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1. Droplet Microfluidic Technology for the Early and Label-Free Isolation of Highly-Glycolytic, Activated T-Cells

   https://doi.org/10.3390/mi13091442  

   Zielke, Claudia ; Gutierrez Ramirez, Adriana J. ; Voss, Kelsey ; Ryan, Maya S. ; Gholizadeh, Azam ; Rathmell, Jeffrey C. ; Abbyad, Paul ( September 2022 , Micromachines)

   A label-free, fixation-free and passive sorting method is presented to isolate activated T-cells shortly after activation and prior to the display of activation surface markers. It uses a recently developed sorting platform dubbed “Sorting by Interfacial Tension” (SIFT) that sorts droplets based on pH. After polyclonal (anti-CD3/CD28 bead) activation and a brief incubation on chip, droplets containing activated T-cells display a lower pH than those containing naive cells due to increased glycolysis. Under specific surfactant conditions, a change in pH can lead to a concurrent increase in droplet interfacial tension. The isolation of activated T-cells on chip is hence achieved as flattened droplets are displaced as they encounter a micro-fabricated trench oriented diagonally with respect to the direction of flow. This technique leads to an enrichment of activated primary CD4+ T-cells to over 95% from an initial mixed population of naive cells and cells activated for as little as 15 min. Moreover, since the pH change is correlated to successful activation, the technique allows the isolation of T-cells with the earliest activation and highest glycolysis, an important feature for the testing of T-cell activation modulators and to determine regulators and predictors of differentiation outcomes. 

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2. Method for Passive Droplet Sorting after Photo-Tagging

   https://doi.org/10.3390/mi11110964  

   Dobson, Chandler ; Zielke, Claudia ; Pan, Ching ; Feit, Cameron ; Abbyad, Paul ( November 2020 , Micromachines)

   null (Ed.)

   We present a method to photo-tag individual microfluidic droplets for latter selection by passive sorting. The use of a specific surfactant leads to the interfacial tension to be very sensitive to droplet pH. The photoexcitation of droplets containing a photoacid, pyranine, leads to a decrease in droplet pH. The concurrent increase in droplet interfacial tension enables the passive selection of irradiated droplets. The technique is used to select individual droplets within a droplet array as illuminated droplets remain in the wells while other droplets are eluted by the flow of the external oil. This method was used to select droplets in an array containing cells at a specific stage of apoptosis. The technique is also adaptable to continuous-flow sorting. By passing confined droplets over a microfabricated trench positioned diagonally in relation to the direction of flow, photo-tagged droplets were directed toward a different chip exit based on their lateral movement. The technique can be performed on a conventional fluorescence microscope and uncouples the observation and selection of droplets, thus enabling the selection on a large variety of signals, or based on qualitative user-defined features. 

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3. Mass Activated Droplet Sorting (MADS) Enables High‐Throughput Screening of Enzymatic Reactions at Nanoliter Scale

   https://doi.org/10.1002/anie.201913203  

   Holland‐Moritz, Daniel A. ; Wismer, Michael K. ; Mann, Benjamin F. ; Farasat, Iman ; Devine, Paul ; Guetschow, Erik D. ; Mangion, Ian ; Welch, Christopher J. ; Moore, Jeffrey C. ; Sun, Shuwen ; et al ( January 2020 , Angewandte Chemie International Edition)

   Microfluidic droplet sorting enables the high‐throughput screening and selection of water‐in‐oil microreactors at speeds and volumes unparalleled by traditional well‐plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for high‐throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESI‐MS). Droplets are split, one portion is analyzed by ESI‐MS, and the second portion is sorted based on the MS result. Throughput of 0.7 samples s⁻¹is achieved with 98 % accuracy using a self‐correcting and adaptive sorting algorithm. We use the system to screen ≈15 000 samples in 6 h and demonstrate its utility by sorting 25 nL droplets containing transaminase expressed in vitro. Label‐free ESI‐MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.

    

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4. Mass Activated Droplet Sorting (MADS) Enables High‐Throughput Screening of Enzymatic Reactions at Nanoliter Scale

   https://doi.org/10.1002/ange.201913203  

   Holland‐Moritz, Daniel A. ; Wismer, Michael K. ; Mann, Benjamin F. ; Farasat, Iman ; Devine, Paul ; Guetschow, Erik D. ; Mangion, Ian ; Welch, Christopher J. ; Moore, Jeffrey C. ; Sun, Shuwen ; et al ( January 2020 , Angewandte Chemie)

   Microfluidic droplet sorting enables the high‐throughput screening and selection of water‐in‐oil microreactors at speeds and volumes unparalleled by traditional well‐plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for high‐throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESI‐MS). Droplets are split, one portion is analyzed by ESI‐MS, and the second portion is sorted based on the MS result. Throughput of 0.7 samples s⁻¹is achieved with 98 % accuracy using a self‐correcting and adaptive sorting algorithm. We use the system to screen ≈15 000 samples in 6 h and demonstrate its utility by sorting 25 nL droplets containing transaminase expressed in vitro. Label‐free ESI‐MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.

    

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5. A High-Throughput Microfluidic Cell Sorter Using a Three-Dimensional Coupled Hydrodynamic-Dielectrophoretic Pre-Focusing Module

   https://doi.org/10.3390/mi14101813  

   Aghaamoo, Mohammad ; Cardenas-Benitez, Braulio ; Lee, Abraham P. ( October 2023 , Micromachines)

   Beskok, A. (Ed.)

   Dielectrophoresis (DEP) is a powerful tool for label-free sorting of cells, even those with subtle differences in morphological and dielectric properties. Nevertheless, a major limitation is that most existing DEP techniques can efficiently sort cells only at low throughputs (<1 mL h−1). Here, we demonstrate that the integration of a three-dimensional (3D) coupled hydrodynamic-DEP cell pre-focusing module upstream of the main DEP sorting region enables cell sorting with a 10-fold increase in throughput compared to conventional DEP approaches. To better understand the key principles and requirements for high-throughput cell separation, we present a comprehensive theoretical model to study the scaling of hydrodynamic and electrostatic forces on cells at high flow rate regimes. Based on the model, we show that the critical cell-to-electrode distance needs to be ≤10 µm for efficient cell sorting in our proposed microfluidic platform, especially at flow rates ≥ 1 mL h−1. Based on those findings, a computational fluid dynamics model and particle tracking analysis were developed to find optimum operation parameters (e.g., flow rate ratios and electric fields) of the coupled hydrodynamic-DEP 3D focusing module. Using these optimum parameters, we experimentally demonstrate live/dead K562 cell sorting at rates as high as 10 mL h−1 (>150,000 cells min−1) with 90% separation purity, 85% cell recovery, and no negative impact on cell viability.

    

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