Real-Time Signal Analysis with Wider Dynamic Range and Enhanced Sensitivity in Multiplex Colorimetric Immunoassays Using Encoded Hydrogel Microparticles.

The simultaneous quantification of multiple proteins is crucial for accurate medical diagnostics. A promising technology, the multiplex colorimetric immunoassay using encoded hydrogel microparticles, has garnered attention, due to its simplicity and multiplex capabilities. However, it encounters challenges related to its dynamic range, as it relies solely on the colorimetric signal analysis of encoded hydrogel microparticles at the specific time point (i.e., end-point analysis). This necessitates the precise determination of the optimal time point for the termination of the colorimetric reaction. In this study, we introduce real-time signal analysis to quantify proteins by observing the continuous colorimetric signal change within the encoded hydrogel microparticles. Real-time signal analysis measures the "slope", the rate of the colorimetric signal generation, by focusing on the kinetics of the accumulation of colorimetric products instead of the colorimetric signal that appears at the end point. By developing a deep learning-based automatic analysis program that automatically reads the code of the graphically encoded hydrogel microparticles and obtains the slope by continuously tracking the colorimetric signal, we achieved high accuracy and high throughput analysis. This technology has secured a dynamic range more than twice as wide as that of the conventional end-point signal analysis, simultaneously achieving a sensitivity that is 4-10 times higher. Finally, as a demonstration of application, we performed multiplex colorimetric immunoassays using real-time signal analysis covering a wide concentration range of protein targets associated with pre-eclampsia.

Multiplex genotyping of SNPs in genomic DNA via hydrogel-based assay mediated with MutS and polyethylene glycol.

The simultaneous genotyping of multiple single nucleotide polymorphisms (SNPs) in genomic DNA derived from organisms holds significant potential for applications such as precision medicine and food product authentication. However, conventional assay technologies including qPCR-based techniques, microarrays, and hydrogel-based assays face limitations in efficient multiplexing of SNPs, particularly for large-size DNA beyond kilobase scales, due to constraints in multiplex capability, specificity, or sensitivity. In this study, a hydrogel-based multiplex SNP genotyping platform specifically designed for genomic DNA is presented. This platform integrates the ligation detection reaction (LDR) and rolling circle amplification (RCA) techniques within a hydrogel-based multiplex sensing system, enabling adaptable and sensitive SNP genotyping for genomic DNA. To enhance the specificity of the assay, MutS protein and polyethylene glycol are introduced into the protocol, reducing the non-specific ligation and RCA reactions synergistically. With significant specificity improvement of over 10-fold, three types of SNPs within an artificially constructed ∼1000 bp double-stranded DNA (dsDNA) are successfully genotyped with double-digit picomolar sensitivity. Furthermore, the practical applicability of the developed process for the origin identification of raw materials is demonstrated by genotyping three types of SNPs within genomic DNA obtained from two closely related plant species, Korean ginseng (Panax ginseng) and American ginseng (Panax quinquefolius), containing ca. 3.5 gigabase genome size. Of notable significance, this study marks the premiere achievement in PCR-free multiplex genotyping of SNPs in genomic DNA using a single fluorophore.

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

Graphically encoded hydrogel microparticle (HMP)-based bioassay is a diagnostic tool characterized by exceptional multiplex detectability and robust sensitivity and specificity. Specifically, deep learning enables highly fast and accurate analyses of HMPs with diverse graphical codes. However, previous related studies have found the use of plain particles as data to be disadvantageous for accurate analyses of HMPs loaded with functional nanomaterials. Furthermore, the manual data annotation method used in existing approaches is highly labor-intensive and time-consuming. In this study, we present an efficient deep-learning-based analysis of encoded HMPs with diverse graphical codes and functional nanomaterials, utilizing the auto-annotation and synthetic data mixing methods for model training. The auto-annotation enhanced the throughput of dataset preparation up to 0.11 s/image. Using synthetic data mixing, a mean average precision of 0.88 was achieved in the analysis of encoded HMPs with magnetic nanoparticles, representing an approximately twofold improvement over the standard method. To evaluate the practical applicability of the proposed automatic analysis strategy, a single-image analysis was performed after the triplex immunoassay for the preeclampsia-related protein biomarkers. Finally, we accomplished a processing throughput of 0.353 s per sample for analyzing the result image.

Controlled growth factor delivery via a degradable poly(lactic acid) hydrogel microcarrier synthesized using degassed micromolding lithography.

Controlled and targeted delivery of growth factors to biological environments is important for tissue regeneration. Polylactic acid (PLA) hydrogel microparticles are attractive carriers for the delivery of therapeutic cargoes based on their superior biocompatibility and biodegradability, uniform encapsulation of cargoes, and non-requirement of organic solvents during particle synthesis. In this study, we newly present controlled growth factor delivery utilizing PLA-based hydrogel microcarriers synthesized via degassed micromolding lithography (DML). Based on the direct gelation procedure from the single-phase aqueous precursor in DML, bovine serum albumin, a model protein of growth factor, and fibroblast growth factor were encapsulated into microparticles with uniform distribution. In addition, by tuning the monomer concentration and adding a hydrolytically stable crosslinker, the release of encapsulated cargoes was efficiently controlled and extended to 2 weeks. Finally, we demonstrated the biological activity of encapsulated FGF-2 in PLA-based microparticles using a fibroblast proliferation assay.

High-Resolution Surface Replication of Living Organisms using Air-Through-Precursor Suction-Augmented Replica Molding.

Replica molding is widely used to reproduce the surface microstructures that provide living organisms with distinct and useful functions. However, the existing methods are limited by the low resolution resulting from the air trapped in the structures during precursor solution loading. This study investigated replica molding with an air-through-precursor suction (APS) process, which used a degassed polydimethylsiloxane substrate to remove the trapped air through the precursor solution. The liquid loading times are characterized using a model template, and air suction that is up to 36 times faster can be achieved using the APS process relative to a conventional method. Using APS replica molding, biocompatible replicates from human fingerprints and gecko skin are fabricated using only a 3 min precursor solution loading step. Owing to the enhanced and reproducible resolution from APS replica molding, for the first time, the structural changes in the foot of a living gecko at the microscale can be observed when standing on a horizontal or vertical surface.

Encoded hydrogel microparticles with universal mismatch-incorporated DNA probes for highly specific multiplex detection of SNPs.

Hydrogel microparticle-based nucleic acid assays are an attractive detection platform based on their multiplexing capabilities with high sensitivity and specificity. A particular area of interest is single-nucleotide polymorphism (SNP) sensing, where multiple SNPs should be identified in a highly reliable yet economical manner. However, hydrogel microparticles leveraging probe-target hybridization as a key mechanism are hampered by small duplex stability differences arising from single base-pair mismatch. We have developed encoded hydrogel microparticles with DNA probes tailored for multiplex SNP detection. Within the DNA probes, we adopt a widely used base analog (5-nitroindole) so that it substitutes one of the base sequences among DNA probes. The effects of the modification of the probes' structure on SNP sensing has been tested from multiple perspectives, such as specificity, sensitivity, and available assay temperatures at a given ionic strength. We have validated that our hydrogel microparticles exhibit much higher specificity for a single base-pair mismatch with minimal reduction in sensitivity. Our particles can also detect multiple SNPs located in different target strands, which is a significant challenge for conventional particles.

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

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.

Rapid colorimetric analysis of multiple microRNAs using encoded hydrogel microparticles.

microRNAs (miRNAs) have attracted much attention as potential biomarkers for the diagnosis of various fatal diseases. With increasing interest in miRNA detection at practical sites, colorimetric bead-based assays have garnered much attention, because these allow for simple analysis with cheap and portable devices. Among them, the encoded hydrogel microparticle-based colorimetric miRNA assay is considered as one of the promising techniques, due to its strengths, such as large multiplex capacity, acceptable sensitivity, and simple analysis. However, it still imposes a limitation in terms of the assay time, particularly the colorimetric reaction time, which is too long, making the practical application of the assay difficult and undermining its detection accuracy. In this work, we present a rapid colorimetric assay based on encoded hydrogel microparticles, which exhibits a significant decrease in the colorimetric reaction time due to two factors: (1) an increase in the number of enzymes bound to hydrogel microparticles via a post-synthesis functionalization method, and (2) an elevation in the enzyme reaction temperature during colorimetric labeling. We obtained a comparable sensitivity of the colorimetric assay with three different miRNA targets, even with a shortened colorimetric reaction time. Furthermore, we validated that our colorimetric detection method is suitable for multiplex miRNA detection, owing to its low cross-reactivity.

Discontinuous Dewetting in a Degassed Mold for Fabrication of Homogeneous Polymeric Microparticles.

Discontinuous dewetting (DD) is an attractive technique that enables the production of large liquid arrays in microwells and is applicable to the synthesis of anisotropic microparticles with complex morphologies. However, such loading of liquids into microwells presents a significant challenge, as the liquids used in this technique should exhibit low mold surface wettability. This study introduces DD in a degassed mold (DM), a simple yet powerful technique that achieves uniform loading of microparticle precursors into large microwell arrays within 1 min. Using this technique, hydrogel microparticles are produced by different polymerization mechanisms with various shapes and sizes, ranging from a few micrometers to hundreds of micrometers. Hydrophobic oil microparticles are produced by the simple plasma treatment of the DM, and agarose microparticles encapsulating bovine serum albumin (in a well-dispersed state) are produced by submerging the DM in fluorinated oil. To demonstrate additional functionality of microparticles using this technique, high concentrations of magnetic nanoparticles are loaded into microparticles for particle-based immunoassays performed in a microwell plate, and the immunoassay performance is comparable to that of ELISA.

Photopolymerization-Based Synthesis of Uniform Magnetic Hydrogels and Colorimetric Glucose Detection.

Magnetic hydrogels have been commonly used in biomedical applications. As magnetite nanoparticles (MNPs) exhibit peroxidase enzyme-like activity, magnetic hydrogels have been actively used as signal transducers for biomedical assays. Droplet microfluidics, which uses photoinitiated polymerization, is a preferred method for the synthesis of magnetic hydrogels. However, light absorption by MNPs makes it difficult to obtain fully polymerized and homogeneous magnetic hydrogels through photoinitiated polymerization. Several methods have been reported to address this issue, but few studies have focused on investigating the light absorption properties of photoinitiators. In this study, we developed a simple method for the synthesis of poly(ethylene glycol) (PEG)-based uniform magnetic hydrogels that exploits the high ultraviolet absorption of a photoinitiator. Additionally, we investigated this effect on shape deformation and structural uniformity of the synthesized magnetic hydrogels. Two different photoinitiators, Darocur 1173 and lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP), with significantly different UV absorption properties were evaluated based on the synthesis of magnetic hydrogels. The magnetic characteristics of the PEG-stabilized MNPs in hydrogels were investigated with a vibrating sample magnetometer. Finally, the colorimetric detection of hydrogen peroxide and glucose was conducted based on the enzyme-like property of MNPs and repeated several times to observe the catalytic activity of the magnetic hydrogels.

Phosphorylcholine-based encoded hydrogel microparticles with enhanced fouling resistance for multiplex immunoassays.

Due to the growing interest in multiplex protein detection, encoded hydrogel microparticles have received attention as a possible path to high performance multiplex immunoassays through a combination of high multiplexing capability and enhanced binding kinetics. However, their practical operation in real complex samples is still limited because polyethylene glycol, which is the main component of hydrogel particles, suffers from oxidative damage and relatively high fouling properties in biochemical solutions. Here, we introduce poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-based encoded hydrogel microparticles to perform fouling-resistant multiplex immunoassays, where the anti-fouling characteristics are attributed to the zwitterionic PMPC. By applying a newly developed molding lithography technique, viscous PMPCs with low reactivity were successfully incorporated into the hydrogel network while maintaining uniformity and rigidity for use in multiplex immunoassays. Non-specific protein adsorption on the PMPC particles was reduced by about 37.5% compared to that of conventional PEG particles, which leads to better assay sensitivity. We also validate the multiplex capability of the PMPC particles by performing multiplex detection of two target proteins. Furthermore, we verify that the PMPC particles have a 70% enhancement in anti-fouling characteristics compared to PEG particles in human platelet-rich plasma, potentiating a practical immunoassay platform for clinical diagnosis.

Direct Conjugation of Streptavidin to Encoded Hydrogel Microparticles for Multiplex Biomolecule Detection with Rapid Probe-Set Modification.

Encoded hydrogel microparticles synthesized via flow lithography have drawn attention for multiplex biomarker detection due to their high multiplex capability and solution-like hybridization kinetics. However, the current methods for preparing particles cannot achieve a flexible, rapid probe-set modification, which is necessary for the production of various combinations of target panels in clinical diagnosis. In order to accomplish the unmet needs, streptavidin was incorporated into the encoded hydrogel microparticles to take advantage of the rapid streptavidin-biotin interactions that can be used in probe-set modification. However, the existing methods suffer from low efficiency of streptavidin conjugation, cause undesirable deformation of particles, and impair the assay capability. Here, we present a simple and powerful method to conjugate streptavidin to the encoded hydrogel microparticles for better assay performance and rapid probe-set modification. Streptavidin was directly conjugated to the encoded hydrogel microparticles using the aza-Michael addition click reaction, which can proceed in mild, aqueous condition without catalysts. A highly flexible and sensitive assay was developed to quantify DNA and proteins using streptavidin-conjugated encoded hydrogel microparticles. We also validated the potential applications of our particles conducting multiplex detection of cancer-related miRNAs.