Time-resolved fluorescence of a new europium-chelate complex: demonstration of highly sensitive detection of protein and DNA samples

Saha, Ashis; Kross, Kathryn; Kloszewski, Edmond D.; Upson, Donald A.; Toner, John L.; Snow, Robert A.; Black, Chrostpher D. V.; Desai, Vinay C.

doi:10.1021/ja00076a088

Cited by 142 publications

(76 citation statements)

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“…[1][2][3][4][5][6] One of the more recent developments is the use of time-resolved fluorescence (TRF) assays, in which a long fluorescence lifetime is utilized to distinguish the probe fluorescence from any other short-lived background emission. [7][8][9][10] Another independent trend is the use of fluorescent peptide substrates, which are based on intramolecular probe/quencher contact, [11][12][13][14] rather than on quenching by conventional fluorescence resonance energy transfer (FRET). [15][16][17][18] Herein, we introduce an assay type that combines the assets of time-resolved fluorescence detection with the advantages of collision-induced quenching and the use of a highly biocompatible fluorophore:…”

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confidence: 99%

Nanosecond Time‐Resolved Fluorescence Protease Assays

et al. 2006

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Arguably, fluorescence-based assays are on the rise and, stepby-step, will replace alternative radioactive or absorptionbased assays on a laboratory microscale as well in highthroughput screening (HTS) for the pharmaceutical industry. [1][2][3][4][5][6] One of the more recent developments is the use of time-resolved fluorescence (TRF) assays, in which a long fluorescence lifetime is utilized to distinguish the probe fluorescence from any other short-lived background emission. [7][8][9][10] Another independent trend is the use of fluorescent peptide substrates, which are based on intramolecular probe/quencher contact, [11][12][13][14] rather than on quenching by conventional fluorescence resonance energy transfer (FRET). [15][16][17][18] Herein, we introduce an assay type that combines the assets of time-resolved fluorescence detection with the advantages of collision-induced quenching and the use of a highly biocompatible fluorophore:We have previously introduced DBO derivatives as an unconventional class of fluorescent probes; they have been termed fluorazophores.[19] The most remarkable feature is their exceedingly long fluorescence lifetime (up to 1 ms, l max = 430 nm, f f = 0.26), [20] which we have previously exploited for mechanistic investigations in the area of supramolecular and biomolecular chemistry, namely for the determination of the association kinetics of water-soluble host molecules, [21][22][23][24][25] the diffusional properties of antioxidants in membrane models, [26][27][28] and the kinetics of oligonucleotide and polypeptide folding. [29][30][31] This study describes an implementation of these basic research efforts for fluorescence-based assays with immediate biochemical relevance for studies on enzymatic activity and drug discovery.TRF assays have become popular for lanthanide emitters. Their fluorescence, which should be more generally referred to as luminescence due to the nature of the electronic transitions involved, has lifetimes in the ms range that can be easily detected, given the presence of suitable enhancement agents or chelation, and differentiated from background emission. [7][8][9][10] This background is ubiquitous and originates from unconverted substrate, scattered excitation light, sample container materials, enzyme cofactors, impurities, and, most relevant for inhibitor screening and HTS, library compounds to be screened for biological activity. [5] When employing genuine fluorescence of organic chromophores, fluorescence lifetimes above 100 ns and time gates in the nanosecond range are required to allow the same measurement mode, which we refer to as Nano-TRF. Resting on the possibility of laser excitation, highly sensitive detection, and electronic time gating, the pertinent lifetime regime is sufficiently long to allow an accurate discrimination from unconverted substrate as well as background. DBO presents the first organic fluorophore to be utilized for Nano-TRF assays, and DBO-labeled asparagine ("Dbo"), which was previously synthesized in Fmoc-protected form, [29] c...

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Nanosecond Time‐Resolved Fluorescence Protease Assays

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“…The PA spectra of m ol ecular electro n tra nsiti ons for the sam pl e wi th Eu(I I I) are i n goo d agreement wi th the energy l evels of thi s i on i n a larg e D TP A-l ik e compl ex [8]. The gro und sta te of Eu i on i s spl it i nto seven com p onents ( { ) due to the Ùrst order spin{ orbi t coupl i ng wi th 6.…”

Section: R Esu L T S An D Discussionmentioning

confidence: 54%

“…For exam pl e, gadol i ni um(I I I) chelates ha ve b een under i ntense scruti ny for thei r use as contra st agents f or magneti c resona nce i m aging appl icati ons [5,6]. Euro pi um (I I I) compl exes are used as pro bes for l abel ing of D NA [7] and pro tei ns [8], and i n l um i nescence resonance energy tra nsf er studi es [9]. Ano ther use of l antha ni de(I I I) compl exes i s i n the sequence-speciÙc cleavage of nucl eic acids [10].…”

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confidence: 99%

Charge Transfer and f-f Transitions Studied by Photoacoustic Spectroscopy of [R(NO₃)₂(PicBH)₂]NO₃and [R(NO₃)₃(PicBH)₂] Complexes (R - Rare Earth Ion)

et al. 2003

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“…23,24 In the search for more sensitive markers, some chelates of lanthanides had already been obtained and tested in various biological assay models. [25][26][27] Time-resolved fluoroimmunometric assay (TR-FIA) is a method based on the luminescence emission that is measured after a delay has elapsed from a pulsed excitation, enabling the short-lived background fluorescence to be excluded. 28 Soini et.…”

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confidence: 99%

A fluorescent-labeled microcystin-LR terbium cryptate

Oliveira¹,

Nova²,

Alves-Jr³

et al. 2006

J. Braz. Chem. Soc.

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com o grupo éster do criptato de térbio. A formação do produto foi acompanhada por cromatografia líquida de alta eficiência (CLAE) em 238 nm e 310 nm. A presença de microcistina-LR na molécula marcada foi confirmada através de ensaio com enzima ligada a imunoabsorvente (ELISA) e por reação protéica com ácido bicincônico. O espectro de luminescência do criptato e da molécula conjugada também foram confirmados.A new fluorescent labeled compound of microcystin-LR with terbium cryptate was obtained by initial conjugation of microcystins-LR with aminoethanethiol followed by the reaction with the ester group of terbium cryptate. The product formation was followed by high performance liquid chromatography (HPLC) at 238 nm and 310 nm. The presence of microcystin-LR in the labeled molecule was confirmed by enzyme linked immunosorbent assay ELISA and by protein reaction with bicinhonic acid. Luminescence spectra of cryptate and the conjugated molecule were carried through as well. Keywords: terbium cryptate, microcystin-LR, cyanobacteria IntroductionMicrocystins are a group of stable heptapeptide hepatotoxins produced by freshwater cyanobacteria genera that lower the water quality leading to an increase in the risk of intoxication for animals and humans. Species of genera Microcystis, Anabaena, Aphanizomenon, Planktothrix (Oscillatoria), and Nostoc are frequently described as producers of microcystins. 1 The eutrophication of ponds, rivers and other freshwater ecosystems allows cyanobacteria to flourish, leading to contamination of the water. Microcystins are potent inhibitors of protein phosphatases 1 (PP 1 ) and 2A (PP 2A ), which are regulatory enzymes present in the cytosol of mammalian cells. 3 The general structure of microcystins is composed of cyclo-(D-Ala-X-D-MeAsp-ZAdda-D-Glu-Mdha), where X and Z could be various kinds of L-amino acids; D-MeAsp is D-erythro-β-methylaspartic acid; Mdha is N-methyldehydroalanine; and Adda is the unusual amino acid 2S,3S,8S,9S-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4E,6E-dienoic acid. 4 The most extensively studied form is microcystin-LR that contains L-leucine and L-arginine in the two main variant positions. In the search for more sensitive markers, some chelates of lanthanides had already been obtained and tested in various biological assay models. [25][26][27] Time-resolved fluoroimmunometric assay (TR-FIA) is a method based on the luminescence emission that is measured after a delay has elapsed from a pulsed excitation, enabling the short-lived background fluorescence to be excluded. 28Soini et. al.29 labeled anti-rabbit IgG with europium chelate as a model to detect rabbit IgG for human smooth muscle myosin in a histological section; Bonin et al. 30 used a complex of europium and samarium for the detection of diphteria antitoxins in serum using the ELISA method and Xu et al.31 labeled anti-β-LH and monoclonal anti-β-FSH antibodies with europium and samarium, respectively. The use of luminescent lanthanide as a substitute for labeled enzyme (e.g. horseradish peroxidas...

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Time-resolved fluorescence of a new europium-chelate complex: demonstration of highly sensitive detection of protein and DNA samples

Cited by 142 publications

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Nanosecond Time‐Resolved Fluorescence Protease Assays

Nanosecond Time‐Resolved Fluorescence Protease Assays

Charge Transfer and f-f Transitions Studied by Photoacoustic Spectroscopy of [R(NO₃)₂(PicBH)₂]NO₃and [R(NO₃)₃(PicBH)₂] Complexes (R - Rare Earth Ion)

A fluorescent-labeled microcystin-LR terbium cryptate

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Time-resolved fluorescence of a new europium-chelate complex: demonstration of highly sensitive detection of protein and DNA samples

Cited by 142 publications

References 0 publications

Nanosecond Time‐Resolved Fluorescence Protease Assays

Nanosecond Time‐Resolved Fluorescence Protease Assays

Charge Transfer and f-f Transitions Studied by Photoacoustic Spectroscopy of [R(NO3)2(PicBH)2]NO3and [R(NO3)3(PicBH)2] Complexes (R - Rare Earth Ion)

A fluorescent-labeled microcystin-LR terbium cryptate

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Charge Transfer and f-f Transitions Studied by Photoacoustic Spectroscopy of [R(NO₃)₂(PicBH)₂]NO₃and [R(NO₃)₃(PicBH)₂] Complexes (R - Rare Earth Ion)