DNA embedding update

May 16, 2019

by Kil Ho Lee

QDs are inorganic nanoparticles that are coated with surface capping agent known as ligands. Depending on the type of ligand, QDs can be soluble either in an organic solvent (i.e. chloroform) or in an aqueous solvent (i.e. water). For biological applications, QDs need to be soluble in an aqueous environment. Therefore, the ligands must be suitable to render QDs stable in an aqueous solvent, to enable their use as imaging agents for STORM.

We have been using quantum dot CdSe/ZnS having octadecylamine (ODA) as the capping ligand. A ligand exchange process is necessary to achieve water solubility. Ligand exchange involves the replacement of the hydrophobic octadecylamine with hydrophilic mercaptopropionic acid (MPA). Mercaptopropionic Acid is suitable for transfer from organic (i.e. chloroform, hexane) phases to the aqueous (i.e. water) phase. Our previous reports described process optimization to successfully replace the original octadecylamine (ODA) ligands on the QD surface with 3-MPA.

The previously optimized ligand exchange technique has not been working with a new batch of QDs. Hence, in this report, we took a step back and attempted to fine-tune our aqueous transfer procedures to make them more robust.

 

Results:

  1. Ligand exchange experiments:

The following set of experiments were conducted using QD from Ocean Nanotech.

We tried to apply the ligand exchange process for a new batch of QD (Emission peak at 533 nm) using the same ratio of MPA to QD, following the same experimental procedure been used for previous experimentation.

Figure 1. Fluorescence spectra of MPA coated Aqueous QD (Ocean Nanotech)

 

From figure 1, it can be clearly seen that QD exhibit very low fluorescence signal. Such a low fluorescence intensity output clearly indicates that the ligand exchange procedure did not work well with this new batch of QD and that the QD surface is not passivated enough. From the results of the 2nd trial. It can be seen that the QD emission maxima has shifted to a higher wavelength indicating the formation of aggregates and hence, explaining decreased fluorescence output.

Figure 2. QD aggregation during the embedding process

 

Embedding procedure requires a pH level of 12.2. However, when the QD surface is not well passivated with MPA ligands (after the ligand exchange process), QDs tend to aggregate and crash out of the aqueous solution. A similar observation was reported when this new batch of MPA coated QD was used in the embedding process.

Quality of QD is dependent on a number of factors including the procedure followed for synthesis, reaction, and surrounding temperature, humidity, etc. Therefore, we see variabilities in QD quality from batch to batch and vendor to vendor [1]. We have tried different QD batches from the same vendor (Ocean Nanotech), and the procedure is not robust enough to be adapted for different batches.

Further, we tried to apply the same ligand exchange protocol to QDs (Emission maxima – 544 nm) procured from NN labs.

 

The following set of experiments were conducted using QD from NN Labs.

Figure 3. Fluorescence spectra of MPA coated Aqueous QD (NN Labs)

 

The ligand exchange process worked very well for NN Labs QD indicating the inherent differences in the QD supplied by different vendors. The MPA coated QD exhibited a fluorescent intensity maxima of 8 x 105 1/s, indicating successful passivation of the QD surface with MPA ligands.

The original stock of MPA coated NN Labs QD was diluted 10 times its original concentration for use in embedding experiments. However, diluted QDs were not colloidally stable at a lower concentration and aggregated standing overnight. The capping ligand used to passivate the QD surface is in reversible equilibrium with free ligands in solution. In addition to passivating the QD surface, ligands also provide steric repulsion, thereby keeping the nanoparticles colloidally stable. When the MPA coated QD stock was diluted with water, the reversible equilibrium kinetics is disrupted. With the QDs diluted, the equilibrium free ligand concentration is greatly reduced. This develops a driving force for ligands to dissociate from the surface, thereby decreasing the ligand density on the nanoparticle. Decreasing ligand density leads to ineffective passivation of the surface, manifesting itself in the form of nanoparticle aggregates. Due to such dynamic interactions between MPA and QDs, ligand-exchanged QDs tend to aggregate over time in an aqueous environment.

Figure 4. Colloidal stability of diluted MPA coated QD from NN-Labs (0.2 mg/ml)

 

  1. DNA embedding experiments:

Table 1. Materials and conditions used for DNA Embedding/PBS (100x dilution) washing

 

QD (0.02 mg/ml)

DNA (8 μM)

Heating

Washing buffer

QD:DNA Ratio

Control 1

100 μL

100 μL

X

PBS buffer 100X dilution

1/200

Sample 1

100 μL

100 μL

0

PBS buffer 100X dilution

1/200

Control 2

100 μL

200 μL

X

PBS buffer 100X dilution

1/400

Sample 2

100 μL

200 μL

0

PBS buffer 100X dilution

1/400

Control 3

100 μL

300 μL

X

PBS buffer 100X dilution

1/600

Sample 3

100 μL

300 μL

0

PBS buffer 100X dilution

1/600

 

Figure 5. Fluorescence spectra of QDs and Cy5 terminated ssDNA from DNA-embedded QDs purified using PBS buffer (100x dilution)

 

The fluorescence emissions of QDs and Cy5 terminated ssDNA showed two distinct peaks at 550 nm (QD) and 664 nm (Cy5). Sample 1, 2 and 3 were subjected to the embedding procedure with the proper set of their respective controls. The embedded samples have ssDNA and MPA along with the enlarged QD shell formed during the embedding process. PBS 100X provide an ionic environment that minimizes the electrostatic repulsion between the negatively charged ssDNA strands. This reduction of electrostatic repulsion between nanoparticles coupled with an increased number of centrifugation steps at higher rotation speeds leads to embedded QD sticking to the centrifugal filter device. Hence, we see a reduced fluorescence output for all the embedded samples.

Future Work: The Ligand exchange protocol works well with QDs procured from NN Labs. However, the colloidal stability of MPA coated NN-Labs QDs presents an issue on dilution with water. Further experiments are required to study the effect of dilution on the stability of MPA coated QD. Experiments will be conducted using a series of diluted MPA coated QD, with fluorescence measured at regular time periods. Freshly prepared MPA coated NN-labs QD seem to have a decent fluorescent output. With proper optimization of the ligand exchange protocol, we will be able to develop a well-passivated QD which is robust enough to provide decent fluorescence output after embedding as well.

 

References: [1] Kil Ho Lee, Thomas Porter, Jessica O. Winter, Fluorescence loss of commercial aqueous quantum dots during preparation for bioimaging, MRS communications (2019). DOI: 10.1557/mrc.2019.4