Author(s): Dario Brambilla, Federica Panico, Lorenzo Zarini, Alessandro Mussida, Anna M. Ferretti, Mete Aslan, Selim Ünlü and Marcella Chiari

Biosensors

2024, 14(7), 319

https://doi.org/10.3390/bios14070319

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.

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Author(s): Laura Sola, Laura Abdel Mallak, Francesco Damin, Alessandro Mussida, Dario Brambilla and Marcella Chiari

Micromachines 14(2), 302

https://doi.org/10.3390/mi14020302

We report here a deep investigation into the effect of the concentration of a polymeric coating’s functional groups on probe density immobilization with the aim of establishing the optimal formulation to be implemented in specific microarray applications. It is widely known that the ideal performance of a microarray strictly depends on the way probes are tethered to the surface since it influences the way they interact with the complementary target. The N, N-dimethylacrylamide-based polymeric coating introduced by our research group in 2004 has already proven to offer great flexibility for the customization of surface properties; here, we demonstrate that it also represents the perfect scaffold for the modulation of probe grafting. With this aim in mind, polymers with increasing concentrations of N-acryloyloxysuccinimide (NAS) were synthesized and the coating procedure optimized accordingly. These were then tested not only in DNA microarray assays, but also using protein probes (with different MWs) to establish which formulation improves the assay performance in specific applications. The flexibility of this polymeric platform allowed us also to investigate a different immobilization chemistry—specifically, click chemistry reactions, thanks to the insertion of azide groups into the polymer chains—and to evaluate possible differences generated by this modification.

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Author(s): Dario Brambilla, Federica Panico, M. Selim Unlu, Marcella Chiari

CHEMRXIV 29 November 2023, Version 1

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.

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Author(s): D. Brambilla, F. Panico, A. Mussida, L. Sola, F. Damin, M. Chiari

3rd EVIta Symposium 2023 (Urbino, 13-15 September 2023)

Extracellular vesicles (EVs) emerged as a powerful source of biomarkers. Unfortunately, a single platform capable of interrogating the complex biology of EVs does not exist. EVs are insufficiently characterized to support their clinical implications. Immunoaffinity approaches offer unmatched selectivity and the possibility to separate subpopulations of EVs. However, current immunoaffinity bead-based assays lack solutions for the release of intact EVs, limiting immunoaffinity isolation’s range of applications. We introduce a novel approach that integrates two fundamental steps for EV analysis: i) affinity isolation of target EVs from plasma, and ii) digital, multiparametric analysis of EVs. Antibodies that target a specific EV subpopulation are conjugated to an oligonucleotide composed of two domains: a streptavidin aptamer region, and a “barcode” sequence. The aptamer is used for the reversible immobilization of antibodies on magnetic beads. Upon incubation with biotin, which competes for streptavidin with the aptamer, the conjugates are released from beads. The barcode sequence is used for the recapture of EVs-antibody complexes on the surface of a DNA microarray allowing multiplexed detection. The proposed barcoding strategy provides the following advantage: i) efficient EV release in mild conditions, preserving EVs integrity, ii) flexibility and customizable capture of a large number of EV targets, iii) unique features for integrated in-line EV isolation and detection.