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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.


Selective bioconjugation reaction scheme of hydrazide QDs (QD-hydrazide) to aldehyde-containing IgG antibodies (IgG-CHO). The reaction involves a) mild periodate oxidation of glycosylated IgG, yielding IgG-CHO; b) synthesis of QD-hydrazide via derivatization of QD-COOH with EDC/ADH; and c) conjugation of QD-hydrazide with IgG-CHO via formation of hydrazone linkage to yield QD-IgG.
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Figure 3: Selective bioconjugation reaction scheme of hydrazide QDs (QD-hydrazide) to aldehyde-containing IgG antibodies (IgG-CHO). The reaction involves a) mild periodate oxidation of glycosylated IgG, yielding IgG-CHO; b) synthesis of QD-hydrazide via derivatization of QD-COOH with EDC/ADH; and c) conjugation of QD-hydrazide with IgG-CHO via formation of hydrazone linkage to yield QD-IgG.

Mentions: Reductive amination is a bioconjugation technique popular in the labeling of glycoproteins. Taking advantage of the polysaccharide chains within the Fc region of an antibody, it can allow bioconjugation to occur relatively far away from the antigen binding site. Through oxidation (using sodium periodate) of the carbohydrate hydroxyls, the aldehydes formed are highly reactive toward primary amines and hydrazides [9]. This makes QD-NH2 or QD-COOH (derivatized with adipic acid dihydrazide (ADH)) suitable candidates for conjugation [9]. In addition, selective bioconjugation can occur without a proceeding reduction reaction, thus retaining the integrity of the antibody (Figure 3).


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)

Selective bioconjugation reaction scheme of hydrazide QDs (QD-hydrazide) to aldehyde-containing IgG antibodies (IgG-CHO). The reaction involves a) mild periodate oxidation of glycosylated IgG, yielding IgG-CHO; b) synthesis of QD-hydrazide via derivatization of QD-COOH with EDC/ADH; and c) conjugation of QD-hydrazide with IgG-CHO via formation of hydrazone linkage to yield QD-IgG.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Selective bioconjugation reaction scheme of hydrazide QDs (QD-hydrazide) to aldehyde-containing IgG antibodies (IgG-CHO). The reaction involves a) mild periodate oxidation of glycosylated IgG, yielding IgG-CHO; b) synthesis of QD-hydrazide via derivatization of QD-COOH with EDC/ADH; and c) conjugation of QD-hydrazide with IgG-CHO via formation of hydrazone linkage to yield QD-IgG.
Mentions: Reductive amination is a bioconjugation technique popular in the labeling of glycoproteins. Taking advantage of the polysaccharide chains within the Fc region of an antibody, it can allow bioconjugation to occur relatively far away from the antigen binding site. Through oxidation (using sodium periodate) of the carbohydrate hydroxyls, the aldehydes formed are highly reactive toward primary amines and hydrazides [9]. This makes QD-NH2 or QD-COOH (derivatized with adipic acid dihydrazide (ADH)) suitable candidates for conjugation [9]. In addition, selective bioconjugation can occur without a proceeding reduction reaction, thus retaining the integrity of the antibody (Figure 3).

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.