A Simple, Pipette-free, and Power-free Device for Virus Nucleic Acid Detection

A portable, inexpensive, and easy-to-manufacture microfluidic device is developed for the detection of SARS-CoV-2 dsDNA fragments. In this device, four reaction chambers separated by carbon fiber rods are pre-loaded with isothermal amplification and CRISPR-Cas12a reagents. The reaction is carried out by simply pulling the rods, without the need for manual pipetting. To facilitate power-free pathogen detection, the entire detection is designed to be heated with a disposable hand warmer. After the CRISPR reaction, the fluorescence signal generated by positive samples is identified by naked eye, using an inexpensive flashlight. This simple and sensitive device will serve as a new model for the next-generation viral diagnostics in either hospital or resource-limited settings. © 2023 SPIE.

A Multiplexed SERS Microassay for Accurate Detection of SARS-CoV-2 and Variants of Concern.

Severe acute respiratory syndrome coronavirus 2 variants play an important role in predicting patient outcome during postinfection, and with growing fears of COVID-19 reservoirs in domestic and wild animals, it is necessary to adapt detection systems for variant detection. However, variant-specific detection remains challenging. Surface-enhanced Raman scattering is a sensitive and multiplexing technique that allows the simultaneous detection of multiple targets for accurate identification. Here we propose the development of a multiplex SERS microassay to detect both the spike and nucleocapsid structural proteins of SARS-CoV-2. The designed SERS microassay integrates gold-silver hollow nanobox barcodes and electrohydrodynamically induced nanomixing which in combination enables highly specific and sensitive detection of SARS-CoV-2 and the S-protein epitopes to delineate between ancestral prevariant strains with the newer variants of concern, Delta and Omicron. The microassay allows detection from as low as 20 virus/µL and 50 pg/mL RBD protein and can clearly identify the virus among infected versus healthy nasopharyngeal swabs, with the potential to identify between variants. The detection of both S- and N-proteins of SARS-CoV-2 and the differentiation of variants on the SERS microassay can aid the early detection of COVID-19 to reduce transmission rates and lead into adequate treatments for those severely affected by the virus.

Mass production system for RNA-loaded lipid nanoparticles using piling up microfluidic devices

Microfluidic devices are widely used in lipid nanoparticle (LNP)-based vaccines and nanomedicine research. These devices should be stiff enough to withstand the high flow rate for the mass production of LNPs, and malleable enough to use when fabricating complicated microchannel or micromixer structures, such as staggering herringbone micromixers. Due to the limitations of the available fabrication methods, optimal microfluidic devices have not yet been developed. In this study, we report the development of a glass-based microfluidic device based on the invasive Lipid Nanoparticle Production (iLiNP) device® reported previously. The LNP size controllability of glass-based iLiNP device was similar to that of the poly(dimethylsiloxane) (PDMS)-based iLiNP device, and the glass-iLiNP device was used for mRNA-loaded LNP production with ionizable lipids used for COVID-19 mRNA vaccines. We also demonstrate a piling- and numbering-up strategy based on glass-iLiNP device. The iLiNP unit composed of five-layered microchannels was fabricated by piling-up each glass-iLiNP device followed by parallelization (numbering-up) for the mass production of LNPs. This iLiNP system can produce LNPs with sizes ranging between 20 and 60 nm at a flow rate of 20–50 mL/min, and its performance is comparable to that of the commercially available microfluidic system like NanoAssemblr®.

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.

Lipid nanoparticles employed in mRNA-based COVID-19 vaccines: an overview of materials and processes used for development and production

In the light of the recommended application of the third dose, both public and professional community would benefit from a detailed report on the technological advances behind the developed messenger ribonucleic acid (mRNA) based COVID-19 vaccines. Although many vaccine developers are yet to reveal their precise formulations, it is apparent they are founded on nanotechnology platforms similar to the one successfully used for registered drug Onpattro™ (INN: patisiran). Optimal encapsulation of mRNA requires the presence of four lipids: an ionizable cationic lipid, a polyethylene-glycol (PEG)-lipid, a neutral phospholipid and cholesterol. Together with other excipients (mainly buffers, osmolytes and cryoprotectives), they enable the formation of lipid nanoparticles (LNPs) using rapid-mixing microfluidic or T-junction systems. However, some limitations of thermostability testing protocols, coupled with the companies’ more or less cautious approach to predicting vaccine stability, led to rigorous storage conditions:-15° to-25°C or even-60° to-80°C. Nevertheless, some inventors recently announced their mRNA-LNP based vaccine candidates to be stable at both 25° and 37°C for a week. Within the formulation design space, further optimization of the ionizable lipids should be expected, especially in the direction of increasing their branching and optimizing pKa values, ultimately leading to the second generation of mRNA-LNP COVID-19 vaccines. © 2022, Pharmaceutical Association of Serbia. All rights reserved.

Integration of RT-LAMP and Microfluidic Technology for Detection of SARS-CoV-2 in Wastewater as an Advanced Point-of-Care Platform.

Development of lab-on-a-chip (LOC) system based on integration of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and microfluidic technology is expected to speed up SARS-CoV-2 diagnostics allowing early intervention. In the current work, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and RT-LAMP assays were performed on extracted RNA of seven wastewater samples from COVID-19 hotspots. RT­LAMP assay was also performed on wastewater samples without RNA extraction. Current detection of SARS-CoV-2 is mainly by RT-qPCR of ORF (ORF1ab) and N genes so we targeted both to find the best target gene for SARS-CoV-2 detection. We also performed RT-LAMP with/without RNA extraction inside microfluidic device to target both genes. Positivity rates of RT-qPCR and RT-LAMP performed on extracted RNA were 100.0% (7/7) and 85.7% (6/7), respectively. RT-qPCR results revealed that all 7 wastewater samples were positive for N gene (Ct range 37-39), and negative for ORF1ab, suggesting that N gene could be the best target gene for SARS-CoV-2 detection. RT-LAMP of N and ORF (ORF1a) genes performed on wastewater samples without RNA extraction indicated that all 7 samples remains pink (negative). The color remains pink in all microchannels except microchannels which subjected to RT-LAMP for targeting N region after RNA extraction (yellow color) in 6 out of 7 samples. This study shows that SARS-CoV-2 was successfully detected from wastewater samples using RT-LAMP in microfluidic chips. This study brings the novelty involving the use of wastewater samples for detection of SARS-CoV-2 without previous virus concentration and with/without RNA extraction.

A Facile Single-Phase-Fluid-Driven Bubble Microfluidic Generator for Potential Detection of Viruses Suspended in Air

Microfluidics devices have widely been employed to prepare monodispersed microbubbles/droplets, which have promising applications in biomedical engineering, biosensor detection, drug delivery, etc. However, the current reported microfluidic devices need to control at least two-phase fluids to make microbubbles/droplets. Additionally, it seems to be difficult to make monodispersed microbubbles from the ambient air using currently reported microfluidic structures. Here, we present a facile approach to making monodispersed microbubbles directly from the ambient air by driving single-phase fluid. The reported single-phase-fluid microfluidic (SPFM) device has a typical co-flow structure, while the adjacent space between the injection tube and the collection tube is open to the air. The flow condition inside the SPFM device was systematically studied. By adjusting the flow rate of the single-phase fluid, bubbles were generated, the sizes of which could be tuned precisely. This facile bubble generator may have significant potential as a detection sensor in detecting viruses in spread droplets or haze particles in ambient air.

Advances in Nucleic Acid Amplification-Based Microfluidic Devices for Clinical Microbial Detection

Accurate and timely detection of infectious pathogens is urgently needed for disease treatment and control of possible outbreaks worldwide. Conventional methods for pathogen detection are usually time-consuming and labor-intensive. Novel strategies for the identification of pathogenic nucleic acids are necessary for practical application. The advent of microfluidic technology and microfluidic devices has offered advanced and miniaturized tools to rapidly screen microorganisms, improving many drawbacks of conventional nucleic acid amplification-based methods. In this review, we summarize advances in the microfluidic approach to detect pathogens based on nucleic acid amplification. We survey microfluidic platforms performing two major types of nucleic acid amplification strategies, namely, polymerase chain reaction (PCR) and isothermal nucleic acid amplification. We also provide an overview of nucleic acid amplification-based platforms including studies and commercialized products for SARS-CoV-2 detection. Technologically, we focus on the design of the microfluidic devices, the selected methods for sample preparation, nucleic acid amplification techniques, and endpoint analysis. We also compare features such as analysis time, sensitivity, and specificity of different platforms. The first section of the review discusses methods used in microfluidic devices for upstream clinical sample preparation. The second section covers the design, operation, and applications of PCR-based microfluidic devices. The third section reviews two common types of isothermal nucleic acid amplification methods (loop-mediated isothermal amplification and recombinase polymerase amplification) performed in microfluidic systems. The fourth section introduces microfluidic applications for nucleic acid amplification-based detection of SARS-CoV-2. Finally, the review concludes with the importance of full integration and quantitative analysis for clinical microbial identification.

Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery.

In 2021, mRNA vaccines against COVID-19 were approved by the Food and Drug Administration. mRNA vaccines are important for preventing severe COVID-19 and returning to normal life. The development of RNA-delivery technology, including mRNA vaccines, has been investigated worldwide for ~30 years. Lipid nanoparticles (LNPs) are a breakthrough technology that stably delivers RNA to target organs, and RNA-loaded LNP-based nanomedicines have been studied for the development of vaccines and nanomedicines for RNA-, gene-, and cell-based therapies. Recently, microfluidic devices and technologies have attracted attention for the production of LNPs, particularly RNA-loaded LNPs. Microfluidics provides many advantages for RNA-loaded LNP production, including precise LNP size controllability, high reproducibility, high-throughput optimization of LNP formulation, and continuous LNP-production processes. In this review, we summarize microfluidic-based RNA-loaded LNP production and its applications in RNA-based therapy and genome editing.