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

Paolo Antonio Netti,Alessia Mazzarotta,Edmondo Battista,Filippo Causa,Tania Mariastella Caputo

doi:10.3390/s21227671

Abstract

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.

Highlights

Many analytical methods have been developed so far with the aim to identify oligonucleotide biomarkers in human fluids [1]
Abstract: 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 signal is generated following the hybridization of the target molecule with the probe, avoiding all the additional procedures currently required of manipulation, separation, washing, mixing and reading [3,4,5]

Summary

Introduction

Many analytical methods have been developed so far with the aim to identify oligonucleotide biomarkers in human fluids [1]. The ideal biosensor would combine the advantages of assays performed in solution with those obtained on solid surfaces To achieve this goal, it is necessary to optimize the probe design and density, but above all, to fine tune the environment in which probes are immobilized. Since the environment of the 3D network influences the biosensor sensitivity and specificity, it must be as much as possible close to the ideal solution For this purpose, the nature of the materials has a pivotal role, affecting both the diffusion of the molecules and the probe/target hybridization [20]. In particular, reduce the costs, synthesis time and reagent consumption compared to the conventional techniques, achieving a large production of monodisperse microparticles per hour (about 105 particles) [24] Based on these considerations, in this study we have designed 3D one step functionalized hydrogel microparticles by microfluidic for microRNA (miRNA or miR) detection. The 3D-PEG-hydrogels microparticles can be comprised into mix and read bioassays as they proved the ability to detect miRNA directly in human serum, just mixing microparticles with the sample and reading the fluorescence turn-off

Fabrication of Engineered Microparticles

Microparticles Characterization

Probe Design and Hydrogel-Beads Based Assay Set-Up

Assay Specificity

Diffusion Studies

Human-miR-143-3p Detection in PBS and Human Serum

Findings

Conclusions