Difference between revisions of "Team:Freiburg/Measurement"

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<h2> Measurement</h2>
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<h4>Note</h4>
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<p>In order to be considered for the <a href="https://2015.igem.org/Judging/Awards#SpecialPrizes">Best Measurement Approach award</a>, you must fill out this page.</p>
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<div class="content_box results_page">  
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<h1>Results of Our Self-Built iRIf Device</h1>
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<div class="level1">
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 +
<p>
 +
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.
 +
</p>
 
</div>
 
</div>
  
 +
  <h1>Detection of Antigen-Antibody binding using our selfmade device</h1>
  
<p>There are a lot of exciting Parts in the Registry, but many Parts have still not been characterized. Synthetic Biology needs great measurement approaches for characterizing new parts, and efficient new methods for characterizing many parts at once. If you've done something exciting in the area of Measurement, describe it here!</p>
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<div class="level1">
 +
  <div class="image_box left">
 +
    <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/c/cc/Freiburg_results-result_device_20150818_preview.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>
 +
  </div>
  
  
  
<h4>Inspiration</h4>
+
  <p>
<p>You can look at what other teams did to get some inspiration! <br />
+
  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.
Here are a few examples:</p>
+
  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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</p>
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<div class="image_box right" style="clear:both;">
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  <div class="thumb2 trien" style="width:410px">
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    <div class="thumbinner">
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      <a href="https://static.igem.org/mediawiki/2015/c/c9/Freiburg_Device_Prototype_setup.jpg" title="Freiburgs first iRIf prototype" class="media lightbox_trigger"><img alt="Freiburgs first iRIf prototype" src="https://static.igem.org/mediawiki/2015/1/14/Freiburg_Device_Prototype_setup_preview.jpg" width="400"/></a>
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      <div class="thumbcaption">
 +
      <strong>Figure 2:</strong> Picture of our first iRIf device prototype. Note that the Nikon camera was replaced with a Canon 50D.
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        </div>
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      </div>
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  </div>
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</div>
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<p>
 +
  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.
 +
  </p>
 +
 
 +
<div class="accordion" style="margin-top:100px">
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  <div class="accordion-section">
 +
    <a class="accordion-section-title" href="#accordion-2">Spot analysis by hand: A step by step guidance</a>
 +
    <div id="accordion-2" class="accordion-section-content" style="display:none; padding:15px;">
 +
<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>
 +
<ol>
 +
<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> 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> Go to "Image" then "Adjust" and click on "Brightness/Contrast..." and play around with "brightness" and "contrast" till the spots get visible.</li>
 +
<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>
 +
<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>
 +
</ol>
 +
<p>
 +
Remember that signals normally differ by about 0.1 - 0.8 percent depending on how strong your binding is.
 +
</p>
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<span class="anchor" id="howto_anchor"></span>
 +
<h1 class="left" id="how_to_build_your_own_device">How To Build Your Own iRIf Device</h1>
 +
 
 +
 
 +
<div class="image_box right"> 
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  <div style="margin:0 auto 0 auto; width:500px">
 +
    <a class="media lightbox_trigger" href="https://static.igem.org/mediawiki/2015/b/b7/Freiburg_Own_Device_Foto.jpg" title="Freiburgs self-made iRIf device">
 +
      <img alt="Picture of the Freiburg device" src="https://static.igem.org/mediawiki/2015/2/2e/Freiburg_Own_Device_Foto_preview.jpg" width="500"/>
 +
    </a>
 +
  </div>
 +
</div>
 +
 
 +
<p>
 +
  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.
 +
</p>
 +
 
 +
<h2 class="left">General Principle</h2>
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 +
<div class="level2">
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      <div class="thumbinner">
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        <a class="media lightbox_trigger" href="https://static.igem.org/mediawiki/2015/e/ed/Freiburg_results-device_build_composition.jpg" title="results:device_build_composition.jpg">
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          <img alt="" src="https://static.igem.org/mediawiki/2015/e/ed/Freiburg_results-device_build_composition.jpg" width="500"/>
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        </a>
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        <div class="thumbcaption">
 +
          <strong>Figure 3: Our device from top.</strong>
 +
        </div>
 +
      </div>
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    </div>
 +
 
 +
  </div> <!-- end image_box -->
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 +
  <p>
 +
  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.
 +
  </p>
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</div>
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<!-- EDIT4 SECTION "General principle" [2000-2617] -->
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<h2 class="sectionedit5">Construction Guidance</h2>
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<div class="level2">
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<div class="accordion">
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  <div class="accordion-section">
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    <a class="accordion-section-title" href="#accordion-1">To rebuild our iRIf device, we used the following parts:</a>
 +
    <div id="accordion-1" class="accordion-section-content" style="display:none; padding:15px;">
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      <div class="table sectionedit6">
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      <table class="inline">
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        <tr class="row0">
 +
        <th class="col0"> Part </th><th class="col1"> Description </th><th class="col2"> Manufacturer / Part name </th><th class="col3"> Price[USD]</th>
 +
        </tr>
 +
        <tr class="row1">
 +
        <td class="col0">A fairly strong LED</td><td class="col1"> In our case a 3W green 520 nm LED</td><td class="col2">Cree XP-E on star circuit board</td><td class="col3">10</td>
 +
        </tr>
 +
        <tr class="row2">
 +
        <td class="col0">A cooling element</td><td class="col1">Used to prevent the LED from overheating</td><td class="col2">Fischer Elektronik ICK S</td><td class="col3">9</td>
 +
        </tr>
 +
        <tr class="row3">
 +
        <td class="col0">A constant-current-source</td><td class="col1">To ensure static LED light (no flickering)</td><td class="col2">Roschwege GmbH KSQ-3W</td><td class="col3">15</td>
 +
        </tr>
 +
        <tr class="row4">
 +
        <td class="col0">An AC/DC rectifier</td><td class="col1">Used to grant direct current to the LED</td><td class="col2">Goobay 67951</td><td class="col3">16</td>
 +
        </tr>
 +
        <tr class="row5">
 +
        <td class="col0">Two optical lenses</td><td class="col1">Here we used two identical 60 mm lenses</td><td class="col2">Thorlabs AC254-060-A</td><td class="col3">160</td>
 +
        </tr>
 +
        <tr class="row6">
 +
        <td class="col0">A camera</td><td class="col1">We used a SLR camera</td><td class="col2">Canon 550D</td><td class="col3">200-400</td>
 +
        </tr>
 +
        <tr class="row7">
 +
        <td class="col0">A microfluidics chamber</td><td class="col1">Our chamber consists of an iRIf slide attached to a PDMS flow cell</td><td class="col2">Made ourselves</td><td class="col3"></td>
 +
        </tr>
 +
        <tr class="row8">
 +
        <td class="col0">Magnets</td><td class="col1">Used to attach the flow cell to the device</td><td class="col2">5 mm Neodymium magnets</td><td class="col3">3</td>
 +
        </tr>
 +
        <tr class="row9">
 +
        <td class="col0">A small syringe</td><td class="col1">We used a regular 10 ml syringe</td><td class="col2">BRAUN INJEKT 10 ml</td><td class="col3">&lt; 1</td>
 +
        </tr>
 +
        <tr class="row10">
 +
        <td class="col0">A microfluidic tube</td><td class="col1"> Used to connect the flow chamber with the syringe</td><td class="col2">-</td><td class="col3">&lt; 1</td>
 +
        </tr>
 +
        <tr class="row11">
 +
        <td class="col0">A case for the device</td><td class="col1"> 5 mm thick acrylic glass cutouts to hold the parts in place</td><td class="col2"> Ordered at <a class="urlextern" href="http://formulor.com" rel="nofollow" target="_blank" title="http://formulor.com">http://formulor.com</a></td><td class="col3">100</td>
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        </tr>
 +
      </table></div>
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    <div class="image_box center">
 +
      <div class="thumb2 trien" style="width:610px"><div class="thumbinner"><a class="media lightbox_trigger" href="https://static.igem.org/mediawiki/2015/2/24/Freiburg_results-led_and_magnets_and_flowcell.png" title="results:led_and_magnets_and_flowcell.png"><img alt="Some parts needed to build your own iRIf device" class="mediabox2" src="https://static.igem.org/mediawiki/2015/2/24/Freiburg_results-led_and_magnets_and_flowcell.png" width="600"/></a>
 +
        <div class="thumbcaption">
 +
        <strong>Figure 3:</strong> (A) The LED glued to the cooling element with thermal adhesive; (B) The 10 magnets, each 5x5x5 mm as well as a PDMS flow chamber (depth of the flow chamber: 30 µm); (C) One of the lenses used in the setup</div></div></div>
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    </div>
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    </div><!-- end accordion-section-content -->
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  </div><!-- end accordion-section -->
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</div> <!-- end accordion -->
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 +
<p>
 +
  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).
 +
</p>
 +
  <div class="image_box right">
 +
    <div class="thumb2 trien" style="width:410px"><div class="thumbinner"><a class="media lightbox_trigger" href="https://static.igem.org/mediawiki/2015/6/69/Freiburg_results-device_3d_perp.png" title="results:device_3d_perp.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/6/69/Freiburg_results-device_3d_perp.png" width="400"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/6/69/Freiburg_results-device_3d_perp.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div><strong>Figure 4:</strong> 3D model of our device, built from the vector files used to order the parts.</div></div></div>
 +
  </div>
 +
  <div class="image_box right">
 +
    <div class="thumb2 trien" style="width:410px;padding-top:10px"><div class="thumbinner"><a class="media lightbox_trigger" href="https://static.igem.org/mediawiki/2015/8/88/Freiburg_results-device_3d_no_walls.png" title="results:device_3d_no_walls.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/8/88/Freiburg_results-device_3d_no_walls.png" width="400"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/8/88/Freiburg_results-device_3d_no_walls.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div><strong>Figure 5: The 3D model of our device without walls and top part.</strong> 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.</div></div></div>
 +
  </div>
 +
 
 +
<p>
 +
  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.
 +
 
 +
</p>
 +
 
 +
<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>
 +
 
 +
<div class="image_box left">
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    <div class="thumb2 trien" style="width:410px">
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      <div class="thumbinner">
 +
        <a href="https://static.igem.org/mediawiki/2015/4/4c/Freiburg_results-device_parts_vector.png" class="lightbox_trigger" title="results:device_parts_vector.png"><img alt="Freiburg iRIf device parts" src="https://static.igem.org/mediawiki/2015/4/4c/Freiburg_results-device_parts_vector.png" width="400"/></a>
 +
        <div class="thumbcaption">
 +
          <strong>Figure 6</strong>: 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 <a href="#device_download_links_anchor">download section</a> to downloaded the vector graphic files.</div>
 +
        </div>
 +
    </div>
 +
  </div>
 +
  <p>
 +
  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.
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</p>
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<div class="flexbox">
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  <div class="image_box right">
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    <div class="thumb2 trien" style="width:410px">
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      <div class="thumbinner">
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        <a href="https://static.igem.org/mediawiki/2015/2/27/Freiburg_Device_Flowcell_chamber_syringe.jpg" class="lightbox_trigger" title="Flow cell attachment"><img alt="Freiburg iRIf device flow cell attachment" src="https://static.igem.org/mediawiki/2015/0/08/Freiburg_Device_Flowcell_chamber_syringe_preview.jpg" width="400"/></a>
 +
        <div class="thumbcaption">
 +
          <strong>Figure 7</strong> - 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.
 +
        </div>
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      </div>
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    </div>
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  </div>
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    <div class="thumb2 trien" style="width:390px">
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        <a href="https://static.igem.org/mediawiki/2015/c/c0/Freiburg_flowchamber_mounted_unmounted.png" class="lightbox_trigger" title="Mounted and unmounted flow-chamber in our iRIf device"><img alt="Freiburg iRIf device flow chamber mounted and unmounted" src="https://static.igem.org/mediawiki/2015/1/1f/Freiburg_flowchamber_mounted_unmounted_preview.png" width="390"/></a>
 +
        <div class="thumbcaption">
 +
          <strong>Figure 8:</strong> The device with and without the mounted flow chamber.
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        </div>
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        <a href="https://static.igem.org/mediawiki/2015/0/00/Freiburg_Masken_M25-M33-1.jpg" class="lightbox_trigger" title="PDMS flow chamber"><img alt="Freiburg iRIf device flow cell" src="https://static.igem.org/mediawiki/2015/0/00/Freiburg_Masken_M25-M33-1.jpg" width="400"/></a>
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          <strong>Figure 9:</strong> Layout used to produce the wafer of our PDMS flow cell.
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<h2 class="left">Download section</h2>
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      <a href="https://static.igem.org/mediawiki/2015/2/2f/Freiburg_iRIf_Device_Manual.pdf" target="_blank"><img alt="Manual for building your own DiaCHIP" src="https://static.igem.org/mediawiki/2015/c/c9/Freiburg_diachip_device_manual_preview.png" width="300"/></a>
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        Click on the image to download a manual which will show you how to mount the casing of the device.
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<li><a href="https://2014.igem.org/Team:Aachen">2014 Aachen  </a></li>
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<li><a href="https://2014.igem.org/Team:Valencia_Biocampus">2014 Valencia Biocampus</a></li>
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    <a href="https://static.igem.org/mediawiki/2015/0/04/Freiburg_Device_Template_SVG.zip">Blueprint of the casing for cutting out your own parts - SVG </a>
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    <a href="https://static.igem.org/mediawiki/2015/3/32/Freiburg_Device_Template_Illustrator.zip">Blueprint of the casing for cutting out your own parts - Adobe Illustrator</a>
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    <a href="https://static.igem.org/mediawiki/2015/2/2f/Freiburg_iRIf_Device_Manual.pdf">PDF manual showing how to build your own device</a>
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<h2 class="left">Legal notice</h2>
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<p>
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The iRIf detection method is patented.
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<a href="http://biametrics.com/en/"  target="_blank"> Biametrics </a>  
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and associated persons own patents concerning the detection principle.
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These patents are:
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<a href="http://www.google.com/patents/EP1805502A1?cl=en&hl=de"  target="_blank">  Method for examining physical, chemical and biochemical interactions </a> (DE102004051098.9, DE102005015030.6, EP05797776.1) 
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<a href="http://www.google.com/patents/DE102007038797A1?cl=en&hl=de"  target="_blank">  Study of molecular interactions on and / or in thin layers </a> (DE102007038797.2)
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<a href="http://www.google.com/patents/DE102009019711A1?cl=en&hl=de"  target="_blank">  Method and apparatus for determining reflection coefficients to filter arrangement with thin layers </a> (DE102009019711.7)
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<a href="http://www.google.com/patents/DE102009019476A1?cl=en&hl=de"  target="_blank">  Again Detectable support for optical measuring methods </a> (DE102009019476.2)
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Revision as of 00:15, 19 September 2015

""

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: