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| <img src="https://static.igem.org/mediawiki/2020/c/cd/T--CCU_Taiwan--Poster_Modeling1.png"> | | <img src="https://static.igem.org/mediawiki/2020/c/cd/T--CCU_Taiwan--Poster_Modeling1.png"> |
| <img src="https://static.igem.org/mediawiki/2020/0/0d/T--CCU_Taiwan--Poster_Modeling2.png"> | | <img src="https://static.igem.org/mediawiki/2020/0/0d/T--CCU_Taiwan--Poster_Modeling2.png"> |
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| <h3>Interaction between Gold Nanoparticles</h3> | | <h3>Interaction between Gold Nanoparticles</h3> |
| <b>Purpose:</b> | | <b>Purpose:</b> |
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| <li>Absorbent pad (cellulose fibers)</li> | | <li>Absorbent pad (cellulose fibers)</li> |
| </ol> | | </ol> |
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| The conjugate pad contains the gold nanoparticles (AuNPs) coupled with the first PTRS (PTRS-1), in which PTRS-1 is designed to interact with the virions. The second PTRS (PTRS-2) is mounted on the test line to capture the virion. E proteins are conjugated on the control line, which should always interact with PTRS-1, to ensure the kit functions normally. | | The conjugate pad contains the gold nanoparticles (AuNPs) coupled with the first PTRS (PTRS-1), in which PTRS-1 is designed to interact with the virions. The second PTRS (PTRS-2) is mounted on the test line to capture the virion. E proteins are conjugated on the control line, which should always interact with PTRS-1, to ensure the kit functions normally. |
| <br> | | <br> |
| <img src="https://static.igem.org/mediawiki/2020/1/13/T--CCU_Taiwan--Poster_Detection_kit-1.gif"> | | <img src="https://static.igem.org/mediawiki/2020/1/13/T--CCU_Taiwan--Poster_Detection_kit-1.gif"> |
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| <h4>Sample without Dengue Virions:</h4> | | <h4>Sample without Dengue Virions:</h4> |
| The AuNPs will not accumulate on the test line, and no color change will be found. The AuNPs will aggregate on control line via the interaction of PTRS-1 and E protein, so the control line turns red. | | The AuNPs will not accumulate on the test line, and no color change will be found. The AuNPs will aggregate on control line via the interaction of PTRS-1 and E protein, so the control line turns red. |
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| <h4>Sample with Dengue Virions:</h4> | | <h4>Sample with Dengue Virions:</h4> |
| The AuNPs bound to the virions via PTRS-1 will accumulate on the test line, and the residual AuNPs will aggregate on the control line. Both the test and control lines will be red. | | The AuNPs bound to the virions via PTRS-1 will accumulate on the test line, and the residual AuNPs will aggregate on the control line. Both the test and control lines will be red. |
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| <h4>Current Progress of DENDETX:</h4> | | <h4>Current Progress of DENDETX:</h4> |
| We have shown that we are able to conjugate peptides/proteins to the AuNP, control line, and test line. | | We have shown that we are able to conjugate peptides/proteins to the AuNP, control line, and test line. |
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| <br><br> | | <br><br> |
| <h4>Detection Kit</h4> | | <h4>Detection Kit</h4> |
− | The AuNPs were first modified with MUA/MCH so that carboxyl groups are exposed on the surface of the particles. Then, peptides with the amine sidechains could be conjugated to the AuNPs using the EDC/NHS approach. Similarly, the glass fiber membranes, the substrate of the test and control lines, were modified with CES to form a carboxyl surface, which allows us to employ the EDC/NHS approach to conjugate with peptides or proteins. | + | The AuNPs were first modified with MUA/MCH, and then, peptides with the amine sidechains could be conjugated to the AuNPs using the EDC/NHS approach. Similarly, the glass fiber membranes, the substrate of the test and control lines, were modified with CES, which allows us to employ the EDC/NHS approach to conjugate with peptides or proteins. |
| <br><br> | | <br><br> |
− | As a proof of concept that we are able to form the covalent bonds between the primary amines (from the PTRS) and AuNPs, we tried to conjugate the DNA primers with the modified AuNPs. We found that with the DNA conjugation, the Raman signals have a significant decrease (Figure 1). Although we have no model to explain this effect, we believe it resulted from interactions with the DNA, suggesting DNA can bind to AuNPs. | + | As a proof of concept that we are able to form the covalent bonds between the primary amines (from the PTRS) and AuNPs, we tried to conjugate the DNA primers with the modified AuNPs. We found that with the DNA conjugation, the Raman signals have a significant decrease. Although we have no model to explain this effect, we believe it resulted from interactions with the DNA, suggesting DNA can bind to AuNPs. |
| <br><br> | | <br><br> |
− | We expressed green fluorescent protein (GFP) as a mock E protein to show that after modification the glass fiber membrane can bind to the primary amines (side-chain amines) from a peptide or protein. The intensity of membrane reaction with 0.5x and 1x GFP supernatant are shown in Figure 2. The intensity was about twice as strong in the 1x supernatant as the 0.5x one, suggesting the conjugation experiments were successful. | + | We expressed green fluorescent protein (GFP) as a mock E protein to show that after modification the glass fiber membrane can bind to the primary amines (side-chain amines) from a peptide or protein. The intensities of emission correlate to the concentration of GFP using in the reactions, suggesting the conjugation experiments were successful. |
| <br> | | <br> |
| <img src="https://static.igem.org/mediawiki/2020/2/2a/T--CCU_Taiwan--Poster_Results5.png"> | | <img src="https://static.igem.org/mediawiki/2020/2/2a/T--CCU_Taiwan--Poster_Results5.png"> |
| + | <br> |
| <b>Abbreviations:</b> | | <b>Abbreviations:</b> |
| MUA: 11-mercaptoundecanoic acid; | | MUA: 11-mercaptoundecanoic acid; |
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| <h4>Interviews</h4> | | <h4>Interviews</h4> |
| We wanted to shape our project from different perspectives to achieve socially responsible research. Thus, we consulted with government officials, experts, and academic specialists to consider the application of our project in society. From the interviews, we understood the process and the standards for verification for medical devices from the Taiwan Food and Drug Administration (TFDA). Moreover, we learned about pretreatment of blood sample from The Dengue Prevention and Control Center. We also comprehended the modification of gold nanoparticles after consulting with experts in the Department of Chemistry and Biochemistry at National Chung Cheng University. | | We wanted to shape our project from different perspectives to achieve socially responsible research. Thus, we consulted with government officials, experts, and academic specialists to consider the application of our project in society. From the interviews, we understood the process and the standards for verification for medical devices from the Taiwan Food and Drug Administration (TFDA). Moreover, we learned about pretreatment of blood sample from The Dengue Prevention and Control Center. We also comprehended the modification of gold nanoparticles after consulting with experts in the Department of Chemistry and Biochemistry at National Chung Cheng University. |
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| <h4>Public Survey</h4> | | <h4>Public Survey</h4> |
| We wanted to find out how much the public knows about dengue and their demands for a dengue virus detection kit. Thus, we created a questionnaire and received 248 questionnaires by sharing the it online and distributing it in the community. | | We wanted to find out how much the public knows about dengue and their demands for a dengue virus detection kit. Thus, we created a questionnaire and received 248 questionnaires by sharing the it online and distributing it in the community. |
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| The results showed nearly 70% of people did not know about severe dengue and nearly 70% did not know what kind of treatment is required for dengue fever. This shows that the public in Taiwan lack common understanding about dengue, which leads to low awareness about the severity of this disease in Taiwan. This urged us to conduct outreach and education programs to popularize knowledge of dengue fever. | | The results showed nearly 70% of people did not know about severe dengue and nearly 70% did not know what kind of treatment is required for dengue fever. This shows that the public in Taiwan lack common understanding about dengue, which leads to low awareness about the severity of this disease in Taiwan. This urged us to conduct outreach and education programs to popularize knowledge of dengue fever. |
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− | From the survey we also determined that our detection kit meets their needs, as it should have high sensitivity during the first five days of symptoms, which is when people would consult a doctor.
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| <br> | | <br> |
| + | From the survey we also determined that our detection kit meets their needs, as it should have high sensitivity during the first five days of symptoms, which is when people would consult a doctor. |
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| <h4>Education</h4> | | <h4>Education</h4> |
| <b>Podcast</b> | | <b>Podcast</b> |