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

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<div class="kommentar_stefan"> Korrigiert Stefan </div>
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<div class="kommentar_stefan"> Korrigiert Stefan, Günter, Carina </div>
 
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The observation and quantification of protein-protein or other molecular interactions is a very difficult task and requires expensive and complicated equipment. Therefore we designed and <a href="#howto_anchor">constructed a cheap and easy to use device </a> that can easily be reconstructed by the iGEM community. Using our device we were able to measure <a href="https://2015.igem.org/wiki/index.php?title=Team:Freiburg/Results#device_results_anchor">antigen-antibody interactions</a> and reproduced an experiment performed with the professional device. All in all, we made it easy for everyone to perform professional binding experiments.
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The observation and quantification of protein-protein or other molecular interactions is a very difficult task and requires expensive and complicated equipment. Therefore, we designed and <a href="#howto_anchor">constructed a cheap and easy to use device </a> that can easily be reconstructed by the iGEM community. Using our device we were able to measure <a href="https://2015.igem.org/wiki/index.php?title=Team:Freiburg/Results#device_results_anchor">antigen-antibody interactions</a> and reproduced an experiment performed with the professional device. All in all, we made it easy for everyone to perform professional binding experiments.
 
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   In order to test our device we immobilized rabbit-derived proteins on an iRIf slide in distinct spots (figure 1 D). These proteins were polyclonal antibodies against HCV (Hepatitis C Virus) raised in rabbit. They were detected using anti-rabbit antibodies that bind specifically to the constant regions of all rabbid derived antibodies. We used them as interaction partners in a series of experiments to reproduce a measurement previously performed in the professional device.
+
   In order to test our device, we immobilized rabbit-derived proteins on an iRIf slide in distinct spots (figure 1 D). These proteins were polyclonal antibodies against HCV (Hepatitis C Virus) raised in rabbit. They were detected using anti-rabbit antibodies that bind specifically to the constant regions of all rabbit derived antibodies. 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 with BSA [10 mg/ml].
+
   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 onto the slide in an alternating pattern. 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 acquire pictures at an interval of five seconds. After flushing the chamber with buffer, the exposure time was set to obtain apprx. 80% saturation (Fig. 1 A & B).
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   The camera used for the measurement was a Canon 50D. It was set to automatically acquire pictures at an interval of five seconds. After flushing the chamber with buffer, the exposure time was set to obtain apprx. 80% saturation (Fig. 1 A & B).
  
 
The antibody solution was pipetted into the flow chamber without the use of a microfluidic pump. Instead, a syringe was loaded with 500 µl [5 µg/ml] anti-rabbit antibody solution (diluted in 1xPBS) and slowly released into the binding chamber of the device by gently dispensing it from the syringe.
 
The antibody solution was pipetted into the flow chamber without the use of a microfluidic pump. Instead, a syringe was loaded with 500 µl [5 µg/ml] anti-rabbit antibody solution (diluted in 1xPBS) and slowly released into the binding chamber of the device by gently dispensing it from the syringe.
  
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   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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   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 no or negligible unspecific binding.
 
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     <a class="accordion-section-title" href="#accordion-2">Spot analysis by hand: A step by step guidance</a>
 
     <a class="accordion-section-title" href="#accordion-2">Spot analysis by hand: A step by step guidance</a>
 
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<p> This step by step guidance shows you how to determine endpoint intensities of the binding experiment as shown Figure 1C. Have a look at our <a href="https://2015.igem.org/Team:Freiburg/Project/iRIf">iRIF</a> page to get all the background informations about the detection method.</p>
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<p> This step by step guidance shows you how to determine endpoint intensities of the binding experiment as shown figure 1C. Have a look at our <a href="https://2015.igem.org/Team:Freiburg/Project/iRIf">iRIF</a> page to get all the background information about the detection method.</p>
 
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<li> During your iRIF experiment you take two pictures. One before the binding event and one after the binding event. Open both pictures in <a href="http://imagej.nih.gov/ij/">ImageJ</a>. </li>
 
<li> During your iRIF experiment you take two pictures. One before the binding event and one after the binding event. Open both pictures in <a href="http://imagej.nih.gov/ij/">ImageJ</a>. </li>
 
<li> Go to "Process" and click on "Image Calculator..." </li>
 
<li> Go to "Process" and click on "Image Calculator..." </li>
 
<li> Set "Image1" to the image after the binding event and "Image2" to the image before the binding event. Choose "divide" as operation. Activate the "32-bit (float) result" and click "ok". A quotient picture will be calculated and opened. </li>
 
<li> Set "Image1" to the image after the binding event and "Image2" to the image before the binding event. Choose "divide" as operation. Activate the "32-bit (float) result" and click "ok". A quotient picture will be calculated and opened. </li>
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<li> Go to "Image" then "Adjust" and klick on "Brightness/Contrast..." and play around with "brightness" and "contrast" till the spots get visible.</li>
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<li> Go to "Image" then "Adjust" and click on "Brightness/Contrast..." and play around with "brightness" and "contrast" till the spots get visible.</li>
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<li> Mark the spots using the "oval" selection tool and press "T" on your keybord. Do this for every spot. Do not foret to make a selection of the background. </li>
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<li> Mark the spots using the "oval" selection tool and press "T" on your keyboard. Repeat this for every spot. Do not forget to make a selection of the background. </li>
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<li> Go to the ROI-manager that poped up when you first pushed T and click on "Deselect" and then on "Measure". The results of your Measurment will pop up. You can save them in Excel.</li>
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<li> Go to the ROI-manager that popped up when you first pushed T and click on "Deselect" and then on "Measure". The results of your measurement will pop up. You can save them in Excel.</li>
 
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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. Therefore the DiaCHIP can be used even in areas where high-tech laboratories are inaccessible.
+
   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. Therefore the DiaCHIP can even be used in areas where high-tech laboratories are inaccessible.
 
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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.  
 
   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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   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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   Next, the light hits the iRIf slide where it is reflected (under the same angle as it hits the slide) and enters a second lens that projects an image of the slide onto the CCD chip of the camera.
 
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   A major problem that we encountered when building the device from scratch was to ensure that all components are at the proper distance and angle to each other. Correct alignment 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 challenge by designing a case for the device that ensures the right placement of the components inside the device. We calculated all distances between the LED, lenses, camera and slide using the laws of geometrical optics and drew an vector graphic blueprint for our device. Next, we constructed a digital 3D model of the casing based on the vector blueprint to avoid an expensive and time-consuming trial and error process (Figure 4).
+
   A major problem that we encountered when building the device from scratch was to ensure that all components are at the proper distance and angle to each other. Correct alignment 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 challenge by designing a case for the device that ensures the right placement of the components inside the device. We calculated all distances between the LED, lenses, camera and slide using the laws of geometrical optics and drew a vector graphic blueprint for our device. Next, we constructed a digital 3D model of the casing based on the vector blueprint to avoid an expensive and time-consuming trial and error process (Figure 4).
 
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   The casing is designed to keep all the necessary parts (lenses, LED) in place safely during a measurement, but allows them to be removed to facilitate transportation of the device (i.e. to the Giant Jamboree). Figure 5 illustrates the parts that hold the lenses and LED in place.
+
   The casing is designed to keep all the necessary parts (lenses, LED) safely in place during a measurement, but allows them to be removed to facilitate transportation of the device (i.e. to the Giant Jamboree). Figure 5 illustrates the parts that hold the lenses and LED in place.
  
 
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   After setup of the 3D model of our device, we created a vector graphic of all required parts in a 2D plane. Using this vector graphic, we ordered the parts at <a href="http://www.formulor.com" target="_blank">Formulor</a>, a service which uses laser cutting to cut out parts from acrylic glass and other materials. The cutting laser is guided by the paths defined in the vector graphic template. The template is shown in figure 6, and may be  <a href="#device_download_links_anchor">downloaded</a> and used by everyone to build their own device. To allow easy mounting of the casing, we also provide an easy to understand <a href="https://static.igem.org/mediawiki/2015/2/2f/Freiburg_iRIf_Device_Manual.pdf" target="_blank">manual</a>. One advantage of acrylic glass is that the parts can be glued together easily using a few drops of acetone. The vector graphic also contains a template for the parts which are necessary to attach the glass and PDMS slide to the device. They should be cut from 1 mm thick acrylic glass. The microfluidic tubes can be glued to these parts (figure 7) and attached to the device with the magnets (figure 8)</p>
+
   Subsequently to the setup of the 3D model of our device, we created a vector graphic of all required parts in a 2D plane. Using this vector graphic, we ordered the parts at <a href="http://www.formulor.com" target="_blank">Formulor</a>, a service which uses laser cutting to cut out parts from acrylic glass and other materials. The cutting laser is guided by the paths defined in the vector graphic template. The template is shown in figure 6, and may be  <a href="#device_download_links_anchor">downloaded</a> and used by everyone for building their own device. To allow easy mounting of the casing, we also provide an easy to understand <a href="https://static.igem.org/mediawiki/2015/2/2f/Freiburg_iRIf_Device_Manual.pdf" target="_blank">manual</a>. One advantage of acrylic glass is that the parts can be glued together easily using a few drops of acetone. The vector graphic also contains a template for the parts which are necessary to attach the glass and PDMS slide to the device. They should be cut from 1 mm thick acrylic glass. The microfluidic tubes can be glued to these parts (figure 7) and attached to the device with the magnets (figure 8)</p>
  
 
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Revision as of 22:44, 18 September 2015

""

Korrigiert Stefan, Günter, Carina

Results of Our Self-Built iRIf Device

The observation and quantification of protein-protein or other molecular interactions is a very difficult task and requires expensive and complicated equipment. Therefore, we designed and constructed a cheap and easy to use device that can easily be reconstructed by the iGEM community. Using our device we were able to measure antigen-antibody interactions and reproduced an experiment performed with the professional device. All in all, we made it easy for everyone to perform professional binding experiments.

Detection of Antigen-Antibody binding using our selfmade device

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

In order to test our device, we immobilized rabbit-derived proteins on an iRIf slide in distinct spots (figure 1 D). These proteins were polyclonal antibodies against HCV (Hepatitis C Virus) raised in rabbit. They were detected using anti-rabbit antibodies that bind specifically to the constant regions of all rabbit derived antibodies. 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 onto the slide in an alternating pattern. 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. It was set to automatically acquire pictures at an interval of five seconds. After flushing the chamber with buffer, the exposure time was set to obtain apprx. 80% saturation (Fig. 1 A & B). The antibody solution was pipetted into the flow chamber without the use of a microfluidic pump. Instead, a syringe was loaded with 500 µl [5 µg/ml] anti-rabbit antibody solution (diluted in 1xPBS) 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 no or negligible unspecific binding.

Spot analysis by hand: A step by step guidance

How To Build Your Own iRIf 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. Therefore the DiaCHIP can even be used 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 (under the same angle as it hits the slide) and enters a second lens that projects an image of the slide onto the CCD chip of the camera.

Construction Guidance

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

A major problem that we encountered when building the device from scratch was to ensure that all components are at the proper distance and angle to each other. Correct alignment 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 challenge by designing a case for the device that ensures the right placement of the components inside the device. We calculated all distances between the LED, lenses, camera and slide using the laws of geometrical optics and drew a vector graphic blueprint for our device. Next, we constructed a digital 3D model of the casing based on the vector blueprint to avoid an expensive 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 the cooling element and LED in place; Blue: platforms for holding the lenses in place; Gray: rear end wall where the flow chamber is attached to. The slits in the top and bottom part where the magnets need to be attached.

The casing is designed to keep all the necessary parts (lenses, LED) safely in place during a measurement, but allows them to be removed to facilitate transportation of the device (i.e. to the Giant Jamboree). Figure 5 illustrates the parts that hold the lenses and LED in place.

Subsequently to the setup of the 3D model of our device, we created a vector graphic of all required parts in a 2D plane. Using this vector graphic, we ordered the parts at Formulor, a service which uses laser cutting to cut out parts from acrylic glass and other materials. The cutting laser is guided by the paths defined in the vector graphic template. The template is shown in figure 6, and may be downloaded and used by everyone for building their own device. To allow easy mounting of the casing, we also provide an easy to understand manual. One advantage of acrylic glass is that the parts can be glued together easily using a few drops of acetone. The vector graphic also contains a template for the parts which are necessary to attach the glass and PDMS slide to the device. They should be cut from 1 mm thick acrylic glass. The microfluidic tubes can be glued to these parts (figure 7) and 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 is the PDMS flow chamber since it requires a microstructured silicon wafer, which can only be produced in a cleanroom environment. The template used for our PDMS flow cell is shown in figure 9. If your facility has an engineering department, there is a high chance that a cleanroom is present. Ask the personnel in charge if they can help you out with producing your flow cell. If you already use another type of microfluidic flow chamber, 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, separated by thin tape. Note that our device was not primarily build to support such a chamber 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 (red) flow cell - center: a syringe attached to a pipette tip, used to inject antibody solutions into the flow chamber - right: the part of our device which connects the flow chamber, the syringe and the iRIf device.
Freiburg iRIf device flow chamber mounted and unmounted
Figure 8: The device with and without the mounted flow chamber.
Freiburg iRIf device flow cell
Figure 9: Layout used to produce the wafer of our PDMS flow cell.

Legal notice

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