Content of DENDETX and Materials Used:
- Sample pad (cellulose fibers)
- Conjugate pad (glass fibers)
- Test line (glass fibers)
- Control line (glass fibers)
- Absorbent pad (cellulose fibers)
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.
Sample without Dengue Virions:
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.
Sample with Dengue Virions:
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.
Method:
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.
Results:
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.
Figure 1. The Raman spectra of DCC/NHS modified AuNPs (yellow), and DCC/NHS modified AuNPs conjugated with 1 μM (blue) and 0.1 μM (red) DNA primers.
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.
Figure 2. Fluorescence from modified glass fiber membranes reacting with 1x GFP stock, modified glass fiber membranes reacting with 0.5x GFP stock, and non-modified glass fiber membranes (control).
Current Progress of DENDETX:
We have shown that we are able to conjugate peptides/proteins to the AuNP, control line, and test line.
Abbreviations:
MUA: 11-mercaptoundecanoic acid;
MCH: 6-hydroxy-1-hexanethiol;
EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;
NHS: N-hydroxysuccinimide;
DCC: N,N’-dicyclohexylcarbodiimide;
MHA: 16-mercaptohexadecanoic acid;
SB thiol: 1-(2-sulfosulfanylethylamino)tetradecane