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

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.

1. Cleaning cover glass substrate
   1. 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 it without any splash.
   2. Take the rack out of the NaOH solution and rinse the cover glass using water for ten times. Keep the cover glass in the pure water.
      NOTE: Discard the alkaline waste to the designated tank. The NaOH solution can be recycled to the original bottle and used for up to five times.
   3. Dry each piece of cover glass with an air blow gun.
   4. Bake the dried cover glass on a hot plate at 200 °C for 5 min. Keep the orientation of the upper side of the glass substrate consistent during the following handling processes.
2. Silanization of the glass surface
   1. Reset the cleaned cover glass on a dry staining rack.
   2. Add 150 mL of ethanol into a PTFE beaker. Use a needle (22 G x 70 mm) equipped 1 mL syringe to draw 0.075 mL of (3-aminopropyl)triethoxysilane and immediately inject it to the ethanol (i.e., 0.05 vol %). Pull and push the syringe several times to rinse the inside of the syringe. Stir the solution with the needle to make it homogeneous.
      NOTE: (3-aminopropyl)triethoxysilane can be stored at room temperature and 1 atm pressure for two years with a tight sealing. Absolute ethanol should be tightly sealed after use. We do not recommend using any expired reagents.
   3. Incubate the cover glass in the 0.05 vol % aminosilane solution for 1 h at RT.
   4. Rinse the cover glass using pure water for five times. Keep the cover glass in the pure water.
      NOTE: Discard the waste to the designated tank.
   5. Dry each piece of cover glass with the air blow gun.
   6. Place all the dried cover glass on aluminum foil. Bake the cover glass on the hot plate at 80 °C for 5 min.
3. Spin-coating CYTOP perfluoropolymer
   1. Place the cover glass on a customized vacuum chuck of a spin coater (Figure 1A). Turn on the vacuum switch to fix the glass substrate.
      NOTE: The design of the vacuum chuck is crucial for a uniform coating of viscous polymer on the rectangular (24 mm × 32 mm) and thin (0.13-0.17 mm) substrate (Figure 1A). We found that a rectangular sample stage with multiple holes (48 holes, each with 1 mm diameter) connecting to the vacuum channel worked better than the round stage with a single central hole did.
   2. Drop 70-90 µL of type-A CYTOP polymer at the center of the glass substrate. Immediately spin-coat the polymer (Figure 1B).
      NOTE: The spin-coating condition (3,400 rpm, 30 s) provides 3 μm thickness. Use the micropipette designed for viscous liquid. Hold the micropipette upright to make the polymer drop near-perfectly circle on the substrate. An air bubble is often generated inside the pipette tip as the plunger moves downward. Do not drop the last drop of CYTOP with the bubble.
   3. Pick up the coated glass with fingers holding corners of the glass substrate, and place it on aluminum foil.
   4. Repeat steps 1.3.1-1.3.3 to spin-coat all the remaining pieces of the cover glass.
   5. Bake the coated glass at 80 °C for 30 min and then at 200 °C for 1 h.
      NOTE: This processing fully cures the CYTOP and covalently bond the CYTOP to the glass surface. Cover the baking area with a lid made of aluminum foil to avoid potential dust during the long-time baking process if needed.
   6. Remove the CYTOP-coated cover glass from the hotplate and cool down to RT. Carefully pick up each piece of the coated glass and inspect it to see if it is properly coated. Discard the substrate exhibiting inhomogeneous coating.
      NOTE: The surface of a nicely coated CYTOP should look flat without irregular rainbow-like patterns (Figure 1C). Visual inspection from a proper angle can rapidly judge the flatness as well as the coating quality. The protocol can be paused here.
4. Spin-coating photoresist
   1. Place the CYTOP-coated cover glass on the vacuum chuck (see step 1.3.1) of the spin coater. Turn on the vacuum switch to fix the cover glass.
   2. Drop 0.2-0.3 mL of photoresist at the center of the substrate. Immediately spin-coat the photoresist at 6,000 rpm for 60 s.
      NOTE: Do not drop the last drop of the photoresist with bubbles. If the spin coater supports higher spin speed, the spin speed could be up to 7,500 rpm to achieve a thinner coating. The low surface energy of CYTOP prevents most of the photoresists from being adhered to its surface. If AZ P4903 photoresist is not available, AZ P4620 photoresist is a good alternative.
   3. Pick up the coated substrate with two fingers holding corners of the substrate. Wipe off the excess photoresist near the substrate edge (often called as edge bead removal or EBR) using an ethanol-soaked clean wiper (Figure 1D). Place the substrate on the aluminum foil.
      NOTE: Acetone is NOT recommended for EBR.
   4. Repeat steps 1.4.1-1.4.3 to spin-coat the photoresist for the remaining pieces of CYTOP-coated cover glass. Pick up every piece of the coated glass and inspect it to see if it is properly coated.
      NOTE: The surface of a nicely coated photoresist should look flat without irregular patterns (Figure 1D). Visual inspection from a proper angle can rapidly judge the flatness as well as the coating quality. The substrate exhibiting inhomogeneous coating can be recycled through washing with acetone, 2-propanol, and H₂O in sequence, and reused.
   5. Bake the coated substrate at 110 °C for 5 min on the hotplate.
   6. Remove the coated substrate from the hotplate and cool down to RT. Let stand for 30 min at a relative humidity of 40%-60% for rehydration of the photoresist.
      NOTE: H₂O is necessary for the following photochemical reaction. The baked photoresist lost the H₂O content inside the photoresist. The rehydration process allows H₂O to be absorbed from the air. Relative humidity lower than 20% or higher than 80% is not suitable for the rehydration process.
5. Photolithography
   1. During the waiting time of rehydration (see step 1.4.6), start up the mask aligner. Warm up the light source. Load the chrome photomask.
      NOTE: Follow the instruction manual to operate the mask aligner. The photomask can be fabricated via electron beam (EB) lithography if the EB facility is available. Alternatively, the photomask can be outsourced to a local company.
   2. Load the rehydrated substrate (after finishing step 1.4.6) on the chuck (Figure 1E).
   3. Set exposure parameters. Expose the substrate for 25 s with a UV intensity of 13 mW/cm² (h-line) in the vacuum-contact mode. Then unload the substrate and set it on the cover glass staining rack (the same rack used in step 1.1.1).
   4. Repeat steps 1.5.2-1.5.3 to expose the remaining pieces of the rehydrated substrate.
6. Development
   1. Develop the substrate for 5 min to dissolve the exposed photoresist (Figure 1F). During which, gently shake the rack in the developer once at the timepoint of 4 minute.
      NOTE: The developer was AZ 300 MIF. Alternative alkaline developers (e.g., AZ 400 K) are generally applicable for the development but should require optimization of development conditions (e.g., time, temperature, agitation). Do NOT use glass beakers as the developer container.
   2. Rinse the substrate using pure water for ten times. Keep the cover glass in the pure water.
      NOTE: Discard the developer to the designated tank.
   3. Thoroughly dry every piece of substrate with the air blow gun.
7. Reactive-ion etching
   1. Place the dried substrate (from step 1.6.3) in the reaction chamber of the reactive-ion etching (RIE) machine.
      NOTE: According to the maintenance rule of the facility, the RIE machine can be either always on or shut down every time. If the switch was off, turn on the switch according to the manual.
   2. Etch the photoresist-uncovered CYTOP with the O₂ plasma (Figure 1G).
      NOTE: The RIE condition was as follows. O₂ gas flow rate: 50 sccm; chamber pressure: 10.0 Pa; RF power: 50 W; etching time: 27 min. The etching time was optimized for completely etching 3 μm thickness of CYTOP.
   3. Pick up every piece of substrate from the reaction chamber using the tweezer and place them on the cover glass staining rack.
      NOTE: According to the maintenance rule of the facility, the reaction chamber may require a short O₂ plasma cleaning process (e.g., etching time: 5 min) to keep the reaction chamber clean after every run.
8. Removing photoresist and cleaning
   1. Sonicate the substrate in acetone for 5 min at RT to dissolve the remaining photoresist.
   2. Sonicate the substrate in 2-propanol for 5 min at RT.
   3. Rinse the substrate using pure water for five times. Sonicate the substrate for 5 min at RT. Rinse the sample using pure water for another five times. Keep the substrate in pure water.
   4. Dry every piece of substrate with the air blow gun (Figure 1H).
      NOTE: The substrate can be stored in a plastic Petri dish. The fabricated substrate is structurally stable at RT for at least one year. The protocol can be paused here.
   5. Characterize the surface profile of the as-prepared substrate by three-dimensional (3D) laser scanning confocal microscopy, white light interferometry (or coherence scanning interferometry), or scanning electron microscopy. Use the respective software to measure the diameter and depth of the resulting microchambers.
      NOTE: The sample can be reused for other experiments after the characterization with the non-contact and non-destructive 3D laser scanning confocal microscope or white light interferometer. The protocol can be paused here.

2. Preparation of polydimethylsiloxane (PDMS) microchannel

NOTE: Do NOT wear latex gloves to handle PDMS. Instead, wear polyethylene (PE) gloves.

1. Making the microchannel master
   1. Cut a double-coated adhesive Kapton film tape into a defined (3 mm x 19 mm) microchannel shape using a desktop cutter.
      NOTE: The Kapton tape was No. 7602 #25 (Teraoka Seisakusho), resulting in a channel height of 135 μm. The STIKA desktop cutter uses a plugin (available from Roland website: https://www.rolanddga.com) of Adobe Illustrator to run the cutting according to the drawing in the Adobe Illustrator file. The protocol can be paused here.
   2. Stick every cut Kapton tape on the flat bottom of a Petri dish. The standard 90 mm Petri dish can accommodate 12 pieces of the tape.
      NOTE: The relief structure serves as a master for the replica molding of PDMS. Press the tape surface gently with a tweezer if there are any air bubbles sandwiched between the tape and the Petri dish substrate. Alternatively, classical soft-lithography can be applied to prepare the master on a silicon wafer³⁸. The protocol can be paused here.
2. Curing PDMS resin in the tape-patterned Petri dish
   1. Wear new PE gloves, and transfer 2.5 g of curing agent of SYLGARD 184 silicone elastomer using a disposable plastic pipette into the specified plastic beaker (Figure 2A).
   2. Change into new gloves, and transfer 25 g of the prepolymer base of SYLGARD 184 silicone elastomer using a disposable 50 mL syringe into the above plastic beaker.
      NOTE: Change the gloves herein to prevent the potential cross-contamination of the curing agent to the base.
   3. Use a deaeration mixer to mix and deaerate the mixture (program: 3 min mixing followed by 2 min deaeration).
      NOTE: If the deaeration mixer is not available, a manually agitated (for about 15 min) mixture can be degassed in a vacuum chamber.
   4. Pour the PDMS mixture into the tape-patterned Petri dish (Figure 2B). Set the Petri dish in a mini vacuum chamber and deaerate the PDMS mixture for 1-3 h (Figure 2C).
      NOTE: If any air remains in the PDMS mixture after 1 h, take the Petri dish out of the vacuum chamber, break the air bubble using an air blower, and then put the Petri dish back in the vacuum chamber and continue the deaeration process.
   5. Place the Petri dish in an oven at 60 °C overnight to cure the PDMS (Figure 2D).
      NOTE: Although increasing the temperature can shorten the curing time, the maximum temperature that can be tolerated by the polystyrene Petri dish without physical deformation is about 60 °C.
3. Replica molding of PDMS channel
   1. Peel off the cured PDMS elastomer from the Petri dish (Figure 2E).
   2. Cut off every piece of PDMS channel blocks using a flat-cable cutter (Figure 2F).
   3. Place the PDMS elastomer on a cutting mat facing channel side up. Punch a hole at each end of the channel using a biopsy punch (Figure 2G).
   4. Clean the surface of the PDMS channel with Scotch tape. Then wrap the PDMS resin in another piece of Scotch tape to keep it clean before use. Store the prepared PDMS channel in a clean Petri dish.
      NOTE: The protocol can be paused here.

3. Assembly of femtoliter microchamber array device

1. Place some pieces of water-soaked clean wipers along the inside wall of a Petri dish to make a simplified humidifying chamber. Place the PDMS resin covered by the Scotch tape in the humidifying chamber and seal the chamber using paraffin film. Incubate for at least 3 h but no longer than a day.
   NOTE: PDMS is a porous material that allows gas to pass across the resin³⁹. The porous structure of PDMS leads to an absorption of water molecules from the surroundings until reaching an equilibrium. This pre-treatment fills the pores of PDMS with water vapor and can significantly repress the evaporation of aqueous droplets adjacent to the edge of the PDMS channel.
2. Take the Scotch tape off the PDMS resin. Position the PDMS channel on the microchamber array area of the substrate (Figure 2H).
   NOTE: The PDMS resin reversibly adheres to the CYTOP surface. Press the PDMS block gently with a tweezer to remove any air bubbles in between the PDMS resin and the substrate if existed.
3. Insert a 200 μL non-filtered pipette tip to one (as the outlet) of the holes of the PDMS channel.
   NOTE: The adhesion strength between PDMS and CYTOP is limited. To avoid physical deformation of the PDMS resin as well as detachment of the PDMS channel off the surface, do not insert the pipette tip too deep.

4. Loading reaction solution to the assembled device

1. Put an aluminum microtube stand on the ice. Prechill the flush oil (ASAHIKLIN AE-3000 oil mixed with 0.1 wt % SURFLON S-386 surfactant) in a 1.5 mL microtube on the aluminum stand.
2. Put another aluminum block on ice.
3. Preparation of CFPS reaction solution
   1. Prepare the CFPS reaction solution in a PCR tube. Mix every aliquot component of the CFPS kit, a diluted template DNA (as exemplified herein by fluorescent protein mNeonGreen⁴⁰ and Escherichia coli alkaline phosphatase⁴¹) solution, and other necessary components according to the specific needs (e.g., RNase inhibitor, fluorogenic substrate, chaperone).
      NOTE: The template DNA used for CFPS must be prepared according to the instruction of the corresponding CFPS kit. This demonstration applied a T7-based expression system⁴². Because the reagent consumption in the FemDA device is quite small, 10 μL is large enough to fill the entire PDMS channel.
   2. For mNeonGreen fluorescent protein synthesis, mix the components in the following order: add 2.7 μL of nuclease-free H₂O to a 6 μL aliquot of solution A (from the CFPS kit); add 0.3 μL of RNase inhibitor; add 1.5 μL of diluted template DNA solution; briefly mix and transfer the mixture to a 4.5 μL aliquot of solution B (from the CFPS kit).
      NOTE: The total volume is 15 μL. As the quantity of every aliquot is proportional to the total volume of the final mixture, a total volume less than 10 μL may result in a far too small volume (< 0.2 μL) of some component aliquot.
   3. For alkaline phosphatase synthesis, mix the components in the following order: add 1.2 μL of H₂O to 6 µL of solution A; add 0.3 µL of RNase inhibitor; add 0.6 μL of disulfide bond enhancer 1 and 2 (from a kit); add 0.3 μL of 5 mM 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP); add 1.5 μL of DNA solution; briefly mix and transfer the mixture to 4.5 μL of solution B.
      NOTE: In the assay of alkaline phosphatase, the fluorogenic substrate DiFMUP can produce highly fluorescent 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU) upon enzymatic cleavage of the terminal phosphate group.
4. Draw up 10 μL of the mixture using a 200 μL non-filtered pipette tip. Insert the pipette tip to the inlet hole (refer to step 3.3) of the PDMS channel. Push down on the plunger to inject the solution to the channel until it overflows from the outlet (where another pipette tip has already been inserted at step 3.3).
5. Separate the pipette from the pipette tip and keep these two pipette tips still inserted to the inlet and outlet.
   NOTE: Avoid generating air bubbles during the pipetting. Do not leave the thumb from the plunger until separating the pipette from the pipette tip to prevent the backflow of the solution inside the PDMS channel.

5. Generating femtoliter droplet array (FemDA) for CFPS

1. Transfer the whole set of the assembled device to the pre-chilled aluminum block (see step 4.2). Carefully confirm that the microchamber array area quickly turns from translucent (Figure 2I) to transparent (Figure 2J).
   NOTE: The CFPS reaction solution cannot straightly enter into the microchambers. The solubility of air in water is in inverse proportion to the temperature. The trapped air will dissolve into the solution after the device was moved from RT to the low temperature. The transparency change is a convenient visual indicator to judge whether the solution enters into the microchambers.
2. Draw 30 μL of the pre-chilled flush oil (see step 4.1), and immediately transfer the oil into the inlet-inserted pipette tip from its upper opening. The flush oil moves into the channel and extrudes the excess reaction solution located outside the microchambers. Every microchamber is isolated by the flush oil.
   NOTE: The moving interface between the reaction solution and the flush oil is visible, which helps the people to observe the movement of the fluid inside the channel. The extruded solution can be collected into the previous tube, stored at 4 °C, and reused for another FemDA device or a parallel bulk reaction (in the microtube or a microtiter plate).
3. Pre-draw 30 μL of sealing oil (Fomblin Y25) to a 200 μL filtered pipette tip. Hang the pipette upright somewhere.
4. Simultaneously remove the two inserted pipette tips (see steps 3.3 and 4.4) from the device. Immediately move the device from the aluminum block to parafilm.
   NOTE: Use a tweezer to fix (but keep away from the channel area) the device on the aluminum block during the period of removing the pipette tips.
5. Immediately insert the ready pipette tip containing the sealing oil (see step 5.3) into the inlet of the PDMS channel, and inject the oil into the channel until it overflows from the outlet.
   NOTE: Carry out this step as soon as moving the device to RT. To prevent the backflow inside the channel, do not leave the thumb from the plunger until the sealing oil overflows from the outlet. The flush oil evaporates immediately after moving out of the outlet of the channel, while the following sealing oil accumulates in the outlet because of its extremely low evaporation loss.
6. Separate the pipette from the pipette tip and then remove the pipette tip from the device. Clean the PDMS surface using a piece of the clean wiper and then apply a new drop of sealing oil to the inlet and outlet, respectively. The femtoliter droplets are sealed in individual microchambers by the oil.
   NOTE: Use a tweezer to fix (but keep away from the channel area) the PDMS channel on the substrate during the period of removing the micropipette tip.
7. Wipe the moisture accumulated on the lower surface of the cover glass. The as-prepared FemDA device is now ready for incubation and microscopy imaging.

6. Microscopy imaging

1. Start up an inverted fluorescence microscope. Wait for the stabilization (cooling) of the camera. Use a 60x or 100x oil immersion objective lens. Set the FemDA device on the motorized stage. Set the imaging parameters.
   NOTE: The imaging parameters include the file path, imaging channels, filters, exposure times (in general, 100 ms is enough; shorter or longer time is also possible according to the specification of the camera), and light intensity.
2. Find the focal plane. Adjust the z-axis position of the objective lens and fix the focal plane using an internal autofocus system. Set the region of interest (ROI; i.e., x, y-axis) to be imaged.
   NOTE: The major microscope manufacturers (Nikon, Olympus, Zeiss, Leica) all provide the option to integrate the autofocus system with the microscope. This autofocus system is necessary for keeping the focal plane constant during the automatic multi-area imaging. The ROIs cover the FemDA area in the PDMS channel.
3. Capture the fluorescence images. Capture a single frame of a defocused bright-field (BF) image for every ROI. Do NOT change the order and the coordinates of the ROIs when imaging the different channels.

7. Image data analysis

NOTE: Analyze the image data using a homemade plugin (named “FemDA”) based on Fiji (http://fiji.sc) to extract the fluorescence intensity of each droplet⁴³. Install the correct version of Fiji according to the operating system. Fiji supports most image file formats (e.g., the nd2 file from Nikon microscope, czi file from Zeiss microscope) using a build-in plugin “Bio-Formats.”

1. Plugin installation
   1. Copy and paste the plugin file (which is available upon inquiry to the corresponding author or via the following institutional link: https://fbox.jamstec.go.jp/public/FcsQwAsMKIqA9j0B2MJwLUMeHGsxp09hAkAFdVhQIDkZ) into Fiji’s “plugins” folder.
   2. Start the Fiji software; the plugin “FemDA” can be found in the drop-down menu “Plugins” of Fiji.
2. Image data preprocessing
   1. Open the defocused BF image (see step 6.3). Click Process | Subtract Background… to remove smooth continuous backgrounds of every frame of the image data (Figure 4B). Click Process | Filters | Median to reduce the noise of every frame of the image data (Figure 4C).
   2. Click Image | Adjust | Threshold to check and separate the foreground (indicating the microchambers) from the background (indicating the area outside the microchambers) of every frame of the image data (magnified inserts in Figure 4A-C).
      NOTE: Select a proper algorithm from the drop-down list in the Threshold window so that only the microchamber areas, as well as the edges of every microchamber, can be identified and highlighted as the foreground. In particular, most of the highlighted edges must be continuous without gaps.
3. Droplet coordinate determination
   1. Click Plugins | FemDA | FemDA Analysis to open the graphic user interface (GUI) of the customized plugin (upper half of Figure 4D).
   2. Input the approximate minimum and maximum numbers of pixels of a single microchamber. Input the expected minimum and maximum circularity (0: arbitrary shape; 1: perfect circle) of the microchambers. Input the frame number of the start frame and the end frame.
   3. Click Generate ROI on the GUI to detect every microchamber, and the successfully detected ROIs are shown in a popup window ROITable.
   4. Go back to the window FemDA Analysis and click the button Apply ROI mask to check if the microchambers over the frames were properly detected (lower left of Figure 4D). If no, modify some of them and repeat clicking Generate ROI and Apply ROI mask. If yes, go to the next step.
      NOTE: There may be a very few microchambers that cannot be well identified as the ROI (see the lower half of Figure 4D) by any algorithm of the Threshold because of the insufficient contrast between the foreground and the background. It is not problematic in the statistical viewpoint.
4. Fluorescence intensity extraction
   1. Open the fluorescence image and bring it to the front. Click Apply ROI mask again to apply the determined ROIs onto the fluorescence image (lower right of Figure 4D).
   2. Select either One-Shot for analyzing an end-point image (as exemplified with the mNeonGreen data) or Time-Lapse for analyzing a time-course data (as exemplified with the alkaline phosphatase data).
   3. Input 100 in the box Number of top pixels means that only the top 100 pixels of each ROI will be used to calculate the mean intensity of the corresponding droplet. If input 0 in the Number of top pixels, all pixels of each ROI will be used to calculate the mean intensity of the corresponding droplet.
      NOTE: There may be a drift of several pixels between the BF and the fluorescence images, which means some pixels within the ROIs may belong to the background of the fluorescence image. Hence, the plugin provides an option to specify the number of top intensity-ranked pixels for the calculation of the mean intensity of every droplet.
   4. For the analysis of the end-point image (i.e., select One-Shot at step 7.4.2), click button Measure intensity to calculate the mean intensities of all detected droplets. The ROITable is updated with the new data from the fluorescence image. Meanwhile, a histogram is displayed in a new popup window Histogram (Figure 4E).
      NOTE: Changing the bin size in the box Bin Number can optimize the display of the histogram. A fitting to a sum of Gaussian distributions can be accomplished in the developed GUI. The popup Log window of Fiji displays the fitting results. Alternatively, export the data by clicking the button save as text and carry out various statistical analyses with other software (Figure 4F, G).
   5. For the analysis of the time-course image (i.e., select Time-Lapse at step 7.4.2), the popup window is Intensity Time Course instead of Histogram, which contains a histogram corresponding to a specific time-point and a time-intensity plot (Figure 5). The textual data can also be exported using the File menu of the popup window.