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Quicker, more efficient diagnosis method, using hepatitis #scichallenge2017


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This is my project for the #scichallenge2017. It's a quicker diagnosis method for poverty stricken countries using hepatitis to demonstrate how my method works. Blaise Cloran

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Quicker, more efficient diagnosis method, using hepatitis #scichallenge2017

  2. 2. MY EXPERIMENT My experiment is a quicker more efficient diagnosis for poverty stricken countries using the wonders of silk fibroin. It uses an ELISA test as the main form of diagnosis and silk fibroin to add other qualities such as transportation without refrigeration. Silk fibroin was also a successful base for my project as it had the perfect arrangement of particles and sheets, which I explain more in ‘the process of my experiment’. What basically happens is instead of antigens and antibodies binding in the wells that come provided in the ELISA kit, they instead bind on the silk fibroin, which allows them to soak more efficiently and be transported without any cooling. The diagnosis is very quick and is done under two hours depending on the illness, some may be shorter, but in this case when using the disease hepatitis it takes two hours. My hypothesis is, will a quicker more efficient way of diagnosis, stop the outbreak of epidemics in poverty stricken countries. Like I said earlier my experiment can be presented in two ways, the basic ELISA kit or on a square of silk fibroin, it is quite small which means it can be easily transported in large quantities. I went on to later develop my idea into a care package with wound care, diagnosis and tissue regeneration, all aimed to help developing countries who need cheaper and more efficient equipment. How it works is that, it will all be sent over to the targeted country, the materials (silk fibroin) for the wound care and tissue regeneration will be provided and can be used straight away, chemicals can also be sent over without refrigeration if they are put into the silk fibroin. Then lastly the ELISA test will be included and ready to use, there will be instructions that I have simplified
  3. 3. MY AIMS When I first started my experiment I wanted it to have a real world application and I wanted it to help. I had many small aims when in the process of research, testing and just about everything to do with my experiment and then I also had much bigger aims for the final result. My experiment was everything to me, I thought about it all the time and worked on it 24/7, so it was obvious that the aims I had set myself HAD to be achieved otherwise I would be distraught. My first aim was to build a proper picture of what I wanted my experiment to be. After about a month of research and a lot of trial and error, I finally came up with a plausible idea, but it wasn’t complete and ready for me to move on yet. My second aim was to draw reliable results that backed up my writing, which the ELISA testing and the mini experiments did. This gave my experiment a higher chance of being used in the world and it made it a lot more interesting to not only read about but to also do, I loved getting properly involved with the science of my idea, not just backing it up with theory. My third aim was for it to help, and by making my experiment for developing countries it defiantly is. The project is easy and works perfectly for those without lots of medical equipment. It made my heart swell when my idea had pieced together to achieve this aim. My fourth aim was to build on my idea and make it more interesting which the wound care and tissue regeneration helps me to do. It shaped my experiment into a care package that gives so much more medical depth and help towards 3rd world countries. And my fifth and final aim was to finish and make it perfect. I wanted a project that I could be proud of and one that was completed, with school work and other clubs I would sometimes leave my work for days and it was hard to start up again when I had reached a block but I knew that by achieving my final aim it would be more than rewarding.
  4. 4. ABOUT ME • My name is Blaise Cloran, I’m 13 years old and live in west Sussex. I’ve been crazy about science ever since I can remember! I love its logic and possibilities. • My favourite things are playing tennis with my friends and finding new information that never ceases to fascinate me. I’m a tutor and a competitive chess player, always trying to be better than I was yesterday. • I have been accepted into a NASA course and a surgery course and it was then when I really found my calling! After being at the surgery camp I was fascinated by medical discovery and started looking into what grabbed my attention most, diseases. I loved finding about epidemics and chronic illness but at the same time it was horrific to see the deaths they had caused, so decided I wanted to help. My good friend recently started studying medicine and I was in awe of her, I’m always looking for ways I can advance my school work and with her inspiring me I wanted to make something different and something that would make a difference, I was bored of pen and paper I wanted an idea, an experiment! • I spent forever looking into different epidemics and seeing how I could stop the death toll, I couldn’t cure them but I could help them from spreading. I stumbled across hepatitis and saw that the diagnosis takes 3-12weeks! By then its progressed into a serious epidemic, I did lots of research and saw that the disease focused on antibodies and antigens. I decided to create a quick, inexpensive diagnosis that can be transported without refrigeration. It’s also very simple!
  5. 5. STAGE 1 I’ve always loved science but naturally different topics have stood out to me more than others, especially disease. I was fascinated. The idea of tiny organisms wiping out thousands of people, gripped me, we learnt about antibiotic resistance, vaccines and pathogens in school and I decided to look more into the subject of bacteria. I loved reading books about disease, fiction and non-fiction, so from an early stage I had decided that I wanted to be an epidemiologist, which made my research all the more enjoyable. As I looked more into the growth and spread of disease I saw that crucial epidemics mostly spread because people can’t stop them, and this is because they don’t have the facilities or equipment to even try. That’s why developing countries have the highest death tolls when a new illness becomes apparent. I knew I couldn’t cure a disease, it would be too much of a challenge and I wanted an idea that I could actually carry out and test. Therefore I decided to look more into diagnosis, after researching current techniques I realised that diagnosis for many diseases is slow and takes a lot of expensive equipment. This is where I thought that using 3rd world countries as a base would be perfect, as that is where diagnosis is needed most. After finally deciding that I wanted to use diagnosis as my experiment idea I started to look very closely into how I could create a new diagnosis method, I looked into syringe injected chemicals but unfortunately I had no clue where to start with this. At this point I thought that maybe things would get easier if I focused on one particular disease instead of spreading myself too thin. I wanted an epidemic that effected thousands of people as that way I would achieve one of my first aims; to help. I spent many lunch times in the library looking at different illnesses but nearly all of them were too specific, making it extremely hard to even think about a possible diagnosis. It was driving me crazy not being able to find anything so my friends decided to help, we created the ‘who could find the deadliest disease first’ game, it was loads of fun, even though we did get some funny looks when one of us shouted out ‘chronic heart disease’. Even after five of us working together, I still couldn’t find my perfect disease, so I put I hold on it and hoped it would come to me.
  6. 6. STAGE 2 After deciding to put a hold on finding a disease for my experiment, I thought it would be best to go back and try to figure out a diagnosis method. The current diagnosis methods are long, take a lot of equipment and are expensive, this gave me lots of things to try and conquer. Another problem with the current methods are that they are extremely inconvenient for developing countries, these countries do not have the equipment or the facilities to even have a go at these current methods. This leads to serious diseases spreading like wild fire, I started to research in depths about current diagnosis techniques and this is what I found out, depending on the illness there can be many different types, blood tests which need sterilized syringes and test results can take more than a week to come back depending on the condition, the use of a blood test is also risky if not done by a trained professional as they needle has to be put in exactly the right blood channel, to avoid disruptions and invalid results. There are two main types of blood tests used in medical facilities, clinical blood panels include a basic metabolic panel or a complete blood count. Basic metabolic panel is a blood test consisting of a set of seven or eight biochemical tests and is commonly used here in the UK. This method is efficient for our country and other urban places but in 3rd world countries it doesn’t work as well. The complete blood cell count or full blood exam, is a blood panel that gives information about the cells in a patient's blood, such as the cell count for each cell type and the concentrations of various proteins and minerals, it can be also used for patients who may need a blood transfusion. This small insight into the common types of blood tests show that they are not just a needle going in, they are a lot, lot more. Some results are needed to be analysed on computer technology which is inaccessible for most run down places. I later went on to research scans, urine tests and lots more, most if not all had complications which meant I couldn’t just improve one I had to make my own, but that’s more fun right? It didn’t take me long to find out about ELISA tests, I have always been interested in enzymes and their protein capabilities, so after I looked into enzymes I soon found the ELISA method.
  7. 7. STAGE 3 This is the stage of my experiment where I look into depth about the ELISA tests and do lots of research on my hopefully new diagnosis method. When I first stumbled across the ELISA test I thought it was perfect and nearly stopped there. But after doing a bit more research I found out that they weren’t as simple as they seemed. An ELISA test is an enzyme-linked immunosorbent assay, it’s a test that uses antibodies and colour change to identify a substance, in my case a disease, and how it works is that antigens from the sample are attached to a surface. Then, another specific antibody is applied over the surface so it can bind to the antigen. This antibody is linked to an enzyme, and, in the final step, a substance containing the enzyme's substrate is added. The reaction produces a detectable sign, normally a colour change in the substrate. The kit uses the layout of wells as a base for the chemicals to be injected into. As the test is antibody and antigen based I needed an example disease to fit this criteria, this narrowed down the illnesses I could choose from. I looked harder into antigen and antibody related diseases and decided on hepatitis, hepatitis can be inflammation of the liver due to alcohol abuse or drugs and it can also most commonly be a virus. There are five main types of viral hepatitis, type A, B, C, D, and E. Hepatitis A and E are mainly spread by contaminated food and water. Both hepatitis B and hepatitis C are commonly spread through infected blood and can be passed on by needles. In 2015 chronic hepatitis B affected about 343 million people, which is shocking. The diagnosis takes 3-12 weeks depending on the cause and type, by then its developed into a serious epidemic. Hepatitis has specific antigens and antibodies present when infected with the disease, this makes it perfect for the ELISA format. At this point I had finally decided my disease and my technique of diagnosis which meant my project was becoming a clearer picture, I looked into getting hold of the ELISA test and found out it was extremely hard to find, and some of them were very expensive. I was heartbroken, if I couldn’t test my idea then I couldn’t finish it! I asked my school and some of my older, academic friends but none of them knew anything about getting hold of it. I kept trying, I rang industries up and asked loads of questions but I just couldn’t get hold of it, I lost hope for quite a long time and focused on other school topics until one day I decided to have a look on this website that sold research supplies and to my luck it had a hepatitis B antigen ELISA kit, for a reasonable price! I ordered it straight away and couldn’t wait to start testing.
  8. 8. STAGE 4 After ordering my ELISA test, I was extremely excited. I still had quite a bit of time to wait though before my test arrived and this was the point that I discovered silk fibroin and it’s amazing qualities. I thought that using the basic ELISA format wouldn’t have the array of aspects that I needed to reach my aims and make my experiment successful. So I looked into different bases which would add important and useful characteristics to my experiment. During my brain storming for my experiment idea I came up with other ideas such as wound care and tissue regeneration which I later added into my final project. From looking into all these things I studied a lot of materials, so I had a somewhere to start when looking for my ELISA base. I have always been interested in one of the strongest man made materials; spider silk. And I wondered if I could somehow incorporate this into my experiment, but after researching spiders silk wasn’t going to work, it wasn’t strong enough to support the chemicals. Never the less when looking into silk I discovered the protein silk fibroin. Silk fibroin is an insoluble protein found in silk created by spiders, the fibroin protein has many layers of beta sheets and its structure is made up of the amino acid sequence (Gly-Ser-Gly-Ala-Gly-Ala). The high glycine content allows for tight arrangement of the sheets, which contributes to silk's hard structure and strength. Because of this the material can be used in biomedicine and textile manufacture and is perfect for the use of my design. There are lots of recipes on how to make silk fibroin and if my experiment was ever needed in bulk it could be printed by a 3D printer. Not only this, but the silk fibroin possesses stabilising properties, and therefore reagents become chemically stable when mixed into the silk, which allows them to stay chemically active and that means that the chemicals can then be transported without refrigeration. When I discovered this I was over the moon! It was perfect, it made it even easier for my design to be used in 3rd world countries as now the facilities didn’t even have to have refrigeration sites. It also opened up a whole new aspect to my project which I could develop using this material. The chemicals could be transported without refrigeration too, and as my dad builds planes for a living I know how much of a hassle it can be trying to kept substances cold on flights, the manufacturer could be on the other side of the world and it wouldn’t be a problem.
  9. 9. STAGE 5 Once I had found out about silk fibroin there was no going back, I had found a vital component in my experiment, and everything was finally fitting together. I decided to start testing and finding out more about silk fibroin. I purchased silk cocoons and followed the recipe I found on the internet, it was quite long but I completed it eventually. After I had made the silk fibroin I didn’t really know what do with it as my ELISA test hadn’t arrived yet, I thought of putting apple juice on it and seeing if it stayed cold but I didn’t want to waste the little amount I had, so I left that idea till later. I decided to look into the colour regent aspect of my experiment as it would show the positive or negative result, I tested out food colouring on normal silk first just to see the way it stained, I then went on to look into universal indicators and made my own using cabbage, I looked at how both of these soaked into the silk fibroin and how well they stained. They didn’t have the right properties to be used with my idea and I decided to use the colour substance that binds in with the substrate in the ELISA kit instead. Because I hadn’t used much of my silk fibroin I went ahead with the apple juice idea, I bought commercially available apple juice and stored it in the fridge overnight to make sure it was defiantly cold, I then, the next morning I took it out and placed a sample on the silk fibroin, I measured the temperature with a thermometer and recorded it. I next left it for a couple of hours, when I came back to check the temperature, to my delight it was exactly the same. To make sure I had left it for long enough I kept it the same overnight, and when I came back in the morning nothing had changed. I was pleased that the apple juice had become chemically stable and that it had ‘broken the cold chain’, this meant that I could put faith in the silk fibroin. To keep the mini experiment a fair test and safe, I kept all conditions the same and took safety precautions, this included wearing gloves.
  10. 10. STAGE 6 This is the best stage of my experiment by far! The actual testing of the ELISA kit, I was extremely excited when it arrived, I had waited and searched for months to get it. I was still worried that I would open it and everything would be destroyed but thank gosh it wasn’t. I put it straight in our fridge becau e it had to be stored between 2-8 degrees and started reading the manual and brief. It was quite complicated so I decided to wait a few days before I started any testing. After collecting other bits and pieces that I needed for the experiment, I decided to test it the following Sunday morning. The first step was letting all the chemicals reach room temperature, which meant letting them sit for about half an hour, while I prepared the wells. The kit came with two sets of one hundred number stickers and I needed to use six, three for the negative tests, two for the positive and one for the blank. Next I added 50ul of positive control, and 50ul of negative control to their wells and the HBsAg specimen. And then I added 50ul HRP conjugate to all of the wells except the blank. And mixed gently by tapping the plate. After this I put the test into a water bath at 37 degrees for 60 minutes. . When the test had finished in the water bath, I washed each plate five times with the diluted wash buffer, each time allowing each well to soak for 30-60 seconds, before turning the plate down onto blotting paper to get rid of any remainders. Next I added 50ul of chromogen A and 50ul of chromogen B to each well including the blank, and then I mixed by tapping gently. After mixing I put the test into the water bath for 15 minutes at 37 degrees, covering with a lid to avoid light. When I took the test out of the water bath the positive wells had turned blue, this was because of the enzymatic reaction between the chromogen solutions and the HRP conjugate. To stop the reaction I dispensed 50ul of stopping solution into each well. The positive wells turned yellow at this point. After completing the test I went on to measure the results, I calibrated the plate reader with the blank well the read the absorbance at 450nm. In the concept of my design a colour regent will be used to measure the wells and the next step that I did is unnecessary, but even though I saw the colour change in the positive wells, I needed to do some more calculations to make sure it was a fair and accurate test. If needed this can be done when using my design. To measure negative results, specimen giving less than the cut off value (C.O.<1)show that there is no hepatitis B present and that no surface antigen has been detected. To measure positive results, specimen giving an absorbance equal or greater than the cut of value (C.O.>_1), indicate that hepatitis B virus surface antigen has been detected using DAI HBsAg ELISA. If specimen have absorbance of cut off ratio between 0.9 and 1.1 are considered border line and more testing is needed. My results showed the three negative wells to be negative with less than the cut of value and my positive with differing equal/more than the cut of value. The results were very pleasing and shows that my method is accurate.
  11. 11. STAGE 7 Once I had finished the ELISA testing I had basically finished my project, but I wanted to expand it and make it a broader, more diverse topic of research/experiments. By this stage I had gotten into the final of the big bang fair, and I was very, very excited and all I wanted to do was work on my project. I decided to look more into the amazing properties of silk fibroin as I couldn’t get over the amount of use this protein had. Just to recap, silk fibroin is an insoluble protein found in silk created by spiders and the larvae of Bombyx mori. If you look back to the previous stage four, I talk a lot about silk fibroin. I have always been fascinated by tissue regeneration, it’s a futuristic part of medicine and that’s why I love it. I focused on tissue regeneration in humans as that is an ever evolving topic, and one that could contribute to the help that my experiment is giving to developing countries. Tissue regeneration is complex and a procedure that needs everything to be exactly right, and finding the right material for the new tissue isn’t easy. Regeneration of tissues using cells and scaffolds is a perfect approach in the repair of tissues and the treatment of organ failure. My proposal is that silk fibroin could be an effective scaffold in this process, silk fibroin has lots of strength, elasticity and mammalian cell compatibility, and all of these make silk fibroin a possible material for tissue regeneration. Tissue regeneration requires a material that mimics the old cellular tissues, other materials such as polymers and metals are commonly used for tissue scaffolds but they have limitations, which opens a gap for a new material to be used. Because protein is already a component of natural tissue it is a safe choice when looking into scaffolds, that’s what drew me to silk fibroin in the first place, the temperature control aspect of silk fibroin could also be extremely useful and open up new roads for the treatment of organ failure. Silk is also a material that doesn’t become easily infected and is easy to clean, both of these things are vital for this kind of process. Furthermore silk possesses a large molecular weight with other heavy modular hydrophobic domains, silk fibroin is made of a heavy and light chain linked together by a disulphide bond, this organisation of silk fibroin determines its biomaterial properties. Through my research and work I see silk fibroin as a perfect material for tissue regeneration and the treatment of organ failure, obviously I couldn’t test this theory as I don’t have access to organs and I’m not permitted to work on any living body part. But I tried the best with what I had to make a strong report, and think that by including it in my experiment it adds more depth. At this point I see my experiment becoming almost a medical care package for developing countries, it has a diagnosis system and the possible materials for tissue regeneration and wound care (which I will talk about in stage eight).
  12. 12. STAGE 8 In my previous stage I talked a lot about the wide variety of things that silk fibroin can do/help to do. And is this stage I go on to look at its other capabilities, as silk fibroin is a perfect component for biomedical engineering it made sense to look more into how it benefits that area and take that information away to look at how it could help something similar. Of course in the previous stage I had done lots and lots of research into its properties and how it helped tissue regeneration and the treatment of organ failure, after looking at my notes and screenshots of useful texts I decided on looking into wound care. Wound care is a massive part of daily medicine as crucial wounds have been rising in the last few years due to the ongoing mass of terrorist attacks and the population growth which leads to more cars on the road and therefore more car accidents. Chronic wounds are especially difficult to treat, they are wounds that take a long time to heal and are very painful. If they aren’t treated properly the patient can blead to death, this puts a lot of pressure on the medical team and the treatment that is needed to work. Silk has been known as a material that has good soaking properties and a material that helps the skin, it is often used as a natural face mask, because of silk fibroins mammalian cell structure it is a diverse material, mammalian cells are cells with no cell wall and are bounded by only plasma membrane, they are extremely detailed and hard to study. Silk fibroin also has no effect on the immune system. From my research and comparing others experiments I have concluded that silk fibroin does help wound care, its wonderful properties and b sheet arrangement allow it to reduce the wound size, increasing the healing rate. It is also possible that silk fibroin nanofibers could have an effect on cell attachment. When doing my research for this topic I looked at resent studies that showed that silk fibroin from Bombyx mori silk worms had a high cell attachment and growth of fibroblast cells compared with collagen. Collagen is the main protein in the extracellular space in many tissues that are present in animals. Therefore making the most abundant protein in mammals. Because of this information silk fibroin could promote collagen synthesis. As the process of collagen synthesis is a slow and detailed one silk fibroin used this way could cause a real breakthrough and could then be carried on in veterinary. To conclude, the use of silk fibroin as a blending material for wound care would be a huge help to medical treatment in that area, it would speed up the time of the skin healing and have no effect on the immune system. I have added this aspect of research to my project and along with the ELISA diagnosis kit and the tissue scaffold, I have created the perfect medical care package for developing countries, it’s not only a new diagnosis method it’s a whole new aspect of medical equipment and materials.
  13. 13. STAGE 9 Now we are into the final stages of my experiment and everything is piecing together, which means writing up the final documents and getting them ready for the fair. I ordered silk cocoons online and made a trip to my local hobby craft to get stand supplies and card to mount my work on. I really enjoyed picking out colours and designs, although I have no idea what I’m going to do with wooden ELISA letters afterwards though… After this I got thinking about maybe making a computer program for my results as I thought it would be a nice touch to my project and add a whole new element. I looked into some software and starting jotting down ideas and planning. I also wasn’t too sure at first how the silk fibroin part of my experiment was going to link to the ELISA test but after looking at its format (the ELISA test) I knew that it would be quite easy to replace the wells with the silk fibroin. I was really pleased when I found out that it was all going to work, I kept doubting everything and looking for faults but finally it looked like it was all going to be a suitable experiment.
  14. 14. STAGE 10 After finishing my ELISA test I had a feeling that I could do more with my project. I have always had a love for coding, and I know I enjoy it because I will start in the morning on a new program, look up and find it’s nearly ten at night! I started to experiment on a program called python, seeing what I could do and how I was going to present my idea. After a few attempts with trial and error I settled on a format that when you clicked run it would change the colours of the wells as if you were doing the experiment. My first step was producing the square ELISA base which was quite simple as all I had to do was set the y and x axis, after this I went on to fill the square with ellipses to represent the ELISA wells, that would eventually change colour. Next I set the base colour that was present in the first stage of my experiment and went on to make it change colour into the reds and pinks seen in the negative and positive wells, (which is all shown in the pictures of the code). It wasn’t easy to get the colours to change as changing one thing would change everything! After getting the pinks and reds I started on the later enzymatic reactions. It took a while to find the code for yellow or blue as I hadn’t used python much before, but after finding it I was nearly finished. It’s quite hard to explain the code as most of it you need to see to understand, but I have included a copy and pictures. As the frame count goes up it causes the colours to change, this creates an accurate representation of the real experiment. After finishing the code I decided to print out pictures of the program and code as I don’t have access to a laptop for the fair. You can also use a print function to print out the code in the black part at the bottom of the screen. If you multiply the frame count variable by another number, this makes the animation faster or slower for each ellipse.
  15. 15. REAL WORLD APPLICATION My whole experiment is designed to help. I really wanted to make a difference and the end result will! My project will help thousands all over the world who don’t have access to medical equipment or hospitals, it makes everything so much easier in the process of trying help developing countries. There no longer has to be refrigeration facilities for any medicine to be used as the silk fibroin will stabilise the chemicals, there also is no need for trained professionals or excessive use of equipment. My design is small, cheap and easy. Not only that but it’s quick too so the break out of epidemics will decrease, my care packages can be used in local hospitals or even homes. In places like Africa there are no fancy, sterilised units that villagers can quickly be treated at, people will que for hours just to be sent away, whereas with my package the time it takes to diagnose someone will be half, meaning that twice as many people can be helped. And bigger procedures such as tissue regeneration will maybe become applicable due to the use of silk fibroin. It’s also extremely easy for any wound to become infected in these areas, the silk fibroin can be used as a quick, effective dressing or, in larger cases, used to treat chronic wound care. I strongly believe that my care package is going to change the lives of sick and struggling people all over the world.
  16. 16. MEDICAL ADVANTAGES My experiment has lots of medical advantages, I spent months making sure it was perfect. The biggest one is that it allows what once was a complicated task, to know be quick and easy. It does this by allowing these tasks to be carried out without lots of medical equipment or trained professionals. This then goes on to free up time in the hospitals, which as we all know, is well needed. The silk fibroin that is also in the care package contributes to not only wound care, tissue regeneration and chemical transport, it contributes to quick dressings on smaller cuts, bandages, the use of cleaning medical tools, and pretty much almost just about everything! All these advantages will help save lives in so many countries, medical equipment is scarce in more places than thought about and it isn’t easy to raise money to get them sent over. And even if the area has everything they need but just want a quicker method then it can be used for that too. The ELISA test will allow for nearly all diagnoses to be done in the same way, which will help doctors do it quickly and accurately as there is no longer lots of different things to remember. Furthermore, my experiment can be used on planes and ambulances too, as it frees up space and allows for the chemicals to be transported without refrigeration.
  17. 17. PICTURES