Current rapid testing kits have been made to identify or recognize diseases by mixing a sample with a substance and creating a chemical reaction. These antibody and antigen rapid kits are revolutionising, with technologies that can provide results within a few seconds, however, these aren’t able to detect mutations, a main cause of many genetic diseases. On the other hand, rapid tests such as molecular rapid tests are made to identify a target genetic sequence. However, these tests are advanced, often requiring high-end laboratory equipment, preventing them from being made into a kit such as something like an antigen test. Despite the complexity, most genetic related tests have proven to be the most accurate and they’ve become an extremely useful way to test for many genetic disorders. Molecular rapid tests are essentially the first step to meet the goal of our project. If a “target gene” identifying kit could be manufactured at a cheap cost and produced in large quantities many patients would be saved due to the early detection of a disease.

              As of current day there are over 7000 genetic diseases, many of which could end up being fatal and severely harming a patient. Among these disorders is sickle cell disease, where red blood cells begin to take the shape of a crescent or sickle and increase its vulnerability and chances of being destroyed. This lack of red blood cells is called anemia and it (especially when inherited) often leads to death among other things including heart failure, increased blood loss, and increased heart rate. If the disease becomes severe the only way to cure it is through a stem-cell or bone marrow transplant that is incredibly risky and not recommended unless the disorder is extreme. However, when sickle cell disease or anemia is detected soon it can be cured through vaccines, blood transfusions, gene therapy, etc. This shows why early detection is crucial, not only for this genetic disorder, but almost all. Sickle cell disease is commonly caused by a defective hemoglobin protein (what carries the oxygen and CO2 in red blood cells) or specifically a mutation in the beta globin gene/HBB gene. With the rapid testing kit we are attempting to create, we will hopefully be able to detect the mutation without screening necessary. Our findings could then be applied to other genetic disorders and potentially lead to early detection for diseases becoming common.

              Overall, our project aims to design a rapid testing kit, using simple technologies or advanced methods scaled down, that would be able to detect sickle cell disease early in patients. We are hoping that with our new technology, all patients carrying sickle cell disease will have it detected before it becomes highly severe and their chances of survival will vastly increase. Additionally, we are intending for our genetic rapid testing kit to become usable on other diseases meaning that it would eventually become a versatile device that can save patients with several different heritable disorders.

 

Research:

What is a rapid testing kit and how do they work?

               A rapid testing kit is a medical tool that can quickly assess a person for a condition or disease. These kits involve the use of a sample and typically some form of chemical reaction to display the results. There are 3 types of rapid tests: antigen, antibody and molecular, each of which are used to identify different things that would result in a disease. 

               Antigens are small structures made of proteins, peptides or polysaccharides that stick off of all cells and pathogens. Peptides are short chains of amino acids or the bases of proteins and polysaccharides are the bases of carbohydrates. B lymphocyte cells are special types of immune cells that recognize foreign antigens called non-self antigens. Each antigen has a unique structural shape that the cells recognize using B cell receptors (BCRs) on their membrane. Once an antigen is acknowledged to be a non-self antigen, several antibodies/immunoglobulins are made and released to attach onto the specific antigen it was made for, until more help can come. The immune system then sends T lymphocyte cells and a chemical called cytokines that kill the antigen forever. Antigens are the reason you can’t get blood from other blood types, since your body won’t recognize the antigens as self antigens and attack the new cells. However, they’re still incredibly useful to let your body know of a pathogen and save you from many diseases. An antigen test works by examining a bodily fluid like blood or saliva for the antigens of a virus. The sample is mixed with a small bit of liquid to suspend it and create the analyte. This is then placed in the cartridge of the antigen test and absorbed up the testing strip. The analyte is what hydrates the nitrocellulose test strip and activates the antibodies. The test strip is made of nitrocellulose membrane as it immobilizes proteins and allows for the antibody to look for antigens. There are 2 types of antibodies on the strip, one is mixed with a component that will visually display the results such as gold colloid, and the other (known as a test antibody) is immobilized along the test line. As the analyte flows past the test lines, any target antigens will bind with the test antibodies, and the free flowing antibodies (those mixed with the colloid) will be caught at the same spot. If enough gold colloid antibodies get stuck at the test line it will change colors, providing a positive result.

               The second type of rapid tests are antibody testing kits that look for target antibodies specific to antigens of a certain virus or pathogen. Antibodies are what the immune system produces when a non-self antigen is detected, and they are what destroy antigens. Antibodies are made using a long process that starts with the B lymphocytes first detecting the antigen. As previously mentioned the B cells then attach to the antigen using BCRs or a type of membrane bound immunoglobulin. Then with the “approval” of T lymphocytes, the B cells start a process called clonal expansion where they divide and clone into plasma cells (a type of white blood cell). These plasma cells contain the DNA which is used to make antibodies. The DNA is split and read by RNA polymerase in a process called transcription to make mRNA. The mRNA is then sent to the ribosome where it is read 3 bases at a time, where the corresponding amino acid is released and a chain of amino acids is produced. This chain is folded into a complex shape in the golgi apparatus or ER that is the protein or antibody. An antibody consists of 4 polypeptide chains, 2 light chains and 2 heavy chains connected by disulfide bonds to create the Y shape. The light chains are either kappa or lambda (differentiated by their amino acid sequence) and the heavy chains are made up of one of the 5 types of immunoglobulin; IgA, IgG, IgM, IgE or IgD. A rapid antibody test works by looking for target antibodies that correspond to a pathogen of a specific virus in the sample. These tests are used to see if a patient already has a virus and has developed antigens to fight against it. Typically the tests have 3 test lines, depending on how many antibodies are being searched for and what the virus is, the “C” line or control line, the “G” line which looks for IgG antibodies and the “M” line which looks for ImM antibodies. Once the sample is placed in the cartridge, the nitrocellulose filters the solid parts of blood from the liquid parts, allowing the plasma to flow through. Nitrocellulose is incredibly useful as it filters the sample, is extremely absorbent and therefore consistently spreads the sample, can easily bind to biomolecules placed in the test, and can immobilize specific proteins. Once the sample is filtered any present antibodies will bind to corresponding antigens carrying gold colloid in the test. The new compounds will then flow to the test lines and be captured by monoclonal antibodies (mAbs) or laboratory-made antibodies. If enough antigens are present at the test line then the gold colloid will become visible. If IgM antibodies are present that means the virus is recent since they are your body's immediate defense against pathogens, but if IgG are present then it’s likely the virus isn’t new. The control line will become visible if the test is correctly executed and it’s properly working: usually works by immobilizing a certain protein.

               The third type of rapid test is a molecular one that works by detecting the RNA or DNA of a specific pathogen. Molecular rapid tests use different technologies including PCR, CRISPR and LAMP, however these haven’t been developed into a “kit” and require clinical testing. 

PCR (polymerase chain reaction) is the most common method used which works by amplifying the target DNA. This process contains 3 main steps per cycle (each cycle every strand of DNA is duplicated); denaturation, annealing and elongation. First, the sample, primer, nucleotide bases, and polymerase are placed into the thermal cycler. Once inside, denaturation takes place, which is when the temperature rises to from 90-95°C causing the DNA to separate into 2 strands. Then annealing occurs and the temperatures decrease to 50-60°C allowing the primers to attach onto the corresponding separated strands. A primer is a small chain of nucleotides (usually about 18-25) that attach onto the target spot of DNA. Not only do the primers detect the target DNA, but they create a starting point for the polymerase to start. Next elongation takes place and temperatures rise to 70°C which is the ideal temperature for polymerase to synthesize DNA. The Taq polymerase (a polymerase naturally adapted to high temperatures since it’s found in the hot springs) attaches to the 3’ end of the primer, building a new strand in the 5’ to 3’ direction with excess nucleotides, using  the original DNA as a template. Once 2 daughter strands of DNA are made the cycle ends, and the second cycle begins with denaturation again. This process repeats until enough copies of the target DNA are made for scientists to be able to analyze them. PCR is similar to how DNA is replicated in the body except primers are made by an enzyme called primase and the DNA is separated into 2 strands by an enzyme known as helicase. 

               The second type of technology used in molecular testing is CRISPR which is commonly used for gene editing, but can be used to detect certain DNA. CRISPR was inspired by a naturally occurring process used in bacteria to fend off reappearing viruses. It starts by having CAS proteins (CRISPR associated proteins) cut a spacer segment from the viral DNA and embed it into the host's DNA. CRISPR (clustered regularly interspaced short palindromic repeats) is used to indicate regions of DNA where the top strand will read the same forwards (in base pairs) as the bottom strand reads backwards, however these palindromic segments and interspaced meaning they have spacer parts throughout the strand which is what is put in the bacteria’s DNA. Then the DNA undergoes transcription in which RNA strands are made. Then microbes cut the RNA strands into shorter parts called crRNA and tracrRNA that connect and attach themselves to CRISPR associated proteins such as CAS9, which makes straight cuts, CAS12, which makes staggered cuts, and CAS 4, which cuts RNA. This protein then looks around and connects to free-floating genetic material until it finds something that matches the virus. The protein, acting as molecular scissors, finally cuts the DNA and destroys the virus. Scientists have been able to combine crRNA and tracrRNA to make a guide RNA that directs the CAS protein to any target nucleic acid. Researchers also discovered a CAS 13 protein that has “collateral cleavage” meaning it cuts a nearby piece of RNA instead of the target one. They then adapted this protein and developed a nucleic acid detection technology called SHERLOCK where the CAS 13 cuts a nearby RNA reporter instead of the target. Typically the report RNA is mixed with a fluorescent substance so once it’s cut it will release a detectable fluorescent signal. 

               The other common molecular testing method is LAMP or loop-mediated isothermal amplification. This process works similar to PCR, as a polymerase enzyme amplifies the DNA, however it’s done at a constant temperature about 60-65C (making it isothermal). In LAMP a strand-displacing polymerase is used, typically Bst DNA polymerase, which splits the DNA as it synthesizes it, meaning it isn’t necessary for the temperature to increase. LAMP also uses 4-6 primers, the 4 primers typically used are the forward inner primer, the forward outer primer, the backward inner primer and the backward outer primer. Additionally there are 6 regions on the target DNA that the primers use to bind to; F3c, F2c, F1c, B1, B2, B3, in the 3’ to 5’ direction. The “F” stands for forwards, “B” for backwards and “c” for complementary so F1c is complementary to F1, B1c is complementary to B1and so forth. Firstly, the forward inner primer attaches its F2 region to the F2c region of the 3’ to 5’ strand on the target DNA and the polymerase starts synthesizing a new strand. As this happens the polymerase displaces the second strand of the DNA for the same process to occur. Next the forward outer primer similarly binds to the strand and displaces the product made from the first primer as it creates a new strand. Then this whole process is repeated on the product strand, but backwards, starting with the backwards inner primer and moving onto the backwards outer primer. The structure of the two inner primers create a complementary overhang for the F1 and B1 regions of the final product, and this creates two loops on either end of the strand or a short dumbbell shape. At this point several replicas of the target DNA have been made with only the final product having a loop structure. As the process repeats, more complex structures are created with various loops. Overall, LAMP is still quite a new process that has several advantages over PCR, being faster and occurring at a constant temperature. Once enough copies of the target DNA have been synthesized, amplification of a strand can be detected through ph indicators or fluorescent dyes.

 

How does genetic testing vary from other disease testing?

               Genetic or molecular testing focuses on looking for specific nucleic acids, such as RNA and DNA, related to a disease, whereas most other testing looks for antibodies, antigens, or pathogens of a disease. In genetic testing, blood, saliva, hair, skin or other tissues are examined to look for changes in their genetic material. Changes or mutations in genes can result in various types of genetic disorders which is why these tests are extremely important. They can diagnose a genetic/inherited condition, help start safe treatments, allow an individual to know what may be harmful to them, and aid in planning ahead for future family. A single mutation can affect a part of a gene or a whole gene and may lead to various malfunctioning tissues or cells. A mutation is a change of nucleotide sequence in a DNA strand that can occur during cell division, transcription or due to exposure to mutagens. Our body is usually able to repair mutations or turn off the mutated gene with specialized enzymes, but sometimes these mutations go on to produce abnormal or no protein. Since these mutations can be heritable and cause disorders, genetic testing is incredibly important. There are 3 main types of genetic testing; cytogenetic, that examines whole chromosomes, biochemical, to examine protein made by a gene, and molecular, to search for specific mutations in a target DNA sequence.

               Cytogenetic tests examine chromosomes to determine if any of them have structural abnormalities or an unusual amount of chromosomes (aneuploidy). Aneuploidy is when a cell has extra or missing chromosomes, leading to disorders including Down syndrome, Edward’s syndrome and Turner syndrome. Structural abnormalities of chromosomes include duplication where a part of the chromosome is repeated, deletion where a part of the chromosome is missing, translocation where information from two chromosomes are exchanged, insertion where information from another chromosome is added and finally inversion when a part of a chromosome is inverted. A common method used for cytogenetic tests is fluorescent in situ hybridization (FISH) that uses a probe mixed with fluorescent dyes to attach to a target DNA sequence. This technique can be incredibly useful however it does not examine all chromosomes and identifies a specific complementary sequence, limiting its abilities.

               Biochemical tests study unusual enzymes or proteins in the body made by certain genes. They would, for instance, look for an enzyme that’s typically missing in individuals with a certain disease. With a sample of blood, urine, spinal fluids, amniotic (the innermost membrane around an embryo) fluid, or other tissue samples, scientists can search for dysfunctional proteins or the mutated genes causing them. They can be found through electrophoresis when an electrical charge is passed through a matrix, typically a gel substance, and then the samples are placed inside wells at the negative end. As a current passes through the gel, the charged smaller molecules migrate farther through and larger or uncharged molecules won’t move as much. The samples are mixed with fluorescent dyes such as ethidium bromide before being loaded, so after the process is done, different bands can be seen through the gel. The different bands help identify absent or abnormal enzymes, based on their size and conductivity. Biochemical tests also include enzyme assays that can determine the absence or presence of a certain enzyme in a sample as well as the amount of the enzyme. Enzymes are a type of protein used to speed up the reaction process of turning substrates into products by “facilitating” the chemical reaction and reducing activation energy. Unlike other proteins or nucleic acids, enzymes can be detected through the acceleration of their reactions and the amount of product they create. Enzyme activity depends on the temperature, ph, nature, and the strength of ions, meaning that due to diversity and needs of different enzymes, enzyme assays are typically only capable of testing for specific enzymes. Essentially, enzyme assays work by determining the amount and speed of activity of an enzyme in their optimal conditions. This is done using various techniques such as spectrophotometry and fluorometry to measure the substrate and product concentration throughout a period of time, allowing for the quantification and identification of an enzyme.

               The third type of genetic tests are molecular tests, which search specifically for mutations in RNA or DNA that may cause a disease or disorder. These include advanced technologies including CRISPR, LAMP, and PCR, involving amplifying and searching for target DNA sequences (explained in paragraphs 5, 6, and 7 of question one).

               Testing made for other diseases tends to search for symptoms, or parts of pathogens like antigens and antibodies. On the other hand, genetic testing looks for mutations or abnormalities that may result in a disease. Genetic tests such as FISH, gel electrophoresis and PCR identify inherited conditions and predict possible diseases someone can obtain due to a mutation. Advantages of genetic tests include being highly accurate, providing insight into what treatments will or won’t work, and identifying the exact cause. However, they have limitations as well, being expensive and unable to detect environmental causes. Similarly tests made for other diseases, that look for symptoms and pathogens, have their own advantages; quickly and affordably diagnosing, being able to monitor progression of the disease (in some cases), and detecting diseases caused by various things. Disadvantages of other disease testing includes not identifying genetic causes, as well as not being able to identify potentially harmful mutations.

 

What methods have been developed for sickle cell disease testing and why haven’t they been developed into a rapid kit?

               Several methods have been developed to test for sickle cell disease including 4 of the main ones; hemoglobin electrophoresis, SickleDex (chemically testing), PCR and DNA sequencing, and high performance liquid chromatography (HPLC). These haven’t been made into rapid testing kits because of their complexity and price. Many of these require laboratory facilities and expensive equipment meaning it isn’t feasible for them to be made into low-cost tests that can be used at home. Along with this the cheap tests aren't as accurate and to get consistent, accurate results advanced techniques are necessary. Another challenge to overcome is that even if these tests were available for a cheap price they may not be safe to conduct in a household setting without scientific supervision or proper protection. Additionally, sickle cell disease lacks a biomarker like antigens, which most tests can easily detect, making it even harder for these kits to realistically be formed into an at-home rapid testing kit. 

               The first method used for sickle cell testing is hemoglobin electrophoresis, a test used to measure levels and types of hemoglobin in the body. Hemoglobin is a protein found in blood used to transport oxygen and waste to and from different tissues. Hemoglobin is made of 4 chains, 2 beta and 2 alpha, each with heme groups for iron, oxygen and waste to bind to. Sickle cell disease is caused by a mutation in one of the beta polypeptide chains leading to a type of hemoglobin known as hemoglobin S (HbS). This is typically absent in the body, and can be detected by putting a blood sample through gel electrophoresis. Gel Electrophoresis is one the most frequent ways used by scientists to compare 2 or more fragments of macromolecules using forensic science. A macromolecule is a large molecule such as types of protein, RNA and DNA. Gel Electrophoresis is a process, in which you insert a macromolecule into a tray of agarose gel and compare the difference between how far the pieces of the macromolecule traveled. When placed inside the electric field the hemoglobin will be attracted towards the positive electrode, since hemoglobin is naturally negatively charged because of its amino acids including glutamic and aspartic acids. When you place the hemoglobin in the agarose gel and connect it to a circuit, the molecules will move to the positive end, as long as the gel contains a buffer solution. The buffer solution keeps the gel conductive by carrying ions through it and maintaining a pH or an acidic level. Agarose gel has microscopic holes in it which filter the hemoglobin meaning that at the end, the smaller and more positively charged segments of hemoglobin will have traveled farther than the larger, more negatively charged segments. Since hemoglobin is red in color a loading dye isn’t actually necessary to make the sample visible, however, it weighs down the sample keeping it near the bottom of the gel and away from the surface. Usually, dyes like xylene cyanol and bromophenol blue are used, as well as glycerol or sucrose to add extra weight. The results will be bands, and the distance of each band determines the type of hemoglobin present. The Hbs mutation causing sickle cell disease is when adenine replaces thymine in condon 6 of the beta globin chain, making the normal glutamic amino acid get replaced by a valine amino acid in position 6. Valine acid is less negatively charged glutamic acid, which is why during hemoglobin electrophoresis the HbS protein would travel a lot less farther than the HbA protein. The reason these haven’t been developed into a rapid test kit is due to the amount of time it takes (usually hours), requires costly equipment, and human interpretation may be incorrect. 

               The second method used for testing sickle cell disease is SickleDex that works by mixing a blood sample with a chemical solution where red blood cells containing hemoglobin S clump together and become visibly cloudy. The test first uses saponin, a chemical commonly obtained from plants to lyse the red blood cells or disintegrate their cell membrane. This is because saponin has 2 main parts hydrophilic (water attracting) and hydrophobic (fat attracting), and the hydrophobic part likes to bind to cholesterol. Cholesterol plays an important part in stabilizing the lipid bilayer part of a cell membrane and so when the hydrophobic section reacts with the cholesterol, the cell membrane is disrupted and pores or micelles are created. These pores allow ions, water molecules, and hemoglobin to flow through the cell creating an osmotic imbalance, leading to more movement and eventually the rupture of the membrane. Hemoglobin as well as other molecules being released into the surrounding area is known as hemolysis. Once the hemoglobin is released, sodium hydrosulfite then reduces it by giving it electrons since it’s a reducing agent. A reducing agent is a chemical species that gives its electrons to an oxidizing agent, which in this case is the iron in the heme group. For the HbA nothing occurs, but for the HbS mutation the molecules start to polymerize. In HbA the iron is kept the same and nothing changes in the structure of the hemoglobin unless it started off as ferric (Fe³⁺) in which case it would be reduced to the normal Fe²⁺. It’s different for the HbS molecule for several reasons. Firstly, hemoglobin has 2 conformations; the relaxed (R) state where it easily binds to oxygen, and the tension (T) state where it’s more likely to release oxygen. As previously stated HbS is a mutation in the beta chain of a hemoglobin molecule in which glutamic acid is replaced by valine acid in position 6. This causes a hydrophobic patch on the surface of the hemoglobin molecule; the hydrophobic patch makes the molecule want to interact with other hemoglobin S molecules. When in the R state HbS molecules bury the hydrophobic patch, therefore preventing polymerization, when in the T state however HbS molecules release oxygen making them deoxygenated and bringing the hydrophobic patch to an exposed location of the surface. This makes the molecules prone to polymerization and causes the HbS molecules to stick together. The sodium hydrosulfite promotes polymerization by “facilitating” the interactions between different HbS molecules. The collecting of the hemoglobin causes the solution to become cloudy, resulting in a positive test, whereas the solution would be clear with HbA. If placed on paper the positive results will have a centered area of molecules, while a negative result wouldn’t. These haven’t been developed into a rapid testing kit because it’s more of a screening device and would likely need further testing (since it’s difficult to differentiate HbAS and HbSS or trait and disease) as well as it’s hard to obtain consistent results especially in unreliable environments. 

               These are just 2 of the many ways developed to test for sickle cell disease, but most of the remaining methods require advanced technologies and laboratory settings. It wouldn’t be realistically possible to develop these rapid testing kits that can quickly, easily and simply be used at home.

 

What is sickle cell disease?

               Sickle cell disease is an inherited blood disorder caused by mutations which leads to irregular red blood cells. Normal red blood cells are round and are able to move freely all over the body. SCD is where they are shaped like sickles or crescent moons, the blood cells then clump together and can’t move within blood vessels, blocking blood flow, causing pain and infections. SCD complications occur because it blocks blood flow to specific organs which can lead to worsen complications like strokes, acute chest syndrome, organ damage, disabilities and even in some cases, premature death. The disease is seen mostly in non-hispanic black, african american, mediterranean descendants, and asians. 

               SCD is caused by a mutation in the HBB gene. The HBB gene gives the directions for creating a protein called the beta goblin, a component of a larger protein called hemoglobin located in red blood cells. The mutation leads to the production of an abnormal form of hemoglobin, hemoglobin S (HbS). Beta Globin is a chain of 147 amino acids, in sickle cell anemia, the gene is mutated, one base dna changes from A to T, resulting in the protein still consisting of 147 amino acids but because of the single base mutation, the 6th amino acid is valine rather than glutamic acid. Valine is an essential amino acid that helps with muscle repair and energy. It has a non-polar, hydrophobic side chain; valine is found in foods like meat, fish and beans. Glutamic acid has a polar, acidic side chain. It’s non essential meaning your body can produce it, glutamic acid acts as a neurotransmitter, helping nerve cells in the brain to send and receive information. Valine is hydrophobic which disrupts the structure of the protein, causing hemoglobin molecules to stick together, forming fibers that distort the red blood cells into sickle shapes. There are 4 types of SCD, Sickle cell anemia(SS), Sickle Hemoglobin-C Disease (SC), Sickle Beta-Plus Thalassemia and Sickle Beta-Zero Thalassemia, Sickle cell anemia being the most common. 

               Sickle Hemoglobin-C Disease is a mild form of sickle cell anemia, the disease causes pain because red blood cells break down faster than they should. Usually, blood cells live up to 120 days, but sickle cells die within 10-20 days. Symptoms of this disease include, Anemia, pain, infections, eyes and spleen. You could be low on blood which may lead to exhaustion or weakness but anemia isn't a big problem. Children with Hb S/C disease have higher chances of pneumonia, where there is an infection in either one or both air sacs of their lungs. The air sacs are filled with fluid or pus which leads to coughing, fevers, chills, and trouble breathing. Pneumonia is very serious towards infants, and people older than the age of 65, health issues or weakened immune systems. 

 

How would early detection benefit sickle cell disease patients?

               Sickle cell disease that is not a curable disease but can be managed. Early detection in sickle cell disease can be highly beneficial to the individuals and lower any risks of complications. SCD can worsen over time, so it’s best that it’s identified as soon as possible, if a child is diagnosed early, they can know how to manage that disease, taking medications, and avoiding any activities that may trigger life threatening catastrophes. Early detection can reduce the severity, and probability of infections, and other diseases, such as, the flu, pneumonia, meningitis, strokes, acute chest syndrome,and organ or tissue damage. Although it can not completely prevent those things happening to the individuals, it can reduce the chances,  

               When SCD is detected later, it can be harder to manage, and more severe problems can arise. Organs such as the lungs, spleen, kidney and heart can be damaged as well as vision loss or problems. The spleen filters blood, removing old cells, and produces new blood cells, protecting the body from infections. If it’s infected, it can result in an enlarged spleen, known as splenomegaly, which increases the risk of more infections and reduces the amount of red and white blood cells in your bloodstream. Blocked blood vessels can also lead to strokes, scientists have found that children have a much higher chance of getting strokes from blood blockage than adults. So it’s important diseases are detected as early as possible.  It can also lead to kidney issue damage over time, 

               When detected early, they get vaccinations and antibiotics, some including flu vaccinations, Haemophilus influenzae type b, Pneumococcal vaccine, Hepatitis A and B vaccines, and penicillin, which helps fight against viruses. Penicillin is an antibiotic which kills off bacteria by breaking down their cell walls, but Penicillin does not work on viral viruses like a cold or flu. Early detection also ensures proper nutrition, growth, and guidance for the individuals. There will also be less pain, lower chances of infections or organ failure and it also increases the patients life expectancy.

 

What is the most common way to identify mutations and why are they so common?

               There are various ways of identifying mutations, some including, DNA sequencing, DNA hybridization, Restriction enzyme digestion, Allele-specific Polymerase Chain Reaction, Allele-specific amplification, and Ligation. All of these are easily able to detect mutations, and are the most common methods used by scientists to detect mutations.

               DNA hybridization is a technique used to join single strands of DNA together to form a double-stranded molecule. It occurs due to the specific pairing rules, A-T, C-G, it allows for scientists to identify and analyze specific sequences. It works by taking double-stranded DNA from two samples, then allowing the single strands to re-annel into double stranded DNA. DNA re-annel is the process of reforming a double-stranded DNA molecule from single strands that have been separated. The stronger the hybrid strands are, the more similar the original DNA samples were. DNA hybridization helps detect mutations by using a DNA probe, which is a single stranded DNA or RNA segment that is used to find specific sequences, that is designed specifically for a target sequence, if there is a mutation existing in that sequence then the probe won't bind as much or not at all, allowing scientists to identify the mutation based off the binding strength compared to normal sequences.

               Restriction enzyme digestion is a process that uses enzymes to cut specific DNA sequences. The enzymes cut the DNA into a specific sequence, they act like scissors, once the enzymes find the sequence, it breaks the phosphodiester bond the DNA backbone at specific points , creating DNA fragments A nucleoside is a combination of a sugar and a nitrogen base, when it’s attached to a phosphate group, its referred as a nucleotide. The phosphate group is made of phosphoric acid, (H3PO4), when it’s attached it will result in a loss of two hydrogen atoms. When a phosphoric acid reacts with two hydroxyl groups (-OH) of two other molecules, the phosphate group loses gen atom, (H), a water molecule is released (HO), then two ester bonds are formed. Phosphate bonds are usually found in the backbone of DNA or RNA. After creating dna fragments, it can be used for research purposes, gene cloning, or dna fingerprinting. When cutting the enzymes, if there was a mutation in the sequence, the enzyme would not be able to cut that area, resulting in a different DNA fragment pattern when analyzed. 

               Polymerase Chain Reaction is a method where it copies a specific DNA segment, it’s an incredibly fast and efficient way to make billions of copies of DNA samples. There are multiple steps to this method, first, is denaturation, where DNA is heated to high temperatures breaking the hydrogen bonds, separating the double strands into single strands. Then is 

               Annealing, when the temperature lowers, allows the primers, (single-stranded DNA sequences) to bind their sequences onto the single stranded DNA. Next is Extension, the temperatures are slightly raised, allowing it to add the DNA strands by adding nucleotides from the primer. Allele specific Polymerase Chain Reaction (AS-PCR) is a mutation method using PCR. It works by using two primers, one that binds to the normal DNA, the other binds only if a mutation is present. If amplification occurs with the mutation specific primer, then there is a mutation.

 

Design 1:

Our first model is similar to that of a SickleDex kit, using a solution combining saponin and a non-toxic substitute for sodium hydrosulfite to be mixed with the sample and display results. If HbS is present then it should precipitate in the solution or cause it to become cloudy because of its structure and non-polar characteristics that make it clump together. If HbA is present in the sample, meaning that the customer has no risk of having the disease, then the solution will remain clear because glutamic acid is already protonated and when placed in an acid it becomes neutral. Along with this glutamic acid has a different side chain structure than valine, so it doesn’t have the hydrophobic patches that valine uses to precipitate in the solution. 

This graph shows how we’d expect the results to turn out if we were to repeat the test and rank the visibility of each solution from a scale of 1-5, with one being barely visible and 5 being no difference in the visibility. Our prediction was that the solution would become cloudy and hard to see through when the HbS positive sample is placed in and it would become slightly harder to see through when the HbA negative sample is placed in because of the red color from the hemoglobin.

We designed this kit to be composed of 2 main parts, the solution, and the testing tube. 

The first part is the solution needing 2 substances, one to initiate lysis and the other to act as a reducing agent for the hemoglobin molecules. For the substance initiating lysis we decided on using saponin, a relatively cheap compound that breaks apart red blood cells. We found a couple resources varying from prices around $1.50 per gram to around $0.50 per gram. Based on our research hemolysis can be detected with a minimum concentration of 6µg/mL (or 6e-6g/mL) with full hemolysis occurring at 12µg/mL (or 12e-6g/mL). Since we want to make sure that the saponin will fully lyse all the red blood cells, but we also don't want to waste money on unnecessarily concentrated solutions; we thought that using a 1% concentration would be ideal. The second part of the solution in SickleDex uses sodium hydrosulfite as a reducing agent to deoxygenate hemoglobin, however, this is a toxic compound that can irritate the skin and eyes and be deadly if swallowed. The reason sodium hydrosulfite is such an efficient reducing agent is because of all the electrons it has. We found that ascorbic acid(vitamin C) also readily donates electrons to recipient electrons. In similar experiments involving vitamin C, typically a low concentration is used so we believe that 3% should be sufficient. Ascorbic acid is however more polar than sodium hydrosulfite due to the hydroxyl groups (-OH) meaning that may be less sensitive and not make as much of a difference in the HbS. Ascorbic acid can be found in the form of powder in vitamin C supplements yet it's quite expensive with 500mg costing around $3 and 1 gram costing about $6. After combining both products, you will get the final solution that you should need 2mL of during the actual test. This means that the amount of needed solution would be worth ¢2 of saponin and ¢12 of vitamin C.

The second section of this design is the test tube to hold the sample and the solution while they mix and until the results are displayed. The test tube part would come with the solution already inside it for simplicity, and have a line to indicate how much of a blood sample needs to be added. To first make the test tube we needed to find the volume to make sure there'd be enough room for the solution and sample as well as extra space for mixing. We decided that it would have a radius of 0.5 inches and a height of 3 inches. To calculate the volume we split the tube into 2 parts, the hemisphere at the bottom and the cylindrical part. To calculate the area of the hemisphere we did simple calculations: 

V = 2/3 π r3  Replacing r with 0.5 we got V= 2/3 π 0.125 ≈ 0.26"3

Then calculating the cylindrical area we did:

V = π r2 h  Replacing r and h with true values we got  V = 2π 0.25 ≈ 1.57"3

This provided us with a total volume of 1.83"3  Cubic inches can be converted into mL by multiplying by 16.387 mL/cubic inches so by applying the calculations to this we get 

V = 1.83 x 16.387 ≈ 29.99mL

As only 2mL of the solution is needed, 30mL is more than enough room to hold the solution while still providing room to mix it with the sample. Only 20µl of the blood sample is needed for the solution to be able to detect hemoglobin S. We also ensured to add a lid to the test tube, so the customer can thoroughly mix the solution with the sample and rest it somewhere while waiting for any present haemoglobin S to group up. The cost to create one of these test tubes in plastic would be around ¢40 bringing the total price to ¢54. Yet of course we still need to account for costs of labor, packaging, shipping, and instructions, likely adding up to at most a couple dollars per individual kit. This means that this design of the rapid test would cost under 15 dollars, while being incredibly simple, straightforward, and safe.

 

 

Design 2:

Our second design is inspired by hemoglobin electrophoresis, where a sample is placed through a gel matrix in a circuit, and different types of hemoglobin are separated by their conductivity and size. This kit is a bit more complicated than the last and requires 4 total parts the agar gel matrix, the wires & electrodes, the buffer solution, and finally the loading dye.

First is the agar gel matrix and it's container, this will have to be a certain legnth to clearly and most effictively show where the bands of the HbS and HbA samples should end. We decided to use a 1% agar concentration to save cost and speed up the process, since a higher concentration would result in a thicker gel and make the hemoglobin travel through at a slower pace. The prices per gram of agar varyed; being cheaper online than in stores, but the average minimum was $0.13 and the maximum was $0.30. Additionally we made gel around 5cm long to ensure speration between both HbS and HbA while being short to speed up the process. We then needed to calculate the average time it would take for the hemoglobin sample to reach the end of the kit, we were calulating the speed of the HbA sample since that one needs to travel the furthest. First we needed to caluclate stregnth of the electric field which can be found through:

E = V / D Replacing V and D we would get 45V / 5cm = 9 V / cm

Where V is the voltage and D is the distance it's between the anode and the cathode. Then we need to calculate the velocity using the formula v = µ (E) where µ is the mobility of the hemoglobin molecule through the gel and E is the electric field. For the sake of this calculation we'll say that the average mobility of hemoglobin A is 5 x 10^-4 cm² / v-s, this is a estimate since the type of buffer solution or loading dye may effect the speed, but this is a good average for 1% agar gel. Using the formula we get

V =  5 x 10^-4 cm² / v-s (9 V / cm) = 45 x 10^-4 cm / s 

Then finally you can calculate the time it would take using:

T = L / v Replacing the values of L and v we get T = 5 cm / 0.00045 cm / s = 11111.11...s ≈ 3h

Where L is the legnth of the gel and v is the velocity. We get to final number of 3 hours which is quite long for a rapid testing kit, but the process is likely to be sped up by the buffer solution and loading dye. Knowing this we can label the end of the kit with HbA showing that if the band didn't reach the end of the kit within 3 hours and stopped halfway, it's likely there's presence of HbS in your blood. However, if there are 2 bands, one at the end and one in the middle this will mean you have sickle cell trait, which doesn't affect you or mean you have the disease. The timing of the kit tooks us many attempts to perfect, starting off with a low voltage of 9V and long distance of 10cm this design originally took around 61.7 hours, which we understood was far too long. Increasing the voltage a bit at a time, we managed to get to 7 hours, but we still felt this far too long especially for a "rapid" testing kit. We didn't want to increase the voltage passed 45V since we knew that coulds result in hazards and issues if a customer was to touch the gel while the process was taking place. This is when we decided to reduce the distance by 5cm which managed to significantly reduce the time the test would. We felt that though 3 hours is still quite a long time for the kit, we knew other factors would impact and likely speed up the rate at which the hemoglobin was traveling. Additonally, we made sure to design the kit to be 2 cm deep to allow room for the bufffer solution. We also decided that the kit doesn't need to be too wide since only 1 well is needed for the sample we thought 3 cm would be a good size. This brings the total volume of the kit to 30cm² (not including wireis or additional parts) and since the gel will only be about 1cm thick the volume of gel to 15cm².

The second part of the kit is wires and electrodes which will be connected to the inside of the kit for effectiveness and a clean look on the overall kit. The electrodes are what allow the current to reach the gel and travel through it, they go at either end of the gel matrix and connect to the power source. We connected the wires to electrodes and brought through the inisde of kit, making sure there's enough to easily reach the batteries. We decided that this kit would need five 9V batteries to speed up the electrophoresis and pull the hemoglobin through at a faster rate. The overall cost of this section can vary from $0.50 to $1.00 depending on the source of the copper wires with insulation.

The third major section of this kit is the buffer solution which we decided will be supplied in a seperate container to be added over the gel during the time of the test. The purpose of the buffer solution is to keep the gel conductive throughout so that the electricity is able to pull the sample through to the anode. Along with this the buffer solution mantains a stable Ph during the process, which is important since different Ph levels can change the structure of some molecules. We knew you need just enough buffer solution to cover the surface of the gel so we calculated us to need to cover 3cm³ with the buffer solution in terms of volume, if converted to grams we estimated we'd need around 3mL (likely less). We wanted to account for any spill or accidents which is why added a bit more, and to ensure it touroughly covers all of the gel, accounting the inside of the wells. Looking at prices from different cells we were able to get an average cost of $0.30 for 3mL of a buffer solution (TAE or TBE).

The final part of this design is the loading dye, which consists of glyerol / glycerin to weigh down the sample and keep at the bottom of the gel. Looking at different kits we were inspired by the antigen Covid 19 rapid test kit that provided little packets of a solution and decided this would be the best way to pakage this. We found we'd just need a small amount of glucose would suffice to weigh down the blood sample so we only need around a 20% concentration when mixed with the sample. This means that since about 3µg of the sample is needed per well you would only need 0.6µg of glycercol. To get this into mililiters you would use the fromula:

V = M / D substituing M and D you get V = 0.0000006g / 1.26g / mL ≈ 4.76 x 10^-7 mL

Where V is the volume, M is the mass and D is the density of the glycerol which is 1.26g / mL at room tempreture or 25 degrees celcius. 0.000000476 is an extremely small amount of glycerin so the cost is almot immerausable, costing less than ¢0 which is why we decided not to include the sot of this as a part of the kit. The glycerin would also come with a small amount of saponin to lyse the red blood cells while not affecting the density too much. Combining the costs we got to an average of $1.20 to make the kit not including costs of shipping, packaging, or labor bringing this kit to a total cost of under $15 as well.

Test 1 (SickleDex):

Hypothesis:

We predict that the positive (albumin) sample will percipitate or become cloudy in ascorbic acid solution, while the negative (HbA) sample will remain the same.

Materials:

For the first test we needed Ascorbic Acid/Vitamin C tablets, Water, Test tubes, Mortar and Pestel, Eggs, and a HbA sample.

 

Controlled Variables: Amount of Ascorbic Acid/Vitamin C, Type of Water (distilled), Type of Test tubes, Amount of Sample, Amount  of solution

Manipulated Variables: Eggs (Positive control), HbA sample (Negative control)

Responding Variables: Visibility of solution for the HbA sample and egg whites

Procedure: 

1. First, make a 1% ascorbic acid solution by crushing one 1000mg vitamin C tablets and mixing it with 100mL of water.

2. Next, you need to measure out 2mL of the solution into 2 different containers or test tubes.

3. Then, crack and carefully extract egg whites from the egg.

4. After that, add 20µL of egg whites to the solution and thouroughly mix it, set a stopwatch or timer to keep track of time for that sample

6. Now you need to get a small bit of blood, 20µL, and crush/grind it to lyse the cells and release hemoglobin (due to lack of saponin).

7. Then you can add the blood to the second container of solution, thoroughly mix it and again set a stopwatch or timer to keep track of time for that sample.

8. After about 5-10 minutes the egg whites should begin to percipitate (would take less time for an actual HbS sample) and become visibly clouldy in the container

9. The blood sample solution should remain clear.

 

Test 2 (Hemoglobin Electrophoresis):

Hypothesis:

We predict that the negative (Hba) sample will travel farther than the positive (albumin) sample because it is less negatively charged and should therefore be pulled towards the positive end less.

Materials:

For this test we needed: Glycerin, UV pen, Water, Container, UV light, Baking soda, 9-volt batteries, Wires, Agarose Powder, Scissors, Cardboard, Egg, HbA sample, and Paper clips.

 

Controlled Variables: Amount of Glycerin, Amount of flourcent dye, Type of Water (distilled), Amount of Agarose Powder, Voltage, Size of well, Size of sample, Amount of Buffer Solution

Manipulated Variables: Eggs (Positive control), HbA sample (Negative control)

Responding Variables: Distance traveled by egg whites and HbA sample

Procedure:

1. First, you need to make 2 electrodes which allow the electricity to reach the gel. To this you need to unravel metal paperclips and bend one end so it can hook over the side of the box. The part of the paperclip not bent should be around the width of the box, so once hooked over it can almost reach the other end. Place one at the top of the box for your negative elctrode and one at the bottom for the positive electrode.

2.  Then, connect all 5 of the 9-volt batteries by attatching the positive terminal of one to the negative terminal of another. All these batteries combined will make your battery pack.

3. Next, you'll have to connect the negative terminal and the negative electrode using one wires. Repeat this step for the postivie electrode and terminal as well. Make sure not to complete the circuit by having the electrodes touch as they will result in a short circuit and can be extremely dangerous.

4. After that you'll have to cut out a comb from cardboard to create wells when forming the gel. The edges of the comb should be slightly longer than the width of the container so they can rest on the sides while the gel sets. The comb should have 2 teeth, that are a fairly long distance apart, and each should be slightly shorter than the depth of the box so they aren't touching the bottom when the combing is resting on the container.

5. Now you'll have to make a 200mL buffer solution that's used in the agarose gel and on top while running the test. The buffer solution has a concentration of 1% baking soda meaning for 200mL of water you ned to add 2g of baking soda.

6. Then, you have to make a 1% agarose gel by combining 1g of the agarose powder with 100mL of the buffer solution. Once you mix them together you have to heat it in a microwave at 15 second intervals until it starts to bubble. Every 15 seconds you should take it out and stir it, once it's ready it will look translucent.

7. Next, you need to place the comb into the container at the negative end with at least a 0.5cm gap between the comb and the end of the box. After doing so pour the new agarose solution into the container (make sure you remove the electrodes before doing so) and wait for it to set. This should take approximately 30 minutes.

8. While waiting for the gel to solidify you need to make your loading dye. To do this you first need to remove the nib from the bottom of the UV pen. Then holding it right side up, the ink should begin to drip out of the pen. Mix equal amounts of the ink from the pen and glycerin to create the loading dye.

9. After that, you can prepare your positive control sample or egg whites. You'll need to crack an egg into a bowl and carefully extract the egg whites into a seperate container. Then with a syringe you need to take a small amount of the egg whites around 3µg and mix it with some of the loading dye. The amount loading dye should be 20% of the egg whites sample as adding too much will interfere with the egg whites and change how far it travels.

10. Now you'll need to prepare the positive control sample, to do so get 3µg of normal blood and crush it. This is to lyse the cells and release the hemogloblin for the electrophoresis. Then repeat what you did with the egg whites and mix in the appropriate amount of loading dye. Make sure at the time you're doing this the gel is almost set as the sample can easily dry out.

11. By this time the agarose gel should have solidified so you can add in the left over buffer solution from step 5. You only need to add enough of the buffer solution for the gel to be fully submerged.

12. Then you can gently remove the comb from the gel. The comb should have made wells in the agarose gel for you to put the samples in.

13. Next, you need to make 2 cuts at each end of the chamber and the you can place the electrodes in. Make sure that the negative electrode is attatched to the negative terminal and is going into the top of the chamber where the wells are and vice-versa.

14. Then drop in the egg white sample into one of the wells and the HbA one into another. If the ciruit is working, bubbles should begin to form around the electrode. After about 3 hours the results should appear when a UV light is shone on them and the egg whites should have  migrated a lot less than the HbA sample.

Test 1 (SickleDex):

              We did the test inspired by a SickleDex kit 2 times to ensure accuracy and it was successful all three rounds. We placed both the HbA solution and the egg whites into the ascorbic acid around the same time and after thoroughly mixing it and leaving it to settle for around 5 minutes the egg whites began to precipitate in the solution. 

               This image of the second test clearly shows how the egg white solution (on the left) became slightly more cloudy, and more importantly how the egg whites precipitated and formed a “clump” in the liquid. However, the HbA solution (on the right) was left clear and had almost no reaction when placed in the solution. The same occurred with the first test and the hemoglobin A didn’t make much of a difference when placed in the solution.This met our expectations and predictions showing that our design was accurate and worked. Overall, this kit proved to be a highly reliable design that would work as one of the first genetic rapid testing kits for sickle cell disease due to its simplicity, speed and cost.

              There were of course limitations and sources of error with this and we aren’t sure whether it’s 100% accurate due to several external factors. These include not being able to access a hemoglobin S solution, as well as not being able to get saponin and having to do manual lysis. While egg whites were a good substitute and model of HbS we can’t be sure how different the true molecule would have reacted in the vitamin C solution. 

              In the ascorbic acid, the egg whites went through denaturation which is the unfolding of a protein due to a change in its structure. The structure of proteins can have up to 4 major parts including the primary structure, secondary structure, tertiary structure, and the quaternary structure. Albumin (the main protein in egg whites) goes until the tertiary structure, which is made up of a complex arrangement of only α helix structures and rarely contains β plated sheets unlike hemoglobin (more detail in method). When denaturation occurs in albumin the hydrogen bonds holding together the secondary and tertiary structures are broken apart. Resulting in a physical change and causing the protein to unfold and clump together, but there is no chemical change in the primary structure or amino acid sequence. The secondary structure is what makes up the helix chains, stabilized through the hydrogen bonds of 1 amino acid carbonyl oxygen atom and the amide hydrogen atoms of 4 other amino acids. A carbonyl oxygen atom is any compound with carbon and oxygen while an amide hydrogen atom is an atom with carbon, nitrogen and oxygen. Hydrogen bonds also play a major role in the tertiary structure or the 3 dimensional folding structure of proteins. The side chains of the amino acids are held together by strong hydrogen bonds made from polar covalent bonds. This is when an electronegative oxygen or nitrogen attaches itself to a hydrogen that’s already connected to another oxygen or nitrogen atom. Hydrogen bonds play and extremely important part in holding together the secondary and tertiary structure of proteins including albumin, and when placed in an acid like ascorbic acid protonation occurs and H+ adds an extra proton to one of the atoms or molecules already there; disrupting the hydrogen bond. This leads to the protein falling apart and then precipitating through denaturation as previously mentioned, with no chemical changes made.

Albumin Structure:

On the other hand, when hemoglobin is placed in the ascorbic acid it precipitates due to a chemical change not a physical change.The hemoglobin S solution becomes cloudy due to the reducing agent in the solution. As stated before, the reducing agent (in this case ascorbic acid) caused the valine amino acid to enter a tensed state and caused its hydrophobic patch to move to the surface. This makes it prone to polymerization where monomers combine to form a larger molecule called a polymer, hence the precipitation or cloudy appearance. There are 4 types of amino acids: polar, non-polar, acidic, and basic. Glutamic acid is acidic because of the carboxylic acid group in its side chain (-COOH), and at a physiological pH (the average pH of the body) it’s capable of gaining an extra electron with H+ making it -COO-, a negatively charged compound. Valine is a non-polar amino acid because its side chain is made of only carbon and hydrogen atoms which both have similar electronegativity meaning that no one atom has a stronger pull on the shared electron, therefore making it non-polar. This hydrocarbon side chain additionally makes valine aliphatic as well as hydrophobic or insoluble in water and simply avoid polar compounds. This is why after being deoxygenated and the hydrophobic patches are brought to the surface they group together; to minimize contact with the water around it. The valine molecules sticking together is what would cause the cloudiness in the ascorbic acid solution. As you can see albumin and hemoglobin S are both similar in structure, being proteins and made of primarily α helix chains, however when placed in the solution they both precipitate for very different changes; the albumin due to loss in structure and breaking of hydrogen bonds, while the hemoglobin S because of its chemical form leading to long rigid polymers.

These pictures show the structure of glutamic acid and valine. You can clearly see that the glutamic side chain contains a carboxylic acid group where as valine's side chain contains an isopropyl group.

               Another limitation we experienced was not being able to access saponin and therefore having to do manual lysis to release hemoglobin from the red blood cells. As stated before saponin releases hemoglobin by attaching to the cholesterol, a major part of the lipid membrane, and creating pores within the membrane. Saponins are glycosides meaning they have a sugar part (glycone) and a non sugar tail part (aglycone). The glycone part is known as hydrophilic (polar) and the aglycone part is known as hydrophobic (non-polar). Similar to how valine is attracted to other valine molecules, the hydrophobic tail segment of saponins are made of a long carbon chain making it non-polar attracted to cholesterol since they’re also non-polar (made up of 4 hydrocarbon rings and a hydrocarbon tail). As the phrase “like dissolves like” suggests, the saponin breaks down lipid membrane by dissolving the cholesterol, which works because they are of like polarity. This process is very gentle, unlike manual lysis that will likely end up disrupting the structure of the hemoglobin itself. Additionally, manual lysis may result in inconsistent samples because there were probably different numbers of cells that got lysed each round. All in all, manual lysis may have resulted in the HbA samples having inconsistencies or damaged hemoglobin, whereas saponin would have been more uniform and reliable.

The graph above shows how we rated the visibility of each sample for all test rounds on a scale of 1-5, 1 being barely visible and 5 meaning no change in the visibility. As shown, the HbA or the negative samples were far more visible when mixed with the solution than the egg whites or positive samples. When we compare this to our theoretical graphs, or expectations you can see that the results are relatively similar with the positive samples being much harder to see through than the negative samples. This acts as evidence that our design did in fact work, in terms of materials, quantities and results. We thought this kit was a great prototype, and would work really well if actually manufactured and sold as a rapid testing kit, because of how cheap it was to produce and how easy it was to make, use, and analyse the results.

 

Test 2 (Hemoglobin Electrophoresis):

               This test was inspired by hemoglobin electrophoresis, a process that compares macromolecules like protein and nucleic acids by putting them through a circuit and testing their conductivity and size. We had to repeat this test once, because of limitations as well as human error; we had to slightly modify the test to better work with egg whites to create a fair model of hemoglobin electrophoresis. For the first test round we had to wait much longer than the recommended time for the agar to solidify, which was 30 minutes, but after 30 minutes, it was still very watery. About an hour or so later did it actually turn into a jelly like solution.

 

               The image on the left is what it looks like with UV light and the right is without it. As you can see the samples are still slightly visible without the UV light, but mixing the fluorescent dyes helped us keep track of them as they traveled through the gel. There were some challenges that we faced when conducting this experiment for the first time. We were not sure if the gel consistency was right. The agarose gel sat for much longer than it needed to properly solidify, leading us to question if the gel was right. If it was too soft, the gel would lose its shape, causing the hemoglobin to move around or travel through too quickly, if it was too thick, it would make it hard for the samples to migrate. Based on our results of the first test round, the gel was a decent consistency, allowing the samples to migrate through, but it was still a bit too liquidy. Since the egg whites already contain smaller molecules compared to the HbA sample this made it travel way too fast and it ended up reaching the end of the gel before the HbA. To fix it we doubled the gel concentration to 2% agarose powder, and this ended up working much better, leading to a firmer gel that took an appropriate time to set. The thicker concentration of the gel aided in slowing down the egg whites during the second test, making the test more reliant on the charge of the proteins rather than the size of them. This helped make our model more accurate as HbS molecules are the same size as HbA molecules meaning that theoretically, it would be impartial for the test to compare the proteins based on size.

               The voltage that we used for the first test was 45V, from five 9V batteries. This was too much voltage and resulted in burnt electrodes, burnt bubbles coming up to the surface, blocking the view, and even the gel itself getting slightly burnt. We reduced the volts to 27V and it worked much better than 45V.  The first time we had excessive volts in comparison to the amount of gel and the size of the gel chamber. A balance between the voltage and the gel must be met for fast and efficient migration of protein fragments. It also caused the gel to melt and soften due to overheating. With the lower voltage, we avoided the issue of burning or melting the agarose gel. Less voltage means less heat, resulting in a slower but controlled migration of the hemoglobin samples, leading to more distinct bands. By using 27V, the heat was minimized, leading to fewer bubbles and risks of burning the gel. Although if we went any lower in voltage, this would’ve led to poor results because the migration of the samples would be slowed by far too much, or the samples would not have travelled far enough. The fragments would also not separate properly because the agar gel is not strong and doesn't have enough force to drive them. Along with this, a lower voltage would cause a prolonged time for the electrophoresis to work. However, even though lower voltage is much safer than higher voltage, it reduces the efficiency of the experiment. 

After our samples were loaded into the wells, we applied an electric current to the gel. The proteins are negatively charged because of their side chains, so when the current is applied, it will move towards the positive electrode. The glycerol ensures that the DNA stays within the well at the bottom and does not come out, and when the electric current is applied, the DNA from both samples will move towards the positive end, (opposite charges attract). In our first experiment, we observed that hemoglobin electrophoresis fragments separated based on their sizes. This is because agar gel is made up of small pores, when an electric current is applied, the hemoglobin fragments pass through these small pores. Smaller fragments pass through much faster than big ones because they don’t get stuck in the pores, unlike the larger fragments that take a longer time because they have to squeeze through. During the second experiment the electrophoresis separated the protein fragments based on their charge because of the thicker gel concentrations. 

               Like the last design this test also had a couple limitations; the main one being unable to access HbS and replacing it with albumin. While both HbS and albumin/egg whites are proteins with negative charges their size and current vary. Albumin is negatively charged due to the amino acids it’s made of which are mostly glutamic acid or aspartic acid. These are both acidic amino acids because of their negatively charged side chains. As previously stated, acidic amino acids are considered an acid because of the carboxyl group in their side chains (-COOH) and as mentioned when in a physiological pH they are forced to lose an H+ proton. This results in the oxygen gaining an electron and becoming negatively charged, creating a new compound -COO-. This is why albumin is a negatively charged protein and worked for this experiment. HbA is negatively charged for the same reason, containing glutamic acid. HbS is also negatively charged which is why it works in hemoglobin however, it’s significantly less negatively charged than the albumin. Therefore, the albumin ended up traveling much farther than it should have. Along with this albumin is made up of smaller molecules than hemoglobin, increasing the rate at which it traveled through the gel. 

The graph above shows our results from the hemoglobin electrophoresis tests, and as you can see the results of the first round are inaccurate. The negative sample ended up traveling a smaller distance than the albumin which isn’t what should theoretically happen. To accommodate for the egg white solution containing smaller molecules and more negatively charged, we used a higher concentration of agarose gel. After making adjustments the tests worked as we expected and our hypothesis was correct. The yellow and green lines are the results of both our second and third test since they had very similar results and we thought it would be hard to display them seperately.

 

Conclusion:       

               After analyzing each of the tests based on their simplicity, speed, and cost we were able to conclude that design 1 based on the SickleDex rapid kit is the best, most efficient, kit out of all 3 we designed. The PCR kit required higher end technologies and thermal equipment, not only instantly increasing the price, but also the complexity. Most people wouldn’t be able to operate one of these machines with the higher end technology and it additionally would take a long time to amplify enough hemoglobin to test. It’s important to remember that PCR only amplifies protein/DNA making several copies of it to be used for testing, but it doesn’t test the protein itself meaning it would require additional parts on top of the main machine. Explaining how to use the heating chamber and packaging plus safely shipping the advanced products would be difficult challenges to overcome. The hemoglobin electrophoresis inspired test worked incredibly well and was the most accurate out of the 3 designs, however when testing the kit we realized the various different steps it would take for someone to correctly execute the test. There are several things that the kit would demand a customer to do including adding the buffer solution to the gel, mixing the blood sample with the loading dye, connecting the kit to a very specific amount of volts and carefully adding the sample to the gel. As you may see, there is an extremely large amount of room for error by the customer. The kit would be quite complex and require them to do many procedures that could easily go wrong. Not only did the other 2 designs being too advanced, complicated, and costly make us choose the 1st design as the best rapid kit, but the first kit has several advantages. This kit is straightforward with minimal room for error, increasing the possibilities of accurate results. It was also extremely easy to create, showing that producing it wouldn’t be much of an issue either. Compared to the other 2 designs, this rapid kit was extremely fast, providing results in 15 minutes, and it was the cheapest out of the 3 models making it perfect as a rapid test. This model was successful both test rounds, proving it’s reliable and would likely work the same on hemoglobin. Even though this design can’t differentiate between sickle cell disease and sickle cell trait, additional testing is almost always needed after rapid tests, and knowledge of sickle cell trait is extremely important for a patient’s family. Overall, the 1st design inspired by the SickleDex one really stood out to us, because of its almost endless advantages and the simple design that made it convenient and efficient such as those rapid tests already existing.

               Both tests met our expectations and hypotheses about what would happen when the samples are placed in the test. The first design was instantly successful, and remained so during the second test as well, providing evidence to support that it wasn’t luck or chance. As we predicted in our hypothesis, the egg white sample precipitated while the HbA sample solution stayed transparent. The second design didn’t work during the first test round because we didn’t account for the difference in molecule size between albumin and HbS. However, after making necessary changes to accommodate for egg whites and make the test as accurate as possible we were able to successfully gain results that met our hypothesis. Just as we assumed, the HbA sample traveled much farther through the gel than the egg whites due to their negative charges (after changing the process for the albumin). If we were to do this innovation again we would definitely seek out more help from professionals to try to access a hemoglobin S solution and accurately test both of our designs. Along with this we would try and truly make the rapid kits to see if our calculations are accurate in terms of size, speed, and cost. We hope that scientists that specialize in this field can give us advice in the future and help us actually produce these kits and fix any flaws they may contain.

 

Sources of Error:

               There are multiple areas in which we could’ve had errors in. Crushing the vitamin C tablets unevenly could have led to having an inaccurate vitamin C concentration which may lead to incorrect results of eggs precipitating faster than they should. If the vitamin concentration is too low, then it can lead to a false negative, even when there is a HbS present, it would take much longer to show or not show at all. Along with this, the vitamin C tablets we used weren’t pure ascorbic acid, meaning they included other ingredients; carbohydrate gum, microcrystalline cellulose, hydroxypropyl cellulose, and stearic acid. These ingredients may have interfered with the reaction taking place with the albumin due their chemical structure. Based on our research most of these are polar meaning that it shouldn’t precipitate when in the acid, however stearic acid is non-polar and contains -COOH the carboxyl group that HbS contains. This means that this acid may have precipitated in the solution and interfered with the denaturation of the albumin. Another potential error is the two metal paper clips meeting, if they happen to touch each other in the gel, it can cause a short circuit because one paper clip is connected to the positive electrode and the other paper clip is connected to the negative electrode. So when they touch each other, the two paper clips the electricity will flow through the paper clips instead of through the gel, meaning now electricity will go through the gel, leading to no migration in the samples. Cross contamination is another source of error, using the same pipette tip for both the negative and positive sample will lead to inaccurate results. Finally, human error is a large source of error in our experiments and we have made mistakes in terms of measurements, calculations, etc, which could have affected our results.

 

Application:

Since our kit was proven to truly work we hope it can be used to diagnose other genetic diseases and be made versatile to test for several disorders at once. Several other genetic diseases are caused for similar reasons so by changing a couple aspects of the kits could be truly beneficial in increasing the rate of diagnosis. The purpose of this project was to help those without immediate access to medical attention have enough time to seek the facilities they need in the event they have sickle cell disease. As we stated in our problem, there are over 7000 identified genetic diseases and almost none of these have efficient ways to test for them in a home setting. We’ve found several antigen rapid testing kits that detect antigens found on the pathogens of a specific virus, by using antibodies. We also came across several antibody rapid testing kits that detect the antibodies your immune system produces to defend your body against a disease. These designs were simple, cheap, and effective which is why they were able to be purchased and used by the public at home. Yet no one has made these for any genetic diseases that are inherited and cause a mutation. There are advanced technologies such as CRISPR or newborn screening devices, but nothing that an average person would be able to use. Therefore we want to extend on our project by changing the properties of our kit to identify other mutations or diseases in general. The hemoglobin electrophoresis test, for instance, could easily be modified to find another disease, by looking at the electronegativity of the mutation causing that disease, and changing the marking lines on the kit. Overall, we hope to spread our invention and make it adaptable to quickly look for one of the many genetic diseases. There are around 80 million people directly affected by genetic diseases every year, and they’re a main cause of deaths which is why early detection could make a true difference. Therefore, we want to work with more experienced experts in this field who could help us actually manufacture these kits to be sent out to stores where people are more likely to purchase them. This will also help the general public understand more about genetic diseases, their symptoms, and their effects which would be greatly beneficial in the case of an emergency.

 

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We would like to thank our CYSF coordinators, Mrs. Rheinstein and Mr. Lahoda, for helping us come this far, taking the time to organize this for everyone and encouraging us to take initiative to make the best project we can. We would also like to acknowledge our families and friends for supporting and guiding us this whole time as well as helping us prepare for this project. Our families played a huge part in helping us get together and meet up to work on this project with minimal difficulties. And most importantly, we would like to thank our judges and the organizers of CYSF for making it possible for us to have this opportunity and experience it to the fullest.