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.

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 Microfluidic Diagnostic Device Capable of Autonomous Sample Mixing and Dispensing for the Simultaneous Genetic Detection of Multiple Plant Viruses.

As an efficient approach to risk management in agriculture, the elimination of losses due to plant diseases and insect pests is one of the most important and urgent technological challenges for improving the crop yield. Therefore, we have developed a polydimethylsiloxane (PDMS)-based microfluidic device for the multiplex genetic diagnosis of plant diseases and pests. It offers unique features, such as rapid detection, portability, simplicity, and the low-cost genetic diagnosis of a wide variety of plant viruses. In this study, to realize such a diagnostic device, we developed a method for the autonomous dispensing of fluid into a microchamber array, which was integrated with a set of three passive stop valves with different burst pressures (referred to as phaseguides) to facilitate precise fluid handling. Additionally, we estimated the mixing efficiencies of several types of passive mixers (referred to as chaotic mixers), which were integrated into a microchannel, through experimental and computational analyses. We first demonstrated the ability of the fabricated diagnostic devices to detect DNA-based plant viruses from an infected tomato crop based on the loop-mediated isothermal amplification (LAMP) method. Moreover, we demonstrated the simultaneous detection of RNA-based plant viruses, which can infect cucurbits, by using the reverse transcription LAMP (RT-LAMP) method. The multiplex RT-LAMP assays revealed that multiple RNA viruses extracted from diseased cucumber leaves were successfully detected within 60 min, without any cross-contamination between reaction microchambers, on our diagnostic device.