Latest revision as of 06:56, 20 November 2015

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Main 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 that specifically binds our target proteins. Combined in a microfluidic chamber we produced a protein array that can be flushed with a serum sample Label-free detection of antigen-antibody binding can be done in real-time by the interferometric method called iRIf (imaging Reflectometric Interference). Finally we built our own detection device in a simplified and cost-efficient manner and thus, making 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

Figure 1: Quotient picture of GFP and tetanus antigen spots. Human blood serum of an immunized person against tetanus followed by an anti-human antibody was flushed over the chip. A & B: Tetanus spotsshow binding of anti-tetanus and anti-human antibody, respectively; C & D: GFP spots used as negative control.

To test the DiaCHIP under real-life conditions, we analysed the blood serum of a person that has been immunized against tetanus for the presence of anti-tetanus antibodies. First we confirmed the binding of antibodies present in the blood to our purified tetanus antigen by ELISA. Then we spotted the antigen and GFP, which both were expressed in E.coli and purified by His-tag affinity purification, on a PDITC surface. The much stronger binding of blood components to the tetanus antigen spots compared to GFP spots indicates specific interactions. By confirming that the bound blood components are human antibodies with anti-human, we successfully demonstrated that we are able to detect antibody binding even in complex samples. ▼ Detailed Description.

Figure 2: Western Blot of His-tagged C. tetani antigen with anti His-tag HRP Conjugate. The soluble fraction of the overexpressed protein was used for this Western Blot. The expected molecular weight is 50 kDa. Anti- His Tag HRP conjugated antibody was used in a 1:1000 dilution in blocking buffer.

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 E. coli and purified from whole cell lysate. To verify the successful purification of the antigen, Western Blot analysis was performed (figure 2). The usage of an antibody against 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⁾.

Figure 3: ELISA for the tetanus antigen with blood samples taken after the immunization. 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-Streptavidin to bBSA. The grey lines show the corresponding negative controls.

We performed an ELISA to investigate, whether the blood samples we obtained contain antibodies that bind specific to our purified tetanus antigen. Therefore, we spotted tetanus antigen and bBSA of the same concentration (12.5 µg/mL) and 2 times 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 Streptavidin (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 was measured in a plate reader over time (figure 3). The absorption signal for tetanus is even higher than the positive control, which illustrates the successful binding of antibodies from the blood to tetanus antigen.

Figure 4: Binding curves for the detection of the binding of anti-tetanus antibodies from blood serum via iRIf shown in figure 1.

After the validation of the antigen antibody binding for tetanus by ELISA we wanted to see if we can detect this interaction also with iRIf. Therefore we first expressed our Clostridium tetani antigen and GFP in E.coli. The purified His-tagged proteins then were spotted on a PDITC surface. The slide with the immobilized proteins was flushed with human blood serum, and anti-human antibody. Strong binding was detected for the tetanus antigen spots, while the GFP spots only exhibited minimal binding (see figure 1A-D). This indicates that components contained in the blood serum bind specifically to the tetanus antigen. The subsequent binding of anti-human antibody to the tetanus antigen spots (figure 4) confirms the presents and binding of human antibodies to the spotted tetanus antigens. These results successfully demonstrate that it is possible to detect the binding of antibodies from blood serum to antigens immobilized, on a surface.

Figure 5: Quotient picture of iRIf measurement. Line A was flushed with serum taken before vaccination against tetanus. Line B was flushed with serum taken 2 weeks after vaccination. Spots from left to right show: GFP, neg. control, Tetanus antigen.

With an additional experiment we wanted to see if we can differentiate the vaccination status of patients with our system. Therefore we used blood serum samples, taken before and two weeks after immunization against tetanus. A slide with immobilized tetanus antigen and GFP was prepared (figure 5). It was consecutively flushed with both blood sera. The blood serum taken after vaccination exhibits a much more intense binding to the tetanus spot than the serum taken before binding. Unfortunately we did not had enough anti-human antibody to proof further the presents of bound human antibodies from blood serum.

Figure 6: Quotient pictures of slides flushed with blood serum taken before and after vaccination. (A) Schematic pattern of proteins immobilized on a glass slide. (B) Quotient picture of iRIf measurement flushed with anti-GFP and blood serum taken before vaccination . (C) Quotient picture of iRIf measurement in which the slide was flushed with anti-GFP and blood serum five weeks after vaccination.

While the outcome of this first experiment looked promising for our future application to determine the vaccination status of patients, further experimentation did not confirm these initial results. We were still able to detect binding of both blood sera to the tetanus antigen, but no difference in binding intensity was visible. To test the assumption that the used blood was not fresh enough and therefore lost some of its binding capabilities a fresh blood sample five weeks after vaccination was taken for the experiment (See figure 6B and C). To determine whether the identification of the vaccination status is possible with our system further experimentation needs to be done.

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

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

Our self-made cell-free expression mix, the DiaMIX, in combination with specific glass surfaces allows the expression and immobilization of target proteins in a distinct manner. The DiaMIX, after expressing His- tagged GFP, was spotted onto a Ni-NTA coated glass slide side-by-side with conventionally purified His-GFP. Even after several washing steps it could be shown that the proteins maintain stable on the surface. This is shown via an iRIf measurement: Antibodies against GFP are flushed over the slide to detect GFP that is bound to the slide. The specific binding of anti-GFP antibodies to the control spot (purified GFP) and the cellfree expressed GFP confirms the success of cellfree GFP expression and its immobilization on the surface.
▼ Detailed Description.

We could successfully show that the cell-free expression of His-GFP results in functional GFP proteins that are bound by anti-GFP antibody during an iRIf measurement. 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 His-GFP was then spotted on an iRIf slide with a Ni-NTA surface by hand (figure 1). The spots were incubated on the slide overnight.

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

This specific surface allows binding of the expressed His-GFP 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 an anti-GFP antibody solution, to analyze the binding to the cell-free expressed His-GFP. As a positive control we spotted a purified His-GFP onto the surface as well as DiaMIX that did not contain any DNA as negative control figure 2).
Find out more about the preparation of the DiaCHIP by producing a protein microarray from a DNA template using cell-free expression.

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

Figure 2: Measurement of cell-free expressed GFP on Ni- NTA surface. The presence of GFP is detected with GFP antibodies within an iRIf measurement The spot on the right is the cell-free expressed His-GFP protein spotted onto the Ni-NTA surface. The spot on the left shows the positive control, His-GFP expressed in E. coli, purified and spotted onto the Ni-NTA surface. The negative control represents the DiaMIX without DNA. A clear iRIf signal is detected for the positive control and the cellfree expressed GFP.

Figure 3: Binding curve of anti-GFP binding to cell-free expressed His-GFP 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.

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Building Our Low-Cost DiaCHIP Measuring Device

Figure 1: 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, an 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 everyone 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 side.

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) antibody were used as antigens in this experiment. Note that the anti-HCV antibody 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 with 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 was run 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 favorable light conditions.

The quotient pictures show all intensity shifts that occurred during the measurement. Two left spots: GFP spots at which an intensity shift occurred during anti-GFP flush. Right spot: anti-HCV antibody (derived from rabbit), at which an intensity shift occurred during the flush with anti-Rabbit.

Figure 4: Comparison of two quotient pictures of our DiaCHIP with the professional setup. Left: Professional device; Right: Our device. Both quotient pictures show the binding events that occurred during an iRIf measurement with anti-GFP at the GFP spots.

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 DiaCHIP 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 led to a more stable light situation, however the signal strength dropped as a consequence. Figure 3 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. An evaluation of how well our device performs in comparison with the professional device was not performed, mainly due to time constraints. However our results hint that we get comparable results for strong binding such as GFP/anti-GFP, as can be seen in figure 4.

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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 are 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 (figure 1). ▼ Detailed description.

Figure 2: Cell-free expressed His-GFP spotted on a PDITC slide. Cell-free expression was performed for 2 hours at 37°C. The positive control was purified His-GFP, 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. 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. Using a Ni-NTA covered glass slide (figure 1) we could increase the amount of bound cell-free expressed His-GFP compared to an unspecific surface (figure 2). In both experiments 1.5 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 in a seperate tube prior to pipetting the samples by hand onto the surfaces and incubated on the slide overnight. Before measurment in the iRIf device, the optical detection method we use, the slides were blocked with BSA and then flushed with anti-GFP antibodies. An increase in light intensity 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 His-GFP is higher on the Ni-NTA surface.

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Producing Our Cell-free Expression Mix

Figure 1: Cell-free expression of our DiaMIX in comparison to a commercial kit over time. Measurements were performed every minute for 120 minutes (Excitation: 480 nm; Emission: 520nm). Reactions were performed in triplicates and normalized to air. Negative controls were subtracted as background. Corridors represent the standard deviation. A propagation of error and smoothening of the values was conducted.

A key feature of our DiaCHIP is the on-demand antigen production via cell-free expression. Thereby, a fixed DNA template is copied into our desired antigen, which in turn can bind to our specific surface. Our expression mix called DiaMIX was established from scratch. It is based on an E. coli lysate. In a direct comparison with a commercial product our mix showed comparable expression rates, indicating its performance capability (figure 1). ▼ Detailed Description.

Our DiaMIX is a complex system consisting of several individually adjusted components that allow us to make use of the cells own production system, without the tedious drawbacks of normal cell-based protein expression. As a main component of the DiaMIX the E. coli lysate contains the necessary transcription and translation apperatus. The lysate is complemented by additives needed for energy regeneration, buffering, and generation of the protein. An overview of cell-free expression systems and its components can be found on our project pages.
As ultimate evaluation, we compared the DiaMIX to a commercial kit as seen in figure 1.

Both expression systems successfully expressed the applied vector.
Furthermore, the cell-free expression of GFP with our cell-free mix could be verified in an iRIf measurement.
To obtain these final results, a lot of optimization and testing had to be done. Want to know more about how we established the DiaMIX? 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 can 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 in rabbit serum

Detection of anti- Salmonella

Detection of Multiple Binding Events

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

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+        var diag_right_circ = document.getElementById("salmonella");
+        diag_left_circ.style.left = assembly_width*0.145 + "px";
+        diag_left_circ.style.top = assembly_height*0.661 + "px";
+        diag_mid_circ.style.left = assembly_width*0.544 + "px";
+        diag_mid_circ.style.top = assembly_height*0.509 + "px";
+        diag_right_circ.style.left = assembly_width*0.811 + "px";
+        diag_right_circ.style.top = assembly_height*0.712 + "px";
+        $(".circle").css("width", ""+assembly_width/9);
+        $(".circle").css("height", ""+assembly_width/9);
+        $(".circle").css("border-radius", "100px");
+        $('.hovertext').css({"font-size": 120*assembly_width/900 + "%"});
+      }
+</script>
 <div class="content_box">
-<div id="anti_tetanus_results">
+  <div class="result_box">
-	<div class="floatbox right">
+    <h1 style="text-align:right">Main Results</h1>
-		<h2>
+    <p>
-			We successfully diagnosed anti-Tetanus antibodies in human blood serum
+      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> that specifically binds our target proteins.
-		</h2>
+Combined in a microfluidic chamber we produced a protein array that can be flushed with a serum sample Label-free detection of antigen-antibody binding can be done in real-time by the interferometric method called iRIf (imaging Reflectometric Interference).  Finally we <a href="#device_results_anchor">built our own detection device</a> in a simplified and cost-efficient manner and thus, making it available for future iGEM Teams.
-		<p>
+      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.
-			Check out this video showing how the antibodies in the serum bind our tetanus antigens on the DiaChip
+    </p>
-		</p>
+  </div>
-		<div class="image_box right">
-			<img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png" width="100%"></img>
-		</div>
-	</div>
+<span id="anti_tetanus_results_anchor" class="anchor"></span>
+  <div class="result_box" id="anti-tetanus_results">
+    <h1 class="left">Detection of Anti-Tetanus Antibodies in Human Blood Serum</h1>
-	<div class="floatbox left">
+<div class="image_box right">
-		<p>
+ <div class="thumb2 trien" style="width:250px">
-		We took a persons blood before and after a tetanus vaccination and screened for a tetanus infection.
+                	<div class="thumbinner">
-		An immunized person has antibodies against tetanus antigens - we bound spots of our purified tetanus antigens onto the specific surface of our chip. We then incubated the DiaChip with 33 µl of the blood serum for half an hour and measured the results.
+                    	<a href="https://static.igem.org/mediawiki/2015/c/c3/Freiburg_20150822_Quot_GFP_Tet_ABCD.png" class="lightbox_trigger">
+                  	  <img src="https://static.igem.org/mediawiki/2015/c/c3/Freiburg_20150822_Quot_GFP_Tet_ABCD.png" width="250px">
+                    		<div class="thumbcaption">
+                   		 </a>
+                       		<p><strong>Figure 1: Quotient picture of GFP and tetanus antigen spots. Human blood serum of an immunized person against tetanus followed by an anti-human antibody was flushed over the chip. </strong> A & B: Tetanus spotsshow binding of anti-tetanus and anti-human antibody, respectively; C & D: GFP spots used as negative control.</p>
+                   		 </div>
+               	 </div>
+</div>
+</div>
-		Take a look at the video and see for yourself how good our DiaChip performed.
+    <p>
+To test the DiaCHIP under real-life conditions, we analysed the blood serum of a person that has been immunized against tetanus for the presence of anti-tetanus antibodies. First we confirmed the binding of antibodies present in the blood to our purified tetanus antigen by ELISA. Then we spotted the antigen and GFP, which both were expressed in E.coli and purified by His-tag affinity purification, on a PDITC surface. The much stronger binding of blood components to the tetanus antigen spots compared to GFP spots indicates specific interactions. By confirming that the bound blood components are human antibodies with anti-human, we successfully demonstrated that we are able to detect antibody binding even in complex samples.
+      <a class="details_box_trigger" href="#details_box-1">▼ Detailed Description.</a>
+    </p>
+  </div>
-		And remember: that this isn’t an ELISA, we don’t need no expensive secondary Antibodies!
+  <div id="details_box-1" class="details_boxes">
-		</p>
-	</div>
+<div class="image_box left">
+ <div class="thumb2 trien" style="width:300px">
+                	<div class="thumbinner">
+                    	<a href="https://static.igem.org/mediawiki/2015/5/5b/Freiburg_tetanus_westernblot_protpur.png" class="lightbox_trigger">
+                  	  <img src="https://static.igem.org/mediawiki/2015/5/5b/Freiburg_tetanus_westernblot_protpur.png" width="250px">  </a>
+                    		<div class="thumbcaption">
+                       		<p><strong>Figure 2: Western Blot of His-tagged C. tetani antigen with anti His-tag HRP Conjugate.</strong> The soluble fraction of the overexpressed protein was used for this Western Blot. The expected molecular weight is 50 kDa. Anti- His Tag HRP conjugated antibody was used in a 1:1000 dilution in blocking buffer.</p>
+                   		 </div>
+               	 </div>
+</div>
 </div>
+    <p>
+     The <a href="http://parts.igem.org/Part:BBa_K1621003"target="_blank">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>E. coli</i> and purified from whole cell lysate. To verify the successful purification of the antigen, Western Blot analysis was performed (figure 2). The usage of an antibody against 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>
+    </p>
-<div id="agfp_results">
+<div class="image_box right">
-	<div class="floatbox right">
+ <div class="thumb2 trien" style="width:250px">
-		<h2>
+                	<div class="thumbinner">
-			We measured the binding of anti-GFP to cell-free expressed GFP in blood serum
+                    	<a href="https://static.igem.org/mediawiki/2015/f/f0/Freiburg_labjournal-surchem-20150902_elisa.png" class="lightbox_trigger">
-		</h2>
+                  	  <img src="https://static.igem.org/mediawiki/2015/f/f0/Freiburg_labjournal-surchem-20150902_elisa.png" width="250px">
-		<p>
+                    		<div class="thumbcaption">
-		We expressed GFP with our own cell-free expression system and bound it to the surface of our DiaChip. We than ran some rabbit blood serum over the chip which we had spiked with anti-GFP antibodies. The results show that our cell-free proteins can be measured even in a complex solution such as blood serum. </p>
+                   		 </a>
-	</div>
+                       		<p><strong>Figure 3: ELISA for the tetanus antigen with blood samples taken after the immunization.</strong> 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-Streptavidin to bBSA. The grey lines show the corresponding negative controls.</p>
-	<div class="floatbox left">
+                   		 </div>
-		<div class="imagebox left">
+               	 </div>
-			<img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png" width="100%"></img>
+</div>
-		</div>
-	</div>
 </div>
-<div id="device_results">
+    <p>We performed an ELISA to investigate, whether the blood samples we obtained contain antibodies that bind specific to our purified tetanus antigen. Therefore, we spotted tetanus antigen and bBSA of the same concentration (12.5&nbsp;µg/mL) and 2 times 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 Streptavidin (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 was measured in a plate reader over time (figure 3). The absorption signal for tetanus is even higher than the positive control, which illustrates the successful binding of antibodies from the blood to tetanus antigen.
-	<h1>
+    </p>
-		We built our very own, low-cost DiaChip device - and can measure with it!
-	</h1>
+<div class="image_box left">
-	<div class="flexbox">
+ <div class="thumb2 trien" style="width:300px">
-		<img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png" width="80%"></img>
+                	<div class="thumbinner">
-	</div>
+                    	<a href="https://static.igem.org/mediawiki/2015/2/26/Freiburg_Tetanusserum_Bindekurve.png" class="lightbox_trigger">
-	<p>
+                  	  <img src="https://static.igem.org/mediawiki/2015/2/26/Freiburg_Tetanusserum_Bindekurve.png">
-	A green LED, two lenses and a regular SLR Camera is basically all you need to build your own iRIf device.
+                    		<div class="thumbcaption">
-	</p>
+                   		 </a>
+                       		<p><strong>Figure 4:  Binding curves for the detection of the binding of anti-tetanus antibodies from blood serum via iRIf shown in figure 1.</strong> </p>
+                   		 </div>
+               	 </div>
 </div>
 </div>
-<div class="content_box">
+    <p>
+        After the validation of the antigen antibody binding for tetanus by ELISA we wanted to see if we can detect this interaction also with iRIf. Therefore we first expressed our <i>Clostridium tetani</i> antigen and GFP in <i>E.coli</i>. The purified <a href="https://2015.igem.org/Team:Freiburg/Project/DNA_Engineering#detailed_cloning_anchor">His-tagged</a> proteins then were spotted on a PDITC surface. The slide with the immobilized proteins was flushed with human blood serum, and anti-human antibody. Strong binding was detected for the tetanus antigen spots, while the GFP spots only exhibited minimal binding (see figure 1A-D). This indicates that components contained in the blood serum bind specifically to the tetanus antigen. The subsequent binding of anti-human antibody to the tetanus antigen spots (figure 4) confirms the presents and binding of human antibodies to the spotted tetanus antigens. These results successfully demonstrate that it is possible to detect the binding of antibodies from blood serum to antigens immobilized, on a surface.
+    </p>
-<div class="flexbox">
+<div class="image_box right">
-<a href="https://2015.igem.org/Team:Freiburg/Results/Assembling">
+ <div class="thumb2 trien" style="width:250px">
-<img src="https://static.igem.org/mediawiki/2015/0/0f/Freiburg_homepage_chip_blood.png" width="100%">
+                	<div class="thumbinner">
-</a>
+                    	<a href="https://static.igem.org/mediawiki/2015/5/57/Freiburg_tetanus_quotion_picture_59a.png" class="lightbox_trigger">
+                  	  <img src="https://static.igem.org/mediawiki/2015/5/57/Freiburg_tetanus_quotion_picture_59a.png" width="250px">
+                    		<div class="thumbcaption">
+                   		 </a>
+                       		<p><strong>Figure 5: Quotient picture of iRIf measurement.</strong> Line A was flushed with serum taken before vaccination against tetanus. Line B was flushed with serum taken 2 weeks after vaccination. Spots from left to right show: GFP, neg. control, Tetanus antigen. </p>
+                   		 </div>
+               	 </div>
+</div>
+</div>
-<a href="https://2015.igem.org/Team:Freiburg/Results/Diagnostics">
+  <p>
-<img src="https://static.igem.org/mediawiki/2015/0/0f/Freiburg_homepage_chip_blood.png" width="100%">
+       With an additional experiment we wanted to see if we can differentiate the vaccination status of patients with our system. Therefore we used blood serum samples, taken before and two weeks after immunization against tetanus. A slide with immobilized tetanus antigen and GFP was prepared (figure 5). It was consecutively flushed with both blood sera.  The blood serum taken after vaccination exhibits a much more intense binding to the tetanus spot than the serum taken before binding. Unfortunately we did not had enough anti-human antibody to proof further the presents of bound human antibodies from blood serum.</br>
-</a>
+<div class="image_box left">
+ <div class="thumb2 trien" style="width:300px">
+                	<div class="thumbinner">
+                    <a href="https://static.igem.org/mediawiki/2015/9/94/Freiburg_Tetanus_iRIf_measurement_quotient_pics_14.09.15_incl_illustration.png" class="lightbox_trigger">
+                  	  <img src="https://static.igem.org/mediawiki/2015/9/94/Freiburg_Tetanus_iRIf_measurement_quotient_pics_14.09.15_incl_illustration.png" width="250px">
+                    		<div class="thumbcaption">
+                   		 </a>
+                       		<p><strong>Figure 6: Quotient pictures of slides flushed with blood serum taken before and after vaccination.</strong> (A) Schematic pattern of proteins immobilized on a glass slide. (B) Quotient picture of iRIf measurement flushed with anti-GFP and blood serum taken before vaccination . (C) Quotient picture of iRIf measurement in which the slide was flushed with anti-GFP and blood serum five weeks after vaccination. </p>
+                   		 </div>
+               	 </div>
+</div>
 </div>
-<p> Click on one of the images to get further insight how we build up our DiaCHIP </p>
+<p>While the outcome of this first experiment looked promising for our future application to determine the vaccination status of patients, further experimentation did not confirm these initial results. We were still able to detect binding of both blood sera to the tetanus antigen, but no difference in binding intensity was visible. To test the assumption that the used blood was not fresh enough and therefore lost some of its binding capabilities a fresh blood sample five weeks after vaccination was taken for the experiment (See figure 6B and C).
+To determine whether the identification of the vaccination status is possible with our system further experimentation needs to be done.
+</p>
+<div>
+        <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>
+    <div class="footnotes">
+      <div class="fn"><sup><a class="fn_bot" href="#fnt__1" id="fn__1" name="fn__1">1)</a></sup>
+        <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>
+      </div>
+    </div>
+    <a class="closeButton" href="#anti_tetanus_results">▲ Close Box</a>
+  </div>
 </div>
+<span id="agfp_results_anchor" class="anchor"></span>
+  <div class="result_box" id="agfp_results">
+<p></p>
+     <h1 class="left">
+        Detection of Anti-GFP Antibodies using Cell-Free Expressed GFP on
+Ni-NTA
+    </h1>
+    <div class="image_box left">
+      <div class="thumbinner"style="width:300px;">
+        <meta http-equiv="X-UA-Compatible" content="IE=Edge"/>
+        <video width="300" height="300"
+poster="https://static.igem.org/mediawiki/2015/9/93/Freiburg_20150819_NiNTA_cellfr
+ee_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.
+        </video>
+        <div class="thumbcaption"><strong>Video 2: Binding of anti-GFP
+antibodies to His-GFP on a Ni-NTA surface.</strong> Anti-GFP antibody binds
+to purified His-GFP and cell-free expressed HIs-GFP. Control spots
+consisting of cell-free expression mix without DNA template show no binding.
+        </div>
+      </div>
+    </div>
+    <p>
+      Our self-made cell-free expression mix, the DiaMIX, in combination
+with specific glass surfaces allows the expression and immobilization of
+target proteins in a distinct manner. The DiaMIX, after expressing His-
+tagged GFP, was spotted onto a Ni-NTA coated glass slide side-by-side with
+conventionally purified His-GFP. Even after several washing steps it could
+be shown that the proteins maintain stable on the surface. This is shown via
+an iRIf measurement: Antibodies against GFP are flushed over the slide to
+detect GFP that is bound to the slide. The specific binding of anti-GFP
+antibodies to the control spot (purified GFP) and the cellfree expressed GFP
+confirms the success of cellfree GFP expression and its immobilization on
+the surface.<br>
+      <a class="details_box_trigger" href="#details_box-2">▼ Detailed
+Description.</a>
+    </p>
+  </div>
+  <div id="details_box-2" class="details_boxes">
+    <p>
+      We could successfully show that the cell-free expression of His-GFP
+results in functional GFP proteins that are bound by anti-GFP antibody
+during an iRIf measurement.
+      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 His-GFP was then spotted on an iRIf slide
+with a Ni-NTA surface by hand (figure 1).
+      The spots were incubated on the slide overnight.
+    </p>
+    <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>
+    <p>
+      This specific surface allows binding of the expressed His-GFP 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/Spotting"target="_blank"> blocking and washing protocol</a> to prevent further unspecific binding.
+      The iRIf slide was then flushed with an anti-GFP antibody solution, to analyze the binding to the cell-free expressed His-GFP. As a positive control we spotted a purified His-GFP onto the surface as well as DiaMIX that did not contain any DNA as negative control figure 2).
+      <br>
+  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.
+</p>
+<p>
+The measurement shows a high signal for the cell-free expressed His-GFP and the positive control as can also be seen in the respective binding curve shown in figure 3.
+</p>
+     <div class="flexbox">
+      <div class="thumb2 trien" style="width:350px">
+        <div class="thumbinner">
+          <a
+href="https://static.igem.org/mediawiki/2015/7/7d/Freiburg_cellfreeNiNta3spots.png
+" class="lightbox_trigger">
+            <img
+src="https://static.igem.org/mediawiki/2015/7/7d/Freiburg_cellfreeNiNta3spots.png"
+style="width:300px">
+          </a>
+          <div class="thumbcaption">
+            <strong>Figure 2: Measurement of cell-free expressed GFP on Ni-
+NTA surface. The presence of GFP is detected with GFP antibodies within an
+iRIf measurement</strong> The spot on the right is the cell-free expressed
+His-GFP protein spotted onto the Ni-NTA surface. The spot on the left shows
+the positive control, His-GFP expressed in <i>E. coli</i>, purified and
+spotted onto the Ni-NTA surface. The negative control represents the DiaMIX
+without DNA. A clear iRIf signal is detected for the positive control and
+the cellfree expressed GFP.
+          </div>
+        </div>
+      </div>
+      <div class="thumb2 trien" style="width:410px">
+        <div class="thumbinner">
+          <a class="lightbox_trigger"
+href="https://static.igem.org/mediawiki/2015/0/07/Freiburg_cellfreeNiNtamain.png">
+            <img alt="" class="mediabox2"
+src="https://static.igem.org/mediawiki/2015/0/07/Freiburg_cellfreeNiNtamain.png"
+width="400">
+          </a>
+          <div class="thumbcaption">
+            <strong>Figure 3: Binding curve of anti-GFP binding to cell-free
+expressed His-GFP 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>
+      </div>
+    </div>
+    <p>
+      <a class="closeButton" href="#agfp_results">▲ Close Box</a>
+    </p>
+  </div>
+<span id="device_results_anchor" class="anchor"></span>
+  <div class="result_box" id="device_results">
+<p></p>
+    <h1 class="left">
+        Building Our Low-Cost DiaCHIP Measuring Device
+    </h1>
+    <div class="image_box right">
+        <div class="thumb2 trien" style="width:250px">
+          <div class="thumbinner">
+              <a
+href="https://static.igem.org/mediawiki/2015/b/b7/Freiburg_Own_Device_Foto.jpg">
+              <img
+src="https://static.igem.org/mediawiki/2015/2/2e/Freiburg_Own_Device_Foto_preview.
+jpg"></img>
+              </a>
+              <div class="thumbcaption">
+                  <strong>Figure 1: Functional and low-cost variant of the
+measuring device.</strong>
+              </div>
+          </div>
+        </div>
+    </div>
+    <p>
+      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, an 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 everyone to download and use.
+      <a class="details_box_trigger" href="#details_box-3">▼ Detailed
+Description.</a>
+    </p>
+  </div>
+  <div id="details_box-3" class="details_boxes">
+    <div class="image_box left">
+      <div class="thumbinner"style="width:324px;">
+        <meta http-equiv="X-UA-Compatible" content="IE=Edge"/>
+        <video width="324" height="300"
+poster="https://static.igem.org/mediawiki/2015/8/80/Freiburg_iRIf_measurement_devi
+ce_preview.jpg" controls>
+        <source
+src="https://static.igem.org/mediawiki/2015/7/77/Freiburg_20150911_DEVICE_GFP_RABB
+IT.mp4" type="video/mp4">
+        Your browser does not support the video tag.
+        </video>
+        <div class="thumbcaption"><strong>Video 1: Demonstration of the
+capability of our iRIf device at measuring antibody-antigen
+interaction.</strong> GFP was spotted on the left side, rabbit proteins
+(anti-HCV antibodies from rabbit) on the right side.
+        </div>
+      </div>
+    </div>
+    <p>
+      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) antibody were used as
+antigens in this experiment. Note that the anti-HCV antibody 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 with 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 was run 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.
+    </p>
+    <div class="image_box left">
+        <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_R
+abbit_LUT_0985_1003_preview.png"></img>
+                </a>
+                <div class="thumbcaption">
+                  <strong>Figure 3: Quotient picture of the same measurement
+with favorable light conditions.</strong> <p>
+The quotient pictures show all intensity shifts that occurred during the
+measurement. Two left spots: GFP spots at which an intensity shift occurred
+during anti-GFP flush. Right spot: anti-HCV antibody (derived from rabbit),
+at which an intensity shift occurred during the flush with anti-Rabbit.
+<p>
+                </div>
+            </div>
+        </div>
+    </div>
+    <div class="image_box right">
+        <div class="thumb2 trien" style="width:250px">
+            <div class="thumbinner">
+                <a
+href="https://static.igem.org/mediawiki/2015/3/38/Freiburg_comparison_professional
+_ourDevice.png">
+                    <img
+src="https://static.igem.org/mediawiki/2015/3/38/Freiburg_comparison_professional_
+ourDevice.png"></img>
+                </a>
+                <div class="thumbcaption">
+                  <strong>Figure 4: Comparison of two quotient pictures of
+our DiaCHIP with the professional setup.</strong> Left: Professional device;
+Right: Our device.
+Both quotient pictures show the binding events that occurred during an iRIf
+measurement with anti-GFP at the GFP spots.
+                </div>
+            </div>
+        </div>
+    </div>
+    <p>
+      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 DiaCHIP 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 led to a more stable
+light situation, however the signal strength dropped as a consequence.
+Figure 3 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. An evaluation of how well our device performs in comparison with the
+professional device was not performed, mainly due to time constraints.
+However our results hint that we get comparable results for strong binding
+such as GFP/anti-GFP, as can be seen in figure 4.
+    </p>
+    <a class="closeButton" href="#device_results">▲ Close Box</a>
+  </div>
+  <div class="result_box" id="specific_surfaces">
+<p></p>
+    <h1 class="left">
+        Establishing a Highly Specific Ni-NTA Surface
+    </h1>
+    <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/2015/6/65/Freiburg_GFP_CF_NiNTA_preview.png
+" width="300px">
+                </a>
+                <div class="thumbcaption">
+                    <p><strong>Figure 1: Cell-free expressed GFP-His spotted
+on a Ni-NTA slide.</strong> 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>
+    <p>
+      Since there are 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
+(figure 1).
+      <a class="details_box_trigger" href="#details_box-4">▼ Detailed
+description.</a>
+    </p>
+  </div>
+  <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><strong>Figure 2: Cell-free expressed His-GFP spotted on
+a PDITC slide.</strong> Cell-free expression was performed for 2 hours at
+°C. The positive control was purified His-GFP, the negative control was a
+sample of cell-free expression mix not containing DNA. </p>
+            </div>
+        </div>
+        </div>
+    </div>
+    <p>
+      For cell-free protein expression lots of different proteins, like
+RNA-polymerases, ribosomes and other <i>E. coli</i> proteins are essential.
+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. Using a Ni-NTA covered glass slide (figure 1)
+we could increase the amount of bound cell-free expressed His-GFP compared
+to an unspecific surface (figure 2). In both experiments 1.5 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
+in a seperate tube prior to pipetting the samples by hand onto the surfaces
+and incubated on the slide overnight. Before measurment in the iRIf device,
+the optical detection method we use, the slides were blocked with BSA and
+then flushed with anti-GFP antibodies. An increase in light intensity 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 His-GFP is higher on the Ni-NTA surface.
+    </p>
+    <a class="closeButton" href="#specific_surfaces">▲ Close Box</a>
+  </div>
+  <div class="result_box" id="own_mix">
+<p></p>
+    <h1 class="left">
+        Producing Our Cell-free Expression Mix
+    </h1>
+<div class="image_box right">
+        <div class="thumb2 trien" style="width:450px">
+          <div class="thumbinner">
+              <a
+href="https://static.igem.org/mediawiki/2015/e/e3/Freiburg_comparison_cellfree_mix
+es.png">
+              <img
+src="https://static.igem.org/mediawiki/2015/0/03/Freiburg_comparison_cellfree_mixe
+s_preview.png"></img>
+              </a>
+              <div class="thumbcaption">
+                  <strong> Figure 1: Cell-free expression of our DiaMIX in
+comparison to a commercial kit over time. </strong> Measurements were
+performed every minute for 120 minutes (Excitation: 480 nm; Emission:
+nm). Reactions were performed in triplicates and normalized to air.
+Negative controls were subtracted as background. Corridors represent the
+standard deviation. A propagation of error and smoothening of the values was
+conducted.
+              </div>
+          </div>
+        </div>
+    </div>
+   <p>
+        A key feature of our DiaCHIP is the on-demand antigen production via
+cell-free expression. Thereby, a fixed DNA template is copied into our
+desired antigen, which in turn can bind to our specific surface. Our
+expression mix called DiaMIX was established from scratch. It is based on an
+<i>E. coli</i> lysate. In a direct comparison with a commercial product our
+mix showed comparable expression rates, indicating its performance
+capability (figure 1).
+        <a class="details_box_trigger" href="#details_box-5">▼ Detailed
+Description.</a>
+    </p>
+  </div>
+  <div id="details_box-5" class="details_boxes">
+      <p>
+        Our DiaMIX is a complex system consisting of several individually
+adjusted components that allow us to make use of the cells own production
+system, without the tedious drawbacks of normal cell-based protein
+expression.
+As a main component of the DiaMIX the <i>E. coli</i> lysate contains the
+necessary transcription and translation apperatus. The lysate is
+complemented by additives needed for energy regeneration, buffering, and
+generation of the protein. An overview of cell-free expression systems and
+its components can be found on our <a class=”wikilink1”
+href=https://2015.igem.org/Team:Freiburg/Project/Cellfree_Expression>project
+pages</a>. <br/>
+        As ultimate evaluation, we compared the DiaMIX to a commercial kit
+as seen in figure 1. <br/>
+</p>
+<p>
+        Both expression systems successfully expressed the applied
+vector.<br/>
+        Furthermore, the cell-free expression of GFP with our cell-free mix
+could be verified 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. Want to know more about how we established the DiaMIX? 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>
+<span id="explore_DiaCHIP_anchor" class="anchor"></span>
+<div class="content_box results_page" id="explore_DiaCHIP">
+  <h1 style="text-align:center">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 can 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" class="left">Assembling the DiaCHIP</h2>
+  <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 id="assembly_image"
+src="https://static.igem.org/mediawiki/2015/f/f9/Freiburg_150912_Resultsoverviewgr
+afik_quer_DNAonPDMS_CF_Surface_RJ.png" width="100%">
+      </div>
+  </div>
+  <h2 class="left">Diagnosis of Antigens</h2>
+</script>
+  <div class="flexbox">
+      <div id="Diagnosis" class="link_image">
+          <a
+href="https://2015.igem.org/Team:Freiburg/Results/Diagnostics#agfp"
+id="multbind" class="circle"></a>
+          <div class="hovertext" id="multbind_label">Specific Detection in
+rabbit serum</div>
+         <a
+href="https://2015.igem.org/Team:Freiburg/Results/Diagnostics#salmonella"
+id="salmonella" class="circle"></a>
+          <div class="hovertext" id="salmonella_label">Detection of anti-
+Salmonella</div>
+          <a
+href="https://2015.igem.org/Team:Freiburg/Results/Diagnostics#multiple"
+id="irif" class="circle"></a>
+          <div class="hovertext" id="irif_label">Detection of Multiple Binding Events</div>
+          <img id="diagnosis_image"
+src="https://static.igem.org/mediawiki/2015/5/55/Freiburg_150912_Resultsoverviewgr
+afik_Tet_Sal_GFP_RJ.png" width="100%">
+      </div>
+  </div>
+  <p> Click on one of the images to get a further insight on how we build
+our DiaCHIP </p>
 </div>
-</html>
+</html>
 <!-- wiki content ends here -->
 {{Freiburg/wiki_content_end}}

Difference between revisions of "Team:Freiburg/Results"

Latest revision as of 06:56, 20 November 2015

Main Results

Detection of Anti-Tetanus Antibodies in Human Blood Serum

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

Building Our Low-Cost DiaCHIP Measuring Device

Establishing a Highly Specific Ni-NTA Surface

Producing Our Cell-free Expression Mix

Explore the DiaCHIP

Assembling the DiaCHIP

Diagnosis of Antigens