A bulk sub-femtoliter in vitro compartmentalization system using super-fine electrosprays.

Bineet Sharma,Yuzuru Takamura,Manish Biyani,Tatsuya Shimoda

doi:10.1038/srep26257

Bineet Sharma, Yuzuru Takamura + Show 2 more

Open Access

https://doi.org/10.1038/srep26257

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Abstract

The extreme miniaturization of biological and chemical assays in aqueous-droplet compartments enables spatiotemporal control for large-scale parallel experimentation and can thus permit new capabilities for “digitizing” directed molecular evolution methodologies. We report a remarkably facile bulk method to generate mega-scale monodisperse sub-femtoliter aqueous droplets by electrospray, using a prototype head with super-fine inkjet technology. Moreover, the electrostatic inkjet nozzle that injects the aqueous phase when immersed within an immiscible phase (an optimized oil/surfactant mixture) has the advantage of generating cell-like sub-femtoliter compartments for biomolecule encapsulation and successive biological and chemical reactions. Sub-femtoliter droplets of both liquid (water-in-oil, volumes ranging from 0.2 to 6.4 fL) and gel bead (agarose-in-oil, volume ranging from 0.3 to 15.6 fL) compartments with average sizes of 1.3 μm and 1.5 μm, respectively, were successfully generated using an inkjet nozzle at a speed of more than 105 droplets per second. We demonstrated the applicability of this system by synthesizing fluorescent proteins using a cell-free expression system inside electrosprayed sub-femtoliter droplets at an accelerated rate, thereby extending the utility of in vitro compartmentalization with improved analytical performance for a top-down artificial cellular system.

Highlights

Orders of magnitude larger than the upper limit of the microfluidic droplet numbers
The production rate of an in vitro gene expression system was reported to be inversely proportional to the radius of the droplets[4,19] and was strongly affected by the nature of the oil/surfactant used for droplet generation in bulk emulsion[20,21]
We developed a simple new platform that combines immersed electrospray with in vitro compartmentalization (IVC) by using super-fine inkjet technology (SIJ Technology, Inc.) for the ultra-rapid generation of water-in-oil droplets

Summary

Introduction

Orders of magnitude larger than the upper limit of the microfluidic droplet numbers. In a different approach, microfabricated arrays of pico/femtoliter chambers using quartz or poly(dimethylsiloxane) (PDMS) have been developed but again are technically limited to low throughput (on a kilo-to-mega scale)[15,16,17,18]. The electrospray is caused by an electric force, which acts on the surface of a liquid at a capillary nozzle outlet to form the Taylor cone; when the electric field force exceeds the surface tension, a jet elongates into a fine filament that spontaneously breaks up into fine and relatively monodisperse droplets that are much smaller than the orifice diameter This technique has been widely recognized, including by a Nobel Prize in chemistry; its use in an immersed mode is still limited, mainly because of a lack of knowledge about the phenomenon when the dispersing fluid is immersed in another immiscible liquid[23]. The methodologies developed here are straightforward to set up and should be useful for quantifying the yield of active proteins and for controlling gene expression noise in in vitro droplets for directed molecular evolution studies

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Results

Conclusion