Single-particle Interferometric Reflectance Imaging Sensor Research Articles

Biofabricated osteoblast-derived nanovesicles as extracellular vesicle mimics for bone repair

Osteoblast-derived extracellular vesicles (EVs) have demonstrated therapeutic utility for bone repair as transporters of key biomolecules capable of accelerating biomineralisation and tissue repair. The clinical translation of these biologically derived nanoparticles, however remains limited due to scalability, heterogeneity and standardisation issues. Herein we investigate the generation of nanovesicles (NVs) from mineralising osteoblasts by extrusion directly compared against natural EV counterparts from the same parental cells.Mineralising osteoblast-derived EVs (MO-EVs) were isolated via ultracentrifugation from cell culture media. The parental osteoblasts were then processed via serial extrusion to <200 nm. EVs and NVs were characterised by comparing their size, concentration and morphology. The presence of tetraspanin markers was detected by Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS). Osteoblasts viability and metabolic activity was assessed after both EV and NV-treatment before comparing their mineralising potency via alizarin red staining.EVs and NVs exhibited similar diameters of approximately 100 nm with a vesicular morphology. EVs were found to be richer in proteins and exhibited a significantly more negative ζ-potential compared to NVs. SP-IRIS analysis confirmed the presence of CD9, CD63 and CD81 in EVs. At both 1 and 10 μg/mL, EVs and NVs reduced the metabolic activity of osteoblasts, however, this was not associated with any cytotoxic effects. The biomineralisation study performed using osteoblasts showed that only EVs significantly increased mineral deposition (p < 0.05) compared to untreated control.In this study, we have established for the first time the biofabrication of cell-derived nanovesicles as a promising alternative to extracellular vesicles derived from mineralising osteoblasts.

Abstract 3633: Multi-parametric characterization of extracellular vesicles in the aqueous humor of uveal melanoma eyes undergoing brachytherapy

Abstract Introduction: Aqueous humor (AH), fluid in the anterior chamber of the eye, is a liquid biopsy source of tumor information in uveal melanoma (UM). Extracellular vesicles (EVs) have recently been characterized in the AH of retinoblastoma eyes, however, have not been investigated in UM. EVs are small membrane-enclosed particles secreted by virtually every cell type with a role in intercellular communications. We hypothesize that tumor-derived EVs are detectable in the AH of UM eyes and that understanding the type of vesicles present may help us better understand their function, particularly for cancer. Small extracellular vesicles (sEVs) have been shown to promote tumorigenesis, treatment resistance, and metastasis, leading to the discovery of clinically-relevant biomarkers for multiple cancer types. As the diversity and role of sEVs in the AH of UM patients has never previously been reported, this project is a first step towards investigating new biomarkers of clinical utility. Our aim is to use single particle analysis to characterize sEV subpopulations in the AH of UM patients by quantifying their size, concentration, and phenotypes based on cell surface markers, specifically tetraspanin co-expression patterns. Methods: sEVs were analyzed from paired pre- and post-brachytherapy AH samples collected from 19 UM patients. AH samples from 5 glaucoma patients served as the control cohort. Unprocessed AH underwent Nanoparticle Tracking Analysis for size quantification and Single Particle-Interferometric Reflectance Imaging Sensor analysis for fluorescent particle count and tetraspanin immunophenotyping; counts were subsequently converted to percentages for analysis. Statistics were performed using Mann-Whitney U, ANOVA, and Tukey’s tests. Results: AH derived from UM eyes (pre-brachytherapy) displayed a broader sEV size distribution than glaucoma eyes, with more diverse sEV subpopulations. Both tumor and nontumor AH samples demonstrated a dominant CD63+ sEV subpopulation. A significantly higher percentage of CD63/81+ sEVs (P= 0.0217) and CD9/63/81+ sEVs (P= 0.0146) were found in post-brachytherapy samples compared to pre-brachytherapy. Conclusions: sEV subpopulations in UM eyes demonstrate increased heterogeneity in comparison to nontumor eyes. Our results are consistent with prior studies reporting that the CD63+ sEV subtype is an AH-specific biomarker enriched across multiple ocular diseases. The increase in CD63/81+ sEVs and CD9/63/81+ sEVs after brachytherapy is presumably a result of radiation-induced release of these vesicles, suggesting a tumor-derived origin of these subpopulations. CD63/81+ sEV subpopulation was previously reported to also be a tumor-derived subpopulation in AH of retinoblastoma eyes. Further study of the cargo carried by these sEV subpopulations may uncover molecular mechanisms pivotal to UM and other ocular cancers. Citation Format: Shreya Sirivolu, Chen-Ching Peng, Paolo Neviani, Jesse L. Berry, Liya Xu. Multi-parametric characterization of extracellular vesicles in the aqueous humor of uveal melanoma eyes undergoing brachytherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3633.

CD63/81 Small Extracellular Vesicles in the Aqueous Humor are Retinoblastoma Associated.

Although biopsy is contraindicated in retinoblastoma (RB), the aqueous humor (AH) is a robust liquid biopsy source of molecular tumor information, facilitating biomarker discovery. Small extracellular vesicles (sEVs), promising biomarker candidates across multiple cancers, were recently identified in RB AH, but relationships between sEVs and RB clinical features are unknown. We analyzed sEVs in 37 AH samples from 18 RB eyes of varying International Intraocular Retinoblastoma Classification (IIRC) groups and explored clinical correlations. Ten samples were collected at diagnosis (DX) and 27 during treatment (Tx). Unprocessed AH underwent Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) analysis for fluorescent particle count and tetraspanin immunophenotyping; counts were subsequentially converted to percentages for analysis. Comparing DX and Tx samples, a higher percentage of CD63/81+ sEVs was found in DX AH (16.3 ± 11.6% vs. 5.49 ± 3.67% P = 0.0009), with a more homogenous mono-CD63+ sEV population seen in Tx AH (43.5 ± 14.7% vs. 28.8 ± 9.38%, P = 0.0073). Among DX samples, CD63/81+ sEVs were most abundant in group E eyes (n = 2) compared to group D (n = 6) by count (2.75 × 105 ± 3.40 × 105 vs. 5.95 × 103 ± 8.16 × 103, P = 0.0006), and to group A+B (n = 2) by count (2.75 × 105 ± 3.40 × 105 vs. 2.73 × 102 ± 2.59 × 102, P = 0.0096) and percentage (32.1 ± 7.98% vs. 7.79 ± 0.02%, P = 0.0187). CD63/81+ sEVs enrich AH from RB eyes before treatment and those with more significant tumor burden, suggesting they are tumor-derived. Future research into their cargo may reveal mechanisms of cellular communication via sEVs in RB and novel biomarkers.

Solid-Phase Optical Sensing Techniques for Sensitive Virus Detection.

Viral infections can pose a major threat to public health by causing serious illness, leading to pandemics, and burdening healthcare systems. The global spread of such infections causes disruptions to every aspect of life including business, education, and social life. Fast and accurate diagnosis of viral infections has significant implications for saving lives, preventing the spread of the diseases, and minimizing social and economic damages. Polymerase chain reaction (PCR)-based techniques are commonly used to detect viruses in the clinic. However, PCR has several drawbacks, as highlighted during the recent COVID-19 pandemic, such as long processing times and the requirement for sophisticated laboratory instruments. Therefore, there is an urgent need for fast and accurate techniques for virus detection. For this purpose, a variety of biosensor systems are being developed to provide rapid, sensitive, and high-throughput viral diagnostic platforms, enabling quick diagnosis and efficient control of the virus's spread. Optical devices, in particular, are of great interest due to their advantages such as high sensitivity and direct readout. The current review discusses solid-phase optical sensing techniques for virus detection, including fluorescence-based sensors, surface plasmon resonance (SPR), surface-enhanced Raman scattering (SERS), optical resonators, and interferometry-based platforms. Then, we focus on an interferometric biosensor developed by our group, the single-particle interferometric reflectance imaging sensor (SP-IRIS), which has the capability to visualize single nanoparticles, to demonstrate its application for digital virus detection.

Abstract 6717: Extracellular vesicles in diagnostic retinoblastoma aqueous humor correlate with disease stage and clinical outcome: a pilot study

Abstract Introduction: Retinoblastoma (RB), a pediatric retinal malignancy, is unique in that it cannot be biopsied, impeding biomarker discovery. Aqueous humor (AH) is an ocular fluid that is an established source of tumor information, serving as a surrogate liquid biopsy. Extracellular vesicles (EVs) are promising biomarker candidates, and were recently established in RB AH. EVs are membrane-enclosed secretory vesicles carrying bioactive molecular information, which can be phenotyped using validated tetraspanin protein markers. Currently, relationships between EVs and RB clinical features are unknown, but establishing such relationships is necessary to determine the utility of EVs as RB biomarkers. Methods: We quantified and phenotyped EVs in 37 AH samples from 18 RB eyes with varying International Intraocular Retinoblastoma Classification (IIRC) stages and explored clinical correlations. Samples were collected from 10 eyes at diagnosis (DX) and 27 eyes during treatment (Tx). By outcome, 6 DX eyes were enucleated and 4 were salvaged. DX samples were collected from one A, one B, six D and two E eyes by IIRC group. 10μL of unprocessed AH underwent Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) (Exoview R100) analysis for fluorescent-based particle count and immunophenotyping of tetraspanin expression (CD63/81/9). Counts were converted to percentages. Statistics were done with Mann-Whitney U, ANOVA, and Tukey’s tests. Results: Samples from DX eyes (n=10) contained a higher number of EVs than samples from Tx eyes (n=27) (1.68 x 107 ± 4.28 x 107 per mL vs. 2.10x 106 ± 1.33 x 106 per mL) by SP-IRIS analysis. By percentage, DX samples were enriched with CD63/81+ EVs (P&amp;lt;0.001) while Tx samples had more homogenous mono-CD63+ dominant EV populations (P=0.007). Enrichment of CD63/81+ EVs was significantly higher in enucleated AH (n=6) than salvaged AH (n=4) when compared by count (P=0.038) and marginally higher when compared by percentage (P=0.067). The CD9/81+ EV population percentage was significant in salvaged eyes (P=0.01). By IIRC, CD63/81+ EVs were most abundant in Group E eyes (n= 2) when compared to Group D eyes (n=6) by count (P&amp;lt;0.001), and when compared to Group A+B eyes (n=2) by count (P=0.01) and percentage (P=0.019). Group A+B eyes had a larger percentage of CD9/81+ EVs than did Group E eyes (P=0.043). Conclusions: EV quantity in RB AH decreases with treatment, and phenotypic expression profiles vary by treatment status and clinical outcome. TheCD63/81+ EV subpopulation is higher in eyes with advanced Group E disease and those requiring enucleation, while the CD9/81+ subpopulation is higher in eyes with less advanced Group A+B disease and those salvaged with treatment. Our data suggests that EV phenotypes correlate with RB clinical features, and further research into these subpopulations offers the prospect of establishing critical RB biomarkers. Citation Format: Sarah Pike, Chen-Ching Peng, Paolo Neviani, Jesse L. Berry, Liya Xu. Extracellular vesicles in diagnostic retinoblastoma aqueous humor correlate with disease stage and clinical outcome: a pilot study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6717.

A high-throughput single-particle imaging platform for antibody characterization and a novel competition assay for therapeutic antibodies

Monoclonal antibodies (mAbs) play an important role in diagnostics and therapy of infectious diseases. Here we utilize a single-particle interferometric reflectance imaging sensor (SP-IRIS) for screening 30 mAbs against Ebola, Sudan, and Lassa viruses (EBOV, SUDV, and LASV) to find out the ideal capture antibodies for whole virus detection using recombinant vesicular stomatitis virus (rVSV) models expressing surface glycoproteins (GPs) of EBOV, SUDV, and LASV. We also make use of the binding properties on SP-IRIS to develop a model for mapping the antibody epitopes on the GP structure. mAbs that bind to mucin-like domain or glycan cap of the EBOV surface GP show the highest signal on SP-IRIS, followed by mAbs that target the GP1-GP2 interface at the base domain. These antibodies were shown to be highly efficacious against EBOV infection in non-human primates in previous studies. For LASV detection, 8.9F antibody showed the best performance on SP-IRIS. This antibody binds to a unique region on the surface GP compared to other 15 mAbs tested. In addition, we demonstrate a novel antibody competition assay using SP-IRIS and rVSV-EBOV models to reveal the competition between mAbs in three successful therapeutic mAb cocktails against EBOV infection. We provide an explanation as to why ZMapp cocktail has higher efficacy compared to the other two cocktails by showing that three mAbs in this cocktail (13C6, 2G4, 4G7) do not compete with each other for binding to EBOV GP. In fact, the binding of 13C6 enhances the binding of 2G4 and 4G7 antibodies. Our results establish SP-IRIS as a versatile tool that can provide high-throughput screening of mAbs, multiplexed and sensitive detection of viruses, and evaluation of therapeutic antibody cocktails.

Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts.

Receptor activator of nuclear factor-kappa B (RANK)-containing extracellular vesicles (EVs) bind RANK-Ligand (RANKL) on osteoblasts, and thereby simultaneously inhibit bone resorption and promote bone formation. Because of this, they are attractive candidates for therapeutic bone anabolic agents. Previously, RANK was detected in 1 in every 36 EVs from osteoclasts by immunogold electron microscopy. Here, we have sought to characterize the subpopulation of EVs from osteoclasts that contains RANK in more detail. The tetraspanins CD9 and CD81 were localized in osteoclasts by immunofluorescence. EVs were visualized by transmission electron microscopy. A Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and immunoaffinity isolations examined whether RANK is enriched in specific types of EVs. Immunofluorescence showed CD9 was mostly on or near the plasma membrane of osteoclasts. In contrast, CD81 was localized deeper in the osteoclast's cytosolic vesicular network. By interferometry, both CD9 and CD81 positive EVs from osteoclasts were small (56-83 nm in diameter), consistent with electron microscopy. The CD9 and CD81 EV populations were mostly distinct, and only 22% of the EVs contained both markers. RANK was detected by SP-IRIS in 2%-4% of the CD9-containing EVs, but not in CD81-positive EVs, from mature osteoclasts. Immunomagnetic isolation of CD9-containing EVs from conditioned media of osteoclasts removed most of the RANK. A trace amount of RANK was isolated with CD81. RANK was enriched in a subset of the CD9-positive EVs. The current study provides the first report of selective localization of RANK in subsets of EVs.

Detailed Characterization of Small Extracellular Vesicles from Different Cell Types Based on Tetraspanin Composition by ExoView R100 Platform.

Small extracellular vesicles (sEV) hold enormous potential as biomarkers, drug carriers, and therapeutic agents. However, due to previous limitations in the phenotypic characterization of sEV at the single vesicle level, knowledge of cell type-specific sEV signatures remains sparse. With the introduction of next-generation sEV analysis devices, such as the single-particle interferometric reflectance imaging sensor (SP-IRIS)-based ExoView R100 platform, single sEV analyses are now possible. While the tetraspanins CD9, CD63, and CD81 were generally considered pan-sEV markers, it became clear that sEV of different cell types contain several combinations and amounts of these proteins on their surfaces. To gain better insight into the complexity and heterogeneity of sEV, we used the ExoView R100 platform to analyze the CD9/CD63/CD81 phenotype of sEV released by different cell types at a single sEV level. We demonstrated that these surface markers are sufficient to distinguish cell-type-specific sEV phenotypes. Furthermore, we recognized that tetraspanin composition in some sEV populations does not follow a random pattern. Notably, the tetraspanin distribution of sEV derived from mesenchymal stem cells (MSCs) alters depending on cell culture conditions. Overall, our data provide an overview of the cell-specific characteristics of sEV populations, which will increase the understanding of sEV physiology and improve the development of new sEV-based therapeutic approaches.

Quantitative analysis of exosomes in the aqueous humor of Korean patients with pseudoexfoliation glaucoma

We aimed to quantitatively analyze the exosome and its cargo in individual aqueous humor (AH) samples from pseudoexfoliation (PEX) glaucoma patients compared to controls using a novel detection platform. We investigated the size distribution and measured the quantitative exosome particle counts in each AH sample. AH (80–120 µL) was obtained during cataract surgery or glaucoma filtering surgery from 12 Korean subjects (six with PEX glaucoma and six age-matched controls). The mean size of the exosomes was 58.9 ± 18.5 nm measured by a tangential flow filtration system using single-particle interferometric reflectance imaging sensor. Exosome particle count in each CD 63, CD 81, and CD9 spot was significantly greater in PEX glaucoma than in controls in total, CD 63, CD9, syntenin, and scattering(all p < 0.003). The CD63 spot showed a particle count of 8319.1 ± 797.7 in PEX glaucoma patients and 4786.8 ± 1302.1 in controls (p = 1.88E−11). Individual fluorescent capture spot images also revealed denser exosome particles in PEX patients than in controls. Syntenin, indicating exosomal origin, was detected in all AH samples. Exosomes differentially detected in AH suggest the possible role of exosomes in the pathogenesis of PEX glaucoma.

Abstract 3416: Clearance of tumor-derived extracellular vesicle heterogeneity in aqueous humor after chemotherapy in retinoblastoma eyes

Abstract Introduction: Aqueous humor (AH), the clear fluid in front of the eye, maintains the pressure and vitality of ocular tissues. This fluid is accessible via the cornea which enables use of AH as a liquid biopsy for intraocular disease. Retinoblastoma (Rb), an intraocular cancer in children, is unique in that direct tumor biopsy is prohibited, thus liquid biopsy has great clinical potential. cfDNA in the AH as a biomarker for Rb patients has been investigated and extracellular vesicles (EVs) are detectable in the AH from adults. However, AH EVs in Rb have previously never been explored. We know very little about the heterogeneity of AH EV populations in ocular cancers. Materials and Methods: 27 AH samples from 19 patients from 13 tumor-free eyes and 11 Rb eyes were collected. Rb eyes were further divided into treatment-naïve (Rb_Tn) and treatment-active (Rb_Tx) subgroups. Unprocessed AH samples were subjected to Nanoparticle Tracking Analysis (NTA) (Nanosight NS300) for size distribution and concentration, and to Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) (Exoview R100) for fluorescent-based immunophenotyping of EV marker expression (CD63, CD81, and CD9). Results: NTA demonstrated the concentration of AH EVs is 3.11 x 109-1.38 x 1010 vesicles/mL; majority sized 76.8-103 nm. Study group comparisons showed that non-tumor containing eyes had a smaller nanoparticle mean size compared to Rb containing eyes (P = 0.002). More EVs were significantly detected in Rb_Tn containing eyes compared to Rb_Tx containing eyes (P = 0.022), suggesting the possible presence of tumor-derived EVs in Rb_Tn which were subsequently eliminated by treatment. SP-IRIS revealed distinct patterns of tetraspanin expression of AH small EVs (sEVs). Mono-CD63+ EVs were identified to be the most dominant sEV subpopulation from AH across non-tumor and Rb_Tx eyes. However, more diverse sEV subpopulation profile was detected in Rb_Tn AH samples. Significantly lower percentage of mono-CD63+ EVs could be determined in Rb_Tn eyes compared to Rb_Tx eyes (70.3% vs. 96.1%, P = 0.001). A significant accumulation of CD9/CD63 (4.3% vs. 1.1%, P = 0.035), CD63/CD81 (20.7% vs. 2.0%, P = 0.012) and CD9/CD63/CD81 (4.8% vs. 0.8%, P = 0.022) subpopulations in Rb_Tn was also observed. An enriched mono-CD63+ sEV subpopulation identified in AH indicates this is a potential AH-specific biomarker. In the setting of Rb there was a more heterogeneous population of sEVs which normalized with treatment. Conclusions: Small EVs are readily detectable in unprocessed AH with a dominant mono-CD63+ subpopulation in AH regardless of pediatric eye disease states. Tetraspanin colocalization analysis indicates the clearance of retinoblastoma-derived EV heterogeneity by chemotherapy. These novel finding suggests a potential clinical application of measurement of sEV subpopulations in AH samples to monitor response to therapy. Citation Format: Chen-Ching Peng, Deborah Im, Shreya Sirivolu, Bibiana Reiser, Aaron Nagiel, Paolo Neviani, Liya Xu, Jesse L. Berry. Clearance of tumor-derived extracellular vesicle heterogeneity in aqueous humor after chemotherapy in retinoblastoma eyes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3416.

Single vesicle analysis of aqueous humor in pediatric ocular diseases reveals eye specific CD63‐dominant subpopulations

Aqueous humor (AH), the clear fluid in front of the eye, maintains the pressure and vitality of ocular tissues. This fluid is accessible via the clear cornea which enables use of AH as a liquid biopsy source of biomarkers for intraocular disease. Extracellular vesicles are detectable in the AH and small extracellular vesicles (sEVs) are present in the AH from adults. However, EVs in AH from pediatric eyes in vivo have never previously been explored. We know very little about the heterogeneity of AH EV populations in ocular disease. Twenty-seven processing-free AH samples from 19 patients across four different pediatric ocular diseases were subjected to Nanoparticle Tracking Analysis (NTA) and Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) analysis. NTA demonstrated the concentration of AH EV/EPs is 3.11 × 109-1.38 × 1010 particles/ml; the majority sized 76.8-103 nm. SP-IRIS revealed distinct patterns of tetraspanin expression of AH sEVs. An enriched mono-CD63+ sEV subpopulation identified in AH indicates this is a potential AH-specific biomarker. In the setting of retinoblastoma there was a more heterogeneous population of sEVs which normalized with treatment. This suggests a potential clinical application of direct measurement of sEV subpopulations in AH samples to monitor successful tumor response to therapy.

Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics.

Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different biological molecules, including protein, lipid, DNA, and RNA, and are postulated to facilitate cell-to-cell communication in diverse tissues and organs. Recently, EVs have attracted significant attention as biomarkers for diagnostics and therapeutic agents for various diseases. Many methods have been developed for EV characterization. However, current methods for EV analysis all have different limitations. Thus, developing efficient and effective methods for EV isolation and characterization remains one of the crucial steps for this cutting-edge research field as it matures. Here, we provide a detailed protocol outlining a single-particle interferometric reflectance imaging sensor (SP-IRIS), as a method that is capable of detecting and characterizing EVs from unpurified biological sources and purified EVs by other methodologies. This advanced technique can be used for multi-level and comprehensive measurements for the analysis of EV size, EV count, EV phenotype, and biomarker colocalization.

Configurable Digital Virus Counter on Robust Universal DNA Chips.

Here, we demonstrate real-time multiplexed virus detection by applying a DNA-directed antibody immobilization technique in a single-particle interferometric reflectance imaging sensor (SP-IRIS). In this technique, the biosensor chip surface spotted with different DNA sequences is converted to a multiplexed antibody array by flowing antibody-DNA conjugates and allowing for specific DNA-DNA hybridization. The resulting antibody array is shown to detect three different recombinant vesicular stomatitis viruses (rVSVs), which are genetically engineered to express surface glycoproteins of Ebola, Marburg, and Lassa viruses in real time in a disposable microfluidic cartridge. We also show that this method can be modified to produce a single-step, homogeneous assay format by mixing the antibody-DNA conjugates with the virus sample in the solution phase prior to incubation in the microfluidic cartridge, eliminating the antibody immobilization step. This homogenous approach achieved detection of the model Ebola virus, rVSV-EBOV, at a concentration of 100 PFU/mL in 1 h. Finally, we demonstrate the feasibility of this homogeneous technique as a rapid test using a passive microfluidic cartridge. A concentration of 104 PFU/mL was detectable under 10 min for the rVSV-Ebola virus. Utilizing DNA microarrays for antibody-based diagnostics is an alternative approach to antibody microarrays and offers advantages such as configurable sensor surface, long-term storage ability, and decreased antibody use. We believe that these properties will make SP-IRIS a versatile and robust platform for point-of-care diagnostics applications.

Interferometric Detection and Enumeration of Viral Particles using Si-Based Microfluidics

Single-particle interferometric reflectance imaging sensor enables optical visualization and characterization of individual nanoparticles without any labels. Using this technique, we have shown end-point and real-time detection of viral particles using laminate-based active and passive cartridge configurations. Here, we present a new concept for low-cost microfluidic integration of the sensor chips into compact cartridges through utilization of readily available silicon fabrication technologies. This new cartridge configuration will allow simultaneous detection of individual virus binding events on a 9-spot microarray, and provide the needed simplicity and robustness for routine real-time operation for discrete detection of viral particles in a multiplex format.

DNA-Directed Antibody Immobilization for Robust Protein Microarrays: Application to Single Particle Detection 'DNA-Directed Antibody Immobilization.

Protein microarrays are emerging tools which have become very powerful in multiplexed detection technologies. A variety of proteins can be immobilized on a sensor chip allowing for multiplexed diagnostics. Therefore, various types of analyte in a small volume of sample can be detected simultaneously. Protein immobilization is a crucial step for creating a robust and sensitive protein microarray-based detection system. In order to achieve a successful protein immobilization and preserve the activity of the proteins after immobilization, DNA-directed immobilization is a promising technique. Here, we present the design and the use of DNA-directed immobilized (DDI) antibodies in fabrication of robust protein microarrays. We focus on application of protein microarrays for capturing and detecting nanoparticles such as intact viruses. Experimental results on Single-particle interferometric reflectance imaging sensor (SP-IRIS) are used to validate the advantages of the DDI method.

Robust Visualization and Discrimination of Nanoparticles by Interferometric Imaging.

Single-molecule and single-nanoparticle biosensors are a growing frontier in diagnostics. Digital biosensors are those which enumerate all specifically immobilized biomolecules or biological nanoparticles, and thereby achieve limits of detection usually beyond the reach of ensemble measurements. Here we review modern optical techniques for single nanoparticle detection and describe the single-particle interferometric reflectance imaging sensor (SP-IRIS). We present challenges associated with reliably detecting faint nanoparticles with SP-IRIS, and describe image acquisition processes and software modifications to address them. Specifically, we describe a image acquisition processing method for the discrimination and accurate counting of nanoparticles that greatly reduces both the number of false positives and false negatives. These engineering improvements are critical steps in the translation of SP-IRIS towards applications in medical diagnostics.

Digital Detection of Exosomes by Interferometric Imaging.

Exosomes, which are membranous nanovesicles, are actively released by cells and have been attributed to roles in cell-cell communication, cancer metastasis, and early disease diagnostics. The small size (30–100 nm) along with low refractive index contrast of exosomes makes direct characterization and phenotypical classification very difficult. In this work we present a method based on Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows multiplexed phenotyping and digital counting of various populations of individual exosomes (>50 nm) captured on a microarray-based solid phase chip. We demonstrate these characterization concepts using purified exosomes from a HEK 293 cell culture. As a demonstration of clinical utility, we characterize exosomes directly from human cerebrospinal fluid (hCSF). Our interferometric imaging method could capture, from a very small hCSF volume (20 uL), nanoparticles that have a size compatible with exosomes, using antibodies directed against tetraspanins. With this unprecedented capability, we foresee revolutionary implications in the clinical field with improvements in diagnosis and stratification of patients affected by different disorders.

DNA-Directed Antibody Immobilization for Enhanced Detection of Single Viral Pathogens.

Here, we describe the use of DNA-conjugated antibodies for rapid and sensitive detection of whole viruses using a single-particle interferometric reflectance imaging sensor (SP-IRIS), a simple, label-free biosensor capable of imaging individual nanoparticles. First, we characterize the elevation of the antibodies conjugated to a DNA sequence on a three-dimensional (3-D) polymeric surface using a fluorescence axial localization technique, spectral self-interference fluorescence microscopy (SSFM). Our results indicate that using DNA linkers results in significant elevation of the antibodies on the 3-D polymeric surface. We subsequently show the specific detection of pseudotyped vesicular stomatitis virus (VSV) as a model virus on SP-IRIS platform. We demonstrate that DNA-conjugated antibodies improve the capture efficiency by achieving the maximal virus capture for an antibody density as low as 0.72 ng/mm(2), whereas for unmodified antibody, the optimal virus capture requires six times greater antibody density on the sensor surface. We also show that using DNA conjugated anti-EBOV GP (Ebola virus glycoprotein) improves the sensitivity of EBOV-GP carrying VSV detection compared to directly immobilized antibodies. Furthermore, utilizing a DNA surface for conversion to an antibody array offers an easier manufacturing process by replacing the antibody printing step with DNA printing. The DNA-directed immobilization technique also has the added advantages of programmable sensor surface generation based on the need and resistance to high temperatures required for microfluidic device fabrication. These capabilities improve the existing SP-IRIS technology, resulting in a more robust and versatile platform, ideal for point-of-care diagnostics applications.

Digital sensing and sizing of vesicular stomatitis virus pseudotypes in complex media: a model for Ebola and Marburg detection.

Rapid, sensitive, and direct label-free capture and characterization of nanoparticles from complex media such as blood or serum will broadly impact medicine and the life sciences. We demonstrate identification of virus particles in complex samples for replication-competent wild-type vesicular stomatitis virus (VSV), defective VSV, and Ebola- and Marburg-pseudotyped VSV with high sensitivity and specificity. Size discrimination of the imaged nanoparticles (virions) allows differentiation between modified viruses having different genome lengths and facilitates a reduction in the counting of nonspecifically bound particles to achieve a limit-of-detection (LOD) of 5 × 10(3) pfu/mL for the Ebola and Marburg VSV pseudotypes. We demonstrate the simultaneous detection of multiple viruses in a single sample (composed of serum or whole blood) for screening applications and uncompromised detection capabilities in samples contaminated with high levels of bacteria. By employing affinity-based capture, size discrimination, and a "digital" detection scheme to count single virus particles, we show that a robust and sensitive virus/nanoparticle sensing assay can be established for targets in complex samples. The nanoparticle microscopy system is termed the Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and is capable of high-throughput and rapid sizing of large numbers of biological nanoparticles on an antibody microarray for research and diagnostic applications.

An Interferometric Reflectance Imaging Sensor for Point of Care Viral Diagnostics

The use of in vitro diagnostic devices is transitioning from the laboratory to the primary care setting to address early disease detection needs. Time critical viral diagnoses are often made without support due to the experimental time required in today's standard tests. Available rapid point of care (POC) viral tests are less reliable, requiring a follow-on confirmatory test before conclusions can be drawn. The development of a reliable POC viral test for the primary care setting would decrease the time for diagnosis leading to a lower chance of transmission and improve recovery. The single particle interferometric reflectance imaging sensor (SP-IRIS) has been shown to be a sensitive and specific-detection platform in serum and whole blood. This paper presents a step towards a POC viral assay through a SP-IRIS prototype with automated data acquisition and analysis and a simple, easy-to-use software interface. Decreasing operation complexity highlights the potential of SP-IRIS as a sensitive and specific POC diagnostic tool. With the integration of a microfluidic cartridge, this automated instrument will allow an untrained user to run a sample-to-answer viral assay in the POC setting.