1.Through research and reading scientific articles, we discovered the mechanism of DNAzyme to detect heavy metal ions and it incorporates perfectly with our project aim.
2.Accompanied with the HTDC technology, 8-17 DNAzyme incorporated into the mechanism and it was one of the main DNAzymes we tested upon.
3.Besides the 8-17 DNAzyme, we also designed three other DNAzymes: GR-5, S9, and F8-X.
1.DNAzymes are DNA oligonucleotides that are capable of performing a specific chemical reaction, often but not always catalytic. On the substrate strand, we incorporated the fluorophore and the fluorescence quencher, in which the fluorescent signal is being repressed. However, if we incorporate heavy metal into the DNAzyme structure, the substrate strand will be cleaved and the fluorescent signal can be detected.
Fig.2.2.1 Schematic diagram of DNAzyme reaction
Table 2.2.2 DNAzyme Design
Fig.2.2.3 8-17 DNAzyme 2D structure
The 2-D structure of 8-17 DNAzyme is shown below.
Fig. 2.2.4 8-17 DNAzyme detection limit testing
8-17 DNAzyme shows high efficiency in detecting heavy metal ions, and its specificity towards Pb2+ is demonstrated. The detection lower limit for Pb2+ is 0.01 μM.
Fig. 2.2.5 F8-X DNAzyme 2D structure
Fig. 2.2.6 F8-X H2O2 concentration testing
According to an article, H2O2 might promote the efficiency of F8-X DNAzyme. So we conducted this experiment and found that the best concentration of H2O2 is 1000 μM.
Fig. 2.2.7 1000 μM H2O2 testing
From this experiment, we determined that F8-X’s detection limit for Cu2+ is 0.01 μM (micro mole).
Fig. 2.2.8 F8-X Specificity testing
We have determined that F8-X DNAzyme has specificity for Cu2+.
F8-X DNAzyme shows high efficiency in detecting heavy metal ions, and its specificity towards Cu2+ is demonstrated.
Because of the extensive length of GR-5 DNAzyme, it is very difficult to incorporate the fluorescence group into the sequence. Therefore we decided to directly combine GR-5 with Cas12a system.
Fig. 2.2.9 GR-5 DNAzyme 2D structure
Fig. 2.2.10 GR-5 Specificity testing
The testing for GR-5 DNAzyme failed. Its specificity for lead ion could not be determined.
Through testing, we came to the conclusion that GR-5 is incapable of detecting heavy metal ions after combining to Cas12a system, so we chose not to use it for our project.
Same with GR-5, S9 is also incapable of adding the fluorescence group onto the sequence and just incorporates with Cas12a.
Fig. 2.2.11 S9 DNAzyme 2D structure
Fig. 2.2.12 S9 Specificity testing
We have determined that S9 DNAzyme has specificity for Mn2+.
Fig. 2.2.13 S9 Mn2+ detection limit testing
From this experiment, we find that S9’s detection limit for Mn2+ is 100 μM (micro mole).
The combination testing of S9 and Cas12a is successful. We proved S9's specificity towards Mn2+ and determined its detection limit for Mn2+, which is 100 μM.
DNAzyme Experiment Conclusion
Of four DNAzymes we tested upon, 8-17 DNAzyme is tested individually, GR-5 and S9 DNAzyme are tested by combining to Cas12a system, F8-X DNAzyme has both individual testings and combination testings. For 8-17 DNAzyme, its detection limit of Pb2+ is 0.01 μM. For GR-5 DNAzyme, its combination testings have failed. We believe that the added Pb2+ might affect the activity of Cas12a nuclease. For S9 DNAzyme, its combination testings are successful. It has specificity for Pb2+, and its detection limit of Pb2+ is 100 μM. For F8-X DNAzyme, its individual testings are successful. It has specificity for Cu2+, and its detection limit of Cu2+ is 0.01 μM. In contrast, the F8-X and Cas12a combination testings have failed. We believe that the added Cu2+ might affect the activity if Cas12a nuclease.
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