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Capillary electrophoresis for the characterization of quantum dots after non-selective or selective bioconjugation with antibodies for immunoassay.

Pereira M, Lai EP - J Nanobiotechnology (2008)

Bottom Line: The migration times of these conjugates were determined in comparison to their non-conjugated QD relatives based upon their charge-to-size ratio values.Together, both QDs and CE-LIF can be applied as a sensitive technique for the detection of biological molecules.This work will contribute to the advancements in applying nanotechnology for molecular diagnosis in medical field.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, Ottawa-Carleton Chemistry Institute, Carleton University, Ottawa, ON K1S 5B6, Canada. edward_lai@carleton.ca.

ABSTRACT
Capillary electrophoresis coupled with laser-induced fluorescence was used for the characterization of quantum dots and their conjugates to biological molecules. The CE-LIF was laboratory-built and capable of injection (hydrodynamic and electrokinetic) from sample volumes as low as 4 muL via the use of a modified micro-fluidic chip platform. Commercially available quantum dots were bioconjugated to proteins and immunoglobulins through the use of established techniques (non-selective and selective). Non-selective techniques involved the use of EDCHCl/sulfo-NHS for the conjugation of BSA and myoglobin to carboxylic acid-functionalized quantum dots. Selective techniques involved 1) the use of heterobifunctional crosslinker, sulfo-SMCC, for the conjugation of partially reduced IgG to amine-functionalized quantum dots, and 2) the conjugation of periodate-oxidized IgGs to hydrazide-functionalized quantum dots. The migration times of these conjugates were determined in comparison to their non-conjugated QD relatives based upon their charge-to-size ratio values. The performance of capillary electrophoresis in characterizing immunoconjugates of quantum dot-labeled IgGs was also evaluated. Together, both QDs and CE-LIF can be applied as a sensitive technique for the detection of biological molecules. This work will contribute to the advancements in applying nanotechnology for molecular diagnosis in medical field.

No MeSH data available.


Related in: MedlinePlus

Overlapping electropherograms illustrating QD-NH2 (1), QD-hydrazide (2), and QD-COOH (3). CE buffer electrolyte used was 50 mM borate, pH 9.2. Gravity injection performed by elevating inlet capillary 7 cm for 5 s. Applied voltage for CE separation was 28 kV. Capillary temperature maintained at 20°C. Excitation source and detection wavelength was 473 nm and 620 nm, respectively.
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Figure 9: Overlapping electropherograms illustrating QD-NH2 (1), QD-hydrazide (2), and QD-COOH (3). CE buffer electrolyte used was 50 mM borate, pH 9.2. Gravity injection performed by elevating inlet capillary 7 cm for 5 s. Applied voltage for CE separation was 28 kV. Capillary temperature maintained at 20°C. Excitation source and detection wavelength was 473 nm and 620 nm, respectively.

Mentions: Figure 9 illustrates overlapping electropherograms of QD-hydrazide (2) in comparison to QD-NH2 (1) and QD-COOH (3). Their characteristic migration times can be attributed to the pKa of the functional group expressed on the QD relative to the pH of the CE separation buffer (9.2). Alkylated primary amines and carboxylic acids have measured pKa ~10, and ~4.5, respectively. Thus, the effect of the CE separation buffer pH allows the QD-NH2 to exhibit a net positive charge due to protonation of the primary amines. However, the QD-COOH will be completely ionized, exhibiting a net negative charge. Figure 8 can show a distinct change in migration time between QD-NH2 and QD-COOH. Hydrazides have remarkably low pKa values (~2.5), thus QD-hydrazides will be deprotonated during CE separation and exhibit a net-neutral charge. Again, this can be observed in figure 8 as QD-hydrazide migrates intermediate of the positively- and negatively- charged QDs. The small differences in migration times between QDs with substantially different charged residues on their surfaces can be attributed to the very large size of the particles that greatly influence their migration. Suppression of the EOF may improve resolution by means of increased electrophoretic contributions from QD-biomolecule conjugates [33].


Capillary electrophoresis for the characterization of quantum dots after non-selective or selective bioconjugation with antibodies for immunoassay.

Pereira M, Lai EP - J Nanobiotechnology (2008)

Overlapping electropherograms illustrating QD-NH2 (1), QD-hydrazide (2), and QD-COOH (3). CE buffer electrolyte used was 50 mM borate, pH 9.2. Gravity injection performed by elevating inlet capillary 7 cm for 5 s. Applied voltage for CE separation was 28 kV. Capillary temperature maintained at 20°C. Excitation source and detection wavelength was 473 nm and 620 nm, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2567343&req=5

Figure 9: Overlapping electropherograms illustrating QD-NH2 (1), QD-hydrazide (2), and QD-COOH (3). CE buffer electrolyte used was 50 mM borate, pH 9.2. Gravity injection performed by elevating inlet capillary 7 cm for 5 s. Applied voltage for CE separation was 28 kV. Capillary temperature maintained at 20°C. Excitation source and detection wavelength was 473 nm and 620 nm, respectively.
Mentions: Figure 9 illustrates overlapping electropherograms of QD-hydrazide (2) in comparison to QD-NH2 (1) and QD-COOH (3). Their characteristic migration times can be attributed to the pKa of the functional group expressed on the QD relative to the pH of the CE separation buffer (9.2). Alkylated primary amines and carboxylic acids have measured pKa ~10, and ~4.5, respectively. Thus, the effect of the CE separation buffer pH allows the QD-NH2 to exhibit a net positive charge due to protonation of the primary amines. However, the QD-COOH will be completely ionized, exhibiting a net negative charge. Figure 8 can show a distinct change in migration time between QD-NH2 and QD-COOH. Hydrazides have remarkably low pKa values (~2.5), thus QD-hydrazides will be deprotonated during CE separation and exhibit a net-neutral charge. Again, this can be observed in figure 8 as QD-hydrazide migrates intermediate of the positively- and negatively- charged QDs. The small differences in migration times between QDs with substantially different charged residues on their surfaces can be attributed to the very large size of the particles that greatly influence their migration. Suppression of the EOF may improve resolution by means of increased electrophoretic contributions from QD-biomolecule conjugates [33].

Bottom Line: The migration times of these conjugates were determined in comparison to their non-conjugated QD relatives based upon their charge-to-size ratio values.Together, both QDs and CE-LIF can be applied as a sensitive technique for the detection of biological molecules.This work will contribute to the advancements in applying nanotechnology for molecular diagnosis in medical field.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, Ottawa-Carleton Chemistry Institute, Carleton University, Ottawa, ON K1S 5B6, Canada. edward_lai@carleton.ca.

ABSTRACT
Capillary electrophoresis coupled with laser-induced fluorescence was used for the characterization of quantum dots and their conjugates to biological molecules. The CE-LIF was laboratory-built and capable of injection (hydrodynamic and electrokinetic) from sample volumes as low as 4 muL via the use of a modified micro-fluidic chip platform. Commercially available quantum dots were bioconjugated to proteins and immunoglobulins through the use of established techniques (non-selective and selective). Non-selective techniques involved the use of EDCHCl/sulfo-NHS for the conjugation of BSA and myoglobin to carboxylic acid-functionalized quantum dots. Selective techniques involved 1) the use of heterobifunctional crosslinker, sulfo-SMCC, for the conjugation of partially reduced IgG to amine-functionalized quantum dots, and 2) the conjugation of periodate-oxidized IgGs to hydrazide-functionalized quantum dots. The migration times of these conjugates were determined in comparison to their non-conjugated QD relatives based upon their charge-to-size ratio values. The performance of capillary electrophoresis in characterizing immunoconjugates of quantum dot-labeled IgGs was also evaluated. Together, both QDs and CE-LIF can be applied as a sensitive technique for the detection of biological molecules. This work will contribute to the advancements in applying nanotechnology for molecular diagnosis in medical field.

No MeSH data available.


Related in: MedlinePlus