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.

The authors have nothing to disclose.

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 quality of the resulting substrate. Given the generally short working distance of the objective lens with high magnifications (see step 6.1), the thin cover glass must be used. A high-quality spin-coating on a thin and flexible substrate is generally tricky. We have not yet tested all possible designs but have found that the multi-hole vacuum chuck with the same rectangular dimension did work well for the spin-coating. It is important to drop the CYTOP polymer at the center of the cover glass substrate and immediately start the spin-coating (see step 1.3.2). The degree of the difficulty is less related to the shape of the substrate but the viscosity of the polymer. The CYTOP product line offers several different concentrations of the commercially available package. The accompanying diluent in the package can also be used to dilute the original product to a lower viscosity if needed. CYTOP 816 that we used in the experiment is the commercial product with the highest concentration capable of stably dissolving the polymer in the solvent. The thicker coating requires lower spin speed or multiple rounds of coating and curing. A plot of characteristic thickness vs. spin speed curve is highly recommended when using a new spin coater in a new environment.
Second, the appropriate humidity of the cleanroom is crucial for ideal photolithography as well as the complete removal of the photoresist at the specified position. The photochemical reaction in photolithography uses H2O as one of the reactants. However, the softbake at the temperature higher than the boiling temperature of H2O removes H2O content from the coated photoresist. The &#8220;dried&#8221; photoresist must take time to absorb H2O again from the air (see step 1.4.6). This point is often overlooked. Given that many laboratories may only have some simplified cleanroom facilities without humidity control, the humidity would fluctuate dramatically over the season or be affected by the weather. A low-cost solution is to use a home-use humidifier or dehumidifier in the cleanroom. The fully exposed CYTOP layer after development can be selectively and fully removed by RIE, fully exposing the glass bottom. Eventually, the hybrid structure of the hydrophilic glass bottom and the hydrophobic CYTOP sidewall is important for stably trapping aqueous solution to the femtoliter space.
Third, the newly found oils (ASAHIKLIN AE-3000, Fomblin Y25) and surfactant (SURFLON S-386) showed unprecedented sealing performance for the fluoropolymer reactor13. The injection of the flush oil must be carried out on the chilled flat metal block (see step 5.2); otherwise, droplets near the inlet of the channel cannot be formed. The injection of the second sealing oil can be carried out at RT (see step 5.5). These oils and surfactants have never been used in biological research before. No better alternatives have been found for now. To expand the arsenal of the useful combination of oils and surfactants for droplet preparation, the new member of the oils (or the similar ones in the same product line) and the surfactant (or the similar ones in the same product line) are worthy of trying to be applied in microfluidic droplet systems.
Here are two additional points to notice. One is the fact that there is a time lag among ROIs during the microscopy imaging. The total imaging time for one cycle is mainly dependent on the array size and the magnification of the objective lens. The exposure time, shutter speed, and the moving speed of the motorized stage are also minor factors. As a rule of thumb, imaging 106 droplets at 60&#215; magnification would at least require 10-20 minutes. This inevitable time lag introduces some errors in data analysis, which should always be taken into careful consideration. The other one is the fact that the total number of droplets on a single substrate would not exceed 108&#8211;109, even increasing the density of the droplets or reducing the individual size of the droplet. This is because the size of the glass substrate that can be handled by the mask aligner (for 4-inch wafers) is limited. Further increasing the size of the cover glass is not recommendable because a large, thin, and fragile glass substrate is hard to handle.
FemDA can be considered as a super miniaturized microtiter plate. Any biochemical reactions that have been carried out in 96-well microtiter plates could be tried to conduct using FemDA. It can surely be used for enzyme activity measurement without complicated protein purification. It could also be compatible with digital polymerase chain reaction (digital PCR) and digital enzyme-linked immunosorbent assay (digital ELISA)31,49. The small volume, large array, and high stability would bring some advantages over existing digital bioassay methods. The FemDA system capable of resolving different numbers of template DNA molecules in femtoliter droplets has already shown the power on accurate protein screening13. FemDA is a new in vitro compartmentalization system capable of rapidly evolving a variety of enzymes. With the advances in bioinformatics and library construction techniques, the era of a prompt on-demand creation of high-performance proteins will be surely coming.

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.&lt;br/&gt; 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 &amp;times; 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.&lt;br/&gt; 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.&lt;br/&gt; 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 &amp;times; 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&lt;br/&gt; 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 &amp;times; 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 droplets was verified by encapsulating fluorescent solution in the microchambers (Figure 3B). The fluorescence intensity extracted using the developed software is a good indicator of the droplet size. The CV of the fluorescence intensity, 3%, reflected the narrow distribution of the droplet size over the entire array (Figure 3C). The reconstructed 3D image from confocal microscopy also showed the consistent droplet volume over time (Figure 3D, Supplementary Movie 1)44. In comparison, a widely used FC-40 oil generated the droplets exhibiting a severalfold difference in the fluorescence intensity45. The formed droplets in FemDA were stable at RT for at least 24 hours (Figure 3E). The high quality of the droplets eventually formed the basis of quantitative measurement.
High-throughput data necessitate high-throughput data-analyzing tools46,47. The developed plugin greatly simplified and speeded up the image data analysis. Based on the theory of digital image processing48, the defocused BF image can be binarized and used to provide the coordinate information of every droplet after background correction and noise reduction (Figure 4A-C). This idea made the precise localization of dark droplets possible.
The fluorescent protein mNeonGreen was synthesized in FemDA. Template DNA with a concentration of 0.05 molecules per droplet was randomly distributed into each droplet to initiate the protein synthesis with coupled cell-free transcription and translation reactions. Because the average number of DNA molecules per droplet was smaller than 1, some droplets contained zero template DNA, while others contained one or more DNA molecules. After 6 hours of incubation at RT, the end-point image was captured using the microscope. The stack image data was analyzed by the developed software with the aid of the concurrent defocused BF image (Figure 4A-E). The fluorescence intensity of each droplet is a measure of the protein yield in the corresponding droplet. The histogram of the fluorescence intensities showed a discrete distribution and was well fitted by a sum of Gaussian distributions of equal peak-to-peak intervals (Figure 4F), which strongly suggested an occupancy of different numbers of DNA molecules per droplet. Similar to the repeated coin-flipping game, the number of independent random events that occur is mathematically described by the Poisson distribution. The probability of occurrence of droplets containing different numbers (up to 3 in this example) of DNA molecules was a perfect fit to a Poisson distribution (P = e-&#955;&#160;&#8226; &#955;k&#160;/ k!, where &#955; is the expected average number of DNA molecules per droplet and k is the actual number of DNA molecules in a droplet) with an average of 0.05 DNA molecules per droplet (Figure 4G)31, as expected for a random distribution of DNA molecules. Because the loading concentration of the template DNA solution was the same as the final fitted &#955; (i.e., 0.05), the CFPS reaction efficiency in our FemDA was 100% (or near 100%). In other words, a single DNA molecule is enough to trigger the CFPS reaction in the femtoliter droplet efficiently.
The CFPS reaction of alkaline phosphatase was recorded every 5 minutes. The developed software also supports analyzing the time-course data (Figure&#160;5). The coupled fluorogenic reaction showed a similar discrete distribution of the fluorescence intensity of the droplets at earlier time-points. The histogram results also verified an occupancy of different numbers of DNA molecules in the droplet. The fluorescence intensity eventually converged to a value along with the gradual depletion of the fluorogenic substrate DiFMUP.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60945/60945fig01.jpg" /> 
Figure 1: Microfabrication process of the ultrahigh-density microchamber array substrate.&#160;(A) Technical drawing of the customized vacuum chuck. Unit: mm. (B) CYTOP film thickness vs. spin speed data. (C) Spin-coating of the perfluoropolymer CYTOP on a silanized glass substrate. The photographs showed a good example of homogenous coating and a bad example of inhomogeneous coating, respectively. The black arrow indicated the specific position of the inhomogeneous CYTOP film. (D) Spin-coating of photoresist on the CYTOP-coated substrate. After the spin-coating, the photoresist near the substrate edge must be removed using an ethanol-soaked clean wiper (middle photograph). The photographs showed a good example of homogenous coating and a bad example of inhomogeneous coating, respectively. The white arrow indicated the specific position of the inhomogeneous photoresist film. (E) Exposure of the coated photoresist using a mask aligner. (F) Development of the exposed photoresist in a developer. The exposed part of the photoresist is soluble in the developer solution. (G) Reactive-ion etching of CYTOP. The uncovered CYTOP was removed by O2 plasma. (H) Removal of the photoresist mask. The photoresist was removed by acetone. The substrate was cleaned using 2-propanol and H2O. <a href="https://www.jove.com/files/ftp_upload/60945/60945fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60945/60945fig02.jpg" /> 
Figure 2: Preparation and use of the microchamber array device.&#160;(A) Weighing the curing agent and prepolymer of PDMS. (B) Pouring the mixed and deaerated mixture into a tape-patterned Petri dish. There were some newly generated air bubbles in the polymer mixture. (C) Deaerating the mixture again in a vacuum chamber. The air bubbles were rising and burst on the top surface. (D) The deaerated and cured PDMS resin. (E) Peeling off the cured PDMS elastomer from the Petri dish. (F) Cutting off every piece of PDMS channel blocks using a flat-cable cutter. (G) Punching holes at each end of the PDMS channel using a biopsy punch. (H) The assembled device. The PDMS resin can adhere to the CYTOP surface. (I) Translucent microchamber array area inside the PDMS channel filled with the aqueous solution before chilling. (J) Transparent microchamber array area inside the PDMS channel after chilling. <a href="https://www.jove.com/files/ftp_upload/60945/60945fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60945/60945fig03.jpg" /> 
Figure 3: Ultra-uniform and ultra-stable femtoliter droplets.&#160;(A) 3D laser scanning confocal microscopy imaging for the fabricated substrate. The cylindrical microchambers in the example image showed a height of 3 &#956;m and a diameter of 4 &#956;m. (B) Uniform femtoliter droplets over a large area of the planar array. Only a partial area of the entire array was shown herein. A fluorescent solution (10 &#956;M ATTO-514) was sealed in each droplet. (C) The size distribution of the droplets over an entire single array. The fluorescence intensity was used as an indicator of the droplet size. The CV was only 3%. (D) Volumetric measurement using confocal z-stack time-course data. The volume of droplets over the array was given by the microscope software (NIS-Elements, Nikon). (E) Fluorescence recovery after photobleaching. After the first frame, several droplets were completely photobleached using a confined laser beam of a confocal microscope. Their fluorescence intensity was recorded for 24 h, and no fluorescence recovery was observed (red line). The fluorescence intensity of other non-photobleached droplets in the same field of view was recorded in the black line. Error bars (translucent colors) were 1 SD for every time-point. <a href="https://www.jove.com/files/ftp_upload/60945/60945fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60945/60945fig4v2.jpg" /> 
Figure 4: Analysis procedure of the end-point microscopic image data.&#160;The defocused bright-field image was used to extract the coordinate of every microchambers/droplets. Based on the intensity difference between the edge of microchambers (as foreground) and other areas (as background), the continuous and near-circular edge can be extracted from the background. Because of the uneven background distribution across the field of view (A), some image pre-processing is generally required to improve the quality of the binarizing output. After subtracting background (B), the background of every frame was uniformized. Empirically, the input value in the &#8220;Rolling ball radius&#8221; (step 1) was 20-50 for 2048 pixel &#215; 2048 pixel images and 10-20 for 512 pixel &#215; 512 pixel images. The larger the value, the shorter the processing time. To reduce the noise in the background-subtracted image, apply a median filter to the image (C). In general, the input value in the Radius (step 3) was 1-2 pixels. As shown in the magnified insert, the background-subtracted and filtered image can be nicely binarized using the build-in threshold plugin so that the foreground corresponding to the edges as well as the region of interests (ROIs) can be accurately recognized. The ROIs detection for all frames of the image was carried out by using the installed homemade plugin FemDA Analysis (D). The pixel size (steps 5 and 6) and circularity (steps 7 and 8) of ROIs, the frames that we want to put into the calculation (steps 9 and 10) were manually defined according to the actual data. After clicking Generate ROI (step 11) and waiting, the coordinate of every ROI across the input frames was determined. After clicking the Apply ROI mask (step 12), every detected ROI was enclosed and highlighted by a yellow line. After opening the fluorescence image and clicking the Apply ROI mask again, the determined ROIs mask was applied to the fluorescence image. The Number of top pixels (step 14) specified the number of top intensity-ranked pixels of each ROI for the calculation of the mean intensity of the respective droplets. After clicking Measure intensity (step 15) and waiting, the histogram was generated (E). The histogram can be fitted with a sum of Gaussian distributions using the parameters available in the histogram window. Alternatively, the mean intensity data can be exported to a text file (step 16) and analyzed by other software (F). (G) The probability of occurrence of droplets containing different numbers of DNA molecules in the given array. The histogram (grey color) was nicely fitted by a Poisson distribution (red dashed line) with an average of 0.05 DNA molecules per droplet, as expected for a random distribution of DNA molecules. <a href="https://www.jove.com/files/ftp_upload/60945/60945fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60945/60945fig05.jpg" /> 
Figure 5: Analysis of the time-course microscopic image data.&#160;The detected ROIs (following the steps 1-12 of Figure 4) was directly applied to the time-course data. In step 13 of Figure 4, select Time-lapse and input the actual time-interval (in minutes) between adjacent frames in Time-interval [min]. After clicking Measure intensity (step 15 of Figure 4) and waiting, the time-intensity plot and the histogram (the same as Figure 4E) were generated. The Hist position specified the time-point of the histogram, which was shown as a vertical red line. The plugin also supports specifying colors for every trace line. Yellow: 0 DNA; blue was 1 DNA; red: 2 DNA; black: 3 DNA. The time-course data can also be exported to a text file. <a href="https://www.jove.com/files/ftp_upload/60945/60945fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Movie 1.&#160; <a href="https://www.jove.com/files/ftp_upload/60945/MovieS1.avi" target="_blank">Please click here to download this movie.</a>

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 ...

a-femtoliter-droplet-array-for-massively-parallel-protein-synthesis

Nanyang Technological University

Research

JoVE Journal

Chemistry

10.2K Views.  Japan Agency for Marine-Earth Science and Technology (JAMSTEC). 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.

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

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

Cancer Research

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Bioengineering

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Genetics

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 106x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 &#181;g of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.

The study describes a protocol for creating large (&#181;g-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins ...

Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA templates for CFPS will further enable the technology to reach its full potential, decreasing the experimental time by eliminating the steps of cloning, transformation, and plasmid extraction. Linear DNA can be rapidly and easily amplified by PCR to obtain high concentrations of the template, avoiding potential in vivo expression toxicity. However, linear DNA templates are rapidly degraded by exonucleases that are naturally present in the cell extracts. There are several strategies that have been proposed to tackle this problem, such as adding nuclease inhibitors or chemical modification of linear DNA ends for protection. All these strategies cost extra time and resources and are yet to obtain near-plasmid levels of protein expression. A detailed protocol for an alternative strategy is presented here for using linear DNA templates for CFPS. By using cell extracts from exonuclease-deficient knockout cells, linear DNA templates remain intact without requiring any end-modifications. We present the preparation steps of cell lysate from Escherichia coli BL21 Rosetta2 &#916;recBCD strain by sonication lysis and buffer calibration for Mg-glutamate (Mg-glu) and K-glutamate (K-glu) specifically for linear DNA. This method is able to achieve protein expression levels comparable to that from plasmid DNA in E. coli CFPS.

Presented here is a protocol for the preparation and buffer calibration of cell extracts from exonuclease V knockout strains of Escherichia coli BL21 Rosetta2 (&#916;recBCD and &#916;recB). This is a fast, easy, and direct approach for expression in cell-free protein synthesis systems using linear DNA templates.

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA ...