Design of Optically Encoded Microspheres of Different Sizes for Multiplexed Flow Cytometry

Abstract

The development of innovative fluorescence detection methods based on optically encoded microspheres of different colors and sizes is an important area of multiplexed detection and diagnosis of various diseases. For example, the xMAP technology of the American company Luminex employs polystyrene microspheres labeled with two or three organic fluorophores at different ratios, each with its unique spectral characteristics. Although xMAP can detect as many as 80 proteins or DNA sequences in a single test system, the need to use special equipment, produced only by the same company, for multiplexed analysis, as well as inherent disadvantages of organic fluorophores (a large Stokes shift and photobleaching under laser excitation) limit the applications of this technology. The objectives of this study were to design and fabricate microspheres encoded with semiconductor quantum dots. Carboxylated melamine-formaldehyde microspheres of three sizes were optically encoded with quantum dots of two colors immobilized between layers of oppositely charged polyelectrolytes on the surface of the microspheres. As a result, six populations of microspheres with different sizes and/or unique optical codes were obtained, characterized by a long-term stability and homogeneity in aqueous solutions. Analysis of the microspheres by the dynamic light scattering, epifluorescence microscopy, and flow cytometry methods showed their suitability for multiplexed analysis. At the same time, the use of quantum dots for optical encoding makes it possible to exclude photodegradation of the signal and to excite all quantum dot populations at the same wavelength of radiation, with effective separation of signals from the microspheres into different channels of a standard flow cytometer.