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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.


Possible unfavorable polymer formation during following bioconjugation steps. a) QD-hydrazide synthesis from QD-COOH, and b) QD-IgG bioconjugation from QD-hydrazide and IgG-CHO.
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Figure 8: Possible unfavorable polymer formation during following bioconjugation steps. a) QD-hydrazide synthesis from QD-COOH, and b) QD-IgG bioconjugation from QD-hydrazide and IgG-CHO.

Mentions: Conjugation of IgG-CHO with QD-NH2 is possible using reductive amination. However, the drawback is the degree of uncontrollability of the resulting conjugate, as undesirable IgG-IgG crosslinking can occur through the presence of primary amines on the IgGs surface. Thus, conjugating IgG-CHO with QDs functionalized with hydrazides was reasoned to be more selective as conjugation is occurring exclusively on the polysaccharide chain. However, since commercially obtainable QDs are typically functionalized with carboxylic acids or amines, a derivatization was required. Derivatization was performed on QD-COOH and involved the use of EDCHCl in the presence of the bis-hydrazide compound, ADH, yielding relatively stable hydrazide-functionalized QDs (QD-hydrazide). The drawback is that ADH, being is homobifunctional crosslinker, can introduce undesirable side reactions. As both functional groups on the crosslinker are identical, they each have the potential of reacting with the same QD, resulting in a closed ring structure that can essentially inactivate that particular region of the QD. However, it is suspected that the spacer arm of the crosslinker lacks the length required to form such a ring structure. Another more likely scenario involves the cross-reaction between a derivatized QD (QD-hydrazide) with an underivatized QD (QD-COOH). This uncontrolled reaction can lead to the undesirable formation of a QD-QD polymer (Figure 8a), but is believed to be minimized when using ADH in excessive quantities during the derivatization.


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)

Possible unfavorable polymer formation during following bioconjugation steps. a) QD-hydrazide synthesis from QD-COOH, and b) QD-IgG bioconjugation from QD-hydrazide and IgG-CHO.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Possible unfavorable polymer formation during following bioconjugation steps. a) QD-hydrazide synthesis from QD-COOH, and b) QD-IgG bioconjugation from QD-hydrazide and IgG-CHO.
Mentions: Conjugation of IgG-CHO with QD-NH2 is possible using reductive amination. However, the drawback is the degree of uncontrollability of the resulting conjugate, as undesirable IgG-IgG crosslinking can occur through the presence of primary amines on the IgGs surface. Thus, conjugating IgG-CHO with QDs functionalized with hydrazides was reasoned to be more selective as conjugation is occurring exclusively on the polysaccharide chain. However, since commercially obtainable QDs are typically functionalized with carboxylic acids or amines, a derivatization was required. Derivatization was performed on QD-COOH and involved the use of EDCHCl in the presence of the bis-hydrazide compound, ADH, yielding relatively stable hydrazide-functionalized QDs (QD-hydrazide). The drawback is that ADH, being is homobifunctional crosslinker, can introduce undesirable side reactions. As both functional groups on the crosslinker are identical, they each have the potential of reacting with the same QD, resulting in a closed ring structure that can essentially inactivate that particular region of the QD. However, it is suspected that the spacer arm of the crosslinker lacks the length required to form such a ring structure. Another more likely scenario involves the cross-reaction between a derivatized QD (QD-hydrazide) with an underivatized QD (QD-COOH). This uncontrolled reaction can lead to the undesirable formation of a QD-QD polymer (Figure 8a), but is believed to be minimized when using ADH in excessive quantities during the derivatization.

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.