Highly Magnetized Encoded Hydrogel Microparticles with Enhanced Rinsing Capabilities for Efficient microRNA Detection

Abstract

Encoded hydrogel microparticles mounting DNA probes are powerful tools for high-performance microRNA (miRNA) detection in terms of sensitivity, specificity, and multiplex detection capability. However, several particle rinsing steps in the assay procedure present challenges for rapid and efficient detection. To overcome this limitation, we encapsulated dense magnetic nanoparticles to reduce the rinsing steps and duration via magnetic separation. A large number of magnetic nanoparticles were encapsulated into hydrogel microparticles based on a discontinuous dewetting technique combined with degassed micromolding lithography. In addition, we attached DNA probes targeting three types of miRNAs related to preeclampsia to magnetically encoded hydrogel microparticles by post-synthesis conjugation and achieved sensitivity comparable to that of conventional nonmagnetic encoded hydrogel microparticles. To demonstrate the multiplex capability of magnetically encoded hydrogel microparticles while maintaining the advantages of the simplified rinsing process when addressing multiple samples, we conducted a triplex detection of preeclampsia-related miRNAs. In conclusion, the introduction of magnetically encoded hydrogel microparticles not only allowed efficient miRNA detection but also provided comparable sensitivity and multiplexed detectability to conventional nonmagnetic encoded hydrogel microparticles.

Direct functionalization of cell‐adhesion promoters to hydrogel microparticles synthesized by stop‐flow lithography

“…PEG 200 was added as a porogen to allow the efficient penetration and diffusion of macromolecules in the hydrogel structure during the conjugation process. It has been proved that PEG 200 can induce porosity in the hydrogel composed of monomers with similar concentration and molecular weight to the current study enough to penetrate the molecules with molecular weight similar to‐ or higher than PLL used in this study (15–30 kDa) 10,21 . When the flowing precursor along with microchannel was halted, the UV light with a 365 nm wavelength was exposed to the precursor through a graphically perforated photomask.…”

“…The detailed calculation procedures and related datas were presented in the supporting information and Figure S2. These unreacted acrylate groups are highly reactive with various functionalized molecules through chemical reactions, including Michael addition reactions 10,16,24 . In particular, considering that various cell‐adhesion promoters (e.g., PLL, fibronectin, collagen, and dopamine) contain amine groups in their molecular structures, the aza‐Michael addition of cell‐adhesion promoter molecules to microparticles can be directly achieved without requiring the preconjugation of linker molecules 2 …”

“…To synthesize microparticles as a platform for cellular microenvironments, diverse fabrication techniques can be employed, including flow lithography, 7,8 replica molding, 9,10 electrohydrodynamic spraying, 11 and emulsion polymerization based on microfluidics or batch reactors 12,13 . Among these processes, stop‐flow lithography (SFL), one of the representative flow lithographic techniques, is a powerful method for the production of cellular microenvironment structures with tunability on particle size and shape and the production of porous structures for the penetration and diffusion of various biomolecules 8,14 .…”

“…To avoid using additional linker molecules and complicated preconjugation steps, the direct conjugation of cell‐adhesion promoter molecules containing amine groups can be an alternative by inducing the aza‐Michael reaction between unreacted acrylate groups in the particles and the nucleophilic amine groups in the cell‐adhesion promoters. Recently, there has been high interest in utilizing unreacted acrylate groups in PEG crosslinkers during polymerization for a high‐performance and simple bio‐conjugation process, in which the remnant acrylate groups enable Michael addition reactions with amine and thiol groups in various biomolecules 10,16,24,25 . It has already been demonstrated that the unreacted acrylate group‐based functionalization methods have powerful advantages, such as the high conjugation capacity, 25 simplicity of process because of the unnecessity of linker molecules, 26 and prevention of the aggregation of biomolecules inside the gel structures 16 .…”

Highly Flexible Deep-Learning-Based Automatic Analysis for Graphically Encoded Hydrogel Microparticles

“…The incorporation of said nanomaterials into encoded HMPs may yield diverse advantages in the bioassay. For instance, magnetic nanoparticles (MNPs) may serve to simplify the buffer exchanging step, ,, enabling the separation of particles by magnetic forces instead of labor-intensive centrifugation methods. Furthermore, colorimetric labeling with nanoprecipitates inside HMPs facilitates field diagnoses with a portable microscope (e.g., a USB microscope), avoiding the requirement of complex and bulky fluorescence-based optical equipment. ,, However, these incorporated nanomaterials cause difficulties in deep-learning-based analyses, as they possess wide ranges of textures and colors. − Consequently, the conventional learning process is insufficient in the analysis of such particles since it uses the clearly transparent encoded HMPs for dataset preparation.…”