Project Description
Mild traumatic injury, mTBI, is a neurological disorder that causes impaired cognitive function, and affects a person's ability to function independently. More than 100.000 people are affected in Sweden, and millions of others worldwide. mTBIs can occur at any age due to falls, accidents, sport injuries etc. The disease is characterised by increased levels of amyloid-beta and tau proteins. There is currently a gap in early detection of neurodegeneration related to mTBIs. By using our biosensor AptaTear, in the future, a quick, non-invasive first screening can be done to understand the severeness.
We hope to make it easier to detect mTBIs so that it is not missed. We also hope that this will be an easy tool to use for sports coaches and others, so that it can be detected fast. We also aim to create a cheaper method of testing, as the current detection mechanisms are very costly, and need to be performed by a medical professional.
Our solution is to create an aptamer-based biosensor to detect tau protein levels in patient-derived tears. Once the aptamer is bound to tau, an amplification occurs using a rolling circle mechanism and in the amplification process, protons will be released and thereby cause a pH change that can be visibly detected by the naked eye. This kit will be user-friendly, non-invasive, and enable earlier detection than other methods, which is essential for people suffering from these disorders.
Tau has long since been proven to be actively excreted in response to neurodegeneration. It was our task to use synthetic biology to detect tau in tears. We did research about different detection methods and found a synthetic biology detection method called Loop-mediated isothermal Amplification (LAMP), coupled to Rolling Circle Amplification (RCA). In a study done by the University of California San Diego, these two techniques were combined to detect short DNA sequences [1]. A recognition site will be attached to the Tau aptamer using a linker. This recognition site can then be detected using RCA-LAMP. When the aptamer construct is added to the sample containing the tau protein, the aptamer will bind to the tau protein. A padlock probe, which is made up of DNA that has been circularised, will bind using complementary base pairs to a recognition section on the aptamer construct. Two primers, called FIP and BIP, will then be added, which causes short DNA sequences to be produced through replication. This production is what makes amplification of our “signal” possible. The production of DNA sequences will cause a pH change and through a pH indicator we can get a visible colour signal.
The customer will receive an antibody coated test tube, as well as washing buffer, aptamer, the ligation mix, and amplification mix.
Let’s take a look at what the user of this product needs to do. First the antibody coated test tube is taken and the tears from the patient are added. The tube is then washed using a washing buffer, and aptamer is added. The tube is then incubated at room temperature for 1 hour. The tube is then washed again and then the ligation mix is added. The tube is then left to incubate at room temperature for 1 hour. The amplification mix is then added and the tube is left to incubate for 1 hour at 65 ℃. If the liquid turned yellow from its original pink colour, then the result is positive and the patient has sustained a head injury. If the liquid retains its original pink colour then the test is negative.
Our long term goal is to continue the development of the biosensor to make it possible to use it to detect other neurodegenerative diseases. This could lead to starting a company which makes these biosensors so that they can be easily accessible and used in health care.