Out of the available methods of encoding, fluorescence-based optical encoding is most favored owing to their ease of identification, high sensitivity, and signal localization. To perform the multiplex bio-detection, the beads are normally encoded with unique signatures for easy identification of analyte bound to the specific bead that targets it. Compared to planar arrays, bead-based bioassays are endowed with advantages such as large surface area-to-volume ratio, efficient binding kinetics, higher flexibility to perform a wide range of tests, easy preparation and handling of samples. It can be performed in two different formats: traditional planar arrays or beads-based bioassays. Multiplexed bio-detection allows simultaneous detection of multiple biomolecules in a single run and is important for applications in different fields such as high-throughput drug screening, genotyping, proteomics, disease diagnosis, and environmental engineering. The system was also demonstrated for multiplexed detection of both human C-reactive protein (hCRP) and HSA protein by immobilizing anti-hCRP antibodies on green UCNPs. Probe antibodies were covalently immobilized on red UCNPs-encoded microbeads for specific capture of human serum albumin (HSA) as a model protein. UCNPs emitting blue color were used to label the reporter antibody.
Multiple color codes were generated by mixing UCNPs emitting red and green colors at different ratios prior to encapsulation. In this study, droplet microfluidics-assisted UCNPs-encoded microbeads of uniform shape, size, and fluorescence were prepared. Here, PEGDA microbeads surface was coated with silica followed by carboxyl modification without compromising the fluorescence intensity of UCNPs. Methods to functionalize the surface of PEGDA microbeads (acrylic acid incorporation, polydopamine coating) reported thus far quench the fluorescence of UCNPs. Additionally, PEGDA microbeads lack functionality for probe antibodies conjugation on their surface. Hence, we utilized droplet microfluidics to obtain encoded microbeads of uniform size, shape, and UCNPs distribution inside. However, to prepare UCNPs-encoded microbeads, currently used swelling-based encapsulation leads to non-uniformity, which is undesirable for fluorescence-based multiplexing. Here, we developed a new multiplexed detection system using UCNPs for encoding poly(ethylene glycol) diacrylate (PEGDA) microbeads as well as for labeling reporter antibody. Compared to organic dye-based commercially available Luminex’s xMAP technology, upconversion nanoparticles (UCNPs) are better alternatives due to their large anti-Stokes shift, photostability, nil background, and single wavelength excitation. Fluorescently encoded microbeads are in demand for multiplexed applications in different fields.
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