This dissertation primarily focuses on biomimicry, the term coming from the Greek words bios meaning life in Greek and mimesis meaning to imitate, a field that seeks to mimic natural mechanisms, structures, and functions to exploit them into several scientific applications. We have been exploring nature-inspired catecholamine-based biopolymers to straightforwardly develop molecularly imprinted polymers (MIPs), mimetic receptors, for bioanalytical and diagnostics applications. From a more comprehensive standpoint, this dissertation addressed the broad need for simple, cost- effective, and accurate catecholamine-based assays, using animal-free reagents. The general structure of this dissertation is explained herein along with an overview of the research goals. Chapter 1 is devoted to the description of MIPs design and to how they are synthesized. The Chapter gives a brief outline of molecular imprinting technology (MIT) along with recent MIPs synthesis progresses, focusing on the selection of the template molecule, a critical factor to assemble efficient receptor mimics. Chapter 2 deals with catechol-derived biopolymers, chiefly focusing on polydopamine (PDA) and polynorepinephrine (PNE), which are becoming increasingly appreciated as soft, sustainable, versatile, and biocompatible materials able to address challenging tasks. Chapter 3 reports on the use of MIPs for the detection of the small peptide, namely gonadorelin, in biological specimens, i.e., human urines. This is the main purpose of the PhD research study which is part of a larger project entitled “New analytical approaches aimed at tackling doping in sports: development of optical biosensors for the analysis of peptide hormones through molecularly imprinted polymers” funded by the Italian Ministry of Health. First, an optical, label-free, and real time sensing strategy was developed for the detection of gonadorelin using Surface Plasmon Resonance (SPR) transduction. In addition, a portable test was settled, motivated by the lack of decentralized rapid gonadorelin assays for quick decision-making and extensive athlete monitoring before and during competitions. More in detail, a biomimetic enzyme-linked immunosorbent assay (BELISA) was developed onto disposable microplates aiming to cut testing costs and time that are usually required for gonadorelin detection (e.g., mass spectrometry). The detection of gonadorelin through a MIP-based bioanalytical approach has been a very ambitious goal, as no point-of-care devices and only a few monoclonal antibodies are commercialized targeting this analyte. The MIP based approach is able to cheaply measure the drug levels directly from human urine specimens by using a small sample volume (in order of microliters). More specifically, a polynorepinephrine (PNE) based MIP was first designed for targeting gonadorelin, and then it was employed as a receptor element in an SPR-based optical sensing platform. A competitive bioanalytical label-free assay has been built over the MIP for gonadorelin quantification in urine samples. After this, the second task of the project involved the scaling down of the competitive assay, i.e., BELISA, into a portable and simple platform to analyze human urine. The strategy developed was validated by mass spectrometric analysis. Urine samples and LC-MS/MS equipment were available at Pisa University Hospital’s Clinical Pathology Lab, partner of the project. Very soon, the journey across MIPs will continue in the direction of nano- MIPs, which we foresee could be further used as advantageous alternatives to antibodies, both in vitro diagnostics and in vivo therapeutic applications. Chapter 4 is, thus, about the preliminary development of catecholamine- based nanoparticles which will be implemented in the MIPs nanotechnological applications. Chapter 5 describes how catecholamines may be exploited in colorimetric microplate-based bioassays to screen different analytes, in addition to their use as functional monomers in mimetics (MIPs) synthesis. In this case, the redox PDA properties, and the capability to build up coating, non-imprinted material, were exploited for analytical purposes. Two colorimetric tests for molecular diagnostics were developed and applied respectively to analyze serum albumin, a biomarker for kidney function, in human fluids (urine) and to preliminarily screen hypochlorous acid, a key determinant for neurodegenerative disorders. Chapter 6 summarizes the pithiest points of the PhD research studies, discussed in the preceding chapters, and introduces considerations for future work on the topic.

Neurotransmitters-derived biopolymers for future diagnostics and bioanalysis / Francesca Torrini. - (2022).

Neurotransmitters-derived biopolymers for future diagnostics and bioanalysis

Francesca Torrini
2022

Abstract

This dissertation primarily focuses on biomimicry, the term coming from the Greek words bios meaning life in Greek and mimesis meaning to imitate, a field that seeks to mimic natural mechanisms, structures, and functions to exploit them into several scientific applications. We have been exploring nature-inspired catecholamine-based biopolymers to straightforwardly develop molecularly imprinted polymers (MIPs), mimetic receptors, for bioanalytical and diagnostics applications. From a more comprehensive standpoint, this dissertation addressed the broad need for simple, cost- effective, and accurate catecholamine-based assays, using animal-free reagents. The general structure of this dissertation is explained herein along with an overview of the research goals. Chapter 1 is devoted to the description of MIPs design and to how they are synthesized. The Chapter gives a brief outline of molecular imprinting technology (MIT) along with recent MIPs synthesis progresses, focusing on the selection of the template molecule, a critical factor to assemble efficient receptor mimics. Chapter 2 deals with catechol-derived biopolymers, chiefly focusing on polydopamine (PDA) and polynorepinephrine (PNE), which are becoming increasingly appreciated as soft, sustainable, versatile, and biocompatible materials able to address challenging tasks. Chapter 3 reports on the use of MIPs for the detection of the small peptide, namely gonadorelin, in biological specimens, i.e., human urines. This is the main purpose of the PhD research study which is part of a larger project entitled “New analytical approaches aimed at tackling doping in sports: development of optical biosensors for the analysis of peptide hormones through molecularly imprinted polymers” funded by the Italian Ministry of Health. First, an optical, label-free, and real time sensing strategy was developed for the detection of gonadorelin using Surface Plasmon Resonance (SPR) transduction. In addition, a portable test was settled, motivated by the lack of decentralized rapid gonadorelin assays for quick decision-making and extensive athlete monitoring before and during competitions. More in detail, a biomimetic enzyme-linked immunosorbent assay (BELISA) was developed onto disposable microplates aiming to cut testing costs and time that are usually required for gonadorelin detection (e.g., mass spectrometry). The detection of gonadorelin through a MIP-based bioanalytical approach has been a very ambitious goal, as no point-of-care devices and only a few monoclonal antibodies are commercialized targeting this analyte. The MIP based approach is able to cheaply measure the drug levels directly from human urine specimens by using a small sample volume (in order of microliters). More specifically, a polynorepinephrine (PNE) based MIP was first designed for targeting gonadorelin, and then it was employed as a receptor element in an SPR-based optical sensing platform. A competitive bioanalytical label-free assay has been built over the MIP for gonadorelin quantification in urine samples. After this, the second task of the project involved the scaling down of the competitive assay, i.e., BELISA, into a portable and simple platform to analyze human urine. The strategy developed was validated by mass spectrometric analysis. Urine samples and LC-MS/MS equipment were available at Pisa University Hospital’s Clinical Pathology Lab, partner of the project. Very soon, the journey across MIPs will continue in the direction of nano- MIPs, which we foresee could be further used as advantageous alternatives to antibodies, both in vitro diagnostics and in vivo therapeutic applications. Chapter 4 is, thus, about the preliminary development of catecholamine- based nanoparticles which will be implemented in the MIPs nanotechnological applications. Chapter 5 describes how catecholamines may be exploited in colorimetric microplate-based bioassays to screen different analytes, in addition to their use as functional monomers in mimetics (MIPs) synthesis. In this case, the redox PDA properties, and the capability to build up coating, non-imprinted material, were exploited for analytical purposes. Two colorimetric tests for molecular diagnostics were developed and applied respectively to analyze serum albumin, a biomarker for kidney function, in human fluids (urine) and to preliminarily screen hypochlorous acid, a key determinant for neurodegenerative disorders. Chapter 6 summarizes the pithiest points of the PhD research studies, discussed in the preceding chapters, and introduces considerations for future work on the topic.
Maria Minunni, Simona Scarano
Francesca Torrini
File in questo prodotto:
File Dimensione Formato  
Torrini Francesca PhDTesi.pdf

embargo fino al 01/03/2023

Descrizione: Tesi di dottorato
Tipologia: Tesi di dottorato
Licenza: DRM non definito
Dimensione 8.05 MB
Formato Adobe PDF
8.05 MB Adobe PDF   Visualizza/Apri   Richiedi una copia

I documenti in FLORE sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2158/1259939
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact