This protocol has been designed as an example of nanolithography using FluidFM®. Here we print a solution containing a fluorescent molecule, 4’,6-diamidino-2-phenylindole (DAPI), on a hydrophilic glass surface coated with DNA.

While the protocol was developed with the FluidFM BOT it can also be used with FluidFM ADD-ON solutions.

Material

• Glass bottom dish WillCo-dish® (Willco Wells, GWSB-5040)
• Cytosurge® Nano Pipette, 300nm aperture, 2N/m specified stiffness
• DAPI dihydrochloride solution in water (Sigma® D9542)
• 90% Glycerol solution
• 5mg/ml Salmon sperm DNA solution in water (Sigma® 31149)
• 0.2% gelatine in water
• ddH2O
• Sigmacote® (Sigma® SL2-25ML)
• 0.2µm filters

Sample preparation:

Probe preparation

Probe coating

The surface of the Nano Pipette needs to be hydrophobically coated. We use Sigmacote®, a silicone based solution, according to Silicone coating protocol.

Reservoir filling

Freshly prepare a 10% glycerol, 1mg/ml DAPI solution in ddH2O. Dispense 1µl of this solution into the probe reservoir, according to "Probe preparation" instructions. In order to avoid drying of the solution, it is strongly recommended to load the probe on the BOT as fast as possible after this step.

Dish preparation

Surface treatment

Willco-Dish® need to be treated to make the glass hydrophilic, and therefore allowing them to bind DNA. Here we propose a treatment using gelatine. Other surface treatment procedures, like Plasma treatment, can also be applied.

1. Cover the bottom of the dish with a 0.2% gelatine solution (filtrated).
2. Wait 15 minutes.
3. Pour out the gelatine.
4. Let the surface of the dish dry out. The dish is ready for coating with DNA.

DNA coating (Optional)

Because DAPI binds strongly to A-T rich regions of DNA, coating of the dish with DNA will therefore help the deposition of DAPI solution during printing. This step is optional as gelatine will also bind DAPI molecules.

However, if using another hydrophilic treatment such as plasma treatment, DNA coating of the dish is strongly recommended.

DAPI has a maximum emission wavelength at 461 nm when bound to DNA, 488 nm when not bound to DNA.

1. Prepare a 5 mg/ml DNA solution.
2. Filter the solution through 0.20 µm pore.
3. Cover the surface of the Willco-Dish® with the filtered DNA solution (1 ml is sufficient).
4. Pour out the DNA solution, and let the Willco-Dish® dry out.

Procedure

Prepare system

• Turn on the BOT, microscope and the computer
• Open the software
• Load the probe into the probe holder in the right port of the BOT. 
  
• Load the coated Willco-Dish® in the left port of the AFM. Make sure that the dish is tightly fixed on the holder. 
  
• Perform the Prepare system workflow.
• System is now ready. Proceed to the printing, either through the "Line printing" workflow or through the "Print from file" workflow

A. Line Printing:

Use the "Line printing workflow" from the software.

Recommended parameters for printing lines as thin as possible:

• Setpoint: 20mV

• Idle Pressure: 0mbar

• Approach speed: 10µm/s

• Retract distance: 20µm

• Rotation: 0

• Line configuration: Line count: 5; Line length: 100µm; Line shift: 10µm, Print speed: 10µm/s; Pressure 20 mbar

• Press the “Ok” button and observe the printing:

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B. Print from file

It is possible to use a predefined pattern, by creating a file with xy coordinates, printing speed and pressure. The BOT will print line between each given point in the file. Each parameter must be separated by a coma.

For each point (i.e. each line in the file):

• First column set the x position in µm.
• Second column set the y position in µm.
• Third column set the speed for printing in µm/s.
• The last column set the pressure in mbar.
• Also, there are special control instructions to e.g. retract and approach.

Use the "Print from file" workflow from the software and select your file. Press the Ok button to start printing.

Some examples are shown below: