Evaluation of an identification method for the SARS-CoV-2 Delta variant based on the amplification-refractory mutation system.

The Delta variant of SARS-CoV-2 dominated the COVID-19 pandemic due to its high viral replication capacity and immune evasion, causing massive outbreaks of cases, hospitalizations, and deaths. Currently, variant identification is performed mainly by sequencing. However, the high requirements for equipment and operators as well as its high cost have limited its application in underdeveloped regions. To achieve an economical and rapid method of variant identification suitable for undeveloped areas, we applied an amplification-refractory mutation system (ARMS) based on PCR for the detection of novel coronavirus variants. The results showed that this method could be finished in 90 min and detect as few as 500 copies/mL and not react with SARS-Coronavirus, influenza A H1N1(2009), and other cross-pathogens or be influenced by fresh human blood, α- interferon, and other interfering substances. In a set of double-blind trials, tests of 262 samples obtained from patients confirmed with Delta variant infection revealed that our method was able to accurately identify the Delta variant with high sensitivity and specificity. In conclusion, the ARMS-PCR method applied in Delta variant identification is rapid, sensitive, specific, economical, and suitable for undeveloped areas. In our future study, ARMS-PCR will be further applied in the identification of other variants, such as Omicron.

High-throughput and high-dimensional single-cell analysis of antigen-specific CD8+ T cells.

Although critical to T cell function, antigen specificity is often omitted in high-throughput multiomics-based T cell profiling due to technical challenges. We describe a high-dimensional, tetramer-associated T cell antigen receptor (TCR) sequencing (TetTCR-SeqHD) method to simultaneously profile cognate antigen specificities, TCR sequences, targeted gene expression and surface-protein expression from tens of thousands of single cells. Using human polyclonal CD8+ T cells with known antigen specificity and TCR sequences, we demonstrate over 98% precision for detecting the correct antigen specificity. We also evaluate gene expression and phenotypic differences among antigen-specific CD8+ T cells and characterize phenotype signatures of influenza- and Epstein-Barr virus-specific CD8+ T cells that are unique to their pathogen targets. Moreover, with the high-throughput capacity of profiling hundreds of antigens simultaneously, we apply TetTCR-SeqHD to identify antigens that preferentially enrich cognate CD8+ T cells in patients with type 1 diabetes compared to healthy controls and discover a TCR that cross-reacts with diabetes-related and microbiome antigens. TetTCR-SeqHD is a powerful approach for profiling T cell responses in humans and mice.

Electroenzymatic choline sensing at near the theoretical performance limit.

A high performance, electroenzymatic microsensor for choline based on choline oxidase (ChOx) immobilized on Pt coated with permselective polymer layers has been created that exhibits sensitivity approaching the theoretical performance limit. Sensor construction was guided by simulations performed with a detailed mathematical model. Implantable microsensors with an array of electroenzymatic sensing sites provide a means to record concentration changes of choline, an effective surrogate for acetylcholine due to its very rapid turnover in the brain, and other neurochemicals in vivo. However, electroenzymatic sensors generally have insufficient sensitivity and response time to monitor neurotransmitter signaling on the millisecond timescale with cellular-level spatial resolution. Model simulations suggested that choline sensor performance can be improved significantly by optimizing immobilized ChOx layer thickness and minimizing the thicknesses of permselective polymer coatings as well. Electroenzymatic choline sensors constructed with a ∼5 µm-thick crosslinked ChOx layer atop 200 nm-thick permselective films (poly(m-phenylenediamine) and Nafion) exhibited unprecedented sensitivity and response time of 660 ± 40 nA µM-1 cm-2 at 37 °C and 0.36 ± 0.05 s, respectively, while maintaining excellent selectivity. Such performance characteristics provide greater flexibility in the design of microelectrode array (MEA) probes with near cellular-scale sensing sites arranged in more dense arrays. Also, faster response times enable better resolution of transient acetylcholine signals and better correlation of these events with electrophysiological recordings so as to advance study of brain function.

Electroenzymatic glutamate sensing at near the theoretical performance limit.

The sensitivity and response time of glutamate sensors based on glutamate oxidase immobilized on planar platinum microelectrodes have been improved to near the theoretical performance limits predicted by a detailed mathematical model. Microprobes with an array of electroenzymatic sensing sites have emerged as useful tools for the monitoring of glutamate and other neurotransmitters in vivo; and implemented as such, they can be used to study many complex neurological diseases and disorders including Parkinson's disease and drug addiction. However, less than optimal sensitivity and response time has limited the spatiotemporal resolution of these promising research tools. A mathematical model has guided systematic improvement of an electroenzymatic glutamate microsensor constructed with a 1-2 µm-thick crosslinked glutamate oxidase layer and underlying permselective coating of polyphenylenediamine and Nafion reduced to less than 200 nm thick. These design modifications led to a nearly 6-fold improvement in sensitivity to 320 ± 20 nA µM-1 cm-2 at 37 °C and a ∼10-fold reduction in response time to 80 ± 10 ms. Importantly, the sensitivity and response times were attained while maintaining a low detection limit and excellent selectivity. Direct measurement of the transport properties of the enzyme and polymer layers used to create the biosensors enabled improvement of the mathematical model as well. Subsequent model simulations indicated that the performance characteristics achieved with the optimized biosensors approach the theoretical limits predicted for devices of this construction. Such high-performance glutamate biosensors will be more effective in vivo at a size closer to cellular dimension and will enable better correlation of glutamate signaling events with electrical recordings.

Corrosion inhibition of X65 steel in sulfuric acid by two food flavorants 2-isobutylthiazole and 1-(1,3-Thiazol-2-yl) ethanone as the green environmental corrosion inhibitors: Combination of experimental and theoretical researches.

Food flavors of 2-isobutylthiazole (ITT) and 1-(1,3-Thiazol-2-yl)ethanone (TEO) for the corrosion inhibition of X65 steel in H2SO4 were studied by electrochemical methods, atomic force microscopy (AFM), scanning electron microscopy (SEM) and theoretical calculations. Electrochemical experiments show that ITT and TEO can effectively inhibit the corrosion of cathode and anode of X65 steel, and they are mixed-type corrosion inhibitors. Surface topography analysis (SEM and AFM) also visually demonstrate that ITT and TEO form an effective barrier film on the X65 steel surface to isolate the corrosive medium. Theoretical calculations profoundly explain the inhibition mechanism of ITT and TEO at the molecular level. In addition, the adsorption behavior of ITT and TEO on the surface of X65 steel is consistent with Langmuir isotherm adsorption. The results of experimental and theoretical studies have shown that the inhibition effect of TEO is better than ITT for X65 in 0.5 M H2SO4.