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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

Non-resolved electropherogram of mixture QD-hydrazide (1) and whole antibody-conjugated QDs (QD-IgG) (2). Sharp peaks (3) observed at the migration time of QD-IgG are attributed to buffer additive (BSA) cross-reacting with the antigen-binding site of the IgG. IgG used for conjugation was rabbit anti-human albumin. CE buffer electrolyte used was 50 mM borate (pH 9.2), 0.1% BSA. Gravity injection performed by elevating inlet capillary 7 cm for 5 s. Applied voltage for CE separation was 25 kV. Capillary temperature maintained at 20°C. Excitation source and detection wavelength was 473 nm and 620 nm, respectively.
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Figure 10: Non-resolved electropherogram of mixture QD-hydrazide (1) and whole antibody-conjugated QDs (QD-IgG) (2). Sharp peaks (3) observed at the migration time of QD-IgG are attributed to buffer additive (BSA) cross-reacting with the antigen-binding site of the IgG. IgG used for conjugation was rabbit anti-human albumin. CE buffer electrolyte used was 50 mM borate (pH 9.2), 0.1% BSA. Gravity injection performed by elevating inlet capillary 7 cm for 5 s. Applied voltage for CE separation was 25 kV. Capillary temperature maintained at 20°C. Excitation source and detection wavelength was 473 nm and 620 nm, respectively.

Mentions: Figure 10 illustrates the CE separation of QD-hydrazide (1) and their conjugation to whole anti-human albumin IgG-CHO (QD-IgG) (2). The separation is not baseline resolved but can be distinguished by the vertical line separating the two peaks. The QD-IgG, not purified by size-exclusion or dialysis, retains a considerable amount of unconjugated IgG in the sample. This resulted in significant changes in EOF, peak shape, and resolution due to protein-capillary wall adsorption. In addition, the lack of baseline separation could be attributed to the whole IgG exerting a reduced negative charge influence when conjugated to QD-hydrazide. To reduce the effects of protein-capillary wall interaction, a 0.1% BSA additive was included in the CE separation buffer. However, due to the similarities between BSA and the IgG immunogen, human serum albumin (HSA), cross-reactivity may have occurred. The cross-reactivity, leading to a non-specific immunoconjugate (QD-IgG-BSA) may be observed in the electropherogram as sharp spikes, unresolved from the QD-IgG peak.


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-resolved electropherogram of mixture QD-hydrazide (1) and whole antibody-conjugated QDs (QD-IgG) (2). Sharp peaks (3) observed at the migration time of QD-IgG are attributed to buffer additive (BSA) cross-reacting with the antigen-binding site of the IgG. IgG used for conjugation was rabbit anti-human albumin. CE buffer electrolyte used was 50 mM borate (pH 9.2), 0.1% BSA. Gravity injection performed by elevating inlet capillary 7 cm for 5 s. Applied voltage for CE separation was 25 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 10: Non-resolved electropherogram of mixture QD-hydrazide (1) and whole antibody-conjugated QDs (QD-IgG) (2). Sharp peaks (3) observed at the migration time of QD-IgG are attributed to buffer additive (BSA) cross-reacting with the antigen-binding site of the IgG. IgG used for conjugation was rabbit anti-human albumin. CE buffer electrolyte used was 50 mM borate (pH 9.2), 0.1% BSA. Gravity injection performed by elevating inlet capillary 7 cm for 5 s. Applied voltage for CE separation was 25 kV. Capillary temperature maintained at 20°C. Excitation source and detection wavelength was 473 nm and 620 nm, respectively.
Mentions: Figure 10 illustrates the CE separation of QD-hydrazide (1) and their conjugation to whole anti-human albumin IgG-CHO (QD-IgG) (2). The separation is not baseline resolved but can be distinguished by the vertical line separating the two peaks. The QD-IgG, not purified by size-exclusion or dialysis, retains a considerable amount of unconjugated IgG in the sample. This resulted in significant changes in EOF, peak shape, and resolution due to protein-capillary wall adsorption. In addition, the lack of baseline separation could be attributed to the whole IgG exerting a reduced negative charge influence when conjugated to QD-hydrazide. To reduce the effects of protein-capillary wall interaction, a 0.1% BSA additive was included in the CE separation buffer. However, due to the similarities between BSA and the IgG immunogen, human serum albumin (HSA), cross-reactivity may have occurred. The cross-reactivity, leading to a non-specific immunoconjugate (QD-IgG-BSA) may be observed in the electropherogram as sharp spikes, unresolved from the QD-IgG peak.

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