Team:Vanderbilt MF/Project

E. chrono
Methods:
Our team utilized a unique microfluidic development process that emphasizes low cost and rapid prototyping. Through the use of open source design software and cheap manufacturing methods we were able to fabricate microfluidic devices in a cost and time efficient manner. It is our hope that these methods can be utilized by other iGEM teams who are interested in incorporating microfluidics into their projects without the hassle of complex design and fabrication techniques.

Microfluidic Computer Aided Design
Several programs were used by members on the team to design and ultimately fabricate microfluidic devices. Initially, traditional computer aided design software packages were used such as AutoCAD and Solidworks. These packages provide advanced features which designers can wield to make complex devices with varying functions such as mixing and trapping cells. Alternative software packages were explored such as the open source vector graphics software Inkscape which proved to be easy to use in the design of a microfluidic device. Inkscape is an open source analogue to Adobe Illustrator, a common vector graphics editing software. Inkscape is easy to use and with a few online tutorials, ideas for microfluidic designs can be turned into realities.
Microfluidic Prototyping using a Vinyl Cutter
In order to rapidly prototype microfluidic devices of different geometries and designs, our team explored fabrication techniques that bypassed the complicated photolithography process that is standard for microfluidic devices in research laboratories. Photolithography has the benefit of achieving very fine resolution and fine details for pinpoint fluid manipulation, but it is detracted from its lengthy creation process. The time scale from device design to fabrication of a working device using photolithography is on the order of 2-3 days and uses a variety of expensive and highly technical equipment that is not readily accessible to many iGEM teams.

Our team focused on bypassing the entire photolithography process by using a commercially available arts and crafts vinyl cutter (Silhouette) to produce device masters. Arts and crafts vinyl cutters are typically used to craft complex shapes and figures for use in decorations. A vinyl cutter has a resolution on the order of 250 microns which means that the devices made using this technique are considerably larger than those made using standard photolithography techniques. While the devices obtained from vinyl cutting are larger, for many synthetic biology applications this is not an issue. For the purpose of E. chrono, the size of the channels simply affected the total amount of fluid needing to be pumped into the device. As mentioned previously, the main benefit of using a vinyl cutter is the rapid production of microfluidic masters. A vinyl cutter is able to take a device design to a master and ultimately a device in roughly an hour. By simplifying and speeding up the design and fabrication process, vinyl cutting can allow iGEM teams with no microfluidic knowledge to rapidly develop their own devices for testing. Using an open source design software such as Inkscape allows a team to rapidly formulate and then test a microfluidic device within an afternoon.

The process for using a Silhouette vinyl cutter to fabricate a microfluidic device is outlined below.

Converting a Design to a Vinyl Master
1) Import the design for microfluidic device into Silhouette Studio, the vinyl cutting software used to make designs. Note: for Design process, see Microfluidic Device Design instructions
2) With device inside Silhouette, check to make sure dimensions are within the vinyl cutter resolution limits. No feature of the device should be smaller than 250 microns, this rule applies predominantly to channel widths.
3) Place vinyl paper inside printer and load the paper in place
4) Place the blade setting to 2
5) On the right hand side there is are several options for vinyl cutting, click Include Edges and then submit the job to be printed.
6) Once the device is cut onto a vinyl sheet, using a razor blade peel away the surrounding vinyl so that only the cut out is left
7) With tweezers and razor blade, pick up the cutout and place on a glass slide
8) Flatten the cutout onto the glass slide, this creates the “master” with which microfluidic devices can be manufactured Fabricating a PDMS device using a Vinyl Master
9) Prepare a PDMS solution with 10:1 ratio of PDMS to epoxy and mix well
10) Pour the PDMS mixture on top of the master, making sure that the PDMS spreads out over the whole petri dish and covers the master evenly.
11) Place the petri dish with PDMS inside a vacuum chamber and suction the chamber for 20 minutes to allow the air bubbles inside the PDMS to flow out
12) Once the bubbles have been removed from the PDMS, place the Petri dish in a warm incubator to cure for 8 hours or overnight.
13) In order to remove the device, a razor blade or scalpel can be used to cut out the device from the surrounding PDMS inside the petri dish.
14) The vinyl master can be removed from the bottom of the device and either thrown away or reused for fabrication of more devices
15) Take a 1/16” OD syringe tip and use it to “punch” a hole inside the microfluidic inlet channel and inside the microfluidic outlet channel
16) Once the device holes have been punched, the device can be plasma bonded to a glass slide or coverslip by treating the bottom of the microfluidic device (where the channels are open) with a plasma wand or plasma oven for 45 seconds and then bonding to a plasma treated glass slide.

The final device will consist of a PDMS slab with microfluidic geometries embedded on the bottom of the slab. Fluids can be run through the device by either directly pipetting them through the inlet or by affixing microfluidic tubing with 1/16” OD to the inlet and pumping fluids in using a fluid pump such as a syringe pump or peristaltic pump.