Engineering quantum dot calibration standards for quantitative fluorescent profiling

Abstract

Fluorescence-based tools, in particular optimized fluorophores, offer useful approaches to map cellular heterogeneity. Applying quantum dot (Qdot) technology towards heterogeneity profiling would be a novel approach for characterizing cellular dispersion and requires sensitive calibration standards. To this end, we have employed biotin–streptavidin chemistry to design improved quantitative, Qdot calibration beads. These calibration beads include commercially available Innovator's Tool Kit (ITK)-streptavidin Qdots (Qdots) conjugated to biotin coated polystyrene beads, providing a laboratory-accessible approach for quantitative calibration. We have engineered Qdot calibration beads at 525 nm, 565 nm, 605 nm, 655 nm, and 705 nm emission spectra. These beads provide calibration standards within the 0.0072–0.72 nM Qdot range, corresponding to an initial estimation of 800–80 000 Qdots per bead. We measured the proportion of Qdot loss during bead isolation steps and determined an accurate relationship between theoretical Qdot levels and actual Qdot levels using inductively coupled plasma mass spectrometry (ICP-MS). A linear relationship between fluorescence and Qdot number was observed with estimated concentrations of 1.13 × 10⁻¹⁴ μg Cd/Qdot 525, 2.34 × 10⁻¹⁴ μg Cd/Qdot 565, 1.49 × 10⁻¹³ μg Cd/Qdot 605, 1.99 × 10⁻¹³ μg Cd/Qdot 655 and 1.65 × 10⁻¹³ μg Cd/Qdot 705. We also report the difference in concentration estimates between the fluorescence and ICP-MS methods. This study establishes the optimal conditions for preparing Qdot calibration beads from commercially available Qdots for quantitative, biophotonic applications including measuring cell surface heterogeneity.