S.tar, by iGEM Technion 2016

Hardware

Introduction

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

Fig. 1: The geometry of the commercial fluidic chip

Table 1: Bill of materials for commercial chip

The chip is made of plastic and the bottom is closed by attaching a glass slide to it. .

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 relatively shallow, forcing the use of a high concentration of bacteria to get a visible signal, this proves to be a problem as storing a large amount of bacteria in a confined space might cause oxygen shortage that will harm bacterial motility. .

We devised new solutions to confronts those problems:
- Design a novel chip, based on the commercial chip but with unique changes to its geometry for improved performance.
- Design a quantitative test, a device built especially for detecting a change in bacterial concentration in the chip. This device is much more sensitive than the naked eye.

Chip redesigned

We designed a new chip with the following improvements:

(a) 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 fix the diffusion source at a permanent place for all of our experiments.

(b).Shaping the channel as a funnel in order to concentrate the bacteria even further as they move away from chemo-repellents (from left to right).

(c) 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.

Fabricating the PDMS chip

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

The PDMS was then fabricated according to the following steps:

1. Design a two part mold using SolidWorks software- cover and base.
2. Print the mold using Ultimaker 2 Etentended+ 3D printer.
3. Mix the polymer base and curing agent at 10:1 weight ratio, respectively. Then, fill the mold with the mix.
4. Place the mold inside a desiccator to degas for 2 hours.
5. Bake the mold at 70 C for 3 hours.
6. Carefully take off the mold’s cover and then cut out the PDMS chip.
7. Attach the PDMS chip to a thin cover glass (0.3 mm) using silicon glue*.
Overview of the fabrication process:
1. A two parts mold was designed using SolidWorks software: cover and base.
2. The mold was printed using Ultimaker 2 Etentended+ 3D printer.
3. The polymer base and curing agent were mixed at a ratio of 10:1 (with the advantage to the polymer base). The mix was then poured into the mold.
4. The mold was placed inside a desiccator to degas for 2 hours and then baked at 70 C for 3 hours.
5. The ready PDMS chip was removed from the mold and attached to a thin cover glass (0.3 mm) using silicon glue.

Table 2: Here there should be a table

The foollowing scheme describes the mentioned process:

Fig. 4: PDMS chip fabrication process

Designing the mold:
The mold is comprised of two parts which together create a unique geometry and allow 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

Table ###: Here there should be a table

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