Single cell multiplexed assay for proteolytic activity using droplet microfluidics

Ng, Ethan; Miller, Miles A.; Jing, Tengyang; Chen, Chia-Hung

doi:10.1016/j.bios.2016.03.002

Cited by 64 publications

(48 citation statements)

References 33 publications

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“…This platform was developed to measure single‐cell secretions by encapsulating single cells and customized chemical sensors. For example, cell‐secreted enzyme activities have been investigated by conducting fluorescence resonance energy transfer (FRET)‐based sensors within droplets for biological sample profiling and diagnosis in a high throughput manner . However, most of the droplet‐based approaches only largely focused on metabolomic or FRET‐based detections since heterogeneous immunoassays that require washing steps are not well suited to the droplet format .…”

Section: Introductionmentioning

confidence: 99%

“…For example, cell‐secreted enzyme activities have been investigated by conducting fluorescence resonance energy transfer (FRET)‐based sensors within droplets for biological sample profiling and diagnosis in a high throughput manner . However, most of the droplet‐based approaches only largely focused on metabolomic or FRET‐based detections since heterogeneous immunoassays that require washing steps are not well suited to the droplet format . Although immunoassay format in droplets has been demonstrated using magnetic relocation approaches for immunoglobulin G (IgG)‐secreting cells, it suffers from high fluorescence background due to the absence of washing steps, resulting in low sensitivity.…”

Section: Introductionmentioning

confidence: 99%

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Smart Hydrogel Microfluidics for Single‐Cell Multiplexed Secretomic Analysis with High Sensitivity

Hsu

Wei

Guo

et al. 2018

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Secreted proteins determine a range of cellular functionalities correlated with human health and disease progression. Because of cell heterogeneity, it is essential to measure low abundant protein secretions from individual cells to determine single‐cell activities. In this study, an integrated platform consisting of smart hydrogel immunosensors for the sensitive detection of single‐cell secretions is developed. A single cell and smart hydrogel microparticles are encapsulated within a droplet. After incubation, target secreted proteins from the cell are captured in the smart hydrogel particle for immunoassay. The temperature‐induced volume phase transition of the hydrogel biosensor allows the concentration of analytes within the gel matrix to increase, enabling high‐sensitivity measurements. Distinct heterogeneity for live cell secretions is determined from 6000 cells within 1 h. This method is tested for low abundant essential secretions, such as interleukin‐6, interleukin‐8, and monocyte chemoattractant protein‐1 secretions of both suspended cells (HL60) and adherent cells (MCF7 and MDA‐MB‐231). This platform is highly flexible and can be used to simultaneously measure a wide range of clinically relevant cellular secretions; it thus represents a novel tool for precise biological assays.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Smart Hydrogel Microfluidics for Single‐Cell Multiplexed Secretomic Analysis with High Sensitivity

Hsu

Wei

Guo

et al. 2018

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“…In part to address such challenges, droplet microfluidics recently emerged as an approach for single-cell isolation, detection, and analysis (Kaminski et al, 2016; Yan et al, 2016). Similar to microchambers and microchannels, microfluidic droplets enable volume reduction, high local signal-to-background ratio, and accelerated time to detection (Boedicker et al, 2008; Ng et al, 2016; Tushar D. Rane et al, 2012). Critically, droplet-based platforms employ passive “flow-focusing” structures to generate droplets that can stochastically encapsulate single bacteria at around 100 to 1000 Hz and detection modules that can exclude empty droplets from analysis.…”

Section: Introductionmentioning

confidence: 99%

Accelerating bacterial growth detection and antimicrobial susceptibility assessment in integrated picoliter droplet platform

Kaushik

Hsieh

Chen

et al. 2017

Biosensors and Bioelectronics

113

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There remains an urgent need for rapid diagnostic methods that can evaluate antibiotic resistance for pathogenic bacteria in order to deliver targeted antibiotic treatments. Toward this end, we present a rapid and integrated single-cell biosensing platform, termed dropFAST, for bacterial growth detection and antimicrobial susceptibility assessment. DropFAST utilizes a rapid resazurin-based fluorescent growth assay coupled with stochastic confinement of bacteria in 20 pL droplets to detect signal from growing bacteria after 1 h incubation, equivalent to 2–3 bacterial replications. Full integration of droplet generation, incubation, and detection into a single, uninterrupted stream also renders this platform uniquely suitable for in-line bacterial phenotypic growth assessment. To illustrate the concept of rapid digital antimicrobial susceptibility assessment, we employ the dropFAST platform to evaluate the antibacterial effect of gentamicin on E. coli growth.

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“…7,8 Furthermore, droplet dimensions are especially suitable for analysis of small biological samples or even single cells, and adjusting operation times to handle variable numbers of droplets accommodates a range of sample volumes. 9–11 Notable recent work leverages droplet microfluidic systems as miniaturized instruments for immunoassays, 12,13 epigenetic analysis, 14 and directed molecular evolution, 15 among other applications.…”

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confidence: 99%

K-Channel: A Multifunctional Architecture for Dynamically Reconfigurable Sample Processing in Droplet Microfluidics

Doonan

Bailey

2017

Anal. Chem.

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By rapidly creating libraries of thousands of unique, miniaturized reactors, droplet microfluidics provides a powerful method for automating high-throughput chemical analysis. In order to engineer in-droplet assays, microfluidic devices must add reagents into droplets, remove fluid from droplets, and perform other necessary operations, each typically provided by a unique, specialized geometry. Unfortunately, modifying device performance or changing operations usually requires re-engineering the device among these specialized geometries, a time-consuming and costly process when optimizing in-droplet assays. To address this challenge in implementing droplet chemistry, we have developed the “K-channel,” which couples a cross-channel flow to the segmented droplet flow to enable a range of operations on passing droplets. K-channels perform reagent injection (0–100% of droplet volume), fluid extraction (0–50% of droplet volume), and droplet splitting (1:1–1:5 daughter droplet ratio). Instead of modifying device dimensions or channel configuration, adjusting external conditions, such as applied pressure and electric field, selects the K-channel process and tunes its magnitude. Finally, interfacing a device-embedded magnet allows selective capture of 96% of droplet-encapsulated superparamagnetic beads during 1:1 droplet splitting events at ~400 Hz. Addition of a second K-channel for injection (after the droplet splitting K-channel) enables integrated washing of magnetic beads within rapidly-moving droplets. Ultimately, the K-channel provides an exciting opportunity to perform many useful droplet operations across a range of magnitudes without requiring architectural modifications. Therefore, we envision the K-channel as a versatile, easy to use microfluidic component enabling diverse, in-droplet (bio)chemical manipulations.

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Single cell multiplexed assay for proteolytic activity using droplet microfluidics

Cited by 64 publications

References 33 publications

Smart Hydrogel Microfluidics for Single‐Cell Multiplexed Secretomic Analysis with High Sensitivity

Smart Hydrogel Microfluidics for Single‐Cell Multiplexed Secretomic Analysis with High Sensitivity

Accelerating bacterial growth detection and antimicrobial susceptibility assessment in integrated picoliter droplet platform

K-Channel: A Multifunctional Architecture for Dynamically Reconfigurable Sample Processing in Droplet Microfluidics

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