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Persistent post-breeding induced endometritis (PPBIE) is considered a major cause of subfertility in mares. It consists of persistent or delayed uterine inflammation in susceptible mares. There are many options for the treatment of PPBIE, but in this study, a novel approach aimed at preventing the onset of PPBIE was investigated. Stallion semen was supplemented with extracellular vesicles derived from amniotic mesenchymal stromal cells (AMSC-EVs) at the time of insemination to prevent or limit the development of PPBIE. Before use in mares, a dose–response curve was produced to evaluate the effect of AMSC-EVs on spermatozoa, and an optimal concentration of 400 × 106 EVs with 10 × 106 spermatozoa/mL was identified. At this concentration, sperm mobility parameters were not negatively affected. Sixteen susceptible mares were enrolled and inseminated with semen (n = 8; control group) or with semen supplemented with EVs (n = 8; EV group). The supplementation of AMSC-EVs to semen resulted in a reduction in polymorphonuclear neutrophil (PMN) infiltration as well as intrauterine fluid accumulation (IUF; p < 0.05). There was a significant reduction in intrauterine cytokine levels (p < 0.05) for TNF-α and IL-6 and an increase in anti-inflammatory IL-10 in mares in the EV group, suggesting successful modulation of the post-insemination inflammatory response. This procedure may be useful for mares susceptible to PPBIE.

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.

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.

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.

The widely overlapping physicochemical properties of lipoproteins (LPs) and extracellular vesicles (EVs) represents one of the main obstacles for the isolation and characterization of these pervasive biogenic lipid nanoparticles. We herein present the application of an atomic force microscopy (AFM)-based quantitative morphometry assay to the rapid nanomechanical screening of mixed LPs and EVs samples.The method can determine the diameter and the mechanical stiffness of hundreds of individual nanometric objects within few hours. The obtained diameters are in quantitative accord with those measured via cryo-electron microscopy (cryo-EM); the assignment of a specific nanomechanical readout to each object enables the simultaneous discrimination of co-isolated EVs and LPs even if they have overlapping size distributions. EVs and all classes of LPs are shown to be characterized by specific combinations of diameter and stiffness, thus making it possible to estimate their relative abundance in EV/LP mixed samples in terms of stoichiometric ratio, surface area and volume. As a side finding, we show how the mechanical behaviour of specific LP classes is correlated to distinctive structural features revealed by cryo-EM. To the best of our knowledge, these results represent the first systematic single-particle mechanical investigation of lipoproteins.The described approach is label-free, single-step and relatively quick to perform. Importantly, it can be used to analyze samples which prove very challenging to assess with several established techniques due to ensemble-averaging, low sensibility to small particles, or both, thus providing a very useful tool for quickly assessing the purity of EV/LP isolates including plasma- and serum-derived preparations.

Tangential flow filtration membrane systems are employed for the isolation and concentration of extracellular vesicles. However, interfacial interactions between the membrane surface and species influence the flux and membrane performance. Here we propose a strategy aimed at introducing functional ligands over the membrane surface to improve the separation process through combined size-exclusion and affinity-based mechanisms, avoiding the binding of contaminants and other non-target molecules. Polysulfone membranes were modified by a nanometric coating of differently functionalized copolymers with the dual purpose of limiting non-specific interactions while promoting the chemoselective conjugation of a membrane-sensing peptide ligand (BPt) for lipid nanovesicles capture. Copoly azide polymer coating positively affects the physico-chemical properties of the membrane, improving filtration performance and antifouling capacity. A decrease of the flux decline ratio from 38.7±3.9% to 21.2±2.4% and an increase of the ratio of protein permeate concentration (Cp) to the respective feed concentration (Cf) to values of 0.97 was measured after coating the membrane with c-(DMA-N3-BP-MAPS) highlighting its capability to reduce protein adsorption. In addition, the BPt-functionalized membrane displayed a high capturing efficiency towards synthetic liposomes which, notably, can be promptly released upon mild treatment with a divalent cation solution. Overall, our work integrates conventional TFF principles with affinity-based isolation, broadening TFF perspective applications.

Surface chemistry is one of the most crucial aspects for microarray modality biosensor development. As a matter of fact, the immobilization capability of the functionalized surface is one of the limiting factors for the final yield of the binding reaction. In this work, we locally deposited many reactive polymers on a single solid support, allowing for a direct comparison of functionality of probes immobilized on different polymers and demonstrating a new way of multiplexing. Our goal was to investigate the immobilization efficiency of reactive polymers, as well as the resulting affinity of the molecular probes, in a single experiment. This idea was demonstrated by spotting a large number of different reactive polymers on an untreated Si/SiO2 chip, and depositing the same molecular probe on all the spots immediately after. This method proved to be efficient and could be used as an initial qualitative assay to decide which functionalization better suits a certain application. We also showed that the localized functionalization method is applicable to proteins as well as oligonucleotides. Moreover, by means of real-time binding measurements performed with the Interferometric Reflectance Imaging Sensor (IRIS), we demonstrated that this functionalization technique is comparable to the uniformity of classical flat-coating solution. The comparison between the binding curves that were obtained from different polymer spots with the same probe allowed us to decide which polymers would work better to immobilize a model protein, α-Lactalbumin, as well as a peptide extracted from the latter, namely LAC-1. The final outcome is promising, and it highlights the multiplexing power of this method: first, it allows to characterize dozens of polymers at once, within a single 60-minutes experiment. Secondly, it removes the limitation, related to coated surfaces, that only molecules with the same functional groups can be tethered to the same solid support. By applying this innovative protocol, there is no more restriction on the type of molecules that can be studied simultaneously and immobilization for each molecular probe can be individually optimized.

Inflammatory response following SARS-CoV-2 infection results in substantial increase of amounts of intravascular pro-coagulant extracellular vesicles (EVs) expressingtissue factor(CD142) on their surface. CD142-EV turned out to be useful as diagnostic biomarker in COVID-19patients. Here we aimed at studying the prognostic capacity of CD142-EV in SARS-CoV-2 infection. Expression of CD142-EV was evaluated in 261 subjects admitted tohospitalfor pneumonia and with a positive molecular test for SARS-CoV-2. The study population consisted of a discovery cohort of selected patients (n=60) and an independent validation cohort including unselected consecutive enrolled patients (n=201). CD142-EV levels were correlated with post-hospitalization course of the disease and compared to the clinically available 4C Mortality Score as referral. CD142-EV showed a reliable performance to predict patient prognosis in the discovery cohort (AUC=0.906) with an accuracy of 81.7%, that was confirmed in the validation cohort (AUC=0.736). Kaplan-Meier curves highlighted a high discrimination power in unselected subjects with CD142-EV being able to stratify the majority of patients according to their prognosis. We obtained a comparable accuracy, being not inferior in terms of prediction of patients' prognosis and risk of mortality, with 4C Mortality Score. The expression of surface vesicular CD142 and its reliability as prognostic marker was technically validated using different immunocapture strategies and assays. The detection of CD142 on EV surface gains considerable interest asrisk stratificationtool to support clinical decision making in COVID-19.

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.

Unlike humans and many other mammalian species, conventional in vitro fertilization (IVF) in equine species is not successful. To mimic in vitro equine spermatozoon-oviduct interaction as close as possible to that which occurs in vivo, extracellular vesicles (EVs) secreted by the female genital tract were used. Three female genital tracts were collected at slaughterhouse from mares in late estrus. Ipsilateral proximal and apical horn endometrial explants were digested with collagenase and trypsin and cells obtained were cultured on insert system to allow their polarization. Ipsilateral oviducts were squeezed out to obtain spheroids. To produce EVs, proximal and apical horn endometrial cells and oviductal spheroids were cultured for three days in serum free medium. To trace interaction between spermatozoa and EVs by fluorescence microscopy, EVs were differently labeled. Pooled samples of ejaculated spermatozoa from three stallions were incubated in capacitating medium (CM) for 6 h and to induce hyperactivation for other 6 h in CM supplemented with different kind of EVs alone or in combination. A control was performed in absence of EVs. Sperm were assessed for motility by CASA system, EV incorporation by confocal microscopy and acrosomal reaction (AR) by staining with FITC-PNA/PI. In vitro fertilization was performed, and presumed zygotes were subjected to chromatin configuration. The results show that incorporation of EVs of the proximal horn does not take place, while apical horn EVs are incorporated in the head of the spermatozoon in 4 h. The EVs of oviductal spheroids are incorporated in the middle tract in 1 h. The rate of AR with EVs of the apical horn and oviductal spheroids were respectively 50.25% and 57.14%. When these EVs were added in combination, the rate of AR was 71.42%. In the control, the rate of AR was of 15%. After in vitro fertilization, 44% of oocytes showed male and female pronuclei, whereas no fertilization is obtained in the control. In conclusion, EVs from apical horn and oviduct could be involved in cell trafficking during equine semen hyperactivation, and their possible use in vitro could facilitate the development of equine reproductive biotechnologies.

Bioprobes immobilization methods that elevate the probes from the substrate are generally preferred in microarray technology because they prevent steric limitations during the hybridization of the target to probes. A versatile approach to control the thickness of a polymeric coating based on click chemistry to obtain covalently linked layer-by-layer coatings for surface functionalization is presented. By alternating cycles of coating using copolymers bearing click groups, the thickness of the film increases, while remaining functional and stable. Click chemistry reactions provide numerous advantages over standard conjugation procedures typically used in microarrays. They include: quantitative yields and insensitivity of the reaction to pH and hydrolysis. Moreover, click reactions do not interfere with organic groups naturally present in DNA, proteins and peptides such as amino and carboxyl groups allowing orthogonal and chemoselective probe immobilization. In addition to the formation of multilayers, click reactions allow to bind biomolecules to polymer chains generating so-called polymeric probes, which are then immobilized on microarray supports. In a microarray assay of clinical relevance, this methodology provides a miniaturized, tri-dimensional multilayer with higher density of capture probe, improved hybridization efficiency and sensitivity.

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.

Due to significant advances in computational biology, protein prediction, together with antigen and epitope design, have rapidly moved from conventional methods, based on experimental approaches, to in silico-based bioinformatics methods. In this context, we report a reverse vaccinology study that identified a panel of 104 candidate antigens from the Gram-negative bacterial pathogen Burkholderia pseudomallei, which is responsible for the disease melioidosis. B. pseudomallei can cause fatal sepsis in endemic populations in the tropical regions of the world and treatment with antibiotics is mostly ineffective. With the aim of identifying potential vaccine candidates, we report the experimental validation of predicted antigen and type I fimbrial subunit, BPSL1626, which we show is able to recognize and bind human antibodies from the sera of Burkholderia infected patients and to stimulate T-lymphocytes in vitro. The prerequisite for a melioidosis vaccine, in fact, is that both antibody- and cell-mediated immune responses must be triggered. In order to reveal potential antigenic regions of the protein that may aid immunogen re-design, we also report the crystal structure of BPSL1626 at 1.9 Å resolution on which structure-based epitope predictions were based. Overall, our data suggest that BPSL1626 and three epitope regions here-identified can represent viable candidates as potential antigenic molecules.

BackgroundAmyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem motor neuron disease for which currently there is no effective treatment. There is an urgent need to identify biomarkers to tackle the disease’s complexity and help in early diagnosis, prognosis, and therapy. Extracellular vesicles (EVs) are nanostructures released by any cell type into body fluids. Their biophysical and biochemical characteristics vary with the parent cell’s physiological and pathological state and make them an attractive source of multidimensional data for patient classification and stratification.MethodsWe analyzed plasma-derived EVs of ALS patients (n = 106) and controls (n = 96), and SOD1G93Aand TDP-43Q331Kmouse models of ALS. We purified plasma EVs by nickel-based isolation, characterized their EV size distribution and morphology respectively by nanotracking analysis and transmission electron microscopy, and analyzed EV markers and protein cargos by Western blot and proteomics. We used machine learning techniques to predict diagnosis and prognosis.ResultsOur procedure resulted in high-yield isolation of intact and polydisperse plasma EVs, with minimal lipoprotein contamination. EVs in the plasma of ALS patients and the two mouse models of ALS had a distinctive size distribution and lower HSP90 levels compared to the controls. In terms of disease progression, the levels of cyclophilin A with the EV size distribution distinguished fast and slow disease progressors, a possibly new means for patient stratification. Immuno-electron microscopy also suggested that phosphorylated TDP-43 is not an intravesicular cargo of plasma-derived EVs.ConclusionsOur analysis unmasked features in plasma EVs of ALS patients with potential straightforward clinical application. We conceived an innovative mathematical model based on machine learning which, by integrating EV size distribution data with protein cargoes, gave very high prediction rates for disease diagnosis and prognosis.

We developed a biosensing system for serological detection of viruses based on the impedance variation between gold microelectrodes upon the capture of the target antibodies hybridized with nanobeads for signal amplification. The microfluidic platform core features a Differential Impedance Sensing (DIS) architecture between a reference and an active sensor able to reach nanoparticle resolution of few tens. The biosensor, functionalized with a copoly layer housing a synthetic peptide probe, has shown a limit of detection (LOD) below 100 pg/mL using a model IgG antibody spiked in a buffer. The biosensor was also tested with human serum samples for quantitative counts of anti-Dengue Virus antibodies, reaching a sensitivity that outperforms commercial ELISA kit. The system is perfectly suited to be easily reconfigured for novel probes by simply modifying the preparation of the biosensor chip surface, thus addressing a wide range of pathogens and diseases with clinically relevant concentrations for rapid immunoassays in a point of care setting.

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.

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.

BackgroundAmyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem motor neuron disease for which currently there is no effective treatment. There is an urgent need to identify biomarkers to tackle the disease’s complexity and help in early diagnosis, prognosis, and therapy. Extracellular vesicles (EVs) are nanostructures released by any cell type into body fluids. Their biophysical and biochemical characteristics vary with the parent cell’s physiological and pathological state and make them an attractive source of multidimensional data for patient classification and stratification.MethodsWe analyzed plasma-derived EVs of ALS patients (n= 106) and controls (n=96), and SOD1G93A and TDP-43Q331K mouse models of ALS. We purified plasma EVs by nickel-based isolation, characterized their EV size distribution and morphology respectively by nanotracking analysis and transmission electron microscopy, and analyzed EV markers and protein cargos by Western blot and proteomics. We used machine learning techniques to predict diagnosis and prognosis.ResultsOur procedure resulted in high-yield isolation of intact and polydisperse plasma EVs, with minimal lipoprotein contamination. There were more particles in the plasma of ALS patients and the two mouse models of ALS while their average diameter was smaller. HSP90 was differentially represented in ALS patients and mice compared to the controls. In terms of disease progression, the levels of cyclophilin A, with the EV size distribution, distinguished fast and slow disease progressors, suggesting a new means for patient stratification. We also measured the levels of phosphorylated TDP-43 and showed that is not an intravesicular cargo of plasma-derived EVs.ConclusionsOur analysis unmasked features in plasma EVs of ALS patients with potential straightforward clinical application. We conceived an innovative mathematical model based on machine learning which, by integrating EV size distribution data with protein cargoes, gave very high prediction rates for disease diagnosis and prognosis.

The interest towards aptamer-based surfaces in the field of bioaffinity assays is constantly growing. A crucial aspect is related to the ability of maintaining the high specificity of these molecules once immobilized on the surface. In this article we compare the immobilization of aptamers on a silanized silicon nitride surface as well as on a soft polymeric layer. An innovative immobilization approach, based on a copolymer, N-dimethylacrylamide-N-acryloyloxysuccinimide-3-(trimethoxysilyl)propylmethacrylate (DMA-NAS-MAPS) coating, able to crosslink itself to the substrate and to bind amino-modified biomolecules is proposed. Comparing this coating with a more classical functionalization process based on silane chemistry, we propose that the better results obtained on the polymeric layer are due to an increased binding efficiency of the aptamers bound to a soft material. The high specificity of immobilized DNA-aptamers is demonstrated using two sequences specific for thrombin detection and a non-sense sequence as negative control. The coating provides higher sensitivity compared to classical self-assembled silane coatings, probably due to a better mobility of bound aptamers. The aptamer immobilization on both surfaces was characterized and optimized using atomic force microscopy, X-ray photoelectron spectroscopy, contact angle and fluorescence microscopy. The comparison between the two different functionalizations highlights the better performances of the copolymer coating in terms of protein recognition, demonstrating thrombin detection down to 0.011 nM in buffer solution and 4.9 nM in complete human serum. Moreover, the localized immobilization of the aptameric sequences, utilized in this work, suggests the possibility of employing this platform also for multianalyte detection.

Background Identifying physiologically relevant binding partners of amyloid-β (Aβ) that modulate in vivo fibril formation may yield new insights into Alzheimer's disease (AD) etiology. Human cathelicidin peptide, LL-37, is an innate immune effector and modulator, ubiquitous in human tissues and expressed in myriad cell types. Objective We present in vitro experimental evidence and discuss findings supporting a novel hypothesis that LL-37 binds to Aβ42 and can modulate Aβ fibril formation. Methods Specific interactions between LL-37 and Aβ (with Aβ in different aggregation states, assessed by capillary electrophoresis) were demonstrated by surface plasmon resonance imaging (SPRi). Morphological and structural changes were investigated by transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy. Neuroinflammatory and cytotoxic effects of LL-37 alone, Aβ42 alone, and LL-37/Aβ complexes were evaluated in human microglia and neuroblastoma cell lines (SH-SY5Y). Results SPRi shows binding specificity between LL-37 and Aβ, while TEM shows that LL-37 inhibits Aβ42 fibril formation, particularly Aβ's ability to form long, straight fibrils characteristic of AD. CD reveals that LL-37 prevents Aβ42 from adopting its typical β-type secondary structure. Microglia-mediated toxicities of LL-37 and Aβ42 to neurons are greatly attenuated when the two peptides are co-incubated prior to addition. We discuss the complementary biophysical characteristics and AD-related biological activities of these two peptides. Conclusion Based on this body of evidence, we propose that LL-37 and Aβ42 may be natural binding partners, which implies that balanced (or unbalanced) spatiotemporal expression of the two peptides could impact AD initiation and progression.

Background Microarray-based component-resolved diagnostics (CRD) has become an accepted tool to detect allergen specific IgE-sensitization towards hundreds of allergens in parallel from one drop of serum. Nevertheless specificity and sensitivity as well as a simultaneous detection of allergen specific IgG4, as a potential parameter for tolerance development, remain to be optimized. Objective We applied the recently introduced silicon chip coated with a functional polymer named copoly(DMA-NAS-MAPS) to the simultaneous detection of food allergen specific IgE and IgG4, and compared it with ImmunoCAP and ImmunoCAP ISAC. Inter- and intra- slide variation, linearity of signal and working range, sensitivity and application of internal calibrations for IgE and IgG4 were assessed. Methods Native and recombinant allergenic proteins from hen's egg and cow's milk were spotted on silicon chips coated with copoly(DMA-NAS-MAPS) along with known concentrations for human IgE and IgG4. A serum pool and 105 patient samples were assessed quantitatively and semi-quantitatively with the ImmunoCAP and ImmunoCAP ISAC and correlated with IgE- and IgG4-specific fluorescence on silicon microarrays. Results Allergen specific IgE and IgG4 were detected in parallel using two fluorescent dyes with no crosstalk. Results from the ImmunoCAP correlated better with microarray fluorescence than with ImmunoCAP ISAC except for the allergen ovomucoid. The working range of the silicon microarray for total hen's egg specific IgE was comparable to the range of 0.1 to >100 kUA/l of the ImmunoCAP system, whereas for total cow's milk the silicon microarray was less sensitive. Detectable allergen specific IgG4 could be determined only for low concentrations, but still correlated positively with ImmunoCAP results. Conclusions We confirmed the ability of the polymer coated silicon microarray to be comparably sensitive to the ImmunoCAP ISAC for various food allergens. This suggests that the copoly(DMA-NAS-MAPS) microarray is a low cost, self-producible alternative to the commercial ImmunoCAP ISAC in allergy research. This article is protected by copyright. All rights reserved.

In recent years, the development of portable platforms for performing fast and point-of-care analyses has drawn considerable attention for their wide variety of applications in life science. In this framework, tools combining magnetoresistive biosensors with magnetic markers have been widely studied in order to detect concentrations of specific molecules, demonstrating high sensitivity and ease of integration with conventional electronics. In this work, first, we develop a protocol for efficient hybridization of natural DNA; then, we show the detection of hybridization events involving natural DNA, namely genomic DNA extracted from the pathogenic bacterium Listeria monocytogenes, via a compact magnetic tunneling junction (MTJ)-based biosensing apparatus. The platform comprises dedicated portable electronic and microfluidic setups, enabling point-of-care biological assays. A sensitivity below the nM range is demonstrated. This work constitutes a step forward towards the development of portable lab-on-chip platforms, for the multiplexed detection of pathogenic health threats in food and food processing environment.

Although the traditional strategy of developing general medical treatments for heterogeneous patient populations has a well-established track record, the acknowledgment that one-size-does-not-fit-all is pushing health-care to enter a new era of tailored interventions. The advent of precision medicine is fueled by the high-throughput analysis of individual DNA variants and mRNA expression profiles. However, due to the role of proteins in providing a more direct view of disease states than genomics alone, the ability to comprehensively analyze protein alterations and post translational modifications (PTMs) is a necessary step to unravel disease mechanisms, develop novel biomarkers and targeted therapies. Protein and peptide microarrays can play a major role in this frame, due to high-throughput, low sample consumption and wide applicability. Here, their current role and potentialities are discussed through the review of some promising applications in the fields of PTMs analysis, enzyme screening, high-content immune-profiling and the phenotyping of extracellular vesicles.

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 bioprobes upon nonspecific adsorption onto cellulose substrates. Here, we address this issue by introducing a simple polymer-based strategy to obtain clickable cellulosic 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.

The mosquito-borne viral disease caused by the Dengue virus is an expanding global threat. Diagnosis in low-resource-settings and epidemiological surveillance urgently requires new immunoprobes for serological tests. Structure-based epitope prediction is an efficient method to design diagnostic peptidic probes able to reveal specific antibodies elicited in response to infections in patients&rsquo, sera. In this study, we focused on the Dengue viral envelope protein (E), computational analyses ranging from extensive Molecular Dynamics (MD) simulations and energy-decomposition-based prediction of potentially immunoreactive regions identified putative epitope sequences. Interestingly, one such epitope showed internal dynamic and energetic properties markedly different from those of other predicted sequences. The epitope was thus synthesized as a linear peptide, modified for chemoselective immobilization on microarrays and used in a serological assay to discriminate Dengue-infected individuals from healthy controls. The synthetic epitope probe showed a diagnostic performance comparable to that of the full antigen in terms of specificity and sensitivity. Given the high level of sequence identity among different flaviviruses, the epitope was immune-reactive towards Zika-infected sera as well. The results are discussed in the context of the quest for new possible structure-dynamics-based rules for the prediction of the immunoreactivity of selected antigenic regions with potential pan-flavivirus immunodiagnostic capacity.

Biosensing analytical platforms rely on the intimate structure–function relationship of immobilized probes. In this context, hydrogels are appealing semi-wet systems to locally confine biomolecules while preserving their structural integrity and function. Yet, limitations imposed by biomolecule diffusion rates or fabrication difficulties still hamper their broad application. Here, using a self-assembling peptide, a printable and self-adhesive hydrogel was obtained and applied to fabricate arrays of localized bio-functional 3D microenvironments on analytical interfaces. This soft matrix represents a robust and versatile material, allowing fast and selective tuning of analyte diffusion, which is exploited here to run in-gel immunoassays under solution-like conditions in an unprecedented (

Many cells of the nervous system have been shown to release exosomes, a subclass of secreted vesicles of endosomal origin capable of transferring biomolecules among cells: this transfer modality represents a novel physiological form of intercellular communication between neural cells. Herein, we demonstrated that progranulin (PGRN), a protein targeted to the classical secretory pathway, is also secreted in association with exosomes by human primary fibroblasts. Moreover, we demonstrated that null mutations in the progranulin gene (GRN), a major cause of frontotemporal dementia, strongly reduce the number of released exosomes and alter their composition. In vitro GRN silencing in SHSY-5Y cells confirmed a role of PGRN in the control of exosome release. It is believed that depletion of PGRN in the brain might cause neurodegeneration in GRN-associated frontotemporal dementia. We demonstrated that, along with shortage of the circulating PGRN, GRN null mutations alter intercellular communication. Thus, a better understanding of the role played by exosomes in GRN-associated neurodegeneration is crucial for the development of novel therapies for these diseases.

Antigen immunoreactivity is often determined by surface regions defined by the 3D juxtapositions of amino acids stretches that are not continuous in the linear sequence. As such, mimicking an antigen immunoreactivity by means of putative linear peptide epitopes for diagnostic purposes is not trivial. Here we present a straightforward and robust method to extend the reach of immune-diagnostic probes design by copresenting peptides belonging to the same antigenic surface. In this case study focused on a computationally predicted Zika virus NS1 protein putative antigenic region, we reached a diagnostic confidence by the oriented and spatially controlled coimmobilization of peptide sequences found adjacent within the protein fold, that cooperatively interacted to provide enhanced immunoreactivity with respect to single linear epitopes. Through our method, we were able to differentiate Zika infected individuals from healthy controls. Remarkably, our strategy fits well with the requirements to build high-throughput screening platforms of linear and mixed peptide libraries, and it could possibly facilitate the rapid identification of conformational immunoreactive regions.

Structure-based epitope prediction drives the design of diagnostic peptidic probes to reveal specific antibodies elicited in response to infections. We previously identified a highly immunoreactive epitope from the peptidoglycan-associated lipoprotein (Pal) antigen from Burkholderia pseudomallei, which could also diagnose Burkholderia cepacia infections. Here, considering the high phylogenetic conservation within Burkholderia species, we ask whether cross reactivity can be reciprocally displayed by the synthetic epitope from B. cenocepacia. We perform comparative analyses of the conformational preferences and diagnostic performances of the corresponding epitopes from the two Burkholderia species when presented in the context of the full-length proteins or as isolated peptides. The effects of conformation on the diagnostic potential and cross-reactivity of Pal peptide epitopes are rationalized on the basis of the 1.8 angstrom crystal structure of B. cenocepacia Pal and through computational analyses. Our results are discussed in the context of designing new diagnostic molecules for the early detection of infectious diseases.