Detection of Antigen Protein by Using Fluorescence Cross‐Correlation Spectroscopy and Quantum‐Dot‐Labeled Antibodies

Fujii, Fumihiko; Kinjo, Masataka

doi:10.1002/cbic.200700399

Cited by 14 publications

(13 citation statements)

References 50 publications

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“…There are many applications of FCCS to detect bimolecular interactions in solution [21][22][23]. However, quantitative measurements of interactions are also important in the living cell environment.…”

Section: Discussionmentioning

confidence: 99%

Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell

Tiwari

Mikuni

Muto

et al. 2013

Biochemical and Biophysical Research Communications

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Section: Discussionmentioning

confidence: 99%

Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell

Tiwari

Mikuni

Muto

et al. 2013

Biochemical and Biophysical Research Communications

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“…FCS monitors the change of the diffusion time (or molecular size) though the interaction cannot be distinguished if the diffusion time is decreased or not changed due to the change of the molecular shape. For demonstration, PEI-QDs and rhodamine green-labeled 500 bp linear DNA (RG-500bp) were mixed at various ratios, and then FCCS measurements were carried out with single-laser excitation at 488 nm (Fujii et al, 2007). Fluorescence emissions were collected from two channels for rhodamine green and QD.…”

Section: Monitoring Of Dna/pei Complex Formation In Vitromentioning

confidence: 99%

Fluorescence Cross-Correlation Spectroscopy for Real-Time Monitoring of Exogenous DNA Behavior in Living Cells

Sasaki¹,

Kinjo²

2011

Gene Therapy Applications

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“…Recently, fluorescence correlation spectroscopy ͑FCS͒, a single molecule technique, has been used for single molecule kinetics and biomolecular interaction studies in living cells, 6 antigen detection, 7 and freely diffusing gold nanorod in vivo imaging. 8 Liu et al demonstrated in a recent study that prostate-specific antigens can be detected by dynamic light scattering ͑DLS͒ with a detection limit of 0.5 ng/ mL in neat buffers.…”

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confidence: 99%

Ultrasensitive protein detection in blood serum using gold nanoparticle probes by single molecule spectroscopy

2009

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Abstract.A one-step rapid and ultrasensitive immunoassay capable of detecting proteins in blood serum is developed using gold nanoprobes and fluorescence correlation spectroscopy ͑FCS͒. In this approach we take advantage of the inherent photoluminescence property of gold nanoparticles ͑GNPs͒ to develop a fluorophore-free assay to observe binding entities by monitoring the diffusion of bound versus unbound molecules in a limited confocal volume. 40-nm GNPs conjugated separately with rabbit anti-IgG ͑Fc͒ and goat anti-IgG ͑Fab͒ when incubated in blood serum containing IgG forms a sandwich structure constituting dimers and oligomers that can be differentiated by to detect IgG in blood serum at a limit of detection ͑LOD͒ of 5 pg/ml. Immunoassays play a prominent role in clinical diagnostics, which are based on specific molecular recognition of antigens by antibodies. Although many advances such as electrochemical biosensors, 1 surface-enhanced Raman scattering 2 have enhanced the limit of detection ͑LOD͒, the quest for a simpler and more sensitive assay format persists. It is known that gold nanoparticles ͑GNP͒ posses an enhanced degree of absorption and scattering due to the surface plasmon oscillation of the electron cloud in metal particles to yield light emission several orders of magnitude compared to some of the conventional dyes. 3 The strong photon luminescence, light scattering property, and biocompatibility of GNP have been exploited for cell imaging 4 and photothermal therapy. 5 Past work on GNP photon luminescence has focused mostly on qualitative application of microscopic imaging and assessment. Our motivation here is to develop a quantitative assay for the very first time in blood serum using fluorescence correlation spectroscopy at a single particle level.Recently, fluorescence correlation spectroscopy ͑FCS͒, a single molecule technique, has been used for single molecule kinetics and biomolecular interaction studies in living cells, 6 antigen detection, 7 and freely diffusing gold nanorod in vivo imaging.8 Liu et al. demonstrated in a recent study that prostate-specific antigens can be detected by dynamic light scattering ͑DLS͒ with a detection limit of 0.5 ng/ mL in neat buffers. 9 We hypothesize that if GNP could be used as labels instead of fluorophores, then the present FCS technique could be adapted to observe the diffusion mode of not only a single gold nanoparticle but also higher-order aggregates of GNP. Because the diffusion time of GNPs passing through the confocal volume increases on formation of aggregates constituting oligomers, by studying the diffusion properties of gold particle-based oligomers from autocorrelation analysis, a highly sensitive FCS-based immunoassay could be developed. In addition to that, we also show that diffusion and hydrodynamic diameter of GNP and their aggregates could be consistently monitored in blood serum for quantification.We report on the development of a one-step assay to detect human IgG as an antigen protein spiked in 20% dog blood serum using GNP conju...

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Detection of Antigen Protein by Using Fluorescence Cross‐Correlation Spectroscopy and Quantum‐Dot‐Labeled Antibodies

Cited by 14 publications

References 50 publications

Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell

Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell

Fluorescence Cross-Correlation Spectroscopy for Real-Time Monitoring of Exogenous DNA Behavior in Living Cells

Ultrasensitive protein detection in blood serum using gold nanoparticle probes by single molecule spectroscopy

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