Part | Description | Manufacturer / Partname |
---|---|---|
A fairly strong LED | In our case a 3W green 520nm LED | Cree XP-E on star circuit board |
A cooling element | Used to prevent the LED from overheating | Fischer Elektronik ICK S |
A constant-current-source | To ensure static LED light (no flickering) | Roschwege GmbH KSQ-3W |
An AC/DC rectifier | Used to grant direct current to the LED | Goobay 67951 |
Two optical lenses | Here we used two identical 60mm lenses | Thorlabs AC254-060-A |
A camera | We used a SLR camera | Canon 550D |
A microfluidics chamber | Our chamber consists of an iRIf slide attached to a PDMS flow chamber | Made ourselves |
Magnets | Used to attach the flow-cell to the device | 5mm Neodymium magnets |
A small syringe | We used a regular 10 ml syringe | BRAUN INJEKT 10ml |
A microfluidic tube | Used to connect the flow chamber with the syringe | - |
A case for the device | 5 mm thick acrylic glass cutouts to hold the parts in place | Ordered at http://formulor.com |
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Revision as of 05:07, 16 September 2015
![](https://static.igem.org/mediawiki/2015/8/85/Freiburg_wiki_background_2_parted_left.png)
![](https://static.igem.org/mediawiki/2015/f/f4/Freiburg_wiki_background_2_parted_right.png)
Here you'll see what we were able to measure with the very first prototype of our own device. To see the results of our final device, please refer to the essential results page. A detailed explanation of how to build your own, low-priced iRIf device can be found on the bottom of this page.
For testing our device we bound proteins derived from rabbits onto an iRIf slide in distinct spots (Fig. 1 D). The proteins used were polyclonal anti-HCV (hepatitis C virus) antibodies derived from rabbit, which we then aimed to detect with anti-rabbit antibodies. We used the rabbit/anti-rabbit couple in this series of experiments was to reproduce a successful measurement with this binding couple which we previously performed in our regular measuring device. The binding layer on the iRIf slide consisted of an APTES/PDITC surface. The spots on the slide were made by pipetting 3 µl (500 µg/ml) rabbit-anti-HCV protein and 3 µl (1 mg/ml) BSA in an alternating pattern onto the slide. After incubation, the slide was blocked for 30 min in BSA solution.
The camera used for the measurement was a Canon 50D. The camera was set to automatically take pictures at an interval of 5 seconds. The exposure time was set so that the pixels in the image were approx. 80% of maximum light saturation before the solution was flown onto the chip (Fig. 1 C & D). The antibody solution was pipetted into the flow-chamber without the use of any microfluidics device. Instead a syringe was loaded with 500 µl [5 µg/ml] anti-rabbit antibody solution (diluted in PBS) and slowly released into the binding chamber of the device by gently dispensing the solution from the syringe. As can be seen in Fig. 1 C, the quotient picture clearly showed binding of anti-rabbit to the rabbit protein spots. The BSA control-spots showed none or negligible unspecific binding.
In this section we'll swho you how to build your own iRIf device from scratch, using affordable, low-tech material. The design focused on creating a device that is both low-priced and portable, demonstrating the potential for the DiaCHIP to be used even in rural areas where high-tech laboratories are poorly accessible.
General principle
As can be seen in Fig. 2, the basic setup is fairly simple. Light from an LED enters a lense where the light rays are made parallel. To achieve this, the distance from LED to the lense has to be exactly one focal length. The light then hits the iRIf slide, where it gets reflected (reflecting at the same angle it hit the slide) and enters a second lense, whose purpose is to project a sharp image of the slide onto the CCD chip of the camera.
Construction guidance
A major problem that we confronted when building the device from scratch was to assure that all components are at the exact distance and angle to each other. This is crucial as a slight misplacement of a component may lead to lower signal strengh, blurred images or in the worst case no signal at all. This can be difficult since our device doesn't rely on straight angles. We overcame this problem by designing a case for the device that ensures the right placement of the components in the device. This was archieved by calculating all the distances beforehand using simple physical law of optics. We realized this by drawing an exact vector graphic blueprint for our device. We then constructed a digital 3D model of the casing based on the vector blueprint, to avoid a costly and time-consuming trial and error process (Figure 4).
![](https://static.igem.org/mediawiki/2015/8/88/Freiburg_results-device_3d_no_walls.png)
We designed the casing so that all the necessary parts (lenses, LED) are held in the correct position safely during the measurement, but remain removable to grant easy transportation of the device (i.e. to the giant jamboree). Fig. 5 illustrates the parts that hold the lenses and LED in place.
After assuring that the 3D model of our device was set-up correctly, we created a vector graphic file which layed out all the parts needed for the casing in a 2D plain. Using this vector graphic file, we ordered the parts at Formulor, a service which lasers out parts from acrylic glass using a vector graphic as a template. The vector graphic template is shown in Figure 6, and may be downloaded and used by everyone to build their own device. To allow easy mounting of the casing we also provide an easy to understand manual. One advantage of using acrylic glass is that the parts can be glued together easily using a few drops of acetone, fusing the parts together. The vector graphic file used to laser our the casing parts also contains a template for the parts which are necessary to attach the glass and PDMS slide to the device. These parts have to be lasered out of 1 mm thick acrylic glass. Once they are lasered out, the microfluidic tubes can be glued to the parts (Figure 7). Once this is completed, these parts can be attached to the device with the magnets (Figure 8)
The most difficult part to build by oneself is the PDMS flow-chamber. For producing such flowchambers, a wafer has to be made in a cleanroom. The template used for our PDMS flow-cell is shown in Fig. 9. If your facility has an engineering department, there's a high chance that a cleanroom is present. Ask the personel in charge if they can help you out with producing your flow-cell. If you already use another type of microfluidic flow chambers, our device should still be compatible with it, though some adaptions might be necessary. If you have no possibility of creating a flow chamber, you might want to consider building a very simplified DIY flow-chambers from two glass slides, separeted by thin duct tape. Note that our device was not primarily build to support such a chamber though and would need appropriate modifications.
![Freiburg iRIf device parts](https://static.igem.org/mediawiki/2015/4/4c/Freiburg_results-device_parts_vector.png)
Download section
Legal notice
The iRIf detection method is patented. Biametrics and associated persons own patents concerning the detection principle. These patents are:
- Method for examining physical, chemical and biochemical interactions (DE102004051098.9, DE102005015030.6, EP05797776.1)
- Study of molecular interactions on and / or in thin layers (DE102007038797.2)
- Method and apparatus for determining reflection coefficients to filter arrangement with thin layers (DE102009019711.7)
- Again Detectable support for optical measuring methods (DE102009019476.2)