A microfluidic-based quantitative analysis system for the multiplexed genetic diagnosis of human viral infections using colorimetric loop-mediated isothermal amplification.

In this study, a microfluidic-based system utilizing colorimetric loop-mediated isothermal amplification (LAMP) is introduced for the quantitative analysis of nucleic acid targets. This system offers a user-friendly and cost-effective platform for the multiplexed genetic diagnosis of various infectious diseases across multiple samples. It includes time-lapse imaging equipment for capturing images of the microfluidic device during the LAMP assay and a hue-based quantitative analysis software to analyze the LAMP reaction, streamlining diagnostic procedures. An electric pipette was used to simplify the loading of samples and LAMP reagents into the device, allowing easy operation even by untrained individuals. The hue-based analysis software employs efficient image processing and post-processing techniques to calculate DNA amplification curves based on color changes in multiple reaction chambers. This software automates several tasks, such as identifying reaction chamber areas from time-lapse images, quantifying color information within each chamber, correcting baselines of DNA amplification curves, fitting experimental data to theoretical curves, and determining the threshold time for each curve. To validate the developed system, conventional off-chip LAMP assays were conducted with a 25 µL reaction mixture in 0.2 mL polymerase chain reaction (PCR) tubes using a real-time turbidimeter. The results indicated that the threshold time obtained using the colorimetric LAMP assay in the developed system is comparable to that obtained with real-time turbidity measurements in PCR tubes, demonstrating the system's capability for quantitative analysis of target nucleic acids, including those from human herpesviruses.

Automatic microdispenser-integrated multiplex enzyme-linked immunosorbent assay device with autonomously driven centrifugal microfluidic system.

In this study, we established the control and design theory of an autonomously driven dispenser at a steady rotation speed and proposed a dispenser-integrated multiplex enzyme-linked immunosorbent assay (ELISA) device. In establishing the theory of the dispenser, we estimated the flow rate in the dispenser and the applied pressure onto the passive valves, so that the suitable burst pressure of the valves and flow rate could be designed. The dispenser-integrated multiplex ELISA device has the potential to perform flow control for executing an ELISA of 6 samples/standards per chip or 18 samples/standards per compact disk by just steadily rotating a chip. In the immunoassay evaluation of the device using mouse IgG detection, it was confirmed that the device could assay 5 µL of several standards in just 30 min without nonspecific reactions, and although this system has a high limit of detection (LOD, 63.4-164 pg mL-1) it is equal to that of manual assay with a titer plate. The device can be fabricated by transferring the microchannel pattern from a mold without complex assembly or alignment, and it can control the liquid operation by just steadily rotating. Thus, the device system developed will contribute to reducing the cost of fabricating chips and control equipment for ELISA systems. Consequently, a compact, portable, and low-cost ELISA system for point-of-care testing is expected to be realized.

Visible Pulsed Laser-Assisted Selective Killing of Cancer Cells with PVP-Capped Plasmonic Gold Nanostars.

A new generation of nanoscale photosensitizer agents has improved photothermal capabilities, which has increased the impact of photothermal treatments (PTTs) in cancer therapy. Gold nanostars (GNS) are promising for more efficient and less invasive PTTs than gold nanoparticles. However, the combination of GNS and visible pulsed lasers remains unexplored. This article reports the use of a 532 nm nanosecond pulse laser and polyvinylpyrrolidone (PVP)-capped GNS to kill cancer cells with location-specific exposure. Biocompatible GNS were synthesized via a simple method and were characterized under FESEM, UV-visible spectroscopy, XRD analysis, and particle size analysis. GNS were incubated over a layer of cancer cells that were grown in a glass Petri dish. A nanosecond pulsed laser was irradiated on the cell layer, and cell death was verified via propidium iodide (PI) staining. We assessed the effectiveness of single-pulse spot irradiation and multiple-pulse laser scanning irradiation in inducing cell death. Since the site of cell killing can be accurately chosen with a nanosecond pulse laser, this technique will help minimize damage to the cells around the target cells.

Automated detection of patterned single-cells within hydrogel using deep learning.

Single-cell analysis has been widely used in various biomedical engineering applications, ranging from cancer diagnostics, and immune response monitoring to drug screening. Single-cell isolation is fundamental for observing single-cell activities and an automatic finding method of accurate and reliable cell detection with few possible human errors is also essential. This paper reports trapping single cells into photo patternable hydrogel microwell arrays and isolating them. Additionally, we present an object detection-based DL algorithm that detects single cells in microwell arrays and predicts the presence of cells in resource-limited environments at the highest possible mAP (mean average precision) of 0.989 with an average inference time of 0.06 s. This algorithm leads to the enhancement of the high-throughput single-cell analysis, establishing high detection precision and reduced experimentation time.

Mixing Performance of a Planar Asymmetric Contraction-and-Expansion Micromixer.

Micromixers are one of the critical components in microfluidic devices. They significantly affect the efficiency and sensitivity of microfluidics-based lab-on-a-chip systems. This study introduces an efficient micromixer with a simple geometrical feature that enables easy incorporation in a microchannel network without compromising the original design of microfluidic devices. The study proposes a newly designed planar passive micromixer, termed a planar asymmetric contraction-and-expansion (P-ACE) micromixer, with asymmetric vertical obstacle structures. Numerical simulation and experimental investigation revealed that the optimally designed P-ACE micromixer exhibited a high mixing efficiency of 80% or more within a microchannel length of 10 mm over a wide range of Reynolds numbers (0.13 ≤ Re ≤ 13), eventually attaining approximately 90% mixing efficiency within a 20 mm microchannel length. The highly asymmetric geometric features of the P-ACE micromixers enhance mixing because of their synergistic effects. The flow velocities and directions of the two fluids change differently while alternately crossing the longitudinal centerline of the microchannel, with the obstacle structures asymmetrically arranged on both sidewalls of the rectangular microchannel. This flow behavior increases the interfacial contact area between the two fluids, thus promoting effective mixing in the P-ACE micromixer. Further, the pressure drops in the P-ACE micromixers were experimentally investigated and compared with those in a serpentine micromixer with a perfectly symmetric mixing unit.

A microfluidic diagnostic device with air plug-in valves for the simultaneous genetic detection of various food allergens.

The identification of accidental allergen contamination in processed foods is crucial for risk management strategies in the food processing industry to effectively prevent food allergy incidents. Here, we propose a newly designed passive stop valve with high pressure resistance performance termed an "air plug-in valve" to further improve microfluidic devices for the detection of target nucleic acids. By implementing the air plug-in valve as a permanent stop valve, a maximal allowable flow rate of 70 µL/min could be achieved for sequential liquid dispensing into an array of 10 microchambers, which is 14 times higher than that achieved with the previous valve arrangement using single-faced stop valves. Additionally, we demonstrate the simultaneous detection of multiple food allergens (wheat, buckwheat, and peanut) based on the colorimetric loop-mediated isothermal amplification assay using our diagnostic device with 10 microchambers compactly arranged in a 20-mm-diameter circle. After running the assays at 60 °C for 60 min, any combination of the three types of food allergens and tea plant, which were used as positive and negative control samples, respectively, yielded correct test results, without any cross-contamination among the microchambers. Thus, our diagnostic device will provide a rapid and easy sample-to-answer platform for ensuring food safety and security.

A method of sequential liquid dispensing for the multiplexed genetic diagnosis of viral infections in a microfluidic device.

In this study, we introduce polydimethylsiloxane (PDMS)-based microfluidic devices capable of sequential dispensing of samples into multiple reaction microchambers in a single operation to provide a fast and easy sample-to-answer platform for multiplexed genetic diagnosis of multiple viral infectious diseases. This approach utilizes the loop-mediated isothermal amplification (LAMP) method to amplify and detect specific nucleic acid (DNA/RNA) targets. We present a microfluidic flow control theory for sequential liquid dispensing phenomena, which provides design guidelines for device optimization. The device specifications, such as the possible dispensing number and maximal allowable flow rate, can be theoretically designed by optimizing the geometric dimensions of the microchannels and a pair of passive stop valves integrated into each microchamber together with the water contact angles of the materials used to fabricate the microfluidic devices. In addition, a passive stop valve with a vertical-type phaseguide structure was designed to improve device performance. We could simultaneously diagnose coronavirus disease 2019 (COVID-19) and other infectious diseases, such as severe acute respiratory syndrome (SARS), seasonal influenza A, and pandemic influenza A (H1N1) 2009. The colorimetric reverse transcription LAMP (RT-LAMP) assay suggests that the four viral infectious diseases can be detected within 30 min using a hue-based quantitative analysis, and the naked eye using our microfluidic devices.

A lab in a bento box: an autonomous centrifugal microfluidic system for an enzyme-linked immunosorbent assay.

In this paper, we report on the demonstration of a portable immunoassay system consisting of a small centrifugal microfluidic device driver (bento box) and a centrifugal microfluidic device made of polypropylene and fabricated by injection molding. The bento box consists of a cheap DC motor and an Arduino microcontroller. It has a simple structure and is the size of a bento box, that is, 150 × 150 × 100 (W × D × H) mm3. The developed device can automatically execute an enzyme-linked immunosorbent assay (ELISA) process under a steady rotating condition because it was designed based on the principle of CLOCK, which we previously presented. Here, we first executed an ELISA using a system consisting of the bento box and a device made of polydimethylsiloxane (PDMS) and compared it with a servo-controlled device driver. It was confirmed that the results of the bento box were consistent with those of the servo-controlled device driver. The limit of detection (LOD) using the bento box was 0.759 ng ml-1. Therefore, the controllability of the bento box was demonstrated. Next, we evaluated the injection-molded device through multi-step fluid control. We confirmed, through real-time observation of the device, that accurate flow control in the designed ELISA procedure was executed. Lastly, ELISA was employed for the measurements of mouse IgG using the system consisting of the bento box and the polypropylene device. The system performed all fluidic controls within 12 min; we confirmed the specificity of the system, and the LOD was 0.320 ng ml-1.