Polyacrylamide gel electrophoresis of semiconductor quantum dots and their bioconjugates: materials characterization and physical insights from spectrofluorimetric detection

Abstract

Colloidal semiconductor quantum dot (QD) nanocrystals have ideal fluorescence properties for bioanalysis and bioimaging, but these materials must be functionalized with an inorganic shell, organic ligand or polymer coating, and conjugated with biomolecules to be useful in such applications. Several different analytical techniques are used to characterize QDs and their multiple layers of functionalization. Here, we revisit poly(acrylamide) gel electrophoresis (PAGE), which has been scarcely used for the characterization of QDs and their bioconjugates in deference to the routine use of agarose gel electrophoresis. We implemented PAGE in a novel "stubby" capillary format with spectrofluorimetric detection, the combination of which enabled more rapid and more detailed characterization of QDs than was possible with both poly(acrylamide) and agarose slab gels. Correlations between the peak photoluminescence (PL) emission wavelength and electropherogram peaks, especially when combined with Ferguson analysis, provided new and significant insight into the key factors that determine the electrophoretic mobility of QDs, and helped to resolve heterogeneity and sub-populations in ensembles of QDs. The method was useful for characterization of the inorganic core/shell nanocrystals, their organic ligand and polymer coatings, and their final bioconjugates, the latter of which were in the form of peptide and protein conjugates. With further development and optimization, we anticipate that capillary PAGE with spectrofluorimetric detection will become a valuable addition to the toolbox of characterization techniques suitable for QDs, their bioconjugates, and other nanoparticle materials as well.

Aqueous Synthesis of DNA-Functionalized Near-Infrared AgInS2/ZnS Core/Shell Quantum Dots

“…As an additional technique, agarose gel electrophoresis was used to characterize the DNA-conjugated QDs. In fact, this technique, particularly relevant for the separation and purification of biomolecules, is also applied for QDs as a routine technique to reveal the presence of DNA or other biomolecules before and after functionalization. ,,− For comparison, cQDs, csQDs, and csQD-DNA conjugates were measured, taking the former two samples as the control. Under an electric field of 120 V, all types of QDs migrated toward the anode because DNA and GSH surface ligands are negatively charged (Figure c).…”

(Bio)conjugation Strategies Applied to Fluorescent Semiconductor Quantum Dots

“…By comparing the electrophoretic mobility of the components alone (biomolecules or QDs) in relation to the conjugates, it is possible to evaluate the interaction between QDs and biomolecules. 116 This technique presents, as principal disadvantages, the analyses of few samples at a time and, in general, it is laborious as well as non-quantitative. 115 The polyacrylamide gel electrophoresis (PAGE), schematized in Figure 8a, is mostly indicated for conjugation confirmation and/or evaluation studies involving protein-conjugated QDs.…”

Spectrotemporal characterization of photoluminescent silicon nanocrystals and their energy transfer to dyes

“…Spectroelectrophoresis measurements were made as described previously. 42,43 Additional details for all PL measurements are provided in the ESI. †…”