A DNA framework-based dual signal amplification biosensor for portable detection of SARS-CoV-2 and its mutations.

We developed a rapid and accurate biosensor to detect SARS-CoV-2 and distinguish its mutations. Benefitting from a DNA framework-modified ordered interface and a dual signal amplification strategy, our biosensor could detect SARS-CoV-2 with a detection limit down to 10 fM. It performed well on pseudo virus and SARS-CoV-2 RNA standard materials, revealing the potential application in disease diagnosis and spread, in combination with a home-made smartphone.

HTLV-1 infection of donor-derived T cells might promote acute graft-versus-host disease following liver transplantation.

Acute graft versus host disease (aGVHD) is a rare, but severe complication of liver transplantation (LT). It is caused by the activation of donor immune cells in the graft against the host shortly after transplantation, but the contributing pathogenic factors remain unclear. Here we show that human T cell lymphotropic virus type I (HTLV-1) infection of donor T cells is highly associated with aGVHD following LT. The presence of HTLV-1 in peripheral blood and tissue samples from a discovery cohort of 7 aGVHD patients and 17 control patients is assessed with hybridization probes (TargetSeq), mass cytometry (CyTOF), and multiplex immunohistology (IMC). All 7 of our aGVHD patients display detectable HTLV-1 Tax signals by IMC. We identify donor-derived cells based on a Y chromosome-specific genetic marker, EIF1AY. Thus, we confirm the presence of CD4+Tax+EIF1AY+ T cells and Tax+CD68+EIF1AY+ antigen-presenting cells, indicating HTLV-1 infection of donor immune cells. In an independent cohort of 400 patients, we verify that HTLV-1 prevalence correlates with aGVHD incidence, while none of the control viruses shows significant associations. Our findings thus provide new insights into the aetio-pathology of liver-transplantation-associated aGVHD and raise the possibility of preventing aGVHD prior to transplantation.

Integrated microfluidic single-cell immunoblotting chip enables high-throughput isolation, enrichment and direct protein analysis of circulating tumor cells.

Effective capture and analysis of a single circulating tumor cell (CTC) is instrumental for early diagnosis and personalized therapy of tumors. However, due to their extremely low abundance and susceptibility to interference from other cells, high-throughput isolation, enrichment, and single-cell-level functional protein analysis of CTCs within one integrated system remains a major challenge. Herein, we present an integrated multifunctional microfluidic system for highly efficient and label-free CTC isolation, CTC enrichment, and single-cell immunoblotting (ieSCI). The ieSCI-chip is a multilayer microfluidic system that combines an inertia force-based cell sorter with a membrane filter for label-free CTC separation and enrichment and a thin layer of a photoactive polyacrylamide gel with microwell arrays at the bottom of the chamber for single-cell immunoblotting. The ieSCI-chip successfully identified a subgroup of apoptosis-negative (Bax-negative) cells, which traditional bulk analysis did not detect, from cisplatin-treated cells. Furthermore, we demonstrated the clinical application of the ieSCI-chip with blood samples from breast cancer patients for personalized CTC epithelial-to-mesenchymal transition (EMT) analysis. The expression level of a tumor cell marker (EpCAM) can be directly determined in isolated CTCs at the single-cell level, and the therapeutic response to anticancer drugs can be simultaneously monitored. Therefore, the ieSCI-chip provides a promising clinical translational tool for clinical drug response monitoring and personalized regimen development.

CRISPR/Cas12a Powered DNA Framework-Supported Electrochemical Biosensing Platform for Ultrasensitive Nucleic Acid Analysis.

Nucleic acid analysis using ultrasensitive and simple methods is critically important for the early-stage diagnosis and treatment of diseases. The CRISPR/Cas proteins, guided by a single-stranded RNA have shown incredible capability for sequence-specific targeting and detection. Herein, in order to improve and expand the application of CRISPR/Cas technology to the electrochemical interface-based nucleic acids analysis, the authors develop a CRISPR/Cas12a powered DNA framework-supported electrochemical biosensing platform via the cis and trans cleavage of Cas12a on the heterogeneous carbon interface (the existing publications which commonly adopted trans-cleavage). Their solid-liquid interface is first immobilized by 3D tetrahedral framework nucleic acids (FNAs) with specific DNA recognition probe. Based on the recognition of the complementary target through protospacer adjacent motif (PAM) confirmation and CRISPR-derived RNA (crRNA) matching, the easily formed Cas12a/crRNA duplex can get access to the interface, and the cis and trans cleavage of Cas12a can be easily activated. In combination with the enzyme catalyzed reaction, they achieved an ultralow limit of detection (LOD) of 100 fm in HPV-16 detection without pre-amplification. Furthermore, the platform is compatible with a spike-in human serum sample and has superior stability. Thus, their reported platform offers a practical, versatile, and amplification-free toolbox for ultrasensitive nucleic acid analysis.

One-pot pre-coated interface proximity extension assay for ultrasensitive co-detection of anti-SARS-CoV-2 antibodies and viral RNA.

In the field of in vitro diagnostics, detection of nucleic acids and proteins from biological samples is typically performed with independent platforms; however, co-detection remains a major technical challenge. Specifically, during the coronavirus disease 2019 (COVID-19) pandemic, the ability to simultaneously detect viral RNA and human antibodies would prove highly useful for efficient diagnosis and disease course management. Herein, we present a multiplex one-pot pre-coated interface proximity extension (OPIPE) assay that facilitates the simultaneous recognition of antibodies using a pre-coated antigen interface and a pair of anti-antibodies labeled with oligonucleotides. Following anti-antibody-bound nucleic acid chain extension to form templates in proximity, antibody signals can be amplified, together with that of targeted RNA, via a reverse transcription-polymerase chain reaction. Using four-color fluorescent TaqMan probes, we demonstrate the co-detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies and viral nucleic acids in a single bio-complex sample, including nucleocapsid protein-specific IgG and IgM, and the RNA fragments of RdRp and E genes. The serum detection limit for this platform is 100 fg/mL (0.67 fM) for the anti-SARS-CoV-2 antibody and 10 copies/µL for viral RNA. The OPIPE assay offers a practical and affordable solution for ultrasensitive co-detection of nucleic acids and antibodies from the same trace biological sample without the additional requirement of complicated equipment.

Application of Microfluidics in Single-cell Manipulation, Omics and Drug Development.

BACKGROUND: Cell heterogeneity exists among different tissues, even in the same type of cells. Cell heterogeneity leads to a difference in cell size, functions, biological activity, and for cancer cells it causes different drug responses and resistance. Meanwhile, microfluidics is a promising tool for single-cell research to reveal cell heterogeneity. METHODS: Through literature research conducted over the past ten years on microfluidics, we summarize and introduce the application of microfluidics in single-cell separation and manipulation, featuring techniques, such as acoustic manipulation, optical manipulation, single-cell trapping, and patterning, as well as single-cell omics including singlecell genomics, single-cell transcriptomics, single-cell proteome, single-cell metabolome, and drug development. RESULTS: Microfluidics is a flexible, precise tool, and it is easy to integrate with different functions. Firstly, it can be used as an important tool to separate rare but important cells according to the cell`s biological or physical properties. Secondly, microfluidics can provide the possibility of single-cell omics. Thirdly, microfluidics can be used in drug development, specifically in drug delivery and drug combination. Meanwhile, droplet microfluidics has gradually become the most powerful tool to encapsulate single-cells with other reagents for DNA, RNA, or protein analysis. CONCLUSION: Microfluidics is a robust platform technology that is able to accomplish rare cell separation, efficient single-cell omics analysis and provide a platform for drug development and drug delivery.