Proof of concept of using a membrane-sensing peptide for sEVs affinity-based isolation.

Introduction: One main limitation in biomarker studies using EVs is the lack of a suitable isolation method rendering high yield and purity samples in a quick and easily standardized procedure. Here we report an affinity isolation method with a membrane-sensing peptide (MSP) derived from bradykinin. Methods: We designed a protocol based on agarose beads carrying cation chelates to specifically bind to the 6His-tagged membrane-sensing peptide. This approach presents several advantages: 1) cation-carrying agaroses are widely used and standardized for His-tagged protein isolation, 2) the affinity protocol can be performed in small volumes, feasible and manageable for clinical routine and 3) elution with imidazole or EDTA allows a gentle and easy recovery without EV damage, facilitating subsequent characterization and functional analyses. Results: The optimized final procedure incubates 0.5 mg of peptide for 10 min with 10 µL of Long-arm Cobalt agarose before an overnight incubation with concentrated cell conditioned medium. EV downstream analyses can be directly performed on the agarose beads adding lysis or nucleic-acid extraction buffers, or gently eluted with imidazole or EDTA, rendering a fully competent EV preparation. Discussion: This new isolation methodology is based on the recognition of general membrane characteristics independent of surface markers. It is thus unbiased and can be used in any species EV sample, even in samples from animal or plant species against which no suitable antibodies exist. Being an affinity method, the sample handling protocol is very simple, less time-consuming, does not require specialized equipment and can be easily introduced in a clinical automated routine. We demonstrated the high purity and yield of the method in comparison with other commercially available kits. This method can also be scale up or down, with the possibility of analyzing very low amounts of sample, and it is compatible with any downstream analyses thanks to the gentle elution procedure.

Hybrid Peptide-Agarose Hydrogels for 3D Immunoassays.

Recent advances in biosensing analytical platforms have brought relevant outcomes for novel diagnostic and therapy-oriented applications. In this context, 3D droplet microarrays, where hydrogels are used as matrices to stably entrap biomolecules onto analytical surfaces, potentially provide relevant advantages over conventional 2D assays, such as increased loading capacity, lower nonspecific binding, and enhanced signal-to-noise ratio. Here, we describe a hybrid hydrogel composed of a self-assembling peptide and commercial agarose (AG) as a suitable matrix for 3D microarray bioassays. The hybrid hydrogel is printable and self-adhesive and allows analyte diffusion. As a showcase example, we describe its application in a diagnostic immunoassay for the detection of SARS-CoV-2 infection.

Epitope Mapping on Microarrays Highlights a Sequence on the N Protein with Strong Immune Response in SARS-CoV-2 Patients.

In SARS-CoV-2 pandemic scenario, the identification of rapid methods to detect antibodies against coronavirus has been a wide and urgent issue. Epitope mapping on peptide microarrays is a rapid way to identify sequences with a high immunoreactivity. The process begins with a proteome-wide screening, based on immune affinity; the use of a high-density microarray is followed by a validation phase, where a restricted panel of probes is tested using peptide microarrays; peptide sequences are immobilized through a click-based strategy.COVID-19-positive sera are tested and immuno-domains regions are identified on SARS-CoV-2 spike (S), nucleocapsid (N) protein, and Orf1ab polyprotein. An epitope on N protein (region 155-171) provided good diagnostic performance in discriminating COVID-19-positive vs. healthy individuals. Using this sequence, 92% sensitivity and 100% specificity are reached for IgG detection in COVID-19 samples, and no cross-reactivity with common cold coronaviruses is detected. Overall, epitope 155-171 from N protein represents a promising candidate for further development and rapid implementation in serological tests.

Membrane-Sensing Peptides for Extracellular Vesicle Analysis.

Analytical platforms for small extracellular vesicle (sEV) high-throughput analysis are highly desirable. These bionanoparticles present fairly distinctive lipid membrane features including high curvature, lipid-packing defects, and a relative abundance in lipids. sEV membrane could be considered as a "universal" marker, complementary or alternative to traditional surface-associated proteins. Here, we describe the use of membrane-sensing peptides as a new, highly efficient ligand to directly integrate sEV capturing and analysis on a microarray platform.

Composite Peptide-Agarose Hydrogels for Robust and High-Sensitivity 3D Immunoassays.

Canonical immunoassays rely on highly sensitive and specific capturing of circulating biomarkers by interacting biomolecular baits. In this frame, bioprobe immobilization in spatially discrete three-dimensional (3D) spots onto analytical surfaces by hydrogel encapsulation was shown to provide relevant advantages over conventional two-dimensional (2D) platforms. Yet, the broad application of 3D systems is still hampered by hurdles in matching their straightforward fabrication with optimal functional properties. Herein, we report on a composite hydrogel obtained by combining a self-assembling peptide (namely, Q3 peptide) with low-temperature gelling agarose that is proved to have simple and robust application in the fabrication of microdroplet arrays, overcoming hurdles and limitations commonly associated with 3D hydrogel assays. We demonstrate the real-case scenario feasibility of our 3D system in the profiling of Covid-19 patients' serum IgG immunoreactivity, which showed remarkably improved signal-to-noise ratio over canonical assays in the 2D format and exquisite specificity. Overall, the new two-component hydrogel widens the perspectives of hydrogel-based arrays and represents a step forward towards their routine use in analytical practices.

Self-Assembling Peptide Hydrogels for 3D Microarrays.

Recent advances in biosensing analytical platforms have brought relevant outcomes for novel diagnostic and therapy-oriented applications. In this context, hydrogels have emerged as appealing matrices to locally confine biomolecules onto sensing surfaces under solution mimetic conditions, preserving their structural integrity and function. Here, we describe the application of a self-assembling peptide hydrogel as a suitable matrix for 3D microarray bioassays. The hydrogel is printable and self-adhesive and allows for fast analyte diffusion. As a showcase example, we describe its application in a diagnostic immunoassay for the detection of arbovirus infection.

Membrane-binding peptides for extracellular vesicles on-chip analysis.

Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a "universal" marker, alternative or complementary to traditional, characteristic, surface-associated proteins. Here, we introduce the use of membrane-sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label-free, single-particle counting and sizing, and by fluorescence co-localisation immune staining with fluorescent anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis.

Chemical Perturbation of Oncogenic Protein Folding: from the Prediction of Locally Unstable Structures to the Design of Disruptors of Hsp90-Client Interactions.

Protein folding quality control in cells requires the activity of a class of proteins known as molecular chaperones. Heat shock protein-90 (Hsp90), a multidomain ATP driven molecular machine, is a prime representative of this family of proteins. Interactions between Hsp90, its co-chaperones, and client proteins have been shown to be important in facilitating the correct folding and activation of clients. Hsp90 levels and functions are elevated in tumor cells. Here, we computationally predict the regions on the native structures of clients c-Abl, c-Src, Cdk4, B-Raf and Glucocorticoid Receptor, that have the highest probability of undergoing local unfolding, despite being ordered in their native structures. Such regions represent potential ideal interaction points with the Hsp90-system. We synthesize mimics spanning these regions and confirm their interaction with partners of the Hsp90 complex (Hsp90, Cdc37 and Aha1) by Nuclear Magnetic Resonance (NMR). Designed mimics selectively disrupt the association of their respective clients with the Hsp90 machinery, leaving unrelated clients unperturbed and causing apoptosis in cancer cells. Overall, selective targeting of Hsp90 protein-protein interactions is achieved without causing indiscriminate degradation of all clients, setting the stage for the development of therapeutics based on specific chaperone:client perturbation.

Clickable cellulosic surfaces for peptide-based bioassays.

The use of peptides in paper-based analytics is a highly appealing field, yet it suffers from severe limitations. This is mostly due to the loss of effective target recognition properties of this relatively small probes upon nonspecific adsorption onto cellulose substrates. Here we address this issue by introducing a simple polymer-based strategy to obtain clickable cellulose surfaces, that we exploited for the chemoselective bioconjugation of peptide bioprobes. Our method largely outperformed standard adsorption-based immobilization strategy in a challenging, real case immunoassay, namely the diagnostic discrimination of Zika + individuals from healthy controls. Of note, the clickable polymeric coating not only allows efficient peptides bioconjugation, but it provides favorable anti-fouling properties to the cellulosic support. We envisage our strategy to broaden the repertoire of cellulosic materials manipulation and promote a renewed interest in peptide-based paper bioassays.

Acid Catalyzed Formation of C⁻C and C⁻S Bonds via Excited State Proton Transfer.

The behavior of 2-naphthol and 7-bromo-2-naphthol as organic photoacids are exploited in organic synthesis for the preparation of benzyl sulfides (using a trichloroacetimidate derivative as the starting substrate) and polycyclic amines via acid catalyzed condensation of 1,2,3,4-tetrahydroisoquinoline with aldehydes.