Team:BIT/Design/Biosenor
Abstract
Cancer is one of the three major killers that threatened mankind since the 21st century. The reason is that it is not taken seriously at the beginning of cancer until it is detected in the middle and late stages, resulting in a very low cure rate. Therefore, early detection becomes a decisive factor to improve the cure rate of cancer. In this project, we use alpha-fetoprotein (AFP)as an example, using the factor that AFP can specifically bind to the aptamer, to achieve a signal conversion that using a small molecule (lysine) to replace AFP content in serum, just like equivalent substitutions in mathematics, to achieve the early detection of liver cancer. This system can be extended to all of the disease detection, which use protein as biomarkers.
Introduction
Technical significance：
AFP is a major fetal plasma protein. Serum AFP is always low expressed in healthy adults, but often high expressed in nearly 75% HCC patients with more than 500 ng/ml.AFP is a liver cancer associated protein and has long been utilized as a serum tumor biomarker of disease progression.
Clinical significance：
Monitoring the level of molecular markers of disease contributes to the early diagnosis of disease, the monitoring of treatment and the prognosis of the disease, which is also a great significance for clinic. However, the molecular markers concentration in our body is very low, making it difficult to analysis and detect directly.
The conventional method is ELISA and other antibody-based techniques to enrich low-abundance molecular markers. ELISA which is based on "antigen-antibody reaction" of the detection method is a little bit expensive. Time and temperature are having a greater impact for its detection.
The purpose of this project is to establish a detection platform suitable for most molecular markers. The platform is low cost, simple to operate and has strong anti - interference ability.
This project uses the AP273 with the highest binding to AFP and the complementary chain paired with the aptamer.
Aptamer is an oligonucleotide sequence or short polypeptide obtained by in vitro screening. Aptamer, as an alternative to the antibody, can bind to the corresponding ligand with high affinity and strong specificity. Its low price, easy to screen synthesis, the nature of stability and other advantages are widely used in the detection of molecular markers of disease. Its presence provides a new way to quickly and efficiently identify bio-makers.
principle
First of all, we select an aptamers AP273, which could best affinity for AFP. We modified a biotin at the end of its 5’ end, which could affinity for streptavidin. Because of the presence of streptavidin-modified beads, aptamer has been locked firmly on beads. At the same time, a complementary chain was synthesized in its protein binding site. We modified an amino at the 3’ end of the complementary chain which can be combined with lysine protected by BOC anhydride to link lysine to the complementary chain. Because Van der Waals forces between AFP and aptamers is stronger than hydrogen bond, due to the action of magnet lying at the bottom of the reaction system, aptamers are separated from complementary chain, one at the bottom, the other in the supernatant. At this point, with the action of trypsin, lysine can be separated from the complementary chain, and start his adventure.
