Influenza A viruses are often present in environmental and clinical samples at concentrations below the limit of detection (LOD) of molecular diagnostics. Here we report an integrated microfluidic preconcentration and nucleic amplification system (μFPNAS) which enables both preconcentration of influenza A virus H1N1 (H1N1) and amplification of its viral RNA, thereby lowering LOD for H1N1. H1N1 virus particles were first magnetically preconcentrated using magnetic nanoparticles conjugated with an antibody specific for the virus. Their isolated RNA was amplified to cDNA through thermocycling in a trapezoidal chamber of the μFPNAS. A detection limit as low as 100 TCID50 (50% tissue culture infective dose) in saliva can be obtained within 2 hours. These results suggest that the LOD of molecular diagnostics for virus can be lowered by systematically combining immunomagnetic separation and reverse transcriptase-polymerase chain reaction (RT-PCR) in one microfluidic device.

Molecular diagnostics for sepsis is still a challenge due to the presence of compounds that interfere with gene amplification and bacteria at concentrations lower than the limit of detection (LOD). Here, we report on the development of a 3D printed modular microfluidic device (3DpmμFD) that preconcentrates bacteria of interest in whole blood and purifies their genomic DNA (gDNA). It is composed of a W-shaped microchannel and a conical microchamber. Bacteria of interest are magnetically captured from blood in the device with antibody conjugated magnetic nanoparticles (Ab-MNPs) at 5 mL/min in the W-shaped microchannel, while purified gDNA of the preconcentrated bacteria is obtained with magnetic silica beads (MSBs) at 2 mL/min in the conical microchamber. The conical microchamber was designed to be connected to the microchannel after the capturing process using a 3D-printed rotary valve to minimize the exposure of the MSBs to interfering compounds in blood. The pretreatment process of spiked blood (2.5 mL) can be effectively completed within about 50 min. With the 3DpmμFD, the LOD for the target microorganism Escherichia coli O157:H7 measured by both polymerase chain reaction (PCR) with electrophoresis and quantitative PCR was 10 colony forming unit (CFU) per mL of whole blood. The results suggest that our method lowers the LOD of molecular diagnostics for pathogens in blood by providing bacterial gDNA at high purity and concentration.

Circulating tumor cells (CTCs) are an indicator of metastatic progression and relapse. Since non-CTC cells such as red blood cells outnumber CTCs in the blood, the separation and enrichment of CTCs is key to improving their detection sensitivity. The ATP luminescence assay can measure intracellular ATP to detect cells quickly but has not yet been used for CTC detection in the blood because extracellular ATP in the blood, derived from non-CTCs, interferes with the measurement. Herein, we report on the improvement of the ATP luminescence assay for the detection of CTCs by separating and concentrating CTCs in the blood using a 3D printed immunomagnetic concentrator (3DPIC). Because of its high-aspect-ratio structure and resistance to high flow rates, 3DPIC allows cancer cells in 10 mL to be concentrated 100 times within minutes. This enables the ATP luminescence assay to detect as low as 10 cells in blood, thereby being about 10 times more sensitive than when commercial kits are used for CTC concentration. This is the first time that the ATP luminescence assay was used for the detection of cancer cells in blood. These results demonstrate the feasibility of 3DPIC as a concentrator to improve the detection limit of the ATP luminescence assay for the detection of CTCs.

Face masks are one of the currently available options for preventing the transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has caused the 2019 pandemic. However, with the increasing demand for protection, face masks are becoming limited in stock, and the concerned individuals and healthcare workers from many countries are now facing the issue of the reuse of potentially contaminated masks. Although various technologies already exist for the sterilization of medical equipment, most of them are not applicable for eliminating virus from face masks. Thus, there is an urgent need to develop a fast and easy method of disinfecting contaminated face masks. In this study, using a human coronavirus (HCoV-229E) as a surrogate for SARS-CoV-2 contamination on face masks, we show that the virus loses its infectivity to a human cell line (MRC-5) when exposed for a short period of time (1 min) to ozone gas produced by a dielectric barrier discharge plasma generator. Scanning electron microscopy and particulate filtration efficiency (PFE) tests revealed that there was no structural or functional deterioration observed in the face masks even after they underwent excessive exposure to ozone (five 1-minute exposures). Interestingly, for face masks exposed to ozone gas for 5 min, the amplification of HCoV-229E RNA by reverse transcription polymerase chain reaction suggested a loss of infectivity under the effect of ozone, primarily owing to the damage caused to viral envelopes or envelope proteins. Ozone gas is a strong oxidizing agent with the ability to kill viruses on hard-to-reach surfaces, including the fabric structure of face masks. These results suggest that it may be possible to rapidly disinfect contaminated face masks using a plasma generator in a well-ventilated place.