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Revision as of 15:43, 18 October 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*.
The foollowing scheme describes the mentioned process:
Fig. 4: PDMS chip fabrication process
*Traditionally, bonding PDMS to glass is done by plasma treatment. Our 3D printed mold resulted in PDMS chips with relatively rough surface finish, forcing us to use other methods.
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 3).
- 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 were able to make a few prototypes of the PDMS chip. The extraction of the chip was relatively easy and without any visible cracks or deformations. The chip still needed to be punctured in the entry slots, duo to spaces between the molds. Also, attaching a glass slide to the PDMS needed to be done carefully, as the glass is thin and brittle.
The fluidic factory 3D printer did not produce us a usable chip. The channels kept collapsing while printing the model. Despite not achieving a usable chip, we believe that this technology shows a lot of promise.
Quantitative test for bacterial concentration
Principle of Operation
Our system uses a photo sensor to measure the intensity of a light beam transmitted through the chip. The measurement process is as follows: A yellow LED emits light at 585-595 [nm] on the chip, with the bacteria inside absorbing a portion of the light. The light transmitted through the chip reaches the photo sensor which outputs an analog signal. This signal is then translated to a digital signal and fed to the computer. The end result is a graph of the output voltage as a function of time.
The output voltage can be compared to the bacterial concentration as shown in section 3.3.
Fig. XX: Schematic diagram of the quantitative system
The system requires two measurements. The first measurement is a blank meant to calibrate the system. This measurement is done on a chip containing only motility buffer (control). The second measurement is for the bacterial solution.
To avoid undesired light reflections, we have designed a dedicated black box as shown in Fig 9, to house the chip and the electrical circuits discussed below.
The electrical circuits
The system consists of two independent electrical circuits as shown in Fig XX. The blue circuit contains a resistor of 10 [kΩ], a potentiometer, a LED and Arduino as a constant voltage source. The red circuit contains a photoresistor LDR that is sensitive to 600 [nm] wavelength, a resistor of 1[MΩ] and an Arduino controller. The Arduino supplies constant voltage to both circuits and measures the voltage that falls on the 1[MΩ] resistor.
Fig. XX: Schematic diagram of the two electrical circuits.
Computer data system
The Arduino controller collects samples of the voltage that falls on the 1[MΩ] resistor. The voltage is converted to a digital signal and fed to the computer. The computer data system is based on a Matlab GUI . Note that the Arduino I/O toolbox needs to install.
When running the Matlab code, the window shown in Fig 11 pops up.
Fig. XX: The system's user interface
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.
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
For the matlab code, see: !@#$%^&^%$
References:
1. Calloway, D. (1997). Beer-Lambert Law. Journal of Chemical Education, 74(7), 744. http://doi.org/10.1021/ed074p744.3
