Herein we describe the development of a mix-read bioassay based on a three-dimensional (3D) poly ethylene glycol-(PEG)-hydrogel microparticles for the detection of oligonucleotides in complex media. The key steps of hydrogels synthesis and molecular recognition in a 3D polymer network are elucidated. The design of the DNA probes and their density in polymer network were opportunely optimized. Furthermore, the diffusion into the polymer was tuned adjusting the polymer concentration and consequently the characteristic mesh size. Upon parameters optimization, 3D-PEG-hydrogels were synthetized in a microfluidic system and provided with fluorescent probe. Target detection occurred by double strand displacement assay associated to fluorescence depletion within the hydrogel microparticle. Proposed 3D-PEG-hydrogel microparticles were designed for miR-143-3p detection. Results showed 3D-hydrogel microparticles with working range comprise between 10-6-10-12 M, had limit of detection of 30 pM and good specificity. Moreover, due to the anti-fouling properties of PEG-hydrogel, the target detection occurred in human serum with performance comparable to that in buffer. Due to the approach versatility, such design could be easily adapted to other short oligonucleotides detection.
Keywords: 3D recognition; hydrogel microparticles; miRNA detection; mix-read bioassay.