Extracellular vesicles selective capture by peptide-functionalized hollow fiber membranes.

Recently, membrane devices and processes have been applied for the separation and concentration of subcellular components such as extracellular vesicles (EVs), which play a diagnostic and therapeutic role in many pathological conditions. However, the separation and isolation of specific EV populations from other components found in biological fluids is still challenging. Here, we developed a peptide-functionalized hollow fiber (HF) membrane module to achieve the separation and enrichment of highly pure EVs derived from the culture media of human cardiac progenitor cells. The strategy is based on the functionalization of PSf HF membrane module with BPt, a peptide sequence able to bind nanovesicles characterized by highly curved membranes. HF membranes were modified by a nanometric coating with a copoly azide polymer to limit non-specific interactions and to enable the conjugation with peptide ligand by click chemistry reaction. The BPt-functionalized module was integrated into a TFF process to facilitate the design, rationalization, and optimization of EV isolation. This integration combined size-based transport of species with specific membrane sensing ligands. The TFF integrated BPt-functionalized membrane module demonstrated the ability to selectively capture EVs with diameter < 200 nm into the lumen of fibers while effectively removing contaminants such as albumin. The captured and released EVs contain the common markers including CD63, CD81, CD9 and syntenin-1. Moreover, they maintained a round shape morphology and structural integrity highlighting that this approach enables EVs concentration and purification with low shear stress. Additionally, it achieved the removal of contaminants such as albumin with high reliability and reproducibility, reaching a removal of 93%.

Serum antibody fingerprinting of SARS-CoV-2 variants in infected and vaccinated subjects by label-free microarray biosensor.

Both viral infection and vaccination affect the antibody repertoire of a person. Here, we demonstrate that the analysis of serum antibodies generates information not only on the virus type that caused the infection but also on the specific virus variant. We developed a rapid multiplex assay providing a fingerprint of serum antibodies against five different SARS-CoV-2 variants based on a microarray of virus antigens immobilized on the surface of a label-free reflectometric biosensor. We analyzed serum from the plasma of convalescent subjects and vaccinated volunteers and extracted individual antibody profiles of both total immunoglobulin Ig and IgA fractions. We found that Ig level profiles were strongly correlated with the specific variant of infection or vaccination and that vaccinated subjects displayed a larger quantity of total Ig and a lower fraction of IgA relative to the population of convalescent unvaccinated subjects.

Changes in glial cell activation and extracellular vesicles production precede the onset of disease symptoms in transgenic hSOD1G93A pigs.

SOD1 gene is associated with progressive motor neuron degeneration in the familiar forms of amyotrophic lateral sclerosis. Although studies on mutant human SOD1 transgenic rodent models have provided important insights into disease pathogenesis, they have not led to the discovery of early biomarkers or effective therapies in human disease. The recent generation of a transgenic swine model expressing the human pathological hSOD1G93A gene, which recapitulates the course of human disease, represents an interesting tool for the identification of early disease mechanisms and diagnostic biomarkers. Here, we analyze the activation state of CNS cells in transgenic pigs during the disease course and investigate whether changes in neuronal and glial cell activation state can be reflected by the amount of extracellular vesicles they release in biological fluids. To assess the activation state of neural cells, we performed a biochemical characterization of neurons and glial cells in the spinal cords of hSOD1G93A pigs during the disease course. Quantification of EVs of CNS cell origin was performed in cerebrospinal fluid and plasma of transgenic pigs at different disease stages by Western blot and peptide microarray analyses. We report an early activation of oligodendrocytes in hSOD1G93A transgenic tissue followed by astrocyte and microglia activation, especially in animals with motor symptoms. At late asymptomatic stage, EV production from astrocytes and microglia is increased in the cerebrospinal fluid, but not in the plasma, of transgenic pigs reflecting donor cell activation in the spinal cord. Estimation of EV production by biochemical analyses is corroborated by direct quantification of neuron- and microglia-derived EVs in the cerebrospinal fluid by a Membrane Sensing Peptide enabled on-chip analysis that provides fast results and low sample consumption. Collectively, our data indicate that alteration in astrocytic EV production precedes the onset of disease symptoms in the hSODG93A swine model, mirroring donor cell activation in the spinal cord, and suggest that EV measurements from the cells first activated in the ALS pig model, i.e. OPCs, may further improve early disease detection.

International Society for Extracellular Vesicles Workshop. QuantitatEVs: multiscale analyses, from bulk to single extracellular vesicle.

The 'QuantitatEVs: multiscale analyses, from bulk to single vesicle' workshop aimed to discuss quantitative strategies and harmonized wet and computational approaches toward the comprehensive analysis of extracellular vesicles (EVs) from bulk to single vesicle analyses with a special focus on emerging technologies. The workshop covered the key issues in the quantitative analysis of different EV-associated molecular components and EV biophysical features, which are considered the core of EV-associated biomarker discovery and validation for their clinical translation. The in-person-only workshop was held in Trento, Italy, from January 31st to February 2nd, 2023, and continued in Milan on February 3rd with "Next Generation EVs", a satellite event dedicated to early career researchers (ECR). This report summarizes the main topics and outcomes of the workshop.

Digital Detection of Single Virus Particles by Multi-Spot, Label-Free Imaging Biosensor on Anti-Reflective Glass.

Rapid detection of whole virus particles in biological or environmental samples represents an unmet need for the containment of infectious diseases. Here, an optical device enabling the enumeration of single virion particles binding on antibody or aptamers immobilized on a surface with anti-reflective coating is described. In this regime, nanoparticles adhering to the sensor surface provide localized contributions to the reflected field that become detectable because of their mixing with the interfering waves in the reflection direction. Thus, these settings are exploited to realize a scan-free, label-free, micro-array-type digital assay on a disposable cartridge, in which the virion counting takes place in wide field-of-view imaging. With this approach we could quantify, by enumeration, different variants of SARS-CoV-2 virions interacting with antibodies and aptamers immobilized on different spots. For all tested variants, the aptamers showed larger affinity but lower specificity relative to the antibodies. It is found that the combination of different probes on the same surface enables increasing specificity of detection and dynamic range.

A Fully Integrated Miniaturized Optical Biosensor for Fast and Multiplexing Plasmonic Detection of High- and Low-Molecular-Weight Analytes.

Optical biosensors based on plasmonic sensing schemes combine high sensitivity and selectivity with label-free detection. However, the use of bulky optical components is still hampering the possibility of obtaining miniaturized systems required for analysis in real settings. Here, a fully miniaturized optical biosensor prototype based on plasmonic detection is demonstrated, which enables fast and multiplex sensing of analytes with high- and low molecular weight (80 000 and 582 Da) as quality and safety parameters for milk: a protein (lactoferrin) and an antibiotic (streptomycin). The optical sensor is based on the smart integration of: i) miniaturized organic optoelectronic devices used as light-emitting and light-sensing elements and ii) a functionalized nanostructured plasmonic grating for highly sensitive and specific localized surface plasmon resonance (SPR) detection. The sensor provides quantitative and linear response reaching a limit of detection of 10-4 refractive index units once it is calibrated by standard solutions. Analyte-specific and rapid (15 min long) immunoassay-based detection is demonstrated for both targets. By using a custom algorithm based on principal-component analysis, a linear dose-response curve is constructed which correlates with a limit of detection (LOD) as low as 3.7 µg mL-1 for lactoferrin, thus assessing that the miniaturized optical biosensor is well-aligned with the chosen reference benchtop SPR method.

Dual-Domain Reporter Approach for Multiplex Identification of Major SARS-CoV-2 Variants of Concern in a Microarray-Based Assay.

Since the emergence of the COVID-19 pandemic in December 2019, the SARS-CoV-2 virus continues to evolve into many variants emerging around the world. To enable regular surveillance and timely adjustments in public health interventions, it is of the utmost importance to accurately monitor and track the distribution of variants as rapidly as possible. Genome sequencing is the gold standard for monitoring the evolution of the virus, but it is not cost-effective, rapid and easily accessible. We have developed a microarray-based assay that can distinguish known viral variants present in clinical samples by simultaneously detecting mutations in the Spike protein gene. In this method, the viral nucleic acid, extracted from nasopharyngeal swabs, after RT-PCR, hybridizes in solution with specific dual-domain oligonucleotide reporters. The domains complementary to the Spike protein gene sequence encompassing the mutation form hybrids in solution that are directed by the second domain ("barcode" domain) at specific locations on coated silicon chips. The method utilizes characteristic fluorescence signatures to unequivocally differentiate, in a single assay, different known SARS-CoV-2 variants. In the nasopharyngeal swabs of patients, this multiplex system was able to genotype the variants which have caused waves of infections worldwide, reported by the WHO as being of concern (VOCs), namely Alpha, Beta, Gamma, Delta and Omicron variants.

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.

A Bifunctional Polymeric Coating for the Co-Immobilization of Proteins and Peptides on Microarray Substrates.

The analytical performance of the microarray technique in screening the affinity and reactivity of molecules toward 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, 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 bifunctional copolymer has been obtained by partial post-polymerization modification of the functional groups of a common precursor. This strategy represents a convenient method to reduce the number of analyses, therefore possible systematic or random errors, besides offering a drastic shortage in time, reagents, and costs.

Peptide-Based Sensor and Microfluidic Platform for IgG Antibody Detection by Differential Impedance Sensing.

Serological assays enable infection screening as relatively easy-to-operate approaches compared with standard methods. In addition, to be relevant for early diagnosis, specific antibody detection is important for epidemiological surveillance and quantitative detection has potential significance for evaluating the severity and prognosis of different diseases.Here, we describe the detection process based on differential impedance sensing of IgG antibodies labeled with polystyrene nanoparticles. The electrode differential configuration, the amplification with nanoparticle functionalization, the electronic reading, and the microfluidic protocol allow to reach a limit of detection below 100 pg/mL for commercial IgG antibody spiked in buffer.

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.

Active Opto-Magnetic Biosensing with Silicon Microring Resonators.

Integrated optical biosensors are gaining increasing attention for their exploitation in lab-on-chip platforms. The standard detection method is based on the measurement of the shift of some optical quantity induced by the immobilization of target molecules at the surface of an integrated optical element upon biomolecular recognition. However, this requires the acquisition of said quantity over the whole hybridization process, which can take hours, during which any external perturbation (e.g., temperature and mechanical instability) can seriously affect the measurement and contribute to a sizeable percentage of invalid tests. Here, we present a different assay concept, named Opto-Magnetic biosensing, allowing us to optically measure off-line (i.e., post hybridization) tiny variations of the effective refractive index seen by microring resonators upon immobilization of magnetic nanoparticles labelling target molecules. Bound magnetic nanoparticles are driven in oscillation by an external AC magnetic field and the corresponding modulation of the microring transfer function, due to the effective refractive index dependence on the position of the particles above the ring, is recorded using a lock-in technique. For a model system of DNA biomolecular recognition we reached a lowest detected concentration on the order of 10 pm, and data analysis shows an expected effective refractive index variation limit of detection of 7.5×10-9 RIU, in a measurement time of just a few seconds.

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.

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.

Differential Impedance Sensing platform for high selectivity antibody detection down to few counts: A case study on Dengue Virus.

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.

A bi-functional polymeric coating for the co-immobilization of proteins and peptides on microarray substrates.

The analytical performance of the microarray technique in screening the affinity and reactivity of molecules 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, 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 of a common precursor. A NMR characterization of the copolymer was conducted to quantify the percentage of NAS that has been transformed into azido groups. The polymer was used to coat surfaces onto which both native antibodies and alkyne modified peptides were immobilized, to perform the phenotype characterization of extracellular vesicles (EVs). 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.

Copolymer Coatings for DNA Biosensors: Effect of Charges and Immobilization Chemistries on Yield, Strength and Kinetics of Hybridization.

The physical-chemical properties of the surface of DNA microarrays and biosensors play a fundamental role in their performance, affecting the signal's amplitude and the strength and kinetics of binding. We studied how the interaction parameters vary for hybridization of complementary 23-mer DNA, when the probe strands are immobilized on different copolymers, which coat the surface of an optical, label-free biosensor. Copolymers of N, N-dimethylacrylamide bringing either a different type or density of sites for covalent immobilization of DNA probes, or different backbone charges, were used to functionalize the surface of a Reflective Phantom Interface multispot biosensor made of a glass prism with a silicon dioxide antireflective layer. By analyzing the kinetic hybridization curves at different probe surface densities and target concentrations in solution, we found that all the tested coatings displayed a common association kinetics of about 9 × 104 M-1·s-1 at small probe density, decreasing by one order of magnitude close to the surface saturation of probes. In contrast, both the yield of hybridization and the dissociation kinetics, and hence the equilibrium constant, depend on the type of copolymer coating. Nearly doubled signal amplitudes, although equilibrium dissociation constant was as large as 4 nM, were obtained by immobilizing the probe via click chemistry, whereas amine-based immobilization combined with passivation with diamine carrying positive charges granted much slower dissociation kinetics, yielding an equilibrium dissociation constant as low as 0.5 nM. These results offer quantitative criteria for an optimal selection of surface copolymer coatings, depending on the application.

Decoding distinctive features of plasma extracellular vesicles in amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic 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. METHODS: We 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. RESULTS: Our 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. CONCLUSIONS: Our 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.

Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis.

Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators' safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.

CovidArray: A Microarray-Based Assay with High Sensitivity for the Detection of Sars-Cov-2 in Nasopharyngeal Swabs.

A new coronavirus (SARS-CoV-2) caused the current coronavirus disease (Covid-19) epidemic. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used as the gold standard for clinical detection of SARS-CoV-2. Under ideal conditions, RT-qPCR Covid-19 assays have analytical sensitivity and specificity greater than 95%. However, when the sample panel is enlarged including asymptomatic individuals, the sensitivity decreases and false negatives are reported. Moreover, RT-qPCR requires up to 3-6 h with most of the time involved in RNA extraction from swab samples. We introduce CovidArray, a microarray-based assay, to detect SARS-CoV-2 markers N1 and N2 in the nasopharyngeal swabs. The method is based on solid-phase hybridization of fluorescently-labeled amplicons upon RNA extraction and reverse transcription. This approach combines the physical-optical properties of the silicon substrate with the surface chemistry used to coat the substrate to obtain a diagnostic tool of great sensitivity. Furthermore, we used an innovative approach, RNAGEM, to extract and purify viral RNA in less than 15 min. We correctly assigned 12 nasopharyngeal swabs, previously analyzed by RT-qPCR. Thanks to the CovidArray sensitivity we were able to identify a false-negative sample. CovidArray is the first DNA microarray-based assay to detect viral genes in the swabs. Its high sensitivity and the innovative viral RNA extraction by RNAGEM allows the reduction of both the amount of false-negative results and the total analysis time to about 2 h.