Madrid-OLM

Manufacturing Protocols

One of the most challenging parts of our project has been generating the tools required to experiment.

Although previous iGEM teams have been working with similar techniques, as microfluidics or microvolume pumping, their workflows involved relevant expenses. This fact encouraged us to search for an alternative solution, and share it with the whole iGEM community.

In the first part of these protocols, you can find our experiences with respect to DIY biodevices manufacturing.

The second part puts together the instructions, tips and tricks about assembling our prototypes, polymerization chamber and microfluidic chips.

Every step followed in this section has been documented in our Github. Click on the link and explore all our content!

Chip

1- Mold making

Laser engraver hacking
Laser engraver hacking

Bill Of Materials: You could see a complete BoM here.

Amount of time: 3 hours.

Total costs: 100 €.

The laser usually come with a proprietary software that only allow you to engrave images. As the chips are going to be cuttered as a vector we’ll need to install a new laser firmware (into the laser) to be controlled with other software (into the PC connected to the laser).
1. Check if your laser could be directly flashable (flash = installing the new firmware) in this link. If the answer is yes, skip to the “software workflow” step. If the answer is no, just continue reading.
2. When the Arduino UNO and the CNC Shield with the drivers arrive, substitute the old electronics of the laser with the new ones following the tutorial of the previous link. The software that you will need to follow this tutorial will be:
Software workflow
Software workflow

Bill Of Materials: You could see a complete BoM here.

Amount of time: 2 hours.

Total costs: 0 €.
1. You only need two applications to run your laser engraver:
2. In order to create the shape of your microfluidic chip, you need to have a feasible design. There are many open software applications, as:
3. If the design is made in a software different to Inkscape, you should export the file as a .dxf file (R2013). Inkscape can import this sort of files.
4. Once the design is created and imported into Inkscape, you should place it within the canvas with the required proportions. For NEJE laser engraver, the dimensions of the bed is 38 x 38 mm. You should find how Inkscape work area relates to the effective area of the bed.
5. Once the dimensions have been corrected and fixed, you should convert the lines to a “path” format, clicking in the “path” tab and selecting “stroke to path”. We have tried to omit this step, and it seems it does not matter skip this step.
6. Click on “extensions” tab and select “generate laser gcode”, and set the cutting parameters. These parameters should be set experimentally. Speed, laser power, passes vary depending on the specific tape selected, or the surface where you stick the tape. We encourage you to set your desired parameters, based on your specific scenario.
7. Once the .gcode file has been successfully created, open the Universal Gcode Sender and and set the ports and baud rate (115200 bps).
8. Now you need to set your “Zero machine coordinates”. From “Machine control” tab, move the X- and the Y- to the coordinates that you want to set as (0,0). And click on “Reset Zero”
9. Import the file. (File mode tab -> Browse). And finally send it to the laser engraver.
Calibration
Calibration

Bill Of Materials: You could see a complete BoM here.

Amount of time: 2 hours.

Total costs: 10 €.

There are three factors that you should consider for calibration purposes:
1. Choosing the tape: Depending on which depth of channel suits your needs, you should choose one tape or another. Cutting parameters should be established by each user, according to their particular needs.
2. Calibrating the dimensions of the Inkscape canvas: we highly recommend you to know the correction factors to apply to your design. A good practice should involve (for instance) designing a 10 x 10 mm square in Inkscape and send it to NEJE laser engraver, and measure it afterwards to know exactly how dimensions translate between software and manufacturing hardware.
3. Getting to know the effective work area of bed of the NEJE laser engraver: if you set the laser over the Zero-machine coordinate, and the, in the “command tab”, you insert the command s01, and M3, you should see how the laser turns on. If you click on X+ or Y+, you should be able to navigate over the edges of the bed, and thus, determine (and engrave) them onto the bed or any other surface that you might consider.
Disclaimer: The programs that we have used in these step are not developed by our group and are hosted in our Github just to keep the version that have worked for us.
Cutting
Cutting

Bill Of Materials: You could see a complete BoM here.

Amount of time: Depending on the project

Total costs: less than 5 €.

Although cutting is a pretty straight forward process, please, take this advises into consideration:
1. Mind your eyes: Please, do not hesitate to use the safety glasses devoted to look to the laser.
2. Use your final polymerization surface to support your tape: we have used an acetate sheet that fits in our final polymerization chamber. Ensure your surface are horizontal and without bubbles between the tape and itself. Lifting off the table from the surface in which it has been cuttered may cause the deformation of the original pattern.
3. Chlorine content in many tapes: As many tapes contain a remarkable amount of chlorine, we advise you to cut in a ventilated place and to avoid inhaling this.
4. Enjoy not always you get the results that you are planning. Take the time to set your arrangement and enjoy this process!

Back to Mold making Index

2- PDMS casting

Preparing PDMS
Preparing PDMS

Bill Of Materials: You could see a complete BoM here.

Amount of time: 1 hour.

Total costs: 120 €.
1. Working with PDMS require tidiness and cleanliness. It is mandatory to have a clear work space and dedicated hardware, as pipettes and recipients specifically devoted to mix. You will need gloves and cover your clothes (if you barely do care).
2. Mixing PDMS with the curing agent require that you follow the following weight ratio: 10:1 (PDMS/curing agent).
3. Mix it with the stirrer. Many air bubbles will appear as a result of this. You want to bring them out for the PDMS chip not to be porous.
4. Pour the mix into a recipient that fits into a vacuum chamber
Pump and chamber
Making a vacuum pump and vacuum chamber

Bill Of Materials: You could see a complete BoM here.

Amount of time: 3 hour.

Total costs: 20 €.
1. First you need to modify the compressor into a vacuum pump. To achieve it, we recommend consulting the following video
2. Once you have converted the compressor into a functional vacuum pump, you can create a vacuum chamber. Please, take into account that the vacuum chamber should be able to accommodate the recipient where the PDMS, the curing agent and the army of air bubbles are.
3. You might want to provide your vacuum chamber with a venting valve for pressurizing the chamber alternatively, as we explain in the following point.
4. You should seal every possible leakage with PTFE tape. Air leaks are not desirable, and your duty is to avoid them.
Degassing
Degassing

Bill Of Materials: You could see a complete BoM here.

Amount of time: 30 minutes

Total costs: 0 €.
1. Degassing involves extracting the air embedded in the mix of PDMS and the curing agent. First you need to place the recipient which holds the mixture of PDMS and bubbles inside the vacuum chamber.
2. Turn on the pump. The vacuum pressure provided by the pump is more than enough for our purposes. Leave it for 20 seconds and you will see the bubbles migrating towards the surface.
3. Once a significant amount of bubbles has moved towards the surface, let the airflow inside the vacuum chamber. Many bubbles will burst, and thus the air will be successfully extracted.
4. Repeat the process enough times to remove all the trapped bubbles.
5. Once the PDMS is homogeneous, you can pour the mixture into the polymerization chamber, where you are planning the PDMS to cure.
Polymerization
Polymerization process: curing time and heating

Bill Of Materials: You could see a complete BoM here.

Amount of time: 1 hour.

Total costs: 0 €.
1. This is the final step of the whole process. Once the PDMS is ready to cure, you may want to have the material ready in an hour, or maybe you can leave it curing overnight.
2. If you place the mixture inside a drying oven (65ºC), your PDMS will be cured in just half and an hour.
3. Elaborate a chart with the parameters that suit your needs better.

Back to PDMS casting Index

3- Plasma bonding

Modding a microwave
Modding a microwave

Bill Of Materials: You could see a complete BoM here.

Amount of time: 2 hours.

Total costs: 50 €.

Disclaimer: Modding any devices that plug to the wall socket is very dangerous. If a capacitor is discharged thank to your body, it may result harmful for you. Keep an eye on this.
1. Check the content online, as this or this videos. Many sources of information can be found online. You may find online a better arrangement than the one that we suggest. Although it has worked for us, there is no better understanding than the hands-on experience.
2. Unplug the microwave.
3. Remove the chassis of the microwave.
4. Saw what is necessary for opening a hole that enables the vacuum hose to get into the microwave.
5. Prepare a recipient for the PDMS chip that you want to treat. We drilled a hole into the Borosilicate Glass Narrow Neck Laboratory Bottle and glued a plastic hose adaptor.
6. Connect the hose to both ends: the recipient inside the microwave, and the vacuum pump.
7. Check that the vacuum circuit has no leaks. Plasma needs approximately 1 Pa to pop up. Vacuum pressure is a critical parameter. Make sure there are no leaks within the air circuit.
8. You are ready to proceed to the next step and calibrate the microwave (if it makes sense after all).
Calibrating the microwave
Calibrating the microwave

Bill Of Materials: You could see a complete BoM here.

Amount of time: 2 hours.

Total costs: 1 €.
1. CBefore even thinking about treating the PDMS, make sure that the plasma is generated successfully. You just need to test the whole display without PDMS inside the bottle. Calibrating the microwave means knowing how the it works experimentally, so take a pen and a paper.
2. You should make a chart with some parameters as:
3. Sparks will help to generate the plasma inside the bottle. In this regard, you need to place some aluminum foil inside the bottle. Measuring the required amount of aluminum foil is key for the experiment to succeed. We cut pieces of 20 x 20 mm of aluminum foil and use them as our arbitrary building blocks of aluminum foil.
4. First, you need to get some sparks out of the aluminum foil. If you get no sparks, add more aluminum foil, or increase the power. Take notes about how it behaves.
5. Once you get some sparks it is time to generate plasma. Vacuum pressure is key. 1 Pa was recommended. We were limited by the resolution of our manometer. We could only read 755 mmHg as (negative) relative pressure. We have no certainty about the actual value of the pressure.
6. You can use a glass of water to absorb any excess of energy. Note how water affects the experimental arrangement.
7. Once the treatment is done, the borosilicate bottle should not exceed 150 ºC. Test it with an IR thermometer gun.
8. Once you are happy with the experimental outcome, you should proceed to the next step and bake the PDMS.
Baking the PDMS
Baking the PDMS

Bill Of Materials: You could see a complete BoM here.

Amount of time: 2 hours.

Total costs: 0 €.
1. This protocol repeats the protocol before, with the exception that now we use the PDMS and the glass and analyze the new results.
2. Although plasma will fill the bottle, you need something to separate the PDMS chip and the aluminum foil. You do not want the PDMS to burn, as it will notoriously change its properties. Glass will prevent the PDMS to burn.
3. Make sure that PDMS and aluminum foil are separated by a glass plate, or any other glass surfaces that you might have at hand.
4. Follow what you have learned in the previous step and bake it.
5. Once the treatment is done, test the hydrophobicity of the PDMS, placing a drop of water on its surface. A treated PDMS chip should be hydrophilic. And a burnt chip will be absolutely hydrophobic
6. You should elaborate another chart with the relevant information about the process, studying the surface tension of the PDMS with respect to a drop of water.
7. Once the surfaces have been treated, they should be joined together immediately, and leave it a few hours to get the desired effect.
8. Two surfaces have joined when the bonding is strong enough to be broken before the surfaces are split up.

Back to Plasma bonding Index

4- Closing the microfluidic circuit

Choosing pump, tubing and connectors
Choosing the pump, the tubing and the connectors

Bill Of Materials: You could see a complete BoM here.

Amount of time: 2 hours.

Total costs: Less than 50 €.
1. Get some syringes with different volumes. Get many needles. They will be part of your pumping system
2. Choosing the right tubbing in your applications depends on several parameters. The most importants are: dead volume to displace, pressure drop and maximum allowed pressure for the tube. You could see a complete explanation of this parameters here. There are available computational methods that you can use to design your circuit.
3. If you do not want to compute any fluid mechanics parameters, there are many available experimental resources at hand, as butterfly needles with a flexible catheter line. This might suit your needs at a very initial point, and serve for hands-on purposes.
4. Syringes, fittings and needles follow a standard, which name is Luer standard. If the pump is a syringe, you should make sure it follows the same standard. Luer taper has two variants: luer-lock and luer-slip. See more information about mechanical specs here (ISO 80369-7:2016).
5. Once you have choose the tubbing is time for choose the fittings. Fittings are the connection between your circuit and your chip. Fittings will vary from one chip to another:
6. Mind the mechanical forces. If the bonding between PDMS-PDMS is not strong enough, a small torque from the fitting and the tubing may produce leaks in around the input area. PMMA chips are more robust.
7. A DIY way of facing all these issues is adapting your design to inexpensive and common resources. For instance:
8. Once the input is plugged in, the output should be (at least) exposed to ambient pressure when fluid is pumped in. Alternatively, the output could have a negative pressure, exerted by a secondary output pump. You do not want to have a high pressure inside your microfluidic chip. The stiffness of the PDMS is very low and a high air pressure will deform it, affecting the ideal behaviour of the circuit.
9. Once the circuit is closed, you shall start experimenting with your chip.
Troubleshooting the leaks
Troubleshooting the leaks

Bill Of Materials: Not applicable.

Amount of time: Not defined.

Total costs: Not applicable
1. You might find a leak somewhere in your circuit. Depending on where you find it, it may be caused by one factor or another.
2. In the clearance of the input/output: Seal it with PDMS.
3. Inside the chip: in the input/output area: when a needle is inserted, it may push the base PDMS surface and create a bubble. If the two PDMS surfaces are not adhered one to another, the fluid may squeeze between these two surfaces.
4. Inside the chip: in the input/output area: if the needle does not touch the lower part of the PDMS, mind the torque due to the mechanical forces in the input. The resultant force should always be normal to the PDMS surface.
5. Inside the chip: in the input/output area: if the chip is too small or too thin, and the input/output are close to the edges, chip sensitivity increases dramatically. Try to be conservative in this regard and promote your chip stiffness at the very beginning. Further successful attempts could be directed to optimise the dimensions of the chip.
6. Inside the chip:Anywhere. You should check for any overpressure inside the circuit. If the output is closed, the injection pressure will deform the chip and open the most sensitive parts of the chip to leaks. A positive pressure in the input should translate in air flowing through the output.
7. Inside the chip: Anywhere. You should check for any overpressure inside the circuit again. If the output is opened, the overpressure could come from your pump. If the pump is manually controlled, maybe you are pushing too hard. An electromechanical control, as we have developed in our project may result handy at some point.
8. Inside the chip: Anywhere. PDMS is very accurate for casting a mold. If the tape has some imperfections, or the surface is irregular, for instance, it will be copied exactly to your chip. Try to work under controlled circumstances.

Back to Closing circuits Index

Assemblies

1-First Prototype

Documentation: Find it in our Github. Part numbers are specified in this document.

Materials: PMMA adhesive and a multi-tool (metal saw)

Amount of time: less than 5 hours + overnight

Total costs: 30 €

Walls
Glueing the walls
1. There are two different sets of walls: the opaque one (14, 15) (for the microfluidic chip) and the transparent one (3, 4) (for the electronics).
2. You should fix the walls with a PMMA adhesive. The adhesive should be spread on the surfaces that have been laser-cutted. See the red spots on the picture.
Threaded rods
Cutting the threaded rods
1. You need four M5 female legs (23). And then, you need to cut the M5 threaded rods (22) to fit the required dimensions.
2. There are two different pairs of M5 threaded rods: one of these pairs has a length of 60 mm approximately. The other pair has a length of 120 mm approximately.
3. Measure it and cut it with the multi-tool.
4. These four rods will connect the legs (the bottom of the device) and two different floors (the stage of electronics and the microfluidics one). You should cover the other end with a M5 cap nut.
Upper cover
Assembling the upper cover
1. These eight parts (13, 14, 15, 16, 17, 18) should be fixed with PMMA adhesive. They should form a rigid block.
2. Once they are hold on together, they should be manipulated freely to reveal the microfluidic chip or hide it from external light.
Middle platform
Assembling the middle platform
1. The middle plattform only consist of two different pieces (7, 8). Piece 7 is repeated twice. See how the arrangement is rotated 180º to enable electrical cabling and avoid as much light as possible.
2. These three parts can be fixed together using PMMA adhesive.
3. To ensure they are properly aligned, you might want to make use of a 2 mm rod or screws.
Microfluidic chip
Assembling the microfluidic chipm
1. The structure made to hold the PDMS microfluidic chip is made with four pieces (9, 10, 11, 12).
2. They do not need to be fixed permanently together..
3. They are hold on together with four spacers (6). And they need four M2 screws to keep the structure rigid once the 40 x 40 PDMS chip is placed correctly.

Back to Firts Prototype Index

2-Pressure pump

Documentation: Find it in our Github. Part numbers are specified in this document.

Materials:PMMA adhesive, transparent tape, and a multi-tool (metal saw).

Amount of time: less than 5 hours + overnight

Total costs: 30 €

Fix the body
Fixing the body
1. Although it is not mandatory, we recommend the user to join permanently these eleven pieces (16, 17, 18, 19).
2. They are meant to be the fixed part of the pressure pump, that is why they need extra stiffness.
3. Do not forget to use two or more M4 rods for aligning perfectly the pieces, avoiding eventual imperfections. Clearances are a bit tight and rotor axis and spindle rotation have imperfections when rotating.
Spindle and axis of the rotor
Coupling the spindle and the axis of the rotor
1. Fixing the stepper motor to the coupler and the spindle (or the threaded rod in our case) (3, 4, 5) requires patience.
2. You need to avoid misalignments, as far a possible. Designing something is pretty straight forward, but making it work is a sensitive task.
3. Mind the eccentricity of the free end of the threaded rod, and align it as much as possible.
Spindle into the PMMA body
Insert the coupled spindle into the PMMA body and attach the front
1. Once the stepper motor to the coupler and the spindle (3, 4, 5), insert the spindle into the body (16, 17, 18, 19) and cover them with the chassis-front (15).
2. Now you need to fix everything together with a M4 bolt, a washer and a nut. The length of the bolt should be around 45 mm approximately.
Fix the syringe tray
Fix with adhesive some pieces of the syringe tray
1. There are two different sets of pieces that need to be joined together permanently. On the left side you see three pieces of the syringe tray (12, 13, 14) and on the right side there are three pieces of the anti-backlash nut (9, 10, 11).
2. You need to fix together two sets of the anti-backlash nut (9, 10, 11), and only one set of the syringe tray (12, 13, 14).
3. Do it with PMMA adhesive so they hold on together permanently.
Anti-backlash nut, syringe tray
Assembly the anti-backlash nut and the syringe tray
1. Insert the pieces in this order. First one of the two sets of the anti-backlash nut (9, 10, 11).
2. Now insert a M4 nut (6), a M4 washer (7) and a spring (8). Then, another washer and another nut.
3. Now it is turn for the syringe tray (12, 13, 14) and finally, the anti-backlash nut (9, 10, 11).
4. The two sets of anti-backlash nut and the syringe tray should be fixed permanently. Notice that the hexagonal piece of the anti-backlash nut should be perfectly accomodated.
5. There is a easy way of configuring the setup. The spring should push the washer and the nut oppositely. Use the anti-backlash nut to adjust the length of the spring. You should start with the spring absolutely unfolded, and turning the last anti-backlash nut PMMA piece, you should be able to adjust the compression of the spring.
6. After fixing everything with the adhesive, we used a bit of transparent tape to make the mobile part a bit more rigid.
Syringe accommodation
Syringe accommodation clearance
1. Syringe (1, 2) has two problematic points, where they accommodate in the body (16, 17, 18, 19) and in the syringe tray (12, 13, 14).
2. There are clearances due to the difference in the size of the syringe and the available PMMA. These two clearances need to be filled with something to prevent them moving when the pump is activated.

Back to Presure Pump Index

3-Second Prototype

Documentation: Find it in our Github. Part numbers are specified in this document.

Materials: Screwdrivers

Amount of time: less than 4 hours

Total costs: 0 €

Base of the device
Setting the base of the device
1. The first step involves assembling the base (1) to the legs (4) and the M4 spacer (13) to the M4 threaded rod (14). This step is a requirement for further steps.
2. As the distance to the next stage is 38 mm, you could place two M4 spacers (20 mm of length) to set the distance correctly from the beginning.
Fix the spacers
Fix the spacers to the Arduino + drivers base
1. Spacers (7) should be fixed to the base (9) before assembling the rest of the prototype.
2. Spacers should be fixed to the base with M3 nuts.
3. The Arduino and the drivers will be fixed to the spacers with bolts. Cut the bolts to the length of the PMMA base taking into account the threaded distance of the spacer.
Connecting the pumps
Connecting the pumps
1. Lower walls (10, 11, 12) require to be assembled before connecting the pumps to the drivers.
2. The cables of the stepper motors need to be assembled through the walls. The walls do not require to be fixed permanently to the base.
3. Please, note that the small wall that has the biggest hole corresponds to the Arduino connector area. Bear this in mind while assembling this part.
Fix the potentiostat
Fix the potentiostat to the double middle base
1. Join two potentiostat bases (8) together with a M3 spacer (7). You should do it with a M3 bolt. Take into account the thickness of the bases and the threaded space of your spacer.
2. If the potentiostat is a Rodeostat, you should modify the diameter of the holes for them to fit into our spacers. And then, fix it to its place. The orientation should be like the one shown in the picture.
3. After joining both pieces, you should assemble the walls before continuing
Assemble the body
Final assemble the body of the device
1. Parts do not need to be adhered one to another. They just need to be placed right in place, minding the gap between the device base (1) and the arduino + drivers base (9).
2. Instead of placing one spacer (13) above the legs (as shown in the picture), you should place two 20 mm length spacers, as explained in the previous step 1.b.
3. Once the assembly has been made you might want to make stiffer the structure, toppint the threaded rod with four M4 Acorn Hex Cap Nuts.
Fix the pressure pumps
Fix the pressure pumps to the structure
1. Pressure pumps are fixed to the structure using 2 M4 bolts, optional M4 washers and M4 nut.
2. The M4 rods that work as sliders for the pressure pump have a designed housing in the device base (1).

Back to 2º Prototype Index

4-Polymerization chamber

Documentation: Find it in our Github. Part numbers are specified in this document.

Materials: Screwdrivers.

Amount of time: 5 minutes.

Total costs: 6 € per unit.

Assembling the polymerization chamber is straight forward.
Note that, as the input / output are laser cutted as small holes, it might be a guide on how to place the sheet with the laser cutted tape in its required position.
The polymerization chamber is closed, as we want to minimize any deformations due to the interactions between PDMS and the mold.
If you are planning to take the chamber into an oven, mind the temperature that the PMMA can resist without any plastic deformations.

Team:Madrid-OLM/ProManufacturing

Manufacturing Protocols

Chip

1- Mold making

Laser engraver hacking

Software workflow

Calibration

Cutting

2- PDMS casting

Preparing PDMS

Making a vacuum pump and vacuum chamber

Degassing

Polymerization process: curing time and heating

3- Plasma bonding

Modding a microwave

Calibrating the microwave

Baking the PDMS

4- Closing the microfluidic circuit

Choosing the pump, the tubing and the connectors

Troubleshooting the leaks

Assemblies

1-First Prototype

Glueing the walls

Cutting the threaded rods

Assembling the upper cover

Assembling the middle platform

Assembling the microfluidic chipm

2-Pressure pump

Fixing the body

Coupling the spindle and the axis of the rotor

Insert the coupled spindle into the PMMA body and attach the front

Fix with adhesive some pieces of the syringe tray

Assembly the anti-backlash nut and the syringe tray

Syringe accommodation clearance

3-Second Prototype

Setting the base of the device

Fix the spacers to the Arduino + drivers base

Connecting the pumps

Fix the potentiostat to the double middle base

Final assemble the body of the device

Fix the pressure pumps to the structure

4-Polymerization chamber