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Protein a detection based on quantum dots-antibody bioprobe using fluorescence coupled capillary electrophoresis.

Qiu L, Bi Y, Wang C, Li J, Guo P, Li J, He W, Wang J, Jiang P - Int J Mol Sci (2014)

Bottom Line: In this report, fluorescence detection coupled capillary electrophoresis (CE-FL) was used to detect Protein A.Antibody was first labeled with Cy5 and then mixed with quantum dots (QDs) to form QDs-antibody bioprobe.This study provides a new trail of thought for the detection of protein.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Coordination Chemistry, Coordination Chemistry Institute, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China. linqiupjj@gmail.com.

ABSTRACT
In this report, fluorescence detection coupled capillary electrophoresis (CE-FL) was used to detect Protein A. Antibody was first labeled with Cy5 and then mixed with quantum dots (QDs) to form QDs-antibody bioprobe. Further, we observed fluorescence resonance energy transfer (FRET) from QDs donor to Cy5 acceptor. The bioprobe was formed and brought QDs and Cy5 close enough to allow FRET to occur. After adding protein A, the FRET system was broken and caused the FRET signal to decrease. Thus, a new method for the determination of protein A was proposed based on the FRET signal changes. This study provides a new trail of thought for the detection of protein.

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Electropherograms of quantum dots (QDs)-IgG-Cy5 conjugation with detection in 612 nm channel (Black) and 670 nm channel (Red). (a) QDs alone, 2 μM and (b) QDs-IgG-Cy5, 2 μM. CE conditions: 25 mM borate buffer (pH 9.3) at 18 kV. λex = 420 nm.
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f1-ijms-15-01804: Electropherograms of quantum dots (QDs)-IgG-Cy5 conjugation with detection in 612 nm channel (Black) and 670 nm channel (Red). (a) QDs alone, 2 μM and (b) QDs-IgG-Cy5, 2 μM. CE conditions: 25 mM borate buffer (pH 9.3) at 18 kV. λex = 420 nm.

Mentions: In our CE experiments, two signal channels of fiber optic spectrometer with fixed detecting wavelength at 612 ± 10 and 670 ± 10 nm were used to simultaneously collect the fluorescence signal of donor and acceptor. QDs and IgG-Cy5 mixtures were first analysed by CE-FL. QDs-IgG-Cy5 bioprobe was prepared by electrostatic adsorption method. When mixed with QDs in solution, IgG-Cy5 can assemble with QDs. Figure 1 shows the electropherograms of mixing IgG-Cy5 with QDs. It was obvious that CE could efficiently separate the bound and unbound species. The migration time of QDs alone was 500 s (Figure 1, curve a); while for the conjugates (Figure 1, curve b), indicated by a stable species of QDs-IgG-Cy5 in CE-FL with migration time of 230 s, significantly different from QDs. By the location of the emission peak, this peak was known to be caused by the QDs-IgG-Cy5. After the conjugation of IgG-Cy5 and QDs, the surface charge changed and the fluorescence peak moved forward. This implies an ordered assembly. Experimental results also approved that there was cross-talk between the donor and acceptor channel. This indicated that FRET happened between QDs and Cy5.


Protein a detection based on quantum dots-antibody bioprobe using fluorescence coupled capillary electrophoresis.

Qiu L, Bi Y, Wang C, Li J, Guo P, Li J, He W, Wang J, Jiang P - Int J Mol Sci (2014)

Electropherograms of quantum dots (QDs)-IgG-Cy5 conjugation with detection in 612 nm channel (Black) and 670 nm channel (Red). (a) QDs alone, 2 μM and (b) QDs-IgG-Cy5, 2 μM. CE conditions: 25 mM borate buffer (pH 9.3) at 18 kV. λex = 420 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3958821&req=5

f1-ijms-15-01804: Electropherograms of quantum dots (QDs)-IgG-Cy5 conjugation with detection in 612 nm channel (Black) and 670 nm channel (Red). (a) QDs alone, 2 μM and (b) QDs-IgG-Cy5, 2 μM. CE conditions: 25 mM borate buffer (pH 9.3) at 18 kV. λex = 420 nm.
Mentions: In our CE experiments, two signal channels of fiber optic spectrometer with fixed detecting wavelength at 612 ± 10 and 670 ± 10 nm were used to simultaneously collect the fluorescence signal of donor and acceptor. QDs and IgG-Cy5 mixtures were first analysed by CE-FL. QDs-IgG-Cy5 bioprobe was prepared by electrostatic adsorption method. When mixed with QDs in solution, IgG-Cy5 can assemble with QDs. Figure 1 shows the electropherograms of mixing IgG-Cy5 with QDs. It was obvious that CE could efficiently separate the bound and unbound species. The migration time of QDs alone was 500 s (Figure 1, curve a); while for the conjugates (Figure 1, curve b), indicated by a stable species of QDs-IgG-Cy5 in CE-FL with migration time of 230 s, significantly different from QDs. By the location of the emission peak, this peak was known to be caused by the QDs-IgG-Cy5. After the conjugation of IgG-Cy5 and QDs, the surface charge changed and the fluorescence peak moved forward. This implies an ordered assembly. Experimental results also approved that there was cross-talk between the donor and acceptor channel. This indicated that FRET happened between QDs and Cy5.

Bottom Line: In this report, fluorescence detection coupled capillary electrophoresis (CE-FL) was used to detect Protein A.Antibody was first labeled with Cy5 and then mixed with quantum dots (QDs) to form QDs-antibody bioprobe.This study provides a new trail of thought for the detection of protein.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Coordination Chemistry, Coordination Chemistry Institute, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China. linqiupjj@gmail.com.

ABSTRACT
In this report, fluorescence detection coupled capillary electrophoresis (CE-FL) was used to detect Protein A. Antibody was first labeled with Cy5 and then mixed with quantum dots (QDs) to form QDs-antibody bioprobe. Further, we observed fluorescence resonance energy transfer (FRET) from QDs donor to Cy5 acceptor. The bioprobe was formed and brought QDs and Cy5 close enough to allow FRET to occur. After adding protein A, the FRET system was broken and caused the FRET signal to decrease. Thus, a new method for the determination of protein A was proposed based on the FRET signal changes. This study provides a new trail of thought for the detection of protein.

Show MeSH