Towards High‐Throughput Production of Uniformly Encoded Microparticles

Spherical particles conjugated to molecules with affinity for, or able to interact with, an analyte are one of the most convenient tools for parallel multiplex analysis in research and diagnosis. Identification of the different analytes is based on the signal associated to the marker particles, whose size or color is specific. For high-throughput applications, dye-labeled fluorescent microspheres have emerged as a very important alternative to traditional microarrays. They present the possibility of multiplex color detection and are expected to be more flexible in target selection, faster in binding, and less expensive in production, using very small sample volumes with a three-dimensional (3D) configuration. Another advantage of fluorescent microparticle arrays is the possibility of using flow cytometry as a powerful, well-established, fast, sensitive, and accurate detection technique of biomolecular interaction, particularly relevant in very recent cancer-diagnosis findings. The functionality of microbead-based arrays relies heavily on the properties of the microspheres, e.g., their size range, stability, uniformity, and ability to retain the fluorescent dye. Despite all of the efforts aimed at the preparation of labeled, functionalized polymeric beads, a considerable challenge still remains, namely, how to develop and optimize simple methods for the preparation of huge quantities of fluorescently encoded microparticles for demanding applications (e.g., > 10 particles per day and device) with a uniform shape and surface, homogeneous size distribution, and controlled fluorescence properties. Numerous strategies and processes have been developed to produce polymeric microparticles; the determinant step being the drop formation, which fixes the size distribution of the resulting microparticles. Depending on the physical properties of the fluids, different techniques or mechanisms are used to produce monodisperse drops. The first straightforward strategy is the formation of a single drop at a time, as in dripping processes, emulsification membranes, or microfluidic emulsification. Notwithstanding the very uniform microparticles obtained with these techniques, the drop-production rate is very low and the drop diameter scales with the diameter of the capillary or the pore, which makes it difficult to produce microparticles of a few micrometers or less. The second strategy is the formation of numerous drops at a time, as in mixing or stirring processes, with scarce size predictability and homogeneity, or jet-disintegration techniques, in which a wide range of drop sizes is obtained, with different distributions depending on the Reynolds and Weber numbers of the jet. In particular, in laminar-jet disintegration or Rayleigh breakup, the jet breaks up into uniform droplets due to capillary instability. Nevertheless, under real conditions, formation of satellite drops between the main drops, drop coalescence, and the presence of natural disturbances on the jet surface induce wide drop size distributions. On the other hand, monodisperse drops, and consequently monodisperse microparticles, were produced recently using oscillatory stimulation to break the jet up. An acoustic transducer induced a horizontal oscillation at the optimal wavelength, stimulating the fastest growth mode in order to break the jet up into a chain of perfectly monodisperse drops. Despite the perfect control of the size distribution of the droplets, the drop size is determined by the diameter of the nozzle, as the jet diameter scales with the orifice nozzle. Therefore, the production of small microparticles (less than 25 lm in diameter) becomes impossible as the risk of orifice clogging increases with narrow diameter nozzles. Here, we report a versatile and controlled procedure for the production of microparticles, with diameters smaller than 25 lm (typically 5 lm), yielding remarkable size accuracy, with a small size dispersion as well as allowing the selection of surface topology and internal composition. To produce such microparticles, we combined flow-focusing technology in a unique parametrical range, where high productivity and size control are preserved (high Reynolds and moderate/high Weber numbers), with a solvent evaporation/extraction proceC O M M U N IC A IO N S