Parathyroid-on-a-chip simulating parathyroid hormone secretion in response to calcium concentration.

The parathyroid gland is an endocrine organ that plays a crucial role in regulating calcium levels in blood serum through the secretion of parathyroid hormone (PTH). Hypoparathyroidism is a chronic disease that can occur due to parathyroid defects, but due to the difficulty of creating animal models of this disease or obtaining human normal parathyroid cells, the evaluation of parathyroid functionality for drug development is limited. Although parathyroid-like cells that secrete PTH have recently been reported, their functionality may be overestimated using traditional culture methods that lack in vivo similarities, particularly vascularization. To overcome these limitations, we obtained parathyroid organoids from tonsil-derived mesenchymal stem cells (TMSCs) and fabricated a parathyroid-on-a-chip, capable of simulating PTH secretion based on calcium concentration. This chip exhibited differences in PTH secretion according to calcium concentration and secreted PTH within the range of normal serum levels. In addition, branches of organoids, which are difficult to observe in animal models, were observed in this chip. This could serve as a guideline for successful engraftment in implantation therapies in the future.

Size-classified monitoring of ATP bioluminescence for rapid assessment of biological distribution in airborne particulates.

The COVID-19 pandemic ignited massive research into the rapid detection of bioaerosols. In particular, nanotechnology-based detection strategies are proposed as alternatives because of issues in bioaerosol enrichment and lead time for molecular diagnostics; however, the practical implementation of such techniques is still unclear due to obstacles regarding the large research and development effort and investment for the validation. The use of adenosine triphosphate (ATP) bioluminescence (expressed as relative luminescence unit (RLU) per unit volume of air) of airborne particulate matter (PM) to determine the bacterial population as a representative of the total bioaerosols (viruses, bacteria, and fungi) has been raised frequently because of the high reponse speed, resolution, and compatibility with culture-based bioaerosol monitoring. On the other hand, additional engineering attempts are required to confer significance because of the size-classified (bioluminescence for different PM sizes) and specific (bioluminescence per unit PM mass) biological risks of air for providing proper interventions in the case of airborne transmission. In this study, disc-type impactors to cut-off aerosols larger than 1 µm, 2.5 µm, and 10 µm were designed and constructed to collect PM1, PM2.5, and PM10 on sampling swabs. This engineering enabled reliable size-classified bioluminescence signals using a commercial ATP luminometer after just 5 min of air intake. The simultaneous operations of a six-stage Andersen impactor and optical PM spectrometers were conducted to determine the correlations between the resulting RLU and colony forming unit (CFU; from the Andersen impactor) or PM mass concentration (deriving specific bioluminescence).

Modified Aerotaxy for the Plug-in Manufacture of Cell-Penetrating Fenton Nanoagents for Reinforcing Chemodynamic Cancer Therapy.

The assemblies of anisotropic nanomaterials have attracted considerable interest in advanced tumor therapeutics because of the extended surfaces for loading of active molecules and the extraordinary responses to external stimuli for combinatorial therapies. These nanomaterials were usually constructed through templated or seed-mediated hydrothermal reactions, but the lack of uniformity in size and morphology, as well as the process complexities from multiple separation and purification steps, impede their practical use in cancer nanotherapy. Gas-phase epitaxy, also called aerotaxy (AT), has been introduced as an innovative method for the continuous assembly of anisotropic nanomaterials with a uniform distribution. This process does not require expensive crystal substrates and high vacuum conditions. Nevertheless, AT has been used limitedly to build high-aspect-ratio semiconductor nanomaterials. With these considerations, a modified AT was designed for the continuous in-flight assembly of the cell-penetrating Fenton nanoagents (Mn-Fe CaCO3 (AT) and Mn-Fe SiO2 (AT)) in a single-pass gas flow because cellular internalization activity is essential for cancer nanotherapeutics. The modified AT of Mn-Fe CaCO3 and Mn-Fe SiO2 to generate surface nanoroughness significantly enhanced the cellular internalization capability because of the preferential contact mode with the cancer cell membrane for Fenton reaction-induced apoptosis. In addition, it was even workable for doxorubicin (DOX)-resistant cancer cells after DOX loading on the nanoagents. After combining with immune-checkpoint blockers (antiprogrammed death-ligand 1 antibodies), the antitumor effect was improved further with no systemic toxicity as chemo-immuno-chemodynamic combination therapeutics despite the absence of targeting ligands and external stimuli.

Massively parallel direct writing of nanoapertures using multi-optical probes and super-resolution near-fields.

Laser direct-writing enables micro and nanoscale patterning, and is thus widely used for cutting-edge research and industrial applications. Various nanolithography methods, such as near-field, plasmonic, and scanning-probe lithography, are gaining increasing attention because they enable fabrication of high-resolution nanopatterns that are much smaller than the wavelength of light. However, conventional methods are limited by low throughput and scalability, and tend to use electron beams or focused-ion beams to create nanostructures. In this study, we developed a procedure for massively parallel direct writing of nanoapertures using a multi-optical probe system and super-resolution near-fields. A glass micro-Fresnel zone plate array, which is an ultra-precision far-field optical system, was designed and fabricated as the multi-optical probe system. As a chalcogenide phase-change material (PCM), multiple layers of Sb65Se35 were used to generate the super-resolution near-field effect. A nanoaperture was fabricated through direct laser writing on a large-area (200 × 200 mm2) multi-layered PCM. A photoresist nanopattern was fabricated on an 8-inch wafer via near-field nanolithography using the developed nanoaperture and an i-line commercial exposure system. Unlike other methods, this technique allows high-throughput large-area nanolithography and overcomes the gap-control issue between the probe array and the patterning surface.

Non-cytotoxic Dy3+ activated La10W22O81 nanophosphors for UV based cool white LEDs and anticancer applications.

White-light-emitting La10W22O81 (LWO): xDy3+ (0.5 ≤ x ≤ 10 mol%) nanocrystalline phosphors were developed by a facile hydrothermal assisted solid-state reaction. X-ray diffraction (XRD) pattern indicated that the prepared samples adopted orthorhombic crystal structures. The agglomeration of uniform nanorods was identified from the FE-SEM analysis of the optimized LWO: 1.5 mol% Dy3+ nanocrystalline phosphors. Additionally, transmission electron microscope, scanning transmission electron microscopy, selected area electron diffraction, and X-ray photoelectron spectroscopy were employed to explore the surface morphology, size, interplanar distance, and chemical composition with valence states of the LWO: 1.5 mol% Dy3+ phosphors, respectively. By exciting with 387 nm, the LWO: Dy3+ emission spectra showed two intense peaks at 476 nm (4F9/2→6H15/2) and 571 nm (4F9/2→6H13/2) and a shoulder peak at 659 nm (4F9/2→6H11/2). Optimum emission intensity was achieved for 1.5 mol% Dy3+ in the LWO host lattice. The luminescence quenching beyond 1.5 mol% Dy3+ is attributed to the dipole-dipole interactions when the Dy3+ (donor) and Dy3+ (acceptor) ions are at a critical distance of 58.53 Å. Photometric studies were conducted to evaluate the performance and practical applicability of the phosphors. The CIE chromaticity diagram suggests that the LWO: 1.5 mol% Dy3+ nanophosphor conspicuously exhibits cool white light. Therefore, this material could be a promising and potential white light-emitting nanocrystalline phosphor material for white light emitting diodes (LEDs) under near-UV excitation. In addition, the toxicity of the optimized nanophosphor in normal WI-38 lung fibroblast cells and MCF-7 breast cancer cells was examined. Surprisingly, LWO: 1.5 mol% Dy3+ nanophosphor was found to be non-cytotoxic to normal cells, but extremely toxic to cancer cells. Therefore, the nanophosphor materials can be considered potential candidates for biomedical applications, particularly for cancer treatment.

Biocompatible Yb3+/Er3+ Co-activated La2(WO4)3 Upconversion Nanophosphors for Optical Thermometry, Biofluorescent, and Anticancer Agents.

Non-cytotoxic upconversion nanocrystals are preferred candidates because they offer exceptional advantages for numerous applications, ranging from optical thermometry to bioimaging/biomedical applications. In this report, we demonstrate the luminescence characteristics and practical utility of a multifunctional upconversion nanophosphor based on Yb3+/Er3+:La2(WO4)3 (LWO) flakes. Strong upconversion green emission was observed from 6-mol % Er3+-doped LWO nanophosphor flakes excited by a 980 nm laser. We further enhanced the upconversion emission considerably by co-doping LWO nanophosphors with Yb3+/Er3+ to exploit energy migration from Yb3+ to Er3+ ions. The exceptional improvement in upconversion green and near-infrared emission was achieved by Yb3+ ion co-doping up to 6 mol %; beyond 6 mol %, emission intensities remarkably dropped due to concentration quenching. Photometric parameters were evaluated with and without Yb3+ ion-doped LWO nanophosphors, which exhibited a high green color purity of 95.6%, to elucidate their energy transfer mechanism. In addition, temperature-dependent upconversion emission trends were evaluated by analyzing the fluorescence intensity ratio, exhibiting higher temperature sensitivity than that previously reported. This suggests the applicability of our proposed nanophosphors to optical thermometry. As for bioimaging applications, the non-cytotoxicity of the optimized nanophosphor was confirmed based on distinct fluorescence images of a normal fibroblast cell line (L929). Furthermore, we demonstrated the strong cytotoxicity of nanophosphors against human colon cancer (HCT-116) cells. Based on the results, non-cytotoxic Yb3+(6 mol %)/Er3+ (6 mol %):LWO upconversion nanophosphor flakes are expected to be exceptional candidates owing to their extensive suitability to the fields of upconversion lasers, optical thermometry, and biomedical and anticancer applications. The results indicate the potential of upconversion materials in the effective execution of multiple strategic applications.

Direct replication of a glass micro Fresnel zone plate array by laser irradiation using an infrared transmissive mold.

It is not yet possible to fabricate micrometer-scale, glass optical components with nanometer-scale precision. Glass thermal imprinting enhances production efficiency. However, dimensional changes caused by shrinkage are inevitable because of phase transitions. Replication is very difficult when high-level pitch precision is essential. We used an infrared-transparent silicon mold and a CO2 laser to perform replica-type, thermal surface texturing at the nanoscale level; we analyzed a glass Fresnel zone plate array to this end. The Fresnel zone plate array was 10 × 10 mm2 in area and featured a 20 × 20 array. The individual Fresnel zone plate diameter was 500 µm and had 21 rings of minimum linewidth 2.9 µm and height 737 nm.

Bacterial Expression Systems for Enzymatic Activity in Droplet-Based Microfluidics.

Functional screenings in droplet-based microfluidics require the analysis of various types of activities of individual cells. When screening for enzymatic activities, the link between the enzyme of interest and the information-baring molecule, the DNA, must be maintained to relate phenotypes to genotypes. This linkage is crucial in directed evolution experiments or for the screening of natural diversity. Micro-organisms are classically used to express enzymes from nucleic acid sequences. However, little information is available regarding the most suitable expression system for the sensitive detection of enzymatic activity at the single-cell level in droplet-based microfluidics. Here, we compare three different expression systems for l-asparaginase (l-asparagine amidohydrolase, EC 3.5.1.1), an enzyme of therapeutic interest that catalyzes the conversion of l-asparagine to l-aspartic acid and ammonia. We developed three expression vectors to produce and localize l-asparaginase (l-ASNase) in E. coli either in the cytoplasm, on the surface of the inner membrane (display), or in the periplasm. We show that the periplasmic expression is the most optimal strategy combining both a good yield and a good accessibility for the substrate without the need for lysing the cells. We suggest that periplasmic expression may provide a very efficient platform for screening applications at the single-cell level in microfluidics.

Mass fabrication of uniform sized 3D tumor spheroid using high-throughput microfluidic system.

In vivo tumors develop in a three-dimensional manner and have unique and complex characteristics. Physico-biochemical barriers on tumors cause drug resistance and limit drug delivery efficiency. Currently, 2D cancer cell monolayer platforms are frequently used to test the efficiency of new drug materials. However, the monolayer platform generally overestimates drug efficiency because of the absence of physico-biochemical barriers. Many literatures indicated that a 3D tumor spheroid model has very similar characteristics to in vivo tumor models, and studies demonstrated the accurate prediction of drug efficiency using this model. The use of a 3D tumor spheroid model in drug development process remains challenging because of the low generation yield and difficulties in size control. In this study, we developed a droplet-based microfluidic system that can generate cancer cells encapsulated by micro-droplets with very high generation yield (16-20 Hz, 1000 droplets/min). The system can control the number of encapsulated cancer cells in the droplet or diameter of the 3D spheroid model precisely between 50 and 150 µm. Moreover, the formed 3D tumor spheroid model can be cultured for >2 weeks by an additional step of droplet disruption and recollection, and can grow up to 245 µm in diameter.

Fabrication of Glass Microchannel via Glass Imprinting using a Vitreous Carbon Stamp for Flow Focusing Droplet Generator.

This study reports a cost-effective method of replicating glass microfluidic chips using a vitreous carbon (VC) stamp. A glass replica with the required microfluidic microstructures was synthesized without etching. The replication method uses a VC stamp fabricated by combining thermal replication using a furan-based, thermally-curable polymer with carbonization. To test the feasibility of this method, a flow focusing droplet generator with flow-focusing and channel widths of 50 µm and 100 µm, respectively, was successfully fabricated in a soda-lime glass substrate. Deviation between the geometries of the initial shape and the vitreous carbon mold occurred because of shrinkage during the carbonization process, however this effect could be predicted and compensated for. Finally, the monodispersity of the droplets generated by the fabricated microfluidic device was evaluated.

Direct fluorescent labeling for efficient biological assessment of inhalable particles.

Labeling of aerosol particles with a radioactive, magnetic, or optical tracer has been employed to confirm particle localization in cell compartments, which has provided useful evidence for correlating toxic effects of inhaled particles. However, labeling requires several physicochemical steps to identify functionalities of the inner or outer surfaces of particles, and moreover, these steps can cause changes in size, surface charge, and bioactivity of the particles, resulting in misinterpretations regarding their toxic effects. This study addresses this challenging issue with a goal of introducing an efficient strategy for constantly supplying labeled aerosol particles in a single-pass configuration without any pre- or post-physicochemical batch treatments of aerosol particles. Carbon black (CB, simulating combustion-generated soot) or calcium carbonate (CC, simulating brake-wear fragments) particles were constantly produced via spark ablation or bubble bursting, respectively. These minute particles were incorporated with fluorescein isothiocyanate-poly(ethylene glycol) 2-aminoethyl ether acetic acid solution at the orifice of a collison atomizer to fabricate hybrid droplets. The droplets successively entered a diffusion dryer containing 254-nm UV irradiation; therefore, the droplets were dynamically stiffened by UV to form fluorescent probes on particles during solvent extraction in the dryer. Particle size distributions, morphologies, and surface charges before and after labeling were measured to confirm fluorescence labeling without significant changes in the properties. In vitro assays, including confocal imaging, were conducted to confirm the feasibility of the labeling approach without inducing significant differences in bioactivity compared with untreated CB or CC particles.

Design methodology for a confocal imaging system using an objective microlens array with an increased working distance.

In this study, a design methodology for a multi-optical probe confocal imaging system was developed. To develop an imaging system that has the required resolving power and imaging area, this study focused on a design methodology to create a scalable and easy-to-implement confocal imaging system. This system overcomes the limitations of the optical complexities of conventional multi-optical probe confocal imaging systems and the short working distance using a micro-objective lens module composed of two microlens arrays and a telecentric relay optical system. The micro-objective lens module was fabricated on a glass substrate using backside alignment photolithography and thermal reflow processes. To test the feasibility of the developed methodology, an optical system with a resolution of 1 µm/pixel using multi-optical probes with an array size of 10 × 10 was designed and constructed. The developed system provides a 1 mm × 1 mm field of view and a sample scanning range of 100 µm. The optical resolution was evaluated by conducting sample tests using a knife-edge detecting method. The measured lateral resolution of the system was 0.98 µm.

Controlling molecular transport in minimal emulsions.

Emulsions are metastable dispersions in which molecular transport is a major mechanism driving the system towards its state of minimal energy. Determining the underlying mechanisms of molecular transport between droplets is challenging due to the complexity of a typical emulsion system. Here we introduce the concept of 'minimal emulsions', which are controlled emulsions produced using microfluidic tools, simplifying an emulsion down to its minimal set of relevant parameters. We use these minimal emulsions to unravel the fundamentals of transport of small organic molecules in water-in-fluorinated-oil emulsions, a system of great interest for biotechnological applications. Our results are of practical relevance to guarantee a sustainable compartmentalization of compounds in droplet microreactors and to design new strategies for the dynamic control of droplet compositions.

An Interference-Assisted Thermal Bonding Method for the Fabrication of Thermoplastic Microfluidic Devices.

Solutions for the bonding and sealing of micro-channels in the manufacturing process of microfluidic devices are limited; therefore, further technical developments are required to determine these solutions. In this study, a new bonding method for thermoplastic microfluidic devices was developed by combining an interference fit with a thermal treatment at low pressure. This involved a process of first injection molding thermoplastic substrates with a microchannel structure, and then performing bonding experiments at different bonding conditions. The results indicated the successful bonding of microchannels over a wide range of bonding pressures with the help of the interference fit. The study also determined additional advantages of the proposed bonding method by comparing the method with the conventional thermal bonding method.

Parallelized ultra-high throughput microfluidic emulsifier for multiplex kinetic assays.

Droplet-based microfluidic technologies are powerful tools for applications requiring high-throughput, for example, in biochemistry or material sciences. Several systems have been proposed for the high-throughput production of monodisperse emulsions by parallelizing multiple droplet makers. However, these systems have two main limitations: (1) they allow the use of only a single disperse phase; (2) they are based on multiple layer microfabrication techniques. We present here a pipette-and-play solution offering the possibility of manipulating simultaneously 10 different disperse phases on a single layer device. This system allows high-throughput emulsion production using aqueous flow rates of up to 26 ml/h (>110 000 drops/s) leading to emulsions with user-defined complex chemical composition. We demonstrate the multiplex capabilities of our system by measuring the kinetics of ß-galactosidase in droplets using nine different concentrations of a fluorogenic substrate.

The microfluidic puzzle: chip-oriented rapid prototyping.

We demonstrate a new concept for reconfigurable microfluidic devices from elementary functional units. Our approach suppresses the need for patterning, soft molding and bonding when details on a chip have to be modified. Our system has two parts, a base-platform used as a scaffold and functional modules which are combined by 'plug-and-play'. To demonstrate that our system sustains typical pressures in microfluidic experiments, we produce droplets of different sizes using T-junction modules with three different designs assembled successively on a 3 × 3 modular scaffold.

An Amplex Red-based fluorometric and spectrophotometric assay for L-asparaginase using its natural substrate.

We report on the development of a sensitive real-time assay for monitoring the activity of L-asparaginase that hydrolyzes L-asparagine to L-aspartate and ammonia. In this method, L-aspartate is oxidized by L-aspartate oxidase to iminoaspartate and hydrogen peroxide (H2O2), and in the detection step horseradish peroxidase uses H2O2 to convert the colorless, nonfluorescent reagent Amplex Red to the red-colored and highly fluorescent product resorufin. The assay was validated in both the absorbance and the fluorescence modes. We show that, due to its high sensitivity and substrate selectivity, this assay can be used to measure enzymatic activity in human serum containing L-asparaginase.

Micro-optical lens array for fluorescence detection in droplet-based microfluidics.

We demonstrate the design and integration of droplet-based microfluidic devices with microoptical element arrays for enhanced detection of fluorescent signals. We show that the integration of microlenses and mirror surfaces in these devices results in an 8-fold increase in the fluorescence signal and in improved spatial resolution. Using an array of microlenses, massively parallel detection of droplets containing fluorescent dyes was achieved, leading to detection throughputs of about 2000 droplets per second and per lens, parallelized over 625 measurement points.

Ultra-high throughput detection of single cell ß-galactosidase activity in droplets using micro-optical lens array.

We demonstrate the use of a hybrid microfluidic-micro-optical system for the screening of enzymatic activity at the single cell level. Escherichia coli ß-galactosidase activity is revealed by a fluorogenic assay in 100 pl droplets. Individual droplets containing cells are screened by measuring their fluorescence signal using a high-speed camera. The measurement is parallelized over 100 channels equipped with microlenses and analyzed by image processing. A reinjection rate of 1 ml of emulsion per minute was reached corresponding to more than 105 droplets per second, an analytical throughput larger than those obtained using flow cytometry.

Development of a two-chamber process for self-assembling a fluorooctatrichlorosilane monolayer for the nanoimprinting of full-track nanopatterns with a 35 nm half pitch.

The technology of depositing a uniform and stable anti-adhesion layer on a wafer-scale nanostamp is a critical issue in the industrialized nanoimprinting process. The deposition of an anti-adhesion layer involves O2 plasma treatment to modify the stamp surface and the reaction of the monomers with the surface. Although an automated one-chamber system was developed for uniform and stable anti-adhesion layer coating, unwanted molecules are irregularly deposited on a sample during the O2 plasma treatment due to the contamination of the chamber, leading to the degradation of the anti-adhesion properties. In this paper, a two-chamber self-assembled monolayer (SAM) deposition system was proposed to prevent the degradation of the anti-adhesion properties due to contamination. To examine the effectiveness of the proposed system, the contact angles and chemical compositions of the SAM-coated silicon mold prepared using the one- and two-chamber systems were measured and compared. Finally, 4-in nanoimprinting of 35-nm-half-pitch full-track nanopatterns was conducted using a SAM-coated silicon nanomold prepared using the one- and two-chamber systems, and the replication quality was examined.