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.

Download

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.

Download

Author(s): Jerome Nouvel, Gonzalo Bustos-Quevedo, Tony Prinz, Ramsha Masood, George Daaboul, Tanja Gainey-Schleicher, Uwe Wittel, Sophia Chikhladze, Bence Melykuti, Martin Helmstaedter, Karl Winkler, Irina Nazarenko, Gerhard Pütz

Journal of Extracellular Vesicles Volume13, Issue10 October 2024

https://doi.org/10.1002/jev2.70008

Extracellular vesicles (EVs) are valuable targets for liquid biopsy. However, attempts to introduce EV-based biomarkers into clinical practice have not been successful to the extent expected. One of the reasons for this failure is the lack of reliable methods for EV baseline purification from complex biofluids, such as cell-free plasma or serum. Because available one-step approaches for EV isolation are insufficient to purify EVs, the majority of studies on clinical samples were performed either on a mixture of EVs and lipoproteins, whilst the real number of EVs and their individual specific biomarker content remained elusive, or on a low number of samples of sufficient volume to allow elaborate 2-step EV separation by size and density, resulting in a high purity but utmost low recovery. Here we introduce Fast Protein Liquid Chromatography (FPLC) using Superose 6 as a matrix to obtain small EVs from biofluids that are almost free of soluble proteins and lipoproteins. Along with the estimation of a realistic number of small EVs in human samples, we show temporal resolution of the effect of the duration of postprandial phase on the proportion of lipoproteins in purified EVs, suggesting acceptable time frames additionally to the recommendation to use fasting samples for human studies. Furthermore, we assessed a potential value of pure EVs for liquid biopsy, exemplarily examining EV- and tumour-biomarkers in pure FPLC-derived fractions isolated from the serum of patients with pancreatic cancer. Consistent among different techniques, showed the presence of diseases-associated biomarkers in pure EVs, supporting the feasibility of using single-vesicle analysis for liquid biopsy.

Download

Author(s): Marvin Droste, Maija Puhka, Martin E. van Royen, Monica S. Y. Ng, Charles Blijdorp, Gloria Alvarez-Llamas, Francesc E. Borràs, Anja K. Büscher, Benedetta Bussolati, James W. Dear, Juan M. Falcón-Pérez, Bernd Giebel, Cristina Grange, Ewout J. Hoorn, Janne Leivo, Metka Lenassi, Alicia Llorente, Fabrice Lucien, Inge Mertens, Harald Mischak, Desmond Pink, Tobias Tertel, Swasti Tiwari, Dolores Di Vizio, Peter S. T. Yuen, Natasa Zarovni, Guido Jenster, Dylan Burger, Elena S. Martens-Uzunova, Uta Erdbrügger

Journal of Extracellular Vesicles, Volume14, Issue7, July 2025

https://doi.org/10.1002/jev2.70120

Despite remarkable interest in the biomarker potential of urinary extracellular vesicles (uEVs) and the identification of numerous promising candidates, their clinical translation still presents multiple challenges. The opportunities for successful translation are obvious, yet the main roadblocks on the way have hardly been systematically considered and more coordinated approaches are needed to overcome them. In the present review article, we have identified the most relevant roadblocks of clinical translation of urinary EV-based biomarkers and discuss possible solutions to overcome them. These roadblocks are categorized as fundamental and technical but also related to development of novel biomarker assays and clinical acceptance. In addition, hurdles within the regulatory approval process are discussed. It is clear that various roadblocks to clinical translation of urinary EV biomarkers exist; however, they are addressable by promoting rigor and reproducibility as well as collaboration between basic and clinical scientists, clinicians, industry and regulatory bodies. Moreover, knowledge of obstacles for assay development and regulatory requirements should already be considered when developing a new biomarker to maximize the chance of successful translation. This review presents not only a status quo, but also a roadmap for the further development of the field.

Download

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.

Download

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.