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Multiplexed droplet digital recombinase polymerase amplification (MddRPA) for detection of pandemic diseases.

The precise manipulation of the neural stem cell (NSC)-derived neural differentiation is still challenging, and there is a technological barrier to regulate the axonal regeneration in a controlled manner. Here, we developed a microfluidic chip integrated with a microelectrode array as an axonal guidance platform. The microfluidic electrode array chip consisted of two compartments and a bridge microchannel that could isolate and guide the axons. We demonstrated that the NSCs were largely differentiated into neural cells as the electric field was applied to the microfluidic electrode array chip. We also confirmed the synergistic effects of the electrical stimulation (ES) and neurotrophic factor (NF) on axonal outgrowth. This microfluidic electrode array chip can serve as a central nervous system (CNS) model for axonal injury and regeneration. Therefore, it could be a potentially powerful tool for an

For rapid detection of the COVID-19 infection, the digital polymerase chain reaction (dPCR) with higher sensitivity and specificity has been presented as a promising method of point-of-care testing (POCT). Unlike the conventional real-time PCR (qPCR), the dPCR system allows absolute quantification of the target DNA without a calibration curve. Although a number of dPCR systems have previously been reported, most of these previous assays lack multiplexing capabilities. As different variants of COVID-19 have rapidly emerged, there is an urgent need for highly specific multiplexed detection systems. Additionally, the advances in the Internet of Things (IoT) technology have enabled the onsite detection of infectious diseases. Here, we present an IoT-integrated multiplexed dPCR (IM-dPCR) system involving sample compartmentalization, DNA amplification, fluorescence imaging, and quantitative analysis. This IM-dPCR system comprises three modules: a plasmonic heating-based thermal cycler, a multi-color fluorescence imaging set-up, and a firmware control module. Combined with a custom-developed smartphone application built on an IoT platform, the IM-dPCR system enabled automatic processing, data collection, and cloud storage. Using a self-priming microfluidic chip, 9 RNA groups (

Gold nanoparticles (AuNPs) have widely used for various biological applications, such as drug screening, photo-thermal therapy, and biosensing. In particular, the synthesis of AuNPs with narrow size distribution plays an important role in increasing the efficiency of nanoparticle-mediated biosensors. However, the conventional synthesis methods (e.g., citrate reduction method) still suffer from controlling the sizes of the nanoparticles. In this paper, we present the synthesis method of homogeneous AuNPs using a droplet-based microfluidic chip. Prior to experiments, we optimized the size of droplets using a simulation software for stable droplet generation. We demonstrated that the nanoparticles synthesized in our microfluidic chip system showed a narrower size distribution and a higher reproducibility compared to conventional batch synthesis. Furthermore, we observed that the signal of anti-horseradish peroxidase (HRP) was significantly enhanced by the droplet-based microfluidic chip. Therefore, our synthesis method of homogeneous AuNPs could play an important role in improving the efficiency of AuNPs-based sensing signals.

Cell free DNA (cfDNA) is degraded DNA fragments found in the blood plasma of cancer patients. While cfDNA is a good marker for early diagnostics and cancer prognosis, the extraction of cfDNA from whole blood and sample preparation for later sequencing is still challenging. Here, we presented a microfluidic device for the removal of cells from a cfDNA sample in a first step. In a second step, carboxylated magnetic beads were used for cfDNA extraction and purification. Lastly, cfDNA was amplified using a low-power, plasmonic polymerase chain reaction (PCR) system. Using fluorescent-labeled beads, we demonstrated that the separation efficiency for cells was 99% and the mixing efficiency for purification of cfDNA was 94%. Captured cfDNA could be successfully amplified by PCR, as demonstrated by gel electrophoresis. We confirmed that the limit of detection of our microfluidic system was 10 ng/mL.

We developed a microfluidic gradient device to utilize as a drug screening system with human induced pluripotent stem cell (hiPSC)-derived motoneurons. The microfluidic channel was asymmetrically designed to generate the concentration gradients and a micropillar array was used to trap and culture the motoneuron spheroids containing motoneurons for 9 days. We optimized the concentration gradients in the microfluidic device using a computational fluid dynamics (CFD) model. We also observed that the motoneuron spheroid-derived neurite network was generated in response to the concentration gradients of riluzole in the microfluidic device. Therefore, this microfluidic gradient device could be useful for screening of various drugs for neurological disease applications.

A miniaturized polymerase chain reaction (PCR) system is not only important for medical applications in remote areas of developing countries, but also important for testing at ports of entry during global epidemics, such as the current outbreak of the coronavirus. Although there is a large number of PCR sensor systems available for this purpose, there is still a lack of portable digital PCR (dPCR) heating systems. Here, we first demonstrated a portable plasmonic heating-based dPCR system. The device has total dimensions of 9.7 × 5.6 × 4.1 cm and a total power consumption of 4.5 W, allowing for up to 25 dPCR experiments to be conducted on a single charge of a 20 000 mAh external battery. The dPCR system has a maximum heating rate of 10.7 °C s−1 and maximum cooling rate of 8 °C s−1. Target DNA concentrations in the range from 101 ± 1.4 copies per μL to 260 000 ± 20 000 copies per μL could be detected using a poly(dimethylsiloxane) (PDMS) microwell membrane with 22 080 well arrays (20 μm diameter). Furthermore, the heating system was demonstrated using a mass producible poly(methyl methacrylate) PMMA microwell array with 8100 microwell arrays (80 μm diameter). The PMMA microwell array could detect a concentration from 12 ± 0.7 copies per μL to 25 889 ± 737 copies per μL.

Microfluidic-based separation methods have been highlighted for a number of biological applications, such as single cell analysis, disease diagnostics, and therapeutics. Although a number of previous studies have been carried out to minimize the physical damage and chemical deformations of the sample during the separation process, it still remains a challenge. In this paper, we developed a microfluidic device with dual-neodymium magnet-based negative magnetophoresis for the separation of the microparticles and cells. The poly(ethylene oxide) (PEO) was added to the solution to increase the viscoelasticity of the medium which could assist the sorting of the microparticles in the microfluidic device even at low flow rates, while minimizing damage to the cells and microparticles. Following this method, it was possible to separate 10 and 16 μm microparticles with high efficiency of 99 ± 0.1%, and 97 ± 0.8%, respectively. We also demonstrated the separation of glioblastoma cancer cells and neural stem cells (NSCs) in the microfluidic device.

We investigated the algorithm analysis of the bubble generation in a microfluidic device to study the effect of the surface tension and the flow rate on the microbubble size. For the analysis of the surface tension, five different solutions were used: 3.5% brine, mineral oil, 1% polyethylene glycol (PEG) 400, 1% tween 80, and 1% triton X-100. The various flow rates were also employed: 5~15 µL/min for the liquid and 100~200 mL/min for the gas phase. The size of the bubble was measured via the algorithm analysis and the bubble defect was also detected by c chart. We observed that the microbubble size was affected by the flow rates of solution and the gas. Hence, we developed an equation to estimate the size through the flow rate ratio between the solution and gas phase, showing that the microbubble size could be controlled by the liquid properties or the flow rates. Therefore, this algorithm-based microfluidic device could be a powerful tool for generating gas micro-bubbles in a controlled manner.

In past few years, cancer specific targeted drug formulations have shown some hope of improving the therapeutics with minimized associated side effects of chemotherapy. However, the benefits has proven modest due to lack of systems that can be assessed easily from parent drugs while maintaining the same features of cancer associated prodrug activation. We developed a small molecule-based, carboxylesterase responsive theranostic, EDOX, with tumor-specific enzymatic activation for targeted delivery of the anticancer drug, Doxorubicin (Dox), to hepatocellular carcinoma (HCC) cells. Easy synthetic access, physiological stability, tumor-selective activation, and sustained drug release make EDOX an excellent candidate for use as a next-generation drug delivery system for HCC.

The research of fungi is of great importance in a number of fields, such as environmental and healthcare studies. While there are a large number of optical and molecular methods available for characterization and identification of fungi and their spores, their isolation is still conducted using slow and labor-intensive methods. Here, we develop a microfluidic device for the continuous separation of fungal spores from other eukaryotic cells. The spores were separated through the microfluidic device by expanding pinched flow fractionation (PFF) containing the spores, achieving a spatial separation perpendicular to the flow direction according to the spore size. Further branch flow fractionation (BFF) and co-flow of a Newtonian and viscoelastic fluid were used to enhance the separation performance. Using this microfluidic device, we demonstrated the separation of two different types of fungal spores and further separation of fungal spores from eukaryotic cells with a separation efficiency of above 90%. Compared to the existing conventional methods, our microfluidic flow focusing device requires little manual handling and uses small amounts of samples without any pre-treatment steps of the samples.

Shear stress occurs in flowing liquids, especially at the interface of a flowing liquid and a stationary solid phase. Thus, it occurs inside the artery system of the human body, where it is responsible for a number of biological functions. The shear stress level generally remains less than 70 dyne/cm2 in the whole circulatory system, but in the stenotic arteries, which are constricted by 95%, a shear stress greater than 1,000 dyne/cm2 can be reached. Methods of researching the effects of shear stress on cells are of large interest to understand these processes. Here, we show the development of a microfluidic device for generating shear stress gradients. The performance of the shear stress gradient generator was theoretically simulated prior to experiments. Through simple manipulations of the liquid flow, the shape and magnitude of the shear stress gradients can be manipulated. Our microfluidic device consisted of five portions divided by arrays of micropillars. The generated shear stress gradient has five distinct levels at 8.38, 6.55, 4.42, 2.97, and 2.24 dyne/cm2. Thereafter, an application of the microfluidic device was demonstrated testing the effect of shear stress on human umbilical vein endothelial cells.

We developed copper sulfide (CuS)/reduced graphene oxide (rGO)-poly (ethylene glycol) (PEG) nanocomposites for photothermal bonding of a polymethyl methacrylate (PMMA)-based plastic lab-on-a-chip. The noncontact photothermal bonding of PMMA-based plastic labs-on-chip plays an important role in improving the stability and adhesion at a high-temperature as well as minimizing the solution leakage from microchannels when connecting two microfluidic devices. The CuS/rGO-PEG nanocomposites were used to bond a PMMA-based plastic lab-on-a-chip in a short time with a high photothermal effect by a near-infrared (NIR) laser irradiation. After the thermal bonding process, a gap was not generated in the PMMA-based plastic lab-on-a-chip due to the low viscosity and density of the CuS/rGO-PEG nanocomposites. We also evaluated the physical and mechanical properties after the thermal bonding process, showing that there was no solution leakage in PMMA-based plastic lab-on-a-chip during polymerase chain reaction (PCR) thermal cycles. Therefore, the CuS/rGO-PEG nanocomposite could be a potentially useful nanomaterial for non-contact photothermal bonding between the interfaces of plastic module lab-on-a-chip.

In this study, potential protection of chlorophyll a from illumination and oxidation-induced decomposition has been examined using C-phycocyanin (C-PC) aqueous medium. Photo-oxidation resistance of chlorophyll a was monitored in various aqueous media using ultraviolet-visible spectroscopy and direct-infusion atmospheric pressure chemical ionization mass spectrometry analysis. The spectroscopy results showed that chlorophyll a in C-PC medium experienced the lowest rate of conversion to its derivatives, thus, it was demonstrated that chlorophyll a was mostly intact in the C-PC medium. Furthermore, the C-PC treated with chlorophyll a showed the lowest concentrations of malondialdehyde, and chlorophyll a in C-PC medium did not cause serious damage to human liver cells in vitro after intensive illumination. Therefore, we propose a new method of protecting chlorophyll a from photodegradation and oxidation using C-PC aqueous medium.

Electrical stimuli play an important role in regulating the delivery of plasmonic nanomaterials with cancer targeting peptides. Here, we developed an electro-responsive hydrogel-based microfluidic actuator platform for brain tumor targeting and photothermal therapy (PTT) applications. The electro-responsive hydrogels consisted of highly conductive silver nanowires (AgNWs) and biocompatible collagen I gels. We confirmed that an electrically conductive hydrogel could be used as an effective actuator by applying an electrical signal in the microfluidic platform. Furthermore, we successfully demonstrated PTT efficacy for brain tumor cells using targetable Arg-Gly-Asp (RGD) peptide-conjugated gold nanorods (GNRs). Therefore, our electro-responsive hydrogel-based microfluidic actuator platform could be useful for electro-responsive intelligent nanomaterial delivery and PTT applications.

Despite their simplicity, monolayer cell cultures are not able to accurately predict drug behavior in vivo due to their inability to accurately mimic cell-cell and cell-matrix interactions. In contrast, cell spheroids are able to reproduce these interactions and thus would be a viable tool for testing drug behavior. However, the generation of homogenous and reproducible cell spheroids on a large scale is a labor intensive and slow process compared to monolayer cell cultures. Here, we present a droplet-based microfluidic device for the automated, large-scale generation of homogenous cell spheroids in a uniform manner. Using the microfluidic system, the size of the spheroids can be tuned to between 100 and 130 μm with generation frequencies of 70 Hz. We demonstrated the photothermal therapy (PTT) application of brain tumor spheroids generated by the microfluidic device using a reduced graphene oxide-branched polyethyleneimine-polyethylene glycol (rGO-BPEI-PEG) nanocomposite as the PTT agent. Furthermore, we generated uniformly sized neural stem cell (NSC)-derived neurospheres in the droplet-based microfluidic device. We also confirmed that the neurites were regulated by neurotoxins. Therefore, this droplet-based microfluidic device could be a powerful tool for photothermal therapy and drug screening applications. A microfluidic device for the efficient production of three-dimensional ‘spheroid’ cell cultures could accelerate the discovery and testing of therapeutic agents. These small clumps of cells can better recapitulate the physiology of the tissue environment than two-dimensional cultures, but are challenging to produce in a high-throughput fashion. Researchers led by Prof. Bong Geun Chung of Sogang University, Seoul, Korea have now developed a system that can generate 42,000 spheroids/h with minimal user intervention. As an initial demonstration, they produce brain tumor-derived spheroids, and use these to test the cancer-killing efficacy of a therapy based on heated graphene-based nanoparticles. Prof. Chung and colleagues subsequently employ their device to assess the detrimental effects of the neurotoxin thapsigargin on neural stem cell-derived spheroids, highlighting the potential value of this platform for high-throughput cell screening assays.

We developed the microfluidic co-culture platform to study photothermal therapy applications. We conjugated folic acid (FA) to target breast cancer cells using reduced graphene oxide (rGO)-based functional nanomaterials. To characterize the structure of rGO-based nanomaterials, we analyzed the molecular spectrum using UV–visible and Fourier-transform infrared spectroscopy (FT-IR). We demonstrated the effect of rGO-FA-based nanomaterials on photothermal therapy of breast cancer cells in the microfluidic co-culture platform. From the microfluidic co-culture platform with breast cancer cells and human umbilical vein endothelial cells (HUVECs), we observed that the viability of breast cancer cells treated with rGO-FA-based functional nanomaterials was significantly decreased after near-infrared (NIR) laser irradiation. Therefore, this microfluidic co-culture platform could be a potentially powerful tool for studying cancer cell targeting and photothermal therapy.

Even though in vitro co-culture tumor spheroid model plays an important role in screening drug candidates, its wide applications are currently limited due to the lack of reliable and high throughput methods for generating well-defined and 3D complex co-culture structures. Herein, we report the development of a hydrogel microwell array to generate uniform-sized multicellular tumor spheroids. Our developed multicellular tumor spheroids are structurally well-defined, robust and can be easily transferred into the widely used 2D culture substrates while maintaining our designed multicellular 3D-sphere structures. Moreover, to develop effective anti-cancer therapeutics we integrated our recently developed gold-graphene hybrid nanomaterial (Au@GO)-based photothermal cancer therapy into a series of multicellular tumor spheroid co-culture system. The multicellular tumor spheroids were harvested onto a two-dimensional (2D) substrate, under preservation of their three-dimensional (3D) structure, to evaluate the photothermal therapy effectiveness of graphene oxide (GO)-wrapped gold nanoparticles (Au@GO). From the model of co-culture spheroids of HeLa/Ovarian cancer and HeLa/human umbilical vein endothelial cell (HUVEC), we observed that Au@GO nanoparticles displayed selectivity towards the fast-dividing HeLa cells, which could not be observed to this extent in 2D cultures. Overall, our developed uniform-sized 3D multicellular tumor spheroid could be a powerful tool for anticancer drug screening applications.

A new senescence-responsive fluorescent probe (SRP) was developed for the detection of β-galactosidase activity in senescent cells. UV-absorption of t probe SRP at 495 nm was increased in the presence of β-galactosidase. Its fluorescence at λem 545 nm increased ∼27-fold upon incubation with β-galactosidase (0.1 U/mL). The SRP probe is non-toxic and highly chemoselective for β-galactosidase. The high cell viability and chemoselcivity of probe SRP offer it to be a suitable marker for assessing cell senescence in live cells. Using this probe, H2O2-induced cellular senescence of human umbilical vein cells (HUVECs) could be distinguished from normal cells based on the extent of fluorescent labeling of the cells. Moreover, it was preferentially localized in acidic lysosomes. Overall, SRP is a unique chemosensor that can provide preclinical information on cell senescence in vascular endothelial cells.

We report herein a novel surfactant-free synthetic approach for hollow silica nanoparticles (HSN) loaded with Au nanocomposites (Au-HSN) to enhance photothermal properties. Transmission electron microscopy and elemental analysis confirmed the uniform HSN of Au nanoparticles. UV-vis spectroscopy and Fourier transform infrared spectroscopy were performed to demonstrate the dopamine (DA)-hyaluronate (HA) coating on Au-HSN. These nanocomposites dramatically increased up to 48 °C within few seconds under near-infrared irradiation. In addition, these Au-HSN/DA-HA nanoparticles showed the excellent endocytosis against cancer cells without any cytotoxicity. Therefore, it could be a powerful nanocarrier for tumor targeting and photothermal therapy applications.

Nanoparticles have gained large interest in a number of different fields due to their unique properties. In medical applications, for example, magnetic nanoparticles can be used for targeting, imaging, magnetically induced thermotherapy, or for any combination of the three. However, it is still a challenge to obtain narrowly dispersed, reproducible particles through a typical lab-scale synthesis when researching these materials. Here, we present a droplet capillary reactor that can be used for the synthesis of magnetic iron oxide nanoparticles. Compared to conventional batch synthesis, the particles synthesized in our droplet reactor have a narrower size distribution and a higher reproducibility. Furthermore, we demonstrate how the particle size can be changed from 5.2 ± 0.9 nm to 11.8 ± 1.7 nm by changing the reaction temperature and droplet residence time in the droplet capillary reactor.

The inability of neurons to undergo mitosis renders damage to the central or peripheral nervous system. Neural stem cell therapy could provide a path for treating the neurodegenerative diseases. However, reliable and simple tools for the developing and testing neural stem cell therapy are still required. Here, we show the development of a micropillar-based microfluidic device to trap the uniform-sized neurospheres. The neurospheres trapped within micropillar arrays were largely differentiated into neuronal cells, and their neurite networks were observed in the microfluidic device. Compared to conventional cultures on glass slides, the neurite networks generated with this method have a higher reproducibility. Furthermore, we demonstrated the effect of thapsigargin on the neurite networks in the microfluidic device, demonstrating that neural networks exposed to thapsigargin were largely diminished and disconnected from each other. Therefore, this micropillar-based microfluidic device could be a potential tool for screening of neurotoxins.

Understanding the effects of shear stress on mammalian cells is a crucial factor for understanding a number of biological processes and diseases. Here, we show the development of a circular-shaped microfluidic device for the facile generation of shear stress gradients. With this microfluidic device, the effect of shear stress on orientation of human umbilical vein endothelial cells was studied. This microfluidic device, which enables to control the alignment of human umbilical vein endothelial cells within a microchannel, can be a valuable tool to mimic blood vessels.

One of the main challenges regarding treatment of neurodegenerative diseases in the central nervous system (CNS) lies in the inability of neurons to undergo mitosis. Stem cell therapy could provide a possible solution to treat the neurodegenerative diseases. Here, we developed a conductive polyethylene glycol (PEG) hydrogel/silver nanowire (AgNW) composite micropattern-based sensor to direct differentiation of neuronal stem cells (NSCs) and guidance of neurite outgrowth. The ridge micropatterns made from the composite PEG hydrogel/AgNW materials were fabricated on a flexible polyethylene terephthalate (PET) film. Through the combination of these composite materials and the ridge micropatterns, the outgrowth of neurites could be efficiently guided in a controlled manner. Therefore, this conductive PEG hydrogel/AgNW micropattern-based sensor could potentially allow future use of the flexible device as an implant for NSC therapy applications.

Cancer is one of the main causes of morbidity and mortality. Although a number of techniques are available for treatment, these methods still have a number of drawbacks, destroying healthy tissues and cells to cause various side effects. Here we present the synthesis and biological application of a composite nanomaterial, folic acid (FA)-conjugated graphene oxide (GO) nanosheets functionalized with manganese dioxide (MnO2) nanoparticles. While FA-conjugated GO nanosheets can be used for targeted photothermal therapy (PTT) when irradiated with a near infrared (NIR) light, MnO2 nanoparticles degrade hydrogen peroxide (H2O2) in the cancer microenvironment, countering hypoxia. Further the nanoparticles can be used as a contrast agent in MRI imaging. We demonstrated that MnO2-FA-GO nanosheets were uptaken by HeLa cells overexpressing FA receptors to induce NIR irradiation-mediated hyperthermia (35% viability). Therefore, this composite MnO2-FA-GO nanosheet could be a powerful carrier for cancer targeting and PTT applications.

The high incidence of colorectal cancer worldwide is currently a major health concern. Although conventional chemotherapy and surgery are effective to some extent, there is always a risk of relapse due to associated side effects, including post-surgical complications and non-discrimination between cancer and normal cells. In this study, we developed a small molecule-based theranostic system, Gal-Dox, which is preferentially taken up by colon cancer cells through receptor-mediated endocytosis. After cancer-specific activation, the active drug Dox (doxorubicin) is released with a fluorescence turn-on response, allowing both drug localization and site of action to be monitored. The therapeutic potency of Gal-Dox was also evaluated, both in vivo and ex vivo, thus illustrating the potential of Gal-Dox as a colorectal cancer theranostic with great specificity.

Microwell arrays are widely used for the analysis of fluorescent-labelled biomaterials. For rapid detection and automated analysis of microwell arrays, the computational image analysis is required. Support Vector Machines (SVM) can be used for this task. Here, we present a SVM-based approach for the analysis of microwell arrays consisting of three distinct steps: labeling, training for feature selection, and classification into three classes. The three classes are filled, partially filled, and unfilled microwells. Next, the partially filled wells are analyzed by SVM and their tendency towards filled or unfilled tested through applying a Gaussian filter. Through this, all microwells can be categorized as either filled or unfilled by our algorithm. Therefore, this SVM-based computational image analysis allows for an accurate and simple classification of microwell arrays.