Discrimination of the H1N1 and H5N2 Variants of Influenza A Virus Using an Isomeric Sialic Acid-Conjugated Graphene Field-Effect Transistor.

There has been a continuous effort to fabricate a fast, sensitive, and inexpensive system for influenza virus detection to meet the demand for effective screening in point-of-care testing. Herein, we report a sialic acid (SA)-conjugated graphene field-effect transistor (SA-GFET) sensor designed using α2,3-linked sialic acid (3'-SA) and α2,6-linked sialic acid (6'-SA) for the detection and discrimination of the hemagglutinin (HA) protein of the H5N2 and H1N1 viruses. 3'-SA and 6'-SA specific for H5 and H1 influenza were used in the SA-GFET to capture the HA protein of the influenza virus. The net charge of the captured viral sample led to a change in the electrical current of the SA-GFET platform, which could be correlated to the concentration of the viral sample. This SA-GFET platform exhibited a highly sensitive response in the range of 101-106 pfu mL-1, with a limit of detection (LOD) of 101 pfu mL-1 in buffer solution and a response time of approximately 10 s. The selectivity of the SA-GFET platform for the H1N1 and H5N2 influenza viruses was verified by testing analogous respiratory viruses, i.e., influenza B and the spike protein of SARS-CoV-2 and MERS-CoV, on the SA-GFET. Overall, the results demonstrate that the developed dual-channel SA-GFET platform can potentially serve as a highly efficient and sensitive sensing platform for the rapid detection of infectious diseases.

Multifunctional Filter Membranes Based on Self-Assembled Core-Shell Biodegradable Nanofibers for Persistent Electrostatic Filtration through the Triboelectric Effect.

The massive production of polymer-based respiratory masks during the COVID-19 pandemic has rekindled the issue of environmental pollution from nonrecyclable plastic waste. To mitigate this problem, conventional filters should be redesigned with improved filtration performance over the entire operational life while also being naturally degradable at the end. Herein, we developed a functional and biodegradable polymeric filter membrane consisting of a polybutylene adipate terephthalate (PBAT) matrix blended with cetyltrimethylammonium bromide (CTAB) and montmorillonite (MMT) clay, whose surface properties have been modified through cation exchange reactions for good miscibility with PBAT in an organic solvent. Particularly, the spontaneous evolution of a partial core-shell structure (i.e., PBAT core encased by CTAB-MMT shell) during the electrospinning process amplified the triboelectric effect as well as the antibacterial/antiviral activity that was not observed in naive PBAT. Unlike the conventional face mask filter that relies on the electrostatic adsorption mechanism, which deteriorates over time and/or due to external environmental factors, the PBAT@CTAB-MMT nanofiber membrane (NFM)-based filter continuously retains electrostatic charges on the surface due to the triboelectric effect of CTAB-MMT. As a result, the PBAT@CTAB-MMT NFM-based filter showed high filtration efficiencies (98.3%, PM0.3) even at a low differential pressure of 40 Pa or less over its lifetime. Altogether, we not only propose an effective and practical solution to improve the performance of filter membranes while minimizing their environmental footprint but also provide valuable insight into the synergetic functionalities of organic-inorganic hybrid materials for applications beyond filter membranes.

Violacein-embedded nanofiber filters with antiviral and antibacterial activities.

Most respiratory masks are made of fabrics, which only capture the infectious virus carriers into the matrix. However, these contagious viruses stay active for a long duration (∼7 days) within the fabric matrix possibly inducing post-contact transmissions. Moreover, conventional masks are vulnerable to bacterial growth with prolonged exposure to exhaled breaths. Herein, we combined violacein, a naturally-occurring antimicrobial agent, with porous nanofiber membranes to develop a series of functional filters that autonomously sterilizes viruses and bacteria. The violacein-embedded membrane inactivates viruses within 4 h (99.532 % reduction for influenza and 99.999 % for human coronavirus) and bacteria within 2 h (75.5 % reduction). Besides, its nanofiber structure physically filters out the nanoscale (<0.8 µm) and micron-scale (0.8 µm - 3 µm) particulates, providing high filtration efficiencies (99.7 % and 100 % for PM 1.0 and PM 10, respectively) with long-term stability (for 25 days). In addition, violacein provides additional UV-resistant property, which protects the skin from sunlight. The violacein-embedded membrane not only proved the sterile efficacy of microbe extracted pigments for biomedical products but also provided insights to advance the personal protective equipment (PPE) to fight against contagious pathogens.

Diagnostic performance and clinical feasibility of a novel one-step RT-qPCR assay for simultaneous detection of multiple severe acute respiratory syndrome coronaviruses.

Coronavirus disease 2019 (COVID-19) is an acute respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Other coronaviruses (CoVs) can also infect humans, although the majority cause only mild respiratory symptoms. Because early diagnosis of SARS-CoV-2 is critical for preventing further transmission events and improving clinical outcomes, it is important to be able to distinguish SARS-CoV-2 from other SARS-related CoVs in respiratory samples. Therefore, we developed and evaluated a novel reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting the genes encoding the spike (S) and membrane (M) proteins to enable the rapid identification of SARS-CoV-2, including several new circulating variants and other emerging SARS-like CoVs. By analysis of in vitro-transcribed mRNA, we established multiplex RT-qPCR assays capable of detecting 5 × 10° copies/reaction. Using RNA extracted from cell culture supernatants, our multiple simultaneous SARS-CoV-2 assays had a limit of detection of 1 × 10° TCID50/mL and showed no cross-reaction with human CoVs or other respiratory viruses. We also validated our method using human clinical samples from patients with COVID-19 and healthy individuals, including nasal swab and sputum samples. This novel one-step multiplex RT-qPCR assay can be used to improve the laboratory diagnosis of human-pathogenic CoVs, including SARS-CoV-2, and may be useful for the identification of other SARS-like CoVs of zoonotic origin.

A rapid quantitative on-site coronavirus disease 19 serological test.

On-site severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) serological assays allow for timely in-field decisions to be made regarding patient status, also enabling population-wide screening to assist in controlling the coronavirus disease 2019 (COVID-19) pandemic. Here we propose a rapid microfluidic serological assay with two unique functions of nanointerstice filling and digitized flow control, which enable the fast/robust filling of the sample fluid as well as precise regulation of duration and volume of immune reaction. Developed microfluidic assay showed enhanced limit of detection, and 91.67% sensitivity and 100% specificity (n = 152) for clinical samples of SARS CoV-2 patients. The assay enables daily monitoring of IgM/IgG titers and patterns, which could be crucial parameters for convalescence from COVID-19 and provide important insight into how the immune system responds to SARS CoV-2. The developed on-site microfluidic assay presented the mean time for IgM and IgG seroconversions, indicating that these titers plateaued days after seroconversion. The mean duration from day 0 to PCR negativity was 19.4 days (median 20 d, IQR 16-21 d), with higher IgM/IgG titres being observed when PCR positive turns into negative. Simple monitoring of these titres promotes rapid on-site detection and comprehensive understanding of the immune response of COVID-19 patients.

A single snapshot multiplex immunoassay platform utilizing dense test lines based on engineered beads.

Co-circulation of coronavirus disease 2019 (COVID-19) and dengue fever has been reported. Accurate and timely multiplex diagnosis is required to prevent future pandemics. Here, we developed an innovative microfluidic chip that enables a snapshot multiplex immunoassay for timely on-site response and offers unprecedented multiplexing capability with an operating procedure similar to that of lateral flow assays. An open microchannel assembly of individually engineered microbeads was developed to construct nine high-density test lines, which can be imaged in a 1 mm2 field-of-view. Thus, simultaneous detection of multiple antibodies would be achievable in a single high-resolution snapshot. Next, we developed a novel pixel intensity-based imaging process to distinguish effective and non-specific fluorescence signals, thereby improving the reliability of this fluorescence-based immunoassay. Finally, the chip specifically identified and classified random combinations of arbovirus (Zika, dengue, and chikungunya viruses) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies within 30 min. Therefore, we believe that this snapshot multiplex immunoassay chip is a powerful diagnostic tool to control current and future pandemics.