Supramolecular spectrally encoded microgels with double strand probes for absolute and direct miRNA fluorescence detection at high sensitivity

We present novel microgels as a particle-based suspension array for direct and absolute microRNA (miRNA) detection. The microgels feature a flexible molecular architecture, antifouling properties, and enhanced sensitivity with a large dynamic range of detection. Specifically, they possess a core-shell molecular architecture with two different fluorescent dyes for multiplex spectral analyses and are endowed with a fluorescent probe for miRNA detection. Encoding and detection fluorescence signals are distinguishable by nonoverlapping emission spectra. Tunable fluorescence probe conjugation and emission confinement on single microgels allow for ultrasensitive miRNA detection. Indeed, the suspension array has high selectivity and sensitivity with absolute quantification, a detection limit of 10(-15) M, a dynamic range from 10(-9) to 10(-15) M, and higher accuracy than qRT-PCR. The antifouling properties of the microgels also permit the direct measurement of miRNAs in serum, without sample pretreatment or target amplification. A multiplexed assay has been tested for a set of miRNAs chosen as cancer biomarkers

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

Turn-on fluorescence detection of protein by molecularly imprinted hydrogels based on supramolecular assembly of peptide multi-functional blocks

Supramolecular in-cavity target–peptide complex for self-reporting imprinted polymers

Peptide Assisted Imprinting for Turn-On Fluorescence Detection of Proteins

Current limitations in protein imprinting technology above all rely on poor cavity control for sensing purpose [1,2]..

Bioengineering Microgels and Hydrogel Microparticles for Sensing Biomolecular Targets

Hydrogels, and in particular microgels, are playing an increasingly important role in a diverse range of applications due to their hydrophilic, biocompatible, and highly flexible chemical characteristics. On this basis, solution-like environment, non-fouling nature, easy probe accessibility and target diffusion, effective inclusion of reporting moieties can be achieved, making them ideal substrates for bio-sensing applications. In fact, hydrogels are already successfully used in immunoassays as well as sensitive nucleic acid assays, also enabling hydrogel-based suspension arrays. In this review, we discuss key parameters of hydrogels in the form of micron-sized particles to be used in sensing applications, paying attention to the protein and oligonucleotides (i.e., miRNAs) targets as most representative kind of biomarkers

Supramolecular Microgels with Molecular Beacons at the Interface for Ultrasensitive, Amplification-Free, and SNP-Selective miRNA Fluorescence Detection

In this study, a supramolecular structure with femtomolar biorecognition properties is proposed for use in analytical devices. It is obtained by an innovative interface between synthetic hydrogel polymers and molecular beacon (mb) probes. Supramolecularly structured microgels are synthetized with a core-shell architecture with specific dyes polymerized in a desired compartment. Mb probes are opportunely conjugated at the microgel interface so that their recognition mechanism is preserved and their spatial distribution is optimized to avoid crowding effects. The miR-21, a microRNA involved in various biological processes and usually used as a biomarker in early cancer diagnosis, has been selected as the target. The results demonstrate that by tuning the spatial distribution of molecular probes immobilized on the microgel and/or the amount of microgels, the assay shows scalable sensitivity reaching a limit of detection down to about 10 fM, without amplification steps and with detection time as short as 1 h. The assay results specific toward single mutated targets, and it is stable in the presence of high-interfering oligonucleotides concentrations. The miRNA target is also detected in human serum with performances similar to those observed in PBS buffer because of microgel antifouling properties without the need of any surface treatment. All tests were performed in a low sample volume (20 μL). As a result, mb-microgel represents an innovative biosensor to precisely quantify microRNAs in a direct (mix&read), scalable, and selective way. Such an approach paves the way for creating innovative biosensing interfaces with other probes, such as hairpins, aptamers, and PNA

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