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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

Electropherogram of mixture containing QD-COOH (1) and myoglobin-conjugated QDs (QD-Myo) (2). CE buffer electrolyte used was 50 mM borate, pH 9.2. Gravity injection performed by elevating inlet capillary 7 cm for 5 s. Applied voltage for CE separation was 20 kV. Capillary temperature maintained at 20°C. Excitation source and detection wavelength was 473 nm and 620 nm, respectively.
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Figure 5: Electropherogram of mixture containing QD-COOH (1) and myoglobin-conjugated QDs (QD-Myo) (2). CE buffer electrolyte used was 50 mM borate, pH 9.2. Gravity injection performed by elevating inlet capillary 7 cm for 5 s. Applied voltage for CE separation was 20 kV. Capillary temperature maintained at 20°C. Excitation source and detection wavelength was 473 nm and 620 nm, respectively.

Mentions: Figure 5 illustrates the CE separation of QD-COOH (1) with their conjugation to myoglobin (QD-Myo) (2). The migration time of QD-Myo is also longer with respect to QD-COOH. However the differences are not substantial enough for baseline separation to occur. In comparison to QD-BSA, there may be a weakened net negative charge that is present on QD-Myo, since myoglobin has a pI value measured at ~7.2. In addition, there is a considerable size difference between BSA (MW~66 kDa) and Myo (MW~16.7 kDa) that may likely influence the respective conjugate's migration time. As both MW and pI can influence a protein's charge-to-size ratio, their conjugation to polydisperse QDs (each with possibly different binding ratios) will contribute to their respective migration times.


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)

Electropherogram of mixture containing QD-COOH (1) and myoglobin-conjugated QDs (QD-Myo) (2). CE buffer electrolyte used was 50 mM borate, pH 9.2. Gravity injection performed by elevating inlet capillary 7 cm for 5 s. Applied voltage for CE separation was 20 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 5: Electropherogram of mixture containing QD-COOH (1) and myoglobin-conjugated QDs (QD-Myo) (2). CE buffer electrolyte used was 50 mM borate, pH 9.2. Gravity injection performed by elevating inlet capillary 7 cm for 5 s. Applied voltage for CE separation was 20 kV. Capillary temperature maintained at 20°C. Excitation source and detection wavelength was 473 nm and 620 nm, respectively.
Mentions: Figure 5 illustrates the CE separation of QD-COOH (1) with their conjugation to myoglobin (QD-Myo) (2). The migration time of QD-Myo is also longer with respect to QD-COOH. However the differences are not substantial enough for baseline separation to occur. In comparison to QD-BSA, there may be a weakened net negative charge that is present on QD-Myo, since myoglobin has a pI value measured at ~7.2. In addition, there is a considerable size difference between BSA (MW~66 kDa) and Myo (MW~16.7 kDa) that may likely influence the respective conjugate's migration time. As both MW and pI can influence a protein's charge-to-size ratio, their conjugation to polydisperse QDs (each with possibly different binding ratios) will contribute to their respective migration times.

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