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A two-year-old male Pomeranian dog visited the hospital due to side effects of surgical correction for patellar luxation. Stifle joint arthrodesis (SJA) was performed on the patient’s right leg using autologous bone grafting techniques. The right leg’s femur and tibial joint were angled 120-130◦ and the 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. The results were observed for walking and trotting of the right hind limb, evaluated for 27 days after surgery. It was difficult for the patient to walk because weight loading was not performed for 3 days after 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. For trotting, the patient showed intermittent normal steps 5 to 7 days after surgery, but the disruption continued. After 14 days, trotting was possible and movements were shown to be maintained during everyday activities.

Thermoresponsive polymers incorporated with photo-absorbing agents have been widely utilized for controlled drug delivery using light as an external stimulus. However, previously developed thermoresponsive drug carriers have disadvantages such as low biocompatibility and implantation failure. In the present study, gold nanorods (GNRs)-encapsulated poly(N-vinyl caprolactam) (PVCL) (GNR-PVCL) microparticles were synthesized by the stop-flow lithography (SFL) method. The SFL method enabled the fabrication of near-infrared (NIR) light-responsive GNR-PVCL microparticles of uniform size, which can allow localized injection. Doxorubicin (DOX) was encapsulated into GNR-PVCL microparticles to achieve NIR light-responsive anticancer therapy. DOX-loaded GNR-PVCL (DOX-GNR-PVCL) microparticles exhibited NIR light-triggered drug release due to the photothermal effect of GNRs, which increases the local temperature above the volume phase transition temperature of GNR-PVCL microparticles. In addition, DOX-GNR-PVCL exhibited controlled DOX release in response to the periodic irradiation of NIR light. Moreover, we demonstrated the efficient intracellular release of DOX upon NIR light exposure, and thus, NIR light-responsive anticancer activity. This study demonstrates that DOX-GNR-PVCL microparticles have significant potential as implantable drug carriers enabling NIR light-triggered drug release.

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 (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.

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.

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.

The development of photo-triggered drug delivery system is promising for the temporal and spatial control of drug release. However, most of the progress in photothermal drug delivery has been focused on nanosized particles which can be removed from the injected sites by exocytosis. In this study, injectable and implantable near-infrared (NIR) light-responsive microparticles are fabricated using droplet-based microfluidics. The particles are composed of 1-tetradecanol, as a class of non-toxic phase change material, and IR780, as a NIR-absorbing photothermal agent. Doxorubicin, an anticancer drug, is encapsulated in the particles to obtain NIR light-responsive anticancer drug carriers. The photothermal response of IR780 to NIR irradiation facilitates the release of the encapsulated drug through the melting of 1-tetradecanol and demonstrates the NIR light-responsive drug release. This study shows that IR780-encapsulated 1-tetradecanol microparticles fabricated by droplet-based microfluidics have great potential as injectable and implantable drug carriers featuring NIR light-sensitive drug release.

Recent interests in comprehensive protein surveys and protein biomarker studies have led to an increased demand for simultaneous measurement of multiple proteins in a single sample. Among various multiplex techniques, bead-based immunoassays, which use encoded particles attached with capture probes, have demonstrated distinct advantages of fluid-phase kinetics, high precision, and flexible target selection. In particular, encoded hydrogel particles composed of porous, hydrophilic, three-dimensional polymers have received positive attention because they enhance the binding kinetics of proteins, reduce protein denaturation, and increase the loading density of capture probes. Microfluidic techniques have been extensively used to fabricate the encoded hydrogel particles for multiplex immunoassays, enabling mass-production of highly monodisperse particles with complex morphologies in mild synthesis conditions. In this paper, we review microfluidic techniques available for the synthesis of encoded hydrogel particles and the important design parameters that determine the particles’ immunoassay performance. We also discuss currently reported multiplex immunoassay platforms that are based on encoded hydrogel particles.

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&ndash, 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.

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.

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).

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&ndash, 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.

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.

Polydimethylsiloxane (PDMS) microfluidic devices, though they are commonly utilized in microfluidic applications, have several limitations, such as short-term modified surface condition, swelling in the presence of organic solvents, and deformation under high pressure or when built with low aspect ratios. To resolve the restrictions, Norland Optical Adhesive (NOA) has been introduced as an excellent alternative for PDMS. Here, we present a practical protocol for the fabrication of NOA microfluidic devices via a step-wise molding process. Through the indirect molding of NOA on wafers, the damage to the wafers can be significantly reduced. Furthermore, because we use positivepatterned wafers, which are commonly used to fabricate PDMS devices, no additional fabrication of the wafer is required. This simple strategy thus avoids damage to the wafers and simultaneously allows for the mass production of NOA devices without deformation. We also test the performance of NOA devices in oil-in-water droplet production and in a microfluidic process using organic solvents.

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.

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.

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.

Various thermo-responsive polymers have been developed for controlled drug delivery upon the local application of external heat. The development of thermo-responsive polymers with high biocompatibility and tunable thermo-sensitivity is crucial for safe and efficient therapeutic application. In this study, thermo-responsive drug carriers featuring tunable thermo-sensitivities were synthesized using biocompatible poly(N-vinyl caprolactam) (PVCL) and stop-flow lithography. The PVCL-based particles showed selective drug release depending on temperature, illustrating their feasibility for on-demand controlled drug delivery. The volume phase transition temperature (VPTT) of the PVCL-based particles can be adjusted to vary from room temperature to body temperature by controlling their monomer compositions. In addition, modulated drug release was achieved by constructing multicompartments of different thermo-sensitivities within the PVCL particles. To accomplish thermo-responsive anticancer therapy, doxorubicin (DOX) was encapsulated into the PVCL particles as an anticancer drug. The DOX-loaded PVCL particles exhibited both thermo-responsive drug release and anticancer activity. This study demonstrates that thermo-responsive PVCL particles are highly promising carriers for safe and targeted anticancer therapy.

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.

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.