Multiple sclerosis (MS) is a chronic progressive inflammatory demyelinating disorder of the central nervous system. Although the aetiology of MS is not fully understood yet, it is generally defined to be an autoimmune disease, where both genetic and environmental factors play a key role. The identification of reliable biomarkers and the development of assays for their quantification is currently an unmet need for the early and accurate diagnosis of MS. Recently, electronic-based immunosensors, such as electrolyte-gated organic transistors (EGOTs), have emerged as a promising alternative platform for the ultra-sensitive and label-free detection of biological analytes. The intrinsic characteristics of these devices, such as high amplification or low cost of production, place them as the ideal candidates for point-of-care applications. The aim of this thesis was to develop novel immunosensor, exploiting different EGOT architectures for the detection of biomarkers of MS. In particular, two candidate MS biomarkers were investigated: neurofilament light chain (NF-L) and mitochondrial DNA (mtDNA). The EGOTs were endowed with biorecognition capability by functionalizing the gold gate electrode. Several functionalization strategies were investigated, i.e., the immobilization of the biorecognition element via cys-tagged protein-G, or through the formation of a self-assembled monolayer, exploring the biotin-avidin interaction. The functionalization strategies were further validated by cyclic voltammetry, surface plasmon resonance (SPR), fiber optic SPR, and surface plasmon-enhanced fluorescence. In the first part of this thesis, an EGOT-based biosensor was developed for the detection of NF-L. The biosensor demonstrated to selectively detect NF-L in a buffered solution, with an estimated limit of detection of 30 fM. In the second part of the thesis, an EGOT-based biosensor was developed to monitor the hybridization of mtDNA, proving to recognize the complementary mtDNA strand in a wide dynamic range of concentrations, and successfully discriminate it from a non-complementary sequence. The last part of the thesis was focused on the development of EGOT-based biosensors for the detection of NF-L in plasma. The developed biosensor presented optimal operability, even in such a complex medium as plasma, and proved to selectively discriminate NF-L from other proteins present in plasma. The rapid, selective, reproducible, and label-free response provided by the EGOT-based biosensors indicates their potential as a promising alternative strategy for the detection of MS biomarkers.

La sclerosi multipla (SM) è una malattia infiammatoria demielinizzante cronica progressiva del sistema nervoso centrale. La causa di questa patologia non è ancora chiara, anche se si presume un’origine autoimmune, dove la genetica e i fattori ambientali giocano un ruolo importante. Attualmente, l’identificazione di biomarcatori attendibili e lo sviluppo di tecniche per la loro quantificazione è di urgente bisogno per una pronta e accurata diagnosi della SM. Gli immunosensori basati su elettronica organica, come i transistor organici modulati mediante elettrolita (EGOT), hanno recentemente preso piede come un’alternativa promettente per la rilevazione ultra-sensibile e label-free di analiti biologici. Le caratteristiche intrinseche di questi dispositivi, come alta amplificazione, biocompatibilità, e bassi costi di produzione, li rendono candidati ideali per applicazioni cliniche. Lo scopo di questa tesi è lo sviluppo di immunosensori, basati su un’architettura EGOT, per la rilevazione di biomarcatori promettenti per la SM, come il neurofilamento light chain (NF-L) e il DNA mitocondriale (mtDNA). Con lo scopo di rendere capace l’EGOT di riconoscere selettivamente il target analitico, l’elettrodo gate di oro è stato usato come elemento sensibile. Diverse strategie fi funzionalizzazione sono state studiate, come l’immobilizzazione d’anticorpi specifici con orientamento controllato attraverso la proteina-G, o l’immobilizzazione sfruttando l’interazione biotina-avidina. Le strategie di funzionalizzazione sono state ottimizzate e validate mediante tecniche elettrochimiche e spettroscopiche. Nella prima parte della tesi, è stato sviluppato un biosensore EGOT per la rilevazione di NF-L. Il biosensore ha dimostrato di rilevare selettivamente NF-L, anche in presenza di molecole potenzialmente interferenti, con un limite di rilevazione stimato di 30 fM. In aggiunta, un secondo biosensore EGOT è stato sviluppato per monitorare l’ibridizzazione dal mtDNA, dimostrando di riconoscere il filamento di mtDNA complementare presente nella soluzione in un ampio intervallo di concentrazioni, discriminandolo tra filamenti non complementari. L’ultima parte della tesi è basata nello sviluppo di biosensori EGOT per la rivelazione di NF-L in plasma. Il biosensore ha mostrato una ottima operabilità nel plasma e ha dimostrato di discriminare selettivamente molecole di NF-L tra altre proteine presenti nella soluzione. In conclusione, la risposta rapida, selettiva, riproducibile, e label-free, dei biosensori EGOFET, indica il potenziale di questi dispositivi come una strategia alternativa promettente per la rivelazione di biomarcatori di SM.

Biosensori a base EGOT per la Rilevazione di Biomarcatori della Sclerosi Multipla in Campioni di Plasma / Kateryna Solodka , 2023 May 19. 35. ciclo, Anno Accademico 2021/2022.

Biosensori a base EGOT per la Rilevazione di Biomarcatori della Sclerosi Multipla in Campioni di Plasma

SOLODKA, KATERYNA
2023

Abstract

Multiple sclerosis (MS) is a chronic progressive inflammatory demyelinating disorder of the central nervous system. Although the aetiology of MS is not fully understood yet, it is generally defined to be an autoimmune disease, where both genetic and environmental factors play a key role. The identification of reliable biomarkers and the development of assays for their quantification is currently an unmet need for the early and accurate diagnosis of MS. Recently, electronic-based immunosensors, such as electrolyte-gated organic transistors (EGOTs), have emerged as a promising alternative platform for the ultra-sensitive and label-free detection of biological analytes. The intrinsic characteristics of these devices, such as high amplification or low cost of production, place them as the ideal candidates for point-of-care applications. The aim of this thesis was to develop novel immunosensor, exploiting different EGOT architectures for the detection of biomarkers of MS. In particular, two candidate MS biomarkers were investigated: neurofilament light chain (NF-L) and mitochondrial DNA (mtDNA). The EGOTs were endowed with biorecognition capability by functionalizing the gold gate electrode. Several functionalization strategies were investigated, i.e., the immobilization of the biorecognition element via cys-tagged protein-G, or through the formation of a self-assembled monolayer, exploring the biotin-avidin interaction. The functionalization strategies were further validated by cyclic voltammetry, surface plasmon resonance (SPR), fiber optic SPR, and surface plasmon-enhanced fluorescence. In the first part of this thesis, an EGOT-based biosensor was developed for the detection of NF-L. The biosensor demonstrated to selectively detect NF-L in a buffered solution, with an estimated limit of detection of 30 fM. In the second part of the thesis, an EGOT-based biosensor was developed to monitor the hybridization of mtDNA, proving to recognize the complementary mtDNA strand in a wide dynamic range of concentrations, and successfully discriminate it from a non-complementary sequence. The last part of the thesis was focused on the development of EGOT-based biosensors for the detection of NF-L in plasma. The developed biosensor presented optimal operability, even in such a complex medium as plasma, and proved to selectively discriminate NF-L from other proteins present in plasma. The rapid, selective, reproducible, and label-free response provided by the EGOT-based biosensors indicates their potential as a promising alternative strategy for the detection of MS biomarkers.
Electrolyte-Gated Organic Transistor-based Biosensors for Detection of Multiple Sclerosis Biomarkers in Plasma
19-mag-2023
PINTI, Marcello
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/1305588
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