Fluorescence Polarisation Immunoassay

Klein, Christian; Batz, Hans‐Georg; Draeger, B.; Guder, Hans-Joachim; Herrmann, Rupert; Josel, Hans‐Peter; Nägele, Ulrich; Schenk, Roland; Vogt, Bernd

doi:10.1007/978-3-642-77372-3_17

Cited by 7 publications

(5 citation statements)

References 16 publications

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“…We therefore subjected 4-carboxyresorufin ( 11 )56 to the AM ether strategy, using phase-transfer conditions to prepare the isomeric substrates 12 and 13 in 37 and 35% yield, respectively. We used compound 12 in subsequent experiments.…”

Section: Resultsmentioning

confidence: 99%

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Synthesis and utility of fluorogenic acetoxymethyl ethers

2011

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Phenolic fluorophores such as fluorescein, Tokyo Green, resorufin, and their derivatives are workhorses of biological science. Acylating the phenolic hydroxyl group(s) in these fluorophores masks their fluorescence. The ensuing ester is a substrate for cellular esterases, which can restore fluorescence. These esters are, however, notoriously unstable to hydrolysis, severely compromising their utility. The acetoxymethyl (AM) group is an esterase-sensitive motif that can mask polar functionalities in small molecules. Here, we report on the use of AM ether groups to mask phenolic fluorophores. The resulting profluorophores have a desirable combination of low background fluorescence, high chemical stability, and high enzymatic reactivity, both in vitro and in cellulo. These simple phenyl ether-based profluorophores could supplement or supplant the use of phenyl esters for imaging biochemical and biological systems.

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Section: Resultsmentioning

confidence: 99%

“…2′,7′-Difluorofluorescein ( 1b ; ref 12), Tokyo Green ( 7 ; ref 33), and 4-carboxyresorufin ( 11 ; ref 56) were synthesized as described previously. Preactivated, powdered 4-Å molecular sieves from Aldrich were used as received.…”

Section: Methodsmentioning

confidence: 99%

Synthesis and utility of fluorogenic acetoxymethyl ethers

2011

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“…Fluorescence immunoassays are extensively used in medical diagnostics (Gosling, 1980;Vo-Dinh et al, 1993;Hemmila, 1992;Van Dyke and Van Dyke, 1990). Awide variety of approaches have been used including polarization (Klein et al, 1993;Fiore et al, 1988;Dandliker and de Saussure, 1970), resonance energy transfer (RET) (Morrison, 1988;Ullman et al, 1976) and gated "time-resolved" assays based on long lived lanthanide emission (Diamandis, 1988;Lövgren and Pettersson, 1990;Soini, 1984). There is continued development of new approaches to fluorescence immunoassays, including time-resolved assays (Ozinskas et al, 1993;Nithipatikom and McGown, 1987), RET assays for larger molecular weight species (Bruno et al, 2001) and multi-photon excitation (Baker et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Immunoassays based on directional surface plasmon-coupled emission

Matveeva¹,

Gryczyński²,

Gryczyński³

et al. 2004

Journal of Immunological Methods

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We described a new approach to immunoassays using surface plasmon-coupled emission (SPCE). Fluorescence is visually isotropic in space, so that the sensitivity is limited in part by the light collection efficiency. By the use of SPCE, we can efficiently collect the emission and convert it to a cone-like directional beam in a glass substrate. SPCE is the coupling of excited fluorophores with a thin metal film, resulting in radiation of surface plasmons into the higher refractive index media. We used SPCE to develop a model affinity assay using labeled goat anti-rabbit immunoglobulin G (IgG) antibodies against rabbit IgG bound to a 50-nm-thick silver film. Binding of labeled IgG to the surface resulted in increased intensity observed at an angle of 75 degrees from the normal in the glass substrate. The SPCE intensity depends on proximity of the fluorophore to the silver film and does not require a change in quantum yield upon binding. The use of SPCE is shown to provide background suppression because excited fluorophores distant from the silver film do not result in SPCE. Sensitivity and selectivity can be further increased by excitation under conditions of surface plasmon resonance (SPR) because the evanescent field is enhanced by the resonance interaction and excitation is limited to the region near the metal. We believe SPCE will provide a new technology for high sensitivity and selectivity in surface-bound assays and microfluidic systems.

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“…Immunoassay based on fluorescence detection is one approach to high sensitivity detection of biomarkers . − Different fluorescence detection approaches include polarization, − resonance energy transfer, − and time-resolved “gated” assays based on long-lived lanthanide emission . − Currently, new approaches to fluorescence immunoassays are being developed, including multiphoton excitation, − with the emphasis on high-throughput immunoassays. − …”

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confidence: 99%

Myoglobin Immunoassay Utilizing Directional Surface Plasmon-Coupled Emission

Matveeva

Gryczyński

et al. 2004

Anal. Chem.

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We described an immunoassay for the cardiac marker myoglobin on a thin silver mirror surface using surface plasmon-coupled emission (SPCE). SPCE occurs for fluorophores in proximity (within approximately 200 nm) of a thin metal film (in our case, silver) and results in a highly directional radiation through a glass substrate at a well-defined angle from the normal axis. We used the effect of SPCE to develop a myoglobin immunoassay on the silver mirror surface deposited on a glass substrate. Binding of the labeled anti-myoglobin antibodies led to the enhanced fluorescence emission at a specific angle of 72 degrees . The directional and enhanced directional fluorescence emission enables detection of myoglobin over a wide range of concentrations from subnormal to the elevated level of this cardiac marker. Utilizing SPCE allowed us also to demonstrate significant background suppression (from serum or whole blood) in the myoglobin immunoassay. We expect SPCE to become a powerful technique for performing immunoassays for many biomarkers in surface-bound assays.

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Fluorescence Polarisation Immunoassay

Cited by 7 publications

References 16 publications

Synthesis and utility of fluorogenic acetoxymethyl ethers

Synthesis and utility of fluorogenic acetoxymethyl ethers

Immunoassays based on directional surface plasmon-coupled emission

Myoglobin Immunoassay Utilizing Directional Surface Plasmon-Coupled Emission

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