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Sorting cells while maintaining their viability for further processing or analysis is an essential step in a variety of biological processes ranging from early diagnostics to cell therapy. Sorting techniques such as fluorescence-activated cell sorting (FACS) have evolved considerably and provide standard ways of sorting. Nevertheless, the search for compact, integrated, efficient, and high throughput microfluidic sorting platforms continues due to challenges such as cost, cell viability, and biosafety. In our previous work, we introduced a technology with the potential to become such a platform: the bubble-jet sorter. It is a silicon-based sorter chip relying on cell deflection through micro vapor bubble formation. In this work, we present a new version of the sorter chip that emphasizes durability and continuous sorting operation. To characterize the sorter, we first focus on the technical performance and show a sorter lifetime that repeatedly exceeds 80 million actuation cycles. In addition, we show continuous operation at high firing rates, but also discuss limitations due to heat buildup. In a second step, we present continuous sorting runs of millions of beads and CD3 positive T cells at rates surpassing 1000 sorting events per second, while maintaining high purity (>90%) and recovery (>85%). Dedicated viability tests show that the gentle sorting process maintains cell viability in this closed, aerosol-free device. The remarkable combination of high lifetime, sorting rate, and sorting efficiency, along with the potential for on-chip parallelization show the promise of this technology to meet the growing demand for large-scale sample isolation in drug and immunotherapy development.

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Nanoparticles are a promising solution for delivery of a wide range of medicines and vaccines. Optimizing their design depends on being able to resolve, understand, and predict biophysical and therapeutic properties, as a function of design parameters. While existing tools have made great progress, gaps in understanding remain because of the inability to make detailed measurements of multiple correlated properties. Typically, an average measurement is made across a heterogeneous population, obscuring potentially important information. In this work, we develop and apply a method for characterizing nanoparticles with single-particle resolution. We use convex lens-induced confinement (CLiC) microscopy to isolate and quantify the diffusive trajectories and fluorescent intensities of individual nanoparticles trapped in microwells for long times. First, we benchmark detailed measurements of fluorescent polystyrene nanoparticles against prior data to validate our approach. Second, we apply our method to investigate the size and loading properties of lipid nanoparticle (LNP) vehicles containing silencing RNA (siRNA), as a function of lipid formulation, solution pH, and drug-loading. By taking a comprehensive look at the correlation between the intensity and size measurements, we gain insights into LNP structure and how the siRNA is distributed in the LNP. Beyond introducing an analytic for size and loading, this work allows for future studies of dynamics with single-particle resolution, such as LNP fusion and drug-release kinetics. The prime contribution of this work is to better understand the connections between microscopic and macroscopic properties of drug-delivery vehicles, enabling and accelerating their discovery and development.

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New technologies have powered rapid advances in cellular imaging, genomics and phenotypic analysis in life sciences. However, most of these methods operate at sample population levels and provide statistical averages of aggregated data that fail to capture single-cell heterogeneity, complicating drug discovery and development. Here we demonstrate a new single-cell approach based on convex lens-induced confinement (CLiC) microscopy. We validated CLiC on yeast cells, demonstrating subcellular localization with an enhanced signal-to-noise and fluorescent signal detection sensitivity compared with traditional imaging. In the live-cell CLiC assay, cellular proliferation times were consistent with flask culture. Using methotrexate, we provide drug response data showing a fivefold cell size increase following drug exposure. Taken together, CLiC enables high-quality imaging of single-cell drug response and proliferation for extended observation periods.

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Biomolecular condensates formed by liquid-liquid phase separation of proteins and nucleic acids have been recently discovered to be prevalent in biology. These dynamic condensates behave like biochemical reaction vessels, but little is known about their structural organization and biophysical properties, which are likely related to condensate size. Thus, it is critical that we study them on scales found in vivo. However, previous in vitro studies of condensate assembly and physical properties have involved condensates up to 1000 times larger than those found in vivo. Here, we apply confinement microscopy to visualize condensates and control their sizes by creating appropriate confinement length scales relevant to the cell environment. We observe anomalous diffusion of probe particles embedded within confined condensates, as well as heterogeneous dynamics in condensates formed from PEG/dextran and in ribonucleoprotein complexes of RNA and the RNA-binding protein Dhh1. We propose that the observed non-Gaussian dynamics indicate a hopping diffusion mechanism inside condensates. We also observe that, for dextran-rich condensates, but not for ribonucleo condensates, probe particle diffusion depends on condensate size.

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On-chip microvalves regulate electrical and fluidic access to an array of nanopores integrated within microfluidic networks. This configuration allows for on-chip sequestration of biomolecular samples in various flow channels and analysis by independent nanopores.

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Nanopore arrays are fabricated by controlled dielectric breakdown (CBD) in solid-state membranes integrated within polydimethylsiloxane (PDMS) microfluidic devices. This technique enables the scalable production of independently addressable nanopores. By confining the electric field within the microfluidic architecture, nanopore fabrication is precisely localized and electrical noise is significantly reduced. Both DNA and protein molecules are detected to validate the performance of this sensing platform.

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We present gravitational field-flow fractionation and hydrodynamic chromatography of colloids eluting through 18 µm microchannels. Using video microscopy and mesoscopic simulations, we investigate the average retention ratio of colloids with both a large specific weight and neutral buoyancy. We consider the entire range of colloid sizes, including particles that barely fit in the microchannel and nanoscopic particles. Ideal theory predicts four operational modes, from hydrodynamic chromatography to Faxén-mode field-flow fractionation. We experimentally demonstrate, for the first time, the existence of the Faxén-mode field-flow fractionation and the transition from hydrodynamic chromatography to normal-mode field-flow fractionation. Furthermore, video microscopy and simulations show that the retention ratios are largely reduced above the steric-inversion point, causing the variation of the retention ratio in the steric- and Faxén-mode regimes to be suppressed due to increased drag. We demonstrate that theory can accurately predict retention ratios if hydrodynamic interactions with the microchannel walls (wall drag) are added to the ideal theory. Rather than limiting the applicability, these effects allow the microfluidic channel size to be tuned to ensure high selectivity. Our findings indicate that particle velocimetry methods must account for the wall-induced lag when determining flow rates in highly confining systems.

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