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.766466 (INDEX) - Horizon 2020 Framework Programmehttps://s3-eu-west-1.amazonaws.com/itempdf74155353254prod/8976347/Simultaneous_Evaluation_of_Multiple_Microarray_Surface_Chemistries_Through_Real-Time_Interferometric_Imaging_v1.pdfFirst author draf

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.1941195 - National Science Foundation; Boston University; 2027109 - National Science FoundationPublished versio

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.SO766466 (INDEX) - European Union Horizon 2020; NSF iCorps Award n°5242027109 - National Science Foundation; NSF-TT PFI Award n°1941195 - National Science FoundationPublished versio

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.Ignition Program - Boston University; INDEX (766466) - Horizon 2020; iCorps (2027109) - National Science Foundation; PFI-TT (1941195) - National Science FoundationPublished versio

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.Published versio

Interferometric imaging for high sensitivity multiplexed molecular measurements

The diagnostic and pharmaceutical industries rely on tools for characterizing, discovering, and developing bio-molecular interactions. Diagnostic assays require high affinity capture probes and binding specificity for accurate detection of biomarkers. Selection of drug candidates depends on the drug residency time and duration of drug action. Further, biologic drugs can induce anti-drug antibodies, which require characterization to determine the impact on the drug safety and efficacy. Label-free biosensors are an attractive solution for analyzing these and other bio-molecular interactions because they provide information based on the characteristics of the molecules themselves, without disturbing the native biological systems by labeling. While label-free biosensors can analyze a broad range of analytes, small molecular weight analytes (molecular weight < 1kDa) are the most challenging. Affinity measurements for small molecular weight targets require high sensitivity and long-term signal stability. Additional difficulties occur with different liquid refractive indices that result from to temperature, composition, or matrix effects of sensor surfaces. Some solutions utitlize strong solvents to increase the solubility of small molecules, which also alter the refractive index. Moreover, diagnostics require affinity measurements in relevant solutions, of various refractive indices. When a refractive index difference exists between the analyte solution and the wash buffer, a background signal is generated, referred to as the bulk effect, obscuring the small signal due to surface binding in the presence of large fluctuations due to variations of the optical refractive index of the solutions. The signal generated by low molecular weight analytes is small, and conventional wisdom tends toward signal amplification or resonance for detection of these small signals. With this approach, Surface Plasmon Resonance (SPR) has become the gold standard in affinity measurement technologies. SPR is an expensive and complex technology that is highly susceptible to the bulk effect. SPR uses a reference channel to correct for the bulk effect in post-processing, which requires high precision and sophisticated temperature control, further increasing the cost and complexity. Additionally, multiplexing is desirable as it allows for simultaneous measurements of multiple ligands; however, multiplexing is only possible in the imaging modality of SPR, which has lower sensitivity and difficulty with referencing. The Interferometric Reflectance Imaging Sensor (IRIS) is a low-cost, optical label-free bio-molecular interaction analysis technology capable of providing precise binding affinity measurements; however, limitations in sensitivity and usability have previously prevented its widespread adaptation. Overcoming these limitations requires the implementation of automation, compact and easy-to-use instrumentation, and increased sensitivity. Here, we explore methods for improved sensitivity and usability. We achieve noise reduction and elimination of solution artifacts (bulk effect) through engineered illumination uniformity and temporal and spatial image processing. To validate these methods, we experimentally analyze small molecule molecular interactions to demonstrate highly sensitive kinetic binding measurements, independent of solution refractive index.2023-09-24T00:00:00

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

Frequency Selective Hollow-core Negative Curvature Fiber Design via Nest-clad Poling

A frequency selective negative curvature fiber design is proposed for potential optical sensing applications. Loss modulation depth of 11.78 dB and differential loss ratio of 51 between the baseline and filtered wavelengths were achieved

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 of 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 hour) 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 antobodies, each spotted at six different concentrations, simultaneously. The affinity characterization is then analyzed through a model which 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. </p

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 ko f f ) 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