the vibrational fingerprinting of the Raman spectroscopy, respectively. Thus, the inelastic scattering (or Raman scattering) of the light is used to capture so-called Raman active vibrations in presence of an external source of light. On the base of this phenomenon, unique vibration can allow identifying with high selectivity a tremendous number of chemical structures. But, Raman scattering is di per se a rare event, that occurs only when certain circumstances are achieved. In SERS spectroscopy coherent oscillating conductive electrons on the metal surface are used to amplify the vibrational energy by the production of a very confined electromagnetic field at the nanoscale, providing an outcome signal up to 10 times stronger than the initial one. We refer to this effect as localized surface plasmon resonance LSPR, which allows specific nanostructured surfaces to reach molecular resolution too. SERS is also a fast, user-friendly technique, in which these features coupled with the advancement in optic and photonics (bench, potable and palmar instrumentation can be bought nowadays) have created a fertile field for the spreading of SERS applications. Nevertheless, plasmonic surfaces, or more properly called SERS substrates, can be obtained with a simple procedure, e.g by coating on the paper silver nanoparticles, obtained with salt reduction in water. Eventually, SERS spectroscopy is a non-destructive technique allowing measurement in aqueous media. For all these features SERS has been introduced, as a promising tool in precision medicine and biofluid analysis. In this frame, the scope of my thesis has been to find a simple and inexpensive method to extract biochemical information from complex biological matrixes. The simplest way to do SERS biosensing is to use the Label-free SERS approach. Thus, it is possible, accordingly to the sample compositions and affinity for the metal surface, to identify small molecules and more specifically metabolites. This provides a snapshot of a pool of metabolites of which the variation can be used to extract information on the state of illness of patients. For instance, many studies of label-free SERS ha reached interesting results such as in monitoring the therapeutic window and in the classification of patients' state of illness on the base of the relative concentration of small metabolites. In this frame I have developed for my PhD project, a protocol to extract biochemical information from stool samples that were adopted to study for the first time stool samples of the celiac patient (celiac disease can be defined as an uncontrolled immunogenic response to the gluten ingestion), with promising results in assessing the compliance of gluten-free diet patient. On the other hand, we have developed a fast SERS sampling protocol for human serum on the paper that allow achieving reproducible SERS spectra via the centrifugation of few µl of samples spotted on the paper in the presence of silver colloidal Nanoparticles. Thus, it was possible to obtain a fast and reliable SERS protocol to be adopted for sampling and SERS bioanalytic.
BONIFACIO, ALOIS
34
2020/2021
Settore CHIM/07 - Fondamenti Chimici delle Tecnologie
Università degli Studi di Trieste
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/3030740
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