Progress Indicator Animation
New insights into cellular mechanisms are spreading the awareness of heterogeneity between single cells of one organism in the same environment. Therefore, we created a microfluidic chip for cultivation and single cell monitoring. This grants the opportunity of precise quantification and measurements of our systems recognition efficiency. As the manufacturing of these systems is quite complex and cost intensive with standard methods, we tried to use additive manufacturing and quick prototyping methods to find a cheap and effective alternative that meet the expectations. Furthermore, we examined methods for different tasks and organisms.
With the open source character in mind, we also engineered different useful devices to make microfluidic easily accessible for other teams and able to use in other laboratories. The whole concept and the respective single devices are designed to make working with microfluidics easier and faster to process for everyone.
In addition to our main hardware project, we used additive manufacturing for different tasks and problems at our lab and with the open source character in mind, we created diverse helpful objects that can be useful for many teams. The parts are reaching from general lab supplies to mounts and even different models to make explanations more understandable.
With the emerging topic of plastic waste and environmental pollution, our goal was also to reduce the burden that plastic waste causes to the environment. So, we exclusively used only biocompatible and biodegradable plastics for our apertures, mounts and casings.


Advance of Microfluidic

Microfluidic chips on a microscope slide.
New insights into cellular mechanisms are spreading the awareness of heterogeneity between single cells of one organism in the same environment. Current measurement systems and methods often are generating a mean value of the relevant physical quantity. Individual cells in a given population of seemingly uniform cells, for instance, can have different properties when it comes to metabolic products or behavior of the cells. Regarding the specificity of a method like the recognition efficiency of phages, it is crucial to observe cells on a much more individual level for significant data and quantification. We are trying to face these challenges with the advances of modern microfluidic systems. These systems are providing many benefits as they can be operated in single cell resolution. Therefore, it is possible to examine the reaction of a single cell in a given population. For this purpose, one main feature of the chip used for the measurement series has to be the capability of cell cultivation which includes access for inoculation, channels for substrate supply, and sufficient oxygen supply. Furthermore, a transparent material for monitoring the cell growth, movement and behavior as well as conducting measurements is a necessity. We wanted to design a microfluidic system that is able to:
  1. Contain several cells in one chip
  2. Provide sufficient environmental conditions for cell survival and cultivation
  3. Provide a separate growth chamber for a cell population
  4. Provide the opportunity to observe the cells and the cells behavior

As our project is strongly related to fungi, especially S. cerevisiae and A. niger, it is necessary that the microfluidic systems are further capable of cultivating different forms and sizes of fungi. To bypass the intense expenditure of time and the high costs of designing and engineering a microfluidic chip with standard methods, we want to establish a standard procedure for the fabrication of those chips with modern additive manufacturing methods. This way, we want to make microfluidics easy and convenient for everyone.

Advance of Additive Manufacturing

Tabletop FDM 3D printer in action.
We want to use the opportunities and advantages of the emerging field of 3D printing to design and manufacture our microfluidic systems and necessary devices. The main advantages of 3D printing are:
  1. Easy construction of the parts
  2. Fast design and prototyping
  3. Often significantly less expensive than other methods
  4. Tabletop 3D printers are cheap, small and simple to use
  5. 3D printers are commonly used, good support

Furthermore, we want to extend the range of application of 3D printers to be used in biotechnological laboratories and laboratories in general by creating open source products that are needed or helpful in general lab work. One of the main goals is to establish 3D printing in laboratories as an exceedingly fast, easy and cheap method for prototyping as well as manufacturing. All in all, we are trying to meet the open source character of iGEM with providing a simple way to share new applications and hardware parts.

Advance of Combination

Microfluidic and 3D printing are each relatively new fields with a new range of applications for both of them. The combination of 3D printing and microfluidics offers many advantages. We want to use this combination to make:
  1. microfluidics easily accessible by construction of diverse necessary devices leading to decreased costs
  2. the construction of microfluidic chips easy, fast, inexpensive and without the need of special cleanrooms
  3. microfluidic systems simple and fast to share and use as sending microfluidic chips between working groups is quite difficult and can damage the chips

At the end we want to create a standard procedure for microfluidic chip manufacturing that can not only be used for our specific purposes but is also able to cover a broad range of different microfluidic applications. The user should get into the manufacturing steps with ease and should be able to poduce his own microfluidic systems by adapting the standard procedures to his needs.

Wafer Design

The first step to produce a functional microfluidic system is to design a wafer, that is used like a stamp to form the later molding. The wafer has the same structures as the final chip, so it is possible to directly create the channel structures and chambers that are necessary for later functions. Wafers are mainly consisting of a basic ground plate, which has to be preferably plane and smooth, and the required channel structures directly on it. Therefore, a simple design consists of one flat cuboid as the base and many much more smaller cuboids on it representing the later channels in the chip. As a functional design presupposes a profund knowledge of the available methods and techniques we talked to Anton Gillert, an expert in the field of 3D printing. He gave us useful information and considerations about different 3D printing techniques and their special features that we integrated in our project.

Design Programs

User interface of the CAD software "FreeCAD".
The whole design can be made easily with most CAD programs (Computer-Aided Design programs). There are many freeware CAD programs that can be used for this purpose, such as FreeCAD, blender, 3D Builder, AutoCAD and many more. We recommend using FreeCAD or 3D Builder for beginners, as they are quite easy to understand and there are many free online tutorials that can be helpful. However, other CAD programs may also be sufficient and could be used for the wafer design.
The correct export of the made design is considerably important. Finished designs have to be converted into 3D data formats that can be processed for 3D printing later on. Standard data formats for 3D printing are “.stl” and “.obj”. They can be generated by converting or saving the design with the common CAD programs, but in any case, with the listed programs above. Once the design is successfully saved as a printable format it is possible to start the actual printing of the wafer.

Design Procedure

Before starting the construction of the chip, there are a few things that have to be considered to get a functioning design. What to consider (overview):
  1. Which function does the chip have to serve?
  2. Which organisms have to be used/investigated?
  3. Which features are required/preferred?
  4. Which printing technique is required/most suited?

As the very first step it is important to check which function the chip has to serve, as it is one of the main factors that will influence the design and purpose of different elements that will be on the chip later.
Secondly, the organisms of interest will be one of the fundamental criteria. If organisms have to be investigated there are several aspects that have to be considered, such as e.g. height and width of channels and growing chambers, loading of the chip with given organisms and much more. If the system is meant to investigate organisms over a longer time span and especially if the cultivation of organisms is required, it is also very important to check sufficient nutrient and oxygen supply. Different microorganisms could also influence the recommendable printing technique as variations in the needed size of channels and chambers influence the needed precision and accuracy of the printer.
The third main factor are the required and preferred features the chip should have. There are many different features that may be considered. What certainly plays a role at this point will be e.g. how many inlets and outlets, supply channels, growth chambers, etc. are needed.
If all points are considered it is possible to start the main design with the chosen CAD program.


Test Wafer

Test wafer as 3D model.
For testing the procedure of manufacturing the wafer, we designed different test wafer that were used as a reference for the single steps. The shown test wafer features channel like structures that where placed on a rectangular basis. The channels are angled 90° on the half of their length to represent rectangular angles of later structures. That way we were able to simulate how structures on the chips for cultivation will behave in the printing and post-processing steps. Further the channel width is varied to see which resolution is possible in the printing process. The width for the channels are:
  1. 300 µm (inner channel)
  2. 400 µm (middle channel)
  3. 500 µm (outer channel)

Main application for the test wafer was to see how different printing parameters will influence the quality of the printed wafer and how the wafer could be processed after printing. The whole manufacturing process of the microfluidic chip was first conducted and tested as well as optimized on this wafer.

FDM Wafer for Filamentous Fungi

3D models of a wafers designed for the cultivation of filamentous fungi.
This wafer is designed for the construction in a standard FDM/FFF 3D print. The design is oriented on the results and experience we got from the test wafer. It has a similar construction with a rectangular base that underlays the main structure.
The shown wafer was designed for the cultivation of fungi with filamentous growth like A. niger. It features one inlet structure to load the chip with the preculture and maintain fresh medium while cultivation. Further there are two outlet structures of which one is solely for the purpose of loading the chip with the preculture. After the loading the right outlet can be closed by squeezing the respective hose. After that the medium is flushed only through the channel which assures that the fungi are not rinsed out of the growth chamber. The growth chamber is situated on the middle of the chip and constructed as a 2x2 mm large square structure to allow the growth over a long period of time.
Constructing the chip with two outlets has the huge advantage of a simple and reliable loading with cells, without the disadvantage of flushing them out of the chambers because of convection.

DLP Wafer for S. cerevisiae

3D model of a wafer for the cultivation of Saccharomyces cerevisiae.
Unicellular fungi, like S. cerevisiae are rather small in comparison to filamentous fungi. The construction of a wafer for a cultivation chip for those organisms is slightly more complex as the structures need to be smaller and certain aspects have to be regarded. The main design differs not much from the concept of the previous presented wafer. The main features are one inlet, channels to the growth chambers, the growth chambers and three outlets. The outlet in the middle of the wafer is again used for maintaining a certain flow of media through the microfluidic chip to obtain sufficient nutrient supply for the culture. Both outlets on the outside can be closed via squeezing of the respective hose, to stop the flow through the growth chambers and ensure that the cells are remaining in the chamber.
Because very small structures are needed for S. cerevisiae, standard FDM printers can not be used. For precise printing of those microstructures it is crucial to use printing techniques with preferably high resolution, such as DLP printing. Getting the highest possible resolution from those printers, requires some special considerations in the construction process. As the technique is based on the UV illumination of polymers, the structures have to be multiples of the size of one pixel. That ensures the optimal illumination for higher printing quality.

Wafer Manufacturing

Different test wafers printed with DLP and FDM 3D printing.
The wafer manufacturing starts with printing the designed and converted part with a 3D printer. As the printing methods differ remarkably from each other, it is important to know which method is suited best and what to pay attention on.
There are many different printing methods on the market by now with a variety of many diverse techniques. Most important for printing wafers for microfluidic chips are the differences in resolution printing quality and surface quality as well as the usable materials and the potential post-processing steps.
All in all, there is a trend to a much higher resolution and to better details when DLP printing method is used. Although DLP provides a high-quality surface and is quite easy to process after printing. The available home 3D DLP printers are not as cheap as the FDM tabletop printers which is certainly a disadvantage, but it is possible to order DLP printed parts quite fast from professional providers at a reasonable cost.
As a very cheap and easy method it is possible to use FDM/FFF printing as an alternative when slightly larger structures are necessary or sufficient. We decided to use FDM/FFF as a standard method that can be used very easily by many teams and DLP as a suitable method for highest resolution and smallest chip structures which is also easily available for everyone.

Wafer 3D printing

FDM/FFF Printing

Cura user interface with loaded 3D model and settings.
Before printing, the part has to be sliced with a slicing software to make the file understandable for the 3D printer. The slicing software is also needed for preferred printing settings of the part. We used the slicing software “Ultimaker Cura V4.3” for preparing our print and to set the right printing parameters. Cura is a free to use printing software that can be downloaded and used to modify printing parameters and settings for the parts. It also has a user-friendly surface and is quite easy to use as it already comes with standard settings for many common tabletop FDM 3D printers.
To modify the settings, it is important to have the preferred material in mind, as this has an enormous influence on the best suited setting parameters. We chose our material after the following criteria:
  1. Printability
  2. Printing detail
  3. Printing quality
  4. Surface quality
  5. Availability
  6. Environmental compatibility
  7. Price

We talked to Marco Depaoli from the printing material industry to find a suited material for our purpose. The most important criteria for printing wafers are the printability, the details, print quality and surface quality. After the comparison of different materials properties, we decided to use PLA for our wafers, as well as for most of our other parts. PLA is perfectly suited because:
  1. very easy to print (even for beginners)
  2. Delivers good printing detail and quality
  3. Relatively even surface
  4. Excellent availability
  5. Is environmentally compatible (biocompatible material)
  6. One of the most inexpensive materials

Further it is possible to smooth the surface of the material excellently after the printing to get a very even and smooth finish that is suited for the chip manufacturing.
To find suited printer settings for our used materials and find out how height, width and geometry influences the precision of different printed structures, we started with printing test structures.
Construated test structure for printing tests.
3D printed test structure with some stringing between the elevated structures.
If you want to find the optimal printing settings for your material of choice you can use this test obejcts to try out the effects of different parameters.
The test structures are available as .stl files on our Download page.

After we determined our preferred printing settings for the used material and printer, we tried the first printing test with our test wafer. Even if PLA is very easy to print it is possible that warping occurs on certain objects and if the printing parameters are not ideally adjusted. Warping is a certain effect that is quite common in 3D printing and results in warped surfaces of the printed object. It is a result of the shrinkage of the heated material that is cooling down not homogenously because of temperature gradients while printing. This can cause the wafer to be uneven on the surface, which would affect the later processing and usage as mold negatively and cause the microfluidic chip to be leaky after assembly. The following shown prints demonstrate the unfavorable effect of warping of flat objects such as wafers.
Warping on a flat structure.
Two wafers that are warped on their edges.

To overcome warping and get high quality prints there are diverse methods that can be used effectively. One approach to overcome this effect is to print a brim around the object, which can be adjusted in the settings of the slicing software.
We asked Andy Linnas what we could do to prevent warping and get an evenly flat object. His recommended approach was to use printing adhesion products. Coating the surface that is printed on with adhesives lets the first layer of the print stick excellently to the surface of the printing bed, which could prevent warping. Further he underlined the additional advantages that adhesion products would have on our project. Sticking the printed object to the printing bed also assures that it does not move while printing, which provides accurate positioning of the extruder and thereby a precise printing. Further it is easier to remove the print afterwards when it is cooled down, which minimizes the risk of damaging the sensitive structures.
3D printed surface model of the lipoprotein CsgG (Nanopore). On the bottom the brim used for printing is clearly visible.
Two wafer 3D printed with adhesions product coated building plate and without brim.

After adjusting the printing parameters for PLA, it was possible to get perfect printing results with adhesion products or small brim. Small structures could even be printed without any anti warping measures on our heated glass printing bed. Printing the wafer with 100 % infill and extra fine profile (0,06 mm) is suited best for optimal quality and the structures resolution of the wafer.
Important printing parameters and the used values for PLA are shown in the following table.

Printing Parameter Value
Extruder Temperature 219 °C
Printing Bed Temperature 61 °C
Fan Speed 100 %
Flow 105 %
Printing Speed 80 mm/s

Please note that the ideal Parameters are a product of the used material, the manufacturer, filament diameter, the used printer, the used printing bed material and other influences. The optimal printing parameters could differ from the ones stated above.

When printing without brim it is recommended to print a small structure first to clean the nozzle and ensure a constant material flow for the printing of the actual part. A similar effect has the option “print skirt” that can be preset in the slicing software. If selected, the printer will print a certain number of lines around the printing space of the object first. You can see the effect of printing a skirt on the following figure.
Printed brim (one line) around the gelcomb to clean the nozzle and obtain a constant material flow.
Close up view of the beginning and ending of a 3D printed brim around the gelcomb structure. The differences of the layer width at the beginning and ending are clearly visible.
It is clearly to distinguish between the beginning of the print and the actual printing, as the first line is starting very thin and is getting bigger till it reaches the starting point again.

DLP Printing

Table top DLP 3D printer.
DLP (Digital Light Processing) is a certain technique of 3D printing that is based on the polymerization of a liquid photo sensible resin. The polymerization of the fluid resin is started with UV light that is projected on the building plate from a projector on the bottom of the printer. The illuminated area will polymerize and build a solid structure.
Preparing the designed wafer for printing with the DLP technique is generally quite similar to the steps conducted for the process with FDM printers. At first it is important to open the 3D files in a special software that slices the models and makes it understandable for the printer. Functional software for DLP printers can mostly be obtained from the producer of the printer. The software is again also used for the settings of the printer and the printing process. However, unlike the preparation for FDM printing, it is crucial to pay attention on the alignment of the models on the building plate of the printer. For best printing results and to reach the maximal possible resolution, the models have to be matched with the pixel array when setting the layout of the print.
Interface of the slicing software for the DLP 3D printer.
Wafer models correctly aligned to the pixel array of the printers projector on the virtual building plate of the printer.

After adjusting the objects on the pixel array of the layout, the printer can be prepared for the printing process. At first the vat for the resin has to be installed and the resin should be filled in carefully so that no air bubbles emerge. Second, the sweeper will be set into the vat and locked in his mount.
Filling the vat with printing resin.
Installing the sweeper.
Locking the sweeper in its mount.

After preparing the printer the lid is being closed and the printing process can be started. The typical time for printing the wafers is about 10 minutes. As the whole building area is illuminated at once, the number of the printed objects has no influence on the needed time for printing, what makes multiple processing quite efficient and astonishing fast. The limiting factor regarding the printing speed is the number of layers. For the improvement of the printing speed the number of layers can be reduced quite easily by reducing the height of the wafer basis as this is not very important in the later process. Printing speed itself, say the speed of the vat moving, should be set possibly low to get higher printing quality and assure that the layers are not shifted while printing. This could cause fragments on the wafer and cause the channels to be unusable.
Table top DLP 3D printer in action printing new wafer for cultivating.

Once the printing process is finished, the lid can be opened, and the building plate has to be removed. The printed structures can be removed directly after printing from the building plate. Using a razor blade or similar objects can help loosening the structures from the plate. Be careful to do not touch the structures on the surface as this can damage the thin channel structures and cause the chip to be leaky and channels to be sealed.
Printer with opened lid after the printing process. The building plate moved out of the resin.
Removing the building plate to detach the printed structures.
Detaching the printed wafers with a razor blade.
The detached wafers can now be further processed.

Post Processing

After the printing step the wafers have to be processed first before using it to obtain better results at the chip manufacturing step. The processing is depending on the used printing technique.

Post Processing FDM Print

After printing the test wafer, we tried to use it directly for the chip manufacturing without processing it. A big problem of FDM 3D printing is, that the surface is not perfectly even and not smooth enough to use it for the further manufacturing process. Molding these wafers led to an uneven mold, which did not bond exactly to the glass substrate and thereby resulted in leaky microfluidic chips. Therefore, a post processing step is needed to smooth the wafers surface evenly. There are different approaches of smoothing surfaces in the 3D printing sector. One of the most common are:
  1. Grinding
  2. Smoothing step with the hot end of the extruder while printing
  3. Heat smoothing (with hot air)
  4. Smoothing with chemicals

Result of smoothing the test wafer with the smoothing option of the slicing software.
Grinding is out of the question as it is quite irregular and can not be applied on smaller structures. It would as well lead to a relatively rough surface if compared to the surface roughness that is required. The main application of grinding is the optical smoothness or regularity of the surface structure. So, we focused on the other three options.

Using the setting for a smoothing step with the hot end of the extruder after printing the wafer is a comfortable, easy and fast method of smoothing the top layers of printed objects. We tried to print our test wafer with the smoothing step setting, which can be set in the used slicing software. The smoothing of the surface itself turned out quite tolerable. However, the small channel structures were damaged and could not be used anymore. The smoothing option using the hot end is not recommended.

Result of smoothing the wafer with a hot gun.
Another option was the smoothing with hot air that would slightly warm up and melt the upper layers of the object to gain a very smooth surface after cooling down again. We tried to heat the upper layers with a heat gun to receive a homogeneous top layer.
Heating the wafer is quite difficult because it is not possible to heat it equally on every side, which resulted in deformation of the structure. The effect is comparable with warping and caused by the same reasons. The end result was chastening, and the wafer could not be used because it was warped, and the channel structures were bent.
Using heat to smooth the wafer is generally not recommended. Short time heating will cause the chip to warp noticeably. Long time exposure to heat will cause the structures to lose their integrity and to deform consequently.

Finally, we tried to smooth the wafers surface with chemicals. Different organic solvents are reacting with the synthetic polymers of the 3D printed parts. Plastics that are coming in contact with those solvents are getting resolved on the surface. If the solvent is removed from the part the polymer reaches its initially adhesion and is hardening again in the new form. We used this process for the smoothing procedure of our test wafer.
Wafer attached to a wire in the beaker ready for smoothing.
Our used material (PLA) can be solved in trichloromethane. To maintain the original structure without deforming the wafer, trichloromethane is not applied directly on the parts. The organic solvent is heated in a beaker with a magnetic stirrer to 80 °C. After a short time, the solvent will form a vapor phase which can be used to apply it very gently and dosed to the printed wafer.
Our wafer design features little eyelets on each side, which now can be used to attach the wafer to a piece of string or wire to mount it in the beaker and keep it at a fixed position in the vapor phase. We tried how long the wafer can withstand the vapor in the upper area of the vapor phase. After 90 seconds, the PLA was solved and began to deform distinctively. Shortly after, it dripped into the trichloromethane on the bottom of the beaker. Best results were achieved, when the wafer was exposed for about 60 seconds to the vapor of the solvent. After the smoothing in the vapor phase, the wafer is very sensible on the surface as the material is very soft. To remove the remaining solvent on the top layer, the wafer is attached to a mount in the laboratory hood until the solvent is evaporated.
This process has to be performed two times to get a structure that is smooth enough for manufacture functional microfluidic chips. Smoothing only one time will not suffice to reduce the roughness of the surface effectively. Make sure that between the both smoothing steps there is enough time so that the wafer does not get deformed. We achieved good results by resting the wafer for about 10 minutes before starting the second step. The results can be seen in the following figures.
3D printed wafer without smoothing.
3D printed wafer after the second smoothing step.

The structures of the wafer were measured with a stylus profilometer to compare the channel structures and the wafer surface before and after smoothing.
Wafer in the measure area of the stylus proilometer.
Channel structur on the wafer as seen on the screen of the profilometer.
Profile of the 3D printed test wafer without smoothing.
Profile of the 3D printed test wafer after the second smoothing step.

The measured profiles are showing, that the channel structure is much smoother after the treatment with organic solvents. It is also clearly visible, that the rectangular designed channel structure has no sharp edges when being printed. Further the channel structures are rounded through the process as expected.

Post Processing DLP Print

DLP printed wafer after washing steps ready for hardening in the UV chamber.
The post processing of the wafer printed with the DLP printer is much easier and faster than the post processing of the FDM printed wafers as the surface is very even and homogenous through the illumination of the fluid resin. The first step after removing the wafer from the building plate is washing of the surplus fluid resin that is still on the wafers. The wafer can be washed with distilled water. The water should be changed a few times to guarantee that not hardened resin is rinsed of the wafer. If an ultrasonic bath is available, it can be used to accelerate the process. Do not use isopropanol as this could degrade the material and roughens the surface of the wafer.
After washing the wafers carefully, the resin is not perfectly hardened yet. The wafers are illuminated with UV light in an UV chamber for about 15 to 30 minutes. Make sure that the wafers were rinsed carefully with fresh water and that no larger waterdrops are left on the wafers surface. In water solved resin could concentrate on the wafer as the water evaporates and harden afterwards, which will result in stains that can influence the later chip manufacturing. Make sure that all structures on the wafer basis are not touched or damaged through the whole post processing. The outer edge of the wafer can be used to hold it. Using tweezers is recommended.
Surface of a DLP printed wafer with a clearly visible stain because of unsufficient rinsing.
DLP 3D printed wafer after post processing with an even and smooth surface. Tip of a pencil for scale.

Chip Manufacturing

Preparing the wafer for molding and placing them on two sided adhesive tape in the modling vessel.
After the post processing of the wafers, they can be used for the manufacturing of the microfluidic chips. The chip manufacturing is the same for every wafer type, independently of the used printing technique, and starts with preparing the wafers for the molding. For this purpose, the wafers are put in a suited vessel, such as a petri plate. It is quite useful to attach them to the bottom with two-sided adhesive tape to prevent them from moving or swimming to the top when the molding process starts. It is important to work very clean and prevent dirt and dust from getting into the vessel and on your wafer. Working under a laminar flow bench is recommended if available.
For the molding of the wafers we used polydimethylsiloxane, short PDMS, which is a polymer based on silicon. It is easy to use and is suited excellently for the manufacturing of microfluidic chips. The PDMS is mixed freshly before making the mold. Therefore, the base is given into a cup or a beaker. Adding the linker fluid will start the polymerization of the base and create a clear polymer. The linker is mixed in the ratio of 1:10 and the both fluids are stirred firmly for several minutes to get a homogenous mix. Stirring the mixture with a magnetic stirrer will not be effective because of the high viscosity of the fluids. If it is possible, an anchor stirrer can be used, otherwise stirring with a stirring rod by hand for several minutes will be necessary.
Weighing out the base and linker that is used to start the PDMS polymerization.
Mixing the base and linker firmly with a stirring rod.

If the PDMS is mixed sufficiently, it can be poured on the wafers. The wafers should be covered by a few millimeters of the fluid, depending on the desired height of the chips afterwards. Because the polymer will have many air bubbles from stirring it firmly, the vessel with the wafer should be placed in a desiccator to remove the bubbles under vacuum. Every bubble and dirt that is getting into the polymer, when fully polymerized, will be on the chip and can be a disruptive factor for microscopy and the observation of the cells in the completed chips.
Pouring the mixed PDMS on the wafers in the modling vessel.
Molding vessels in a desiccator.

The following figures are visualizing the enormous difference of the poured PDMS on the wafer before and after the treatment in the desiccator. The best results were reached by pumping vacuum multiple times after aerating the desiccator again.
The freshly poured PDMS in the molding vessel before the usage of the desiccator. Many small and big air bubbles are clearly visible.
The PDMS in the molding chamber after the desiccator was used. All air bubbles vanished trhough the process and the mold is absolutely clear.

After using the desiccator all air bubbles should be removed from the PDMS. The molding vessel can now be put in an oven to accelerate the polymerization process and get a solid mold. We heated the mold for two hours at 80 °C to finish the polymerization. When the PDMS is cooled down it is hard enough and can be further processed. The molds of the wafers are cut out with a sharp scalpel and can then be prepared for the last step of the chip manufacturing.
Heating the mold to 80 °C for two hours in the oven.
The mold is cut out on the edge of the wafer with a scalpel.

To prepare the raw chips for the activation in the plasma generator and the bonding afterwards, they have to be removed from the mold by cutting them out on the edge of the wafer. Afterwards they are placed on a black, elastic polymer to punch the holes for the inlets and outlets with a biopsy punch. Best results were achieved when the inside of the chip was placed upwards, because this ensures to punch through the channel, even if the puncher is putted on in an angle.
After punching the holes for the inlets and outlets, the chips and one glass slide, that will serve as substrate for the chip, are washed three times with isopropanol and blown dry with compressed air to remove dirt and residues. The chips are placed with their inner surface upwards on a glass slide and are laid, together with the cleaned glass slide, into the chamber of the plasma generator for plasma activation.
Punching the inlet and outlet structures with the biopsy punch.
Prepared chips in the chamber of the plasma generator for plasma activation of the surfaces.

Plasma activating the surfaces will make them highly hydrophilic and cause them to bond covalently when two activated surfaces are joined. After activating the surfaces of the chips and the glass substrate in the plasma generator, the chips can be laid on the glass substrate which will bond the molded structure to the glass slide. This closes the channel structures on the chip that where molded from the wafer and finishes the chip. Be careful to leave enough space between the single chips when putting them on the same glass slide. Touching edges can prevent the chips from bonding to the substrate at this area, which will result in leaky and thereby unusable chips.

Different versions of the cultivation chips (left) and glass substrate (right) after plasma activation.
The chips were turned around and placed with the activated surface on the glass substrate for bonding (right).

The finally bonded microfluidic chip can now be used for experiments. For this, a syringe is connected to a hose with a cannula on its end, which is inserted into the previously punched inlet or outlet structure. The syringe is placed in a syringe pump and can now be used to generate a continuously flow through the channels on the chips.
Test chips manufactured with FDM (left) and DLP (right). Food color was used for testing the structures and see if the chips had correctly bonded.
Testing the FDM test chip with food color.

User Feedback

User feedback is important to notice the advantages of a novel system or approach. It is also important for improvements of the project. To get some user feedback we asked Julian Schmitz from the working group "Multiscale Bioengineering" of Bielefeld University. Julian has experience in microfluidics and chip manufacturing and can evaluate the production process very accurately. He tested the manufacturing of microfluidic chips with our approach and gave us feedback on the process itself as well as on the useability and fields of application.
The received feedback was very positive. According to Julian the manufacturing was easy and fast. All his manufacturing attempts worked first time and the chips were bonded correctly. Leakiness of the chips or collapsing of the channels could not be observed.
Because of the simple, fast, inexpensive, and highly adaptable process he even considers using our approach for practical trainings of students. Furthermore, our approach could be suited for an upcoming project according to Julian. First trials are already conducted.


A basic setup for cultivating on microfluidic chips as used for our experiments, comprising of syringe pump, microfluidic chip, microscope with camera, and waste tube.
We cultivated the two different organisms Saccharomyces cerevisiae and Aspergillus niger on our microfluidic chips. For each organism we designed a specific wafer to obtain a suited chip and setup for the respective organism. Both of our tested and previous presented methods were used for the manufacturing of the wafers. The wafer for Saccharomyces cerevisiae required smaller structures and a high resolution, which made DLP printing more suited. Creating the wafers with FDM printing would be possible if the printer is equipped with a smaller printing nozzle, and if the settings are optimized. However, reaching the same resolution as DLP printers would not be possible. Printing the wafer for Aspergillus niger could easily be made with a standard FDM printer and is possibly even more suited because the filamentous growth of this fungi can much better be observed in larger structures. Another advantage of larger structures can be the observation over a longer time span and the better reflection of the natural environment of the fungi.

Aspergillus niger

Aspergillus niger was cultivated in a chip manufactured with an FDM printed wafer. The 3D model and the printed version of this are shown in the figures below.
3D model of the used wafer design for the cultivation of Aspergillus niger.
3D printed wafer for the cultivation of Aspergillus niger before smoothing.

Costs for the printing of the wafer are very low and it is finished and useable in nearly no time compared to conventional methods. The costs and time for the single steps are listed below.
Wafer costs
Material Amount Costs/piece Total costs
PLA filament 0.20 m 0.09 $/m 0.02 $
Organic solvent 5 mL 78.61 $/L 0.39 $
Total 0.41 $

Manufacturing time
Step Estimated time
Printing 20 min
Smoothing 45 min
Total time 1 h 05 min

The design of the wafer allowed to load the chip with the preculture quite easily by squeezing the tube coming from the main channel and thereby conducting the flow through the growth chamber. For cultivation the main channel was opened again, and the side channel was closed. That way the flow of the medium was conducted through the main channel, ensuring that the culture will not being washed out of the growth chamber through convection. Nutrient supply was provided by diffusion of the nutrients from the main channel into the growth chamber.
Microfluidic chips for the cultivation of Aspergillus niger with different sizes of growth chambers for testing.

After loading the chip with the preculture, we changed the syringes to pump new media into the channels. We used CM medium with a flow rate of 10 µl/min. For finding the right flow rate for our system, we contacted Dr. Christian Sachs from the “Forschungszentrum Jülich”. He told us that two main factors for selecting the right flow rate are guaranteeing a sufficient nutrient supply through diffusion and technical factors, such as applicable pressure, blocking/leakiness, and how long the reservoir will suffice. Regarding all the factors, we decided to use a flow rate of 10 µl/min after roughly estimating the flow rate needed with a safety factor. This flow rate is comparatively high but suited as the channels structures are relatively big and experiments with flow rates of less than 1 µl/min showed that low flow rates can cause the chip to dry out.
Cultivation of Aspergillus niger in the growth chamber of a microfluidic chip.

The recording of the cultivation of the Aspergillus niger shown in the video was started 24 hours after loading the chip with the preculture. The whole cultivation in the chip was conducted over the duration of 58 hours. However it is possible to cultivate for an even longer time span. Groth and behaviour of the fungi can be observed on a very detailed level and over a long period of time. Clearly visible is the typical growth of A. niger with hyphae and the formation of a pellet like structure at the edge of the chamber where growth started and where it is limited through space. A second fungi can be observed growing in the bottom rigth corner, starting from a small hyphe at the beginning of the video.

All in all, the results of the microfluidic chip, manufactured using 3D printing for the wafer, are as expected. It is easy to print, highly customizable, very inexpensive, and above all it is absolutely functional. The idea and design can very easily be adapted for other organisms and purposes.

Saccharomyces cerevisiae

Saccharomyces cerevisiae was cultivated in a chip manufactured with a DLP printed wafer. The 3D model and the printed version of this are shown in the figures below.
3D model of the used wafer design for the cultivation of Saccharomyces cerevisiae.
3D printed wafer for the cultivation of Saccharomyces cerevisiae. Tip of a pencil for scale.

Costs for the printing of the wafer are very low and it is finished and useable in nearly no time compared to conventional methods. The costs and time for the single steps are listed below. They are according to the used softwares calculations.
Wafer costs
Material Amount Costs/piece Total costs
Casting resin 0.31 mL ca. 200.00 $/L 0.07 $
Total 0.07 $

Manufacturing time
Step Estimated time
Printing 10 min
Washing 30 min
UV illumination 30 min
Total time 1 h 10 min

The design of the wafer allowed to load the chip with the preculture quite easily by squeezing the tube coming from the main channel and thereby conducting the flow through the growth chamber. For cultivation the main channel was opened again, and the side channel was closed. That way the flow of the medium was conducted through the main channel, ensuring that the culture will not being washed out of the growth chamber through convection. Nutrient supply was provided by diffusion of the nutrients from the main channel into the growth chamber.
Loading the chip for cultivation with Saccharomyces cerevisiae. The blue arrow indicates the position of the cells travelling from the main channel through the side channel into the growth chamber.

The figure above shows a section of the microfluidic cultivation chip. On the left side a big vertical structure can be seen, which represents the main channel, used for loading and media supply. The smaller horizontal structure is the side channel that poses as a connection between the main channel and the growth chamber. it is used to load the chip with the preculture and to ensure diffusive nutrient supply. Blue arrow is indicating cells of Saccharomyces cerevisiae that are loaded into the growth chamber using the convection of the stream when the loading setup is used.
Cultivation of Saccharomyces cerevisiae in the growth chamber of a microfluidic chip.

The video from the beginning of the cultivation is showing that the resolution of the observation can be on a single cell level. The culture can be seen on the left side of the growth chamber. The video below is showing the culture after 12 hours.

Cultivation of Saccharomyces cerevisiae in the growth chamber of a microfluidic chip. Images were taken every 10 minutes.

Microfluidic Starter Kit

We recognized that microfluidic is quite complex and that many devices are needed for the manufacturing of the chips and conduction of experiments. Therefore, we created a microfluidic lab starter kit that is open source and can be downloaded for free on our Download page.
The kit comprises of:
  1. 3D models for printable microfluidic chips
  2. UV chamber for hardening the chips
  3. PDMS anchor stirrer
  4. Oven for baking the mold
  5. Syringe pump, needed for loading the chips and cultivating organisms

Every device is quite simple to print, easy to assemble and can get build at a very reasonable cost. We designed these devices to make microfluidics simple and accessible for other teams. The devices are subdivided in single parts that are all working together. The modularity makes the devices highly customizable which contributes largely to the usability by other teams and therefore perfectly reflects the open source character. Devices are designed very compact and small, what makes them highly flexible and arrangeable in the lab without any problems.
All devices were designed after the following criteria:
  1. Modular setup
  2. Easy and fast assembly
  3. Easy to use
  4. cost effective
  5. Space effective
  6. Compact housing

All of our prints were made with PLA and GreenTEC as they are high quality filaments, easy to use, robust and can be acquired at a reasonable price. Another aspect that was quite relevant for us is that PLA is biocompatible and GreenTEC even biodegradable. So, we are able to minimize the environmental impact that is commonly caused by plastics.

The calculation of printing time and costs is mainly based on the calculation of the used slicing software and the following price calculation of the used filaments.
Calculating the costs of printing:

The printing filament can be seen as a large cylinder where its length resembles the height of the cylinder.

And the volume of the cylinder can be calculated with:

With the Volume we can calculate the density:

If we change this formula, we get:



Regarding the filaments respective price of:

The price per meter is:

Converting the price from € to $ the price per meter for the filaments will be 0,09 $/m for PLA and 0,19 $/m for GreenTEC.

The whole kit comprising of UV chamber, PDMS stirrer, oven, and syringe pump can be build for under 50 $ which makes it highly affordable for other teams.
By virtue of the high customizability, all devices can also be adapted to extend their field of the named applications.
Expenses of the Microfluidic Lab Starter Kit
Device Expenses
UV Chamber 2.93 $
PDMS Stirrer 9.91 $
Oven 7.89 $
Syringe Pump 27.43 $
Total 48.16 $


Essential for microfluidics are of course the wafers, which are used in the manufacturing process of the microfluidic chips. The wafers are designed so that they can easily be printed and used.
3D model of the used wafer design for the cultivation of Aspergillus niger.
3D model of the used wafer design for the cultivation of Saccharomyces cerevisiae.

The files for this part are open source and you can download it for free to print this device for your lab.
You can find the files on our Download page.

UV Chamber

When working with a DLP printer to print the wafers, illuminating the freshly printed and washed wafers with UV light is recommended to harden them fully for better results. We designed a small but handy UV chamber that can be 3D printed and assembled quite easily. For printing the case it is recommended to use support structures. They can easily be removed afterwords but the print will come out much better.
Of course the UV chamber can also be used for other illumination applications and if needed, the LEDs can even be changed to receive a more suited light spectrum. Please pay attention on the harmful effects of UV light and wear respective protection gear as precautionary measure.
3D model of the UV chamber for the illumination of the freshly printed and washed wafer.
3D printed UV chamber for the illumination of the freshly printed and washed wafer.

The assembly of the chamber is quite easy. The two openings on the upper sides are used for the integration of the power jack and the switch. The LEDs are put in the frame with the illuminating side into the chamber. The LEDs can be driven with 12 V direct current, four LEDs can therfore be series-wired. Power supply from the power jack should be connected to the switch first as shown in the picture. Last the cover can be put on top to seal the electronics and the slide can be inserted into the chamber.
UV chamber from the top without cover to show the easy to assemble electronics in the inside.

UV chamber costs
Material Amount Costs/piece Total costs
PLA filament 11.57 m 0.09 $/m 1.04 $
UV-LEDs 4 0.14 $ 0.56 $
Switch 1 0.88 $ 0.88 $
Power Jack 1 0.45 $ 0.45 $
Total 2.93 $

Manufacturing time
Step Estimated time
Printing 8 h 20 min
Assembly 30 min
Total time 8 h 50 min

The files for this part are open source and you can download it for free to print this device for your lab.
You can find the files on our Download page.

PDMS Stirrer

PDMS mixing can be exhausting and time consuming, especially when higher amounts are needed. Homogenously mixed PDMS is crucial for high quality microfluidic chips. Because of the high viscosity of PDMS, magnetic stirrers are not suited for mixing. We designed a simple and easy to construct stirrer for mixing the PDMS. It will help to mix homogenously for best results and chips of high quality. Of course the stirrer can also be used for other materials and stirring applications. If another stirrer is preferred, it is also possible to change the anchor on the bottom and place a more suited design for the new application.
The stirrer can be used with standard polystyrene cups or with laboratory beakers. For larger vessels, the case is designed to be easily dismountable and used as a single part that can placed on top of the preferred vessel.
Easy to assemble anchor stirrer for PDMS mixing.
3D printed stirrer. The single parts can be assembled without tools.

For printing the mount, support structures are recommended. All other parts can be printed without support. Assembly is simple and mount, case and cover can be sticked together without screws or glue thanks to the pins that where designed to match the single parts.
PDMS stirrer costs
Material Amount Costs/piece Total costs
PLA filament 26.32 m 0.09 $/m 2.64 $
Motor 1 6.00 $ 6.00 $
Switch 1 0.88 $ 0.88 $
Power supply (battery) 1 0.39 $ 0.39 $
Total 9.91 $

Manufacturing time
Step Estimated time
Printing 17 h 25 min
Assembly 30 min
Total time 17 h 55 min

The files for this part are open source and you can download it for free to print this device for your lab.
You can find the files on our Download page.


To accelerate the polymerization of the PDMS the molding vessels can be heated with the mold in an oven. We designed this small and very simple oven that is printed and assembled quite easy and fast. Pay special attention on the used printing material. We used GreenTEC instead of PLA because it is heat resistant. However, you can use every other material that is heat resistant in the temperature range the oven should be used later. Printing the case turns out best when support structures are used. Filaments other than PLA may have a higher risk of warping of the structures. Warping may be reduced with a sufficient brim or the usage of layer adhesion products.
3D model of a DIY oven to heat the mold for faster polymerization.
3D printed oven for heating the mold in the chip manufacturing process.

Assembly of the oven starts with removing the support structures. The openings in the upper half of the oven are used for integrating the power jack and the power switch. For heating it is possible to use a TEC element or a PTC heater. This can clipped in the mount on the ovns bottom. For temperature control a simple thermostat, e.g. "W1209 Temperature Control Switch", can be used. The temperature switch can be setted very easily following the manual of this data sheet. A suited mount for the temperature sensor is placed behind the mount for the heat module.
Oven costs
Material Amount Costs/piece Total costs
GreenTEC filament 11.58 m 0.17 $/m 2.20 $
TEC element 1 2.14 $ 2.14 $
Thermostat W1209 1 2.22 $ 2.22 $
Switch 1 0.88 $ 0.88 $
Power Jack 1 0.45 $ 0.45 $
Total 7.89 $

Manufacturing time
Step Estimated time
Printing 8 h 25 min
Assembly 45 min
Total time 9 h 10 min

The files for this part are open source and you can download it for free to print this device for your lab.
You can find the files on our Download page.

Syringe Pump

For conducting microfluidic experiments syringe pumps are an essential device to get preculture, media and different other substances through your channels and in your chambers. Professional syringe pumps are very expensive and especially for testing issues often not necessary. This 3D model of a syringe pump is quite easy to print, assemble and use. It is ideal for teams starting with microfluidics.
Compact DIY syringe pump.
3D printed syringe pump with inserted syringe in action.

The syringe pump can be assembled in a short time with nearly no tools.
Single parts of the DIY syringe pump in the right orientation before assembly.

Propulsion of the pump is achieved with a gear box 12 V DC motor, which assures a continuous and steady rotation of the threaded bar. We used a gearbox motor because it is easy to use and to integrate into the pump as a special control module or programming is not necessary.
Standard 12 V DC gearbox motor.
Gearbox motor with opened case. The gears are reducing the speed of the motor and resulting in a higher torque which is needed to guarantee a steady rotation.

Manufacturing costs
Material Amount Costs/piece Total costs
PLA filament 11.30 m 0.09 $/m 1.01 $
Motor 1 11.85 $ 11.85 $
Speed controller (with integrated switches) 1 10.03 $ 10.03 $
Position stop switch 1 0.69 $ 0.69 $
Ball bearing 1 0.45 $ 0.45 $
6 mm roud bar (stainless steel), 10 mm 2 1.09 $ 2.18 $
M6 threaded bar, 10 mm 1 0.77 $ 0.77 $
Power Jack 1 0.45 $ 0.45 $
Total 27,43 $

Syringe pump time
Step Estimated time
Printing 9 h 5 min
Assembly 1 h 10 min
Total time 10 h 15 min

The files for this part are open source and you can download it for free to print this device for your lab.
You can find the files on our Download page.

Rapid Processing in Laboratorys

In many fields there is a huge trend to rapid prototyping and processing. Especially because of the huge advantages, like fast construction and relatively low cost, many things are tested and build with 3D printing. Additive manufacturing, especially 3D printing, is a spreading and vastly growing sector. Many problems that would have taken a long time and a financial impact to solve can easily be solved in a vanishingly small time at comparatively low costs.

Protein Purification Mount

If you need to purificate quite often, maybe think about using this neat mount for up to 5 purification columns. Don't tinker in your lab with different mounts, just print it and get started. The assembly is quite easy and no tools are needed.
Purification mount fully assembled.
The single parts the purification mount consists of in the right orientation.
Purification mount fully assembled with matching equipment for purification.
The files for this part are open source and you can download it for free to print this device for your lab.
You can find the files on our Download page.


Gelcomb (standard version)

Wikifreeze is coming and you need to analyze many samples? We got your back. Use this gelcomb with 25 chambers for higher sample throughput. Download the file and start printing, it won't take long.
Gelcomb for 25 samples. Suited for 2x150 mm gelcomb mounts.
3D printed gelcomb (2x150 mm) with 25 chambers (thickness: 2 mm).
The files for this part are open source and you can download it for free to print this device for your lab.
You can find the files on our Download page.

Gelcomb (slim version)

Do you need a higher resolution of your samples and sharper bands? Try this special gelcomb version with thinner chambers (1 mm thickness).
Gelcomb for 25 samples with slim chambers. Suited for 2x150 mm gelcomb mounts.
3D printed gelcomb (2x150 mm) with 25 slim chambers (thickness: 1 mm).
The files for this part are open source and you can download it for free to print this device for your lab.
You can find the files on our Download page.

Tube Stands

Tube Stand (5 x 50 ml)

Many tubes but too few stands? Don't steal it from your coworkers, nobody likes searching for stands. Print this easy and nice tube stand for up to 5 50 ml tubes and never search for stands again.
Tube stand for 5 standard tubes with a volume of 50 ml.
3D printed tube stand for 5 standard tubes with a volume of 50 ml.
The files for this part are open source and you can download it for free to print this device for your lab.
You can find the files on our Download page.

Single Tube Stand (50 ml)

You only need one tube? Use this single tube stand for 25 ml tubes and save space on your working area. It can also be combined with other Hardware parts for an integrated stand.
Tube stand for one standard tube with a volume of 50 ml.
3D printed tube stand for one standard tube with a volume of 50 ml.
The files for this part are open source and you can download it for free to print this device for your lab.
You can find the files on our Download page.

Solder Stand

You are working on a hardware part and have lots of soldering work to do? Your solder roll is always rolling around and it is difficult to get a nice and clean soldered point? Try out our solder stand. It is perfectly suited to help you with your work and get good results.
Solder stand.
3D printed solder stand.
The files for this part are open source and you can download it for free to print this device for your lab.
You can find the files on our Download page.

Educational purposes

A big focus of our project was to make things very simple and understandable. We had several approaches to communicate synthetic biology and our project. To get more attention from the visitors of our exhibitions, we designed models that could be 3D printed and that helped to communicate complex subjects. We talked to Damian Mallepree about the importance 3D models as exhibits, and got a very positive resonance about this approach. For communicating synthetic biology, especially the nanopore sequencing technology, we designed the CsgG lipoprotein as a ribbon and a surface model that can be inserted into a schematic biomembrane. The 3D printed nanopore was ideal to get attention during the exhibtion. Further, to explain the construction of our wafer design, we created a 40:1 model that can be printed to see the planned structure more detailed.

Nanopore Model

Nanopore sequencing is a very fast and relatively easy method for sequencing big amounts of DNA. One main feature needed is the Nanopore, a small lipoprotein with a channel like structure.
Nanopore structural model (ribbon style).
3D printed Nanopore structural model (ribbon style).
3D printed Nanopore structural model (surface style) in a schematic biomembrane.
Download it here:

Wafer Model

Wafer for microfluidic chips have very small structures on it to form the microfluidic channels. Often they are not visible for the naked eye. Therefore models of those wafers can easily be used to let the structures be seen in detail and thus can remarkably increase the understanding of the structures functions.
3D model of a model wafer for cultivation scaled 40:1.
Download it here: