LAE
The results of TR-PCR indicate the length of TRS varies with the concentration of the primers. We found the typical trend that the lower the concentration, the higher the repeats of the TR-PCR products. However, the product concentration decreases when the primer concentration increases. TRS-110*3 and TRS-151*7, with sizes ranging from 200-500 bp.
The TR-PCR products were taken to AD-PCR. The AD-PCR product of TRS-110*3 TRS-151*7 with sizes ranging from 200-500 bp were extracted and ligated to TA vectors.
E Protein
We expressed E protein to use it in the control line.
The weak bands from 63 to 48 kDa indicate expression of the E protein with the HA tag, which has a size of 61.2 kDa. We further confirmed using Western blot with the anti-His tag antibodies.
CLEC5A
In addition to the required E protein used in our kit, we attempted to express CLEC5A as a model protein to interact with E protein.
The strong bands on 26.6 kDa indicate expression of the CLEC5A extracellular domain with the Myc tag, which is also confirmed using western blot with the anti-His tag antibodies.
Detection Kit
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.
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).
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