December 12, 2018
by Kil Ho Lee
Toward DNA-embedded Quantum Dots: Take VII
So far, we successfully demonstrated ligand exchange and DNA embedding techniques suitable for CdSe/ZnS quantum dots. In the last blog post, DNA embedding procedure was performed using single-stranded DNA (ssDNA) terminated with Cyanine Dye (Cy5). QDs with DNA embedded on the surface emitted green fluorescence (~550 nm) of QDs and red fluorescence (~660 nm) of Cy5-modified DNA.
In this experiment, DNA embedding procedure was repeated to test the effect of (1) Heating, and (2) adding excess 3-MPA.
Lastly, the hybridization of DNA embedded QDs with DNA-modified gold nanoparticles (AuNPs) was performed.
Variable (1): Heating
DNA embedding step requires the elevated temperature because the thermal input facilitates the formation of Zinc/-thiol modified DNA shell. However, previous results suggested decrease QD fluorescence intensity upon incubating QDs at high temperature (90 °C).
To test the effect of heating, QDs with and without the addition of Zn2+, 3-MPA, and ssDNA were incubated in a water bath (90 °C). After incubating the samples for 90 minutes, the samples, including QD controls, were purified using 100kDa centrifugal filter devices (repeated 6000 rpm, 3 min, repeated 3 times). Then, the fluorescence intensities were measured using a fluorometer (Figure 1).
Figure 1. Fluorescence intensities of (A) QDs and DNA embedded QDs, and (B) Cy5-modified DNA incubated in either room temperature (No heating), or 90 °C (Heating).
QDs that were not exposed to elevated temperature (Figure 1(A), black) showed the emission wavelength of 565 nm. QD incubated in a water bath (90 °C) (Figure 1(A), yellow) showed a red-shift (emission: 580 nm) and lower fluorescence intensity than the control. This result indicated the aggregation and reduced stability of QDs because of the heating, as well as the filtration step. The addition of Zinc, 3-MPA, and DNA to QD solution is required for DNA embedding. Comparing QDs dispersed in water with and without all three components for DNA embedding, the red-shift was higher for QDs exposed to elevated temperature than QDs stored in a room temperature. The fluorescence intensity was higher for QDs stored in a room temperature. Both cases showed higher fluorescence intensity compare to QD control. These results suggest that heating, as expected, facilitates the formation of DNA embedded ZnS shell. However, the formation of DNA embedded ZnS shell may occur without heating.
Variable (2): Adding excess 3-MPA
One of the components added to QD solution for DNA embedding is 3-MPA. 3-MPA is a ligand allowing QDs to be soluble in water. During DNA embedding step, ssDNA terminated with thiol (-S-) is the source of S in forming ZnS shell. Because 3-MPA also provides the thiol (-S-) for ZnS shell, adding excess 3-MPA may affect the formation of ssDNA embedded shell.
To test the effect of adding excess 3-MPA, DNA embedding was repeated with QDs incubated in a water bath (90 °C) with and without the excess 3-MPA.
Figure 2. Fluorescence intensities of DNA embedded QDs undergone DNA embedding step with and without the addition of excess 3-MPA
The results showed that the fluorescence intensity is higher when the excess 3-MPA is added (Figure 2(A)). This suggested that the excess 3-MPA helps protecting the surface of QDs.
First attempt to hybridize DNA embedded QDs with AuNPs
In this experiment, the first attempt to hybridize ssDNA (poly T) embedded QDs with ssDNA (poly A) modified AuNPs was performed. We expected that the hybridization of DNA embedded QDs and AuNPs would reduce the fluorescence intensity of QDs via FRET.
Figure 3. Fluorescence intensity of 3-MPA coated QDs (Green) and DNA embedded QDs (Red) incubated with DNA modified AuNPs.
DNA-modified AuNPs were added to 3-MPA QDs in PBS buffer (pH 7.4) and DNA embedded QDs (poly T) in PBS buffer (pH 7.4). They were incubated in a water bath at 40°C for 20 minutes. Then, the fluorescence intensities were measured (Figure 3). The fluorescence intensities showed negligible difference between 3-MPA coated QDs and DNA embedded QDs. This suggest that the hybridization did not work.
However, as reported in a blog post written by Abhilasha Dehankar, DNA coverage in AuNPs was found to be less than desired.
We aim to continue working on optimizing DNA embedding technique to increase the number of DNA embedded on QD surface. Simultaneously, we aim to synthesize AuNP with dense DNA coverage to verify FRET upon the hybridization of QDs and AuNPs.