Generation and Screening of Antigen-Specific Nanobodies from Mammalian Cells Expressing the BCR Repertoire Library Using Droplet-Based Microfluidics.

Nanobodies, also known as VHHs, originate from the serum of Camelidae. Nanobodies have considerable advantages over conventional antibodies, including smaller size, more modifiable, and deeper tissue penetration, making them promising tools for immunotherapy and antibody-drug development. A high-throughput nanobody screening platform is critical to the rapid development of nanobodies. To date, droplet-based microfluidic systems have exhibited improved performance compared to the traditional phage display technology in terms of time and throughput. In realistic situations, however, it is difficult to directly apply the technology to the screening of nanobodies. Requirements of plasma cell enrichment and high cell viability, as well as a lack of related commercial reagents, are leading causes for impeding the development of novel methods. We overcame these obstacles by constructing a eukaryotic display system that secretes nanobodies utilizing homologous recombination and eukaryotic transformation technologies, and the significant advantages are that it is independent of primary cell viability and it does not require plasma cell enrichment in advance. Next, a signal capture system of "SA-beads + Biotin-antigen + nanobody-6 × His + fluorescence-labeled anti-6 × His (secondary antibody)" was designed for precise localization of the eukaryotic-expressed nanobodies in a droplet. Based on this innovation, we screened 293T cells expressing anti-PD-L1 nanobodies with a high positive rate of targeted cells (up to 99.8%). Then, single-cell transcriptomic profiling uncovered the intercellular heterogeneity and BCR sequence of target cells at a single-cell level. The complete complementarity determining region (CDR3) structure was obtained, which was totally consistent with the BCR reference. This study expanded the linkage between microfluidic technology and nanobody applications and also showed potential to accelerate the rapid transformation of nanobodies in the large-scale market.

Deep-dLAMP: Deep Learning-Enabled Polydisperse Emulsion-Based Digital Loop-Mediated Isothermal Amplification.

Digital nucleic acid amplification tests enable absolute quantification of nucleic acids, but the generation of uniform compartments and reading of the fluorescence requires specialized instruments that are costly, limiting their widespread applications. Here, the authors report deep learning-enabled polydisperse emulsion-based digital loop-mediated isothermal amplification (deep-dLAMP) for label-free, low-cost nucleic acid quantification. deep-dLAMP performs LAMP reaction in polydisperse emulsions and uses a deep learning algorithm to segment and determine the occupancy status of each emulsion in images based on precipitated byproducts. The volume and occupancy data of the emulsions are then used to infer the nucleic acid concentration based on the Poisson distribution. deep-dLAMP can accurately predict the sizes and occupancy status of each emulsion and provide accurate measurements of nucleic acid concentrations with a limit of detection of 5.6 copies µl-1 and a dynamic range of 37.2 to 11000 copies µl-1 . In addition, deep-dLAMP shows robust performance under various parameters, such as the vortexing time and image qualities. Leveraging the state-of-the-art deep learning models, deep-dLAMP represents a significant advancement in digital nucleic acid tests by significantly reducing the instrument cost. We envision deep-dLAMP would be readily adopted by biomedical laboratories and be developed into a point-of-care digital nucleic acid test system.

Point-of-Care Blood Coagulation Assay Based on Dynamic Monitoring of Blood Viscosity Using Droplet Microfluidics.

Monitoring of the coagulation function has applications in many clinical settings. Routine coagulation assays in the clinic are sample-consuming and slow in turnaround. Microfluidics provides the opportunity to develop coagulation assays that are applicable in point-of-care settings, but reported works required bulky sample pumping units or costly data acquisition instruments. In this work, we developed a microfluidic coagulation assay with a simple setup and easy operation. The device continuously generated droplets of blood sample and buffer mixture and reported the temporal development of blood viscosity during coagulation based on the color appearance of the resultant droplets. We characterized the relationship between blood viscosity and color appearance of the droplets and performed experiments to validate the assay results. In addition, we developed a prototype analyzer equipped with simple fluid pumping and economical imaging module and obtained similar assay measurements. This assay showed great potential to be developed into a point-of-care coagulation test with practical impact.

Emerging biosensing technologies for improved diagnostics of COVID-19 and future pandemics.

Diagnostic tools play significant roles in the fight against COVID-19 and other pandemics. Existing tests, such as RT-qPCR, have limitations including long assay time, low throughput, inadequate sensitivity, and suboptimal portability. Emerging biosensing technologies hold the promise to develop tests that are rapid, highly sensitive, and suitable for point-of-care testing, which could significantly facilitate the testing of COVID-19. Despite that, practical applications of such biosensors in pandemics have yet to be achieved. In this review, we consolidate the newly developed diagnostic tools for COVID-19 using emerging biosensing technologies and discuss their application promise. In particular, we present nucleic acid tests and antibody tests of COVID-19 based on both conventional and emerging biosensing methods. We then provide perspectives on the existing challenges and potential solutions.