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Bifidobacteria represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest in bifidobacteria as a live biotic therapy, our understanding of colonization, host-microbe interactions, and the health-promoting effects of bifidobacteria is limited. To address these major knowledge gaps, we used a large-scale genetic approach to create a mutant fitness compendium in Bifidobacterium breve. First, we generated a high-density randomly barcoded transposon insertion pool and used it to determine fitness requirements during colonization of germ-free mice and chickens with multiple diets and in response to hundreds of in vitro perturbations. Second, to enable mechanistic investigation, we constructed an ordered collection of insertion strains covering 1,462 genes. We leveraged these tools to reveal community- and diet-specific requirements for colonization and to connect the production of immunomodulatory molecules to growth benefits. These resources will catalyze future investigations of this important beneficial microbe. 

Conventional cuvette-based and microfluidics-based electroporation approaches for bacterial gene delivery have distinct advantages, but they are typically limited to relatively small sample volumes, reducing their utility for applications requiring high throughput such as the generation of mutant libraries. Here, we present a scalable, large-scale bacterial gene delivery approach enabled by a disposable, user-friendly microfluidic electroporation device requiring minimal device fabrication and straightforward operation. We demonstrate that the proposed device can outperform conventional cuvettes in a range of situations, including across Escherichia coli strains with a range of electroporation efficiencies, and we use its large-volume bacterial electroporation capability to generate a library of transposon mutants in the anaerobic gut commensal Bifidobacterium longum . 

Manipulation of microparticles and bio-samples is a critical task in many research and clinical settings. Recently, acoustic based methods have garnered significant attention due to their relatively simple designs, and biocompatible and precise manipulation of small objects. Herein, we introduce a flexural wave based acoustofluidic manipulation platform that utilizes low-frequency (4–6 kHz) commercial buzzers to achieve dynamic particle concentration and translation in an open fluid well. The device has two primary modes of functionality, wherein particles can be concentrated in pressure nodes that are present on the bottom surface of the device, or particles can be trapped and manipulated in streaming vortices within the fluid domain; both of these functions result from flexural mode vibrations that travel from the transducers throughout the device. Throughout our research, we numerically and experimentally explored the wave patterns generated within the device, investigated the particle concentration phenomenon, and utilized a phase difference between the two transducers to achieve precision movement of fluid vortices and the entrapped particle clusters. With its simple, low-cost nature and open fluidic chamber design, this platform can be useful in many biological, biochemical, and biomedical applications, such as tumor spheroid generation and culture, as well as the manipulation of embryos. 

In this article, we demonstrate an acoustofluidic device for cell lysis using the acoustic streaming effects induced by acoustically oscillating sharp-edged structures. The acoustic streaming locally generates high shear forces that can mechanically rupture cell membranes. With the acoustic-streaming-derived shear forces, our acoustofluidic device can perform cell lysis in a continuous, reagent-free manner, with a lysis efficiency of more than 90% over a range of sample flow rates. We demonstrate that our acoustofluidic lysis device works well on both adherent and non-adherent cells. We also validate it using clinically relevant samples such as red blood cells infected with malarial parasites. Additionally, the unique capability of our acoustofluidic device was demonstrated by performing downstream protein analysis and gene profiling without additional washing steps post-lysis. Our device is simple to fabricate and operate while consuming a relatively low volume of samples. These advantages and other features including the reagent-free nature and controllable lysis efficiency make our platform valuable for many biological and biomedical applications, particularly for the development of point-of-care platforms. 

Whether reagents and samples need to be combined to achieve a desired reaction, or precise concentrations of solutions need to be mixed and delivered downstream, thorough mixing remains a critical step in many microfluidics-based biological and chemical assays and analyses. To achieve complete mixing of fluids in microfluidic devices, researchers have utilized novel channel designs or active intervention to facilitate mass transport and exchange of fluids. However, many of these solutions have a major limitation: their design inherently limits their operational throughput; that is, different designs work at specific flow rates, whether that be low or high ranges, but have difficulties outside of their tailored design regimes. In this work, we present an acoustofluidic mixer that is capable of achieving efficient, thorough mixing across a broad range of flow rates (20–2000 μL min −1 ) using a single device. Our mixer combines active acoustofluidic mixing, which is responsible for mixing fluids at lower flow rates, with passive hydrodynamic mixing, which accounts for mixing fluids at higher flow rates. The mechanism, functionality, and performance of our acoustofluidic device are both numerically and experimentally validated. Additionally, the real-world potential of our device is demonstrated by synthesizing polymeric nanoparticles with comparable sizes over a two-order-of-magnitude wide range of flow rates. This device can be valuable in many biochemical, biological, and biomedical applications. For example, using our platform, one may synthesize nanoparticles/nanomaterials at lower flow rates to first identify optimal synthesis conditions without having to waste significant amounts of reagents, and then increase the flow rate to perform high-throughput synthesis using the optimal conditions, all using the same single device and maintaining performance. 

Effectively isolating and categorizing large quantities of Caenorhabditis elegans ( C. elegans ) based on different phenotypes is important for most worm research, especially genetics. Here we present an integrated acoustofluidic chip capable of identifying worms of interest based on expression of a fluorescent protein in a continuous flow and then separate them accordingly in a high-throughput manner. Utilizing planar fiber optics as the detection unit, our acoustofluidic device requires no temporary immobilization of worms for interrogation/detection, thereby improving the throughput. Implementing surface acoustic waves (SAW) as the sorting unit, our device provides a contact-free method to move worms of interest to the desired outlet, thus ensuring the biocompatibility for our chip. Our device can sort worms of different developmental stages (L3 and L4 stage worms) at high throughput and accuracy. For example, L3 worms can be processed at a throughput of around 70 worms per min with a sample purity over 99%, which remains over 90% when the throughput is increased to around 115 worms per min. In our acoustofluidic chip, the time period to complete the detection and sorting of one worm is only 50 ms, which outperforms nearly all existing microfluidics-based worm sorting devices and may be further reduced to achieve higher throughput. 

Extracellular vesicles (EVs) and lipoproteins are abundant and co-exist in blood. Both have been proven to be valuable as diagnostic biomarkers and for therapeutics. However, EVs and lipoproteins are both on the submicron scale and overlap in size distributions. Conventional methods to separate EVs and lipoproteins are inefficient and time-consuming. Here we present an acoustofluidic-based separation technique that is based on the acoustic property differences of EVs and lipoproteins. By using the acoustofluidic technology, EVs and subgroups of lipoproteins are separated in a label-free, contact-free, and continuous manner. With its ability for simple, rapid, efficient, continuous-flow isolation, our acoustofluidic technology could be a valuable tool for health monitoring, disease diagnosis, and personalized medicine.