More than one million uniform from the droplets are produced on the finger-sized substrate. And single DNA molecules are randomly distributed into each droplet. The protein yield is proportional to the number of the molecules in a droplet.
Our technique can be used for a rapid and quantitative measurement of enzyme activity without complicated protein expression and purification. To begin, set a cover glass on a staining rack and place the rack in eight molar sodium hydroxide. Sonicate the cover glass and staining rack for 15 minutes at room temperature.
Remove the staining rack from sodium hydroxide and rinse the cover glass with water 10 times. Dry the cover glass with an air gun. Then bake and dry the cover glass on a hot plate at 200 degree Celsius for five minutes.
Prepare 0.05%aminosilane solution. Immerse the cover glass in this solution and incubate it for one hour at room temperature. Rinse the cover glass five times in pure water.
Then dry the cover glass using an air gun. Bake it on the hot plate at 80 degree Celsius for five minutes. Place the cover glass substrate on a customized vacuum chuck for the spin coater.
Dispense 70 to 90 microliters of type-A CYTOP polymer at the center of the glass substrate. Then immediately spin-coat the polymer. The initial polymer coating damage the quality of the final microchamber array.
Dispense the polymer onto the center of the cover glass without introducing air bubbles. Pick up the coated glass by holding it at the corners and place it on an aluminum foil. Bake it for 30 minutes at 80 degree Celsius and then for another hour at 200 degree Celsius.
Dispense 0.2 to 0.3 milliliters of photoresist at the center of the substrate. Immediately spin-coat the photoresist at 6, 000 rpm for 60 seconds. Use an ethanol-soaked wipe to remove the excess photoresist from the edges of the substrate.
Bake the substrate at 110 degree Celsius for five minutes. To rehydrate the photoresist, let the substrate stand for 30 minutes at a relative humidity of 42 to 60%Load the substrate into the mask aligner and expose it for 25 seconds in a vacuum-contact mode. Then immerse the substrate in developer for five minutes to dissolve the exposed photoresist.
Rinse the substrate 10 times using pure water. Then dry the substrate thoroughly using the air gun. Place the substrate in the reaction chamber of the reactive-ion etching machine and etch the photoresist covered CYTOP with the oxygen plasma.
After etching, remove the remaining photoresist by sonicating the substrate in acetone for five minutes at room temperature. Then sonicate the substrate into propanol for five minutes. Rinse, sonicate, and rinse the substrate again using pure water as described in manuscript.
Then dry the substrate with an air gun. Using a desktop cutter, cut double-coated adhesive Kapton film tape into the defined microchannel shapes. Stick the cut pieces of tape in the bottom of a flat Petri dish to serve as a master for the molding of the PDMS.
Pour the PDMS mixture into the tape-patterned Petri dish. Then place the Petri dish into a mini vacuum chamber and deaerate the PDMS mixture for one to three hours. Then place the Petri dish in an oven at 60 degree Celsius and leave it overnight to cure the PDMS.
After the PDMS has cured, peel the cured elastomer from the Petri dish and use a flat-cable cutter to cut out the PDMS microchannel blocks. Then punch a hole at each end of each microchannel. The previously prepared substrate will have an area for the microchamber array.
Position the PDMS microchannel on this part of the substrate. Then insert a 200 microliter pipet tip into one of the holes in the PDMS microchannel. First, prepare the CFPS reaction solution in a PCR tube.
Then, using a 200 microliter non-filter pipet tip, draw up 10 microliters of the solution. Insert the pipet tip into the inlet hole of the PDMS microchannel and push down on the pipet plunger until the solution overflows from the microchannel outlet. Transfer the FemDA device to a pre-chilled aluminum block.
Confirm that the microchamber array area quickly turns from translucent to transparent. Draw 300 microliters of pre-chilled flush oil and immediately transfer the oil into the pipet tip of the microchannel inlet hole. The flush oil moves into the microchannel and extrudes the excess reaction solution located outside the microchambers.
Simultaneously remove the two inserted pipet tips from the device. Immediately move the device from the aluminum block to paraffin film. Insert the prepared pipet tip containing the sealing oil into the inlet of the PDMS microchannel and inject the oil until it overflows from the outlet.
The femtoliter droplets are sealed in individual microchambers by the oil. Open the defocused BF image. To remove smooth continuous backgrounds from every frame, select Subtract Background from the Process menu.
Enter 20 for the rolling ball radius. Check the Preview box and select Okay. Select Yes when asked whether to process all images.
Next, to reduce the noise, select Process, Filters, Median. Enter 2.0 for the radius in pixels. Check the Preview box and select Okay.
Select Yes when asked whether to process all images. Next, select Image, Adjust, Threshold to separate the images of the microchambers from the background of the image. From the Plugins menu, select FemDA, FemDA Analysis.
Input the approximate minimum and maximum numbers of pixels of a single microchamber. The expected minimum and maximum circularity of the microchambers and the start and end frame numbers, then select Generate ROI to detect the microchambers. The successfully detected ROIs are shown in the popup menu ROITable.
In the FemDA Analysis window, select Apply ROI mask. Examine the image to see whether the microchambers were properly detected. Open the fluorescence image and bring it to the front.
Click Apply ROI mask again to add the ROIs to the fluorescence image. In the FemDA Analysis window, select One-Shot to analyze image end-point data. Enter N for the Number of top pixels to use the top-end pixels of each ROI to calculate the mean intensity of the corresponding droplet.
Then click Measure intensity to calculate the mean intensities of all detected droplets. The ROITable is updated with the new data from the fluorescence image and the histogram is displayed in a new window. Export the data by clicking the button Save as Text.
Fluorescent solution was encapsulated in FemDA microchambers and the fluorescence intensity, which is correlated with droplet size was measured using microscopy. The droplets were found to have a narrow size distribution. The reconstructed 3D image from the confocal microscopy also showed the consistent droplet size over time.
The droplets were stable at room temperature without evaporation loss or cross-contamination among droplets for at least 24-hours. The fluorescent protein mNeonGreen was synthesized in FemDA. And the stack image data was analyzed with the aid of the concurrent defocused bright field image.
Because the fluorescence intensity of each droplet is measured of its protein yield, the histogram strongly suggests unequal numbers of DNA molecules per droplet. The probability of droplets containing different numbers of DNA molecules was a perfect fit to a Poisson distribution. Synthesis of the enzyme alkaline phosphatase was also carried out in FemDA with images captured every five minutes.
The reaction showed a similar discreet distribution of the fluorescence intensity of the droplets at an earlier time points and the histogram results verified that the unequal numbers of the DNA molecules in the droplets. Single DNA molecule encapsulated in individual droplets can be further recovered, amplified, and sequenced make it highest proteins screening possible. A droplet technique featuring either small volume and either high stability allows for rapid and precise molecular diagnosis of cancer diseases with specific biomarkers.
