Size exclusion chromatography as a universal method for the purification of quantum dots bioconjugates

Abstract

The bioconjugation of fluorescent quantum dots (QDs) and purification of QDs bioconjugates are of vital importance in bioapplications. In this paper, we systematically investigated the purification efficiency of QDs bioconjugates and their stability during separation process by using size exclusion chromatography (SEC) technique, fluorescence spectroscopy, and fluorescence correlation spectroscopy (FCS). In this study, commercial QDs and fluorescein isothiocyanate (FITC) were used as labeling probes, and bovine serum albumin (BSA) and antibody (Erbitux) were used as mode samples. The covalently linkage and the electrostatic interaction were used in bioconjugation procedures. We systematically studied the effects of certain factors such as the scales of column, loading volume, elution buffer, and separation media on the purification efficiency of QDs bioconjugates by a home-built SEC device. And highly pure and monodisperse QDs bioconjugates could be obtained by SEC purification twice under the optimized conditions. Furthermore, we investigated the stability of QDs bioconjugates in different conjugation ways and purification conditions by FCS, and found that the stability of bioconjugates were poor when electrostatic binding mode was used. We also observed that Sephacryl S300 HR (separation range for globular proteins: 1 × 10(4) -1.5 × 10(6) Da) was the best choice for purifying the vast majority of QDs-bioconjugates. Our work described here will be constructive to popularization and applications of QDs in life science.

How Quantum Dots Aggregation Enhances Förster Resonant Energy Transfer

“…A separation process, such as size exclusion by gel filtration, to remove molecule excess could overcome this phenomenon and made FRET efficiency independent of biotin/QD ratio when biotin molecules are in excess. Such a technique is difficult to set up due to the small size of QDs and the importance to avoid any damage to ligands . For this reason and with the idea to limit the excess of biotin molecules which inhibits FRET efficiency, the critical biotin/QD ratio B 1 was chosen for all the subsequent measurements.…”

Fluorescence resonance energy transfer between green fluorescent protein and doxorubicin enabled by DNA nanotechnology

“…FRET also called Förster resonance emission transfer is a photophysical process through which electronically excited donor molecules nonradiatively transfers its excitation energy to an acceptor molecule via a long range dipole–dipole interaction . This description is applicable when the two molecules are separated by a distance that is large enough so that the approximation of the molecules as point dipoles is valid . The FRET observed by us can be used for a fast confirmation of the presence of this drug in complex through spectral overlap.…”

Homogeneous immunoassay for the cancer marker alpha-fetoprotein using single wavelength excitation fluorescence cross-correlation spectroscopy and CdSe/ZnS quantum dots and fluorescent dyes as labels

“…And the high-purity and monodisperse QD conjugates were obtained by twice SEC under the optimized conditions. This is described in the reference [26]. A detailed introduction of the samples preparation and purification is provided in the Electronic Supporting Material.…”