## 3 Results and Discussion
### Examination of Thrombin Load by Aptamer-Functionalized DNA Hydrogel  
To capture thrombin, the hydrogels were washed extensively using DNA buffer to eliminate non-cross-linked S-DNA, and then immersed in the absorption medium. As the incubation time increased, the TB molecules would penetrate into the hydrogel network and lead to the concentration decreasing of TB in buffer solution. The protein capture capability of the hydrogels could be obtained though detecting the change of TB concentrations in the absorption medium. As shown in Figure 3A, after 6 h of incubation, the DNA hydrogels with aptamers had an obviously higher thrombin capture capability (approximately 51%) than the DNA hydrogels without aptamers (approximately 13%) due to the specific bind of thrombin to the aptamer in the hydrogel. These results demonstrated that the hydrogels without aptamers had a very low protein capture capacity just because of the physical diffusion effect. As for the hydrogel assembled with aptamers, the capture capacity was determined mainly by the specific affinity between the aptamers and proteins, and secondly by the physical diffusion effect. This suggests that aptamers play a crucial role in capturing protein in the DNA hydrogels. Although the hydrogels without aptamers had a very low protein-capture capacity, the ability of the DNA hydrogel to capture thrombin by the physical diffusion effect is still very important. Since aptamers are integrated into hydrogel matrixes, TB has to firstly permeate through the DNA hydrogel networks, and then bind the aptamer embedded in the matrixes. In order to further understand the ability of the DNA hydrogel to capture thrombin, the two building blocks were investigated at different concentrations of X-DNA at 400 \(\upmu\)M and L-DNA at 800 \(\upmu\)M, X-DNA at 500 \(\upmu\)M and L-DNA at 1000 \(\upmu\)M, X-DNA at 600 \(\upmu\)M and L-DNA at 1200 \(\upmu\)M, X-DNA at 700 \(\upmu\)M and L-DNA at 1400 \(\upmu\)M, and X-DNA at 800 \(\upmu\)M and L-DNA at 1600 \(\upmu\)M, respectively. The concentration of S-DNA was kept at 200 \(\upmu\)M. As shown in Figure 3B, the higher concentration of DNA building units (X-DNA at 800 \(\upmu\)M and L-DNA at 1600 \(\upmu\)M) had a lower protein capture capacity. This could be explained by the higher concentrations of DNA units resulting in the more closely knit and higher cross-linked networks, which limited the diffusion of thrombin into hydrogel. As the concentration of DNA building units decreased, the TB loading amount of the hydrogel increased gradually. We performed \(p\)-value calculations for each pair, and showed that the last two data (X:500-L:1000 and X:400-L:800) are significantly different. Therefore, the hydrogel samples that were formed by 400 \(\upmu\)M of X-DNA and 800 \(\upmu\)M of L-DNA had the highest protein-capture capacity. In addition, in order to avoid wasting more protein, this concentration was chosen for the next experiments in the following sections. Except for the concentration of the DNA building units, the concentration of TB in the absorption medium also affected the capture capability of the hydrogel. The hydrogel samples (assembled from 400 \(\upmu\)M of X-DNA, 800 \(\upmu\)M of L-DNA, and 200 \(\upmu\)M of S-DNA) were respectively immersed into 100 \(\upmu\)L of buffer solution with different concentration of thrombin (0.5 \(\upmu\)M, 1 \(\upmu\)M, 1.5 \(\upmu\)M, 2 \(\upmu\)M, 4 \(\upmu\)M, and 8 \(\upmu\)M). After calculating, the molar ratio of TB to aptamers was respectively 0.25:1, 0.5:1, 0.75:1, 1:1,2:1, and 4:1. The results were shown in Figure 3C; the normalized capture was raised from 1 \(\pm\) 0.11 to 4.23 \(\pm\) 0.26, and the molar ratio of TB to aptamers increased from 0.25:1 to 1:1. We performed \(p\)-value calculations for each pair, and found that when the thrombin concentration was raised continuously from 2 \(\upmu\)M to 8 \(\upmu\)M (namely the molar ratio of TB to aptamers was raised continuously from 1:1 to 4:1), there was no obvious change in the normalized capture of hydrogel.