Enhancing Loop-Mediated Isothermal Amplification through Nonpowered Nanoelectric Preconcentration.

The global threat posed by the COVID-19 pandemic has catalyzed the development of point-of-care (POC) molecular diagnostics. While loop-mediated isothermal amplification (LAMP) stands out as a promising technique among FDA-approved methods, it is occasionally susceptible to a high risk of false positives due to nonspecific amplification of a primer dimer. In this work, we report an enhancing LAMP technique in terms of assay sensitivity and reliability through streamlined integration with a nonpowered nanoelectric preconcentration (NPP). The NPP, serving as a sample preparation tool, enriched the virus concentration in samples prior to the subsequent LAMP assay. This enrichment enabled not only to achieve more sensitive assay but also to shorten the assay time for all tested clinical samples by ∼10 min compared to the conventional LAMP. The shortened assay time suppresses the occurrence of nonspecific amplification by not providing the necessary incubation time, effectively suppressing misidentification by false positives. Utilizing this technique, we also developed a prototype of the POC NPP-LAMP kit. This kit offers a streamlined diagnostic process for nontrained individuals, from the sample enrichment, transfer of the enriched sample to LAMP assays, which facilitates on-site/on-demand diagnosis of SARS-CoV-2. This development holds the potential to contribute toward preventing not only the current outbreak but also future occurrences of pandemic viruses.

Accurate real-time monitoring of fine dust using a densely connected convolutional networks with measured plasma emissions.

Accurate identification and monitoring of fine dust are emerging as a primary global issue for addressing the harmful effects of fine dust on public health. Identifying the source of fine dust is indispensable for ensuring the human lifespan as well as preventing environmental disasters. Here a simple yet effective spark-induced plasma spectroscopy (SIPS) unit combined with deep learning for real-time classification is verified as a fast and precise PM (particulate matter) source identification technique. SIPS promises portable use, label-free detection, source identification, and chemical susceptibility in a single step with acceptable speed and accuracy. In particular, the densely connected convolutional networks (DenseNet) are used with measured spark-induced plasma emission datasets to identify PM sources at above 98%. The identification performance was compared with other common classification methods, and DenseNet with dropouts (30%), optimized batch size (16), and cyclic learning rate training emerged as the most promising source identification method.

Three-Dimensional In Vitro Lymphangiogenesis Model in Tumor Microenvironment.

Lymphangiogenesis is a stage of new lymphatic vessel formation in development and pathology, such as inflammation and tumor metastasis. Physiologically relevant models of lymphatic vessels have been in demand because studies on lymphatic vessels are required for understanding the mechanism of tumor metastasis. In this study, a new three-dimensional lymphangiogenesis model in a tumor microenvironment is proposed, using a newly designed macrofluidic platform. It is verified that controllable biochemical and biomechanical cues, which contribute to lymphangiogenesis, can be applied in this platform. In particular, this model demonstrates that a reconstituted lymphatic vessel has an in vivo-like lymphatic vessel in both physical and biochemical aspects. Since biomechanical stress with a biochemical factor influences robust directional lymphatic sprouting, whether our model closely approximates in vivo, the initial lymphatics in terms of the morphological and genetic signatures is investigated. Furthermore, attempting an incorporation with a tumor spheroid, this study successfully develops a complex tumor microenvironment model for use in lymphangiogenesis and reveals the microenvironment factors that contribute to tumor metastasis. As a first attempt at a coculture model, this reconstituted model is a novel system with a fully three-dimensional structure and can be a powerful tool for pathological drug screening or disease model.

Gamma irradiation exposure for collapsed cell junctions and reduced angiogenesis of 3-D in vitro blood vessels.

During radiotherapy, microenvironments neighboring the tumor are also exposed to gamma irradiation; this results in unexpected side effects. Blood vessels can serve as microenvironments for tumors and they play an important role in providing nutrients to tumors. This is mostly related to tumor progression, metastasis, and relapse after therapy. Many studies have been performed to obtain a better understanding of tumor vasculature after radiotherapy with in vitro models. However, compared to 3-D models, 2-D in vitro endothelial monolayers cannot physiologically reflect in vivo blood vessels. We previously remodeled the extracellular matrix (ECM) hydrogel that enhanced the tight barrier formation of 3-D blood vessels and the vascular endothelial growth factor (VEGF) gradient induced angiogenesis in a microfluidic device. In this study, the blood vessel model is further introduced to understand how gamma irradiation affects the endothelial monolayer. After the gamma irradiation exposure, we observed a collapsed endothelial barrier and a reduced angiogenic potential. Changes in the cell behaviors of the tip and stalk cells were also detected in the angiogenesis model after irradiation, which is difficult to observe in 2-D monolayer models. Therefore, the 3-D in vitro blood vessel model can be used to understand radiation-induced endothelial injuries.

Simulation and Experimental Study of Ion Concentration Polarization Induced Electroconvective Vortex and Particle Movement.

Ion concentration polarization (ICP) has been widely applied in microfluidic systems in pre-concentration, particle separation, and desalination applications. General ICP microfluidic systems have three components (i.e., source, ion-exchange, and buffer), which allow selective ion transport. Recently developed trials to eliminate one of the three components to simplify the system have suffered from decreased performance by the accumulation of unwanted ions. In this paper, we presented a new ICP microfluidic system with only an ion-exchange membrane-coated channel. Numerical investigation on hydrodynamic flow and electric fields with a series of coupled governing equations enabled a strong correlation to experimental investigations on electroconvective vortices and the trajectory of charged particles. This study has significant implications for the development and optimization of ICP microfluidic and electrochemical systems for biomarker concentration and separation to improve sensing reliability and detection limits in analytic chemistry.

Onsite real-time detection of covid-like-virus transmission through air using spark-induced plasma spectroscopy.

In March 2020, COVID-19 was officially classified as a pandemic and as a consequence people have adopted strenuous measures to prevent infection, such as the wearing of PPE and self-quarantining, with no knowledge of when the measures will no longer be necessary. Coronavirus has long been known to be non-infectious when airborne; however, studies are starting to show that the virus can infect through airborne transmission and can remain airborne for a significant period of time. In the present study, a spark-induced plasma spectroscopy was devised to characterize the air propagation of the virus in real-time. The risk of air propagation was evaluated in terms of changes in virus concentration with respect to distance traveled and measurement time. Thus, our study provides a benchmark for performing real-time detection of virus propagation and instantaneous monitoring of coronavirus in the air.

Evaluation of Cell-Penetrating Peptides Using Microfluidic In Vitro 3D Brain Endothelial Barrier.

In drug delivery to the human brain, blood vessels are a significant hurdle because they restrict the entry of most solutes to protect brain. To overcome this hurdle, an in vitro 3D model for brain endothelial barrier is developed using a microfluidic device with hydrogel providing a 3D extracellular matrix scaffold. Using the model, peptides known to utilize receptor-mediated transcytosis are verified, which has been one of the most promising mechanisms for brain-specific penetration. The cytotoxicity and cellular damage to the peptide are investigated and the receptor-mediated transcytosis and brain endothelial specific penetrating abilities of the peptides in a quantitative manner are demonstrated. As a preclinical test, applying the quantification assays conducted in this study are suggested, including the penetrating ability, cytotoxicity, endothelial damage, and receptor specificity. Using this microfluidic device as an in vitro platform for evaluating various brain targeting drugs and drug carrier candidates is also proposed.

Clonorchis sinensis excretory-secretory products increase malignant characteristics of cholangiocarcinoma cells in three-dimensional co-culture with biliary ductal plates.

Clonorchis sinensis is a carcinogenic human liver fluke, prolonged infection which provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma (CCA). These effects are driven by direct physical damage caused by the worms, as well as chemical irritation from their excretory-secretory products (ESPs) in the bile duct and surrounding liver tissues. We investigated the C. sinensis ESP-mediated malignant features of CCA cells (HuCCT1) in a three-dimensional microfluidic culture model that mimics an in vitro tumor microenvironment. This system consisted of a type I collagen extracellular matrix, applied ESPs, GFP-labeled HuCCT1 cells and quiescent biliary ductal plates formed by normal cholangiocytes (H69 cells). HuCCT1 cells were attracted by a gradient of ESPs in a concentration-dependent manner and migrated in the direction of the ESPs. Meanwhile, single cell invasion by HuCCT1 cells increased independently of the direction of the ESP gradient. ESP treatment resulted in elevated secretion of interleukin-6 (IL-6) and transforming growth factor-beta1 (TGF-ß1) by H69 cells and a cadherin switch (decrease in E-cadherin/increase in N-cadherin expression) in HuCCT1 cells, indicating an increase in epithelial-mesenchymal transition-like changes by HuCCT1 cells. Our findings suggest that C. sinensis ESPs promote the progression of CCA in a tumor microenvironment via the interaction between normal cholangiocytes and CCA cells. These observations broaden our understanding of the progression of CCA caused by liver fluke infection and suggest a new approach for the development of chemotherapeutic for this infectious cancer.

Ion concentration polarization for pre-concentration of biological samples without pH change.

In this paper, a method was developed for pre-concentrating large-volume biological samples for subsequent analysis. We previously developed another pre-concentration device, but it unfortunately altered the pH of the sample when an electric field was applied to the sample reservoir. Changes in the pH are not suitable for subsequent antibody-antigen reactions because of the stability issues that arise based on the target molecule's isoelectric point (pI). Here, this problem was overcome using ion concentration polarization (ICP) with a cation selective membrane (Nafion). Phosphate buffered saline was used as a test solution for the sample. The sample was contained in a reservoir that was not affected by the electric field, and an ICP barrier was formed in front of the reservoir. This device could concentrate microliter-scale samples without changing the pH because the biomolecules were blocked from passing through the ICP barrier while the sample (phosphate buffered saline) was drained. A 40 µL sample was successfully pre-concentrated to 20 µL in a single channel device and 10 µL in a dual channel device, resulting in 2.1-fold and 3.3-fold increases, respectively, in influenza hemagglutinin concentrations. These changes in the concentration were confirmed by ELISA.

Fast Sound Source Localization Using Two-Level Search Space Clustering.

Steered response power phase transform (SRP-PHAT) is a method that is widely used for robust sound source localization (SSL). However, since SRP-PHAT searches over a large number of candidate locations, it is too slow to run in real-time for large-scale microphone array systems. In this paper, we propose a robust two-level search space clustering method to speed-up SRP-PHAT-based SSL. The proposed method divides the candidate locations of the sound source into a set of groups and finds a small number of groups that are likely to contain the maximum power location. By searching within the small number of groups, the computational costs are reduced by 61.8% compared to a previously proposed method without loss of accuracy.

Microfluidic in-reservoir pre-concentration using a buffer drain technique.

Pre-concentration methods are essential for detecting low concentrations of influenza virus in biological samples from patients. Here, we describe a new method for draining buffer from solution in the reservoir of a microfluidic device to increase the concentration of virus in the reservoir. Viruses were captured in the reservoir by an ion depletion barrier from connected ion selective microfluidic channels. 75 µl of buffer was successfully drained from a 100 µl sample, resulting in a 4-fold increase in influenza hemagglutinin concentration in the reservoir. The volume of the final concentrated sample was suitable for detection of influenza hemagglutinin by the enzyme-linked immunosorbent assay, demonstrating the usefulness of the developed platform for enhanced sensitivity of virus detection in a conventional analysis.