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.

Elimination of Unreacted Acrylate Double Bonds in the Polymer Networks of Microparticles Synthesized via Flow Lithography.

Flow lithography (FL), a versatile technique used to synthesize anisotropic multifunctional microparticles, has attracted substantial interest, given that the resulting particles with complex geometries and multilayered biochemical functionalities can be used in a wide variety of applications. However, after this process, there are double bonds remaining from the cross-linkable groups of monomers. The unreacted cross-linkable groups can affect the particles' biochemical properties. Here, we verify that the microparticles produced by FL contain a significant number of unreacted acrylate double bonds (UADBs), which could cause irreversible biochemical changes in the particle and pernicious effects to biological systems. We also confirm that the particles contain a considerable number of UADBs, regardless of the various synthetic (lithographic) conditions that can be used in a typical FL process. We present an effective way to eliminate a substantial amount of UADBs after synthesis by linking biochemically inert poly(ethylene glycol) based on click chemistry. We verify that eliminating UADBs by using this click chemistry approach can efficiently resolve problems, such as the occurrence of random reactions and the cytotoxicity of UADBs.

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.

Linker-free antibody conjugation for sensitive hydrogel microparticle-based multiplex immunoassay.

Multiplex immunoassay, or the simultaneous detection of multiple proteins in a single sample, is expected to enable a new level of protein analysis across diverse disciplines, such as medical diagnostics and biomarker discovery. A bead-based assay using graphically encoded hydrogel microparticles synthesized using stop flow lithography has been a promising platform because of its high multiplex capacity and its superior sensitivity and dynamic range compared to the enzyme-linked immunosorbent assay (ELISA). The functionalization of these particles has been dependent on the use of a heterobifunctional linker to conjugate the capture antibodies on the hydrogel. However, the linker chemistry, which is based on linking the primary amine groups of antibodies with acrylate functional groups on the hydrogel monomer, is vulnerable to hydrolysis in aqueous conditions and can potentially damage the antigen binding region of the antibody. In this work, we introduce a new antibody conjugation method that avoids the use of the linker and further enhances the sensitivity of hydrogel microparticle-based immunoassays. Disulfide bonds in antibodies are reduced to liberate free thiols, which can directly bond with the double bonds remaining in the hydrogel after particle synthesis. We characterize the optimal reduction of antibodies for producing the highest detection signal and demonstrate an average two-fold improvement in sensitivity compared to the linker-dependent antibody conjugation method. Lastly, we validate the accuracy and specificity of the multiplex assays with particles conjugated with antibodies using the linker-free method.

Microfluidic Synthesis of pH-Sensitive Multicompartmental Microparticles for Multimodulated Drug Release.

Stimuli-responsive carriers releasing multiple drugs have been researched for synergistic combinatorial cancer treatment with reduced side-effects. However, previously used drug carriers have limitations in encapsulating multiple drug components in a single carrier and releasing each drug independently. In this work, pH-sensitive, multimodulated, anisotropic drug carrier particles are synthesized using an acid-cleavable polymer and stop-flow lithography. The particles exhibit a faster drug release rate at the acidic pH of tumors than at physiological pH, demonstrating their potential for tumor-selective drug release. The drug release rate of the particles can be adjusted by controlling the monomer composition. To accomplish multimodulated drug release, multicompartmental particles are synthesized. The drug release profile of each compartment is programmed by tailoring the monomer composition. These pH-sensitive, multicompartmental particles are promising drug carriers enabling tumor-selective and multimodulated release of multiple drugs for synergistic combination cancer therapy.

Vertically encoded tetragonal hydrogel microparticles for multiplexed detection of miRNAs associated with Alzheimer's disease.

Encoded hydrogel particles have attracted attention in diagnostics as these particles can be used for high-performance multiplexed assays. Here, we present encoded tetragonal hydrogel microparticles for multiplexed detection of miRNAs that are strongly related to Alzheimer's disease (AD). The particles are comprised of vertically distinct code and probe regions, and incorporated with quantum dots (QDs) in the code regions. By virtue of the particle geometry, the particles can be synthesized at a high production rate in vertically stacked micro-flows using hydrodynamic focusing lithography. To detect multiple AD-miRNAs, various code labels to identify the loaded probes are designed by changing wavelengths of QDs, increasing the number of code layers and adjusting the thickness of code layers. The probe regions are incorporated with complementary sequences of target miRNAs, and optimized for accurate and timely detection of AD-miRNAs. For proof of concept, we demonstrate the multiplexed capability of the particles by performing a 3-plexed assay of AD-miRNAs.

Stifle Joint Arthrodesis for Treating Chronic-Osteoarthritis-Affected Dogs.

A two-year-old male Pomeranian dog was presented to a veterinary hospital due to the side effects of a surgical correction for patellar luxation. Stifle joint arthrodesis (SJA) was performed on the patient's right leg using autologous bone-grafting techniques. The right femur and tibial joint were angled 120-130°, and an SJA plate was fixed on the front of the two bones. After performing joint fusion of the right limb, medial-patellar-luxation-(MPL)-corrective surgery was performed to cut the tibial tuberosity on the left leg, and the fixing force was increased using the figure-of-eight-tension-band-wiring technique. Results were recorded regarding the dog's ability to walk and trot in the right hind limb; these results were evaluated for 27 days after surgery. It was difficult for the patient to walk because weight-bearing had not been carried out for 3 days after the surgery; short strides and partial weight bearing were possible 5 to 7 days after surgery. After 10 days, the patient was able to move while bearing weight with a slight disruption. With regard to trotting, the patient showed intermittent normal steps 5 to 7 days after surgery, but the disruption continued. After 14 days, trotting was possible, and it was observed that movement could be maintained during everyday activities.

CRISPR-Enhanced Hydrogel Microparticles for Multiplexed Detection of Nucleic Acids.

CRISPR/Cas systems offer a powerful sensing mechanism to transduce sequence-specific information into amplified analytical signals. However, performing multiplexed CRISPR/Cas assays remains challenging and often requires complex approaches for multiplexed assays. Here, a hydrogel-based CRISPR/Cas12 system termed CLAMP (Cas-Loaded Annotated Micro-Particles) is described. The approach compartmentalizes the CRISPR/Cas reaction in spatially-encoded hydrogel microparticles (HMPs). Each HMP is identifiable by its face code and becomes fluorescent when target DNA is present. The assay is further streamlined by capturing HMPs inside a microfluidic device; the captured particles are then automatically recognized by a machine-learning algorithm. The CLAMP assay is fast, highly sensitive (attomolar detection limits with preamplification), and capable of multiplexing in a single-pot assay. As a proof-of-concept clinical application, CLAMP is applied to detect nucleic acid targets of human papillomavirus in cervical brushing samples.

Whole Blood Viscosity Reference Intervals and Its Correlation with Hematology and Serum Chemistry in Cats Using Scanning Capillary Method.

Whole blood viscosity, a hemorheological factor, is currently used for diagnosis, as it is correlated with various vascular diseases that are difficult to diagnose early with a general blood test. It was determined that it was necessary to set reference intervals for further studies and utilization of whole blood viscosity in cats, a representative companion animal, and this study was conducted. Fifty healthy cats were recruited for the study, and whole blood viscosity, complete blood count, and serum chemistry tests were performed. The reference intervals of whole blood viscosity were 15.169 to 43.684 cP at a shear rate of 1 s-1 reflecting diastole, and 3.524 to 5.544 cP at a shear rate of 300 s-1 reflecting systole. Red blood cells, hematocrit, hemoglobin, white blood cells, and neutrophils in the complete blood count, and total protein, albumin, globulin, and cholesterol in the serum chemistry were significantly correlated with whole blood viscosity. The results of this study set the reference intervals of whole blood viscosity for healthy cats in a wide shear rate range that has not yet been fully established, and its correlation with other blood indicators investigated.

Double Digital Assay for Single Extracellular Vesicle and Single Molecule Detection.

Extracellular vesicles (EVs) have emerged as a promising source of biomarkers for disease diagnosis. However, current diagnostic methods for EVs present formidable challenges, given the low expression levels of biomarkers carried by EV samples, as well as their complex physical and biological properties. Herein, a highly sensitive double digital assay is developed that allows for the absolute quantification of individual molecules from a single EV. Because the relative abundance of proteins is low for a single EV, tyramide signal amplification (TSA) is integrated to increase the fluorescent signal readout for evaluation. With the integrative microfluidic technology, the technology's ability to compartmentalize single EVs is successfully demonstrated, proving the technology's digital partitioning capacity. Then the device is applied to detect single PD-L1 proteins from single EVs derived from a melanoma cell line and it is discovered that there are ≈2.7 molecules expressed per EV, demonstrating the applicability of the system for profiling important prognostic and diagnostic cancer biomarkers for therapy response, metastatic status, and tumor progression. The ability to accurately quantify protein molecules of rare abundance from individual EVs will shed light on the understanding of EV heterogeneity and discovery of EV subtypes as new biomarkers.

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.

Ischiocavernosus Muscle Release for Urethral Obstruction Treatment after Pelvic Symphyseal Distraction Osteotomy in a Cat with Pelvic Stenosis.

Symphyseal distraction osteotomy (SDO) with a polymethyl methacrylate (PMMA) spacer is an effective surgical treatment for cats with pelvic stenosis. This study reports the successful treatment of urethral obstruction due to ischiocavernosus muscle (IM) tension after SDO with a PMMA spacer. A 2-year-old castrated male Korean domestic shorthair feline had megacolon and pelvic canal stenosis. The ratio of the maximal diameter of the colon to the L5 length and the pelvic canal diameter ratio were 1.6 and 0.45, respectively. Pelvic SDO was performed with a PMMA spacer, leading to pelvic canal enlargement (pelvic canal diameter ratio: 0.73). Two days after surgery, dysuria was identified immediately after removing the preoperatively placed urinary catheter. Complete blood counts and serum biochemical profiles were within the reference intervals, and a positive contrast retrograde urethrogram confirmed urethral obstruction at the level of the membranous-pelvic urethra region. Increased tension of the IM leading to a narrowed urethra was suspected as the cause of urethral obstruction. After IM release at the level of origin on the ischium, the patient had an uneventful recovery with spontaneous urination. Muscle release resulted in excellent functional restoration, with no intraoperative or postoperative complications reported during the 12-month long-term follow-up. Therefore, SDO with IM release could be a feasible therapeutic option for severe pelvic stenosis without complications, such as urethral obstruction, in cats.

Comparison between Novice and Experienced Surgeons Performing Corrective Osteotomy with Patient-Specific Guides in Dogs Based on Resulting Position Accuracy.

Corrective osteotomy has been applied to realign and stabilize the bones of dogs with lameness. However, corrective osteotomy for angular deformities requires substantial surgical experience for planning and performing accurate osteotomy. Three-dimensional printed patient-specific guides (3D-PSGs) were developed to overcome perioperative difficulties. In addition, novices can easily use these guides for performing accurate corrective osteotomy. We compared the postoperative results of corrective osteotomy accuracy when using 3D-PSGs in dogs between novice and experienced surgeons. We included eight dogs who underwent corrective osteotomy: three angular deformities of the radius and ulna, three distal femoral osteotomies, one center of rotational angle-based leveling osteotomy, and one corrective osteotomy with stifle arthrodesis. All processes, including 3D bone modeling, production of PSGs, and rehearsal surgery were carried out with computer-aided design software and a 3D-printed bone model. Pre- and postoperative positions following 3D reconstruction were evaluated by radiographs using the 2D/3D registration technique. All patients showed clinical improvement with satisfactory alignment and position. Postoperative accuracy evaluation revealed no significant difference between novice and experienced surgeons. PSGs are thought to be useful for novice surgeons to accurately perform corrective osteotomy in dogs without complications.

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.

Precipitation-based colorimetric multiplex immunoassay in hydrogel particles.

Despite a growing demand for more accessible diagnostic technologies, current methods struggle to simultaneously detect multiple analytes with acceptable sensitivity and portability. Colorimetric assays have been widely used due to their simplicity of signal readout, but the lack of multiplexibility has been a perpetual constraint. Meanwhile, particle-based assays offer multiplex detection by assigning an identity code to each analyte, but they often require lab-based equipment unsuitable for portable diagnostics. Here, by merging the two approaches, this paper reports a colorimetric multiplex immunoassay based on hydrogel microparticles that achieves the best of both worlds. The low-cost portable multiplex assay demonstrates sensitivities as high as and dynamic ranges greater than the lab-based enzyme-linked immunosorbent assay (ELISA). These critical advances are made possible by local precipitation and amplification of insoluble colour dyes inside the hydrogel networks. For the first time, enzymatic accumulation of colour dyes in hydrogel particles is reported and the kinetics of colour development is characterized in this work. By taking advantage of the colour signals in the visible spectrum, the hydrogel microparticles were imaged and analysed using low-cost portable devices. The colorimetric multiplex immunoassay was used to successfully detect three target biomarkers of preeclampsia and validated clinically using healthy and patient-derived plasma samples.

Affinity-Enhanced CTC-Capturing Hydrogel Microparticles Fabricated by Degassed Mold Lithography.

Technologies for the detection and isolation of circulating tumor cells (CTCs) are essential in liquid biopsy, a minimally invasive technique for early diagnosis and medical intervention in cancer patients. A promising method for CTC capture, using an affinity-based approach, is the use of functionalized hydrogel microparticles (MP), which have the advantages of water-like reactivity, biologically compatible materials, and synergy with various analysis platforms. In this paper, we demonstrate the feasibility of CTC capture by hydrogel particles synthesized using a novel method called degassed mold lithography (DML). This technique increases the porosity and functionality of the MPs for effective conjugation with antibodies. Qualitative fluorescence analysis demonstrates that DML produces superior uniformity, integrity, and functionality of the MPs, as compared to conventional stop flow lithography (SFL). Analysis of the fluorescence intensity from porosity-controlled MPs by each reaction step of antibody conjugation elucidates that more antibodies are loaded when the particles are more porous. The feasibility of selective cell capture is demonstrated using breast cancer cell lines. In conclusion, using DML for the synthesis of porous MPs offers a powerful method for improving the cell affinity of the antibody-conjugated MPs.

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.

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.

Post-synthesis functionalized hydrogel microparticles for high performance microRNA detection.

Encoded hydrogel microparticles, synthesized by Stop Flow Lithography (SFL), have shown great potential for microRNA assays for their capability to provide high multiplexing capacity and solution-like hybridization kinetics. However, due to the low conversion of copolymerization during particle synthesis, current hydrogel microparticles can only utilize ∼10% of the input probes that functionalize the particles for miRNA assay. Here, we present a novel method of functionalizing hydrogel microparticles after particle synthesis by utilizing unconverted double bonds remaining inside the hydrogel particles to maximize functional probe incorporation and increase the performance of miRNA assay. This allows covalent bonding of functional probes to the hydrogel network after particle synthesis. Because of the abundance of the unconverted double bonds and accessibility of all probes, the probe density increases about 8.2 times compared to that of particles functionalized during the synthesis. This results lead to an enhanced miRNA assay performance that improves the limit of detection from 4.9 amol to 1.5 amol. In addition, higher specificity and shorter assay time are achieved compared to the previous method. We also demonstrate a potential application of our particles by performing multiplexed miRNA detections in human plasma samples.

Multiplexed immunoassay using post-synthesis functionalized hydrogel microparticles.

In response to a growing demand for simultaneous detection of multiple proteins in a single sample, multiplex immunoassay platforms have emerged at the forefront of proteomic analysis. In particular, detections using graphically encoded hydrogel microparticles synthesized via flow lithography have received attention for integrating a hydrogel, a substrate that can provide enhanced kinetics and high loading capacity, into the bead-based multiplex platform. Currently, the method of microparticle functionalization involves copolymerization of antibodies with the gel during particle synthesis. However, its practical operation is too precarious to be adopted because antibodies are susceptible to aggregation due to incompatibility with hydrophobic photoinitiators used in the photo-induced gel polymerization. In this work, we present a multiplex immunoassay platform that uses encoded hydrogel microparticles that are functionalized after particle synthesis by conjugating antibodies with remnant active groups readily available in the hydrogels. The method not only precludes antibody aggregation but also augments the loading density of the antibodies, which translates into enhanced detection performance. In addition to multiplexing, our platform demonstrates high sensitivity, a broad assay range, and a fast detection rate that outperform the enzyme linked immunosorbent assay (ELISA).