Team:Bielefeld-CeBiTec/Hardware
Hardware
Summary
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.
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.
Aims
Advance of Microfluidic
- Contain several cells in one chip
- Provide sufficient environmental conditions for cell survival and cultivation
- Provide a separate growth chamber for a cell population
- 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
- Easy construction of the parts
- Fast design and prototyping
- Often significantly less expensive than other methods
- Tabletop 3D printers are cheap, small and simple to use
- 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:- microfluidics easily accessible by construction of diverse necessary devices leading to decreased costs
- the construction of microfluidic chips easy, fast, inexpensive and without the need of special cleanrooms
- 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
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):- Which function does the chip have to serve?
- Which organisms have to be used/investigated?
- Which features are required/preferred?
- 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.
Examples
Test Wafer
- 300 µm (inner channel)
- 400 µm (middle channel)
- 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
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
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
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
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:
- Printability
- Printing detail
- Printing quality
- Surface quality
- Availability
- Environmental compatibility
- 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:
- very easy to print (even for beginners)
- Delivers good printing detail and quality
- Relatively even surface
- Excellent availability
- Is environmentally compatible (biocompatible material)
- 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.
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.
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.
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.
DLP Printing
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.
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.
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.
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.
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:- Grinding
- Smoothing step with the hot end of the extruder while printing
- Heat smoothing (with hot air)
- Smoothing with chemicals
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.
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.
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.
The structures of the wafer were measured with a stylus profilometer to compare the channel structures and the wafer surface before and after smoothing.
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
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.
Chip Manufacturing
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.
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.
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.
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.
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.
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.
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.
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.
Cultivation
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.
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.
| 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 $ |
| 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.
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.
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.
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.
| Material | Amount | Costs/piece | Total costs |
|---|---|---|---|
| Casting resin | 0.31 mL | ca. 200.00 $/L | 0.07 $ |
| Total | 0.07 $ |
| 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.
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.
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.
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:
- 3D models for printable microfluidic chips
- UV chamber for hardening the chips
- PDMS anchor stirrer
- Oven for baking the mold
- 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:
- Modular setup
- Easy and fast assembly
- Easy to use
- cost effective
- Space effective
- 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:
With:
Follows:
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.
| Device | Expenses |
|---|---|
| UV Chamber | 2.93 $ |
| PDMS Stirrer | 9.91 $ |
| Oven | 7.89 $ |
| Syringe Pump | 27.43 $ |
| Total | 48.16 $ |
Wafers
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.
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.
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.
| 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 $ |
| 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.
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.
| 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 $ |
| 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.
Oven
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.
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.
| 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 $ |
| 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.
The syringe pump can be assembled in a short time with nearly no tools.
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.
| 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 $ |
| 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.
You can find the files on our Download page.
Gelcombs
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.
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).
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.
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.
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.
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.
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.
