Time-resolved luminescence anisotropy-based detection of immunoglobulin G using long-lifetime Ru(II) complex-labeled protein A

Shimoyama, Takashi; Mahara, Atsushi; Munaka, Tatsuya; Yamagata, Koich; Iwase, Reiko; Yamaoka, Tetsuji; Murakami, Akira

doi:10.1016/j.ab.2004.02.006

Cited by 9 publications

(6 citation statements)

References 11 publications

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“…Luminescent metal complexes of transition metals and lanthanide ions [28][29][30][31][32], have been explored as molecular probes for fluorescence polarisation based measurements [33]. The long luminescence lifetimes of these probes (0.1-2000 μs) are appealing for studying dynamics on these timescales [33], but binding assays require brighter dyes.…”

Section: Introductionmentioning

confidence: 99%

Azadioxatriangulenium: exploring the effect of a 20 ns fluorescence lifetime in fluorescence anisotropy measurements

Bogh

Bora

Rosenberg

et al. 2015

Methods Appl. Fluoresc.

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Azaoxatriangulenium (ADOTA) has been shown to be highly emissive despite a moderate molar absorption coefficient of the primary electronic transition. As a result, the fluorescence lifetime is ~20 ns, longer than all commonly used red fluorescent organic probes. The electronic transitions in ADOTA are highly polarised (r0 = 0.38), which in combination with the long fluorescence lifetime extents the size-range of biomolecular weights that can be detected in fluorescence polarisation-based experiments. Here, the rotational dynamics of bovine serum albumin (BSA) are monitored with three different ADOTA derivatives, differing only in constitution of the reactive linker. A detailed study of the degree of labelling, the steady-state anisotropy, and the time-resolved anisotropy of the three different ADOTA-BSA conjugates are reported. The fluorescence quantum yields (ϕfl) of the free dyes in PBS solution are determined to be ~55%, which is reduced to ~20% in the ADOTA-BSA conjugates. Despite the reduction in ϕfl, a ~20 ns intensity averaged lifetime is maintained, allowing for the rotational dynamics of BSA to be monitored for up to 100 ns. Thus, ADOTA can be used in fluorescence polarisation assays to fill the gap between commonly used organic dyes and the long luminescence lifetime transition metal complexes. This allows for efficient steady-state fluorescence polarisation assays for detecting binding of analytes with molecular weights of up to 100 kDa.

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Section: Introductionmentioning

confidence: 99%

Azadioxatriangulenium: exploring the effect of a 20 ns fluorescence lifetime in fluorescence anisotropy measurements

Bogh

Bora

Rosenberg

et al. 2015

Methods Appl. Fluoresc.

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“…This provides the basis for the straightforward temporal discrimination of shorter-lived autofluorescence and scattered excitation light from label emission with the aid of time-gated measurements, thereby enhancing the sensitivity [69], and enables lifetime-based sensing. Due to their long lifetimes in conjunction with the straightforward excitation and emission in the visible or rarely, even in the NIR, Ru(II) complexes are common probes and labels in lifetime-based assays and (bio)sensors and in fluorescence polarization assays [70]. As the emission lifetimes of Ru(II) complexes are typically oxygensensitive, these species present the most commonly used lifetime-based oxygen sensors [71,72].…”

Section: Comparison Of Chromophoresmentioning

confidence: 99%

Nanocrystals and Nanoparticles Versus Molecular Fluorescent Labels as Reporters for Bioanalysis and the Life Sciences: A Critical Comparison

Resch‐Genger

Grabolle

Nitschke

et al. 2010

Advanced Fluorescence Reporters in Chemistry and Biology II

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At the core of photoluminescence techniques are suitable fluorescent labels and reporters, the spectroscopic properties of which control the limit of detection, the dynamic range, and the potential for multiplexing. Many applications including recent developments in intracellular labeling rely on well established molecular chromophores such as small organic dyes or fluorescent proteins. However, one of the most exciting -but also controversial -advances in reporter technology, the emerging development and application of luminescent nanoparticles with unique optical properties, yet complicated surface chemistry paves new roads for fluorescence imaging and sensing as well as for in vitro and in vivo labeling. Here, we compare and evaluate the differences in physico-chemical properties of common fluorophores, focusing on traditional organic dyes and luminescent nanocrystals with size-dependent features. The ultimate goal is to provide a better understanding of the advantages and limitations of both classes of chromophores, facilitate fluorophore choice for users of fluorescence techniques, and address future challenges in the rational design and manipulation of nanoparticulate labels and probes.

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“…Furthermore, the highly energetic UV excitation wavelengths are not suited for cellular applications as they result in enhanced photodegradation, significant auto-fluorescence and interference from scattering phenomena [1,9,10]. Following excitation at wavelengths outside the blue region of the spectrum, emissive transition metal complexes have been used to detect large biomolecules such as Immunoglobulin G [2,11]. However, the initial anisotropy for such complexes is intrinsically low, decreasing the dynamic range of the measurement.…”

Section: Introductionmentioning

confidence: 99%

Azadioxatriangulenium: a long fluorescence lifetime fluorophore for large biomolecule binding assay

Sørensen

Thyrhaug

Szabelski

et al. 2013

Methods Appl. Fluoresc.

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Of the many optical bioassays available, sensing by fluorescence anisotropy have great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is in the order of 20–200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatics dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecules assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immuniglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time by more than 75 %, and a change in the steady-state anisotropy increase of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay for detecting binding events involving biomolecules of far larger size than what is possible with the other red emitting organic dyes.

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Time-resolved luminescence anisotropy-based detection of immunoglobulin G using long-lifetime Ru(II) complex-labeled protein A

Cited by 9 publications

References 11 publications

Azadioxatriangulenium: exploring the effect of a 20 ns fluorescence lifetime in fluorescence anisotropy measurements

Azadioxatriangulenium: exploring the effect of a 20 ns fluorescence lifetime in fluorescence anisotropy measurements

Nanocrystals and Nanoparticles Versus Molecular Fluorescent Labels as Reporters for Bioanalysis and the Life Sciences: A Critical Comparison

Azadioxatriangulenium: a long fluorescence lifetime fluorophore for large biomolecule binding assay

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