## 3 Results and Discussion
### Examination of Thrombin Release from Aptamer-Functionalized DNA Hydrogel
Along with the increasing CSs concentration (the molar ratio of CSs to aptamers was adjusted from 1:5, 2:5, 3:5, 4:5, and 5:5), cumulative TB release increased from 30% to 81%. The thrombin binding on an aptamer couldn't be dissociated without the CSs (CSs = 0). As a result, thrombin could be released only by the physical diffusion, and the cumulative release was approximately 15%. The results were similar to those shown in Figure 5.  
The amount of base pair obviously influenced the strength of DNA hybridization [44]. Therefore, longer CSs resulted in stronger competition ability. As shown in Figure 6B, when the length of the CSs was eight nucleotides, there was no significant difference compared to the control. It suggested that the short CSs with only eight nucleotides couldn't trigger the thrombin releasing from the aptamer. When the chain length of CSs changed from eight to 12 nucleotides, the thrombin release amount was  
Figure 5: Dissociation dynamics of thrombin triggered by CS15 for different CSs to aptamer ratios: (**A**) CSs:APtamer = 1:5, (**B**) CSs:APtamer = 2:5, (**C**) CSs:APtamer = 3:5, (**D**) CSs:APtamer = 4:5 and (**E**) CSs:APtamer = 1:1. (**F**) The concentrations of TB were calculated using the fitting formula (i.e., \(In(C_{\infty}-C_{t})/(C_{\infty}-C_{0})=kt\)) to get the rate constants (the slopes of the linear curves) of thrombin dissociation. enhanced fourfold. When the length of CSs was completely complementary with the thrombin aptamer (15 nucleotides), the thrombin release amount was increased approximately five times. These results demonstrated that increasing the length of complementation DNA can promote thrombin separation from aptamer-thrombin complexes. In addition to the reasonable design of the CSs length, triggered release can be adjusted by other factors (for example, the triggering time). As shown in Figure 6C, we applied CS15 to treat the DNA hydrogel at hours 0 and 12. The total amount of the released thrombin over 0 h and 12 h were approximately 31% and 51% (the molar ratio of added CSs to aptamers was fixed at 1:4 at each adding time). The hydrogels without CS15 released approximately 14%. The accumulated release of thrombin was approximately 31% for the first triggering (0 h), and approximately 17% for the second triggering (12 h). It could be explained that the first release included two processes, namely the normal physical diffusion of thrombin (14%) and triggered release by CSs (17%). However, for the second release, the release process was only triggered by CSs. This was because most of the TB was bonded to aptamers due to the TB-aptamer interaction force, and only a few TB was free to diffuse out of hydrogel. The physical diffusion process can get an equilibrium within 5 h, as shown in Figure 6C. That's why we got the conclusion that the first triggering time contained 14% \"normal physical diffusion\" and 17% \"CSs-triggered release\". Then, at subsequent triggering times, we attributed 17% of the release only to a CSs-triggered release. Thus, the release amount rests with the feeding amount of CSs. These results indicate that the sequestration of thrombin can be a triggered release by CSs at a determined time point, and the release content can be precisely adjusted through changing the CSs concentrations.