Difference between revisions of "Team:Freiburg/Project/System"
Line 2: | Line 2: | ||
{{Freiburg/Menubar}} | {{Freiburg/Menubar}} | ||
− | {{Freiburg/ | + | {{Team:Freiburg/wiki_content_start_bubble}} |
<html> | <html> | ||
<style> | <style> | ||
Line 117: | Line 117: | ||
</style> | </style> | ||
+ | <script type="text/javascript"> | ||
+ | //===================BEGIN:Amazing Bubble Sidebar========================== | ||
+ | |||
+ | $(document).ready(function(){ | ||
+ | // CHANGE THE FOLLOWING ATTRIBUTES // | ||
+ | var href_text1='https://2015.igem.org/Team:Freiburg/Project/Overview', | ||
+ | // Text2 needs no href as it is the actual page // | ||
+ | img_url='https://static.igem.org/mediawiki/2015/7/76/Freiburg_icon_project_white_03.png', | ||
+ | href_text3='https://2015.igem.org/Team:Freiburg/Design', | ||
+ | // Text1 needs no text as it is a pic // | ||
+ | text2='Project Overview'; | ||
+ | // Text3 needs no text as its always 'next' // | ||
+ | // HOLD ON CHANGING THINGS --JABBERWOCK // | ||
+ | |||
+ | $('#bubble1').attr('href',href_text1); | ||
+ | $('#bubble1_img').attr('src', img_url); | ||
+ | $('#bubble3').attr('href',href_text3); | ||
+ | |||
+ | $('#bubble2').text(text2); | ||
+ | }); | ||
+ | |||
+ | //===================END:Amazing Bubble Sidebar========================== | ||
+ | </script> | ||
<script type="text/javascript"> | <script type="text/javascript"> | ||
Revision as of 21:40, 18 September 2015
![](https://static.igem.org/mediawiki/2015/8/85/Freiburg_wiki_background_2_parted_left.png)
![](https://static.igem.org/mediawiki/2015/f/f4/Freiburg_wiki_background_2_parted_right.png)
The DiaCHIP : Overview
Our DiaCHIP is a novel diagnostic device that offers the possibility for broadband screening for hundreds of diseases simultaneously. It is made up of an antigen array in a microfluidic chamber and can detect diseases via binding of corresponding antibodies. The antigens are fused to a tag and are produced with a cell-free expression mix, using a DNA array as template. The expressed antigens are arranged in an array structure, using a specific surface to stick them to a glass slide. Binding of antibodies can be detected with an optical method, thus providing the possibility of a label-free and real-time analysis.
The Backgrounds of the DiaCHIP
Step 1: Basic Setup of the DiaCHIP
The aim of our DiaCHIP is to screen simultaneously for hundreds of different infectious diseases. We based our system on the detection of antibodies specifically interacting with antigens derived from viruses and bacteria (figure 1). If you get in contact with one of these pathogens your immune system produces antibodies. These bind specifically to their corresponding antigens. The antigens are produced by using a DNA array as template. The binding of antibodies to their corresponding antigens can be detected with a label-free detection method. Our approach is based on two components: a silicone slide where DNA coding for distinct antigenic peptides is immobilized and a glass slide with a specific surface to capture the expressed antigens. Both are about the size of a microscopy slide and form a microfluidic chamber. By adding blood of a patient, antibodies that might be present in the sample due to a disease bind to the corresponding antigens.
Step 2: Cell-Free Expressed Proteins
To enable the production of a protein array consisting of multiple antigens on demand, their expression is mediated by cell-free expression from a template DNA array. This expression system based on bacterial lysate makes the need for genetically engineered organisms to produce every single antigen redundant. The protein array is generated by flushing our cell-free expression mix through the microfluidic setup. Expressing the antigens from the DNA template, the protein array is adaptable to individual requirements exhibiting the same pattern for both arrays. Our system is made up of two slides enabling the antigens to be immobilized on the opposite side of the DNA template inside the microfluidic chamber (figure 2).
Step 3: A Specific Surface is Catching the Expressed Protein
After cell-free expression not only our desired antigens are present within the chamber, but also all other components of the cell-free mix including ribosomes, polymerases and amino acids (figure 3). All these components would bind unspecifically to an activated glass slide, thereby obstructing the binding of the antigens. We designed our DNA constructs in a way that each antigen can easily be fused to specific tags. This enables targeted protein purification on a specific surface. Testing different tag systems, we identified the Ni-NTA-His-tag system to be working best for our purposes. A basic protocol for this specific surface was optimized by ourselves to reduce unspecific binding.
Step 4: The Measurement of Binding Events
![](https://static.igem.org/mediawiki/2015/9/9a/Freiburg_iRiF_overview_RJ_preview.jpg)
Figure 4: Optical detection method. The detection system mainly consists of a camera and an LED and is called iRIf (imaging Reflectometric Interference). Antigen-Antibody interactions can be detected label-free and in real-time. An optical output of such binding events is generated by a minimal change in the thickness of the layer on the slide right at the corresponding antigen spot.
After assembly of the DiaCHIP, a patient’s serum sample can be flushed over the protein array using the same microfluidic system. The binding of antibodies to the corresponding surface causes a minimal change in the thickness of the layer on the slide just at the corresponding antigen spot. This binding can be detected label-free and in real-time using a novel technique called iRIf (imaging Reflectometric Interference) without the need for further labeling. Its main components are a camera, an LED and two lenses. See how we reconstructed the system in a low-budget device.
Step 5: Changing Perspectives - Off to our Results
When illustrating the basic principle of the DiaCHIP, we mainly looked at it from the side. Now it is time to explore our results and see what we actually achieved. Therefore, it is important to have in mind that you are observing the chip from the camera's position, so basically from the top (figure 5). This perspective remains the same in all the iRIf measurements we are showing in the results section.
After months of engineering and optimizing the different components of the DiaCHIP, we are proud to present our results. We reached the highlight of our project with the successful detection of antibodies in our own blood!