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| {{Freiburg/CSS}} | | {{Freiburg/CSS}} |
| {{Freiburg/Menubar}} | | {{Freiburg/Menubar}} |
− | {{Freiburg/wiki_content_start}} | + | {{Team:Freiburg/wiki_content_start_bubble}} |
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| <html> | | <html> |
| <style> | | <style> |
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| </style> | | </style> |
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| + | <script type="text/javascript"> |
| + | //===================BEGIN:Amazing Bubble Sidebar========================== |
| + | |
| + | $(document).ready(function(){ |
| + | // CHANGE THE FOLLOWING ATTRIBUTES // |
| + | var href_text1='https://2015.igem.org/Team:Freiburg/Results', |
| + | // Text2 needs no href as it is the actual page // |
| + | img_url='https://static.igem.org/mediawiki/2015/1/1e/Freiburg_icon_results_white_03.png', |
| + | href_text3='https://2015.igem.org/Team:Freiburg/Results/Immobilization', |
| + | // Text1 needs no text as it is a pic // |
| + | text2='Our Device'; |
| + | // Text3 needs no text as its always 'next' // |
| + | // HOLD ON CHANGING THINGS --JABBERWOCK // |
| + | |
| + | $('#bubble1').attr('href',href_text1); |
| + | $('#bubble1_img').attr('src', img_url); |
| + | $('#bubble3').attr('href',href_text3); |
| + | |
| + | $('#bubble2').text(text2); |
| + | }); |
| + | |
| + | //===================END:Amazing Bubble Sidebar========================== |
| + | </script> |
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| <!DOCTYPE html> | | <!DOCTYPE html> |
| <!-- content goes in here --> | | <!-- content goes in here --> |
− | <div class="content_box results_page"> | + | <div class="content_box"> |
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− | <h1>Results of Our Self-Built Device</h1> | + | <h1 style="text-align:right">Results of Our Self-Built iRIf Device</h1> |
| <div class="level1"> | | <div class="level1"> |
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| <p> | | <p> |
− | The observation of protein-protein or other molecular interaction often 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. | + | 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> | | </p> |
| </div> | | </div> |
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− | <h1>Detection of Antigen-Antibody binding using our selfmade device</h1> | + | <h2>Detection of Antigen-Antibody Binding Using our Selfmade Device</h2> |
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| <div class="level1"> | | <div class="level1"> |
| <div class="image_box left"> | | <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/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> | + | <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> | | </div> |
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| <p> | | <p> |
− | 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. | + | 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 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]. |
| </p> | | </p> |
| <div class="image_box right" style="clear:both;"> | | <div class="image_box right" style="clear:both;"> |
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| </div> | | </div> |
| <p> | | <p> |
− | 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 (Fig. 1 A & B). | + | 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 (figure 1 A & B). |
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− | 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 PBS) 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. | + | 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> | | </p> |
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− | <div class="accordion" margin-top="10px"> | + | <div class="accordion" style="margin-top:100px"> |
| <div class="accordion-section"> | | <div class="accordion-section"> |
− | <a class="accordion-section-title" href="#accordion-1">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> |
− | <div id="accordion-1" class="accordion-section-content" style="display:none; padding:15px;"> | + | <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> | | <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> 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 new 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> |
− | <li> Go to "Image" then "Adjust" and klick on "Brightness/Contrast..." and play around with "brightness" and "contrast" till the spots get visible.</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 keybord. Do this for every spot. Do not foret to make a selection of the background. <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 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> | + | <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> | | </ol> |
| <p> | | <p> |
− | Remember that signals will only differ by about 0.1 - 0.8 percent depending on how strong your binding is. | + | Remember that signals normally differ by about 0.1 - 0.8 percent depending on how strong your binding is. |
| </p> | | </p> |
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| <span class="anchor" id="howto_anchor"></span> | | <span class="anchor" id="howto_anchor"></span> |
− | <h1 class="left" id="how_to_build_your_own_device">How To Build Your Own Device</h1> | + | <h1 class="left" id="how_to_build_your_own_device">How To Build Your Own iRIf Device</h1> |
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| <p> | | <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, demonstrating the potential for the DiaCHIP to 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. |
| </p> | | </p> |
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| <p> | | <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. | | 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. | + | 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> | | </p> |
| </div> | | </div> |
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| <p> | | <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 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). |
| </p> | | </p> |
| <div class="image_box right"> | | <div class="image_box right"> |
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| <p> | | <p> |
− | 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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| </p> | | </p> |
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| <p> | | <p> |
− | 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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| <div class="image_box left"> | | <div class="image_box left"> |
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| <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> | | <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"> | | <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> | + | <strong>Figure 6: An image of the vector file used to order the parts.</strong> 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> | | </div> |
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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> | | <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"> | | <div class="thumbcaption"> |
− | <strong>Figure 7</strong> - left: a flow chamber consisting of an iRIf slide attached to a PDMS 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. | + | <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> | | </div> |
| </div> | | </div> |