Research Update - October - QDot Team

October 10, 2019

by Abhilasha "Abby" Dehankar

October Update – QDot Team

Edited by Faiz Khan & Elizabeth Jergens

 

Goal:  Our goal is to develop photoswitchable QD-DNA-AuNP composites that will enable us to use QDs for STORM imaging because of their controllable stochastic on/off behavior. QD-DNA-AuNP composites are designed to form by hybridization of complementary azobenzene modified single-stranded DNA (ssDNA) attached to QDs and AuNPs. Formation of QD-azoDNA-AuNP will yield quenching of QD fluorescence via a Forster Resonance Energy Transfer mechanism. The QD-azoDNA-AuNP composites are tailored to undergo photoactivated reversible QD fluorescence quenching. When UV light is applied the azobenzene molecules switch from trans to cis conformation, promoting dissociation of the double-stranded DNA complexes. Thus, QDs are detached from AuNPs, restoring QD fluorescence. Whereas, when visible light is applied the azobenzene switches back to trans conformation, promoting hybridization and reattachment of QD-azoDNA-AuNP composites and quenching of QD fluorescence.

Figure 1. QD-DNA-AuNP conjugate reversible photoswitching mechanism. QD fluorescence quenches in the proximity of AuNPs through a mechanism called “Forster resonance energy transfer” and as a result QD fluorescence is quenched when the DNA is hybridized in our QD-DNA-AuNP conjugates. On shining a UV wavelength light (300-400 nm) on the QD-DNA-AuNP conjugates, the trans-azobenzene isomerizes to its cis form. This destabilizes the DNA actuating its dehybridization which re-establishes the QD fluorescence. In contrast, shining visible light (wavelength>400 nm) reverses the entire process and quenches the QD fluorescence

Review of progress-to-date:

1. We successfully transferred organic QDs purchased from NN-labs into the aqueous phase. We achieved this transfer by exchanging the non-polar ligands (Octadecylamine) on the QD surface with polar phytochelatin-3 (PC3) peptides. The resulting QDs (PC3-QDs) are water-soluble and terminated with carboxylate (-COOH) and amine (-NH2) groups on their surfaces.

2. We successfully conjugated -NH2 terminated sulfo-dibenzocyclooctyne (sDBCO) to -COOH terminations on the surface of PC3-QDs to form QD-sDBCO conjugates using commonly-employed carbodiimide chemistry. Carbodiimide chemistry forms an amide (-CONH2-) bond between –COOH groups and -NH2 groups. QD-sDBCO conjugation allowed us to employ “click” chemistry to successfully conjugate ssDNA to PC3-QDs through the attached sDBCO group. To implement this approach, we mixed the QD-sDBCO conjugates with azide (-N3) terminated ssDNA to permit strain promoted alkyne-azide cycloaddition via a click reaction, generating QD-ssDNA conjugates.

3. The synthesized QD-ssDNA conjugates demonstrated increased attraction towards the surfaces as compared to the solution. This was most likely because of the free sDBCO groups on the QD surface that were not passivated by an ssDNA. As a result, the QDs separated out of the solution rendering them unavailable for QD-DNA-AuNP composite formation. We successfully solved the problem of QD dispersion by addition of polyethylene glycol (PEG) molecules in the solution through depletion-based stabilization.

4. We successfully conjugated AuNPs (size-15 nm) with thiol-terminated ssDNA by the formation of Au-S dative bond using an established instantaneous, low pH method.

 

Research Update - Faiz

Conjugation of ssDNA to QD involves the following steps:

  1. QDs are provided by NN Labs in toluene. For the conjugation experiment, we require the QDs to be water-soluble. Hence, the first step includes the replacement of non-polar ligands (Octadecylamine) on the QD surface with polar phytochelatin-3 (PC3) peptides. The resulting QD-PC3 is water-soluble, having both carboxylate (-COOH) and amino groups (-NH2) on their surface.
  2. Attachment of ss-DNA through strain promoted alkyne-azide cycloaddition via a click reaction

 

Figure 2. Phytochelatin-3 (PC3) coated compact QDs

 

Steps of QD-PC3 coating procedure:

  1. NN labs provides QD capped with hydrophobic ligand octadecylamine. This greatly limits its solubility in water. The first step is based on displacing the original octadecylamine with pyridine. Pyridine binds weakly to the QD surface and can be easily displaced by our desired molecule in the next step.
  2. Phytochelatin-3 (PC3) contains multiple thiol (-SH) groups, which eventually attach to the QD surface. After addition of PC3, pH of the mixture is adjusted to 10 by a tetramethyl ammonium hydroxide (TMAH). The change in pH leads to rapid displacement of pyridine with PC3.

 

We were facing problems with the aqueous transfer of QD through PC3 coating. Addition of PC3 to pyridine exchanged QD and subsequent centrifugation did not give complete precipitation of PC3 coated QD. This indicated that there might be a problem with new batch of QD. However, another simultaneous experiment conducted by our undergraduate researcher (Thomas Porter) yielded good result with complete precipitation of QD-PC3 particles. We further investigated each chemical used in the procedure. It was found that the basic solution (TMAH) might have some impurities which created problems for a complete precipitation. A trivial issue of chemical impurity bottlenecked our further photoswitching experiments. 

 

Figure 3. Left: Incomplete precipitation by contaminated TMAH (Tetramethyl ammonium hydroxide); Right: Samples exposed to UV-light.

 

Note: The QD-PC3 obtained from contaminated TMAH is still fluorescent. After separation of the supernatant, the pellet containing QD-PC3 was dispersible in water and still retained fluorescence. In fact, the concentration and fluorescence intensity of both the samples are similar to each other. From the data, it is difficult to comment about the reddish supernatant observed for QD-PC3 made from contaminated TMAH.

Sample

Concentration (µM)

QD-PC3 (Contaminated TMAH)

0.63

QD-PC3 (Pure TMAH)

0.61

 

Figure 4. Fluorescence Intensity of QD-PC3 samples

 

Research Update - Elizabeth

β1 labeling of HepG2 cells was achieved using DNA cages reversibly attached to secondary antibodies. The negative control features DNA cages made with nanoparticles with a surface chemistry of 50% NH2 and 50% Cy3. The cells have not been labeled with primary antibody in this case so there is quite a lot of non-specific binding. The experiments with 50/50 cages and 100% NH2 cages show a similar level of staining as the negative control. The next step is to eliminate the non-specific binding before continuing with the experiments.

 

Figure 5. Images from Labeling experiments