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Essential Results

Our aim was to develop a novel diagnostic tool enabling fast, simultaneous and label-free detection of antibodies in human blood sera. We successfully generated our self-established cell-free expression system based on an E. coli lysate, which revealed an expression efficiency comparable to a commercial kit. To immobilize the expressed proteins, we established a surface specifically binding our target proteins. This system was used to produce a protein array that can be flushed with a serum sample. The binding of antibodies is detected in a label-free manner by an interferometric method called iRIf (imaging reflectometric interference) in real-time. Moreover, we rebuilt the detection device in a simplified and cost-efficient manner and thus, made it available for future iGEM Teams. Using the DiaCHIP, we did not only show the presence of antibodies in distinct solutions but also verified them in complex samples as human blood serum.

Detection of Anti-Tetanus Antibodies in Human Blood Serum

Video 1: Detection of anti-tetanus antibodies in human serum.

To test the DiaCHIP under real-life conditions, we analyzed the blood serum of a vaccinated person for the presence of anti-tetanus antibodies. We compared blood samples from before and after vaccination and could directly detect its effect. To capture the antibodies, the corresponding antigen was expressed in E. coli, purified by His-tag affinity purification and spotted on a specific Ni-NTA surface. By comparing the spotted pattern with the localization of signal as detected by iRIf, the system's specificity even for complex samples was shown. ▼ Detailed Description.

Figure 1: Western Blot analysis of purified tetanus antigen.

Figure 2: Quotient picture of iRIf measurement of human serum sample taken before vaccination.

The antigen used for detection of anti-tetanus antibodies is derived from the tetanus neurotoxin and commonly used for in vitro testing. For preparation of the DiaCHIP, this antigen had to be overexpressed in Escherichia coli and purified from the whole cell lysate. To verify successful purification of the antigen Western Blot analysis was performed (figure 1). The usage of an antibody targeting the N-terminal His-tag revealed a protein with a molecular weight of about 50 kDa. This correlates with the expected molecular weight of the antigen ¹⁾.

Before we performed an iRIf measurement with the blood of the freshly vaccinated person we checked if we could detect an interaction of antibodies present in the blood with our purified tetanus antigen by an ELISA. Therefore, we spotted tetanus antigen and bBSA of the same concentration (12.5 µg/mL) and twice PBS as negative controls. The tetanus antigen and one PBS well were incubated with 5 mg/mL BSA, blood serum and HRP-labeled anti-human for 1 h respectively. The bBSA and the other PBS well were incubated with HRP-labeled Streptavidine (0.5 µg/mL) for 1 h (positive control). After each step the wells were washed with PBS. To visualize the binding, Tetramethylbenzidine was added and the absorption at 650 nm over time was measured in a plate reader. The graph in figure 3 shows that the signal for tetanus is even higher than the positive control, which illustrates the successful binding of antibodies from the blood to tetanus antigens.

Figure 3: ELISA for the tetanus antigen with freshly vaccinated blood. The graph shows the absorption over time for TMB. The red line shows the binding of anti-tetanus antibodies out of the blood serum to purified tetanus antigen and the blue line shows the binding of HRP-Streptavidine to bBSA. The grey lines show the corresponding negative controls.

The purified antigen was immobilized on a Ni-NTA surface that specifically binds His-tagged proteins. Look here to read more about the establishment of this surface. As a positive control, self-purified His-tagged GFP was immobilized at another spot. The reliable detection of anti-GFP was shown before (link). No binding event should be detected at the negative control spot that was covered with bBSA/mCherry (??).

Figure 4: Quotient picture of iRIf measurement of human serum sample taken three???? weeks after vaccination.

PLATZHALTER! Figure 5: Binding curve of the tetanus measurements.

The human serum samples we obtained have been taken before and three? weeks after a patient received a vaccination boost against tetanus. Analysis of the samples was performed using the same array set-up. Figures 2 and 4 show quotient pictures representing changes in the thickness of the layer covering the slide while it was flushed with the respective samples. The negative control spots do not indicate any binding events, while the binding of anti-GFP to the positive control spot verifies that both experiments are reliable. The comparison of the antigen spots shows that we specifically detected anti-tetanus antibodies in human serum in response to vaccination. No binding event was detected by flushing the array with the sample assumed to be negative (figure 2), whereas a distinct spot is visible after flushing it with the positive sample (figure 4). Both measurements in real-time are shown in parallel in video 1.
The increase in relative light intensity at distinct spots over the course of the experiment is visualized in figure 5 and can be correlated with the amount of antibodies binding to the respective antigen spot. This indicates that the DiaCHIP enables quantification of antibody titers in addition to detecting their presence.
Besides tetanus, other antigens of immunological relevance were taken into account. See all the results we obtained in terms of diagnostics.

¹⁾ Rui et al., 2011. Enhanced expression of soluble recombinant tetanus neurotoxin Hc in Escherichia coli as a tetanus vaccine candidate. Immunobiology.

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hier noch rein bringen, dass ihr euren eigenen cell-free mix hergestellt habt und mit diesem auch GFP expremieren könnt und es im array erfolgreich nachweisen könnt
Auch, dass euer Mix low-cost ist, und trotzdem auf dem Niveau (oder besser) eines kommerziellen kits liegt - Community gedanke etc. (ps12092015)

Detection of Anti-GFP Antibodies Using Cell-Free Expressed GFP on Ni-NTA

Video 2: Binding of anti-GFP antibodies to GFP-His on a Ni-NTA surface. Anti-GFP antibodies bind both to purified GFP-His and cell-free expressed GFP-His. Control spots consisting of cell-free expression mix without DNA template show no binding.

Our self-made cell-free expression, the DiaMIX, in combination with specific glass surfaces allows the expression and purification of target proteins in a simple manner. The mix, after expressing His-tagged GFP, was spotted on a Ni-NTA coated glass slide. Even after several washing steps it could be shown that the proteins maintain their antibody binding properties. A GFP antibody solution was flushed over the slide and a specific binding could be detected in iRIf. ▼ Detailed Description.

We could successfully show that the cell-free expression of GFP-His results in functional GFP proteins that are bound by anti-GFP antibodies during an iRIf measurent. The DNA coding for GFP fused to a 10x His-tag was added to the DiaMIX and expression was performed for 2 h. The DiaMIX containing cell-free expressed GFP-His was then spotted on an iRIf slide with a Ni-NTA surface by hand (figure 1). The spots were incubated on the slide over night.

Figure 1: Specific Ni-NTA surface on iRIf glass slide.

This specific surface allows binding of the expressed GFP-His via the His-tag while all the other proteins present in the DiaMIX are not bound to the surface. This step was followed by the blocking and washing protocol to prevent further unspecific binding. The iRIf slide was then flushed with a anti-GFP antibody solution, to analyze the binding to the cell-free expressed GFP-His. As a positive control we spotted a purified GFP-His onto the surface as well as DiaMIX that did not contain any DNA as negative control (figure 2).

Figure 2: The two spots on the right are the cell-free expressed GFP-His proteins spotted onto the Ni-NTA surface. The spot on the left shows the positive control, GFP-His expressed in E. coli, purified and spotted onto the Ni-NTA surface.

The measurement shows a high signal for the cell-free expressed GFP-His and the positive control as can also be seen in the respective binding curve shwown in figure 3.

Figure 3: Binding curve of anti-GFP binding to cell-free expressed GFP-His on a Ni-NTA surface. Cell-free expressed GFP-His was spotted onto a specific Ni-NTA surface and flushed with anti-GFP. The binding curve indicated a binding event at the respective spot.

Find out more about the preparation of the DiaCHIP by producing a protein microarray from a DNA template using cell-free expression. ▲ Close Box

Building Our Low-Cost DiaCHIP Measuring Device

Figure 2: Functional and low-cost variant of the measuring device.

Commercial systems using the iRIf technology (imaging reflectomretric interference) are mostly bulky and expensive machines even though the components they are based on are rather simple. So we decided on building our own device consisting of not much more than two lenses, a LED and a camera. With this system we were able to reliably detect the binding of anti-GFP to GFP, thus confirming a detection sensitivity in the range of protein-protein interactions. To enable future iGEM Teams to profit from this device as we did, we provide all plans and parts necessary to rebuild it on this website for everyonbe to download and use. ▼ Detailed Description.

Video 1: Demonstration of the capability of our iRIf device at measuring antibody-antigen interaction. GFP was spotted on the left side, rabbit proteins (anti-HCV antibodies from rabbit) on the right.

To test our device we aimed at reproducing measurements that we were previously able to perform in our commercial device. For this reason we focused on using two antibodies: anti-GFP and polyclonal anti-rabbit. GFP and rabbit derived anti-HCV (Hepatitis C Virus) antibodies were used as antigens in this experiment. Note that the anti-HCV antibodies only served as binding partners for the anti-rabbit antibodies, since no HCV proteins were used in this experiment. The antigens were spotted onto an iRIf slide whose binding layer consisted of an APTES/PDITC surface. The spots on the slide were produced by pipetting 3.5 µl [1 mg/ml] rabbit-anti-HCV protein and 3.5 µl [1 mg/ml] GFP onto the slide and incubated overnight. After incubation the slide was blocked for 30 min in BSA solution. A Canon 50D camera was used to record the measurement and was set to take one picture every 5 seconds. The exposure time was set in order for the pixels in the image to be approx. 80% of maximum light saturation before the solution was flushed onto the chip. The antibody solutions were pipetted into the flow-chamber without the use of any microfluidic device. Instead, a syringe was loaded with 660 µl [5 µg/ml] anti-GFP antibody solution (diluted in PBS) and connected to the input pipe of our device. The content of the syringe was then slowly released via this pipe into the chamber of the device by gently dispensing the solution from the syringe. When the whole volume ran over the chip, the process was repeated with 660 µl [5 µg/ml] anti-rabbit antibody solution in the same way. The injection of both solutions was performed during approximately 45 minutes.

Figure 3: Quotient picture of the same measurement with favourable light conditions.

Video 1 shows the results of the measurement. Both, binding of anti-GFP and anti-rabbit to the corresponding antigen spots, could be observed. Due to the fact that the experiment was performed on our demonstration device which was built with a transparent casing, fluctuations in surrounding light had a strong, detrimental influence on the measurement quality. To minimize the influence of the unstable surrounding light, the resulting pictures had to be averaged over 10 pictures each. This in turn lead to a more stable light situation, however the signal strengh dropped as a consequence. Figure XY shows a quotient picture of the measurement where the light situation was temporarily appropriate. The problem of surrounding light scattering into the device can of course be overcome using a non-transparent casing.

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Establishing a Highly Specific Ni-NTA Surface

Figure 1: Cell-free expressed GFP-His spotted on a Ni-NTA slide. Cell-free expression was performed for 2 hours at 37°C. The positive control was purified GFP-His, the negative control was a sample of cell-free expression mix not containing DNA.

Since there is a variety of proteins present in the cell-free mix a specific surface on the future protein array is essential. That is why we established our Ni-NTA surface for the immobilization of the antigens on the chip. All expression constructs contain a His-tag fused to the coding sequence, resulting in antigens that can bind specifically to our Ni-NTA surface. Compared to an unspecific surface (PDITC) we could show that this Ni-NTA surface allows efficient binding of the target protein and prevents unspecific binding of other proteins that are present in the cell-free mix. ▼ Detailed description.

Figure 2: Cell-free expressed GFP-His spotted on a PDITC slide. Cell-free expression was performed for 2 hours at 37°C. The positive control was purified GFP-His, the negative control was a sample of cell-free expression mix not containing DNA.

For cell-free protein expression lots of different proteins, like RNA-polymerases, ribosomes and other E. coli proteins, are essential. So during the process of cell-free expression all these proteins are, besides the target protein, also present in the microfluidic chamber. To have a sufficient amount of target protein immobilized on the chip we established a specific surface chemistry on the glass slide. After testing several tag systems we established a Ni-NTA surface because it worked best for us. Using a Ni-NTA covered glass slide (figure 1) we could increase the amount of bound cell-free expressed GFP-His compared to an unspecific surface (figure 2). In both experiments ____2.5 µg?!?____ of self purified GFP-His was used as positive control. The cell-free expression mix without DNA is supposed to result in no target protein and served as negative control. The cell-free reactions were performed for 2 hours at 37°C. The samples were pipetted by hand onto the surfaces and incubated on the slide overnight. In the iRIf device, the optical detection method we used, the slides were blocked with BSA and then flushed with anti-GFP antibodies. An increase in light intensity on the slide where the spotted protein was located represents a binding event. Due to less unspecific binding of proteins to the surface, the interaction of anti-GFP antibodies with cell-free expressed GFP-His is higher on the Ni-NTA surface.

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Producing Our Cell-free Expression Mix

The mechanism we are using to copy a DNA array template into a protein array on demand is based on cell-free expression. During our project we successfully established our cell-free expression system, the DiaMIX, from scratch, starting with an E. coli lysate. With this mix we succeeded in expressing correctly folded GFP and luciferase. Comparison of the DiaMIX with a commercial cell-free expression kit revealed the great potential of our system! ▼ Detailed Description.

Our DiaMIX was prepared based on a protocol of

??? (Ref.)

. It is a very complex system containing many different enzymes and chemicals for special purposes. An overview of cell-free expression systems and its components can be found on themethodology page.
To investigate the efficiency of our self-prepared expression system we performed an experiment comparing it to a commercially available kit. In order to obtain reliable results we used the exact same vector, containing a sequence encoding GFP. Like this we were able to trace the amount of expressed GFP using a plate reader over a period of two hours. As a negative control, both mixes were treated equally in a second sample but did not contain any DNA.
The evaluation is shown in figure 1 and is based on triplicates. The data was normalized to the mean value of the measurement of air. The uncertainty was calculated using the standard deviation. ÜBERARBEITEN JE NACH GRAPH
picture; Figure 1: Cell-free expression of GFP using the DiaMIX and a commercial kit. The reaction was performed in a volume of 50 µl each and monitored every minute at 37°C over a period of two hours. Excitation at 480 nm, emission at 520 nm.
Both expression systems were shown to successfully express the applied vector. There is a clear enhancement of light emission observable at 520nm for both mixes in comparison to the respective negative control. The expression of GFP using the DiaMIX seems to be slightly better than by using the commercial kit. Still, one has to mention the actual purpose of the commercial kit to express linear templates. However, as the basis protocol for our mix was optimized for circular templates, we used a circular vector for the experiment.
Furthermore, as already shown above, we successfully verified the expression of GFP with our cell-free mix in an iRIf measurement.
To obtain these final results, a lot of optimization and testing had to be done. If you want to know more about how we established the DiaMIX for the DiaCHIP, you can find detailed information about the most important experiments on the cell-free expression results page.

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Explore the DiaCHIP

For establishing our device, we optimized all steps from the immobilization of DNA on the silicone of the flow-chamber to the specific binding of the target proteins on the glass slide. This way, we generated protein arrays we could use to detect different antibodies in human and rabbit serum with the iRIf detection method.
Click on the images below to explore our experiments from expression to detection!

Assembling the DiaCHIP

DNA Immobilization

Cell-free Expression

Binding on Surface

Diagnosis of Antigens

Specific Detection of Multiple Binding Events

Detection of Salmonella Single Chain Antibodies

Imaging Reflectometric Interference

Click on one of the images to get a further insight on how we build our DiaCHIP

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-  transition: all 0.4s ease-in-out;
-        border-radius: 15px;
-}
-.cool_image_blink:hover {
+/* ========== Remove Explore the DiaCHIP on small devices =================*/
-  /*box-shadow:
-    inset 0 0 0 110px #0051A2/*rgba(200,95,66, 0.4)*/,
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-}
-.cool_image_blink:hover .cool_content {
+@media screen and (max-width: 600px){
-  opacity: 1;
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+        display: none;
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+    }
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-}
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-.element3 {
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+    }
-     background-position: center;
 }
+/* ========== END: Remove Explore the DiaCHIP on small devices =================*/
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+  color: #C5162F;
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+  font-size: 40px;
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+  line-height: 45px;
 }
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-     background-image: url("https://static.igem.org/mediawiki/2015/5/56/Freiburg_tetanus_home2.png");
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-.mw-content-ltr ul {
+  overflow: hidden;
-    padding: 0;
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+#anti_tetanus_results, #device_results, #agfp_results, #specific_surfaces,#own_mix{
-    line-height: 1em;
+  display: block;
-}*/
+  overflow: hidden;
-.content_box {
-    box-shadow: none;
-    padding: 2% 4%;
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-/* =============== END: Resetting General CSS ==================== */
+*/
+</style>
-/* ============== Animate boxes on start ===========================*/
+<script type="text/javascript">
-/* from https://daneden.github.io/animate.css/ */
+// JQUERY for Details-Boxes
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-  from {
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+</script>
-    opacity: 1;
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-@keyframes zoomIn {
+<div class="kommentar">
-  from {
+mehr Formulierungen wie: we demonstrated..., we (successfully) tested, we achieved...
-    opacity: 0;
+</div>
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+   <div class="result_box">
-   from {
+     <div class="results_headline">Essential Results</div>
-     opacity: 0;
+     <p>
-     -webkit-transform: scale3d(.3, .3, .3);
+      Our aim was to develop a novel diagnostic tool enabling fast, simultaneous and label-free detection of antibodies in human blood sera. We successfully generated our self-established <a href="#own_mix">cell-free expression system</a> based on an <i>E. coli</i> lysate, which revealed an expression efficiency comparable to a commercial kit. To immobilize the expressed proteins, we established a <a href="#specific_surfaces">surface</a> specifically binding our target proteins.
-    transform: scale3d(.3, .3, .3);
+      This system was used to produce a protein array that can be flushed with a serum sample. The binding of antibodies is detected in a label-free manner by an interferometric method called iRIf (imaging reflectometric interference) in real-time. Moreover, we <a href="#device_results">rebuilt the detection device</a> in a simplified and cost-efficient manner and thus, made it available for future iGEM Teams.
-   }
+      Using the DiaCHIP, we did not only show the presence of antibodies in distinct solutions but also verified them in complex samples as human blood serum.
+    </p>
+   </div>
-% {
+   <div class="result_box" id="anti-tetanus_results">
-     -webkit-transform: scale3d(1.1,1.1,1.1);
+     <div class="results_headline headline_left">Detection of Anti-Tetanus Antibodies in Human Blood Serum</div>
-     transform: scale3d(1.1,1.1,1.1);
+    <div class="image_box right">
-  }
+        <div class="thumb2 trien" style="width:250px">
+          <div class="thumbinner">
+            <img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png"></img>
+            <div class="thumbcaption">
+            Video 1: Detection of anti-tetanus antibodies in human serum.
+            </div>
+          </div>
+        </div>
+     </div>
+    <p>
+      To test the DiaCHIP under real-life conditions, we analyzed the blood serum of a vaccinated person for the presence of anti-tetanus antibodies. We compared blood samples from before and after vaccination and could directly detect its effect. To capture the antibodies, the <a href="http://parts.igem.org/Part:BBa_K1621003">corresponding antigen</a> was expressed in <i>E. coli</i>, purified by His-tag affinity purification and spotted on a specific Ni-NTA surface. By comparing the spotted pattern with the localization of signal as detected by iRIf, the system's specificity even for complex samples was shown.
+      <a class="details_box_trigger" href="#details_box-1">▼ Detailed Description.</a>
+    </p>
+    <div id="details_box-1" class="details_boxes">
-% {
+     <div class="image_box left">
-     opacity: 1;
+        <div class="thumb2 trien" style="width:250px"><div class="thumbinner">
-  }
+            <img src="https://static.igem.org/mediawiki/2015/5/5b/Freiburg_tetanus_westernblot_protpur.png" style="width:230px"></img>
-}
+            <div class="thumbcaption">
+            <strong>Figure 1: Western Blot analysis of purified tetanus antigen.</strong>
+            </div>
+        </div>
+        </div>
+    </div>
-.zoomIn {
+    <div class="image_box right">
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-  animation-name: zoomIn;
+            <img src="https://static.igem.org/mediawiki/2015/b/b9/Freiburg_tetanus%28-%29_quotion_picture.png" style="width:230px"></img>
-}
+            <div class="thumbcaption">
+            <strong>Figure 2: Quotient picture of iRIf measurement of human serum sample taken before vaccination.</strong>
+            </div>
+        </div>
+        </div>
+    </div>
-@-webkit-keyframes zoomOut {
+     <p>
-  from {
+      The <a href=”http://parts.igem.org/Part:BBa_K1621003”>antigen</a> used for detection of anti-tetanus antibodies is derived from the tetanus neurotoxin and commonly used for <i>in vitro</i> testing. For preparation of the DiaCHIP, this antigen had to be overexpressed in <i>Escherichia coli</i> and purified from the whole cell lysate. To verify successful purification of the antigen Western Blot analysis was performed (figure 1). The usage of an antibody targeting the N-terminal His-tag revealed a protein with a molecular weight of about 50 kDa. This correlates with the expected molecular weight of the antigen <sup><a class="fn_top" href="#fn__1" id="fnt__1" name="fnt__1">1)</a></sup>. </br>
-     opacity: 1;
+    </p>
-  }
-% {
+     <p>
-     opacity: 0;
+      Before we performed an iRIf measurement with the blood of the freshly vaccinated person we checked if we could detect an interaction of antibodies present in the blood with our purified tetanus antigen by an ELISA.
-    -webkit-transform: scale3d(.3, .3, .3);
+      Therefore, we spotted tetanus antigen and bBSA of the same concentration (12.5&nbsp;µg/mL) and twice PBS as negative controls. The tetanus antigen and one PBS well were incubated with 5&nbsp;mg/mL BSA, blood serum and HRP-labeled anti-human for 1&nbsp;h respectively. The bBSA and the other PBS well were incubated with HRP-labeled Streptavidine (0.5&nbsp;µg/mL) for 1 &nbsp;h (positive control). After each step the wells were washed with PBS. To visualize the binding, Tetramethylbenzidine was added and the absorption at 650&nbsp;nm over time was measured in a plate reader. The graph in figure 3 shows that the signal for tetanus is even higher than the positive control, which illustrates the successful binding of antibodies from the blood to tetanus antigens.
-    transform: scale3d(.3, .3, .3);
+    </p>
-  }
-  to {
-    opacity: 0;
-  }
-}
-@keyframes zoomOut {
+        <div class="image_box left">
-  from {
+            <div class="thumb2 trien" style="width:250px">
-    opacity: 1;
+              <div class="thumbinner">
-  }
+                  <img src="https://static.igem.org/mediawiki/2015/f/f0/Freiburg_labjournal-surchem-20150902_elisa.png" style="width:230px"></img>
+                  <div class="thumbcaption">
+                  <strong>Figure 3: ELISA for the tetanus antigen with freshly vaccinated blood. The graph shows the absorption over time for TMB. The red line shows the binding of anti-tetanus antibodies out of the blood serum to purified tetanus antigen and the blue line shows the binding of HRP-Streptavidine to bBSA. The grey lines show the corresponding negative controls.</strong>
+                  </div>
+              </div>
+            </div>
+        </div>
-% {
+     <p>
-     opacity: 0;
+        The purified antigen was immobilized on a Ni-NTA surface that specifically binds His-tagged proteins. Look<a href=”https://2015.igem.org/Team:Freiburg/Results/Binding”> here</a> to read more about the establishment of this surface. As a positive control, self-purified His-tagged GFP was immobilized at another spot. The reliable detection of anti-GFP was shown before (link). No binding event should be detected at the negative control spot that was covered with <strong>bBSA/mCherry (??)</strong>.
-    -webkit-transform: scale3d(.3, .3, .3);
+    </p>
-    transform: scale3d(.3, .3, .3);
-  }
-  to {
+        <div class="image_box right">
-    opacity: 0;
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-  }
+                <img src="https://static.igem.org/mediawiki/2015/2/26/Freiburg_tetanus%28%2B%29_quotion_picture.png" style="width:230px"></img>
-}
+                <div class="thumbcaption">
+                <strong>Figure 4: Quotient picture of iRIf measurement of human serum sample taken <b>three????</b> weeks after vaccination.</strong>
+                </div>
+            </div>
+            </div>
+        </div>
-.zoomOut {
+        <div class="image_box left">
-  -webkit-animation-name: zoomOut;
+            <div class="thumb2 trien" style="width:250px">
-  animation-name: zoomOut;
+              <div class="thumbinner">
-}
+                <img src="https://static.igem.org/mediawiki/2015/1/16/Freiburg_tetanus_binding_curve.png" style="width:230px">
+              </img>
+                <div class="thumbcaption">
+                <strong>PLATZHALTER! Figure 5: Binding curve of the tetanus measurements.</strong>
+                </div>
+            </div>
+            </div>
+        </div>
+    <p>
+        The human serum samples we obtained have been taken before and <strong>three?</strong> weeks after a patient received a vaccination boost against tetanus. Analysis of the samples was performed using the same array set-up. Figures 2 and 4 show quotient pictures representing changes in the thickness of the layer covering the slide while it was flushed with the respective samples. The negative control spots do not indicate any binding events, while the binding of anti-GFP to the positive control spot verifies that both experiments are reliable. The comparison of the antigen spots shows that we specifically detected anti-tetanus antibodies in human serum in response to vaccination. No binding event was detected by flushing the array with the sample assumed to be negative (figure 2), whereas a distinct spot is visible after flushing it with the positive sample (figure 4). Both measurements in real-time are shown in parallel in video 1.</br>
+        The increase in relative light intensity at distinct spots over the course of the experiment is visualized in figure 5 and can be correlated with the amount of antibodies binding to the respective antigen spot. This indicates that the DiaCHIP enables quantification of antibody titers in addition to detecting their presence.
+        <br>
+        Besides tetanus, other antigens of immunological relevance were taken into account. See all the results we obtained in terms of <a href="https://2015.igem.org/Team:Freiburg/Results/Diagnostics">diagnostics</a>.
+    </p>
-#box1,
+    <div class="footnotes">
-#box2,
+      <div class="fn"><sup><a class="fn_bot" href="#fnt__1" id="fn__1" name="fn__1">1)</a></sup>
-#box3,
+        <a class="urlextern" href="http://ac.els-cdn.com/S0171298510001567/1-s2.0-S0171298510001567-main.pdf?_tid=05754f9e-4f34-11e5-95a1-00000aacb360&amp;acdnat=1440952235_9b35c0a0389b3dbe95d48a4e8a583234" rel="nofollow" target="_Blank" title="http://ac.els-cdn.com/S0171298510001567/1-s2.0-S0171298510001567-main.pdf?_tid=05754f9e-4f34-11e5-95a1-00000aacb360&amp;acdnat=1440952235_9b35c0a0389b3dbe95d48a4e8a583234">Rui et al., 2011. Enhanced expression of soluble recombinant tetanus neurotoxin Hc in Escherichia coli as a tetanus vaccine candidate. Immunobiology.</a>
-#box4,
+      </div>
-#box5,
+    </div>
-#box6,
-#box7,
-#box8,
-#DiaCHIP_logo,
-#DiaCHIP_text {
-  opacity: 0;
-}
-@media screen and (max-width: 1199px) {
+    <a class="closeButton" href="#anti_tetanus_results">▲ Close Box</a>
+    </div>
+  </div>
-.topicboxes{
+   <div class="result_box" id="agfp_results">
-   display: block;
+    <div class="kommentar">
-  width: 230px;                       /* define size of elements */
+      hier noch rein bringen, dass ihr euren eigenen cell-free mix hergestellt habt und mit diesem auch GFP expremieren könnt und es im array erfolgreich nachweisen könnt
-  height: 200px;
+      <br>
-  margin: 20px;
+      Auch, dass euer Mix low-cost ist, und trotzdem auf dem Niveau (oder besser) eines kommerziellen kits liegt - Community gedanke etc.  (ps12092015)
-  font-size: 100%;
+    </div>
-  text-align: center;
-}
-.circle-container li {
+    <div class="results_headline">
-  display: inline-block;
+        Detection of Anti-GFP Antibodies Using Cell-Free Expressed GFP on Ni-NTA
-}
+    </div>
-.circle-container{
-  display: flex;
-  flex-wrap: wrap;
-  justify-content: space-around;
-  margin-bottom: 2em;
-}
-.cool_container{
+    <div class="image_box left">
-  display: block;
+      <div class="thumbinner"style="width:250px;">
-  padding-top: 0;
+        <meta http-equiv="X-UA-Compatible" content="IE=Edge"/>
-  margin: 0;
+        <video width="250" height="250" poster="https://static.igem.org/mediawiki/2015/9/93/Freiburg_20150819_NiNTA_cellfree_antigfp_preview.png" controls>
-}
+          <source src="https://static.igem.org/mediawiki/2015/4/4a/Freiburg_20150819_NiNTA_cellfree_antigfp_WEB.mp4" type="video/mp4">
+          Your browser does not support the HTML5 video tag.
-.cool_centerimage{
+        </video>
-  display: none;
+        <div class="thumbcaption">Video 2: Binding of anti-GFP antibodies to GFP-His on a Ni-NTA surface. Anti-GFP antibodies bind both to purified GFP-His and cell-free expressed GFP-His. Control spots consisting of cell-free expression mix without DNA template show no binding.
-}
+        </div>
+      </div>
+    </div>
-#cool_centerimage_small{
+    <p>
-  display: block;
+      Our self-made cell-free expression, the DiaMIX, in combination with specific glass surfaces allows the expression and purification of target proteins in a simple manner. The mix, after expressing His-tagged GFP, was spotted on a Ni-NTA coated glass slide. Even after several washing steps it could be shown that the proteins maintain their antibody binding properties. A GFP antibody solution was flushed over the slide and a specific binding could be detected in iRIf.
-  margin: 20px;
+      <a class="details_box_trigger" href="#details_box-2">▼ Detailed Description.</a>
-  font-weight: 500;
+    </p>
-}
-.circle-container a {
+    <div id="details_box-2" class="details_boxes">
-  border-radius: 8px;
+    <p>
-  color: #FFF;
+      We could successfully show that the cell-free expression of GFP-His results in functional GFP proteins that are bound by anti-GFP antibodies during an iRIf measurent.
-  background-color: #0051A2;
+      The DNA coding for GFP fused to a 10x His-tag was added to the <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/Cellfree_Expression#diamix" title="DiaMIX">DiaMIX</a> and expression was performed for 2 h. The DiaMIX containing cell-free expressed GFP-His was then spotted on an iRIf slide with a Ni-NTA surface by hand (figure 1).
-  padding: 1px 10px;
+      The spots were incubated on the slide over night.
-  font-size: 100%;
+    </p>
-  border: 5px solid #0051A2;
-}
-}
+    <div class="image_box left">
+      <div class="thumb2 trien" style="width:250px">
+        <div class="thumbinner">
+        <a href="https://static.igem.org/mediawiki/2015/5/5f/Freiburg_files-20150903_ni-nta_surface.png" class="lightbox_trigger">
+          <img src="https://static.igem.org/mediawiki/2015/5/5f/Freiburg_files-20150903_ni-nta_surface.png" style="width:300px">
+        </a>
+        </img>
+        <div class="thumbcaption">
+          <strong>Figure 1: Specific Ni-NTA surface on iRIf glass slide.</strong>
+        </div>
+        </div>
+      </div>
+    </div>
-</style>
+    <p>
+      This specific surface allows binding of the expressed GFP-His via the His-tag while all the other proteins present in the DiaMIX are not bound to the surface.
+      This step was followed by the <a href="https://2015.igem.org/Team:Freiburg/Protocols/iRIf_Measurement">blocking and washing protocol</a> to prevent further unspecific binding.
+      The iRIf slide was then flushed with a anti-GFP antibody solution, to analyze the binding to the cell-free expressed GFP-His. As a positive control we spotted a purified GFP-His onto the surface as well as DiaMIX that did not contain any DNA as negative control (figure 2).
+    </p>
-<script type="text/javascript">
+    <div class="image_box right">
+      <div class="thumb2 trien" style="width:250px">
-// perform the animation
+        <div class="thumbinner">
+          <img src="" style="width:230px"></img>
+          <div class="thumbcaption">
+            <strong>Figure 2: The two spots on the right are the cell-free expressed GFP-His proteins spotted onto the Ni-NTA surface. The spot on the left shows the positive control, GFP-His expressed in <i>E. coli</i>, purified and spotted onto the Ni-NTA surface.</strong>
+          </div>
+        </div>
+      </div>
+    </div>
-$(document).ready(function() {
+     <p>
-     $('#DiaCHIP_logo').delay(1000).queue(function (next) {$(this).css({"animation-name": "zoomIn", "animation-duration": "1s"}).css({"opacity":"1"}); next(); });
+      The measurement shows a high signal for the cell-free expressed GFP-His and the positive control as can also be seen in the respective binding curve shwown in figure 3.
-    $('#box1').delay(2000).queue(function (next) {$(this).css({"animation-name": "zoomIn-Pics", "animation-duration": "1s"}).css({"opacity":"1"}); next(); });
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-});
-</script>
-<div id="cool_centerimage_small">
-    <!--<img src="https://static.igem.org/mediawiki/2015/9/91/Freiburg_homepage_Dia_chip.png" width="250px">-->
-    <p>Modern everyday life is fast...<br>...too fast for spending unnecessary time at the doctor’s and waiting weeks for your test results.<br> Not to mention spending lots of money on numerous serological tests for your diagnosis. Wouldn't life be easier if a single test was sufficient?
-<br>
-This is the reason why we thought about a universally accessible, cost and time efficient diagnostic device and developed the DiaCHIP!
      </p>
-</div>
+    <div class="image_box left">
-<div class="cool_container">
+      <div class="thumb2 trien" style="width:410px">
+         <div class="thumbinner">
-    <ul class='circle-container'>
+           <a class="lightbox_trigger" href="https://static.igem.org/mediawiki/2015/6/6d/Freiburg_2015_freiburg_cellfex_gfp_on_ni-nta_binding_curve.png">  <img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/6/6d/Freiburg_2015_freiburg_cellfex_gfp_on_ni-nta_binding_curve.png" width="400"/>
-        <li><!--6-->
-         <div id="box1" class="topicboxes">
-           <div class="cool_header"><a href='https://2015.igem.org/Team:Freiburg/Project/Diagnostics'>Diagnostics today</a></div>
-          <div class="cool_image_blink element1">
-          <div class="cool_content cool_text_blink">
-            <a href='https://2015.igem.org/Team:Freiburg/Project/Diagnostics' title="Diagnostics today" style="color:#666">Nowadays, medical diagnoses are mainly based on huge amounts of time-consuming and cost intensive serological tests. Read more about the issues we tackled!
            </a>
-           </div>
+           <div class="thumbcaption">
+            <div class="magnify">
+              <a class="internal" href="/igem2015/lib/exe/detail.php?id=results_overview&amp;media=2015_freiburg_cellfex_gfp_on_ni-nta_binding_curve.png" title="vergrößern">
+                <img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/>
+              </a>
+            </div>
+            <strong>Figure 3: Binding curve of anti-GFP binding to cell-free expressed GFP-His on a Ni-NTA surface.</strong> Cell-free expressed GFP-His was spotted onto a specific Ni-NTA surface and flushed with anti-GFP. The binding curve indicated a binding event at the respective spot.
            </div>
          </div>
-        </li>
+      </div>
+    </div>
-        <li><!--7-->
+    <p>
-        <div id="box2" class="topicboxes">
+      Find out more about the <a href="https://2015.igem.org/Team:Freiburg/Results#assembly_pic">preparation</a> of the DiaCHIP by producing a protein microarray from a DNA template using cell-free expression.
-          <div class="cool_header"><a href='https://2015.igem.org/Team:Freiburg/Project/Overview'>The DiaCHIP</a></div>
+      <a class="closeButton" href="#agfp_results">▲ Close Box</a>
-          <div class="cool_image_blink element2">
+    </p>
-          <div class="cool_content cool_text_blink">
+  </div>
-             <a href="https://2015.igem.org/Team:Freiburg/Project/Overview" title="The DiaCHIP" style="color:#666">Simultaneous screening for hundreds of diseases within a few hours? <br> Find out about the DiaCHIP's potential to enlighten future diagnostics!
-             </a>
+  <div class="result_box" id="device_results">
-          </div>
-          </div>
+    <div class="results_headline headline_left">
-        </div>
+         Building Our Low-Cost DiaCHIP Measuring Device
-         </li>
+    </div>
-        <li><!--8-->
+    <div class="image_box right">
-         <div id="box3" class="topicboxes">
+         <div class="thumb2 trien" style="width:250px">
-           <div class="cool_header"><a href='https://2015.igem.org/Team:Freiburg/Project/System'>Our System</a></div>
+           <div class="thumbinner">
-          <div class="cool_image_blink element3">
+              <a href="https://static.igem.org/mediawiki/2015/b/b7/Freiburg_Own_Device_Foto.jpg">
-          <div class="cool_content cool_text_blink">
+              <img src="https://static.igem.org/mediawiki/2015/2/2e/Freiburg_Own_Device_Foto_preview.jpg"></img>
-              <a href='https://2015.igem.org/Team:Freiburg/Project/System' title="Our system" style="color:#666">Generating a protein array on demand via cell-free expression and detecting antigen-antibody interactions label-free and in real-time: The principle behind the DiaCHIP!
                </a>
-          </div>
+              <div class="thumbcaption">
+                  Figure 2: Functional and low-cost variant of the measuring device.
+              </div>
            </div>
          </div>
-        </li>
+    </div>
-        <li><!--1-->
+    <p>
-            <div id="box4" class="topicboxes">
+      Commercial systems using the iRIf technology (imaging reflectomretric interference) are mostly bulky and expensive machines even though the components they are based on are rather simple. So we decided on building our own device consisting of not much more than two lenses, a LED and a camera. With this system we were able to reliably detect the binding of anti-GFP to GFP, thus confirming a detection sensitivity in the range of protein-protein interactions. To enable future iGEM Teams to profit from this device as we did, we provide all plans and parts necessary to rebuild it on this website for everyonbe to download and use.
-              <div class="cool_header"><a href='https://2015.igem.org/Team:Freiburg/Results/Own_Device'>Our Device</a></div>
+      <a class="details_box_trigger" href="#details_box-3">▼ Detailed Description.</a>
-              <div class="cool_image_blink element4">
+    </p>
-              <div class="cool_content cool_text_blink">
-              <a href='https://2015.igem.org/Team:Freiburg/Results/Own_Device' title="Our Device" style="color:#666">We built our simplified and affordable detection device. Check out the step by step instructions!
-              </a>
-              </div>
-             </div>
-           </div>
-        </li>
-        <li><!--2-->
+    <div id="details_box-3" class="details_boxes">
-          <div id="box5" class="topicboxes">
+      <div class="image_box left">
-            <div class="cool_header"><a href='https://2015.igem.org/Team:Freiburg/Project/pOP-vector'>Improved iGEM Backbone</a></div>
+        <div class="thumbinner"style="width:324px;">
-            <div class="cool_image_blink element5">
+          <meta http-equiv="X-UA-Compatible" content="IE=Edge"/>
-            <div class="cool_content cool_text_blink">
+          <video width="324" height="300" poster="https://static.igem.org/mediawiki/2015/8/80/Freiburg_iRIf_measurement_device_preview.jpg" controls>
-            <a href='https://2015.igem.org/Team:Freiburg/Project/pOP-vector' title="pOP-vector" style="color:#666">We provided an improved backbone for protein overexpression meeting all the iGEM standards. <br> See how we expanded the iGEM Registry!
+          <source src="https://static.igem.org/mediawiki/2015/7/77/Freiburg_20150911_DEVICE_GFP_RABBIT.mp4" type="video/mp4">
-            </a>
+          Your browser does not support the video tag.
-            </div>
+          </video>
-            </div>
+          <div class="thumbcaption">Video 1: Demonstration of the capability of our iRIf device at measuring antibody-antigen interaction. GFP was spotted on the left side, rabbit proteins (anti-HCV antibodies from rabbit) on the right.
           </div>
-        </li>
+        </div>
+      </div>
-        <li><!--3-->
+      <p>
-          <div id="box6" class="topicboxes">
+        To test our device we aimed at reproducing measurements that we were previously able to perform in our commercial device. For this reason we focused on using two antibodies: anti-GFP and polyclonal anti-rabbit. GFP and rabbit derived anti-HCV (Hepatitis C Virus) antibodies were used as antigens in this experiment. Note that the anti-HCV antibodies only served as binding partners for the anti-rabbit antibodies, since no HCV proteins were used in this experiment.
-            <div class="cool_header"><a href='https://2015.igem.org/Team:Freiburg/Practices'>Human Practice</a></div>
+        The antigens were spotted onto an iRIf slide whose binding layer consisted of an APTES/PDITC surface. The spots on the slide were produced by pipetting 3.5 µl [1 mg/ml] rabbit-anti-HCV protein and 3.5 µl [1 mg/ml] GFP onto the slide and incubated overnight. After incubation the slide was blocked for 30 min in BSA solution.
-            <div class="cool_image_blink element6">
-            <div class="cool_content cool_text_blink">
-            <a href='https://2015.igem.org/Team:Freiburg/Practices' title="Human Practice" style="color:#666">What do people think about the DiaCHIP and systems based on synthetic biology? <br> Check out the results of our survey and discussions!
-            </a>
-            </div>
-            </div>
-          </div>
-        </li>
-         <li><!--4-->
+         A Canon 50D camera was used to record the measurement and was set to take one picture every 5 seconds. The exposure time was set in order for the pixels in the image to be approx. 80% of maximum light saturation before the solution was flushed onto the chip.
-        <div id="box7" class="topicboxes">
-          <div class="cool_header"><a href='https://2015.igem.org/Team:Freiburg/Results/Modeling'>Modeling</a></div>
+        The antibody solutions were pipetted into the flow-chamber without the use of any microfluidic device. Instead, a syringe was loaded with 660 µl [5 µg/ml] anti-GFP antibody solution (diluted in PBS) and connected to the input pipe of our device. The content of the syringe was then slowly released via this pipe into the chamber of the device by gently dispensing the solution from the syringe. When the whole volume ran over the chip, the process was repeated with 660 µl [5 µg/ml] anti-rabbit antibody solution in the same way. The injection of both solutions was performed during approximately 45 minutes.
-          <div class="cool_image_blink element7">
+      </p>
-          <div class="cool_content cool_text_blink">
-          <a href='https://2015.igem.org/Team:Freiburg/Results/Modeling' title="Modeling" style="color:#666">Modeling the generation of proteins during cell-free expression as well as their diffusion in our system helped us obtaining better results. Get more information here.
+      <div class="image_box left">
-          </a>
+          <div class="thumb2 trien" style="width:250px">
+              <div class="thumbinner">
+                  <a href="https://static.igem.org/mediawiki/2015/2/24/Freiburg_20150908_Device_03_GFP_Rabbit_LUT_0985_1003.png">
+                      <img src="https://static.igem.org/mediawiki/2015/7/71/Freiburg_20150908_Device_03_GFP_Rabbit_LUT_0985_1003_preview.png"></img>
+                  </a>
+              </div>
+          <strong>Figure 3: Quotient picture of the same measurement with favourable light conditions.</strong>
            </div>
-          </div>
+      </div>
-        </div>
-        </li>
-        <li><!--5-->
+      <p>
-          <div id="box8" class="topicboxes">
+        Video 1 shows the results of the measurement. Both, binding of anti-GFP and anti-rabbit to the corresponding antigen spots, could be observed. Due to the fact that the experiment was performed on our demonstration device which was built with a transparent casing, fluctuations in surrounding light had a strong, detrimental influence on the measurement quality. To minimize the influence of the unstable surrounding light, the resulting pictures had to be averaged over 10 pictures each. This in turn lead to a more stable light situation, however the signal strengh dropped as a consequence. Figure XY shows a quotient picture of the measurement where the light situation was temporarily appropriate. The problem of surrounding light scattering into the device can of course be overcome using a non-transparent casing.
-            <div class="cool_header"><a href='https://2015.igem.org/Team:Freiburg/Results'>Blood Serum Analysis</a></div>
+      </p>
-            <div class="cool_image_blink element8">
-             <div class="cool_content cool_text_blink">
+      <a class="closeButton" href="#device_results">▲ Close Box</a>
-                 <a href='https://2015.igem.org/Team:Freiburg/Results' title="Results" style="color:#666">We successfully detected tetanus antibodies in human blood serum and measured binding of GFP antibodies to cell-free expressed GFP. <br> Visit our results page!
+    </div>
+  </div>
+  <div class="result_box" id="specific_surfaces">
+    <div class="results_headline">
+        Establishing a Highly Specific Ni-NTA Surface
+    </div>
+    <div class="image_box left">
+        <div class="thumb2 trien" style="width:310px">
+             <div class="thumbinner">
+                 <a href="https://static.igem.org/mediawiki/2015/0/0b/Freiburg_GFP_CF_NiNTA.png" class="lightbox_trigger">
+                <img src="https://static.igem.org/mediawiki/2015/0/0b/Freiburg_GFP_CF_NiNTA.png" width="300px">
                  </a>
+                <div class="thumbcaption">
+                    <p>Figure 1: Cell-free expressed GFP-His spotted on a Ni-NTA slide. Cell-free expression was performed for 2 hours at 37°C. The positive control was purified GFP-His, the negative control was a sample of cell-free expression mix not containing DNA. </p>
+                </div>
              </div>
-            </div>
+        </div>
+    </div>
+    <p>
+      Since there is a variety of proteins present in the cell-free mix a specific surface on the future protein array is essential. That is why we established our Ni-NTA surface for the immobilization of the antigens on the chip. All expression constructs contain a His-tag fused to the coding sequence, resulting in antigens that can bind specifically to our Ni-NTA surface. Compared to an unspecific surface (PDITC) we could show that this Ni-NTA surface allows efficient binding of the target protein and prevents unspecific binding of other proteins that are present in the cell-free mix.
+      <a class="details_box_trigger" href="#details_box-4">▼ Detailed description.</a>
+    </p>
+    <div id="details_box-4" class="details_boxes">
+      <div class="image_box right">
+          <div class="thumb2 trien" style="width:310px">
+          <div class="thumbinner">
+              <a href="https://static.igem.org/mediawiki/2015/1/1b/Freiburg_GFP_CF_PDITC.png" class="lightbox_trigger">
+              <img src="https://static.igem.org/mediawiki/2015/1/1b/Freiburg_GFP_CF_PDITC.png" width="300px">
+              <div class="thumbcaption">
+              </a>
+                  <p>Figure 2: Cell-free expressed GFP-His spotted on a PDITC slide. Cell-free expression was performed for 2 hours at 37°C. The positive control was purified GFP-His, the negative control was a sample of cell-free expression mix not containing DNA. </p>
+              </div>
            </div>
-        </li>
+          </div>
-    </ul>
+      </div>
-    <div class="cool_centerimage">
+      <p>
-         <div id="DiaCHIP_logo">
+         For cell-free protein expression lots of different proteins, like RNA-polymerases, ribosomes and other <i>E. coli</i> proteins, are essential. So during the process of cell-free expression all these proteins are, besides the target protein, also present in the microfluidic chamber. To have a sufficient amount of target protein immobilized on the chip we established a specific surface chemistry on the glass slide. After testing several <a href="https://2015.igem.org/Team:Freiburg/Results/Surface">tag systems</a> we established a Ni-NTA surface because it worked best for us. Using a Ni-NTA covered glass slide (figure 1) we could increase the amount of bound cell-free expressed GFP-His compared to an unspecific surface (figure 2). In both experiments ____2.5 µg?!?____ of self purified GFP-His was used as positive control. The cell-free expression mix without DNA is supposed to result in no target protein and served as negative control. The cell-free reactions were performed for 2 hours at 37°C. The samples were pipetted by hand onto the surfaces and incubated on the slide overnight. In the iRIf device, the optical detection method we used, the slides were blocked with BSA and then flushed with anti-GFP antibodies. An increase in light intensity on the slide where the spotted protein was located represents a binding event. Due to less unspecific binding of proteins to the surface, the interaction of anti-GFP antibodies with cell-free expressed GFP-His is higher on the Ni-NTA surface.
-          <img  src="https://static.igem.org/mediawiki/2015/9/91/Freiburg_homepage_Dia_chip.png" width="250px">
+      </p>
-        </div>
+      <a class="closeButton" href="#specific_surfaces">▲ Close Box</a>
-        <div id="DiaCHIP_text">
-          <p style="margin-bottom:4%">Modern everyday life is fast...<br>...too fast for spending unnecessary time at the doctor’s and waiting weeks for your test results. <br> Not to mention spending lots of money on numerous serological tests for your diagnosis. Wouldn't life be easier if a single test was sufficient?
-          <br>
-          This is the reason why we thought about a universally accessible, cost and time efficient diagnostic device and developed the DiaCHIP!
-          </p>
-          <div class="intro_button3 subsubmenu"><a href="https://2015.igem.org/Team:Freiburg/Project/Overview" title="Introduction">Introduction</a></div>
-        </div>
      </div>
+  </div>
+  <div class="result_box" id="own_mix">
+    <div class="results_headline">
+        Producing Our Cell-free Expression Mix
+    </div>
+    <p>
+        The mechanism we are using to copy a DNA array template into a protein array on demand is based on cell-free expression. During our project we successfully established our cell-free expression system, the DiaMIX, from scratch, starting with an <i>E. coli</i> lysate. With this mix we succeeded in expressing correctly folded GFP and luciferase. Comparison of the DiaMIX with a commercial cell-free expression kit revealed the great potential of our system!
+        <a class="details_box_trigger" href="#details_box-5">▼ Detailed Description.</a>
+    </p>
+    <div id="details_box-5" class="details_boxes">
+      <p>
+        Our DiaMIX was prepared based on a protocol of <div class="todo_box">??? (Ref.)</div>.
+        It is a very complex system containing many different enzymes and chemicals for special purposes. An overview of cell-free expression systems and its components can be found on the<a class=”wikilink1” href=https://2015.igem.org/Team:Freiburg/Project/Cellfree_Expression>methodology page</a>. <br/>
+        To investigate the efficiency of our self-prepared expression system we performed an experiment comparing it to a commercially available kit. In order to obtain reliable results we used the exact same vector, containing a sequence encoding GFP. Like this we were able to trace the amount of expressed GFP using a plate reader over a period of two hours. As a negative control, both mixes were treated equally in a second sample but did not contain any DNA. <br/>
+        The evaluation is shown in figure 1 and is based on triplicates. <b>The data was normalized to the mean value of the measurement of air. The uncertainty was calculated using the standard deviation. ÜBERARBEITEN JE NACH GRAPH</b> <br/>
+        picture; Figure 1: <b>Cell-free expression of GFP using the DiaMIX and a commercial kit.</b> The reaction was performed in a volume of 50 µl each and monitored every minute at 37°C over a period of two hours. Excitation at 480 nm, emission at 520 nm. <br/>
+        Both expression systems were shown to successfully express the applied vector. There is a clear enhancement of light emission observable at 520nm for both mixes in comparison to the respective negative control. The expression of GFP using the DiaMIX seems to be slightly better than by using the commercial kit. Still, one has to mention the actual purpose of the commercial kit to express linear templates. However, as the basis protocol for our mix was optimized for circular templates, we used a circular vector for the experiment. <br/>
+        Furthermore, as already shown above, we successfully verified the expression of GFP with our cell-free mix in an <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Results#agfp_results" title="agfp_results">iRIf measurement</a>. <br/>
+        To obtain these final results, a lot of optimization and testing had to be done. If you want to know more about how we established the DiaMIX for the DiaCHIP, you can find detailed information about the most important experiments on the <a class=”wikilink1” href="https://2015.igem.org/Team:Freiburg/Results/Cellfree">cell-free expression results page</a>. <br/>
+      </p>
+      <a class="closeButton" href="#own_mix">▲ Close Box</a>
+    </div>
+  </div>
 </div>
+<div class="content_box" id="explore_DiaCHIP">
+  <h1>Explore the DiaCHIP</h1>
+    <p>
+      For establishing our device, we optimized all steps from the immobilization of DNA on the silicone of the flow-chamber to the specific binding of the target proteins on the glass slide. This way, we generated protein arrays we could use to detect different antibodies in human and rabbit serum with the iRIf detection method. <br> Click on the images below to explore our experiments from expression to detection!
+    </p>
+  <h2 id="assembly_pic">Assembling the DiaCHIP</h1>
+  <div class="flexbox">
+      <div id="Assembling" class="link_image">
+          <a href="https://2015.igem.org/Team:Freiburg/Results/Immobilization" id="immobilization" class="circle"></a>
+          <div class="hovertext" id="immobilization_label">DNA Immobilization</div>
+          <a href="https://2015.igem.org/Team:Freiburg/Results/Cellfree" id="cellfree" class="circle"></a>
+          <div class="hovertext" id="cellfree_label">Cell-free Expression</div>
+          <a href="https://2015.igem.org/Team:Freiburg/Results/Surface" id="binding" class="circle"></a>
+          <div class="hovertext" id="binding_label">Binding on Surface</div>
+          <img src="https://static.igem.org/mediawiki/2015/f/f9/Freiburg_150912_Resultsoverviewgrafik_quer_DNAonPDMS_CF_Surface_RJ.png" width="100%">
+      </div>
+  </div>
-<div class="content_box">
+  <h2>Diagnosis of Antigens</h2>
+  <div class="flexbox">
+      <div id="Diagnosis" class="link_image">
+          <a href="https://2015.igem.org/Team:Freiburg/Results/Diagnostics" id="multbind" class="circle"></a>
+          <div class="hovertext" id="multbind_label">Specific Detection of Multiple Binding Events</div>
+          <a href="https://2015.igem.org/Team:Freiburg/Results/Diagnostics" id="salmonella" class="circle"></a>
+          <div class="hovertext" id="salmonella_label">Detection of Salmonella Single Chain Antibodies</div>
+          <a href="https://2015.igem.org/Team:Freiburg/Results/Diagnostics" id="irif" class="circle"></a>
+          <div class="hovertext" id="irif_label">Imaging Reflectometric Interference</div>
+          <img src="https://static.igem.org/mediawiki/2015/5/55/Freiburg_150912_Resultsoverviewgrafik_Tet_Sal_GFP_RJ.png" width="100%">
+      </div>
+  </div>
-<h1 class="sectionedit1">DiaCHIP - Enlightening Diagnostics</h1>
+  <p> Click on one of the images to get a further insight on how we build our DiaCHIP </p>
-<div class="level1">
-<p>
-In modern medicine, fast detection and differentiation of diseases is a crucial and fundamental task. Typical ELISA-based assays are time-consuming and expensive. We propose an advanced procedure for the simultaneous detection of various diseases in a fast and inexpensive manner, the DiaCHIP. Our approach is based on the interaction of antibodies with their respective antigens. Different antigens are immobilized on a protein array generated by cell-free protein expression, using the corresponding DNA array as a template. Placed in a microfluidic chamber, the protein array is incubated with a patient’s blood sample. The interaction between an antibody in the sample and the corresponding immobilized antigen results in a local change of the optical thickness of the surface. This change can be detected using a label-free and real-time measurement technology called iRIf (imaging Reflectometric Interference). Offering simultaneous screening for several diseases, our DiaCHIP has strong potential to improve future diagnostics.
-</p>
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Difference between revisions of "Team:Freiburg/Testpage"

Revision as of 23:13, 16 September 2015

Explore the DiaCHIP

Assembling the DiaCHIP

Diagnosis of Antigens