Peer Reviewed: Analytical Applications of Single-Molecule Detection

Keller, Richard A.; Ambrose, W. P.; Arias, Angela A.; Cai, Hong; Emory, Steven R.; Goodwin, Peter M.; Jett, James H.

doi:10.1021/ac022035i

Cited by 51 publications

(36 citation statements)

References 34 publications

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“…Specifically, 2 to 23 single-molecule measurements, depending on the experimental uncertainty (CV pop ), provides a mean estimate with an associated CV mean within 1% of CV pop (i.e., the CV error limit ϭ ⌬CV Ͻ 1%). 3 The population mean and CV were determined from replicate simulations of data sets containing 10 4 measurements for each value of CV exp . The number of measurements used to determine pop and CV pop , 10 4 , was based on the number required to obtain a 1% CV for Poisson-limited measurements (8) and the typical size of flow cytometry data sets used below.…”

Section: Resultsmentioning

confidence: 99%

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Statistics of single‐molecule measurements: Applications in flow‐cytometry sizing of DNA fragments

Ferris¹,

Habbersett²,

Wolinsky³

et al. 2004

Cytometry Pt A

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Background:The measurement of physical properties from single molecules has been demonstrated. However, the majority of single-molecule studies report values based on relatively large data sets (e.g., N Ͼ 50). While there are studies that report physical quantities based on small sample sets, there has not been a detailed statistical analysis relating sample size to the reliability of derived parameters. Methods: Monte Carlo simulations and multinomial analysis, dependent on quantifiable experimental parameters, were used to determine the minimum number of singlemolecule measurements required to produce an accurate estimate of a population mean. Simulation results were applied to the fluorescence-based sizing of DNA fragments by ultrasensitive flow cytometry (FCM). Results: Our simulations show, for an analytical technique with a 10% CV, that the average of as few as five

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Section: Resultsmentioning

confidence: 99%

“…Notably, the number of single-molecule detection (SMD) studies examining biomolecules has steadily increased in recent years, with several articles in an issue of Science addressing this topic (1). Additional information and recent progress in the field of SMD is summarized in several review articles (2)(3)(4)(5)(6)(7).…”

mentioning

confidence: 99%

Statistics of single‐molecule measurements: Applications in flow‐cytometry sizing of DNA fragments

Ferris¹,

Habbersett²,

Wolinsky³

et al. 2004

Cytometry Pt A

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“…Current commercial flow cytometers are capable of detecting a minimum level of a few hundred fluorescein molecules per particle. Some custom cytometry systems have reported single fluorophore molecule detection, but this level of sensitivity is not widely available (29). With single nanoparticle scattering intensities on the order of 10 5 fluorescein molecules, commercial flow cytometers theoretically can detect single nanoparticle labels (4).…”

Section: Discussionmentioning

confidence: 99%

Plasmonic flow cytometry by immunolabeled nanorods

Crow¹,

Marinakos²,

Cook³

et al. 2010

Cytometry Pt A

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Fluorescence-based flow cytometry measures multiple cellular characteristics, including levels of receptor expression, by assessing the fluorescence intensity from a population of cells whose cell surface receptors are bound by a fluorescently labeled antibody or ligand for that receptor. Functionalized noble metal nanoparticles provide a complementary method of receptor labeling based on plasmonics for population analysis by flow cytometry. The potential benefits of using plasmonic nanoparticles to label cell surface receptors in flow cytometry include scattering intensity from a single particle that is equivalent to fluorescence intensity of 10 5 fluorescein molecules, biocompatibility and low cytotoxicity, and nonquenching optical properties. The large spectral tunability of nanorods also provides convenient access to plasmonic markers with peak surface plasmon resonances ranging from 600 to 2,200 nm, unlike gold nanosphere markers that are limited to visible wavelengths. Gold nanorod-based plasmonic flow cytometry is demonstrated herein by comparing the scattering of cells bound to antiepidermal growth factor receptor (EGFR)-conjugated nanorods to the emission of cells bound to anti-EGFR-conjugated fluorescent labels. EGFR-expressing cells exhibited a statistically significant six-fold increase in scattering when labeled with anti-EGFRconjugated nanorods compared with labeling with IgG1-conjugated nanorods. Large scattering intensities were observed despite using a 1,000-fold lower concentration of nanorod-conjugated antibody relative to the fluorescently labeled antibody. ' 2010International Society for Advancement of Cytometry Key terms flow cytometry; nanorod; plasmonic markers; epidermal growth factor receptor NOBLE metal nanoparticles exhibit unique optical properties that can be exploited for a variety of applications. These properties arise from the interaction of an incident electromagnetic (EM) field with free conduction band electrons in the nanoparticle. The conduction electrons are polarized relative to the heavier positive core ions because of the exciting EM field (1). The charge separation results in a restoring force and subsequent coherent oscillation of the conduction band electrons, sharply enhancing scattering and absorption at specific resonance frequencies. The scattering spectra of nanoparticles are dictated by intrinsic parameters such as their size, shape, and composition, as well as extrinsic factors such as the local refractive index of the surrounding medium. These intrinsic parameters enable the design of gold nanoparticles with peak resonances that can be tuned to specific wavelengths across a relatively wide spectral region (600-2,200 nm) simply by modifying the synthesis protocol to realize the appropriate structural parameters (2,3). Because slight changes in the local refractive index of nanoparticles result in a peak wavelength shift, noble metal nanoparticles are also exquisitely sensitive reporters of their local, nanoscale environment.The unique properties of p...

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“…Scaffold (version Scaffold_3_00_08, Proteome Software Inc.) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm (Keller et al, 2002). Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides.…”

Section: Proteomics Of Dvl3-based Supermolecular Complexesmentioning

confidence: 99%

Proteomic Analysis of Wnt-Dependent Dishevelled-Based Supermolecular Complexes

Yokoyama¹

2012

Proteomics - Human Diseases and Protein Functions

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Peer Reviewed: Analytical Applications of Single-Molecule Detection

Cited by 51 publications

References 34 publications

Statistics of single‐molecule measurements: Applications in flow‐cytometry sizing of DNA fragments

Statistics of single‐molecule measurements: Applications in flow‐cytometry sizing of DNA fragments

Plasmonic flow cytometry by immunolabeled nanorods

Proteomic Analysis of Wnt-Dependent Dishevelled-Based Supermolecular Complexes

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