Several application of protein microarray technology in diagnostics have been published and a limited number of protein microarrays is currently available on the In Vitro Diagnostics (IVD) market. Albeit several advantages, related to the miniaturization, the multiplexing capability and the possibility of integrating the immunoassays in biosensing devices, microarrays may still lack of specificity or sensitivity. To overcome these limitations and expand the use of protein microarray platform in diagnostics, the present PhD research aimed at developing innovative approaches to increase the assay specificity and sensitivity, reaching very low detection limits, that are compatible with the use of the proposed devices in diagnostics. Furthermore, the use of protein microarrays has been applied to the characterization of emerging biomarkers: exosomes. First of all, surface immobilized hydrogels have been investigated as reagent reservoir for microarray reagents. They have been demonstrated to store reagents in a dry form, stable over days, in a format easy to transport and to preserve. Moreover, they also acted as chambers able to physically separate analytes or reagents which may cross-react with proteins on the printed arrays. In this way the solution was prevented from spreading over the surface and the assays provided sentitive performances, comparable to standard static incubations. In further studies, the complementarity of information provided by fluorescence-based, label-free IRIS and SP-IRIS microarray platforms has been applied to develop immunoassays useful in the diagnostics of Neurodegenerative Disorders. Specifically, two different assay formats have been exploited. The first part of the work focused on the development of a classical sandwich immunoassay able to detect physiological concentrations of Amyloid-beta peptides, biomarkers for Alzheimer’s disease, in both artificial cerebrospinal fluid and real human samples. The second study was aimed at extending the concept of protein microarrays to extracellular vesicles (i.e., exosomes) detection through surface antigen-antibodies recognition. In this innovative application, the nanoparticles were detected with label-free IRIS (total biomass measurements) and SP-IRIS (particle counting and size distribution). In addition, individual particles were incubated with gold-labeled antibodies to identify biomarkers expressed on their surface.

DEVELOPMENT OF NOVEL HIGH PERFORMANCE PROTEIN MICROARRAYS FOR DIAGNOSTIC APPLICATIONS / P. Gagni ; tutor: C. De Micheli ; co-tutor: M. Chiari. - : . Università degli Studi di Milano, 2015 Jan 15. ((27. ciclo, Anno Accademico 2014. [10.13130/p-gagni_phd2015-01-15].

DEVELOPMENT OF NOVEL HIGH PERFORMANCE PROTEIN MICROARRAYS FOR DIAGNOSTIC APPLICATIONS

P. Gagni
2015

Abstract

Several application of protein microarray technology in diagnostics have been published and a limited number of protein microarrays is currently available on the In Vitro Diagnostics (IVD) market. Albeit several advantages, related to the miniaturization, the multiplexing capability and the possibility of integrating the immunoassays in biosensing devices, microarrays may still lack of specificity or sensitivity. To overcome these limitations and expand the use of protein microarray platform in diagnostics, the present PhD research aimed at developing innovative approaches to increase the assay specificity and sensitivity, reaching very low detection limits, that are compatible with the use of the proposed devices in diagnostics. Furthermore, the use of protein microarrays has been applied to the characterization of emerging biomarkers: exosomes. First of all, surface immobilized hydrogels have been investigated as reagent reservoir for microarray reagents. They have been demonstrated to store reagents in a dry form, stable over days, in a format easy to transport and to preserve. Moreover, they also acted as chambers able to physically separate analytes or reagents which may cross-react with proteins on the printed arrays. In this way the solution was prevented from spreading over the surface and the assays provided sentitive performances, comparable to standard static incubations. In further studies, the complementarity of information provided by fluorescence-based, label-free IRIS and SP-IRIS microarray platforms has been applied to develop immunoassays useful in the diagnostics of Neurodegenerative Disorders. Specifically, two different assay formats have been exploited. The first part of the work focused on the development of a classical sandwich immunoassay able to detect physiological concentrations of Amyloid-beta peptides, biomarkers for Alzheimer’s disease, in both artificial cerebrospinal fluid and real human samples. The second study was aimed at extending the concept of protein microarrays to extracellular vesicles (i.e., exosomes) detection through surface antigen-antibodies recognition. In this innovative application, the nanoparticles were detected with label-free IRIS (total biomass measurements) and SP-IRIS (particle counting and size distribution). In addition, individual particles were incubated with gold-labeled antibodies to identify biomarkers expressed on their surface.
DE MICHELI, CARLO
protein microarrays; immunoassays; specificity; sensitivity; hydrogel plugs; Amyloid-beta; Exosomes; Neurodegenerative Disorders; fluorescence-based detection; label-free detection; Interferometric Reflectance Imaging Sensor; Single Particle detection.
Settore CHIM/08 - Chimica Farmaceutica
DEVELOPMENT OF NOVEL HIGH PERFORMANCE PROTEIN MICROARRAYS FOR DIAGNOSTIC APPLICATIONS / P. Gagni ; tutor: C. De Micheli ; co-tutor: M. Chiari. - : . Università degli Studi di Milano, 2015 Jan 15. ((27. ciclo, Anno Accademico 2014. [10.13130/p-gagni_phd2015-01-15].
Doctoral Thesis
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Descrizione: Tesi completa di Dottorato di Ricerca in Chimica del Farmaco, XXVII ciclo, Gagni Paola, matricola R09804
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/249493
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