Gold nanoparticles (AuNPs) play a vital role in biotechnology, medicine, and diagnostics due to their unique optical properties. Their conjugation with antibodies, antigens, proteins, or nucleic acids enables precise targeting and enhances biosensing capabilities. Functionalized AuNPs, however, may experience reduced stability, leading to aggregation or loss of functionality, especially in complex biological environments. Additionally, they can show non-specific binding to unintended targets, impairing assay specificity. Within this work, citrate-stabilized and silica-coated AuNPs (GNPs and SiGNPs, respectively) have been coated using N,N-dimethylacrylamide-based copolymers to increase their stability and enable their functionalization with biomolecules. AuNP stability after modification has been assessed by a combination of techniques including spectrophotometric characterization, nanoparticle tracking analysis, transmission electron microscopy and functional microarray tests. Two different copolymers were identified to provide a stable coating of AuNPs while enabling further modification through click chemistry reactions, due to the presence of azide groups in the polymers. Following this experimental design, AuNPs decorated with ssDNA and streptavidin were synthesized and successfully used in a biological assay. In conclusion, a functionalization scheme for AuNPs has been developed that offers ease of modification, often requiring single steps and short incubation time. The obtained functionalized AuNPs offer considerable flexibility, as the functionalization protocol can be personalized to match requirements of multiple assays.

Gold nanoparticles (AuNPs) play a vital role in biotechnology, medicine, and diagnostics due to their unique optical properties. Their conjugation with antibodies, antigens, proteins, or nucleic acids enables precise targeting and enhances biosensing capabilities. Functionalized AuNPs, however, may experience reduced stability, leading to aggregation or loss of functionality, especially in complex biological environments. Additionally, they can show non-specific binding to unintended targets impairing assay specificity. Within this work, citrate-stabilized and silica coated AuNPs (GNPs and SiGNPs, respectively) have been coated using N,N-dimethylacrylamide-based copolymers to increase their stability and enable their functionalization with biomolecules. AuNPs stability after modification has been assessed by a combination of techniques including spectrophotometric characterization, nanoparticle tracking analysis, transmission electron microscopy and functional microarray tests. Two different copolymers were identified to provide a stable coating of AuNPs while enabling further modification through click chemistry reactions, due to the presence of azide groups in the polymers. Following this experimental design, AuNPs decorated with ssDNA and streptavidin were synthesized and successfully used in a biological assay. In conclusion, a functionalization scheme for AuNPs has been developed that offers ease of modification, often requiring single steps and short incubation time. The obtained functionalized AuNPs offer huge flexibility, as the functionalization protocol can be personalized to match requirements of multiple assays.

Stochiometric issues, encompassing both the quantity and heterogeneity of extracellular vesicles (EVs) derived from tumor or other tissues in blood, pose important challenges across various stages of biomarker discovery and detection, affecting the integrity of data, introducing losses and artifacts during blood processing, EV purification, and analysis. These challenges shape the diagnostic utility of EVs especially within the framework of established and emerging methodologies. By addressing these challenges, we aim to delineate crucial parameters and requirements for tumor-specific EV detection, or more precisely, for tumor identification via EV based assays. Our endeavor involves a comprehensive examination of the layers that mask or confound the traceability of EV markers such as nucleic acids and proteins, and focus on "Low abundance - low occupancy" scenario. Finally, we evaluate the advantages vs. limitations of digital technologies over more conventional bulk assays, suggesting that the combined use of both to capture and interpret the EV signals, in particular the EV surface displayed proteins, may ultimately provide quantitative information on their absolute abundance and distribution.

Extracellular vesicles (EVs) are membrane-bound vesicles secreted by cells, exhibiting diverse compositions reflective of their cellular origin. With significant potential as biomarkers for liquid biopsies, EV research has led to various isolation techniques. However, a consensus on the optimal strategy remains elusive. Immunoprecipitation, selectively capturing EVs based on surface markers, is promising but hindered by cost, low yields, and potential damage during release. In this study, we propose an innovative Antibody-Aptamer Conjugate: a three-component separation reagent for the separation of EVs. Combining an EV-specific antibody, a streptavidin-binding aptamer, and a unique barcode DNA sequence, this conjugate serves dual roles, facilitating both EV separation and subsequent multiplexed analysis. We detail the development and validation of the Antibody-Aptamer Conjugate, demonstrating its efficacy in isolating intact EVs from complex samples. The unique barcode DNA sequence enables high-throughput analysis on a DNA microarray chip, addressing limitations of existing methodologies. This approach offers a valid and cost-effective alternative for selective EV isolation and analysis, with implications for diagnostic and therapeutic advancements in liquid biopsy applications.