EV Separation: Release of Intact Extracellular Vesicles Immunocaptured on Magnetic Particles.

Extracellular vesicles (EVs) have attracted considerable interest due to their role in cell-cell communication, disease diagnosis, and drug delivery. Despite their potential in the medical field, there is no consensus on the best method for separating micro- and nanovesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation of a single class of EVs, such as exosomes, is complex because blood and cell culture media contain many nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high-purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles usually requires harsh conditions that hinder their use in certain types of downstream analysis. A novel capture and release approach for small extracellular vesicles (sEVs) is presented based on DNA-directed immobilization of antiCD63 antibody. The flexible DNA linker increases the capture efficiency and allows for releasing EVs by exploiting the endonuclease activity of DNAse I. This separation protocol works under mild conditions, enabling the release of vesicles suitable for analysis by imaging techniques. In this study, sEVs recovered from plasma were characterized by established techniques for EV analysis, including nanoparticle tracking and transmission electron microscopy.

Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics.

Extracellular vesicles (EVs) have attracted significant attention as impactful diagnostic biomarkers, since their properties are closely related to specific clinical conditions. However, designing experiments that involve EVs phenotyping is usually highly challenging and time-consuming, due to laborious optimization steps that require very long or even overnight incubation durations. In this work, we demonstrate label-free, real-time detection, and phenotyping of extracellular vesicles binding to a multiplexed surface. With the ability for label-free kinetic binding measurements using the Interferometric Reflectance Imaging Sensor (IRIS) in a microfluidic chamber, we successfully optimize the capture reaction by tuning various assay conditions (incubation time, flow conditions, surface probe density, and specificity). A single (less than 1 h) experiment allows for characterization of binding affinities of the EVs to multiplexed probes. We demonstrate kinetic characterization of 18 different probe conditions, namely three different antibodies, each spotted at six different concentrations, simultaneously. The affinity characterization is then analyzed through a model that considers the complexity of multivalent binding of large structures to a carpet of probes and therefore introduces a combination of fast and slow association and dissociation parameters. Additionally, our results confirm higher affinity of EVs to aCD81 with respect to aCD9 and aCD63. Single-vesicle imaging measurements corroborate our findings, as well as confirming the EVs nature of the captured particles through fluorescence staining of the EVs membrane and cargo.

Simultaneous evaluation of multiple microarray surface chemistries through real-time interferometric imaging.

Surface chemistry is a crucial aspect for microarray modality biosensor development. The immobilization capability of the functionalized surface is indeed a limiting factor for the final yield of the binding reaction. In this work, we were able to simultaneously compare the functionality of protein ligands that were locally immobilized on different polymers, while on the same solid support, therefore demonstrating a new way of multiplexing. Our goal was to investigate, in a single experiment, both the immobilization efficiency of a group of reactive polymers and the resulting affinity of the tethered molecules. This idea was demonstrated by spotting many reactive polymers on a Si/SiO2 chip and depositing the molecular probes on the spots immediately after. As a proof of concept, we focused on which polymers would better immobilize a model protein (α-Lactalbumin) and a peptide (LAC-1). We successfully showed that this protocol is applicable to proteins and peptides with a good efficiency. By means of real-time binding measurements performed with the interferometric reflectance imaging sensor (IRIS), local functionalization proved to be comparable to the classical flat coating solution. The final outcome highlights the multiplexing power of this method: first, it allows to characterize dozens of polymers at once. 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 protocol, many types of molecules can be studied simultaneously and immobilization for each probe can be individually optimized.

Characterization of Receptor Binding Affinity for Vascular Endothelial Growth Factor with Interferometric Imaging Sensor.

Wet Age-related macular degeneration (AMD) is the leading cause of vision loss in industrialized nations, often resulting in blindness. Biologics, therapeutic agents derived from biological sources, have been effective in AMD, albeit at a high cost. Due to the high cost of AMD treatment, it is critical to determine the binding affinity of biologics to ensure their efficacy and make quantitative comparisons between different drugs. This study evaluates the in vitro VEGF binding affinity of two drugs used for treating wet AMD, monoclonal antibody-based bevacizumab and fusion protein-based aflibercept, performing quantitative binding measurements on an Interferometric Reflectance Imaging Sensor (IRIS) system. Both biologics can inhibit Vascular Endothelial Growth Factor (VEGF). For comparison, the therapeutic molecules were immobilized on to the same support in a microarray format, and their real-time binding interactions with recombinant human VEGF (rhVEGF) were measured using an IRIS. The results indicated that aflibercept exhibited a higher binding affinity to VEGF than bevacizumab, consistent with previous studies using ELISA and SPR. The IRIS system's innovative and cost-effective features, such as silicon-based semiconductor chips for enhanced signal detection and multiplexed analysis capability, offer new prospects in sensor technologies. These attributes make IRISs a promising tool for future applications in the development of therapeutic agents, specifically biologics.

The Effects of Three-Dimensional Ligand Immobilization on Kinetic Measurements in Biosensors.

The field of biosensing is in constant evolution, propelled by the need for sensitive, reliable platforms that provide consistent results, especially in the drug development industry, where small molecule characterization is of uttermost relevance. Kinetic characterization of small biochemicals is particularly challenging, and has required sensor developers to find solutions to compensate for the lack of sensitivity of their instruments. In this regard, surface chemistry plays a crucial role. The ligands need to be efficiently immobilized on the sensor surface, and probe distribution, maintenance of their native structure and efficient diffusion of the analyte to the surface need to be optimized. In order to enhance the signal generated by low molecular weight targets, surface plasmon resonance sensors utilize a high density of probes on the surface by employing a thick dextran matrix, resulting in a three-dimensional, multilayer distribution of molecules. Despite increasing the binding signal, this method can generate artifacts, due to the diffusion dependence of surface binding, affecting the accuracy of measured affinity constants. On the other hand, when working with planar surface chemistries, an incredibly high sensitivity is required for low molecular weight analytes, and furthermore the standard method for immobilizing single layers of molecules based on self-assembled monolayers (SAM) of epoxysilane has been demonstrated to promote protein denaturation, thus being far from ideal. Here, we will give a concise overview of the impact of tridimensional immobilization of ligands on label-free biosensors, mostly focusing on the effect of diffusion on binding affinity constants measurements. We will comment on how multilayering of probes is certainly useful in terms of increasing the sensitivity of the sensor, but can cause steric hindrance, mass transport and other diffusion effects. On the other hand, probe monolayers on epoxysilane chemistries do not undergo diffusion effect but rather other artifacts can occur due to probe distortion. Finally, a combination of tridimensional polymeric chemistry and probe monolayer is presented and reviewed, showing advantages and disadvantages over the other two approaches.

Sensitive and real-time detection of IgG using interferometric reflecting imaging sensor system.

Considering the limitations of well-known traditional detection techniques, innovative research studies have focused on the development of new sensors to offer label-free, highly sensitive, real-time, low-cost, and rapid detection for biomolecular interactions. In this study, we demonstrate immunoglobulin G (IgG) detection in aqueous solutions by using real-time and label-free kinetic measurements of the Interferometric Reflectance Imaging Sensor (IRIS) system. By performing kinetic characterization experiments, the sensor's performance is comprehensively evaluated and a high correlation coefficient value (>0.94) is obtained in the IgG concentration range of 1-50 µg/mL with a low detection limit (0.25 µg/mL or 1.67 nM). Moreover, the highly sensitive imaging system ensures accurate quantification and reliable validation of recorded binding events, offering new perspectives in terms of direct biomarker detection for clinical applications.

Bulk-Effect-Free Method for Binding Kinetic Measurements Enabling Small-Molecule Affinity Characterization.

Optical technologies for label-free detection are an attractive solution for monitoring molecular binding kinetics; however, these techniques measure the changes in the refractive index, making it difficult to distinguish surface binding from a change in the refractive index of the analyte solution in the proximity of the sensor surface. The solution refractive index changes, due to solvents, temperature changes, or pH variations, can create an unwanted background signal known as the bulk effect. Technologies such as biolayer interferometry and surface plasmon resonance offer no bulk-effect compensation, or they alternatively offer a reference channel to correct in postprocessing. Here, we present a virtually bulk-effect-free method, without a reference channel or any computational correction, for measuring kinetic binding using the interferometric reflectance imaging sensor (IRIS), an optical label-free biomolecular interaction analysis tool. Dynamic spectral illumination engineering, through tailored LED contributions, is combined with the IRIS technology to minimize the bulk effect, with the potential to enable kinetic measurements of a broader range of analytes. We demonstrate that the deviation in the reflectivity signal is reduced to ∼8 × 10-6 for a solution change from phosphate-buffered saline (PBS) (n = 1.335) to 1% dimethyl sulfoxide (DMSO) in PBS (n = 1.336). As a proof of concept, we applied the method to a biotin-streptavidin interaction, where biotin (MW = 244.3 Da) was dissolved at a final concentration of 1 µM in a 1% solution of DMSO in PBS and flowed over immobilized streptavidin. Clear binding results were obtained without a reference channel or any computational correction.

Multiplexed, High-Sensitivity Measurements of Antibody Affinity Using Interferometric Reflectance Imaging Sensor.

Anthrax lethal factor (LF) is one of the enzymatic components of the anthrax toxin responsible for the pathogenic responses of the anthrax disease. The ability to screen multiplexed ligands against LF and subsequently estimate the effective kinetic rates (kon and koff) and complementary binding behavior provides critical information useful in diagnostic and therapeutic development for anthrax. Tools such as biolayer interferometry (BLI) and surface plasmon resonance imaging (SPRi) have been developed for this purpose; however, these tools suffer from limitations such as signal jumps when the solution in the chamber is switched or low sensitivity. Here, we present multiplexed antibody affinity measurements obtained by the interferometric reflectance imaging sensor (IRIS), a highly sensitive, label-free optical biosensor, whose stability, simplicity, and imaging modality overcomes many of the limitations of other multiplexed methods. We compare the multiplexed binding results obtained with the IRIS system using two ligands targeting the anthrax lethal factor (LF) against previously published results obtained with more traditional surface plasmon resonance (SPR), which showed consistent results, as well as kinetic information previously unattainable with SPR. Additional exemplary data demonstrating multiplexed binding and the corresponding complementary binding to sequentially injected ligands provides an additional layer of information immediately useful to the researcher.

The Role of Surface Chemistry in the Efficacy of Protein and DNA Microarrays for Label-Free Detection: An Overview.

The importance of microarrays in diagnostics and medicine has drastically increased in the last few years. Nevertheless, the efficiency of a microarray-based assay intrinsically depends on the density and functionality of the biorecognition elements immobilized onto each sensor spot. Recently, researchers have put effort into developing new functionalization strategies and technologies which provide efficient immobilization and stability of any sort of molecule. Here, we present an overview of the most widely used methods of surface functionalization of microarray substrates, as well as the most recent advances in the field, and compare their performance in terms of optimal immobilization of the bioreceptor molecules. We focus on label-free microarrays and, in particular, we aim to describe the impact of surface chemistry on two types of microarray-based sensors: microarrays for single particle imaging and for label-free measurements of binding kinetics. Both protein and DNA microarrays are taken into consideration, and the effect of different polymeric coatings on the molecules' functionalities is critically analyzed.

A Reliable, Label Free Quality Control Method for the Production of DNA Microarrays with Clinical Applications.

The manufacture of a very high-quality microarray support is essential for the adoption of this assay format in clinical routine. In fact, poorly surface-bound probes can affect the diagnostic sensitivity or, in worst cases, lead to false negative results. Here we report on a reliable and easy quality control method for the evaluation of spotted probe properties in a microarray test, based on the Interferometric Reflectance Imaging Sensor (IRIS) system, a high-resolution label free technique able to evaluate the variation of the mass bound to a surface. In particular, we demonstrated that the IRIS analysis of microarray chips immediately after probe immobilization can detect the absence of probes, which recognizably causes a lack of signal when performing a test, with clinical relevance, using fluorescence detection. Moreover, the use of the IRIS technique allowed also to determine the optimal concentration of the probe, that has to be immobilized on the surface, to maximize the target recognition, thus the signal, but to avoid crowding effects. Finally, through this preliminary quality inspection it is possible to highlight differences in the immobilization chemistries. In particular, we have compared NHS ester versus click chemistry reactions using two different surface coatings, demonstrating that, in the diagnostic case used as an example (colorectal cancer) a higher probe density does not reflect a higher binding signal, probably because of a crowding effect.

Highly Multiplexed Label-Free Imaging Sensor for Accurate Quantification of Small-Molecule Binding Kinetics.

Investigating the binding interaction of small molecules to large ligands is a compelling task for the field of drug development, as well as agro-biotechnology, since a common trait of drugs and toxins is often a low molecular weight (MW). Here, we improve the limit of detection of the Interferometric Reflectance Imaging Sensor (IRIS), a label-free, highly multiplexed biosensor, to perform small-molecule screening. In this work, characterization of small molecules binding to immobilized probes in a microarray format is demonstrated, with a limit of detection of 1 pg/mm2 in mass density. First, as a proof of concept to show the impact of spatial and temporal averaging on the system noise, detection of biotin (MW = 244.3 Da) binding to a streptavidin-functionalized chip is performed and the parameters are tuned to achieve maximum signal-to-noise ratio (SNR ≈ 34). The optimized system is then applied to the screening of a 20-multiplexed antibody chip against fumonisin B1 (MW = 721.8 Da), a mycotoxin found in cereal grains. The simultaneously recorded binding curves yield an SNR ≈ 8. Five out of twenty antibodies are also screened against the toxin in a lateral flow assay, obtaining consistent results. With the demonstrated noise characteristics, further sensitivity improvements are expected with the advancement of camera sensor technology.

Instrument-Free Protein Microarray Fabrication for Accurate Affinity Measurements.

Protein microarrays have gained popularity as an attractive tool for various fields, including drug and biomarker development, and diagnostics. Thus, multiplexed binding affinity measurements in microarray format has become crucial. The preparation of microarray-based protein assays relies on precise dispensing of probe solutions to achieve efficient immobilization onto an active surface. The prohibitively high cost of equipment and the need for trained personnel to operate high complexity robotic spotters for microarray fabrication are significant detriments for researchers, especially for small laboratories with limited resources. Here, we present a low-cost, instrument-free dispensing technique by which users who are familiar with micropipetting can manually create multiplexed protein assays that show improved capture efficiency and noise level in comparison to that of the robotically spotted assays. In this study, we compare the efficiency of manually and robotically dispensed α-lactalbumin probe spots by analyzing the binding kinetics obtained from the interaction with anti-α-lactalbumin antibodies, using the interferometric reflectance imaging sensor platform. We show that the protein arrays prepared by micropipette manual spotting meet and exceed the performance of those prepared by state-of-the-art robotic spotters. These instrument-free protein assays have a higher binding signal (~4-fold improvement) and a ~3-fold better signal-to-noise ratio (SNR) in binding curves, when compared to the data acquired by averaging 75 robotic spots corresponding to the same effective sensor surface area. We demonstrate the potential of determining antigen-antibody binding coefficients in a 24-multiplexed chip format with less than 5% measurement error.