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Fluorescence intensity and intermittency as tools for following dopamine bioconjugate processing in living cells.

Khatchadourian R, Bachir A, Clarke SJ, Heyes CD, Wiseman PW, Nadeau JL - J. Biomed. Biotechnol. (2007)

Bottom Line: CdSe/ZnS quantum dots (QDs) conjugated to biomolecules that quench their fluorescence, particularly dopamine, have particular spectral properties that allow determination of the number of conjugates per particle, namely, photoenhancement and photobleaching.In this work, we quantify these properties on a single-particle and ensemble basis in order to evaluate their usefulness as a tool for indicating QD uptake, breakdown, and processing in living cells.This creates a general framework for the use of fluorescence quenching and intermittency to better understand nanoparticle-cell interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, McGill University, 3775 Rue University, 316 Lyman Duff Medical Building, Montréal, Canada.

ABSTRACT
CdSe/ZnS quantum dots (QDs) conjugated to biomolecules that quench their fluorescence, particularly dopamine, have particular spectral properties that allow determination of the number of conjugates per particle, namely, photoenhancement and photobleaching. In this work, we quantify these properties on a single-particle and ensemble basis in order to evaluate their usefulness as a tool for indicating QD uptake, breakdown, and processing in living cells. This creates a general framework for the use of fluorescence quenching and intermittency to better understand nanoparticle-cell interactions.

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Number of bound dopamine molecules as a function of the number of EDC added to the coupling reaction for QD560 (squares) and QD605 (circles) as measured by OPA assay. Data are an average of three experiments with error bars indicating the standard error of the mean.
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fig2: Number of bound dopamine molecules as a function of the number of EDC added to the coupling reaction for QD560 (squares) and QD605 (circles) as measured by OPA assay. Data are an average of three experiments with error bars indicating the standard error of the mean.

Mentions: In this study, we used red-emitting CdSe/ZnS QDs (QD605, emission peak 605 ± 20 nm) forphotoenhancement and blinking studies, and green-emitting QDs (QD560, emissionpeak 560 ± 20 nm) for cellular uptake studies. QDs were conjugated to theneurotransmitter dopamine via the primary amine located on the opposite end ofthe molecule from the redox-active catechol (see Figure 1). The number of bound ligands was quantified in EDC-coupling reactions containing varying concentrations of dopamine and/or of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), using the o-phthaldialdehyde (OPA) assay asdescribed previously [20]. A strong dependence was observed of thenumber of EDCs per QD on the number of dopamine molecules that bound. The number of bound ligands increasedlinearly with the number of EDC molecules until a certain breakpoint and aplateau was reached, which was considered as the saturation point for the QDs (seeFigure 2). Indirectly, we can interpretthe saturation point as an indicator of the number of functional groupsavailable on the surface of the QDs.This appears to be slightly smaller for green QDs than for red, asexpected due to the smaller size of these particles, although it is the samewithin error at the maximum EDC concentration.


Fluorescence intensity and intermittency as tools for following dopamine bioconjugate processing in living cells.

Khatchadourian R, Bachir A, Clarke SJ, Heyes CD, Wiseman PW, Nadeau JL - J. Biomed. Biotechnol. (2007)

Number of bound dopamine molecules as a function of the number of EDC added to the coupling reaction for QD560 (squares) and QD605 (circles) as measured by OPA assay. Data are an average of three experiments with error bars indicating the standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279208&req=5

fig2: Number of bound dopamine molecules as a function of the number of EDC added to the coupling reaction for QD560 (squares) and QD605 (circles) as measured by OPA assay. Data are an average of three experiments with error bars indicating the standard error of the mean.
Mentions: In this study, we used red-emitting CdSe/ZnS QDs (QD605, emission peak 605 ± 20 nm) forphotoenhancement and blinking studies, and green-emitting QDs (QD560, emissionpeak 560 ± 20 nm) for cellular uptake studies. QDs were conjugated to theneurotransmitter dopamine via the primary amine located on the opposite end ofthe molecule from the redox-active catechol (see Figure 1). The number of bound ligands was quantified in EDC-coupling reactions containing varying concentrations of dopamine and/or of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), using the o-phthaldialdehyde (OPA) assay asdescribed previously [20]. A strong dependence was observed of thenumber of EDCs per QD on the number of dopamine molecules that bound. The number of bound ligands increasedlinearly with the number of EDC molecules until a certain breakpoint and aplateau was reached, which was considered as the saturation point for the QDs (seeFigure 2). Indirectly, we can interpretthe saturation point as an indicator of the number of functional groupsavailable on the surface of the QDs.This appears to be slightly smaller for green QDs than for red, asexpected due to the smaller size of these particles, although it is the samewithin error at the maximum EDC concentration.

Bottom Line: CdSe/ZnS quantum dots (QDs) conjugated to biomolecules that quench their fluorescence, particularly dopamine, have particular spectral properties that allow determination of the number of conjugates per particle, namely, photoenhancement and photobleaching.In this work, we quantify these properties on a single-particle and ensemble basis in order to evaluate their usefulness as a tool for indicating QD uptake, breakdown, and processing in living cells.This creates a general framework for the use of fluorescence quenching and intermittency to better understand nanoparticle-cell interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, McGill University, 3775 Rue University, 316 Lyman Duff Medical Building, Montréal, Canada.

ABSTRACT
CdSe/ZnS quantum dots (QDs) conjugated to biomolecules that quench their fluorescence, particularly dopamine, have particular spectral properties that allow determination of the number of conjugates per particle, namely, photoenhancement and photobleaching. In this work, we quantify these properties on a single-particle and ensemble basis in order to evaluate their usefulness as a tool for indicating QD uptake, breakdown, and processing in living cells. This creates a general framework for the use of fluorescence quenching and intermittency to better understand nanoparticle-cell interactions.

Show MeSH