Limits...
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.

Show MeSH

Related in: MedlinePlus

Relationship between the number of dopamine molecules bound to the surface of QD605 and the resulting emission intensity.  (a) The quenching observed upon conjugation is nearly logarithmic. Data are an average of three experiments with error bars indicating the standard error of the mean.  (b) Unquenching of QD-dopamine conjugates with varying amounts of coverage under Hg-lamp exposure (QD filter set, see Methods). Each symbol is a data point, with error bars smaller than symbols (n = 3). Bare QDs alone (dashed line) show essentially constant fluorescence over a 2.5-second exposure period; they begin to photobleach near the end.  QDs, to which dopamine was added but not conjugated (no EDC) (open circles), achieve maximum fluorescence within 50 milliseconds. Conjugates with 40 ± 14 DA/particle (filled circles) brighten over a time course of ~700 milliseconds. The conjugates with the greatest coverage (filled squares, 255 ± 14 DA/particle) have just begun to plateau at 2.5 seconds. (c) Confocal laser illumination of samples with 255 ± 14 DA/particle.  Photoenhancement showed a strong dependence upon laser power, with efficient enhancement at 20% but limited or no enhancement at 10%, 50%, and 100% power. Each symbol is a data point, with error bars smaller than symbols (n = 5).  Unconjugated QDs showed similar bleaching curves for all powers tested (shown: 50%).
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2279208&req=5

fig3: Relationship between the number of dopamine molecules bound to the surface of QD605 and the resulting emission intensity. (a) The quenching observed upon conjugation is nearly logarithmic. Data are an average of three experiments with error bars indicating the standard error of the mean. (b) Unquenching of QD-dopamine conjugates with varying amounts of coverage under Hg-lamp exposure (QD filter set, see Methods). Each symbol is a data point, with error bars smaller than symbols (n = 3). Bare QDs alone (dashed line) show essentially constant fluorescence over a 2.5-second exposure period; they begin to photobleach near the end. QDs, to which dopamine was added but not conjugated (no EDC) (open circles), achieve maximum fluorescence within 50 milliseconds. Conjugates with 40 ± 14 DA/particle (filled circles) brighten over a time course of ~700 milliseconds. The conjugates with the greatest coverage (filled squares, 255 ± 14 DA/particle) have just begun to plateau at 2.5 seconds. (c) Confocal laser illumination of samples with 255 ± 14 DA/particle. Photoenhancement showed a strong dependence upon laser power, with efficient enhancement at 20% but limited or no enhancement at 10%, 50%, and 100% power. Each symbol is a data point, with error bars smaller than symbols (n = 5). Unconjugated QDs showed similar bleaching curves for all powers tested (shown: 50%).

Mentions: Manipulating theextent of ligand coverage of the QD surface can be an effective way to modulatefluorescent properties if the ligands can act as energy or electron donors oracceptors to or from the QDs. In ourprevious work, we have shown that dopamine can be used to modulate the emissioncharacteristics of QDs by a mechanism of electron transfer. In Figure 3(a), we demonstrate the effect ofthe number of bound dopamine ligands and the subsequent reduction in theemission intensity of the QDs. Todistinguish this type of quenching from the Stern-Volmer collisional quenching,we purified the conjugates from excess unbound ligand. Our results showed alarge decrease in intensity when a relatively small number of ligands werebound to the surface, owing to the electron transfer from dopamine to the QDs [5].


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)

Relationship between the number of dopamine molecules bound to the surface of QD605 and the resulting emission intensity.  (a) The quenching observed upon conjugation is nearly logarithmic. Data are an average of three experiments with error bars indicating the standard error of the mean.  (b) Unquenching of QD-dopamine conjugates with varying amounts of coverage under Hg-lamp exposure (QD filter set, see Methods). Each symbol is a data point, with error bars smaller than symbols (n = 3). Bare QDs alone (dashed line) show essentially constant fluorescence over a 2.5-second exposure period; they begin to photobleach near the end.  QDs, to which dopamine was added but not conjugated (no EDC) (open circles), achieve maximum fluorescence within 50 milliseconds. Conjugates with 40 ± 14 DA/particle (filled circles) brighten over a time course of ~700 milliseconds. The conjugates with the greatest coverage (filled squares, 255 ± 14 DA/particle) have just begun to plateau at 2.5 seconds. (c) Confocal laser illumination of samples with 255 ± 14 DA/particle.  Photoenhancement showed a strong dependence upon laser power, with efficient enhancement at 20% but limited or no enhancement at 10%, 50%, and 100% power. Each symbol is a data point, with error bars smaller than symbols (n = 5).  Unconjugated QDs showed similar bleaching curves for all powers tested (shown: 50%).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Relationship between the number of dopamine molecules bound to the surface of QD605 and the resulting emission intensity. (a) The quenching observed upon conjugation is nearly logarithmic. Data are an average of three experiments with error bars indicating the standard error of the mean. (b) Unquenching of QD-dopamine conjugates with varying amounts of coverage under Hg-lamp exposure (QD filter set, see Methods). Each symbol is a data point, with error bars smaller than symbols (n = 3). Bare QDs alone (dashed line) show essentially constant fluorescence over a 2.5-second exposure period; they begin to photobleach near the end. QDs, to which dopamine was added but not conjugated (no EDC) (open circles), achieve maximum fluorescence within 50 milliseconds. Conjugates with 40 ± 14 DA/particle (filled circles) brighten over a time course of ~700 milliseconds. The conjugates with the greatest coverage (filled squares, 255 ± 14 DA/particle) have just begun to plateau at 2.5 seconds. (c) Confocal laser illumination of samples with 255 ± 14 DA/particle. Photoenhancement showed a strong dependence upon laser power, with efficient enhancement at 20% but limited or no enhancement at 10%, 50%, and 100% power. Each symbol is a data point, with error bars smaller than symbols (n = 5). Unconjugated QDs showed similar bleaching curves for all powers tested (shown: 50%).
Mentions: Manipulating theextent of ligand coverage of the QD surface can be an effective way to modulatefluorescent properties if the ligands can act as energy or electron donors oracceptors to or from the QDs. In ourprevious work, we have shown that dopamine can be used to modulate the emissioncharacteristics of QDs by a mechanism of electron transfer. In Figure 3(a), we demonstrate the effect ofthe number of bound dopamine ligands and the subsequent reduction in theemission intensity of the QDs. Todistinguish this type of quenching from the Stern-Volmer collisional quenching,we purified the conjugates from excess unbound ligand. Our results showed alarge decrease in intensity when a relatively small number of ligands werebound to the surface, owing to the electron transfer from dopamine to the QDs [5].

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
Related in: MedlinePlus