Team:HK SKHLPSS/Result

Result

1. Gel electrophoresis

From the graph, the first four lanes were loaded with each DNA strand that is responsible in forming nano-structure. The fifth lane was loaded with Lactobacillus target DNA. The eighth lane was loaded with all the single stranded DNAs required in forming nano-structure. The ninth lane was loaded with all the single stranded DNAs and the Lactobacillus target DNA. Since the sixth to eighth lanes were shown to be run slower than other lanes, it reflected that the single stranded DNAs were bound with each other so that they possessed a larger molecular size than the each single stranded DNA.

When all the single stranded DNAs and the Lactobacillus target DNA were mixed together, it was found that the molecular size were the largest among all the lanes in the gel, showing that the nano-structure was formed from four single stranded DNAs and the nano-structure could be successfully bound with the actobacillus target DNA. Hence, the above PAGE result is a crucial proof in the nano-structure formation and the binding between the nano-structure and Lactobacillus target DNA.

2. DNA Peroxidase Assay - check for the specificity of the nano-structure in target DNA sequence

From the graph on the left, the background itself (tube A) has given a low absorbance at 0.092. It is also shown that the nano-structure (tube B) and target DNA (tube C) itself provided a low absorbance at 0.141 and 0.101 respectively. However, the mixture of nano-structure and target DNA sequence (tube D) provided a significantly high absorbance at 0.256.

On the other hand, the presence of random DNA in nano-structure (Tube E) did not show an increase in the absorbance (0.107), indicating that the nano-structure has a high specificity with the target DNA. The above results suggest that the target DNA could be successfully bound with the nano-structure we designed via Tiamat.

3. DNA Peroxidase Assay - check for the relationship between the target DNA sequence concentration and absorbance

From the graph on the right, the absorbance increased from 0.141 to 0.256 when the concentration of target DNA sequence increased from 0 to 500nM. This showed that the higher the concentration of target DNA sequence, the higher the absorbance. The detection limit is as low as 208nM. Plus, the increase in absorbance is directly proportional to the concentration of target DNA sequence.

The next part of our project is to use peroxidase assay to find out whether the DNA inside bacteria we extracted from food sample could be bound with our designed nano-structure. Hence, same nano-structure was used in the next peroxidase assay.

4. Lactobacillus Peroxidase Assay- check for the specificity of the nano-structure in lactobacillus

From the graph on the right, it is clearly seen that the mixture of Lactobacillus and the nano-structure (tube H) gave the highest absorbance (0.164) compared to tube I (the mixture of the nano-structure and E.coli) at 0.128, indicating that the DNA in Lactobacillus could be successfully bound with the nano-structure so that a stable G-quadruplex could be formed to give the peroxidase activity of ABTS. It also showed high specificity could be obtained to detect the presence of Lactobacillus.

5. Lactobacillus Peroxidase Assay - check for the relationship between Lactobacillus concentration and absorbance

The second experiment would like to check for the relationship between the concentration of Lactobacillus and the absorbance at 405nm. The above graph indicated that the absorbance increased from 0.149 to 0.164 when the concentration of Lactobacillus increased from 0 to 40 ug/mL. This showed that the higher the concentration of the Lactobacillus, the higher the absorbance. The detection limit is as low as 15.1ug/mL. Again, the increase in absorbance is also directly proportional to the concentration of Lactobacillus, suggesting that the nano-structure we designed could detect the concentration of Lactobacillus.