Effective purification of DNA embedded QDs

April 1, 2019

by Kil Ho Lee

Introduction

Earlier this year, we posted a blog post (Jan. 15th, 2019: “DNA Embedding – One step closer”) on the challenge associated with purifying free ssDNA after DNA embedding. Briefly, after embedding ssDNA on QD (CdSe/ZnS quantum dots) surface, we have performed the centrifugal filtration to remove excess ssDNA strands. However, the control conditions (QD mixed with ssDNA without applying heat for surface modification) have consistently shown that the centrifugal filtration using distilled water would not wash all the free ssDNA strands. In the same report, we demonstrated the attempts to remove excess ssDNA strands using PBS buffer because the ionic buffer would reduce the electrostatic interaction of negatively charged ssDNA strands, helping individual ssDNA to pass through the filter. However, surprisingly, the centrifugal filtration using PBS buffer (pH 7.2) caused QDs, as well as free ssDNA strands, to pass through the filter. To utilize DNA-embedded QDs for forming a complex with another nanostructure (i.e. gold nanoparticles) via DNA hybridization, we need to make sure free DNA strands are removed from DNA-embedded QDs.  

Objective

In this study, we performed the centrifugal filtration using water and PBS buffer at various concentration. Initially, PBS buffer used was a solution consisting of 0.15 M NaCl and 0.1 M sodium phosphate (pH 7.2). We decided to reduce the buffer concentration by diluting the buffer with distilled water because reducing the ionic strength of PBS buffer may help to retain QDs while removing free ssDNA strands.

Results and Discussion

DNA Embedding and purification via Centrifugal filtration using PBS buffer

Table 1 summarizes the experimental conditions.

Table 1. Materials and conditions used for DNA Embedding

 

QD

(0.3 mg mL-1)

DNA

(100 nM)

Heating

Washing buffer

Control

 QD + DNA

100 µL

200 µL

X

water

Sample 1

100 µL

200 µL

O

PBS buffer

Sample 2

100 µL

200 µL

O

PBS buffer 10x dilution

Sample 3

100 µL

200 µL

O

PBS buffer 100x dilution

Sample 4

100 µL

200 µL

O

PBS buffer 1000x dilution

Sample 5

100 µL

200 µL

O

PBS buffer 10000x dilution

 

After applying DNA embedding protocol to Sample 1 – 5, the fluorescence intensities of QDs and Cy5 terminated ssDNA were collected using a fluorometer (Figure 1).

Figure 1. Fluorescence spectra of QDs and Cy5 terminated ssDNA collected from DNA-embedded QDs purified using PBS buffer

The control sample, which the solution of aqueous QDs mixed with ssDNA strands, was washed using distilled water via centrifugal filtration. The fluorescence emissions of QDs and Cy5 terminated ssDNA showed two distinct peaks at 550 nm (QD) and 664 nm (Cy5). Sample 1 – 5 were DNA embedded QDs that were washed using PBS buffer (pH 7.2) at different concentrations. Without dilution (PBS (1x)), the reduced QD emission showed the lost of QDs through the filter. The same sample showed reduced Cy5 signal from ssDNA compared to the control. These results were consistent with previously reported results.

Samples 2 – 5 were washed using PBS buffer (pH 7.2) at a lower concentration. The fluorescence emission of QDs for these samples was relatively close to the control, which indicated that QDs were not washed through the filter. These samples showed reduced Cy5 signal from ssDNA compared to the control, which suggested the effective free ssDNA removal.

 

Based on these findings, we were confident that we could optimize the filtration step to (1) avoid losing QDs through the filter, and (2) remove excess ssDNA strands. Hence, we performed additional experiments.

DNA Embedding and purification via Centrifugal filtration using PBS buffer

Table 2 summarizes one of the experimental conditions we tested to optimize washing.

Table 2. Materials and conditions used for DNA Embedding/PBS (100x dilution) washing

 

QD

(1.75 mg mL-1)

DNA

(1 μM)

Heating

Washing buffer

Control

 QD + DNA

100 µL

23.58 µL

X

water

Sample 1

100 µL

23.58 µL

O

PBS buffer 100x dilution

Sample 2

100 µL

47.17 µL

O

PBS buffer 100x dilution

Sample 3

100 µL

70.75 µL

O

PBS buffer 100x dilution

In this experiment, we used higher QD concentration of 1.75 mg mL-1 and DNA concentration of 1 μM because we wanted to scale up the embedding protocol to obtain higher fluorescence emission peak for clearly distinguishing Cy5 signals from each sample. Also, we tested 3 samples of increasing QD to DNA ratio.

Figure 2. Fluorescence spectra of QDs and Cy5 terminated ssDNA from DNA-embedded QDs purified using PBS buffer (100x dilution)

The control sample consisted of QD mixed with ssDNA (QD to DNA molar ratio = 24). We expected that washing the control sample would reduce the Cy5 signal from ssDNA, while maintaining relatively high QD fluorescence. Indeed, the fluorescence spectra (Figure 2) showed what we expected.

Sample 1, which had the same QD to DNA ratio as the control sample, a slight reduction in QD fluorescence, and negligible Cy5 signal. This result suggested that most of the excess ssDNA strands were removed and DNA embedding, though it was successful (suggested by the slight red shift in QD emission wavelength), at this particular QD to DNA ratio resulted in only a few ssDNA strands on QD surface.

Sample 2, which had 2x of QD to DNA molar ratio as Sample 1, showed a significant aggregation. We believe this particular sample was ill-processed during the filtration. However, we observed a similar result found in Sample 1.

Sample 3, which had the highest QD to DNA molar ratio tested, showed QD emission as high as the control and Sample 1, and the distinct Cy5 peak after the washing. This result suggested that the effective washing was achieved and a substantially more ssDNA is present on QD surface.

 

Although not included in this post, additional experiments performed suggested that the distinct Cy5 peak observed in Sample 3 was indeed the signal from DNA embedded on the QD surface.

Conclusion

By reducing the concentration of PBS buffer (pH 7.2) we were able to purify DNA-embedded QD from excess ssDNA strands. Specifically, when PBS buffer (consisting of 0.15 M NaCl and 0.1 M sodium phosphate) was diluted by 100x, we were able to obtain DNA-embedded QDs with excess DNA removed from the sample.

So far, we were limited to a relatively low QD to DNA ratio because we weren’t able to purify the excess DNA effectively. However, with better purification technique, we believe that we could increase the number of ssDNA present on the QD surface by increasing QD to DNA ratio further. We plan to continue to optimize the system so that we could achieve DNA hybridization of DNA-embedded QDs with DNA hinges/origami structure and/or gold nanoparticles.