Team:Chalmers-Gothenburg/HP/Silver
Introduction
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The prototype
Initial ideas
From the beginning of the project, the idea to create a prototype that could be used for a potential screening program seemed very inviting. By creating such a device, it would also be necessary to truly consider the potential use of the project. The features found most important in a good final product were safety and ease of use, low rate of misdiagnosis as well as low price and thus availability to many in need.
To get started, a prototype brainstorming meeting enabled different ideas to be discussed and the best alternatives are presented in this paragraph. One conclusion made, was that the accuracy of the test is less dependent on the prototype construction than on the system design, which can be read about in the project description. This part of the wiki focuses on how to make use of the system in the best possible way.
The first question faced in the brainstorming was whether the sensor should be possible to use at home or if it should only be used in a controlled environment such as a hospital. The initial idea was to have a yeast filter which one could exhale through, exposing the yeast to potential VOCs. After careful consideration this alternative was deemed too riskful. For people without proper knowledge, handling genetically modified yeast might pose too much of a risk, especially since the yeast would be close to the mouth. Therefore it would be better if the test was handled at a hospital by personnel with appropriate education. One problem with this idea could be that doctors or nurses generally are not used to working with yeast and thus extra personnel would be needed. Also, with the yeast on a filter it would still be close to the mouth. Since it was decided that simplicity and safety are important characteristics of the device, the conclusion of the initial thoughts was that it would be preferable if doctors or nurses not were required to handle the yeast and that the yeast would not be located on a filter. With this in mind, a new idea took form.
The sample bags
Commercially available sample bags that could be used to collect and store VOCs from the exhaled breath were found after some research. If such bags were used, the risk for patients and doctors to come in contact with yeast would be eliminated. After filling the bags with exhaled air they would be sent to laboratory facilities where incubation with the yeast would be conducted. This idea appeared safer and more practical, but new questions arose. How would the yeast be exposed to the VOCs? How much air would be needed to give enough VOCs for the yeast to be able to sense it?
The concentration of VOCs in the breath is low, in the parts per billion range for both octanal [1] and butanone [2]. The sensitivity of GPCRs is varying. According to Minic et. al [3] RatI7 can sense 5*10^-14 - 5*10^-4 M while Olfr1258 can sense around 5*10^-4 M [4]. Since the amount of VOCs needed is unsure, it is safe to assume that a couple of liters of air would most likely be needed. However, to keep the yeast incubated with that much air requires a large, bulky vessel. This could be solved by creating a recirculation system with the air from the sample bags bubbling through the suspended yeast culture. In that way the yeast could be exposed to all the collected air but only a smaller amount at a time.
Consulting with experts
Meeting with Associate Professor Carl Johan Franzén
To discuss the design of such a recirculation system, Carl Johan Franzén was contacted. Carl Johan is Associate Professor in Bioreaction engineering at Chalmers and has many years of experience in working with yeast. The general idea considered was to have a tube with a specially designed lid that would be compatible with the recirculation system. With the use of a compressor, the VOCs could be pumped through the yeast media. Air from the tube should also be led back to the sample bag through a pipe. With enough recirculation and a small enough vessel the advantage would be that a shaking incubator would not be needed. The discussion also covered whether the system would have a problem with oxygen limitations. Carl Johan gave the advise to calculate how much oxygen that is needed and compare to how much breath contains. If the amount of oxygen in the breath is not enough, it might be possible to dilute the collected breath with oxygen. This solution would require the sensitivity of the receptors to be taken into account to ensure high enough VOC concentration to match the range of recognition for the receptors. A third option would be to switch between the collected air, containing VOCs and pure oxygen. To minimize the oxygen consumption the growth rate of the cells should be kept low, possibly with limiting media.
Carl Johan also thought that the use of a spectrophotometer or a plate reader should be considered since it would allow the amount of cells that turn red to be measured. If it would be possible, the plating of the cells would be avoided. It would, however be more expensive. An advantage of using a plate is the ease of analysis since it does not require additional instrumentation.
Consulting with Professor Per Sunnerhagen
To learn more about the mating process and the ADE2 gene and how it affects the colour of the cells, an email conversation with Professor Per Sunnerhagen at University of Gothenburg was initiated.
He explained that he did not believe oxygen limitation to be a problem since the mating process is not particularly oxygen demanding. Additionally, he mentioned the possibility to see the difference in colour of the cells, in liquid media, if the cell density is high enough. He was not aware of the ability of a spectrophotometer to detect the red colour but gave the advice that ADE2 mutants accumulates a polymer of aminoimidazoleribotide which generates a red-orange fluorescent that can easily be seen in a microscope. That ADE2 mutant fluorescence could be helpful both for designing the prototype but also in the lab, where it might be difficult to distinguish the red colour in a colony if only a few of the cells are stained.
Contact with PExA
In addition to consulting experts, the Swedish market was researched since all inputs are beneficial when developing a suitable screening strategy and prototype for the project. The goal was to find companies and professionals working within the field that could aid in modifying and improving the idea. One company that the iGEM team came in contact with was PExA (Particles in Exhaled Air). The company works with sampling small particles from exhaled air.
Founded in 2014, PExA is a company that have created a new method to collect particles from the lungs. The company divides the method into two big parts, sampling and analysis of particles. The sampling device is non-invasive and easy to use. The patient exhales repeatedly into a mouthpiece connected to the machine and the exhaled particles are then collected on a membrane. Each sample usually consists of ~20 % proteins and ~80 % phospholipids, which are used for analysis. The machine is today involved in different research projects for example the search for new biomarkers for early detection of lung diseases.
Since they are experts in the area of breath analysis and sampling correlated to sickness, a contact was established with PExA and a study visit was scheduled. The aim of the meeting was to learn more about the technique, the market and to get feedback on the iGEM project.
At the meeting the team started with a short introduction about the project and the representative from PExA, Svante Höjer, in charge of the development at the company, held a short presentation of the company. The main part of the meeting focused on discussing the project.
PExA explained how they collect the particles with a Teflon membrane which makes it possible and easy to concentrate the particles at one place. This fact made the team rethink the original sampling idea, which comprised the use of airbags designed for containing VOCs. One of the problems with the airbags is the need for a large amount of air to obtain a high enough concentration of VOCs. This is problematic both due to the constraint put on the patient as well as the inefficiency in the transportation of large volumes of air. Also, the air can only be stored in the bags a limited amount of time, or the VOCs can condense on the walls of the bag. If possible, it would therefore be preferable to instead use a small fiber.
Designing the prototype
After the new idea had arisen, a brainstorming session was held to research the possibilities to use a fiber and to discuss which idea to further work with as well as their advantages and disadvantage.
The conclusion from this session was that the idea with the fiber was the one to be developed. SPME and similar fibers absorbing VOCs are commercially available and could be used for our system. The largest advantage with this idea is that the device would be more manageable and that the fiber would be much easier to transport from a hospital to the laboratory facilities. A potential drawback though would be the extra step needed to extract the VOCs from the fiber in order to expose the yeast to them. Though compared to the recirculation system in the sample bags idea, the extraction step is probably less cumbersome. Available SPME fibers could possibly be used as they are, but a more optimized version would be to prefer.
One idea would be to use a porous fiber as a filter, place this in the device and exhale through it. Important in such a design would be that the fiber-filter is not too compact which would make exhaling through the device difficult. The fiber could be sent to a laboratory facility where the VOCs is extracted and added to the yeast media.
For the design of the prototype, a relatively small device was considered desirable in order to make it easy to use. It would also be good, for both practical and environmental reasons, if as much as possible of the device was reusable to enable easy cleaning. A two-part option was therefore considered. One main part where the fiber-filter could be placed and one mouthpiece to blow through. In this design, only the filter would be disposable.
Another important part of the design is to minimize the impact of the surroundings. Since the system will be very sensitive, it is important to avoid that air, other than the exhaled air from the patient, comes in contact with the fiber. For this purpose, valves in the mouthpiece and in the end of the device could be used. To control the background levels of the VOCs, the test should take place in a controlled hospital or laboration facility with oxygen masks or VOCs free air to inhale if necessary. Since the secretion of VOCs also can depend on the food intake, the patient is restricted not to eat a couple of hours before the test, in order for the result to be reliable .
The printing
With these thoughts in mind the prototype was designed in CAD, as may be seen in figure 1 and 2.
In addition to this, the team also contacted a former iGEM team member David Hansson, now working with his master’s thesis on the department of Systems and Synthetic biology at Chalmers University of Technology. He offered to aid with 3D-printing of the prototype and he printed the first prototype after the provided design.
After some consideration the prototype was deemed slightly too big. The mouthpiece could be too big and therefore hard to blow through and the whole device seemed unnecessary long. David made it shorter and scaled it down and printed it again. This time the prototype was perceived too small for holding in the hand even though the mouthpiece was more suitable. The design was therefore settled on a mixture of these two alternatives. The mouthpiece from the smaller version was extended but the size was otherwise kept. The main part had the same size as in the bigger version but was shortened.
The idea of how to use the device is to place the fiber-filter in the opening, exhale through the device, pause for a minute, exhale through again and repeat this a few times. After that the fiber-filter can be sent to the laboratory facilities where the VOCs are extracted and added to the yeast. The color of the yeast cells are then closely watch and analysed, hoping that the yeast don’t turn red.
References
- [1] Fuchs P, Loeseken C, Schubert J and Miekisch W. (2009). Breath gas aldehydes as biomarkers of lung cancer. International journal of cancer. Journal international du cancer, 126. 2663-70. 10.1002/ijc.24970.
- [2] Fu X-A, Li M, Knipp R J, Nantz M H and Bousamra M. Noninvasive detection of lung cancer using exhaled breath. Cancer Medicine 2014; 3(1): 174–181
- [3] Minic J, Persuy M-A, Godel E, Aioun J, Connerton I, Salesse R, and Pajot-Augy E. Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening. FEBS Journal, 272(2):524-537, 2005
- [4] Suzuki Y and Shimono K. Deciphering the receptor repertoire encoding specific odorants by time-lapse single-cell array cytometry. Scientific Reports, 6(19934):1-9, 2015
