Arrayed Imaging Reflectometry Research Articles

Two Decades of Arrayed Imaging Reflectometry for Sensitive, High-Throughput Biosensing.

Arrayed imaging reflectometry (AIR), first introduced in 2004, is a thin-film interference sensor technique that optimizes optical properties (angle of incidence, polarization, substrate refractive index, and thickness) to create a condition of total destructive interference at the surface of a silicon substrate. The advantages of AIR are its sensitivity, dynamic range, multiplex capability, and high-throughput compatibility. AIR has been used for the detection of antibodies against coronaviruses, influenza viruses, Staphylococcus aureus, and human autoantigens. It has also shown utility in detection of cytokines, with sensitivity comparable to bead-based and ELISA assays. Not limited to antibodies or antigens, mixed aptamer and protein arrays as well as glycan arrays have been employed in AIR for differentiating influenza strains. Mixed arrays using direct and competitive inhibition assays have enabled simultaneous measurement of cytokines and small molecules. Finally, AIR has also been used to measure affinity constants, kinetic and at equilibrium. In this review, we give an overview of AIR biosensing technologies and present the latest AIR advances.

Arrayed Imaging Reflectometry monitoring of anti-viral antibody production throughout vaccination and breakthrough Covid-19.

Immune responses to COVID-19 infection and vaccination are individual and varied. There is a need to understand the timeline of vaccination efficacy against current and yet to be discovered viral mutations. Assessing immunity to SARS-CoV-2 in the context of immunity to other respiratory viruses is also valuable. Here we demonstrate the capability of a fully automated prototype Arrayed Imaging Reflectometry system to perform reliable longitudinal serology against a 34-plex respiratory array. The array contains antigens for respiratory syncytial virus, seasonal influenza, common human coronaviruses, MERS, SARS-CoV-1, and SARS-CoV-2. AIR measures a change in reflectivity due to the binding of serum antibodies to the antigens on the array. Samples were collected from convalescent COVID-19 donors and individuals vaccinated with a two-dose mRNA vaccine regimen. Vaccinated samples were collected prior to the first dose, one week after the first dose, one week after the second dose, and monthly thereafter. Information following booster dose and/or breakthrough infection is included for a subset of subjects. Longitudinal samples of vaccinated individuals demonstrate a rise and fall of SARS-CoV-2 spike antibodies in agreement with general knowledge of the adaptive immune response and other studies. Linear Regression analysis was performed to understand the relationship between antibodies binding to different antigens on the array. Our analysis identified strong correlations between closely related influenza virus strains as well as correlations between SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. A small test of using diluted whole blood from a fingerstick provided clean arrays with antibody binding comparable to serum. Potential applications include assessing immunity in the context of exposure to multiple respiratory viruses, clinical serology, population monitoring to facilitate public health recommendations, and vaccine development against new viruses and virus mutations.

StaphAIR: A Label-Free Antigen Microarray Approach to Detecting Anti-Staphylococcus aureus Antibody Responses in Orthopedic Infections.

Arrayed imaging reflectometry (AIR) is an optical biosensor platform for simple, multiplex measurement of antigen-specific antibody responses in patient blood samples. Here, we report the development of StaphAIR, an 8-plex Staphylococcus aureus antigen array on the AIR platform for profiling antigen-specific anti-S. aureus humoral immune responses. Initial validation experiments with mouse and humanized monoclonal antibodies against the S. aureus autolysin glucosaminidase (Gmd) domain, and subsequent testing with dilution series of pooled positive human serum confirmed analytically robust behavior of the array, with all antigens displaying Langmuir-type dose-response curves. Testing a cohort of 82 patients with S. aureus musculoskeletal infections (MSKI) and 30 healthy individuals enabled discrimination of individual patient responses to different S. aureus antigens, with statistical significance between osteomyelitis patients and controls obtained overall for four individual antigens (IsdA, IsdB, Gmd, and SCIN). Multivariate analyses of the antibody titers obtained from StaphAIR revealed its utility as a potential diagnostic tool for detecting S. aureus MSKI (area under the receiver operating characteristic curve (AUC) > 0.85). We conclude that StaphAIR has utility as a high-throughput immunoassay for studying and diagnosing osteomyelitis in patients.

Label-Free, Multiplex Glycan Microarray Biosensor for Influenza Virus Detection.

Newly emerging influenza viruses adapted from animal species pose significant pandemic threats to public health. An understanding of hemagglutinin (HA) receptor-binding specificity to host receptors is key to studying the adaptation of influenza viruses in humans. This information may be particularly useful for predicting the emergence of a pandemic outbreak. Therefore, high-throughput sensing technologies able to profile HA receptor binding can facilitate studies of influenza virus evolution and adaptation in humans. As a step toward this goal, we have prepared glycan-based receptor analogue microarrays on the Arrayed Imaging Reflectometry (AIR) platform. These arrays demonstrate label-free, multiplex detection and discrimination between human and avian influenza viruses. Microarrays consisting of glycan probes with 2,6 and 2,3 linkages were prepared. After first confirming their ability to capture lectins (carbohydrate-binding proteins) with known specificities, we observed that the arrays were able to discriminate between and quantify human pandemic influenza A/California/07/2009 (H1N1pdm) and avian A/Netherlands/1/2000 (H13N8) influenza viruses, respectively. As the method may be expanded to large numbers of glycans (>100) and virus subtypes (H1-H18), we anticipate it can be applied to systematically evaluate influenza virus adaptation in humans. In turn, this will facilitate global influenza surveillance and serve as a new tool enabling health organizations, governments, research institutes, and laboratories to react quickly in the face of a pandemic outbreak.

Immune Profiling System Redirected in Light of COVID-19

Genetic Engineering & Biotechnology NewsVol. 40, No. 11 DepartmentsImmune Profiling System Redirected in Light of COVID-19Adarza BioSystems has adapted its arrayed imaging reflectometry technology to characterize the human antibody response to SARSCoV-2 and other virusesGail DuttonGail DuttonSearch for more papers by this authorPublished Online:5 Nov 2020https://doi.org/10.1089/gen.40.11.04AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 40Issue 11Nov 2020 Information© 2020 by GEN PublishingTo cite this article:Gail Dutton.Immune Profiling System Redirected in Light of COVID-19.Genetic Engineering & Biotechnology News.Nov 2020.14-15.http://doi.org/10.1089/gen.40.11.04Published in Volume: 40 Issue 11: November 5, 2020PDF download

A Stable Biotin-Streptavidin Surface Enables Multiplex, Label-Free Protein Detection by Aptamer and Aptamer-Protein Arrays Using Arrayed Imaging Reflectometry.

While label-free multiplex sensor technology enables “mixing and matching” of different capture molecules in principle, in practice this has been rarely (if ever) demonstrated. To fill this gap, we developed protocols for the preparation of mixed aptamer-protein arrays on the arrayed imaging reflectometry (AIR) sensing platform using streptavidin as a common attachment point for both biotinylated proteins and aptamers. Doing so required overcoming the noted instability of dried streptavidin monolayers on surfaces. After characterizing this degradation, stable surfaces were obtained using a commercial microarray product. Microarraying through the layer of stabilizer then provided mixed aptamer-antibody arrays. We demonstrate that sensor arrays prepared in this manner are suitable for several probes (thrombin and TGF-β1 aptamers; avi-tagged protein) and targets.

Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients

Detection of antibodies to upper respiratory pathogens is critical to surveillance, assessment of the immune status of individuals, vaccine development, and basic biology. The urgent need for antibody detection tools has proven particularly acute in the COVID-19 era. We report a multiplex label-free antigen microarray on the Arrayed Imaging Reflectometry (AIR) platform for detection of antibodies to SARS-CoV-2, SARS-CoV-1, MERS, three circulating coronavirus strains (HKU1, 229E, OC43) and three strains of influenza. We find that the array is readily able to distinguish uninfected from convalescent COVID-19 subjects, and provides quantitative information about total Ig, as well as IgG- and IgM-specific responses.

Abstract PO-045: Multiplex analysis of coronavirus and influenza antibodies using the ZIVA multiplex platform

Abstract Detection of antibodies to upper respiratory pathogens is critical to surveillance, assessment of the immune status of individuals, vaccine development, and basic biology. The urgent need for antibody detection tools has proven particularly acute in the COVID-19 era. We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon chip, to create a multiplex antibody detection array. ZIVA is a new multiplex protein detection platform that leverages AIR™ technology. We have used ZIVA technology to develop an acute respiratory virus panel capable of detecting the presence of antibodies specific for coronavirus and influenza including, but not limited to, detection of antibodies to SARS-CoV-2, SARS-CoV-1, MERS, three circulating coronavirus strains (HKU1, 229E, OC43), and multiple strains of influenza. We find that the array is readily able to distinguish uninfected from convalescent COVID-19 subjects and provides quantitative information about total IgG, as well as IgG- and IgM-specific responses. Samples may also be evaluated with ZIVA cytokine assays to monitor the cytokine storm in conjunction with viral antibody response. Citation Format: Jon Schmuke, Ben Miller, Linda Meeh, Byran Witherbee. Multiplex analysis of coronavirus and influenza antibodies using the ZIVA multiplex platform [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr PO-045.

Abstract 1127: Adarza's ZIVA multiplex protein detection platform utilizes a label-free technology to simultaneously monitor murine and human inflammation cytokines in a CAR-T model system

Abstract We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon biosensor, to create a multiplex antibody array for the detection of human and murine serum cytokines and inflammatory markers. The biosensor is capable of providing quantitative protein concentrations, with limits of detection in the low picograms per mL. Provided in a standard 96-well microplate format, the label-free nature of the array enables flexible creation of standard or custom panels for development of model systems. Here, we demonstrate a custom 22 plex panel developed in collaboration with WUGEN Inc. to provide multi-species cytokine analysis in humanized CAR-T cell models for the evaluation of biologic product efficacy and safety. Samples from in vitro and in vivo models developed for the evaluation of CAR-T activity were analyzed and demonstrated a novel platform for concurrent cross-species cytokine analysis on a single array. Results are evaluated for sensitivity, precision, and accuracy with ZIVA assays comparable to traditional ELISAs. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the ZIVA instrument, the technology has been designed for the ultimate user-experience. Citation Format: Jon J. Schmuke, Alun Carter, You Zhou, Ernest Mueller, Matt Cooper. Adarza's ZIVA multiplex protein detection platform utilizes a label-free technology to simultaneously monitor murine and human inflammation cytokines in a CAR-T model system [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1127.

Adarza’s ZIVA multiplex protein detection platform demonstrates flexibility with custom targets for monitoring both inflammation and anti-drug antibody response

Abstract We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon chip, to create a multiplex antibody array. ZIVA is a new multiplex protein detection platform that leverages AIR™ technology. Understanding the immunogenicity of a therapeutic protein is critical to the development of bioactive stability and efficacy. ZIVA technology can be customized to monitor for multiple biomarker types including markers of inflammation as well as specific ADA targets. In this example, biosensors were custom printed to monitor for the presence of an anti-drug antibodies generated against a single chain antibody presented on the surface of engineered CAR-T cells. Samples that are positive for ADA response may also be evaluated for presence of neutralizing antibodies (NAB) by evaluating specific controls also included on the biosensors. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the ZIVA instrument, the technology has been designed for the ultimate user-experience.

Adarza’s ZIVA multiplex protein detection platform offers format for ultra-high sensitivity

Abstract We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon chip, to create a multiplex antibody array for the detection of both low and highly abundant targets. ZIVA is a new multiplex protein detection platform that leverages AIR™ technology. Because of the label-free properties, sensitivity can be “fine-tuned” by leveraging a variety of mass enhancing options. We have previously demonstrated assays that respond in the nanogram to picogram/ml sensitivity using one format for enhancement. Herein, we demonstrate a newly developed mass enhancement option that increases sensitivity from very low pg/ml to mid-femtogram/ml levels. A custom designed biosensor with markers for inflammation and other custom needs was used to demonstrate this ultra-high sensitivity format in a multiplex array. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the ZIVA instrument, the technology has been designed for the ultimate user-experience.

Adarza’s ZIVA multiplex protein detection platform utilizes a label-free technology to simultaneously monitor murine and human inflammation cytokines in a CAR-T model system

Abstract We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon biosensor, to create a multiplex antibody array for the detection of human and murine serum cytokines and inflammatory markers. The biosensor is capable of providing quantitative protein concentrations, with limits of detection in the low picograms per mL. The flexibility of this system can also reduce the need to generate a series of sample dilutions in order to get results in a limited detection range by enabling the development of each assay with sensitivity expectations for each target on a chip. Provided in a standard 96-well microplate format, the label-free nature of the array enables flexible creation of standard or custom panels for development of model systems. Here, we demonstrate a custom 22 plex panel developed in collaboration with WUGEN Inc. to provide multi-species cytokine analysis in humanized CAR-T cell models for the evaluation of biologic product efficacy and safety. Samples from in vitro and in vivo models developed for the evaluation of CAR-T activity were analyzed and demonstrated a novel platform for concurrent cross-species cytokine analysis on a single array. Results are evaluated for sensitivity, precision, and accuracy with ZIVA assays comparable to traditional ELISAs. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the ZIVA instrument, the technology has been designed for the ultimate user-experience.

Towards simultaneous quantification of protease inhibitors and inflammatory biomarkers in serum for people living with HIV

Detecting small and big molecules together: simultaneous quantification of protease inhibitor (DRV) and inflammatory biomarker in serum by Arrayed Imaging Reflectometry (AIR).

Adarza’s Ziva technology platform enables highly multiplexed label-free detection of serum analytes

Abstract New multiplex, label-free methods for the sensitive and quantitative detection of serum proteins are highly desirable, as they have the potential to dramatically accelerate biomedical research in the age of multi-omic, big data driven personalized medicine. We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon chip, to create a multiplex antibody array for the detection of human serum cytokines and inflammatory markers. The chip is able to provide quantitative information about protein concentrations, with limits of detection in picograms per mL. Provided in a standard 96-well microplate format, the label-free nature of the array will permit rapid expansion in plex size and enable simple, flexible creation of custom panels as there are no secondary antibodies or labels to cross-react. Here we demonstrate validation parameters of sensitivity, precision, and accuracy for inflammatory markers (>20) in a new multiplex technology suitable for analyzing trends across cytokine and inflammatory marker profiles comparable to traditional ELISAs. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the Ziva instrument, the technology has been designed for the ultimate user-experience.

Crowd on a Chip: Label-Free Human Monoclonal Antibody Arrays for Serotyping Influenza.

Rapid changes in influenza A virus (IAV) antigenicity create challenges in surveillance, disease diagnosis, and vaccine development. Further, serological methods for studying antigenic properties of influenza viruses often rely on animal models and therefore may not fully reflect the dynamics of human immunity. We hypothesized that arrays of human monoclonal antibodies (hmAbs) to influenza could be employed in a pattern-recognition approach to expedite IAV serology and to study the antigenic evolution of newly emerging viruses. Using the multiplex, label-free Arrayed Imaging Reflectometry (AIR) platform, we have demonstrated that such arrays readily discriminated among various subtypes of IAVs, including H1, H3 seasonal strains, and avian-sourced human H7 viruses. Array responses also allowed the first determination of antigenic relationships among IAV strains directly from hmAb responses. Finally, correlation analysis of antibody binding to all tested IAV subtypes allowed efficient identification of broadly reactive clones. In addition to specific applications in the context of understanding influenza biology with potential utility in "universal" flu vaccine development, these studies validate AIR as a platform technology for studying antigenic properties of viruses and also antibody properties in a high-throughput manner. We further anticipate that this approach will facilitate advances in the study of other viral pathogens.

Label-free microarray-based detection of autoantibodies in human serum

Multiplex assays for autoantibodies have shown utility both in research towards understanding the basic biology of autoimmune disease, and as tools for clinical diagnosis. New label-free multiplex analysis methods have the potential to streamline both the process of assay development and assay workflow. We report fabrication and testing of a 5-plex autoantigen microarray using the Arrayed Imaging Reflectometry (AIR) platform. This label-free technology provides rapid, sensitive, and quantitative detection of an arbitrary number of analytes in a standard multiwell format. In this work, we demonstrate that AIR is able to detect antibodies to Ro60, La/SSB, Scl-70, BicD2, and Ro52 in single-donor human serum samples with multiplex results comparable to singleplex ELISA or Luminex assays.

A label-free, multiplex competitive assay for small molecule pollutants.

Understanding the amount of exposure individuals have had to common chemical pollutants critically requires the ability to detect those compounds in a simple, sensitive, and specific manner. Doing so using label-free biosensor technology has proven challenging, however, given the small molecular weight of many pollutants of interest. To address this issue, we report the development of a pollutant microarray based on the label-free arrayed imaging reflectometry (AIR) detection platform. The sensor is able to detect three common environmental contaminants (benzo[a]pyrene, bisphenol A, and acrolein) in human serum via a competitive binding scheme.

A Multiplex Label-Free Approach to Avian Influenza Surveillance and Serology.

Influenza serology has traditionally relied on techniques such as hemagglutination inhibition, microneutralization, and ELISA. These assays are complex, challenging to implement in a format allowing detection of several types of antibody-analyte interactions at once (multiplex), and troublesome to implement in the field. As an alternative, we have developed a hemagglutinin microarray on the Arrayed Imaging Reflectometry (AIR) platform. AIR provides sensitive, rapid, and label-free multiplex detection of targets in complex analyte samples such as serum. In preliminary work, we demonstrated the application of this array to the testing of human samples from a vaccine trial. Here, we report the application of an expanded label-free hemagglutinin microarray to the analysis of avian serum samples. Samples from influenza virus challenge experiments in mallards yielded strong, selective detection of antibodies to the challenge antigen in most cases. Samples acquired in the field from mallards were also analyzed, and compared with viral hemagglutinin inhibition and microneutralization assays. We find that the AIR hemagglutinin microarray can provide a simple and robust alternative to standard methods, offering substantially greater information density from a simple workflow.

Functionalized Polymer Microgel Particles Enable Customizable Production of Label-Free Sensor Arrays.

Probe molecule immobilization onto surfaces is a critical step in the production of many analytical devices, including labeled and label-free microarrays. New methods to increase the density and uniformity of probe deposition have the potential to significantly enhance the ultimate limits of detection and reproducibility. Hydrogel-based materials have been employed in the past to provide a 3D protein-friendly surface for deposition of antibodies and nucleic acids. However, these methods are susceptible to variation during polymerization of the hydrogel scaffold and provide limited opportunities for tuning deposition parameters on an antibody-by-antibody basis. In this work, a versatile hydrogel nanoparticle deposition method was developed for the production of label-free microarrays and tested in the context of antibody-antigen binding. Poly(N-isopropylacrylamide) nanoparticles (PNIPAM) were conjugated to antibodies using an avidin/biotin system and deposited onto surfaces using a noncontact printing system. After drying, these gel spots formed uniform and thin layers <10 nm in height. The conjugates were characterized with dynamic light scattering, scanning electron microscopy, and atomic force microscopy. We tested this format in the context of tumor necrosis factor-alpha (TNF-α) detection via arrayed imaging reflectometry (AIR), a label-free protein microarray method. This method of probe molecule deposition should be generally useful in the production of microarrays for label-free detection.

Examining the Interactions of the Splicing Factor MBNL1 with Target RNA Sequences via a Label-Free, Multiplex Method

The near-ubiquity of the involvement of RNA in crucial biological processes is accepted. It is important, therefore, to study and understand the biophysical principles that regulate the function of RNA and its interactions with other molecules (e.g., proteins and antibiotics). Methods enabling the high-throughput determination of RNA-protein binding kinetics and thermodynamics would greatly accelerate understanding of these interactions. To that end, we describe the development of a real-time biomolecular interaction analysis platform based on arrayed imaging reflectometry (AIR) for multiplex analysis of RNA-protein interactions. We demonstrate the use of aqueous AIR by measuring the binding kinetics between muscleblind-like 1 (MBNL1), a splicing regulator protein that plays a pivotal role in the Myotonic Dystrophies and Huntington's Disease, and several of its RNA targets simultaneously on a microarrayed chip. Using this approach, we observe that the kinetics of MBNL1 binding isolated CUG and repeat CUG RNA sequences (as models for "normal" and "pathogenic" RNA, respectively) are different even though their steady state binding constants are similar. The ability to compare binding kinetics between RNA sequences rapidly and easily may provide insight into the molecular basis of MBNL1-RNA binding, and more generally suggests that AIR can be a powerful tool to enable the label-free, real-time analysis of biomolecular interactions in a high-throughput format.