Engineering of Optically Encoded Microbeads with FRET‐Free Spatially Separated Quantum‐Dot Layers for Multiplexed Assays

Quantum dot (QD) encoded microbeads are emerging for multiplexed analysis of biological markers. The quantitative encoding of microbeads prepared with different concentrations of QDs of different colors suffers from resonance energy transfer from the QDs fluorescing at shorter wavelengths to the QDs fluorescing at longer wavelengths. Here, we used the layer‐by‐layer deposition technique to spatially separate QDs of different colors with several polymer layers so that the distance between them would be larger than the Förster energy transfer radius. We performed fluorescence lifetime measurements to investigate and determine the conditions excluding significant resonance energy transfer between QDs within QD‐encoded microbeads. Additionally, the number of QDs adsorbed onto microbeads was systematically established and multilayer structures of the QD‐encoded microbead shells were characterized by scanning probe nanotomography. Finally, we prepared eight populations of FRET‐free microbeads encoded with QDs of three colors at two intensity levels and demonstrated that all the optical codes are excitable at a single wavelength and may be clearly identified in three channels of a flow cytometer. The developed approach for engineering QD‐encoded microbeads that are free from optical artefacts related to inter‐QD resonance energy transfer paves the way to quantitative QD‐based multiplexed assays. An approach for engineering quantum dot (QD) encoded microbeads that are free from optical artifacts related to inter‐QD resonance energy transfer is presented. The method paves the way to the development of quantitative QD‐based multiplexed assays.