Difference between revisions of "Team:Freiburg/Results/Own Device"

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     <div class="thumb2 trien" style="width:400px"><div class="thumbinner"><a href="https://static.igem.org/mediawiki/2015/f/fc/Freiburg_results-result_device_20150818.jpg" title="results:result_device_20150818.jpg" class="media lightbox_trigger"><img alt="Our device measurement with rabbit proteins" src="https://static.igem.org/mediawiki/2015/f/fc/Freiburg_results-result_device_20150818.jpg" width="400"/></a><div class="thumbcaption"><strong>Figure 1: Binding of anti-rabbit antibodies to rabbit proteins measured in our device.</strong> A: The first picture of the measurement. B: The last picture of the measurement. C: The quotient picture of the first and last picture. D: Schematic illustration of the pattern of the spots on the slide </div></div></div>
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     <div class="thumb2 trien" style="width:400px"><div class="thumbinner"><a href="https://static.igem.org/mediawiki/2015/f/fc/Freiburg_results-result_device_20150818.jpg" title="results:result_device_20150818.jpg" class="media lightbox_trigger"><img alt="Our device measurement with rabbit proteins" src="https://static.igem.org/mediawiki/2015/f/fc/Freiburg_results-result_device_20150818.jpg" width="400"/></a><div class="thumbcaption"><strong>Figure 1: Binding of anti-rabbit antibodies to rabbit proteins measured in our device.</strong> A: The first picture of the measurement. B: The last picture of the measurement. C: The quotient picture of the first and last picture. D: Schematic illustration of the spot pattern on the slide </div></div></div>
 
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   For testing our device we immobilized proteins derived from rabbits on an iRIf slide in distinct spots (figure 1 D). The proteins we used were polyclonal antibodies against HCV (Hepatitis C Virus) derived from rabbits. We aimed to detect these proteins with anti-rabbit antibodies (antibodies which specifically bind the constant region of antibodies dervied from rabbits). We used these interaction partners in this series of experiments to reproduce a successful measurement, which we previously performed in the professional measuring device.
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   For testing our device we immobilized rabbit-derived proteins on an iRIf slide in distinct spots (figure 1 D). The proteins we used were polyclonal antibodies against HCV (Hepatitis C Virus) raised in rabbit. We aimed to detect these proteins with anti-rabbit antibodies (antibodies which specifically bind the constant region of antibodies raised in rabbit). We used them as interaction partners in a series of experiments to reproduce a measurement previously performed in the professional device.
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   The binding layer on the iRIf slide consisted of an APTES/PDITC surface. The spots on the slide were produced 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 o/n the slide was blocked for 30 min in BSA [10 mg/ml] solution.
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   The binding layer on the iRIf slide consisted of an APTES/PDITC surface. The spots on the slide were produced 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 o/n the slide was blocked for 30 min with BSA [10 mg/ml].
 
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   The camera used for the measurement was a Canon 50D. The camera was set to automatically take pictures at an interval of five seconds. The exposure time was set in order for the pixels in the image to have approx. 80% of maximum light saturation before the solution was flushed onto the chip (figure 1 A & B).
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   The camera used for the measurement was a Canon 50D. The camera was set to automatically acquire pictures at an interval of five seconds. Before the solution was flushed over the chip, the exposure time was set to obtain apprx. 80% saturation (figure 1 A & B).
  
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The antibody solution was pipetted into the flowchamber without the use of any microfluidic pump. 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 it from the syringe.
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The antibody solution was pipetted into the flowchamber without the use of a microfluidic pump. 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 it from the syringe.
  
 
   As can be seen in figure 1 C, the quotient picture clearly shows binding of anti-rabbit antibodies to the rabbit protein spots. The BSA control spots show none or negligible unspecific binding.
 
   As can be seen in figure 1 C, the quotient picture clearly shows binding of anti-rabbit antibodies to the rabbit protein spots. The BSA control spots show none or negligible unspecific binding.
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   In this section we will demonstrate how to build your own iRIf device from scratch, using affordable, low-tech material. The design is 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.
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   In this section we demonstrate how to build your own iRIf device from scratch, using affordable, low-tech material. The design is focused on creating a device that is both low-priced and portable, demonstrating the potential for the DiaCHIP to be used even in areas where high-tech laboratories are inaccessible.
 
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           <strong>Figure 3: An illustration showing the exact setup of our device from a top perspective.</strong>
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           <strong>Figure 3: Our device from top.</strong>
 
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   As can be seen in figure 3, the basic setup is fairly simple. Light from an LED enters a lense to obtain a parallel light beam. To achieve this, the distance from the LED to the lense has to be exactly one focal length.  
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   As seen in figure 3, the basic setup is fairly simple. Light from an LED enters a lens to obtain a parallel light beam. To achieve this, the distance from the LED to the lens should be close to one focal length.  
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   The light hits the iRIf slide afterwards where it is reflected (in the same angle as it hits 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.
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   Next, the light hits the iRIf slide where it is reflected (in the same angle as it hits the slide) and enters a second lens that projects an image of the slide on the CCD chip of the camera.
 
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Revision as of 16:56, 17 September 2015

""

Results of Our Self-Built Device

Here you will see what we were able to measure with the very first prototype of our self-built device. To see the results of our final device, please refer to the essential results page. A detailed explanation on how to build your own low-priced iRIf device can be found on the bottom of this page.

Our First Prototype is Able to Detect Antigen-Antibody Binding

Our device measurement with rabbit proteins
Figure 1: Binding of anti-rabbit antibodies to rabbit proteins measured in our device. A: The first picture of the measurement. B: The last picture of the measurement. C: The quotient picture of the first and last picture. D: Schematic illustration of the spot pattern on the slide

For testing our device we immobilized rabbit-derived proteins on an iRIf slide in distinct spots (figure 1 D). The proteins we used were polyclonal antibodies against HCV (Hepatitis C Virus) raised in rabbit. We aimed to detect these proteins with anti-rabbit antibodies (antibodies which specifically bind the constant region of antibodies raised in rabbit). We used them as interaction partners in a series of experiments to reproduce a measurement previously performed in the professional device. The binding layer on the iRIf slide consisted of an APTES/PDITC surface. The spots on the slide were produced 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 o/n the slide was blocked for 30 min with BSA [10 mg/ml].

Freiburgs first iRIf prototype
Figure 2: Picture of our first iRIf device prototype. Note that the Nikon camera was replaced with a Canon 50D.

The camera used for the measurement was a Canon 50D. The camera was set to automatically acquire pictures at an interval of five seconds. Before the solution was flushed over the chip, the exposure time was set to obtain apprx. 80% saturation (figure 1 A & B). The antibody solution was pipetted into the flowchamber without the use of a microfluidic pump. 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 it from the syringe. As can be seen in figure 1 C, the quotient picture clearly shows binding of anti-rabbit antibodies to the rabbit protein spots. The BSA control spots show none or negligible unspecific binding.

How To Build Your Own Device

In this section we demonstrate how to build your own iRIf device from scratch, using affordable, low-tech material. The design is focused on creating a device that is both low-priced and portable, demonstrating the potential for the DiaCHIP to be used even in areas where high-tech laboratories are inaccessible.

General Principle

Figure 3: Our device from top.

As seen in figure 3, the basic setup is fairly simple. Light from an LED enters a lens to obtain a parallel light beam. To achieve this, the distance from the LED to the lens should be close to one focal length. Next, the light hits the iRIf slide where it is reflected (in the same angle as it hits the slide) and enters a second lens that projects an image of the slide on the CCD chip of the camera.

Construction Guidance

To rebuild our iRIf device, we used the following parts:

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 strength, blurred images or, in the worst case, no signal at all. This can be difficult since our device does not rely on straight angles. We overcame this problem by designing a case for the device that ensures the right placement of the components inside the device. This was achieved by calculating all the distances beforehand using physical laws 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).

Figure 4: 3D model of our device, built from the vector files used to order the parts.
Figure 5: The 3D model of our device without walls and top part. Parts have been colorized for clarification - Green: The wall where light exits the device and enters the camera; Pink: a platform holding Cooling-Element+LED in place; Blue: platforms for holding the lenses in place; Gray: rear end wall where the flowchamber is attached to. The slits in the top and bottom part where the magnets have to be fixed

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). Figure 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 uses laser cutting to cut out parts from acrylic glass. The cutting laser is guided by the vector paths defined in the vector graphic 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 cut from 1 mm thick acrylic glass. Once they are cut from the acrylic glass with a laser, 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)

Freiburg iRIf device parts
Figure 6: An image of the vector file used to order the parts. The vector file is used to excise parts of a 5 mm acrylic glass board with a laser. Black lines are cut out by the laser, gray lines/areas are engravings. Refer to the download section to downloaded the vector graphic files.

The most difficult part to build by oneself is the PDMS flowchamber. For producing such flowchambers, a wafer has to be made in a cleanroom. The template used for our PDMS flowcell is shown in figure 9. If your facility has an engineering department, there is a high chance that a cleanroom is present. Ask the personel in charge if they can help you out with producing your flowcell. If you already use another type of microfluidic flowchambers, our device should still be compatible with it, though some adaptions might be necessary. If you have no possibility of creating a flowchamber, you might want to consider building a very simplified DIY flowchambers from two glass slides, separated 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 flow cell attachment
Figure 7 - left: a flow chamber consisting of an iRIf slide attached to a PDMS flowcell - center: a syringe attached to a pipette tip, used to inject antibody solutions into the flowchamber - right: the part of our device which connects the flowchamber, the syringe and the iRIf device.
Freiburg iRIf device flow chamber mounted and unmounted
Figure 8: The device with and without the mounted flowchamber.
Freiburg iRIf device flow cell
Figure 9: Layout used to produce the wafer of our PDMS flowcell.

Legal notice

The iRIf detection method is patented. Biametrics and associated persons own patents concerning the detection principle. These patents are: