Digital microarrays: single-molecule readout with interferometric detection of plasmonic nanorod labels

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and limited dynamic range of traditional fluorescence microarrays compared to other detection techniques have been the technology’s Achilles’ heel and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule (“digital”) regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform’s primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about 3 orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10× objective lens. This approach does not require any chemical signal enhancement such as silver deposition and scans arrays with a throughput similar to commercial fluorescence scanners. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about 6 orders of magnitude directly from a single scan. As a proof-of-concept digital protein microarray assay, we demonstrated detection of hepatitis B virus surface antigen in buffer with a limit of detection of 3.2 pg/mL. More broadly, the technique’s simplicity and high-throughput nature make digital microarrays a flexible platform technology with a wide range of potential applications in biomedical research and clinical diagnostics.The authors wish to thank Oguzhan Avci and Jacob Trueb for thoughtful comments and suggestions regarding numerical optimization of the optical system. This work was funded in part by a research contract with ASELSAN, Inc. and the Wallace H. Coulter Foundation 2010 Coulter Translational Award. (ASELSAN, Inc.; Wallace H. Coulter Foundation Coulter Translational Award)Accepted manuscrip

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

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.This work was supported by Regione Lombardia and Fondazione Cariplo through POR-FESR, project MINER (ID 46875467); Italian Ministry of Health, Ricerca Corrente. This work was partially supported by The Scientific and Technological Research Council of Turkey (grant #113E643). (Regione Lombardia; 46875467 - Fondazione Cariplo through POR-FESR, project MINER; Italian Ministry of Health, Ricerca Corrente; 113E643 - Scientific and Technological Research Council of Turkey)Published versio

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation

Small extracellular vesicles modulated by αVβ3 integrin induce neuroendocrine differentiation in recipient cancer cells

The ability of small extracellular vesicles (sEVs) to reprogram cancer cells is well established. However, the specific sEV components able to mediate aberrant effects in cancer cells have not been characterized. Integrins are major players in mediating sEV functions. We have previously reported that the αVβ3 integrin is detected in sEVs of prostate cancer (PαVβ3rCa) cells and transferred into recipient cells. Here, we investigate whether sEVs from -expressing cells affect tumour growth differently than sEVs from control cells that do not express αVβ3. We compared the ability of sEVs to stimulate tumour growth, using sEVs isolated from PrCa C4-2B cells by iodixanol density gradient and characterized with immunoblotting, nanoparticle tracking analysis, immunocapturing and single vesicle analysis. We incubated PrCa cells with sEVs and injected them subcutaneously into nude mice to measure in vivo tumour growth or analysed in vitro their anchorage-independent growth. Our results demonstrate that a single treatment with sEVs shed from C4-2B cells that express αVβ3, but not from control cells, stimulates tumour growth and induces differentiation of PrCa cells towards a neuroendocrine phenotype, as quantified by increased levels of neuroendocrine markers. In conclusion, the expression of αVβ3 integrin generates sEVs capable of reprogramming cells towards an aggressive phenotype

Audit of therapeutic interventions in inpatient children using two scores: are they evidence-based in developing countries?

BACKGROUND: The evidence base of clinical interventions in paediatric hospitals of developing countries has not been formally assessed. We performed this study to determine the proportion of evidence-based therapeutic interventions in a paediatric referral hospital of a developing country METHODS: The medical records of 167 patients admitted in one-month period were revised. Primary diagnosis and primary therapeutic interventions were determined for each patient. A systematic search was performed to assess the level of evidence for each intervention. Therapeutic interventions were classified using the Ellis score and the Oxford Centre for Evidence Based Medicine Levels of Evidence RESULTS: Any dehydration due to diarrhoea (59 cases) and pneumonia (42 cases) were the most frequent diagnoses. Based on Ellis score, level I evidence supported the primary therapeutic intervention in 21%, level II in 73% and level III in 6% cases. Using the Oxford classification 16%, 8%, 1% and 75% therapeutic interventions corresponded to grades A, B, C, and D recommendations, respectively. Overall, according to Ellis score, 94% interventions were evidence based. However, out of the total, 75% interventions were based on expert opinion or basic sciences. Most children with mild to moderate dehydration (52 cases) were inappropriately treated with slow intravenous fluids, and most children with non-complicated community acquired pneumonia (42 cases) received intravenous antibiotics CONCLUSIONS: Most interventions were inappropriate, despite the availability of effective therapy for several of them. Diarrhoeal dehydration and community acquired pneumonia were the most common diagnoses and were inappropriately managed. Existing effective interventions for dehydration and pneumonia need to be put into practice at referral hospitals of developing countries. For the remaining problems, there is the need to conduct appropriate clinical studies. Caution must be taken when assigning the level of evidence supporting therapeutic interventions, as commonly used classifications may be misleadin

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

Digital Microarrays: Single-Molecule Readout with Interferometric Detection of Plasmonic Nanorod Labels

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and limited dynamic range of traditional fluorescence microarrays compared to other detection techniques have been the technology’s Achilles’ heel and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule (“digital”) regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform’s primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about 3 orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10× objective lens. This approach does not require any chemical signal enhancement such as silver deposition and scans arrays with a throughput similar to commercial fluorescence scanners. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about 6 orders of magnitude directly from a single scan. As a proof-of-concept digital protein microarray assay, we demonstrated detection of hepatitis B virus surface antigen in buffer with a limit of detection of 3.2 pg/mL. More broadly, the technique’s simplicity and high-throughput nature make digital microarrays a flexible platform technology with a wide range of potential applications in biomedical research and clinical diagnostics

Single Nanoparticle Detection for Multiplexed Protein Diagnostics with Attomolar Sensitivity in Serum and Unprocessed Whole Blood

Although biomarkers exist for a range of disease diagnostics, a single low-cost platform exhibiting the required sensitivity, a large dynamic-range and multiplexing capability, and zero sample preparation remains in high demand for a variety of clinical applications. The Interferometric Reflectance Imaging Sensor (IRIS) was utilized to digitally detect and size single gold nanoparticles to identify protein biomarkers in unprocessed serum and blood samples. IRIS is a simple, inexpensive, multiplexed, high-throughput, and label-free optical biosensor that was originally used to quantify biomass captured on a surface with moderate sensitivity. Here we demonstrate detection of β-lactoglobulin, a cow’s milk whey protein spiked in serum (>10 orders of magnitude) and whole blood (>5 orders of magnitude), at attomolar sensitivity. The clinical utility of IRIS was demonstrated by detecting allergen-specific IgE from microliters of characterized human serum and unprocessed whole blood samples by using secondary antibodies against human IgE labeled with 40 nm gold nanoparticles. To the best of our knowledge, this level of sensitivity over a large dynamic range has not been previously demonstrated.IRIS offers four main advantages compared to existing technologies: it (i) detects proteins from attomolar to nanomolar concentrations in unprocessed biological samples, (ii) unambiguously discriminates nanoparticles tags on a robust and physically large sensor area, (iii) detects protein targets with conjugated very small nanoparticle tags (∼40 nm diameter), which minimally affect assay kinetics compared to conventional microparticle tagging methods, and (iv) utilizes components that make the instrument inexpensive, robust, and portable. These features make IRIS an ideal candidate for clinical and diagnostic applications