CoID-LAMP: Color-Encoded, Intelligent Digital LAMP for Multiplex Nucleic Acid Quantification

Multiplex, digital nucleic acid tests have important biomedical applications, but existing methods mostly use fluorescent probes that are target-specific and difficult to optimize, limiting their widespread applications. Here, we report color-encoded, intelligent digital loop-mediated isothermal amplification (CoID-LAMP) for the coidentification of multiple nucleic acid targets. CoID-LAMP supplements different primer solutions with different dyes, generates primer droplets and sample droplets, and collectively pairs these two types of droplets in a microwell array device to perform LAMP. After imaging, the droplet colors were analyzed to decode the primer information, and the precipitate byproducts within droplets were detected to determine the target occupancy and calculate the concentrations. We first established an image analysis pipeline based on a deep learning algorithm for reliable droplet detection and validated the analytical performance in nucleic acid quantification. We then implemented CoID-LAMP using fluorescent dyes as the coding materials and established an 8-plex digital nucleic acid assay, confirming the reliable coding performance and the capability of multiplex nucleic acid quantification. We further implemented CoID-LAMP using brightfield dyes for a 4-plex assay, suggesting that the assay could be realized solely by brightfield imaging with minimal demand on the optics. Leveraging the advantages of droplet microfluidics in multiplexing and deep learning in intelligent image analysis, CoID-LAMP offers a useful tool for multiplex nucleic acid quantification

CoID-LAMP: Color-Encoded, Intelligent Digital LAMP for Multiplex Nucleic Acid Quantification

Multiplex, digital nucleic acid tests have important biomedical applications, but existing methods mostly use fluorescent probes that are target-specific and difficult to optimize, limiting their widespread applications. Here, we report color-encoded, intelligent digital loop-mediated isothermal amplification (CoID-LAMP) for the coidentification of multiple nucleic acid targets. CoID-LAMP supplements different primer solutions with different dyes, generates primer droplets and sample droplets, and collectively pairs these two types of droplets in a microwell array device to perform LAMP. After imaging, the droplet colors were analyzed to decode the primer information, and the precipitate byproducts within droplets were detected to determine the target occupancy and calculate the concentrations. We first established an image analysis pipeline based on a deep learning algorithm for reliable droplet detection and validated the analytical performance in nucleic acid quantification. We then implemented CoID-LAMP using fluorescent dyes as the coding materials and established an 8-plex digital nucleic acid assay, confirming the reliable coding performance and the capability of multiplex nucleic acid quantification. We further implemented CoID-LAMP using brightfield dyes for a 4-plex assay, suggesting that the assay could be realized solely by brightfield imaging with minimal demand on the optics. Leveraging the advantages of droplet microfluidics in multiplexing and deep learning in intelligent image analysis, CoID-LAMP offers a useful tool for multiplex nucleic acid quantification

Controlled Tubular Unit Formation from Collagen Film for Modular Tissue Engineering

Bottom-up or modular tissue engineering is one of the emerging approaches to prepare biomimetic constructs <i>in vitro</i>, involving fabrication of small tissue units as building blocks before assembling them into functional tissue constructs. Herein, we reported a microscale tissue engineering approach to generate tubular tissue units through cellular contractile force induced self-folding of cell-laden collagen films in a controllable manner. Self-folding of cell-laden collagen films was driven by film contraction resulted from intrinsic contractile property of adherent mammalian cells seeded in collagen films. We explored in detail independent effects of collagen gel concentration, cell density, and intrinsic cellular contractility on self-folding and tubular structure formation of cell-laden collagen films. Through both experiments and theoretical modeling, we further demonstrated the effectiveness of integrating ridge array structures onto the backside of collagen films in introducing structural anisotropy and thus controlling self-folding directions of collagen films. Our approach of using ridge array structures to introduce mechanical anisotropy and thus promote tubular tissue unit formation can be extended to other biomaterial systems and thus provide a simple yet effective way to prepare tubular tissue units for modular tissue engineering applications

Carbon Nanotube Strain Sensor Based Hemoretractometer for Blood Coagulation Testing

Coagulation monitoring is essential for perioperative care and thrombosis treatment. However, existing assays for coagulation monitoring have limitations such as a large footprint and complex setup. In this work, we developed a miniaturized device for point-of-care blood coagulation testing by measuring dynamic clot retraction force development during blood clotting. In this device, a blood drop was localized between a protrusion and a flexible force-sensing beam to measure clot retraction force. The beam was featured with micropillar arrays to assist the deposition of carbon nanotube films, which served as a strain sensor to achieve label-free electrical readout of clot retraction force in real time. We characterized mechanical and electrical properties of the force-sensing beam and optimized its design. We further demonstrated that this blood coagulation monitoring device could obtain results that were consistent with those using an imaging method and that the device was capable of differentiating blood samples with different coagulation profiles. Owing to its low fabrication cost, small size, and low consumption of blood samples, the blood coagulation testing device using carbon nanotube strain sensors holds great potential as a point-of-care tool for future coagulation monitoring

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

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

High-Throughput Functional Screening of Antigen-Specific T Cells Based on Droplet Microfluidics at a Single-Cell Level

The lack of an efficient method for the identification of tumor antigen-specific T cell receptors (TCRs) impedes the development of T cell-based cancer immunotherapies. Here, we introduce a droplet-based microfluidic platform for function-based screening and sorting of tumor antigen-specific T cells with high throughput. We built a reporter cell line by co-transducing the TCR library and reporter genes at the downstream of TCR signaling, and reporter cells fluoresced upon functionally binding with antigens. We co-encapsulated reporter cells and antigen-presenting cells in droplets to allow for stimulation on a single-cell level. Functioning reporter cells specific against the antigen were identified in the microfluidic channel based on the fluorescent signals of the droplets, which were immediately sorted out using dielectrophoresis. We validated the reporter system and sorting results using flow cytometry. We then performed single-cell RNA sequencing on the sorted cells to further validate this platform and demonstrate the compatibility with genetic characterizations. Our platform provides a means for precise and efficient T cell immunotherapy, and the droplet-based high-throughput TCR screening method could potentially facilitate immunotherapeutic screening and promote T cell-based anti-tumor therapies

High-Throughput Functional Screening of Antigen-Specific T Cells Based on Droplet Microfluidics at a Single-Cell Level

The lack of an efficient method for the identification of tumor antigen-specific T cell receptors (TCRs) impedes the development of T cell-based cancer immunotherapies. Here, we introduce a droplet-based microfluidic platform for function-based screening and sorting of tumor antigen-specific T cells with high throughput. We built a reporter cell line by co-transducing the TCR library and reporter genes at the downstream of TCR signaling, and reporter cells fluoresced upon functionally binding with antigens. We co-encapsulated reporter cells and antigen-presenting cells in droplets to allow for stimulation on a single-cell level. Functioning reporter cells specific against the antigen were identified in the microfluidic channel based on the fluorescent signals of the droplets, which were immediately sorted out using dielectrophoresis. We validated the reporter system and sorting results using flow cytometry. We then performed single-cell RNA sequencing on the sorted cells to further validate this platform and demonstrate the compatibility with genetic characterizations. Our platform provides a means for precise and efficient T cell immunotherapy, and the droplet-based high-throughput TCR screening method could potentially facilitate immunotherapeutic screening and promote T cell-based anti-tumor therapies

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