Robust Visualization and Discrimination of Nanoparticles by Interferometric Imaging

Trueb, Jacob; Avcı, Oğuzhan; Sevenler, Derin; Connor, John H.; Ünlü, M. Selim

doi:10.1109/jstqe.2016.2639824

Cited by 33 publications

(35 citation statements)

References 36 publications

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“…The conjugated GNRs were diluted to a range of concentrations between 100 attomolar and 100 picomolar, then incubated with the DNA chips for 4 hours, then finally washed and dried. A total of 70 spots were imaged with instrument using both the 10x and 50x objective, and images were analyzed using custom particle analysis software described previously 29 . As anticipated, the 10x objective system accurately enumerated immobilized GNRs when they were few (i.e., 60 GNR per spot of fewer), but systematically under-counted as their number increased (Figure 2d).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2018

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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.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2018

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“…GNRs in each region and timepoint were detected independently. The particle detection method described here is a refinement of methods described earlier (21,28), and has three steps: preprocessing, key point detection and key point filtering. First, a sparse pseudo-median filter is applied to each frame of the z-stack (made available online by others at http://imagejdocu.tudor.lu/doku.php?id=plugin:filte r:fast_filters:start) to estimate the image background.…”

Section: Image Processing and Particle Detectionmentioning

confidence: 99%

Beating the reaction limits of biosensor sensitivity with dynamic tracking of single binding events

Sevenler

Trueb

Ünlü

2019

Proc. Natl. Acad. Sci. U.S.A.

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The clinical need for ultra-sensitive molecular analysis has motivated the development of several endpoint assay technologies capable of single molecule readout. These endpoint assays are now primarily limited by the affinity and specificity of the molecular recognition agents for the analyte of interest. In contrast, a kinetic assay with single molecule readout could distinguish between low abundance, high affinity (specific analyte) and high abundance, low affinity (nonspecific background) binding by measuring the duration of individual binding events at equilibrium. Here we describe such a kinetic assay, in which individual binding events are detected and monitored during sample incubation. This method uses plasmonic gold nanorods and interferometric reflectance imaging to detect thousands of individual binding events across a multiplex solid phase sensor with a large area approaching that of leading bead-based endpoint assay technologies. A dynamic tracking procedure is used to measure the duration of each event. From this, the total rates of binding and de-binding as well as the distribution of binding event durations are determined. We observe a limit of detection of 15 femtomolar for a proof-of-concept synthetic DNA analyte in a 12-plex assay format.

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“…Single biological nanoparticles result in small, diffraction-limited perturbations in the reflected field, between 0.5% (around 60-nm diameter) and 10% (around 150-nm diameter). The excellent flatness and smoothness of the Si-SiO 2 substrate is a critical component for SP-IRIS, because it enables the detection of diffraction-limited spots that are only 0.5% dimmer (or brighter, depending on the objective's plane of focus [24]) than the reference field even in the absence of dynamic measurements. This in turn allows the stage to be scanned, which enables a region of interest much larger than a single field of view.…”

Section: Application Of Interference Reflectance Imaging To Bnp Detecmentioning

confidence: 99%

“…In later studies, a high numerical aperture objective was used to detect the faint interference signal from individual virions. Calculating the size of a particle from the brightness of the resulting spot was nontrivial, because increasing the particle size increases the position of its centroid above the reflecting surface, thereby slightly increasing the optical path length difference between forward-and back-scattered light [24]. Once the substrate film thickness and surface morphology were optimized, individual BNPs ranging from 50 nm to 200 nm were detected by affinitybased capture in various complex media with SP-IRIS [88].…”

Section: Importance Of Surface Morphology In Single Particle Optical mentioning

confidence: 99%

“…They require fluorescence labeling of the single natural nanoparticles, which makes them less applicable for a variety of samples. Moreover, nonspecific binding of the fluorescent labels to the other components in the heterogeneous media or formation of aggregates remains as a challenge in fluorescence detection systems along with the inconstancy of the fluorescence signal independent of the size of the biological nanoparticles [24]. Also, irreversible photobleaching of the fluorescent label limits observation time.…”

Section: Introductionmentioning

confidence: 99%

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Surface chemistry and morphology in single particle optical imaging

et al. 2017

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Biological nanoparticles such as viruses and exosomes are important biomarkers for a range of medical conditions, from infectious diseases to cancer. Biological sensors that detect whole viruses and exosomes with high specificity, yet without additional labeling, are promising because they reduce the complexity of sample preparation and may improve measurement quality by retaining information about nanoscale physical structure of the bio-nanoparticle (BNP). Towards this end, a variety of BNP biosensor technologies have been developed, several of which are capable of enumerating the precise number of detected viruses or exosomes and analyzing physical properties of each individual particle. Optical imaging techniques are promising candidates among broad range of label-free nanoparticle detectors. These imaging BNP sensors detect the binding of single nanoparticles on a flat surface functionalized with a specific capture molecule or an array of multiplexed capture probes. The functionalization step confers all molecular specificity for the sensor's target but can introduce an unforeseen problem; a rough and inhomogeneous surface coating can be a source of noise, as these sensors detect small local changes in optical refractive index. In this paper, we review several optical technologies for label-free BNP detectors with a focus on imaging systems. We compare the surface-imaging methods including dark-field, surface plasmon resonance imaging and interference reflectance imaging. We discuss the importance of ensuring consistently uniform and smooth surface coatings of capture molecules for these types of biosensors and finally summarize several methods that have been developed towards addressing this challenge.

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Robust Visualization and Discrimination of Nanoparticles by Interferometric Imaging

Cited by 33 publications

References 36 publications

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

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

Beating the reaction limits of biosensor sensitivity with dynamic tracking of single binding events

Surface chemistry and morphology in single particle optical imaging

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