The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

Abstract

The use of fluorescence calibration beads has been the hallmark of quantitative flow cytometry. It has enabled the direct comparison of inter-laboratory data as well as quality control in clinical flow cytometry. In this paper we have described a simple method for producing color-generalizable calibration beads based on streptavidin functionalized quantum dots. Based on their broad absorption spectra and relatively narrow emission, that is tunable on the basis of dot-size, quantum dot calibration beads can be made for any fluorophore that matches their emission color. In an earlier publication (1) we characterized the spectroscopic properties of commercial streptavidin functionalized dots (Invitrogen). Here we describe the molecular assembly of these dots on biotinylated beads. The law of mass action is used to readily define the site densities of the dots on the beads. The applicability of these beads is tested against the industry standard, commercial fluorescein calibration beads. The utility of the calibration beads are also herein extended to the characterization surface densities of dot-labeled epidermal growth factor ligands as well as quantitative indicators of the binding of dotlabeled virus particles to cells.

Spectroscopic characterization of streptavidin functionalized quantum dots

“…We attribute this behavior to size heterogeneity and surface defects in the QD525, consistent with tendencies previously described in the literature (16,24,25). The results are discussed in the context of bridging the gap between what is currently known in the physical chemistry literature of quantum dots, and the quantitative needs of assay development in biological applications (26).…”

“…In this study we have examined some spectroscopic properties of commercial streptavidin functionalized dots with the view of advancing a rational basis for developing calibration beads (42,43) based on quantum dots (26). In this work we have relied on the theoretical and experimental framework established by the earlier work of Brus et al (10,11,44,45), Bawendi et al (18,19,22,23,(46)(47)(48)(49) and others (15,24,25,50,51).…”

“…We will now attempt to present a measurement model that provides a framework intended to translate the results of the present spectroscopic characterization to the development of calibration beads as described elsewhere (26). In flow cytometry, the quantitation of fluorophore tagged ligands and receptors, is achieved by the use of standard beads of known calibration (42,43).…”

Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides

“…The surface density of βγ subunits on the beads was determined as previously described [24]. The functional activity of M2 antibodies drops over time, therefore over the course of more than 6 months, we noticed a reduction in the surface density of βγ subunits of 25% [25]. The drop in activity of the M2 did not affect the integrity of the ternary complex assembly because it serves as a simple tether of the βγ subunits to the beads.…”

Dual-color immunofluorescent labeling with quantum dots of the diabetes-associated proteins aldose reductase and Toll-like receptor 4 in the kidneys of diabetic rats

“…17,18 QDs are semiconductor nanocrystals with diameters in the order of 2-10 nm, which emit fluorescence depending on their size. Compared with conventional organic dyes and fluorescent proteins, QDs have unique optical properties such as broad absorption spectra, narrow and symmetrical emission spectra, superior signal brightness, and resistance to photobleaching.…”