Team:DUT China A/Demonstrate

Design

The combination of cells and EpCAM aptamer

In order to capture circulating tumor cells (CTCs) specifically, we choose the aptamer named SYL3C, which specifically combines with the EpCAM receptor overexpressing on the tumor cell membrane. After each end of the three kinds of aptamer is attached to a fluorescent radical group, we begin to verify whether the aptamer could bind to cells specifically. We choose two types of cells: Hela cells with no EpCAM expression and MCF-7 cells with high EpCAM expression. Observation was performed under an inverted fluorescence microscope and as the figures shown below illustrated that Hela cells had no fluorescence, which means, HeLa cells did not bind to any of the three aptamers; MCF-7 cells, however, had a better binding effect on aptamer 1 and 3, but an inferior binding effect on aptamer 2.

Figure 1. The combination of MCF-7 cells and aptamer

Figure 2. The combination of HeLa cells and aptamer

The Formation of DNA Hydrogel Without Cells

1. The formation of circular template In order to examine the successful preparation of the circular template, different samples were characterized by polyacrylamide gel electrophoresis (PAGE) as shown in Fig. 3, ligation products of ssDNA (Lane 2) exhibited a series of dispersive bands with slower migration than that of ssDNA (Lane 1), indicating the formation of circular templates and other byproducts. After the ligation products were treated with Exo I and Exo III, only one bright and well-defined band still existed, proving the complete digestion of ligation byproducts and successful preparation of the circular template (Lane 3).

Figure 3. PAGE image analysis of ssDNA (Lane 1), ligation products of ssDNA by reaction with Primer 3 and T4 DNA ligase (Lane 2), purified circular template by digesting above-mentioned ligation products with Exo I, and Exo III (Lane 3).

2. The formation of DNA Hydrogel For the next we carried out the hydrogel formation experiments with the prepared circular template, and the formation of hydrogen was confirmed by the following experiments. (a) Agarose Gel Electrophoresis Agarose gel electrophoresis was used to evaluate the formation of DNA hydrogel. DNA hydrogel is difficult to migrate through the agarose gel and remain in home position.

Figure 4.AGE image analysis of phi29(+) (Lane 1), phi29(-) (Lane 2). M1, DL500 DNA size marker. M2, DL2000 DNA size marker.

(b) SEM Scanning electron microscopy (SEM) was used to obtain the morphology of the DNA hydrogel. As shown in Figure 4, a large number of hydrogel particles with a diameter of about 2 μm are observed in the buffer, which indicates the hydrogel has actually formed.

Figure 5. SEM graph of the hydrogel sample

(c) DLS Dynamic light scattering (DLS) analysis is used to get the size of the DNA hydrogel. As shown in Figure 5, the size of hydrogel particles is between 100 nm and 3000 nm.

Figure 6: DLS size distribution of DNA hydrogel particle

(d) Observation For the next we centrifuged the sample at 14000rpm for 10min. As shown in Figure 6 that significant volume of hydrogel is formed.

Figure 7. DNA Hydrogel

The Formation of DNA Hydrogel With Cells

To verify the success of our design for cell-specific initiation of hydrogel formation, we performed the following experiment. First, we use GelRed, which is a red dye that only dyes DNA while does not dye living cells. To verify this, we stained HeLa and MCF-7 cells directly with GelRed. The results showed that neither type of the cell was stained.

Figure 8. GelRed performed in living cells

After demonstrating that the aptamer successfully binds to EpCAM positive cells without binding to negative cells, we want to form the DNA hydrogel around the cells. The designed hydrogel-forming system was added, and after 8 hours , it was stained with GelRed. Under an inverted fluorescence microscope, it was observed that a hydrogel was formed around the MCF-7 cells with Aptamer 1 and Aptamer-Linker-Inhibitor-H1, which meant that the system we designed to initiate hydrogels by combining cells with aptamers was successful.