Difference between revisions of "Team:INSA-Lyon/Description"

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                <li><a href="https://2016.igem.org/Team:INSA-Lyon/Description">Description</a></li>
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        <li><a href="#stidetection">STI Detection</a></li>
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        <li><a href="#technochoices">Technological choices</a></li>
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        <li><a href="#approaches">How did we do it?</a></li>
                         <h2 class="wow fadeInUp" data-wow-duration="0.5s" data-wow-delay="0.5s">Gotta Detect Them All</h2>
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        <li><a href="#sourcesinsp">Inspirations and motivations</a></li>
                         <p class="wow fadeInUp" data-wow-duration="0.5s" data-wow-delay="0.75s">A multi-sensor based on aptamers.
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    </ul>
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</div>
                        <a class="read-more-btn wow fadeInUp" data-wow-duration="1s" data-wow-delay="1s" href="#abstract">Read More</a>
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                         <div class="col-md-5 col-md-offset-3"><h5 class="wow fadeInUp" data-wow-duration="0.5s" data-wow-delay="0.5s">DESCRIPTION</h5></div>
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                        <div class="title-area" id="stidetection">
                  <h2 class="title">Project Description</h2>
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                            <h2 class="title">A major concern: STI detection</h2>
                  <span class="line"></span>
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                            <span class="line"></span>
                  <p>Have you ever woken up with no memory of your last party? Your night may have spun out of control and you may have had a risky behavior.
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                      What if you could, in only 5 minutes, have the list of all the sexually transmitted diseases (STDs) you may have caught?
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                            <p>
                      What you would want to know is "Did I get a STD? And if so, which one did I get?! Is it the HIV, the syphilis or the hepatitis B?"
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                                Very early in the project we have decided to build a detection device. Sexually Transmitted Infections quickly appeared as an important problem that needed to be solved. Very few iGEM teams had worked on it, and the currently available commercial solutions did not satisfy us. So we thought it would be great to advance on this particular topic.
                      Very-Bad-Trip is a small paper device that detects biomarkers at a very early state of the infection, and several at once.
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                            </p>
                      It's cheap, easy, and gives you multiple answers very quickly to help you remember what happened.
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                            <p>
                  </p>
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                                Sadly, STIs are still a major public health issue today. Treatments are often started too late resulting in health problems such as sterility.  If prevention is probably the most effective action one can take to prevent the spread of STIs, early detection can help limit their deleterious effect. Moreover, after a risky behavior, the question is: “am I infected with a STI?”. We therefore decided to develop a cheap, easy to use, self-test, addressing contamination for several STIs at once.
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                            </p>
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 +
                            <p><b>Cf Interview Rainer</b> </p>
 +
 
 +
                            <a data-toggle="collapse" data-target="#interviewMore">Read More...</a>
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                                <p>
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                                We had the opportunity to talk about biomarkers with Prof. Rainer Bischoff, a dutch biomarker expert. Here are the different topics we talked about during the interview.
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                                </p>
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                                <p>
 +
                                <strong>How would you define a “good” biomarker? Which characteristics/properties do you require in order to consider a biomarker as a “good” one? Do you think that the “strength” of a biomarker can be different from an individual to another one?
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                                </strong></p>
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                                <p>
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                                Defining a good biomarker starts with asking the right question. What is the biomarker supposed to diagnose/prognosticate? What are the required sensitivity and specificity? Be aware that for population-wide studies you may need a specificity well above 99% in order to avoid false positives. To give an example, if you were to screen 10 million people for a disease and you had 1% false positives (99% specificity) this would still mean 100,000 false positives. The health care system is finding this out ‘’the hard way’’ in cases like colon cancer screening, which incurs an enormous cost for follow-up examinations. So please think carefully what you want to achieve with your biomarker and what the follow up would be.
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                                </p>
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                                <p>
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                                    <strong>Is there a large diversity of biomarkers for a specific disease?</strong>
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                                </p>
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                                <p>
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                                    --> This depends very much on the disease. I believe that infectious diseases are fairly ‘’easy’’ to diagnose, since the infectious agent expresses proteins that are usually not present in the host organism. That way highly specific assays can be developed based on ligand binding assays. This is very much different for multifactorial disease that affect the wider population and that have lifestyle factors, environmental factors interacting with the organism and its inherent genetic make-up, physiology etc. It is highly unlikely to find single, specific biomarkers for such diseases (e.g. cardiovascular disease, COPD) and a trend is going towards ‘’biomarker panels’’. It is fair to say that very little has materialized in this respect, as studies are poorly reproducible, study designs inadequate and methods not properly validated. So these aspects are critical to take into account.
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                                    </strong></p>
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                                    <strong>What kind of techniques do you use in order to discover new biomarkers ? How do you then evaluate their “strength”?
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                                    --> We mainly use LC-MS/MS with a lot of bioinformatics and statistics behind to try and find significant differences between different sets of samples. Validation in separate sets of samples, preferably from different centers, is very important as its proper method validation. Again the ‘’strength’’ of a biomarker depends very much on the context. What would you do with false positives and how important is it to avoid false negatives? Most biomarkers are not stand-alone diagnostics/prognostics but are used in the context of decision-making pipelines, for example based on IHC, CT scans, …
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                                    <strong>Overall, are the best biomarkers for a specific disease concentrated in a particular body fluid (blood, urine, etc) ? Or is it possible to find them in various body fluids?
 +
                                    </strong></p>
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                                <p>--> I think that blood-based body fluids are most widely used in practice, because they are easily obtained. A biomarker may also be found in urine or another body fluid (e.g. CSF) but especially urine levels may be very variable depending on how it was collected and on the metabolic state of the person. Urine is certainly of interest for kidney-related diseases. There is a trend towards so-called extracellular vesicles that circulate in blood for cancer-derived biomarkers, since cancer cells have a more rapid turnover and tend to shed such vesicles (e.g. due to necrosis). However, this is currently at the research stage.
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            <p>Early on, our desire was to search for a cheap, maybe do-it-yourself project of "frugal biology".
 
We rapidly thought of a test on paper. What for? The most documented important health problems are about STDs.
 
Currently available STD self-test kits are expensive (av. $30), based on antigen-antibody detection and only work for one infection at a time. We want to developsomething that combines several detections on one chip, and if possible, something fast, low-cost, and easy to use.
 
  
To get another advantage on what already exists, why not try a more recent and very sensitive technology? We decided to focus our research on detection by aptamers.
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            <p>The diagnosis is based on the interaction between a pathology biomarker (a protein) and a specific DNA sensor (an aptamer). This sensor, anchored on a piece of paper, will enable, from a drop of blood, the direct diagnosis thanks to a fluorescence emission.
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This project uses an innovative nucleic acid technology and a combination of existing parts, but no cells are used in this detection process.
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Cells are only used to produce the genetic construction, using synthetic biology.
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                            <p>Biomarkers detection is a field largely explored both by iGEM teams and industrials. Mostly used techniques rely on antibodies. Developing antibodies-based tests has some drawbacks: lengthy and pricey process of identification and production, short shelf-life… innovations are needed to develop more frugal tests.
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                            <p>Our main innovation relies on the use of aptamers technology. Aptamers are short single stranded nucleic acids. They basically act as antibodies. They have numerous advantages. Compared to antibodies, the processes of selection and production of aptamers are quicker, easier, and cheaper. Aptamers can be synthesized by pure chemistry, whereas antibodies need complex eukaryotic chassis to be produced. Aptamers are an user-friendly technology! They also have the advantage to be easily chemically modifiable. Three kinds of BioBricks were constructed and sent to the registry to build our detection device:
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                                <br/><span class="glyphicon glyphicon-chevron-right"></span>target generators (protein subunits of the HIV-1 Reverse Transcriptase and HBsAg, biomarker of Hepatitis B, deposited parts BBa_K1934060 and BBa_K1934061)
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                                <br/><span class="glyphicon glyphicon-chevron-right"></span>anchor allowing the fixation of the aptamer to the paper: our biological detection system needs to be anchored on paper. An existing fusion protein (part BBa_K1499004) could do that, but it was not characterised, so we improved it and tested it fully.
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                  <h4 class="feat-title">Proof of Concept</h4>
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                            <p>Learn more about <a href="https://2016.igem.org/Team:INSA-Lyon/Aptamers">aptamers</a></p>
                  <p>The proof of concept will be made with ATP and thrombin aptamers.
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Then, we will work on the detection of HIV and hepatitis B.
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We are hoping to gather several different tests on a single paper chip, to scan most common STDs.</p>
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                  <h4 class="feat-title">Finality</h4>
 
                  <p>The goal is to build a simple, fast, eco-friendly, and cheap device that detects the most common STDs, and maybe, the drugs you took.</p>
 
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                  <h4 class="feat-title">Human practices</h4>
 
                  <p>A reflexion about the conditions of use of our device seems necessary: should it be made available as a self-test kit, or should it be used under the control of a specialist? What about the consequences of the diagnosis?
 
We are gathering opinions from experts who daily work with the detection of STDs, in order to know which major diseases necessitate the development of a new, faster and more sensitive detection test, who are the most concerned people in the population,..</p>
 
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                          <h2 class="title">NB: DO NOT FORGET TO USE CONDOMS!</h2>
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                            <h2 class="title">How did we do it? </h2>
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                                Two approaches were developed during this project. First, a detection system based on fluorescence. It worked well, but we realized it could not be used at home. The second approach was a detection system based on latex beads.
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                            A first aptamer is covalently linked to the latex beads. After adding the sample onto the beads, they migrate on a paper strip. On this paper strip there are also complementary aptamers which recognize a different epitope of the biomarker. If the target is present in the sample, it will be taken in sandwich between the aptamers on the beads and the fixed aptamers. A visible black band will progressively appear, due to the beads’ coloration. If the target is not present, beads will be unable to form this sandwich and won't be stopped.
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                                A control is also needed. So after the detection band there is also a control band made with complementary strands of the DNA on the beads. Just by base-pairing the beads will be stopped. They are put in excess for the detection, so even if there is the target in the sample to be analyzed, some beads are going to migrate to the control band.
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                            </p>
  
 +
                            <p>How to fix aptamers on the paper strip? We got inspired by another iGEM project, from the <a href="https://2014.igem.org/Team:StanfordBrownSpelman">2014 Stanford-Brown-Spelman team</a>. They developed a Streptavidin linked to a Cellulose binding domain, fusion protein. We took this idea and pushed it a little further, characterizing their part and building new and more efficient ones. Basically, aptamers are biotinylated, they bind to the Streptavidin part, which is fixed onto cellulose thanks to the cellulose binding domains.
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                            <p>Another important aspect of the project was the design of a real device. If one wants to sell a detection platform, it must not only be the biological part, but also a real device to pack it. So we designed and 3D printed a device. A first model was designed as a prototype to test our paper strips. A second one was designed to be specifically used in real life. It’s a cornerstone for our project, because that’s what people are going to see and use.
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                                <figcaption class="figure-caption">Summary of the project advancement. The fluorescent detection system was functional. But it was not easy to use at home so we oriented toward the latex beads based system. It works in vitro but we still have some difficulties to make it work on paper.</figcaption>
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                            <h2 class="title">Sources of Inspiration and motivations</h2>
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                            Aptamers were not used at all in the labs of our school, but we learned they existed by reading literature. We immediately found out it was a promising technology. We looked further in literature to get some ideas about signal transduction with aptamers. That brought us to the idea of using fluorescence. After some tests, we were a little disappointed, so we went to an international congress about Aptamers in Bordeaux (France). Some researchers, and particularly Dr. Ciara O’Sullivan, brought us to the idea of using nanoparticles covalently linked to the aptamers. This idea seduced us and we immediately started working on it. But instead of using gold nanoparticles like most of the labs we chose to use latex beads. That’s a much more eco-friendly option, plus on a large scale it’s less expensive.
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                            <p>Our major motivation was to provide the world with a good solution to detect STIs. But we soon realized that what we achieved is actually a much more general strategy for developing quick and efficient tests. Indeed we developed a strategy which is extendable to pretty much every target you can think about. So we could imagine to sell the device and paper strips to be put inside the casing as a platform to be adapted with aptamers for each situations. Instead of detecting STIs we could detect water pollutants (heavy metals, pesticides…), other health issues (cancer, bacterial infections...) and food contamination. So we hope some future iGEM teams will follow us on this exciting path!
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Revision as of 02:38, 20 October 2016

iGEM : Description

 
DESCRIPTION

A major concern: STI detection

Very early in the project we have decided to build a detection device. Sexually Transmitted Infections quickly appeared as an important problem that needed to be solved. Very few iGEM teams had worked on it, and the currently available commercial solutions did not satisfy us. So we thought it would be great to advance on this particular topic.

Sadly, STIs are still a major public health issue today. Treatments are often started too late resulting in health problems such as sterility. If prevention is probably the most effective action one can take to prevent the spread of STIs, early detection can help limit their deleterious effect. Moreover, after a risky behavior, the question is: “am I infected with a STI?”. We therefore decided to develop a cheap, easy to use, self-test, addressing contamination for several STIs at once.

Cf Interview Rainer

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We had the opportunity to talk about biomarkers with Prof. Rainer Bischoff, a dutch biomarker expert. Here are the different topics we talked about during the interview.

How would you define a “good” biomarker? Which characteristics/properties do you require in order to consider a biomarker as a “good” one? Do you think that the “strength” of a biomarker can be different from an individual to another one?

Defining a good biomarker starts with asking the right question. What is the biomarker supposed to diagnose/prognosticate? What are the required sensitivity and specificity? Be aware that for population-wide studies you may need a specificity well above 99% in order to avoid false positives. To give an example, if you were to screen 10 million people for a disease and you had 1% false positives (99% specificity) this would still mean 100,000 false positives. The health care system is finding this out ‘’the hard way’’ in cases like colon cancer screening, which incurs an enormous cost for follow-up examinations. So please think carefully what you want to achieve with your biomarker and what the follow up would be.

Is there a large diversity of biomarkers for a specific disease?

--> This depends very much on the disease. I believe that infectious diseases are fairly ‘’easy’’ to diagnose, since the infectious agent expresses proteins that are usually not present in the host organism. That way highly specific assays can be developed based on ligand binding assays. This is very much different for multifactorial disease that affect the wider population and that have lifestyle factors, environmental factors interacting with the organism and its inherent genetic make-up, physiology etc. It is highly unlikely to find single, specific biomarkers for such diseases (e.g. cardiovascular disease, COPD) and a trend is going towards ‘’biomarker panels’’. It is fair to say that very little has materialized in this respect, as studies are poorly reproducible, study designs inadequate and methods not properly validated. So these aspects are critical to take into account.

What kind of techniques do you use in order to discover new biomarkers ? How do you then evaluate their “strength”?

--> We mainly use LC-MS/MS with a lot of bioinformatics and statistics behind to try and find significant differences between different sets of samples. Validation in separate sets of samples, preferably from different centers, is very important as its proper method validation. Again the ‘’strength’’ of a biomarker depends very much on the context. What would you do with false positives and how important is it to avoid false negatives? Most biomarkers are not stand-alone diagnostics/prognostics but are used in the context of decision-making pipelines, for example based on IHC, CT scans, …

Overall, are the best biomarkers for a specific disease concentrated in a particular body fluid (blood, urine, etc) ? Or is it possible to find them in various body fluids?

--> I think that blood-based body fluids are most widely used in practice, because they are easily obtained. A biomarker may also be found in urine or another body fluid (e.g. CSF) but especially urine levels may be very variable depending on how it was collected and on the metabolic state of the person. Urine is certainly of interest for kidney-related diseases. There is a trend towards so-called extracellular vesicles that circulate in blood for cancer-derived biomarkers, since cancer cells have a more rapid turnover and tend to shed such vesicles (e.g. due to necrosis). However, this is currently at the research stage.

Technological choices

Biomarkers detection is a field largely explored both by iGEM teams and industrials. Mostly used techniques rely on antibodies. Developing antibodies-based tests has some drawbacks: lengthy and pricey process of identification and production, short shelf-life… innovations are needed to develop more frugal tests.

Our main innovation relies on the use of aptamers technology. Aptamers are short single stranded nucleic acids. They basically act as antibodies. They have numerous advantages. Compared to antibodies, the processes of selection and production of aptamers are quicker, easier, and cheaper. Aptamers can be synthesized by pure chemistry, whereas antibodies need complex eukaryotic chassis to be produced. Aptamers are an user-friendly technology! They also have the advantage to be easily chemically modifiable. Three kinds of BioBricks were constructed and sent to the registry to build our detection device:
target generators (protein subunits of the HIV-1 Reverse Transcriptase and HBsAg, biomarker of Hepatitis B, deposited parts BBa_K1934060 and BBa_K1934061)
anchor allowing the fixation of the aptamer to the paper: our biological detection system needs to be anchored on paper. An existing fusion protein (part BBa_K1499004) could do that, but it was not characterised, so we improved it and tested it fully.

Learn more about aptamers

How did we do it?

Two approaches were developed during this project. First, a detection system based on fluorescence. It worked well, but we realized it could not be used at home. The second approach was a detection system based on latex beads.

A first aptamer is covalently linked to the latex beads. After adding the sample onto the beads, they migrate on a paper strip. On this paper strip there are also complementary aptamers which recognize a different epitope of the biomarker. If the target is present in the sample, it will be taken in sandwich between the aptamers on the beads and the fixed aptamers. A visible black band will progressively appear, due to the beads’ coloration. If the target is not present, beads will be unable to form this sandwich and won't be stopped.

A control is also needed. So after the detection band there is also a control band made with complementary strands of the DNA on the beads. Just by base-pairing the beads will be stopped. They are put in excess for the detection, so even if there is the target in the sample to be analyzed, some beads are going to migrate to the control band.

How to fix aptamers on the paper strip? We got inspired by another iGEM project, from the 2014 Stanford-Brown-Spelman team. They developed a Streptavidin linked to a Cellulose binding domain, fusion protein. We took this idea and pushed it a little further, characterizing their part and building new and more efficient ones. Basically, aptamers are biotinylated, they bind to the Streptavidin part, which is fixed onto cellulose thanks to the cellulose binding domains.

Another important aspect of the project was the design of a real device. If one wants to sell a detection platform, it must not only be the biological part, but also a real device to pack it. So we designed and 3D printed a device. A first model was designed as a prototype to test our paper strips. A second one was designed to be specifically used in real life. It’s a cornerstone for our project, because that’s what people are going to see and use.

Summary of the project advancement. The fluorescent detection system was functional. But it was not easy to use at home so we oriented toward the latex beads based system. It works in vitro but we still have some difficulties to make it work on paper.

Sources of Inspiration and motivations

Aptamers were not used at all in the labs of our school, but we learned they existed by reading literature. We immediately found out it was a promising technology. We looked further in literature to get some ideas about signal transduction with aptamers. That brought us to the idea of using fluorescence. After some tests, we were a little disappointed, so we went to an international congress about Aptamers in Bordeaux (France). Some researchers, and particularly Dr. Ciara O’Sullivan, brought us to the idea of using nanoparticles covalently linked to the aptamers. This idea seduced us and we immediately started working on it. But instead of using gold nanoparticles like most of the labs we chose to use latex beads. That’s a much more eco-friendly option, plus on a large scale it’s less expensive.

Our major motivation was to provide the world with a good solution to detect STIs. But we soon realized that what we achieved is actually a much more general strategy for developing quick and efficient tests. Indeed we developed a strategy which is extendable to pretty much every target you can think about. So we could imagine to sell the device and paper strips to be put inside the casing as a platform to be adapted with aptamers for each situations. Instead of detecting STIs we could detect water pollutants (heavy metals, pesticides…), other health issues (cancer, bacterial infections...) and food contamination. So we hope some future iGEM teams will follow us on this exciting path!