2013 Final Report
- 1 2013 Team at EPFL
- 2 Introduction
- 3 Aim of the Project
- 4 Motivation
- 5 Background research
- 6 Design Criteria
- 7 Arsenic Prototype 1
- 8 Arsenic Prototype 2
- 9 Data and Analysis
- 10 Conclusions
- 11 Future Directions
2013 Team at EPFL
SV 3rd year Bachelors students, biography from 2013
Emilie Mussard is a student in Life Sciences faculty.
She is particularly interested in the physical processes that drive and hold biological systems; this covers a large panel of study domains!
Later, she wants to work as an engineer on real and useful issues and thinks the project is a first step in this direction.
She also likes transmitting what she knows and is currently employed as a teaching-assistant for 2nd year physics class.
She has a previous formation as a theologian and tries to work interdisciplinarily when it is possible, particularly when facing with ethical issues. Besides her studies, she is also playing music and theatre with some groups.
Romain Equey is a third year life sciences student at epfl.
He is particulary interested by all these approaches we can have in this project. A big mixture of all we learn.
The real world aspect of this project is also important for him. He likes to create things by hand, so here he finds what he is looking for.
Jasmina Rubattel is a 3rd year Life Sciences student from Lausanne.
This real world project appeared to be a good way to mix together the main interesting subjects of her studies (biology, electronics, informatics), while applying theoretical knowledge to build a device available for everyone and share it with the world.
I'm 22 years old and study life science for 2 and half years. I come from Geneva where I study until I get my maturity. Then I move to Lausanne to study life science.
I'm interested in informatics, biology and engineering, so this faculty fits well to my demand. I was informatics teacher-assistant the last semester. I'm very interested in this sensor project because of the versatility of these tools and the large application spectrum.
I'm also very interested in the programming and the open source aspect of this project.I also practice beekeeping for five years and I look after about five bee colonies.
I played oboe at Lausanne's conservatory and I play in the Quipasseparlà orchestra. This year we played Beethoven's 6th symphony and Brahms violin concerto and we made a tour in Florence in Italy. I'm also implicated in the orchestra's administration.
Welcome on our final report page. The aim of this report is to present exhaustively the project we worked on this semester. We were a team of four students, the first set of students working on the project which will, we hope, continue for a while with other teams! As we were the first team, we spent the first six weeks of the semester making background reseaches. Hence, a big part of the final report is about these background researches and we hope it will help our readers understand the solutions we found and the future directions we imagine. The final report presents also the two prototypes we made, how they work, why we choose them and what are the next challenge to improve them. Finally, we will make some conclusions and speak about the future directions we hope the next team to work on.
We wish you nice reading!
Aim of the Project
The project look like making a sensor either for Arsenic detection, or for Coliform bacteria detection. But in fact, the aim of the project is more than just building a biodevice.
First, there is the real aim: building a device that could sense a contaminant in water. In the beginning of the semester, two sort of projects were submitted and we had to choose one. The first is Arsenic detection in water, The second is Coliform bacteria detection in water. Why detect these two things? Because they are a major coumpound of water pollution in the two regions where the people we work with are living. The people in Bangalore (India) are more interested in Arsenic detection, because the presence of Arsenic, from geological source or from industrial pollution, is nocive for human health. The exposure to small doses of Arsenic over a long time lead to diseases like skin troubles, digestive tract problems and cancers. The people in Yogyakarta (Indonesia) were more interested in coliform bacteria detection, because these bacterias coming mostly from fecal contamination are pathogens for human, causing serious diarrhea. Hence in both cases it is important to find a possibility to measure water contamination: it is a public health problem.
Then, we could wonder why building a new device, aren't there already many devices on the market measuring Arsenic or coliform bacteria contamination? Yes, we can find some, but many of these are expensive, or need a long time before having a result, or are not accessible to people like you and us, or are not reliable. The second aim of our project is to build something that is accessible and reproducible by those who could be interested in. This is done by the open-source informations we provide, by the publications we made on our blog, by the use of cheap and accessible materials and understandable protocols. The fact is that, more than exactly measring water pollution, we also want to raise awareness about water quality by allowing the devices for water analysis to be accessible for real people, so they realize how important it is to care about this liquid we need to live. As we finally choose to concentrate on Arsenic detection, we used a bacteria as a sensor that has been engineered by microbiology lab in UNIL to be used easily for humanitarian purposes.
This leads to an other aim of the project: integrate different domains and cooperate with different people. The goal is that we learn how to work as a group and also with people from different backgrounds. The cooperation takes place with the teams in Bangalore and in Yogyakarta, we disscussed with them, shared our experiences and tried to achieve a common purpose. We also worked as a team, learning to cease individualism and to integrate the knowledge of everyone. One aim is also that we disscuss with different scientific persons, because we have to use knowledge from many domains as biology, electronics, optics, legacy, environmental analysis... While cooperating with people, we also have to integrate the different domains we studied into one project. The project is for us the possibility to use for one goal, a dozen of different domains that were previously taught during our studies, and to understand the connexions and interactions between them.
Finally, the goal is also that we go out of the lab and go to the real world. This aim is in the project title: biodesign for a real world! We were all motivated about building a concrete device to face a problem that happens in the real world.
The motivation that drives us is very similar to the aim of the project, because it is really its aim that motivates us to take part in this project. In a way, the aims have been defined by our motivation as much as our motivation was dependant of the aims.
The principal motivation for us to participate in this project is to dispose of a new way to learn. During our studies, we are mostly "fed" by taught knowledge and theorical exercises that aim to give us an understanding of the subject. Participating this semester project, we learned by doing, by trying, by meeting, by searching, by being lost and having problems... And we learned a lot!
At a first glance over the project, we were all motivated by its concrete - real world aspect: going out of theory, in the field, trying to solve a real problem, building a prototype, helping other people and ourselves at the same time. Furthermore, the concrete aspect is accompanied by the necessity of doing with what is at hand. We are not inserted in a giant project to complicated to be understood by one person, but we have to do with what we have in terms of material, of knowledge, of possibilities.
Building a device with what we have also means using all the apllicable knowledge we have. What is motivating in this project is the fact that we need to use different subject we previously studied for one purpose and connect them around one goal. We could use subject as various as basic laboratory techniques, electronics, informatics, signal processing, microbiology, optics... and also human qualities to meet the people we work with.
The first part of our project has been spent making background studies. We had to get information (and integrate it!) about the subjects of our project, but also about basic techniques, technologies or coumpounds we were using. Background research is one of a first point of a design process. Because while designing an experiment or a prototype, we need to make informed choices. We were also lacking many informations, for example optics laws and possibilities that are essential for fluorescence measurement and hene necessary to design our prototypes.
You will find a presentation of the most important subjects that mobilized us for the background studies. These subjects (at least the four first) are important to understand our prototypes.
When we had to choose a direction, between Arsenic and Coliform bacteria detection, we were interested by fluorescence. Indeed, to detect Arsenic, we can use a bacteria engineered in Van der Meer's lab that express fluorescence in response to Arsenic. And we discovered that E.Coli can also be detected using fluorescence (E.Coli detection is used to infer Coliform bacteria presence).
To understand how we worked, we need to know:
- What is fluorescence?
- How is fluorescence related to a bacteria?
- What is GFP and how does it work?
- How do we measure fluorescence?
What is fluorescence?
Something that is fluorescent means that it emits light with a certain wavelength caused by the absorption of light having another specific wavelength. Usually, the exciting wavelength is smaller than the emitted wavelength, hence, the excitation has a higher energy than the emission. The fluorescence can also be caused by another electromagnetic radiation than light.
The basic physical fact is that a photon from the exciting light is absorbed by the fluorescing thing, this absorption causes one electron to change its quantum state because the photon gives the electron the necessary energy to go to a higher state. This electron returns to its ground state after a while, and emits the remaining energy under the form of another photon, which has a different wavelenght than the exciting photon. this produce light that is called fluorescence. The excitation of an electron that relaxes a photon while going back to its ground state is also possible with other types of excitation than light, such as heat or chemical reactions. But in this case, we speak of luminescence.
Many minerals or organic coumpounds fluoresce with UV light. Is it the case of aromatic amino-acids and nucleic acids we find in a bacteria. They act as natural fluorophores, and we can use fluorescence with UV excitation to measure the quantity of such fluorophores in a sample. Knowing the fluorescent emission spectrum of a bacteria, we can infer the quantity of it in a sample.
In the bacteria developped to act as an Arsenic biosensor that we want to use, Arsenic presence is detected by the bacteria and its presence activates a gene that was introduced in the bacteria: the green fluorescent protein. This protein, once expressed, act as a fluorophore and we can measure its activity. Hence, we measure GFP fluorescence and not directly E.Coli fluorescence.
What is GFP and how does it work?
The green fluorescent protein, is a protein native to the jellyfish Aequorea Victoria. It is a 27kDa protein mostly formed with beta-sheet making a sort of tube. It acts as a fluorophore. The wild type absorbs light at 395nm and emits light at 509nm, fluorescing with a green light. It was first isolated in the 1960s, but in 1992, its gene was analyzed and published in Gene.
From this discovery, Gfp has been used for molecular biology studies: it can be attached to another protein and measured to understand where this protein is present and/or active and many other applications. Among these, the gene of GFP is positionned after the promoter activated in our bacteria when it senses Arsenic, allowing its expression when Arsenic is present.
Other developpments have been made to GFP, creating other fluorescent proteins either excitted by another wavelenght or emitting at another wavelenght. Today, a whole rainbow of fluorescent proteins exists. These developpments with GFP were rewarded by a Nobel Prize in 2008.
How do we measure fluorescence?
To quantify the emitted fluorescence of a substance, we have to use a dispositive to measure the quantity of light at emission wavelength. Some fluorometer exist that illuminate a sample with a precise wavelength and collect the emitted light at another specific wavelenght. Other techniques exist that are measuring a fluorescent spectrum either by illluminating the sample with different wavelengths, or by collecting the emitted light at different wavelengths. The problem of the collection, is that we need to collect light being sure that we do not collect the exciting light, which would distort the results.
In our case, we tried to ways of thinking: either collecting all the light and applying analytic filters afterward in order to measure only the light at the wavelenght we are interested in; or applying optic filters before the light collection to be sure the exciting light is not touching the photoreceptor used for collection.
Arduino is an electronic open-source hardware. It consists of a micro-controller. There is also a software to programm the activity of the hardware. As it is simple, based on an open-source community, Arduino can be used by anyone interested by electronics for a wide-range of applications. Their website is here.
The first idea of our project was to use an Arduino for the electronic part of our prototype. Actually, we use the Arduino as a energy source for the led, and, for the second prototype, we use it to gather informations given by the photoreceptor. Hence, we do a very little use of Arduino compared to its wide possibilities. Actually, we make the whole analysis with a computer, but a mid-term goal would be to use more Arduino in order to get rid of the computer and have directly a result from the measures.
Working in a lab is not the same as going in the field and working with a genetically modified organism complicates even more the problem regarding applicable laws. When working in a lab, some basic legal principles exists and must be applied, but they are obvious because they concern security. When going into the field, principles about the safeguard of th environment applied as much as principles about public health. Furthermore, we work with a GMO, a sensitive subject concerned by even more laws. Hence, we have to carefully take care of the legacy applicable to our project even if we have to find which laws concern our project and interpret them because our case is special.
There are also ethical problems we have to face, mostly because we want to work in the environment with a GMO having a antibiotic resistance gene but with a public health purpose.
We did research about laws in Switzerland. The first goal of this research was to understand what we are allowed to do with a GMO in Switzerland and if we will be allowed to do anything with our prototype. As the problem is more complex than jus a yes-or-no answer, we had to examine the questions, the legal framework (swiss and international) and the solutions we could find for our prototype.
The beginning of the work about laws is to specify the questions related to our project. They act as a framework or guidelines during the research. In our case, we defined:
- What are we allowed to do? Particularly, what are we allowed to take in the environment?
- What do we have to pay attention to? What do we have to protect and to what extent goes the protection?
- Will our research be legally usable?
- We work with two domains: the environment in which we use the biosensor and the public health that is affected by Arsenic presence in water and about which we want to raise awareness. Knowing that, which domain do we have to give the priority to?
Having defined the questions, we also observe what are the problems we are facing with our biosensor. Indeed, the concept of our biosensor is to produce fluorescence proportional to Arsenic concentration in water, but this fluorescence is due to a gene from the jellyfish that has been introduced by genetical engineering techniques into the bacteria; this makes the bacteria a GMO. The problems are related to our questions and the laws we imagined to find.
- The bacteria is a GMO -> as it is a sensitive subject, there are many regulations. There are also many fears and opinions in the society about GMOs and we have to pay attention to them.
- There is a gene inducing a resistance to an antibiotic (kanamycin) that was used for the selection of the modified bacterias -> it is potentially dangerous if the gene is transmitted to other pathogenic bacterias because kanamycin is an antibiotic use in human medicine.
- These two points lead to questions about what we can legally/ethically do.
- We also have to think what to do with the waste because it cannot be left in the environment.
Juridically, the framework has basically been set up in order to answer the problems about alimentary GMOs. We also found some laws that treat research with organisms containing some particular paragraphs about genetically modified microorganisms such as the bacteria we use. But these laws are not exactly corresponding to our example because the laws on alimentary GMOs concern organisms that have not any gene with an antibiotic resistance and laws about research with GMOs treat only research that happens in a laboratory.
At an international level, we find two principles that governs the laws related to alimentary GMOs and GMOs in general:
- Precaution principle, mostly used in the European Union (If there is any risk of serious or irreversible damage, the absence of absolute scientific certainty must not serve as pretext to postpone adoption of effective measures to prevent environment degradation. The idea is that we need to proove absence of potential risks and not their presence. Any project must proove scientifically that there are no risks due to their product. A quite difficult work with GMOs.)
- Substantial equivalence principle, mostly used in the USA (This principle is used to regulate production and commercialization of new food products as those derived from biotechnologies
(GMOs). It statuates that if an alimentary coumpound is essentially similar to an existing coumpound, it can be treated the same manner concerning security. This principle applied to a GMOs signify that
if the GMO is substantially equivalent to its conventionnal equivalent, it will be declared as healthy as the conventionnal product. This concept is used among others by the american Food and Drug Administration to appreciate and declare innocuousness of GMOs.)
We then also find protection measures and procedures to get an authorization for a product. In fact, a GMO can be allowed in the environment if it satisfies a range of demands, the procedure to obtain the right to use a GMO in the environment is called an authorization. An authorization procedure involve:
- Security: The product must be securized and not cause damage to human, environment or animals. Tests must be done with the most recent knowledge and technology.
- Free choice: Even if a GMO obtained an authorization, consumers, farmers and factories must have the freedom to choose between a product with or without GMOs.
- Labelling: To maintain free choice, a GMO product must be correctly labelled, so the consumer can take a decision correctly informed.
- Traceability: The consumer must have the information of the route from the producer to the seller.
- But at the same time, the authorization must respect protections measures and can be removed if it has to breach them. The protections measures means:
- Safeguarding clause: Politicians can forbid a product if they estimate it is dangerous or not enough known.
- Coexistence, buffer zones, isolation distances: It must exist a «buffer» zone between GMO culture and non-GMO culture. Distance of this zone is regaulated differently in each country. In Europe, it goes from 15m in Sweden to 800m in Luxembourg.
- Zones sans OGM: A state has the possibility to decide that a zone is to be protected and has to be GMO-free.
In Switzerland, the principal argument is the protection. (The federal office of public health investigate, collaborating with other federal offices, autorisation demands: it gives autorisation only if all risks for health and environment is eliminated.) The confederation also function with a duty to inform, There is a need to communicate to the authorities any research, accident, change...
There are two ordinances and one theme from the Swiss Expert Committee for Biosafety that can be applied to our project. The purpose of these ordinances is to protect humans, animals, the environment and its biologic diversity from threats resulting form the use of organisms, their metabolites and their waste. These ordinance regulate the use of organisms in general but contains also specific paragraphs about genetically modified organisms. We find them here:
- Ordinance 814.911 on the Handling of Organisms in the Environment
- Ordinance 814.912 on Handling Organisms in Contained Systems
- Topic of SECB (Swiss Expert Committee for Biosafety) on transport, import and export of biological substance that contain GMOs
The ordinances have the ability to define and clarify the terms we use in order to define what is concerned by which law. They also emphasize on the necessity to classify the organisms we work with and to make an evaluation of the risks and of the danger. The purpose of the evaluation is the diligence duty, that is: everyone must act with counsciousness and precautions that the situation demands to avoid that ogranisms or their waste put in jeopardy humans, animals and the environment and its sutainable use.
The incidence these ordinances have on our project is that they give restrictions on the way we will build and use our biosensor. Especially:
- Our biosensor bacteria must be confined because of its resistance to kanamycin.
- Confinement = constant existence of a physical or a chemical barrier between the environment and the organism.
- We must think of the sample-holder to respect this compulsory confinement meaning the sample-holder must guarantees that the bacteries inside are never in contact with air, water or anything that will return to the environment. Thinking of that we also need to considerd how to put water in contact with the bacteries without breaking the confinement of the bacteries.
- We must manage the waste in order that they remain confined and are destroyed accordingly with the law (autoclaved or inactivated with 80% alcohol).
- We have to notify the authorities what we are doing at least at the beginning of the activities, and we will need to ask for an authorization to use the prototype in the environment.
- We have to inform if there is any accident
- We have to name someone responsible of the biologic security
In a nutshell, we were looking what we are allowed to do and we discover that the legal demands forces us to think HOW to continue our research and build our prototype.
Furthermore, we have to think not only of the laws but also of the society and its opinions. Because we are working with a GMO and that many people are afraid of the consequence of the use of biotechnologies in the environment, we have to be aware to avoid a potential scandal. It is important to inform people and guarantee the confinement to forestall fears. It is also important to have the support of the authorities and the security department of our schools.
This chapter is adapted from the researches presented on our blog. There is a complete and detailed presentation we used for a specific presentation about this subject here: Legacy about GMO in Switzerland
Having chosen to focus on Arsenic detection, the design criteria are more precise. We also try to not forget E.Coli detection which is linked to Coliform bacteria proportion. We want to create a device to detect Arsenic presence in water but that could also be adapted to E.Coli detection by means of some minor changes. We decide that our device has to present these criteria:
- Portability: We want to have a kit that we can take in the field to do measurements. In the end, the idea is that we can measure easily in the field instead of bringing the field in the lab. The portability will allow the users to take the device and travel with it to the place where the water is. The portability forces us to think not only to the size but to all the components we use, the energy sources they need, and their ability to be manipulated in the field.
- Low-cost: The kit must be affordable for non-occidentalized countries, and also by thoses who care about water quality but are not employed by a big lab. This means either choosing low-cost components or using things that most people already have and use daily.
- Replicability: We have to documentate well enough the prototype building for other people to be able to build it by themselves. The next team will also have to prepare a calibration protocol that is replicable.
Fluorescence kit: We decided to use a bacteria expressing green fluorescent protein when put in presence of Arsenic as a sensor. It implies to find a way to measure the fluorescence and relate it to a quantity of Arsenic. Furthermore, E.Coli can also be detected by fluorescence (but with other wavelenght than GFP). To design a kit measuring fluorescence we need:
- Light-source: Both E.Coli and GFP need to be excited by a specific wavelenght to fluoresce. We have to find the right light-source as well as respect the general design criteria.
- Filter: In order to excitate the sample at a certain wavelenght but collect only the wavelenght emitted by the fluorescing sample, we need an optic filter that will cut the non-wanted wavelenght. The nearer the wavelenght for excitation and emission are one from another, the more difficult it is to find a good filter... and it is even more difficult to find a cheap one!
- Sample-holder: The sample with the collected water and the bacterias has to respect some conditions. It must be transparent to have a possible excitation and a measurable emission. It must act as a confinment for the GMO bacterias acting in the sample. It has to allow oxygen presence for bacteria growth and be made of a material that will not react with Arsenic. It also has to allow the measurement to be done in the right direction (where the GFP emits fluorescence).
- Receptor: The most challenging part is to find a receptor that will transform the fluorescence quantity into a countable data we can relate to the Arsenic presence. This receptor must be sensitive to the emitted wavelenght and the order of magnitude with which it is emitted.
- Data analysis: We need to provide a way to analyze the data the receptor provides. It means both an instrument to quantify Fluorescence presence and the necessary calibrations to relate it to real Arsenic presence.
Data and Analysis
We begin during the semester to test our first prototype with dextran samples. It allows us to try the prototype particularly to know if it can detect different level of fluorescence. We took pictures of different dextran dilution's samples and analysed them.
This table is an output from ImageJ. We see that the MEAN for samples 1 to 3 is really close to the MAX. We observe also a break between the third and fourth mean. This is more visible on Fig. 1.
We see a clear difference between high and low concentrations because the sensibility of the camera photoreceptor is limited. When samples are too concentrated, they emit a too big intensity of green light that burns the picture. In this case, there are too much saturated pixels (value = 255 in Table 1) and they predominate on the picture. It gives a linear function according to the concentration.
This is different with small concentrations. The average of green light intensity is more “real“ and gives another linear function with concentration, because the camera could really sense and quantify the light intensity.
Some weeks later we received strain of eGFP bacteria from Dr. Jan Van der Meer Lab. So first we made some control measurements with a fluorometer. We expect to see an increase of fluorescence related to the decrease of dilution as well as excitation and emission peaks at 488 nm and 509 nm respectively.
This is the fluorescence in function of the dilution for excitation at 488 nm and emission at 525 nm (log scale). The function (y) is linear as expected.
The excitation scan is from 450 to 490 nm and the emission is fixed at 525 nm.
In the Fig.4 the emission scan goes from 490 to 560 nm and the excitation is fixed at 488 nm (in fact between 490 and 515 nm we get overlapping so these data are useless).
For excitation and emission wavelengths, we see that the maximum values are at 488 nm and around 509 nm as expected. The first dilution is the only significant one. The others are too small to be readable.
These measurements show that the bacteria are indeed expressing eGFP and also that fluorescence is dependent of the concentration.
Then we want to use our first device. We made a serial dilution. We plated a part of each dilution in order to count them and test samples in our prototype. We expect to see a rise of light intensity according to a drop of dilution like the first experimentation with the dextran.
After 10-3 the results are the same so it shows us a detection limit at 10-3.
Here both are linear function (but also in log scale) as expected. The second curve (mean x area) is more precise because area change slightly between each measure so the mean is balanced by area. But it is almost the same curve. These graphs confirm the detection limit at 10-3.
We got plates back after a growing time. We used the more diluted sample plate to be able to count the colonies. We used ImageJ in order to make it.
Here is the last dilution (5 x 10-6) in process to be analysed.
After image processing we have only the spots remained. The program counted 259 colonies.
We know that we have a volume of 200µl diluted 200’000 times. So we have 259/200 ≈1.3 bacteria per µl. We multiply per 200'000 and obtain 259'000 bacteria/µl. This is the concentration after the cell culture. We know that the limit for our prototype is at 5x10-3 thus the minimal amount of bacteria needed to be detected is 1300 bacteria per µl.
We try the second prototype with dextran like the first one for calibration. We obtain really good results.
So we see a nice linear curve that confirm the increase of signal according to the concentration. We know that dextran has a greater light intensity than eGFP (about 4 times) and here the signal is very small so it has to be more difficult with eGFP.
Finally we use the arsenic biosensor to reach the first aim of our prototypes. We accomplish that in Van der Meer lab. First we also made a control measurement. We use a preconceived protocol to prepare the biosensor with the help of Lab’s people. Once the biosensor ready we mix it with different concentration of arsenite (ASIII) then incubate them for 2-3 hours.
The unit used here is “the relative fluorescence units (RFU) of the induced samples divided by that of the untreated sample (background fluorescence) after hours incubation.“ as it is said in the protocol. The blue series is in an exponential phase and thus bacteria are fully active. While the red one is in a stationary phase. This is why the blue one is more linear and thus more fluorescent than the red curve.
We also brought some contaminated water samples from Martigny that we knew roughly the range (between 10 to 40 [µg/L]). But the results show less than 0 µg for each sample. After discusion we have learnt that a protocol exists to take arsenic samples and keep them without precipitate arsenic. Thus there was no enought arsenic in solution after one week.
Then we also made a test with our first prototype. Here are the results.
Compare to the dextran or the eGFP Figures this one is not distinct at all. We does not observe a linear curve as expected or as the Fig.7. We had some trouble during the experiment. So it might be this trouble the cause of our fail or our prototype cannot simply measure as precisely as we want.
The second prototype has no interesting results. Because the signal cannot be distinguish from the LED light despite the filter.
Our project was divided in three main parts. First the background research that took a long time (nearly half time) but was important for our knowledge. To know what we were talking about and which direction would we choose. Then once we chose where to go, we made a quite simple device, and after some experiments we realised that some aspects of it had to be improved.
And finally we tested both prototypes with dextran, eGFP-E.Coli and the Arsenic biosensor. And we observed that the more complex the sample was the more difficult it was to get significant results. So we learnt that we need to repeat the experiments and be more strict in the following of the protocol (note everything in the lab-notebook). Thus, we could have determined if the manipulations we did are reproductible, or if we have to take another direction. Another point is that we were not trained to handle bacteria, so we should have been going to the lab more often to practice. We also should have contacted Dr. van der Meer and the team in Sion more rapidly, because we learnt a lot from them and it could have helped us for the direction of our project. For example, we discovered that the reporter bacteria expresses eGFP better if oxygen is present, and we realised we did not really think deeply enough about optics, which is an important part of the Prototype II. After all the tests, we also became aware that for other poeple to reproduce our prototype, it would be important to have more precise mesures with a clear pattern.
In the end, we did not get good results detecting our target with our devices, but we learnt so many things doing this project that we are happy with the conclusion of it. We learnt about team work, collaborating with each other and with people with other backgrounds. We discovered what really meant building a project from the start and that it is not always easy to find a direction and stay focused on it. We could have been faster if the had split the work more precisely, each digging a very specific part, but in that other hand, working together allowed us to be very aware of each steps of the project.
The fact that other people will continue to work on our project and that it could possibly be used in the real-world is something that really motivated us, and we hope that the first researched we did will help and inspire other people in the future.
Due to the results we obtained, and the work we made during the semester, we have many ideas of which future directions could be taken, by us or by the next set of students active for this project.
Future directions are classified in two categories: improvements of our prototypes and general reflexions about the bacteria or the device that measures fluorescence.
- We need to do more experiments with arsenic and bioreporter. First, we need to know to what range of fluorescence our device can measure something. But we also need to know how the bacteria fluoresce and how its concentration distort the signal. Finally, we have to understand the function that relates arsenic concentration to the datas finally processed with our prototypes. During this semester, we analyzed a bit these different questions,but always separately one from another. It means that we have no ideas how the different aspects relate one with another... and the only integrated experiment we did gave no interpretable results as we were not prepared enough and had not sufficient knowledge about that.
- We have to test our LEDs. We have to be sure that we have the right excitation wavelength. Imagine our LEDs are sligthly lighting with a wavelength that pass the optical filter: in that case, the results would be distorted a lot because the enery of the LEDs is far greater than the energy of the fluorescence due to GFP. Furthermore, we have to test which wavelenght is preferred by our fluorescent protein and which wavelength range is the best to both excitate and evitate a contamination of the signal.
- For the second prototype, we have to test filters. As for the LEDs, it is important to eliminate any risk of pollution of the signal by examining if the filters are correctly filtering and to what range. If we cannot eliminate all contamination by light, we have to know the range it occupies in order to function with it.
- For the second prototype, we have to add lenses. The fact is that the light from the fluorescing sample is passing the filters only if the angle with the filter is lower than a certain value. Hence, we are loosing a big part of the signal, and as the signal is tiny, it is a pity. With a lens, we could have more light passing the filter and a better activation of our photoreceptor.
- For the second prototype, we can improve it in order to get rid of the computer: we could have an integer circuit and find a way for the results to be displayed on a screen attached to the circuit.
- For the first prototype, we could improve CHDK use in order to analyze directly the picture in the camera. Such an analysis would allow a faster result and a cheaper and more portable device, because no computer would be needed anymore.
- We could design the first prototype to be used with a smartphone. Indeed, a big part of the population nowadays own a smartphone, so it would respect the accessibility criteria. Furthermore, an application ImageJ (the programm we use for analysis) exists for smartphone. We could also create a whole application, taking the picture and directly measuring the fluorescence intensity.
- If we continue using a picture and ImageJ for the first prototype, we can find a way to improve reception, making it closest to 509nm instead of the range of 497nm to 560nm that serves now for the reception of the signal.
- Finally, we could improve the first prototype to have many samples pictured at the same time. This would be a possibility to compare directly samples between them or compare to a blank value, or a known Arsenic concentration.
General future reflexions:
- There is a big reflexion that need to be made about the sample holder. The sample-holder must cumulate a wide range of properties: confinement, growth environment for bacteria, possibility for the light to enter, excitate and being emitted and collected. We could imagine a bit of everything, such as PDMS microfluidics holder, test-tube, sealed tube, plate, sealed plate... We have seen mostly two possibilities: a microfluidics sample-holder, and a bigger, stationnary sample-holder. Microfuidics brings many advantage for the waste size, the movement to which bacteria are submitted, the signal accessibility. But it also brings difficulties in the creation of a system able to measure fluorescence, and in the precisness needed for the instruments used. A bigger sample-holder as we used does not provide a nice growth environment if we do not move it while bacteria are processing the Arsenic signal. We also have to think to the Oxygen supply during the growth (leaving an empty space) without disturb the measures of the light with bubbles. With a big sample-holder we also have to know the distance the signal is traveling before being measred: it is possible that the signal is lost by diffraction in a to big volume. But a big sample-holder is simplier to manipulate and less expensive to produce. Hence, the sample-holder is a nice piece for reflexion and we hope a best solution than the one we use actually will be found.
- One of our biggest problem in the analysis of fluorescence with the bateria we use is the fact that it is expressing eGFP and that eGFP needs an excitation wavelength that is very near the emission wavelength. This proximity makes the filter design difficult. One possibility would be to engineer the bacteria such that the fluorescent protein expressed in presence of Arsenic reacts with different wavelength. Changing the fluorescent protein would also have a cost advantage. Indeed, finding a LED lighting in UV range is far more difficult and expensive than one in the visible spectrum or in infrarred range. Having a fluorescent protein reacting to excitation at a longer wavelength would be interseting in terms of cost an accessibility of the prototype.
- With GFP expressing bacteria, the analysis of Arsenic concentration in water require nearly three hours. this is already faster than going in a lab and waiting days before having a result. But this delay could also be ameliorated if we tried to use another reporter. This is a project that could last years! One idea could be to use luciferase.
- During the semester, we discovered that working with bacteria is not that simply reproducible. The detection of Arsenic is dependent of the number of bacteria, their ability to react, the phase they are in as much as the concentration of Arsenic itself. Assuming we want to end in a reproducible device measuring Arsenic, we would need to define a function that would allow us to infer Arsenic concentration from the fluorescence quantity. This function would be dependant of the number of bacteria, their survival rate, the temperature, the oxygen and other nutrients available in the sample, the movement and other forces they are submitted to... Finding this function need a long time experimentating and a very big number of data. Otherwise, when measuring, we would always need to have samples used as negative and positive controls, such as a sample with a known concentration of Arsenic, in order to compare to the measured sample and guess its Arsenic concenration. So working with bacteria is not that simple and we could also continue researches to find a more reproducible way to measure Arsenic... which is a new adventure!