Manipulating Femtoliter to Picoliter Droplets by Pins for Single Cell Analysis and Quantitative Biological Assay

Abstract

Herein, we developed an automated and flexible system for performing miniaturized liquid-liquid reactions and assays in the femtoliter to picoliter range, by combining the contact printing and the droplet-based microfluidics techniques. The system mainly consisted of solid pins and an oil-covered hydrophilic micropillar array chip fixed on an automated x- y- z translation stage. A novel droplet manipulation mode called "dipping-depositing-moving" (DDM) was proposed, which was based on the programmable combination of three basic operations, dipping liquids and depositing liquids with the solid pins and moving the two-dimensional oil-covered hydrophilic pillar microchip. With the DDM mode, flexible generation and manipulation of small droplets with volumes down to 179 fL could be achieved. For overcoming the scale phenomenon specially appeared in picoliter-scale droplets, we used a design of water moat to protect the femtoliter to picoliter droplets from volume loss through the cover oil during the droplet generation, manipulation, reaction and assay processes. Moreover, we also developed a precise quantitative method, quantitative droplet dilution method, to accurately measure the volumes of femtoliter to picoliter droplets. To demonstrate its feasibility and adaptability, we applied the present system in the determination of kinetics parameter for matrix metalloproteinases (MMP-9) in 1.81 pL reactors and the measurement the activity of β-galactosidase in single cells (HepG2 cells) in picoliter droplet array. The ultrasmall volumes of the droplet reactors avoided the excessive dilution to the reaction solutions and enabled the highly sensitive measurement of enzyme activity in the single cell level.

Printing Life‐Inspired Subcellular Scale Compartments with Autonomous Molecularly Crowded Confinement

“…For these reasons, it is urgent to provide a strategy to combine crowding and confinement of biomolecular systems in artificial subcellular like fL‐scale compartments. Different approaches can produce compartments at this scale . Direct water‐in‐oil emulsification permits to obtain water droplets dispersed in oil typically varying in a wide range size (from micro‐ to nanoscale) .…”

“…In turn, this can lead to the nonspecific adsorption of biomolecules at the oil–water interface which can potentially affect the biological activity . Several reports have shown the possibility to produce fL‐droplets following printing approaches as different as pin‐printing, ultrafine nozzles printing, hydrodynamic droplet dispensing under electrical field guiding, droplets production within liquid environments, satellite droplets printing, liquid meniscus break‐up in a double‐orifice system, or spontaneous interfacial droplet fragmentation . However, these printing methods suffer from complex experimental set‐ups and require specialized hardware, making difficult their rapid implementation in chemistry laboratories.…”

Microfluidic Single‐Cell Omics Analysis

“…The technique can not only generate a great quantity of droplets within short time, but also afford independent compartments for single‐cell analysis to prevent cross contamination . Up to now, droplet operations including moving, merging, splitting, storing, thermo cycling and emulsion breaking, have been successfully processed, facilitating single‐cell analysis such as component detection and long‐term cell culture . Weitz's group utilized the droplet microfluidics to encapsulate single mammalian cells into 33 pL droplets for secretion detection.…”

Slip-driven microfluidic devices for nucleic acid analysis

“…[11][12][13][14] Moreover, arrays of droplets can be directly printed on a solid substrate with the assistance of pumps or robotic systems. [15][16][17] Since the demonstration of the SlipChip concept in 2009, 18 there have been different slip-driven microfluidic devices presented for a broad spectrum of applications, especially nucleic acid analysis. These microfluidic devices generally consist of at least two modules that can change relative position by a slipping motion to connect and disconnect fluidic paths (Fig.…”