Name: a femtoliter droplet array for massively parallel protein synthesis from single dna molecules
Uploaded: 2020-06-20T13:00:00
Description: Watch this Scientific Journal Video about A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules at JoVE.com

This work was supported by JSPS KAKENHI grant number JP18K14260 and the budget of Japan Agency for Marine-Earth Science and Technology. We thank Shigeru Deguchi (JAMSTEC) and Tetsuro Ikuta (JAMSTEC) for providing the characterization facilities. We thank Ken Takai (JAMSTEC) for commercial software support. The microfabrication was conducted at Takeda Sentanchi Supercleanroom, The University of Tokyo, supported by &#34;Nanotechnology Platform Program&#34; of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, Grant Number JPMXP09F19UT0087.

1. Microfabrication of the femtoliter microchamber array substrate
NOTE: Conduct the following microfabrication experiment in a cleanroom. Wear gloves and a cleanroom suit before entering the cleanroom.
<ol>
	<li>Cleaning cover glass substrate
	<ol>
		<li>Set the cover glass on a cover glass staining rack. Sonicate the cover glass in 8 M sodium hydroxide (NaOH) for 15 min at room temperature (RT). 
		CAUTION: NaOH in high concentrations is highly dangerous to skin and eye. Gently handle...

The highly quantitative measurement based on the highly uniform, stable, and biocompatible droplets in FemDA enabled the discrete distribution, the unique feature of our study differing from others. We systematically optimized and detailed the microfabrication and droplet formation processes in this paper. There are several critical steps in the established protocol.
First, the uniform coating of highly viscous CYTOP polymer on the rectangular thin glass substrate largely determines the qualit...

Researchers use reactors to carry out bio/chemical reactions. There are significant efforts that have been made to reduce the size of the reactor and increase the experimental throughput in order to lower the reagent consumption while improving the work efficiency. Both aspects aim to liberate researchers from a heavy workload, decreasing the cost and speeding up research and development. We have a clear historical roadmap about the development of the reactor technologies from the viewpoint of reaction volumes and throughput: single beakers/flask/test-tubes, milliliter tubes, microliter tubes, microliter 8-tube strips, microliter 96/384/1536-well plate, and microfluidic nanoliter/picoliter/femtoliter reactors1,2,3,4,5,6,7. Analogous to downsizing the feature size of transistors on integrated circuit chips in the semiconductor industry in the past decades, bio/chemical microreactors are going through volume reduction and system integration. Such small-scale tools have had a profound impact on cell-based or cell-free synthetic biology, biomanufacturing, and high-throughput prototyping and screening8,9,10,11,12. This paper describes our recent effort on the development of a unique droplet array technology and demonstrates its application in CFPS13, a fundamental technology for synthetic biology and molecular screening communities14. In particular, we intentionally provide an optimized and low-cost protocol to make the FemDA device accessible to everyone. The low-cost and easy-to-handle protocol for the miniaturized device would contribute to the educational purposes of universities and help spread the technology.
FemDA prepares femtoliter droplets at an ultrahigh density of 106 per 1 cm2 on a planar glass substrate. We coated a hydrophobic polymer, CYTOP15, on the glass substrate and selectively etched (removed) CYTOP at predefined positions to generate a microchamber array on the substrate. Thus, the resulting microchamber is composed of a hydrophobic sidewall (CYTOP) and a hydrophilic bottom (glass). When sequentially flowing water and oil over the patterned surface, the water can be trapped and sealed into the microchambers. The hydrophilic-in-hydrophobic structure is vital for repelling water outside the microchambers, isolating individual microreactors, and retaining a tiny aqueous solution inside the femtoliter space. The unique property was successfully applied for the preparation of water-in-oil droplets and lipid bilayer microcompartments16,17. Compared to the prototype device16, we first optimized the microfabrication process to realize a complete removal of the CYTOP polymer as well as a full exposure of the glass bottom. CYTOP is a special fluoropolymer featuring extremely low surface tension (19 mN/m) lower than that of conventional microreactor materials such as glass, plastics, and silicone. Its good optical, electrical, and chemical performance have already been utilized in surface treatment of microfluidic devices18,19,20,21,22,23,24. In the FemDA system, to achieve good wetting of the oil on the CYTOP surface, the surface tension of the oil must be lower than that of the solid surface25. Otherwise, the liquid oil in contact with the solid surface tends to become spherical rather than spreading over the surface. Besides, we found that some popular perfluorocarbon oils (e.g., 3M FC-40)16 and hydrofluoroether oils (e.g., 3M Novec series) can dissolve CYTOP as a result of the amorphous morphology of CYTOP, which is fatal to quantitative measurement and would be questionable in terms of cross-contamination among droplets. Fortunately, we identified a biocompatible and environmentally friendly oil exhibiting lower (&#60; 19 mN/m) surface tension13. We also found a new surfactant that can dissolve in the selected oil and function in a low concentration (0.1%, at least 10-fold lower than previously reported popular ones26,27)13. The resulting water/oil interface can be stabilized by the surfactant. Because of the high evaporation rate of the oil, following the flush with the oil, we applied another biocompatible and environmentally friendly oil to replace the first one to seal the microchambers. We call the first oil (ASAHIKLIN&#160;AE-3000 with 0.1 wt % SURFLON S-386) the &#8220;flush oil&#8221; and the second oil (Fomblin Y25) the &#8220;sealing oil,&#8221; respectively.
The two-step oil-sealing strategy can realize a robust formation of the femtoliter droplet array within minutes and without sophisticated instrumentation. Due to the evaporation problem, it has been considered challenging to generate microreactors smaller than picoliter volumes28. FemDA addressed this issue by systematically optimizing the materials and processes used for the preparation of microreactors/droplets. Several noteworthy features of the resulting droplets include the high uniformity (or monodispersity), stability, and biocompatibility at the femtoliter scale. The coefficient of variation (CV) of the droplet volume is only 3% (without vignetting correction for the microscopic images), the smallest CV among droplet platforms in the world, which ensures a highly parallel and quantitative measurement. The femtoliter droplet is stable for at least 24 hours without cross-contamination among droplets at room temperature, which is valuable for a reliable time-course measurement. Regarding the biocompatibility, we succeeded in synthesizing various proteins from a single-copy template DNA in the femtoliter droplet, which had previously been considered difficult or inefficient29,30. It would be worthy of elucidating why some proteins capable of being synthesized in the FemDA cannot be synthesized in other droplet systems. FemDA was not merely a technical advancement, but also realized an unprecedentedly quantitative measurement that can correlate the protein yield (as reflected by the fluorescence intensity of the droplet) to the number of template DNA molecules in each droplet. As a result, the histogram of the fluorescence intensity of droplets from FemDA-based CFPS showed a discrete distribution that can be nicely fitted by a sum of Gaussian distributions of equal peak-to-peak intervals. Moreover, the probability of occurrence of droplets containing different numbers of DNA molecules was a perfect fit to a Poisson distribution31. Thus, the different protein yield in each droplet can be normalized based on the discrete distribution. This critical feature allows us to separate the enzymatic activity information from the apparent intensity, that has not been available with other microreactor platforms yet. Existing microfluidic cell/droplet sorting systems are skilled in fully automatic sorting and good at concentrating samples but sometimes can only output a relatively broad or long-tailed histogram in the analytical aspect32,33. Our highly quantitative and biocompatible FemDA system sets a new benchmark and a high analytical standard in the field of microreactor development.
The oils and surfactants that could be used for the preparation of droplets are still very limited34. The combination of ASAHIKLIN AE-3000 and SURFLON S-386 established in FemDA is a new member of the growing arsenal of the physiochemical interface between the aqueous phase and the oil phase13. The new interface in FemDA is physically stable, chemically inert, and biologically compatible with the complex transcription, translation, and post-translational modification machinery for many sorts of proteins13. It would be rather attractive to find a protein that cannot be synthesized in the droplet settings instead. Besides, the cost saving of reagents is more evident in the femtoliter droplet system than that in nanoliter and picoliter reactor systems35,36. In particular, there would often be a large dead volume, which is mainly caused by tubing or external supplies, in microfluidic droplet generation systems but not in our FemDA. The array format is also favored by repeated and detailed microscopic characterization (similar to so-called high-content analysis) for every single reactor37, rather than only a single snapshot for a fast-moving object. The femtoliter scale enabled the integration of over one million reactors on a finger-sized area, while the same number of nanoliter reactors (if it exists) requires over square meter area, which would be undoubtedly impractical to manufacture or use such system.

<table>
	<tbody>
		<tr>
			<td>(3-aminopropyl)triethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>440140</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Terumo</td>
			<td>SS-01T</td>
			<td></td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Kanto Chemical</td>
			<td>EL grade</td>
			<td>EL: for electronic use.</td>
		</tr>
		<tr>
			<td>3D laser scanning confocal microscope</td>
			<td>Lasertec</td>
			<td>OPTELICS HYBRID</td>
			<td>Other similar microscopes (e.g., Keyence VK-X1000, Olympus LEXT OLS5000) are also applicable.</td>
		</tr>
		<tr>
			<td>50 mL syringe</td>
			<td>Terumo</td>
			<td>SS-50LZ</td>
			<td></td>
		</tr>
		<tr>
			<td>6,8-difluoro-4-methylumbelliferyl phosphate</td>
			<td>Thermo Fisher Scientific</td>
			<td>D6567</td>
			<td>Prepare a 5 mM stock solution in dimethyl sulfoxide</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Kanto Chemical</td>
			<td>EL grade</td>
			<td>EL: for electronic use. 
			Purity 99.8%.</td>
		</tr>
		<tr>
			<td>Air blower</td>
			<td>Hozan</td>
			<td>Z-263</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum block</td>
			<td>BIO-BIK</td>
			<td>AB-24M-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum microtube stand</td>
			<td>BIO-BIK</td>
			<td>AB-136C</td>
			<td></td>
		</tr>
		<tr>
			<td>ASAHIKLIN AE-3000</td>
			<td>AGC</td>
			<td>(Test sample)</td>
			<td>Free test sample may be available upon inquiry to AGC.</td>
		</tr>
		<tr>
			<td>BEMCOT PS-2 wiper</td>
			<td>Ozu</td>
			<td>028208</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy punch with plunger</td>
			<td>Kai</td>
			<td>BPP-10F</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Matsunami Glass</td>
			<td>No. 1 (24 mm &#215; 32 mm, 0.13~0.17 mm thickness)</td>
			<td>Size-customized.</td>
		</tr>
		<tr>
			<td>Cover glass staining rack</td>
			<td>Nakayama</td>
			<td>803-131-11</td>
			<td></td>
		</tr>
		<tr>
			<td>CRECIA TechnoWipe clean wiper</td>
			<td>Nippon Paper Crecia</td>
			<td>C100-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutting mat</td>
			<td>GE Healthcare</td>
			<td>WB100020</td>
			<td></td>
		</tr>
		<tr>
			<td>CYTOP</td>
			<td>AGC</td>
			<td>CTL-816AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Deaeration mixer</td>
			<td>Thinky</td>
			<td>AR-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop cutter</td>
			<td>Roland</td>
			<td>STIKA SV-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>AZ Electronic Materials</td>
			<td>AZ 300 MIF</td>
			<td>AZ Electronic Materials was now acquired by Merck. 
			Other alkaline developers may be also applicable but should require optimization of development conditions (time, temperature, etc.)</td>
		</tr>
		<tr>
			<td>Double-coated adhesive Kapton film tape</td>
			<td>Teraoka Seisakusho</td>
			<td>7602 #25</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Kanto Chemical</td>
			<td>EL grade</td>
			<td>EL: for electronic use. 
			Purity 99.5%.</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td></td>
			<td>Version: ImageJ 1.51n</td>
		</tr>
		<tr>
			<td>Flat-cable cutter</td>
			<td>Tokyo-IDEAL</td>
			<td>MT-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fomblin oil</td>
			<td>Solvay</td>
			<td>Y25, or Y25/6</td>
			<td>Free test sample may be available upon inquiry to Solvay. Fomblin Y25/6 is an alternative if Y25 is not readily available.</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>AS ONE</td>
			<td>TH-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection needle</td>
			<td>Terumo</td>
			<td>NN-2270C</td>
			<td>22G &#215; 70 mm</td>
		</tr>
		<tr>
			<td>Inverted fluorescence microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti-E</td>
			<td>Epifluorescence specification, CCD or sCMOS camera, motorized stage, autofocus system, and high NA objective lens are required.</td>
		</tr>
		<tr>
			<td>KaleidaGraph</td>
			<td>Synergy</td>
			<td></td>
			<td>Version: 4.5</td>
		</tr>
		<tr>
			<td>Mask aligner</td>
			<td>SUSS</td>
			<td>MA-6</td>
			<td>Other mask aligners are also applicable as long as the vacuum contact mode is avaliable.</td>
		</tr>
		<tr>
			<td>MICROMAN pipette</td>
			<td>GILSON</td>
			<td>E M250E</td>
			<td>Capillary piston tip: CP250</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>Version: 16.16.15</td>
		</tr>
		<tr>
			<td>Mini vacuum chamber</td>
			<td>AS ONE</td>
			<td>MVP-100MV</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>NIPPON GENE</td>
			<td>316-90101</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Amcor</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tube</td>
			<td>NIPPON Genetics</td>
			<td>FG-021D/SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>AS ONE</td>
			<td>GD90-15</td>
			<td>Diameter 90 mm, height 15 mm.</td>
		</tr>
		<tr>
			<td>Photoresist</td>
			<td>AZ Electronic Materials</td>
			<td>AZ P4903</td>
			<td>AZ Electronic Materials was now acquired by Merck. AZ P4620 is an alternative.</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>BioTek</td>
			<td>POWERSCAN HT</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethelene gloves</td>
			<td>AS ONE</td>
			<td>6-896-02</td>
			<td>Trade name: Saniment.</td>
		</tr>
		<tr>
			<td>PURExpress in vitro protein synthesis kit</td>
			<td>New England Biolabs</td>
			<td>E6800S or E6800L</td>
			<td>For cell-free protein synthesis reaction.</td>
		</tr>
		<tr>
			<td>Reactive-ion etching system</td>
			<td>Samco</td>
			<td>RIE-10NR</td>
			<td>Other RIE systems are also applicable but should require optimization of RIE conditions (gas flow rate, chamber pressure, RF power, etching time, etc.)</td>
		</tr>
		<tr>
			<td>RNase inhibitor</td>
			<td>New England Biolabs</td>
			<td>M0314S</td>
			<td></td>
		</tr>
		<tr>
			<td>Scotch tape</td>
			<td>3M</td>
			<td>810-1-18D</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>194-09575</td>
			<td>8 M concentration; danger.</td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Oshigane</td>
			<td>SC-308</td>
			<td></td>
		</tr>
		<tr>
			<td>SURFLON S-386 surfactant</td>
			<td>AGC</td>
			<td>(Test sample)</td>
			<td>Free test sample may be available upon inquiry to AGC.</td>
		</tr>
		<tr>
			<td>SYLGARD 184 silicone elastomer</td>
			<td>Dow</td>
			<td>Sylgard184</td>
			<td>Chemical composition: polydimethylsiloxane. The default mixing ratio is base : curing agent = 10 : 1 (m/m).</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Ideal-tek</td>
			<td>2WF.SA.1 
			2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>AS ONE</td>
			<td>ASU-2M</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum chuck</td>
			<td>Oshigane</td>
			<td>(Customized)</td>
			<td>Material: delrin; rectangular sample stage with multiple holes (48 holes, each with 1 mm diameter); the size is customzied to fit the size of the cover glass (24 mm &#215; 32 mm).</td>
		</tr>
	</tbody>
</table>

a femtoliter droplet array for massively parallel protein synthesis from single dna molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The microfabrication process consists of substrate cleaning, surface functionalization, CYTOP coating, photolithography, dry etching, photoresist stripping, and final cleaning. Importantly, the presented protocol allowed complete removal of the hydrophobic CYTOP polymer inside the microchambers Figure 3A), producing a highly parallel hydrophilic-in-hydrophobic structure on a standard cover glass substrate. With the aid of the oil sealing protocol, the uniform dimension of the resulting dropl...

Watch this Scientific Journal Video about A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules at JoVE.com

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and ...

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