S.tar, by iGEM Technion 2016

Hardware

FlashLab

We developed our first FlashLab prototypes around on the commercial fluidic chip - IBIDI sticky–Slide I Luer 0.8. The commercial chip is designed mostly for performing cell culture experiments under shear stress, with a custom specific bottom.

Fig. 1: The geometry of the commercial fluidic chip

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The chip is made out of plastic and the bottom can be closed by sticking it to a glass slide.

Fig. 2: Setup of commercial fluidic chip.

The commercial chip worked for preliminary testing but was not idle for our uses: The entry slots are relatively wide, making it difficult to load the sample in a uniform and even fashion. This affects the diffusion of the chemo-repellent in the channel and reduces the overall accuracy of the device. Also, the channel is shallow, forcing the use of a high concentration of bacteria to get a visible signal, this presents a problem as storing a large amount of bacteria in a confined area might cause oxygen shortage that will harm bacterial motility.

We designed a new chip that confronts those problems:
- Reducing the radius of the entry slot will enable a controlled insertion of the sample. The smaller slot will slow down any flow (for example, flow caused by loading a sample from a syringe). Also, this will set the diffusion source at a permanent location on the chip for all of our experiments.
- Shaping the channel as a funnel will cause the bacteria to concentrate as they move away from chemo-repellents (from left to right).
- A deeper channel will result in a darker shade of color in the same bacterial concentration than in the commercial chip, while reducing the risk of oxygen shortage.

Fig. 3: The geometry of the designed fluidic chip.

The new chip was fabricated in two methods: as a PDMS chip and in a Dolomite 3D printer.

PDMS

PDMS is considered the standard for microfluidic fabrication in labs. It is optically clear and in general, inert, non-toxic, and non-flammable.

Overview on the fabrication process:
1. A two parts mold was designed using SolidWorks software: cover and base.
2. Printed the mold using Ultimaker 2 Etentended+ 3D printer.
3. Mixed the polymer base and curing agent at 10:1 weight ratio (with the advantage to the polymer base) and poured into the mold.
4. Put the mold inside a desiccator to degas for 2 hours.
5. Baked at 70 C for 3 hours.
6. Carefully removed the mold’s cover and then cut out the PDMS chip.
7. Stick the PDMS chip into thin cover glass (0.3 mm) using silicon glue.*
*Whan making a PDMS chip.

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The foollowing scheam describes the mentioned process:

Fig. 4: PDMS chip fabrication process

Designing the mold:
The mold is composed out of two parts to create unique geometric and for easier extraction of the PDMS out of the mold.

The base
- The cone on the base of the floor is make the funnel shape of the chip ((a) in figure 2).
- Small slits were made in the walls of the base to position the cover accurately.
- The overall size was determent so the chip will fit on standard microscope cover slide. this will enable us to run experiments under a microscope easily.

Fig. 5: The geometry of the mold

The cover
- Four rods coming out of the sides of the cover for easy extraction of the cover when taking out the PDMS.
- The ramp is to insure that the channel will be inserted inside the PDMS and getting the wanted channel height.
- The cover is made smaller than the base for a good fit and for letting out any gas that might have been caught when inserting it. Those gases, if left in will expend in the oven and cause deformation in the chip.

Fig. 6: The geometry of the cover.

Printing the mold using Ultimaker 2 Etentended+. This 3d printer was chosen because of its high accuracy (X,Y,Z =12.5, 12.5, 5 micron) and due that the fact that the polymer it uses (PLA) can be heated to relatively high temperatures without changing form (TG=60-65 C) and does not reacts to the PDMS. Another benefits of 3D printing are the low price and fast manufacturing time: We printed our mold for about 25$, and it took about 6 hours.

Dolomite Fluidic Factory

Fluidic Factory enables fast prototyping of microfluidic chips, manifolds and connectors using COC (FDA approved, biocompatible, translucent and robust polymer). Printing the chip toke about 3 hours and was made straight from computer model. This technology just came out this year and we are the first iGEM group to ever use it.

Fig. 7: Factory chip fabrication process.

Results: We had some difficulties preventing the channels from collapsing in the "Dolomite" printer. While not achieving a usable chip, we believe that this technology shows a lot of promise.

Quantitative test for bacterial concentration

As the bacteria sense either the repellent or the attractant in a certain direction creating a cluster. Our bacteria may be visible, but at low concentrations the cluster might be difficult to distinguish. Through the bacterial concentration test, it will be possible to determine quantitatively whether a cluster was formed or not.
The system is composed of two independent electrical circuits as illustrated in.

Fig. 8: The electricl circuits. (a) contains a LED a voltage source of 5v, (b) contains a photoresistor, a resistor and a voltage source.

Principle of Operation:
The LED, the chip and the photoresistor are placed in a dark box to prevent undesired light leakages and reflections . The LED emits light at 585-595nm (the specs are in the appendix), which passes through the chip and partially absorbed by the bacteria. The transmitted light reaches to the photoresistor and causes a decrease in its resistance. As a result, the voltage that falls on the photoresistor decreases and similarly the voltage that falls on the resistor increases. Then, the signal produced by the photoresistor is converted to a digital signal that goes to the computer. Finally, the output voltage is displayed with a GUI user-friendly interface as shown in Fig 2.

Fig. 9: GUI-interface.

The left side of the interface is used for testing the system and the right side serves for taking measurments. The user can change some parameters by the GUI interface like the number of samples that the system collects and at which frequency.
GUI displays a graph of the voltage that falls on the resistor as a function of number of samples, which is equivalent to time.
After taking reference, the user can take a measurement. The System reading is the ratio between the average of the sample voltage taken in the measurement divided by the samples voltage average of the reference.

GUI: Gui is a graphical user interface is a type of user interface that allows users to interact with electronic devices through graphical icons and visual indicators such as secondary notation, instead of text-based user interfaces, typed command labels or text navigation.
In order to use GUI, it is required to download Arduino I/O toolbox.
In our project we used matlab as programming language. The matlab code is in the appendix section.

Deriving the relationship between the voltage that falls on the resistor and the O.D of the bacterial solution: According to the voltage divider rule, the voltage that falls on the resistor in the photoresistor circuit (VR) is equal to

According to “Emant”, the relationship between the resistance RL of a typical LDR and the light intensity is:

Where LUX is the light intensity that reaches the photoresistor.br

Combining Equation 1 and Equation 2:

By definition:

Where I₀ is the light intensity emitted from the LED and A is the optical density of the bacterial concentration inside the chip.

From Equation 4 it can be derived that VR decreases as A increases.

Bill of materials

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For the matlab code, see: !@#$%^&^%$