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.Read More...
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.
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.
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!