A workflow for rapid SARS-CoV-2 epitope discovery on peptide microarrays is herein reported. The process started with a proteome-wide screening of immunoreactivity based on the use of a high-density microarray followed by a refinement and validation phase on a restricted panel of probes using microarrays with tailored peptide immobilization through a click-based strategy. Progressively larger, independent cohorts of Covid-19 positive sera were tested in the refinement processes, leading to the identification of immunodominant regions on SARS-CoV-2 spike (S), nucleocapsid (N) protein and Orf1ab polyprotein. A summary study testing 50 serum samples highlighted an epitope of the N protein (region 155–71) providing good diagnostic performance in discriminating Covid-19 positive vs. healthy individuals. Using this epitope, 92% sensitivity and 100% specificity were reached for IgG detection in Covid-19 samples, and no cross-reactivity with common cold coronaviruses was detected. Likewise, IgM immunoreactivity in samples collected within the first month after symptoms onset showed discrimination ability. Overall, epitope 155–171 from N protein represents a promising candidate for further development and rapid implementation in serological tests.

The use of micro- and nanoparticles in biological applications has dramatically grown during the last few decades due to the ease of protocols development and compatibility with microfluidics devices. Particles can be composed by different materials, i.e., polymers, inorganic dielectrics, and metals. Among them, silica is a suitable material for the development of biosensing applications. Depending on their final application, the surface properties of particles, including silica, are tailored by means of chemical modification or polymeric coating. The latter strategy represents a powerful tool to create a hydrophilic environment that enables the functionalization of particles with biomolecules and the further interaction with analytes. Here, the use of MCP-6, a dimethylacrylamide (DMA)-based ter-copolymer, to coat silica microspheres is presented. MCP-6 offers unprecedented ease of coating, imparting silica particles a hydrophilic coating with antifouling properties that is able to provide high-density immobilization of biological probes.

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.

The human parasitic disease Schistosomiasis is caused by the Schistosoma trematode flatworm that infects freshwaters in tropical regions of the world, particularly in Sub-Saharan Africa, South America, and the Far-East. It has also been observed as an emerging disease in Europe, due to increased immigration. In addition to improved therapeutic strategies, it is imperative to develop novel, rapid, and sensitive diagnostic tests that can detect the Schistosoma parasite, allowing timely treatment. Present diagnosis is difficult and involves microscopy-based detection of Schistosoma eggs in the feces. In this context, we present the 3.22 Å resolution crystal structure of the circulating antigen Serine protease inhibitor from S. mansoni (SmSPI), and we describe it as a potential serodiagnostic marker. Moreover, we identify three potential immunoreactive epitopes using in silico-based epitope mapping methods. Here, we confirm effective immune sera reactivity of the recombinant antigen, suggesting the further investigation of the protein and/or its predicted epitopes as serodiagnostic Schistosomiasis biomarkers.

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.

Liquid biopsy approaches are powerful strategies that potentially allow the diagnosis and prognosis of a number of diseases. The field is continuously and rapidly growing, encouraging the discovery of novel predictory biomarkers. Antibodies are usually exploited in sensors to validate biomarker candidates. Unfortunately, the immobilization of antibodies on the surface of sensors represents a challenging task. Immobilization strategies need to be optimized for each antibody, representing a huge obstacle to overcome in the discovery of new biomarkers. Herein we propose a novel strategy for the immobilization of antibodies, based on the use of a streptavidin-binding aptamer. Using this approach it is possible to immobilize antibodies on the surface of sensors with no need for optimization, with the only requirement for antibody to be biotinylated. The proposed strategy potentially paves the way towards a straightforward immobilization of antibodies on biosensors, making their use in biomarker validation more accessible.