Immobilized Droplet Arrays in Thermosetting Oil for Dynamic Proteolytic Assays of Single Cells

Abstract

Matrix metalloproteinases (MMPs) play an important role in tumor progression. The study of dynamic MMPs activity at the single-cell level can dissect tumor heterogeneity in the time domain and facilitate finding out more efficient clinical solutions for tumor treatment. Due to the fluidity of the carrier oil, the existing droplet-based methods for single-cell MMP analysis rarely have the capability to track proteolytic assays in droplets continuously. Therefore, we describe a thermosetting oil for realtime monitoring of MMP assays in droplets, which can immobilize droplets by transforming into solid after droplet generation. The solidification of this oil can be accomplished in 33 min at 37 °C, basing on the hydrosilation of vinyl silicone oil and hydrosilicone oil without other inducers (e.g. UV, Ca 2+ ). Through monitoring the MMP assays of single cells, the reaction rates can be calculated according to real-time fluorescent curves, showing significant cell heterogeneity in MMP activity. Moreover, the dynamic MMP activity reveals that some of the A549 cells transiently secrete MMP. In conclusion, the thermosetting oil enables immobilize droplets to achieve real-time monitoring of single-cell proteolytic activity without impairing the flexibility of droplet microfluidics and has a potential in other cell-based assays for providing dynamic information at high resolutions.

Cell Culture in Microfluidic Droplets

“…For instance, the comparison of the glycerol produced by an adipocyte cell line and adipose-tissue derived cells has revealed a unique secretory oscillatory pattern from the tissue explant . In the same vein, the measurement of the kinetics of ECM proteolysis using FRET-based sensors in droplet revealed intrapopulation heterogeneity in terms of dynamics and level of MMP production within large populations of cancer cells. , …”

Single-cell droplet microfluidics for biomedical applications

“…7B). 135 This method can enable the realtime monitoring of single-cell proteolytic activity without com-…”

“…Secreted products such as matrix metalloproteinases (MMPs) represent promising drug targets and biomarker candidates and provide valuable information for evaluating cancer metastasis and, based on an analysis of their activity, allow for the screening of new drugs to regulate inflammatory responses and tumorigenesis. 133–135 Yu et al reported a microfluidic approach for the detection of MMP9 (a gelatinase subgroup of MMPs) enzyme activity in individual tumor cell droplets using a flow-focusing capillary microfluidic device (Fig. 7A).…”

“…7B). 135 This method can enable the real-time monitoring of single-cell proteolytic activity without compromising the flexibility of the droplet microfluidics, allowing for the calculation of a reaction rate according to a real-time fluorescence curve. The obvious cellular heterogeneities in MMP activity revealed by this process indicate its potential for use in other types of cell-based high-resolution dynamic information analysis.…”

A large-scale pico-droplet array for viable bacteria digital counting and dynamic tracking based on a thermosetting oil

“…Then, the diaphragm, that is, the solidified thermosetting oil with nanochannels between adjacent droplets, is used as a dialysis membrane to allow water to transfer freely between the droplets with different osmotic pressures and deform or even rupture. 41 Therefore, we determine the stability of the thermally solidified droplets by observing the migration of fluo- rescence in the droplets under a fluorescence microscope. After droplet generation, the sealed microfluidic chip was incubated at 37 °C and photographed at 2, 4, 6 and 18 hours to determine the percentage of intact droplets.…”