Hydrogel Microparticles for Fluorescence Detection of miRNA in Mix-Read Bioassay.

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.

One-step scalable fluorescent microgel bioassay for the ultrasensitive detection of endogenous viral miR-US4-5p.

Human cytomegalovirus (hCMV) infection is the leading cause of birth defects in newborns and death in immunosuppressed people. Traditional techniques require time-consuming and costly analyses, and sometimes result in false positive results; thus, a rapid and accurate detection for hCMV infection is necessary. Recently, hcmv-miR-US4-5p was selected as the biomarker for cytomegalovirus diagnosis and follow-up. Herein, we propose a bioassay based on microgels endowed with optical fluorescent oligonucleotide probes for the detection of circulating endogenous hcmv-microRNAs. In particular, a double strand probe, based on the fluorescence recovery after target capture, was conjugated on microgels and the probe density was opportunely optimised. Then, the microgels were directly mixed with the sample. The fluorescence read-out was measured as a function of target concentration at a fixed number of microgels per tube. As a bead-based assay, the performances of optical detection in terms of dynamic working range and limit of detection could be finely tuned by tuning the number of microgels per tube. The limit of detection of the assay could be tuned in the range from 39.1 fM to 156 aM by changing the microgel concentration from 50 µg mL-1 to 0.5 µg mL-1, respectively. The assay results specific for the selected target were stable over a one-year time span and they were not affected by the presence of human serum. Therefore, this bioassay based on microgels might represent a flexible platform that should be able to predict, identify and follow-up several diseases by monitoring freely circulating oligonucleotides in body fluids.

Small Oligonucleotides Detection in Three-Dimensional Polymer Network of DNA-PEG Hydrogels.

The control of the three-dimensional (3D) polymer network structure is important for permselective materials when specific biomolecule detection is needed. Here we investigate conditions to obtain a tailored hydrogel network that combines both molecular filtering and molecular capture capabilities for biosensing applications. Along this line, short oligonucleotide detection in a displacement assay is set within PEGDA hydrogels synthetized by UV radical photopolymerization. To provide insights on the molecular filter capability, diffusion studies of several probes (sulforhodamine G and dextrans) with different hydrodynamic radii were carried out using NMR technique. Moreover, fluorometric analyses of hybridization of DNA oligonucleotides inside PEGDA hydrogels shed light on the mechanisms of recognition in 3D, highlighting that mesh size and crowding effect greatly impact the hybridization mechanism on a polymer network. Finally, we found the best probe density and diffusion transport conditions to allow the specific oligonucleotide capture and detection inside PEGDA hydrogels for oligonucleotide detection and the filtering out of higher molecular weight molecules.