Development of nucleocapsid-specific monoclonal antibodies for SARS-CoV-2 and their ELISA diagnostics on an automatic microfluidic device.

Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has threatened public health globally, and the emergence of viral variants has exacerbated an already precarious situation. To prevent further spread of the virus and determine government action required for virus control, accurate and rapid immunoassays for SARS-CoV-2 diagnosis are urgently needed. In this study, we generated monoclonal antibodies (mAbs) against the SARS-CoV-2 nucleocapsid protein (NP), compared their reactivity using an enzyme-linked immunosorbent assay (ELISA), and selected four mAbs designated 1G6, 3E10, 3F10, and 5B6 which have higher reactivity to NP and viral lysates of SARS-CoV-2 than other mAbs. Using an epitope mapping assay, we identified that 1G6 detected the C-terminal domain of SARS-CoV-2 NP (residues 248-364), while 3E10 and 3F10 bound to the N-terminal domain (residues 47-174) and 3F10 detected the N-arm region (residues 1-46) of SARS-CoV-2 NP. Based on the epitope study and sandwich ELISA, we selected the 1G6 and 3E10 Abs as an optimal Ab pair and applied them for a microfluidics-based point-of-care (POC) ELISA assay to detect the NPs of SARS-CoV-2 and its variants. The integrated and automatic microfluidic system could operate the serial injection of the sample, the washing solution, the HRP-conjugate antibody, and the TMB substrate solution simply by controlling air purge via a single syringe. The proposed Ab pair-equipped microsystem effectively detected the NPs of SARS-CoV-2 variants as well as in clinical samples. Collectively, our proposed platform provides an advanced protein-based diagnostic tool for detecting SARS-CoV-2.

Centrifugal Microfluidic Cell Culture Platform for Physiologically Relevant Virus Infection Studies: A Case Study with HSV-1 Infection of Periodontal Cells.

Static well plates remain the gold standard to study viral infections in vitro, but they cannot accurately mimic dynamic viral infections as they occur in the human body. Therefore, we established a dynamic cell culture platform, based on centrifugal microfluidics, to study viral infections in perfusion. To do so, we used human primary periodontal dental ligament (PDL) cells and herpes simplex virus-1 (HSV-1) as a case study. By microscopy, we confirmed that the PDL cells efficiently attached and grew in the chip. Successful dynamic viral infection of perfused PDL cells was monitored using fluorescent imaging and RT-qPCR-based experiments. Remarkably, viral infection in flow resulted in a gradient of HSV-1-infected cells gradually decreasing from the cell culture chamber entrance towards its end. The perfusion of acyclovir in the chip prevented HSV-1 spreading, demonstrating the usefulness of such a platform for monitoring the effects of antiviral drugs. In addition, the innate antiviral response of PDL cells, measured by interferon gene expression, increased significantly over time in conventional static conditions compared to the perfusion model. These results provide evidence suggesting that dynamic viral infections differ from conventional static infections, which highlights the need for more physiologically relevant in vitro models to study viral infections.

A portable centrifugal genetic analyzer for multiplex detection of feline upper respiratory tract disease pathogens.

We present a portable genetic analyzer with an integrated centrifugal disc which is equipped with a glass-filter extraction column for purifying nucleic acid (NA) and multiple reaction chambers for analyzing major feline upper respiratory tract disease (FURTD) pathogens. We targeted four kinds of FURTD including Feline herpesvirus 1 (FHV), Mycoplasma felis (MPF), Bordetella bronchiseptica (BDB), and Chlamydophila felis (CDF). The portable genetic analyzer consists of a spinning motor, two pairs of Peltier heaters, two Minco heater, fluorescent optics, a touchscreen, and software for data analysis, so loop-mediated isothermal amplification (LAMP) or polymerase chain reaction (PCR) can be performed. The overall size of the genetic analyzer was 28 cm × 28 cm × 26 cm and the weight was 10 kg, which was deliverable for point-of-care testing (POCT). Owing to the sophisticated microchannel design and spinning program, the serial injection of the sample solution, the washing solution, and the elution solution was executed through a glass filter membrane for nucleic acid (NA) extraction, and then the cocktail with the purified genome was aliquoted into 9 reaction chambers for LAMP or PCR. The whole process for the LAMP reaction or the PCR was completed within 1.5 h. The fluorescence profiles by a scanning mode showed the matched results between the LAMP and the PCR.

An Integrated Smartphone-Based Genetic Analyzer for Qualitative and Quantitative Pathogen Detection.

The use of the smartphone is an ideal platform to realize the future point-of-care (POC) diagnostic system. Herein, we propose an integrated smartphone-based genetic analyzer. It consists of a smartphone and an integrated genetic analysis unit (i-Gene), in which the power of the smartphone was utilized for heating the gene amplification reaction, and the camera function was used for imaging the colorimetric change of the reaction for quantitative and multiplex foodborne pathogens. The housing of i-Gene was fabricated by using a 3D printer, which was equipped with a macro lens, white LEDs, a disposable microfluidic chip for loop-mediated isothermal amplification (LAMP), a thin-film heater, and a power booster. The i-Gene was installed on the iPhone in alignment with a camera. The LAMP mixture for Eriochrome Black T (EBT) colorimetric detection was injected into the LAMP chip to identify Escherichia coli O157:H7, Salmonella typhimurium, and Vibrio parahaemolyticus. The proportional-integral-derivative controller-embedded film heater was powered by a 5.0 V power bank to maintain 63 °C for the LAMP reaction. When the LAMP proceeded, the color was changed from violet to blue, which was real-time monitored by the smartphone complementary metal oxide semiconductor camera. The images were transported to the desktop computer via Wi-Fi. The quantitative LAMP profiles were obtained by plotting the ratio of green/red intensity versus the reaction time. We could identify E. coli O157:H7 with a limit of detection of 101 copies/µL within 60 min. Our proposed smartphone-based genetic analyzer offers a portable, simple, rapid, and cost-effective POC platform for future diagnostic markets.

Centrifugal microfluidic device for the high-throughput synthesis of Pd@AuPt core-shell nanoparticles to evaluate the performance of hydrogen peroxide generation.

We propose a novel high-throughput screening platform using a centrifugal microfluidic device for producing combinatorial tri-metallic catalysts. The centrifugal device was designed to perform 60 reactions under different conditions on a single device. As a model to search for an optimal tri-metallic catalyst, we synthesized a variety of Pd@AuPt nanoparticles (NPs), in which Pd nanocubes served as a core, and Au and Pt atoms formed a shell. The centrifugal microfluidic device was etched on the top and bottom sides, in which two zigzag-shaped microchannels were patterned on the top side, and 60 reaction chambers were fabricated on the bottom side. Through the sophisticated zigzag-shaped microchannels, Pt2+ ion and Pd nanocube solutions were injected into the channel in one shot, and the centrifugal force equally and automatically divided the injected solutions into 60 aliquots during the rotation. By controlling the sophisticated channel dimensions and designing the passive valve structure, the Pt2+ ion, Pd nanocube, and Au3+ solutions were loaded into the reaction chamber in sequential order depending on the programmed rotational direction and speed. Therefore, the ratio of Au to Pt to synthesize Pd@AuPt core-shell NPs was changed from 0.028 : 1 to 12 : 1, and accordingly, the resultant 60 types of Pd@AuPt catalysts presented with different ratios of metal atom compositions. Then, we screened the catalytic activity of the Pd@AuPt NPs for generating H2O2 according to the degree of coating of Au and Pt, and the Pd@AuPt catalyst with the Au/Pt ratio at 0.5 turned out to be the most effective.

Point-of-care genetic analysis for multiplex pathogenic bacteria on a fully integrated centrifugal microdevice with a large-volume sample.

We present a fully integrated portable centrifugal microsystem for multiplex detection of food poisoning bacteria with a large volume of sample up to 1 mL. The microsystem consists of a portable genetic analyzer and a fully integrated centrifugal microdevice. The centrifugal microdevice is designed with two units: a 3D printed solution-loading cartridge and a centrifugal microfluidic disc. All the essential solutions for loop-mediated isothermal amplification (LAMP) reaction are stored inside the cartridge, and orderly released into centrifugal microdevice by a rotation program. Each unit of the device is designed with 20 reaction chambers for simultaneous detection of food-borne bacteria in one test. To increase the amount of a sample to 1 mL, we incorporated the super absorbent polymer (SAP) in the waste chamber to absorb the sample and the washing solution during the device operation. The whole process was automatically conducted including designated solution release, bead-based DNA extraction, isothermal gene amplification by Eriochrome Black (EBT)-mediated LAMP reaction, and colorimetric and UV-visible detection of amplicons. The ratio between Abs640nm and Abs570nm was used as a criterion to confirm the positive result, and the result was positive upon the condition of Abs640/Abs570 ≥ 1.0. To demonstrate the pathogenic bacteria detection on our proposed microsystem, we targeted three kinds of bacteria (Escherichia coli O157:H7, Salmonella typhimurium, and Vibrio parahaemolyticus) for monoplex and multiplex detection. The whole process from sample to result was completed within 1 h with a low limit of detection (LOD) of 102 cells/mL.

Nucleic acid diagnostics on the total integrated lab-on-a-disc for point-of-care testing.

Since the emergence of the lab-on-a-chip technology in 1979, a variety of microfluidic devices have been developed and utilized for chemical and biological applications. Among the microfluidic devices, the centrifugal microfluidic device or lab-on-a-disc (LOAD) has advanced remarkably due to simple operation by the rotation, total integration, and high-throughput capability. Moreover, the centrifugal microdevices do not need complex tubing and pumping systems, which render them ideal for point-of-care testing (POCT) system. Owing to these characteristics, the centrifugal microdevices have been extensively used for bio-diagnostics. In particular, molecular diagnostics, which are regarded as an essential method for definite determination of the targets related with diseases, have been widely applied on the LOAD. In this review paper, we focus on the molecular diagnostics on the LOAD. The steps for the molecular diagnostics such as cell lysis, genome purification, gene amplification, amplicon detection, and data analysis can be performed individually or totally on the LOAD. Future directions of the LOAD in the fields of bio-diagnostics is to realize POCT for U-healthcare monitoring. In this context, the latest LOAD strategies for molecular diagnostics are summarized in this review paper, which would provide an insight for future POCT platform.