Fluorescence-based flow-injection determination of biotin and biotinylated compounds

Smith‐Palmer, Truis; Barbarakis, Minas S.; Cynkowski, Tadeausz; Bachas, Leonidas G.

doi:10.1016/0003-2670(93)80328-i

Cited by 20 publications

(9 citation statements)

References 20 publications

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“…Though this increase in emission on biotin binding is not well-understood, it provides an analytical measure of concentration. 25 By this empirical method, a much higher concentration of polymer-displayed biotin is required to attain the same increase in streptavidin-FITC emission relative to free biotin (Supporting Information). The origin of this difference is unclear and may indicate diminished accessibility 26 of the biotinylated polymer terminus or an altered FITC emission environment for the protein-nanoparticle complex relative to the protein-biotin complex.…”

Section: Resultsmentioning

confidence: 99%

Z-Group Ketone Chain Transfer Agents for RAFT Polymer Nanoparticle Modification via Hydrazone Conjugation

et al. 2012

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A ketal-containing trithiocarbonyl compound has been synthesized and characterized as a chain transfer agent (CTA) in Reversible Addition Fragmentation Transfer (RAFT) polymerization. The ketal functionality does not interfere with RAFT polymerization of acrylate monomers, which proceeds as previously reported to yield macro-CTA polymers and block co-polymers. Post-polymerization ketal cleavage revealed ketone functionality at the polar terminus of an amphiphilic block co-polymer. Hydrazone-formation was facile in both organic solution as well as in aqueous buffer where polymer nanoparticle assemblies were formed, indicating a conjugation/end-functionalization yield of 40–50%. Conjugation was verified with fluorescein, biotin and Gd-DOTA derivatives, and though the trithiocarbonate linkage is hydrolytically labile, we observed stable conjugation for several days at pH 7.4. and 37°C. As expected, streptavidin binding to biotinylated polymer micelles was observed, and size-change based relaxivity increases were observed when Gd-DOTA hydrazide was conjugated to polymer micelles. Cell-uptake of fluorescently labeled polymer micelles was also readily tracked by FACS and fluorescence microscopy. These polymer derivatives demonstrate a range of potential theranostic/biotechnological applications for this conveniently accessible keto-CTA, which include ligand-based nanoparticle targeting and fluorescent/MR nanoparticle contrast agents.

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Section: Resultsmentioning

confidence: 99%

Z-Group Ketone Chain Transfer Agents for RAFT Polymer Nanoparticle Modification via Hydrazone Conjugation

et al. 2012

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“…A novel approach is the use of biochemical assays as a detection principle linked on-line to an LC separation column. Bachas and co-workers developed biochemical detection systems for biotin based on the binding of biotin to avidin and/or streptavidin using both labeled streptavidin and noncompetitive dyes. , We have demonstrated the feasibility of an on-line biochemical detection by a bioassay for digoxin in plasma, using fluorescein-labeled antibodies, , and the determination of biotin, using fluorescence-labeled biotin and avidin …”

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confidence: 99%

Reversed-Phase Liquid Chromatography Coupled On-Line to Receptor Affinity Detection Based on the Human Estrogen Receptor

et al. 1996

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On-line coupling of reversed-phase liquid chromatography to a biochemical detection system based on receptor-ligand interactions is described. The receptor affinity detection is performed using a postcolumn reaction detection system with open-tubular reaction coils. In the first step, a solution containing the steroid binding domain of the human estrogen receptor is added to the LC effluent via a mixing union, and the mixture is allowed to react. In the second step, a fluorescent estrogenic ligand, coumestrol, is added to saturate the remaining free binding sites of the estrogen receptor. Both heterogeneous and homogeneous detection systems have been developed. In the former case, free and receptor-bound coumestrol were separated prior to detection by means of a short column containing a restricted-access reversed-phase support. Since free and receptor-bound coumestrol differ significantly in fluorescence intensity, the system can also be operated in the homogeneous mode without requiring a separation step. Using 5 nmol/L of human estrogen receptor, a detection limit of 5 nmol/L was obtained for compounds possessing a high affinity for the estrogen receptor, such as 17 beta-estradiol and diethylstilbestrol. Detection limits for weaker ligands amount to 20 nmol/L. Non-estrogenic steroids such as methyltestosterone or progesterone are not detected at all. The selectivity of the present method is demonstrated for the analysis of urine samples.

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“…An increase in this steepness will shorten the dynamic range. Not surprisingly the dose-response curve for biotin has a very steep slope with a small dynamic range, and detection limits of 5 ϫ 10 Ϫ8 M. Biocytin has been shown to behave similarly to biotin in homogeneous fluorophore-linked binding assays with streptavidin-FITC (24). Because of this similarity, one could propose that biocytin, like biotin, would bind much more strongly to streptactin than Strep-tag II-BAP and that the dose-response curves for biocytin and biotin would be similar on the basis of theoretical predictions outlined in Ref.…”

Section: Resultsmentioning

confidence: 96%

Use of a Biomimetic Peptide in the Design of a Competitive Binding Assay for Biotin and Biotin Analogues

Gregory

Bachas

2001

Analytical Biochemistry

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Fluorescence-based flow-injection determination of biotin and biotinylated compounds

Cited by 20 publications

References 20 publications

Z-Group Ketone Chain Transfer Agents for RAFT Polymer Nanoparticle Modification via Hydrazone Conjugation

Z-Group Ketone Chain Transfer Agents for RAFT Polymer Nanoparticle Modification via Hydrazone Conjugation

Reversed-Phase Liquid Chromatography Coupled On-Line to Receptor Affinity Detection Based on the Human Estrogen Receptor

Use of a Biomimetic Peptide in the Design of a Competitive Binding Assay for Biotin and Biotin Analogues

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