Immobilization of biotinylated antibodies through streptavidin binding aptamer

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

A Bi-Functional Polymeric Coating for the Co-Imobilization of Proteins and Peptides on Microarray Substrates

The analytical performance of the microarray technique in screening the affinity and reactivity of several probes towards a specific target, is highly affected by the coupling chemistry adopted to bind probes to the surface. However, the surface functionality limits the biomolecules that can be attached to the surface to a single type of molecule (DNA, protein, or peptide), thus forcing the execution of separate analyses to compare the performance of different species in recognizing their targets. Here we introduce a new N, N-dimethylacrylamide-based polymeric coating, bearing simultaneously different functionalities (N-acryloyloxysuccinimide and azide groups) to allow an easy and straightforward method to co-immobilize proteins and oriented peptides on the same substrate. The bi-functional copolymer has been obtained by partial post polymerization modification of the functional groups (NAS) of a common precursor. A deep characterization of the copolymer was carried out by means of NMR to quantify the percentage of NAS that has been transformed into azido groups. The polymer was then used to coat surfaces onto which both native antibodies and alkyne modified peptides were immobilized, to perform the phenotype characterization of extracellular vesicles (EVs). Ultimately, this strategy represents a convenient method to reduce the number of analysis, thus possible systematic or random errors, besides offering a drastic shortage in time, reagents and costs

Polymeric Coating of Silica Microspheres for Biological Applications: Suppression of Non-Specific Binding and Functionalization with Biomolecules

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

Optimization of Functional Group Concentration of N, N-Dimethylacrylamide-based Polymeric Coatings and Probe Immobilization for DNA and Protein Microarray Applications

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

SARS-CoV-2 Epitope Mapping on Microarrays Highlights Strong Immune-Response to N Protein Region

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

Structure, Immunoreactivity, and In Silico Epitope Determination of SmSPI S. mansoni Serpin for Immunodiagnostic Application

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 angstrom 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

Structure, Immunoreactivity, and In Silico Epitope Determination of SmSPI S. mansoni Serpin for Immunodiagnostic Application

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