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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: 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.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.


Reaction scheme illustrating hydrolysis of sulfo-SMCC activated of QD-NH2 (QD-maleimide). Hydrolyzed QD-maleimide will contain maleamic acid moiety (QD-maleamic) unreactive towards free sulfhydryls.
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Figure 6: Reaction scheme illustrating hydrolysis of sulfo-SMCC activated of QD-NH2 (QD-maleimide). Hydrolyzed QD-maleimide will contain maleamic acid moiety (QD-maleamic) unreactive towards free sulfhydryls.

Mentions: The use of the heterobifunctional crosslinker sulfo-SMCC allowed for straightforward activation of amine-functionalized QDs (QD-NH2) via a nucleophilic reaction between the active ester on the crosslinker and the amine moiety of the QD. Despite the activated QD (QD-maleimide) being relatively stable at physiological pH, temperature is an important factor to control as higher temperatures (above room temperature) can accelerate hydrolysis reactions. Hydrolysis of the maleimide moiety will form maleamic acid that is unreactive towards free sulfhydryls (Figure 6). Characterization of the hydrolyzed QD-maleimide by CE detected a migration time similar to that for QD-COOH (data not shown).


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)

Reaction scheme illustrating hydrolysis of sulfo-SMCC activated of QD-NH2 (QD-maleimide). Hydrolyzed QD-maleimide will contain maleamic acid moiety (QD-maleamic) unreactive towards free sulfhydryls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Reaction scheme illustrating hydrolysis of sulfo-SMCC activated of QD-NH2 (QD-maleimide). Hydrolyzed QD-maleimide will contain maleamic acid moiety (QD-maleamic) unreactive towards free sulfhydryls.
Mentions: The use of the heterobifunctional crosslinker sulfo-SMCC allowed for straightforward activation of amine-functionalized QDs (QD-NH2) via a nucleophilic reaction between the active ester on the crosslinker and the amine moiety of the QD. Despite the activated QD (QD-maleimide) being relatively stable at physiological pH, temperature is an important factor to control as higher temperatures (above room temperature) can accelerate hydrolysis reactions. Hydrolysis of the maleimide moiety will form maleamic acid that is unreactive towards free sulfhydryls (Figure 6). Characterization of the hydrolyzed QD-maleimide by CE detected a migration time similar to that for QD-COOH (data not shown).

Bottom Line: 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.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.