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

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

Core-shell microgels with controlled structural properties

Core-shell microgels with controlled structural properties: Here we report on the multistep synthesis of fluorescent core-shell microgels with inner and surface controlled composition. The tunability and versatility of this approach was confirmed in each step of the synthesis by confocal imaging, XPS and AFM. In particular, the in situ AFM measurements allowed us to study the swelling behaviour to understand the structural organization of the layers at single particle level. Microgels have gained great attention in the biomedical field for their wide application in diagnostic and drug delivery systems. The bulk properties as well as the surface features of these particular microparticles define their final performance. In particular, multifunctional microgels with complex architectures have been widely used in multiplex assays for their favourable capability to accommodate encoding systems and anchoring groups for probes to capture circulating targets by simply changing synthesis parameters. In this work a limited set of fluorescent encoded poly(ethyleneglycol) based microgels, of size ranging between 0.5 and 1.3μm, with a core-shell architecture were obtained by combining precipitation and seeded polymerizations. Here we demonstrate the possibility of tailoring and controlling the bulk and surface properties according to the synthesis by fluorescence imaging and pH titrations. Concerning the structural characterization, we adopted a method to calculate polymer fraction volumes from AFM images and combined these with equilibrium swelling theory (Peppas-Merrill equation) to determine the mesh size of the microgels. Surface composition was probed by X-ray photoelectron spectroscopy directly on freeze-dried microgels. In such a way we were able to describe the organizations of the different adlayers also in response to pH, highlighting the possibility of some overlap of the adlayers representing physical barriers at the boundaries of each shell

Exploiting olive mill wastewater via thermal conversion of the organic matter into gaseous biofuel. A case study

Olive oil is one excellence of the Italian food industry: around 300 kt yr−1 are produced, creating roughly the same amount of olive mill wastewater (OMW) to be disposed of. The present work describes a process to exploit OMW by converting its organic compounds to valuable gaseous biofuel. A sample OMW was characterized (COD, TOC, solids, and polyphenols) and submitted to membrane filtration tests to concentrate the organic compounds. Based on the results of the ex-periments, a treatment process was outlined: the retentate streams from microfiltration and ul-trafiltration steps were fed to a cracking and a steam reforming reactor, respectively; the obtained syngas streams were then mixed and sent to a methanation reactor. The process was simulated with Aspen Plus (AspenTech©) software, assessing operating conditions and streams compositions: the final biofuel is around 81 mol.% methane, 4 mol.% hydrogen, and 11 mol.% carbon dioxide. The permeate stream cannot be directly disposed of, but both its amount and its polluting charge are greatly reduced. The heat needed by the process, mainly due to the endothermic reactions, can be obtained by burning an amount of olive pomaces, roughly corresponding to one-third of the amount left by olive treatments giving rise to the processed OMW feed

multifunctional microgels for direct multiplexed and high sensitive detection

Abstract Detection of tiny quantities of nucleic acids, protein biomarkers and small organic molecules directly in complex matrices is of key relevance both in biomedical and in environmental fields. Moreover direct quantification of clinically relevant molecules (such as miRNA, DNA or proteins) in plasma is difficult due to its heterogeneous composition and the low amounts of target molecules. As for environmental pollutants (i.e. Aflatoxins), the direct detection is also hampered by the complexity of liquid samples (milk, wine, waste waters) lowering the sensitivities and requiring several steps of targets isolation and purification. Here we present our material platform based on microgels and hydrogel microparticles featuring high flexibility in performing detection and harvesting of different kind of targets in complex solutions