Difference between revisions of "Team:INSA-Lyon/Results"
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| − | + | <li><a href="#blood">Fluorescence in Blood</a></li> | |
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| − | + | <div class="col-md-3 col-md-offset-4"><h5 class="wow fadeInUp" data-wow-duration="0.5s" data-wow-delay="0.5s">RESULTS</h5></div> | |
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| − | + | <div class="title-area" id="resultssummary"> | |
| − | + | <h2 class="title">Early Results</h2> | |
| − | + | <span class="line"></span> | |
| − | + | ||
| − | + | <p> | |
| − | + | The most of our relevant results are described in the <a href="https://2016.igem.org/Team:INSA-Lyon/Proof">Proof of Concept</a> page. | |
| − | + | </p> | |
| − | + | <p> | |
| − | + | Here are some of our first experiment results that guided us through our development, and finally, our results with sexually transmitted infections vectors. | |
| − | + | </p> | |
| − | + | ||
| − | + | <div class="subtitle" id="earlyATP"> | |
| − | + | <h3 class="title">First study of the ATP-aptamer’s affinity in liquid</h3> | |
| − | + | <span class="line"></span> | |
| − | + | <p> | |
| − | + | In this experience, we used some modified aptamers. They were functionalized with a FITC, so they were fluorescent. | |
| − | + | Fluorescence was stopped by the reverse-complement of the aptamer, | |
| − | + | which was holding a quencher: a molecule that switches off the fluorescence by the | |
| − | + | Fluorescent Resonnance Energy Transfert (FRET) phenomenon. | |
| − | + | </p> | |
| − | + | <p> | |
| − | + | We studied how the ability of ATP to chase the quencher away. As the target (ATP) and the quencher are in competition to bind the aptamer, | |
| − | + | the emitted fluorescence is a good estimation of the proportion of aptamers that have linked an ATP. | |
| − | + | </p> | |
| − | + | <figure class="figure"> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/e/e6/T--INSA-Lyon--ATP_aptamer_affinity.png" class="img-responsive center-block img-rounded"> | |
| − | + | <figcaption class="figcaption"><strong>Fig 1.</strong></figcaption> | |
| − | + | </figure> | |
| − | + | <p> | |
| − | + | As can be seen here, there is a good response of the system to ATP. With a Kd of 330 µmol.L<sup>-1</sup> (target concentration able to be fixed by the aptamers). | |
| − | + | This is not particularly efficient. We think that the quenching oligo is disturbing the aptamer, lowering its sensitivity. | |
| − | + | </p> | |
| − | + | <p> | |
| − | + | The next step was to try this on paper. | |
| − | + | </p> | |
| − | + | </div> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | <div class="subtitle" id="blood"> | |
| − | + | <h3 class="title">Study of the impact of various solvents and blood proteins on the fluorescence emitted by fluorescein</h3> | |
| − | + | <span class="line"></span> | |
| − | + | <p> | |
| − | + | In this experience we wanted first to test what is the impact of non-isotonic solvents on the emitted fluorescence. </br> | |
| − | + | We obtained no significant differences of fluorescence emissions between assays when me mixed blood with PBS buffer or with NaCl.</br> | |
| − | + | Although, we tried to compare the fluorescence emissions between blood and plasma. The results were more interesting. | |
| − | + | </p> | |
| − | + | <p> | |
| − | + | Student statistical tests were performed for each concentration of fluorescein. It was concluded that blood filtration to remove especially red blood cells which could mask the | |
| − | + | fluorescence is proven to be very important: the fluorescence emitted is between 2 and 11 times larger in the case of serum compared to whole blood. | |
| − | + | </p> | |
| − | + | <figure class="figure"> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/1/16/T--INSA-Lyon--blood_vs_plasma.png" class="img-responsive center-block img-rounded"> | |
| − | + | <figcaption class="figcaption"><strong>Fig 2.</strong></figcaption> | |
| − | + | </figure> | |
| − | + | </div> | |
| − | + | ||
| − | + | </div> | |
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| − | + | ||
| − | + | <div class="title-area" id="filters"> | |
| − | + | <h3 class="title">Revelation with light filters</h3> | |
| − | + | <span class="line"></span> | |
| − | + | <p> | |
| − | + | As our device is meant to be used at home, we wanted a revelation method that does not imply the use of a UV lamp. | |
| − | + | Inspired by the work of the 2015 iGEM Bielefeld-CeBiTec team on sfGFP (another fluorescent molecule which emission wavelengths and absorption are | |
| − | + | relatively close to those of fluorescein), we decided to test whether a revelation with light filters on the flash and camera of our smartphone would work.</br> | |
| − | + | Images taken with the smartphone equipped with various combinations of filters revealed that the filter on the flash was necessary | |
| − | + | to foresee a fluorescence (see below). Moreover, adding a filter to the lens increases the detection sensitivity. | |
| − | + | Also, it turned out that the sample of fluorescein concentration was a very important factor. | |
| − | + | Above a certain threshold of the concentration, fluorescence is quenched. | |
| − | + | </p> | |
| − | + | ||
| − | + | <figure class="figure"> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/3/3f/T--INSA-Lyon--fluorescein_filters.png" class="img-responsive center-block img-rounded"> | |
| − | + | <figcaption class="figcaption"><strong>Fig 3.</strong></figcaption> | |
| − | + | </figure> | |
| − | + | <p> | |
| − | + | However, on a cellulose paper sheet the fluorescence detection turned out to be much less efficient, even in the optimal conditions that ensure a minimum of quenching. | |
| − | + | </p> | |
| − | + | </div> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | <div class="title-area" id="fluidics"> | |
| − | + | <h3 class="title">Paper strips design</h3> | |
| − | + | <span class="line"></span> | |
| − | + | <p> | |
| − | + | We designed paper strips that could handle our biological system. | |
| − | + | These paper strips contained a filtration pad in glass fiber, a nitrocellulose strip, and an adsorption pad in cellulose. | |
| − | + | </p> | |
| − | + | ||
| − | + | <figure class="figure"> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/8/81/T--INSA-Lyon--paper_design.png" class="img-responsive center-block img-rounded"> | |
| − | + | <figcaption class="figcaption"><strong>Fig 4.</strong></figcaption> | |
| − | + | </figure> | |
| − | + | <p> | |
| − | + | The filtration pad was here to prevent erythrocytes from migrating on the paper strip. It worked, after migration the nitrocellulose remained white and the results could be read. We also tested the microfluidics of this system once in our prototype, until we got good migration results. | |
| − | + | </p> | |
| − | + | <figure class="figure"> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/c/c7/T--INSA-Lyon--paper_fluidics.png" class="img-responsive center-block img-rounded"> | |
| − | + | <figcaption class="figcaption"><strong>Fig 5.</strong></figcaption> | |
| − | + | </figure> | |
| − | + | </div> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | <div class="title-area" id="stis"> | |
| − | + | <h2 class="title">Characterization of Aptamers for real STIs: HIV and HBV</h2> | |
| − | + | <span class="line"></span> | |
| − | + | <p> | |
| − | + | After an intensive work on the human thrombin, we tested HIV-1 Reverse Transcriptase (RT). | |
| − | + | This particular target would allow us to detect HIV infections at a very early stage, when this virus has just penetrated the host. | |
| − | + | At this stage antibodies are produced by the patient’s body yet, | |
| − | + | so conventional methods of detection are not valid. We did an EMSA with the most efficient HIV RT aptamer we could find. | |
| − | + | </p> | |
| − | + | ||
| − | + | <figure class="figure"> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/c/c3/T--INSA-Lyon--EMSA-HIV-RT.png" class="img-responsive center-block img-rounded"> | |
| − | + | <figcaption class="figcaption"><strong>Fig 6.</strong></figcaption> | |
| − | + | </figure> | |
| − | + | <p> | |
| − | + | The curve presents an inflexion point for <strong>[RT] = Kd = 490 nmol.L<sup>-1</sup></strong></br> | |
| − | + | The sensitivity is again quite good, with the same warnings than for thrombin. | |
| − | + | </p> | |
| − | + | <p> | |
| − | + | <em>Important remark</em>: We only have one aptamer to catch the Reverse-Transcriptase. Although our detection procedure requires <strong>two</strong> aptamers, | |
| − | + | and the sensitivity of the method will be limited by the worst Kd between the two aptamers. | |
| − | + | </p> | |
| − | + | <p> | |
| − | + | Then, we tried to do the same with 3 known aptamers against the HBV’s surface antigen (HBsAg). | |
| − | + | </p> | |
| − | + | <figure class="figure"> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/4/46/T--INSA-Lyon--EMSA-HBsAg.png" class="img-responsive center-block img-rounded"> | |
| − | + | <figcaption class="figcaption"><strong>Fig 7.</strong></figcaption> | |
| − | + | </figure> | |
| − | + | <p> | |
| − | + | The sensitivity was worse than in the previous EMSAs with the thrombin and the RT.</br> | |
| − | + | We chose the two best for our detection, but we hope to improve them to get a better sensitivity. | |
| − | + | </p> | |
| − | + | </div> | |
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| − | + | ||
| − | + | <div class="title-area" id="errors"> | |
| − | + | <h2 class="title">Things that did not work as expected...</h2> | |
| − | + | <span class="line"></span> | |
| − | + | <p> | |
| − | + | </br><span class="glyphicon glyphicon-chevron-right"></span>Constructing a part with biobricks by ligation. This often failed. We tested conventional ligation, SLIC… We finally needed to order synthesized sequences. | |
| − | + | </br><span class="glyphicon glyphicon-chevron-right"></span>Testing the device on paper. This could not be achieved because we did not receive the required paper sheets on time. | |
| − | + | </p> | |
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Latest revision as of 16:37, 14 November 2016
Early Results
The most of our relevant results are described in the Proof of Concept page.
Here are some of our first experiment results that guided us through our development, and finally, our results with sexually transmitted infections vectors.
First study of the ATP-aptamer’s affinity in liquid
In this experience, we used some modified aptamers. They were functionalized with a FITC, so they were fluorescent. Fluorescence was stopped by the reverse-complement of the aptamer, which was holding a quencher: a molecule that switches off the fluorescence by the Fluorescent Resonnance Energy Transfert (FRET) phenomenon.
We studied how the ability of ATP to chase the quencher away. As the target (ATP) and the quencher are in competition to bind the aptamer, the emitted fluorescence is a good estimation of the proportion of aptamers that have linked an ATP.
As can be seen here, there is a good response of the system to ATP. With a Kd of 330 µmol.L-1 (target concentration able to be fixed by the aptamers). This is not particularly efficient. We think that the quenching oligo is disturbing the aptamer, lowering its sensitivity.
The next step was to try this on paper.
Study of the impact of various solvents and blood proteins on the fluorescence emitted by fluorescein
In this experience we wanted first to test what is the impact of non-isotonic solvents on the emitted fluorescence. We obtained no significant differences of fluorescence emissions between assays when me mixed blood with PBS buffer or with NaCl. Although, we tried to compare the fluorescence emissions between blood and plasma. The results were more interesting.
Student statistical tests were performed for each concentration of fluorescein. It was concluded that blood filtration to remove especially red blood cells which could mask the fluorescence is proven to be very important: the fluorescence emitted is between 2 and 11 times larger in the case of serum compared to whole blood.
Revelation with light filters
As our device is meant to be used at home, we wanted a revelation method that does not imply the use of a UV lamp. Inspired by the work of the 2015 iGEM Bielefeld-CeBiTec team on sfGFP (another fluorescent molecule which emission wavelengths and absorption are relatively close to those of fluorescein), we decided to test whether a revelation with light filters on the flash and camera of our smartphone would work. Images taken with the smartphone equipped with various combinations of filters revealed that the filter on the flash was necessary to foresee a fluorescence (see below). Moreover, adding a filter to the lens increases the detection sensitivity. Also, it turned out that the sample of fluorescein concentration was a very important factor. Above a certain threshold of the concentration, fluorescence is quenched.
However, on a cellulose paper sheet the fluorescence detection turned out to be much less efficient, even in the optimal conditions that ensure a minimum of quenching.
Paper strips design
We designed paper strips that could handle our biological system. These paper strips contained a filtration pad in glass fiber, a nitrocellulose strip, and an adsorption pad in cellulose.
The filtration pad was here to prevent erythrocytes from migrating on the paper strip. It worked, after migration the nitrocellulose remained white and the results could be read. We also tested the microfluidics of this system once in our prototype, until we got good migration results.
Characterization of Aptamers for real STIs: HIV and HBV
After an intensive work on the human thrombin, we tested HIV-1 Reverse Transcriptase (RT). This particular target would allow us to detect HIV infections at a very early stage, when this virus has just penetrated the host. At this stage antibodies are produced by the patient’s body yet, so conventional methods of detection are not valid. We did an EMSA with the most efficient HIV RT aptamer we could find.
The curve presents an inflexion point for [RT] = Kd = 490 nmol.L-1 The sensitivity is again quite good, with the same warnings than for thrombin.
Important remark: We only have one aptamer to catch the Reverse-Transcriptase. Although our detection procedure requires two aptamers, and the sensitivity of the method will be limited by the worst Kd between the two aptamers.
Then, we tried to do the same with 3 known aptamers against the HBV’s surface antigen (HBsAg).
The sensitivity was worse than in the previous EMSAs with the thrombin and the RT. We chose the two best for our detection, but we hope to improve them to get a better sensitivity.
Things that did not work as expected...
Constructing a part with biobricks by ligation. This often failed. We tested conventional ligation, SLIC… We finally needed to order synthesized sequences. Testing the device on paper. This could not be achieved because we did not receive the required paper sheets on time.
