When we are injured on our daily lives, blood clotting occurs in damaged blood vessels. This blood clotting system is divided into two steps: primary hemostasis and secondary hemostasis. Proper blood clotting is induced by complex signal pathways. It is reported that in particular, secondary hemostasis does not occur in wounds that are smaller than a certain size, and the appearance of blood clotting is greatly different.
However, if this signal pathway is defective, blood clotting does not function well, and various diseases can be caused. One of the diseases is hemophilia. Hemophilia is caused by deficiencies in blood coagulation factors such as factor VIII and factor VIIIIXVWF, which play an important role in the blood clotting cascade. For hemophilia, current treatment is performed by administering a deficient protein as a preparation. Disadvantages of this preparation include the occurrence of inhibitors and the possibility of viral infection. When a coagulation factor is administered, the coagulation factor is regarded as a foreign substance in the body, so an inhibitor of the factor is generated, and thus the effect of the preparation may be weakened. Although there is a definitive treatment for inhibitors, but the treatment is expensive. Also, because a quarter of the currently used preparations are derived from blood donation, the possibility of viral infection cannot be denied.
Therefore, we focused on methods that do not use immune cascade reactions which is induced by blood coagulation factors. By achieving non-linear aggregation that responds to signals above a certain level, it is possible to artificially mimic the blood clotting reaction.
 Christian J. Kastrup, Matthew K. Runyon, Feng Shen, Rustem F. Ismagilov, "Modular chemical mechanism predicts spatiotemporal dynamics of initiation in the complex network of hemostasis", PNAS October 24, 2006 103 (43) 15747-15752, https://doi.org/10.1073/pnas.0605560103
The object of our project is to make a non-linear responsive clotting system which can detect highly low concentration of DNA strands under the isothermal condition.
The novelty of Bit Clot is non-linear aggregation. By adjusting the concentration of templates, drain and exonuclease in PEN DNA toolbox, non-linear aggregation can be achieved, and this enables us to imitate the blood clotting system.
Moreover, it is important to detect highly low concentration of DNA strands. For this detection, which is performed specifically to close the wound accurately, damage associated DNAs released from dead cells are used. By using PEN DNA toolbox, the signal amplification from low concentration of DNA to higher concentration of linker can be accomplished.
Additionally, this reaction proceeds under the isothermal condition. In conventional DNA amplification, high temperature cycles are necessary. However, high temperature cycles are inconvenient in vivo nor in vitro in some case. For example, in experiments imitating blood vessel, accurate detection and quantitative assay at high temperature can be difficult because of cell dying and enzyme deactivation. On the other hand, isothermal amplification and sensing enables us to detect DNA strands without killing cells or protein denaturation. Moreover, there is a merit in terms of engineering. Generally, PCR consumes large amount of energy because it repeats extreme heating and cooling. In contrast, isothermal amplification is a reaction at constant temperature, so power consumption becomes low. Also, it gives us the possibility of new device for amplification of the specific DNA sequence. If isothermal amplification is realized, amplification device can be made of low melting point materials such like plastic.
In this section, we show each step to make our project clearly. We aim to design non-linear beads aggregation system similar to blood clotting by taking the average of DNA autocatalytic system and the aggregation of beads, which we called BIT CLOT system.
BIT...Check the DNA strand non-linear amplification system by using DNA autocatalytic system which we called BIT System. [Step 1]
CLOT...Design the beads, linker, and α to linker so that they can reflect nonlinear α bifurcation. (CLOT System) [Step 2,3]
BIT CLOT...Achieve non-linear beads aggregation depending on the initial concentration of α. (BIT CLOT System) [Step 4]