## 3 Results and Discussion
### Examination of Thrombin Release from Aptamer-Functionalized DNA Hydrogel  
The hydrogel underwent thrombin release tests in the absence of CSs to examine the binding ability between the aptamer and thrombin in the hydrogels. As shown in Figure 4A, more than 80% of the thrombin was released from the hydrogel without S-DNA within 2 h. Similarly, the hydrogel with controlled S-DNA also had the burst release phenomena in the first two hours. In contrast, only 10% of thrombin was released from the S-DNA-functionalized hydrogels in 10 h. These results suggest that aptamers dramatically reduce the burst release and result in the effective sequestration of thrombin in the hydrogel. The controlled thrombin release property of hydrogel was detected in the existence of CSs. A comparison of the interaction force between the aptamer-TB complex and the aptamer-CS duplex was shown in Figure S6. The results showed that CS12 and CS15 could trigger the release of TB, while CS8 could not. In order to prove the ability of the hydrogel to hybridize with the CSs, the DABCYL ((4\({}^{\prime}\)-(4-dimethylaminophenylazo) benzoic acid)-labeled CS15 was designed as shown in Table S1. When the CSs diffused into hydrogel network and hybridized with the FAM-labeled S-DNA, the emission of FAM was quenched through DABCYL on account of the Forster resonance energy transfer (FRET) effect [42; 43]. Therefore, along with the hybridization of aptamers and CSs, the intensity of fluorescence decreased with the increasing incubation time, as shown in Figure 4B(a-d). At the same time, the dissociated TB from aptamers increasingly diffused out of the hydrogels, which resulted in the concentrations of TB in the release medium increasing, as shown in Figure 4B(e). These results demonstrate that CSs are able to penetrate rapidly into the DNA hydrogel to hybridize with aptamers and lead to TB release. Figure 4: **(A)** Thrombin release from different hydrogels in the absence of the CSs. (**B**) Fluorescent images of hydrogel (with FAM-labeled S-DNA) in the existence of 4\({}^{\prime}\)-(4-dimethylaminophenylazo) (DABCYL)-labeled CS15 at different reaction times: (a) 5 min, (b) 2 h, (c) 4 h, and (d) 6 h. (e) Cumulative release of TB at different reaction times. The molar ratio of complementary sequences (CSs) to S-DNA was fixed at 1:1. The dissociation rate of thrombin triggered by CS15 can be fitted to a theoretical dynamic model in accordance with the relation between triggering time and thrombin concentrations. The fitting formula is listed as follows:  
\[ln\frac{C_{\infty}-C_{t}}{C_{\infty}-C_{0}}=kt \tag{1}\]  
where \(C_{0}\) is the initial concentrations of thrombin without CSs, \(C_{t}\) is that at time t, and \(C_{\infty}\) is that when the reaction reaches equilibrium with CSs. Here, \(k\) is a rate constant that corresponds to the dissociation rate of thrombin triggered through CSs. The effect of the concentrations of CSs on the rate constants \(k\) of thrombin dissociation was researched. According to the fitting formula, the dissociation rate corresponded to the slopes of the linear curves. The different concentration of CSs resulted in different dissociation rate constants \(k\). As shown in Figure 5A, when the molar ratio of CSs to aptamers was 1:5, \(k\) was \(-0.2971\). When the molar ratio of CSs to aptamers was raised to 1:1, \(k\) was reduced to \(-0.8082\), as shown in Figure 5E. Comparing the slopes of the curve (Figure 5F), we can conclude that higher concentrations of CSs in the same release time will lead to a lower rate constant (i.e., higher release capability). To further understand the protein-releasing law regulated by CSs, the molar ratio of CSs to aptamer, the length of CSs, and the time of CSs adding were carefully discussed in this work. As shown in Figure 6A, through adjusting concentrations of the CS15, the release amount of the TB could be controlled. The percentage of CSs that was diffused into the hydrogel was shown in Figure S7 and Table S3.