Detecting Biomolecules in Picoliter Vials Using Aequorin Bioluminescence

Crofcheck, Czarena; Grosvenor, Anne L.; Anderson, Kimberly W.; Lumpp, Janet K.; Daunert, Sylvia

doi:10.1021/ac9706786

Cited by 32 publications

(33 citation statements)

References 26 publications

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“…It has been used extensively as a calcium indicator 4 and more recently as a highly sensitive quantitive label in analytical assay systems. [5][6][7] Aequorin consists of the apoaequorin (22,400 Mw), coelenterazine (luminophore) and molecular oxygen. When Ca 2+ binds to the aequorin complex, aequorin undergoes a conformational change, and then coelenterazine is oxidized to coelenteramide, with release of CO 2 and light (λmax ~469 nm).…”

Section: Introductionmentioning

confidence: 99%

Development of Bioluminescence Immunoassay Using Photoprotein, Aequorin and Site-directed Immobilization

2003

Bulletin of the Korean Chemical Society

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The heterogeneous bioluminescence immunoassay for digoxin was developed using photoprotein, native aequorin as a label and the site-directed immobilization technique based on avidin/biotin interaction. Aequorin is a bioluminescence protein, originally isolated from the jellyfish Aequoria Victoria and an attractive label in analytical applications because of sensitive detection due to virtually no background bioluminescent signal. Digoxin is a cardioactive drug, and its therapeutic level in serum is at low concentration with very narrow therapeutic index. The aequorin-digoxigenin conjugates were synthesized by the N-hydroxysuccinimide ester method and characterized in terms of bioluminescent residual activity. The resulting dose-response curve shows that the detection limit is 1.0 × 10 −10 M and a dynamic range is three orders of magnitude, which was obtained by 1.0 × 10 −10 M conjugate and 0.9 µg/mL anti-digoxin antibody. Three structurally similar molecules to digoxin were examined for their cross-reactivity. None of these three compounds showed any crossreactivity with digoxin antibody employed in this study. Standard amounts of digoxin corresponding to the therapeutic range were spiked into the each serum solution. Study of the serum matrix effect indicated that correlation coefficient shows good agreement between luminescence light intensity between in buffer and in serum.

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Section: Introductionmentioning

confidence: 99%

Development of Bioluminescence Immunoassay Using Photoprotein, Aequorin and Site-directed Immobilization

2003

Bulletin of the Korean Chemical Society

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“…The vials have been used in several applications, e.g., as a container for electrokinetic injections of DNA samples [4], as targets for MALDI-TOF-MS analysis of proteins [5], and as reaction vessels for tryptic digests of myoglobin followed by an electrophoretic separation of the obtained peptide map [6]. The concept of using open microvials has also been taken up by other investigators in electrochemical studies [7±11] bioluminescence measurements [12], and as MALDI-TOF-MS targets for oligonucleotide analysis [13] and peptides [14]. Fabrication technologies that have been employed are etching [7,8,13], laser ablation [12] or replica imprinting from micromachined masters [9±11], as well as etching optical fibers [15,16].…”

Section: Introductionmentioning

confidence: 99%

“…The concept of using open microvials has also been taken up by other investigators in electrochemical studies [7±11] bioluminescence measurements [12], and as MALDI-TOF-MS targets for oligonucleotide analysis [13] and peptides [14]. Fabrication technologies that have been employed are etching [7,8,13], laser ablation [12] or replica imprinting from micromachined masters [9±11], as well as etching optical fibers [15,16]. Commercially available assay systems for screening applications have also been miniaturized (200 nL vial volume), using a plate design with 9600 wells in each plate [7].…”

Section: Introductionmentioning

confidence: 99%

Parallel reactions in open chip-based nanovials with continuous compensation for solvent evaporation

2000

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In an earlier report (Litborn, E., Emmer, Å., Roeraade, J., Anal. Chim. Acta 1999, 401, 11—19), we described a technique for performing chemistry in chip‐based vials. A major problem, solvent evaporation, was partially remedied by using a closed humidity chamber. In this paper we report an improved technique for performing parallel reactions in open, 15 nL volume, chip‐based vials. The evaporation of solvent from the reaction fluid was continuously compensated by addition of solvent via an array of microcapillaries. The suitability of the method was demonstrated by performing eight separate peptide maps of myoglobin in parallel, using the three enzymes trypsin, α‐chymotrypsin and endoproteinase Glu‐C. The total amount of myoglobin utilized to perform the eight digests was less than 100 pmol. The corresponding amount of enzymes was ca. 0.1 pmol per reaction. In order to evaluate the operating limits of the technique, a study of the evaporation of solvents from a series of vials with proportionally smaller volumes operated at different temperatures was performed. The results showed that the concept for continuous compensation of solvent evaporation should be applicable to reaction volumes down to 30 pL.

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“…Methods for small volume analyses have been reported using spectroscopic detection systems [1±9]. Other approaches to submicroliter analysis that have been reported include binding assays [10], titrations [11±13], separation techniques (for examples see [14±17]) and a host of electrochemical methods, including those highlighted in this issue of Electroanalysis.…”

Section: Introductionmentioning

confidence: 99%

Effect of Fabrication Factors on Performance of Screen-Printed/Laser Micromachined Electrochemical Nanovials

et al. 2000

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Electrochemical vials with volumes of 2.6 nL and embedded working and reference electrodes have been fabricated by coupling screen‐printing (thick‐film) technology with excimer laser micromachining. The processing parameters were varied to determine their effect on the resulting nanocell. The fabricated systems were analyzed by resistance measurements, surface profilometry, and SEM. The performance of the electrochemical nanovials was evaluated by cyclic voltammetry using hexaamineruthenium(III) chloride. The current limits of thick‐film fabrication were approached in creating very small nanocells using the methods described. The functioning small volume electrochemical cells gave voltammograms that were either peak‐shaped or sigmoidal. The shape of the voltammogram corresponded to the electrochemical surface area determined by chronoamperometry.

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Detecting Biomolecules in Picoliter Vials Using Aequorin Bioluminescence

Cited by 32 publications

References 26 publications

Development of Bioluminescence Immunoassay Using Photoprotein, Aequorin and Site-directed Immobilization

Development of Bioluminescence Immunoassay Using Photoprotein, Aequorin and Site-directed Immobilization

Parallel reactions in open chip-based nanovials with continuous compensation for solvent evaporation

Effect of Fabrication Factors on Performance of Screen-Printed/Laser Micromachined Electrochemical Nanovials

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