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1. Single-Cell Infection of Influenza A Virus Using Drop-Based Microfluidics

   https://doi.org/10.1128/spectrum.00993-22  

   Loveday, Emma Kate; Sanchez, Humberto S.; Thomas, Mallory M.; Chang, Connie B. (October 2022, Microbiology Spectrum)

   Kolawole, Abimbola O. (Ed.)

   ABSTRACT Drop-based microfluidics has revolutionized single-cell studies and can be applied toward analyzing tens of thousands to millions of single cells and their products contained within picoliter-sized drops. Drop-based microfluidics can shed insight into single-cell virology, enabling higher-resolution analysis of cellular and viral heterogeneity during viral infection. In this work, individual A549, MDCK, and siat7e cells were infected with influenza A virus (IAV) and encapsulated into 100-μm-size drops. Initial studies of uninfected cells encapsulated in drops demonstrated high cell viability and drop stability. Cell viability of uninfected cells in the drops remained above 75%, and the average drop radii changed by less than 3% following cell encapsulation and incubation over 24 h. Infection parameters were analyzed over 24 h from individually infected cells in drops. The number of IAV viral genomes and infectious viruses released from A549 and MDCK cells in drops was not significantly different from bulk infection as measured by reverse transcriptase quantitative PCR (RT-qPCR) and plaque assay. The application of drop-based microfluidics in this work expands the capacity to propagate IAV viruses and perform high-throughput analyses of individually infected cells. IMPORTANCE Drop-based microfluidics is a cutting-edge tool in single-cell research. Here, we used drop-based microfluidics to encapsulate thousands of individual cells infected with influenza A virus within picoliter-sized drops. Drop stability, cell loading, and cell viability were quantified from three different cell lines that support influenza A virus propagation. Similar levels of viral progeny as determined by RT-qPCR and plaque assay were observed from encapsulated cells in drops compared to bulk culture. This approach enables the ability to propagate influenza A virus from encapsulated cells, allowing for future high-throughput analysis of single host cell interactions in isolated microenvironments over the course of the viral life cycle. 

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2. DNAzyme-powered nucleic acid release from solid supports

   https://doi.org/10.1039/c9cc07790a  

   Cao, Ting; Wang, Yongcheng; Tao, Ye; Zhang, Lexiang; Zhou, Ying-Lin; Zhang, Xin-Xiang; Heyman, John A.; Weitz, David A. (January 2020, Chemical Communications)

   null (Ed.)

   Here, we demonstrate use of a Mg 2+ -dependent, site-specific DNA enzyme (DNAzyme) to cleave oligos from polyacrylamide gel beads, which is suitable for use in drop-based assays. We show that cleavage efficiency is improved by use of a tandem-repeat cleavage site. We further demonstrate that DNAzyme-released oligos function as primers in reverse transcription of cell-released mRNA. 

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3. Single-cell herpes simplex virus type 1 infection of neurons using drop-based microfluidics reveals heterogeneous replication kinetics

   https://doi.org/10.1126/sciadv.adk9185  

   Fredrikson, Jacob P; Domanico, Luke F; Pratt, Shawna L; Loveday, Emma K; Taylor, Matthew P; Chang, Connie B (March 2024, Science Advances)

   Single-cell analyses of viral infections reveal heterogeneity that is not detected by traditional population-level studies. This study applies drop-based microfluidics to investigate the dynamics of herpes simplex virus type 1 (HSV-1) infection of neurons at the single-cell level. We used micrometer-scale Matrigel beads, termed microgels, to culture individual murine superior cervical ganglia (SCG) neurons or epithelial cells. Microgel-cultured cells are encapsulated in individual media-in-oil droplets with a dual–fluorescent reporter HSV-1, enabling real-time observation of viral gene expression and replication. Infection within drops revealed that the kinetics of initial viral gene expression and replication were dependent on the inoculating dose. Notably, increasing inoculating doses led to earlier onset of viral gene expression and more frequent productive viral replication. These observations provide crucial insights into the complexity of HSV-1 infection in neurons and emphasize the importance of studying single-cell outcomes of viral infection. These techniques for cell culture and infection in drops provide a foundation for future virology and neurobiology investigations. 

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4. INgen: Intracellular Genomic DNA Amplification for Downstream Applications in Sequencing and Sorting

   https://doi.org/10.1101/2025.03.03.641299  

   Crossland, P; Schmidlin, K; Valli-Doherty, I; Brettner, L; Geiler-Samerotte, K (March 2025, bioRxiv)

   Abstract Here, we introduce intracellular genomic amplification (INgen), a method that harnesses the cell membrane as a natural reaction chamber for DNA amplification, enabling downstream sequencing and cell sorting. Unlike traditional single-cell techniques, INgen utilizes a strand-displacing, isothermal polymerase to amplify DNAwithinfixed, permeabilized cells while maintaining the cell’s structural integrity. This approach overcomes challenges associated with both typical single-cell DNA sequencing and hindrances encountered when previously attempting to sequence genetic material from fixed microbial cells, allowing amplification of genomic regions up to 100 kb and sequencing of whole genomes from diverse cell types, includingS. cerevisiae, B. subtilis, andE. coli. Additionally, INgen can be adapted for targeted DNA enrichment using biotinylated primers and for fluorescence-based cell sorting. 

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5. A seed and bridge layer method for inkjet printing of narrow traces on receding ink-substrate combinations

   https://doi.org/10.1088/2058-8585/ad1051  

   Pratt, Nicholas; Rao, Pratap M (December 2023, Flexible and Printed Electronics)

   Abstract Inkjet printing of electronic materials is of interest for digital printing of flexible electronics and sensors, but the width of the inkjet-printed lines is still large, limiting device size and performance. Decreasing the drop volume, increasing the drop spacing, and increasing the ink-substrate contact angle are all approaches by which the line width can be lowered, however these approaches are limited by the nozzle geometry, ink coalescence and bead instabilities, and contact angle hysteresis, respectively. Here we demonstrate a novel approach for stable inkjet printing of very narrow lines on ink-substrate combinations with a high contact angle, utilizing the de-wetting of the ink due to the decreased contact angle hysteresis. After printing and drying an initial layer of disconnected seed drops of silver nanoparticle ink, we print an additional layer of bridging drops of the same ink in between the dried seed drops. The bridging drops expand to touch the dried seed drops and then retract into a line, due to the pinning of the wet ink on the dried seed ink but not on the substrate, forming a continuous silver trace. The trace width is decreased from 60μm with a traditional printing approach down to 12.6μm with this seed-bridge approach. The electrical conductivity of the silver trace is similar to that of a conventionally printed trace. Due to poor adhesion on the print substrate, the trace was transferred to a separate polymer substrate with a simple hot-pressing procedure, which preserves the electrical conductivity of the trace. 

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