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Label-free multiplexed virus detection using spectral reflectance imaging.

ABSTRACT: We demonstrate detection of whole viruses and viral proteins with a new label-free platform based on spectral reflectance imaging. The Interferometric Reflectance Imaging Sensor (IRIS) has been shown to be capable of sensitive protein and DNA detection in a real time and high-throughput format. Vesicular stomatitis virus (VSV) was used as the target for detection as it is well-characterized for protein composition and can be modified to express viral coat proteins from other dangerous, highly pathogenic agents for surrogate detection while remaining a biosafety level 2 agent. We demonstrate specific detection of intact VSV virions achieved with surface-immobilized antibodies acting as capture probes which is confirmed using fluorescence imaging. The limit of detection is confirmed down to 3.5 × 10(5)plaque-forming units/mL (PFUs/mL). To increase specificity in a clinical scenario, both the external glycoprotein and internal viral proteins were simultaneously detected with the same antibody arrays with detergent-disrupted purified VSV and infected cell lysate solutions. Our results show sensitive and specific virus detection with a simple surface chemistry and minimal sample preparation on a quantitative label-free interferometric platform.

SUBMITTER: Lopez CA

PROVIDER: S-EPMC3065545 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Label-free multiplexed virus detection using spectral reflectance imaging.

Lopez Carlos A CA Daaboul George G GG Vedula Rahul S RS Ozkumur Emre E Bergstein David A DA Geisbert Thomas W TW Fawcett Helen E HE Goldberg Bennett B BB Connor John H JH Unlü M Selim MS

Biosensors & bioelectronics 20110122 8

We demonstrate detection of whole viruses and viral proteins with a new label-free platform based on spectral reflectance imaging. The Interferometric Reflectance Imaging Sensor (IRIS) has been shown to be capable of sensitive protein and DNA detection in a real time and high-throughput format. Vesicular stomatitis virus (VSV) was used as the target for detection as it is well-characterized for protein composition and can be modified to express viral coat proteins from other dangerous, highly pa ...[more]

PMID: 21342761

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Project description:IntroductionMelanoma diagnosis traditionally relies on microscopic examination of hematoxylin and eosin (H&E) slides by dermatopathologists to search for specific architectural and cytological features. Unfortunately, no single molecular marker exists to reliably differentiate melanoma from benign lesions such as nevi. This study explored the potential of autofluorescent molecules within tissues to provide molecular fingerprints indicative of degenerated melanocytes in melanoma.MethodsUsing hyperspectral imaging (HSI) and spectral phasor analysis, we investigated autofluorescence patterns in melanoma compared to intradermal nevi. Using UV excitation and a commercial spectral confocal microscope, we acquired label-free HSI data from the whole-slice samples.ResultsOur findings revealed distinct spectral phasor distributions between melanoma and intradermal nevi, with melanoma displaying a broader phasor phase distribution, signifying a more heterogeneous autofluorescence pattern. Notably, longer wavelengths associated with larger phases correlated with regions identified as melanoma by expert dermatopathologists using H&E staining. Quantitative analysis of phase and modulation histograms within the phasor clusters of five melanomas (with Breslow thicknesses ranging from 0.5 mm to 6 mm) and five intradermal nevi consistently highlighted differences between the two groups. We further demonstrated the potential for the discrimination of several melanocytic lesions using center-of-mass comparisons of phase and modulation variables. Remarkably, modulation versus phase center of mass comparisons revealed strong statistical significance among the groups. Additionally, we identified the molecular endogenous markers responsible for tissue autofluorescence, including collagen, elastin, NADH, FAD, and melanin. In melanoma, autofluorescence is characterized by a higher phase contribution, indicating an increase in FAD and melanin in melanocyte nests. In contrast, NADH, elastin, and collagen dominate the autofluorescence of the nevus.DiscussionThis work underscores the potential of autofluorescence and HSI-phasor analysis as valuable tools for quantifying tissue molecular fingerprints, thereby supporting more effective and quantitative melanoma diagnosis.

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Label-free multiplexed virus detection using spectral reflectance imaging.

Publications

Label-free multiplexed virus detection using spectral reflectance imaging.

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