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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.


Non-selective bioconjugation reaction scheme of carboxylated QDs (QD-COOH) to amine-containing proteins. This two-step reaction involves a) the activation of QD-COOH with EDC/sulfo-NHS, resulting in a semi-stable active ester (QD-NHS), and b) the nucleophilic reaction between the QD-NHS and amine-containing protein, forming a QD-protein conjugate via a stable amide bond.
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Figure 1: Non-selective bioconjugation reaction scheme of carboxylated QDs (QD-COOH) to amine-containing proteins. This two-step reaction involves a) the activation of QD-COOH with EDC/sulfo-NHS, resulting in a semi-stable active ester (QD-NHS), and b) the nucleophilic reaction between the QD-NHS and amine-containing protein, forming a QD-protein conjugate via a stable amide bond.

Mentions: QDs, often applied for the labeling of biological molecules (proteins, peptides, antibodies, etc.), require specific techniques for their conjugation [4-7]. The most popular bioconjugation technique involves the use of a zero-length crosslinker, 1-ethyl-3- [3-dimethylaminopropyl]carbodiimide hydrochloride (EDCHCl) [1-4,6,7], in the presence of a hydrophilic active group, N-hydroxysulfosuccinimide (sulfo-NHS) [8], for the formation of a stable amide bond between carboxylic acid-functionalized QDs (QD-COOH) and any biomolecules containing a primary amine [9] (Figure 1).


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)

Non-selective bioconjugation reaction scheme of carboxylated QDs (QD-COOH) to amine-containing proteins. This two-step reaction involves a) the activation of QD-COOH with EDC/sulfo-NHS, resulting in a semi-stable active ester (QD-NHS), and b) the nucleophilic reaction between the QD-NHS and amine-containing protein, forming a QD-protein conjugate via a stable amide bond.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Non-selective bioconjugation reaction scheme of carboxylated QDs (QD-COOH) to amine-containing proteins. This two-step reaction involves a) the activation of QD-COOH with EDC/sulfo-NHS, resulting in a semi-stable active ester (QD-NHS), and b) the nucleophilic reaction between the QD-NHS and amine-containing protein, forming a QD-protein conjugate via a stable amide bond.
Mentions: QDs, often applied for the labeling of biological molecules (proteins, peptides, antibodies, etc.), require specific techniques for their conjugation [4-7]. The most popular bioconjugation technique involves the use of a zero-length crosslinker, 1-ethyl-3- [3-dimethylaminopropyl]carbodiimide hydrochloride (EDCHCl) [1-4,6,7], in the presence of a hydrophilic active group, N-hydroxysulfosuccinimide (sulfo-NHS) [8], for the formation of a stable amide bond between carboxylic acid-functionalized QDs (QD-COOH) and any biomolecules containing a primary amine [9] (Figure 1).

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.