Quantum dot (QD) – Deoxyribonucleic Acid (DNA) – Gold nanoparticle (AuNP) composite - Part IV

May 15, 2019

by Abhilasha "Abby" Dehankar

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  SEQ Figure \* ARABIC 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.

Monthly Update:

For this month’s report, we come bearing good news! After solving the setback of QD dispersion last month, we attempted the QD-DNA-AuNP composite formation again and we were successful. We were able to synthesize the QD-DNA-AuNP composites reproducibly for different batches. Successful formation of QD-DNA-AuNP composites was indicated by the reduced fluorescence of QDs as a result of FRET-based quenching (Figure 1). We also tested the functionality of the QD-DNA-AuNP composites by thermal actuation. Briefly, for thermal actuation, we heated the QD-DNA-AuNP composite solution to de-hybridize the DNA causing the QDs to emit higher fluorescence (On-state), followed by cooling of the solution to re-hybridize the DNA diminishing the fluorescence of QDs (Off-state). This process is similar to that described in Figure 1, except that instead of azobenzene photo-actuation, a thermal actuation was employed. In the following section, we will dive into the details of the performed experiments, their results, data analysis, and future steps.

As mentioned above, QD-DNA-AuNP composite or mixture of complementary QD-ssDNA and AuNP-ssDNA composites (C) results in reduced QD fluorescence. The decrease in fluorescence of C is generally referred in comparison to free QD-ssDNA conjugates (Q) or a mixture of non-complementary QD-ssDNA conjugates and AuNP-ssDNA conjugates (NC) because of the absence of FRET-based interaction of QDs with AuNP in the latter two. Therefore, we compared the fluorescence of Q, NC, and C to prove the success of QD-DNA-AuNP composite formation. For the experiment, we prepared the following three samples:

1. Q: QD-ssDNA conjugates dissolved in 2 wt% polyethylene glycol, 20 kDa (PEG20K) in PBS at the final concentration of 3 nM.

2. NC: QD-ssDNA conjugates and non-complementary AuNP-ssDNA conjugates (both dissolved in PEG20K in PBS) mixed at the ratio of 2:1 with final QD concentration of 3 nM.

3. C: QD-ssDNA conjugates and complementary AuNP-ssDNA conjugates (both dissolved in PEG20K in PBS) mixed at the ratio of 2:1 with final QD concentration of 3 nM.

We observed an obvious decrease in the fluorescence of QDs (Fig 2) in sample C as compared to both Q (~90% decrease) and NC (~75% decrease), clearly indicating the successful formation of QD-DNA-AuNP composite. Interestingly, despite the absence of FRET-based AuNP interactions in the NC sample, we observed a significant decrease of QD fluorescence in NC as compared to Q (~50% decrease). We performed multiple replicates of the experiment above to ensure the reproducibility of results. Fig 3a displays the results of all the replicates in the form of a bar graph where each bar represents the normalized fluorescence intensity at 560 nm of that sample.

Figure 2: Fluorescence spectra of QDs in sample Q (QD-ssDNA conjugates: blue), NC (QD-ssDNA conjugates + non-complementary AuNP-ssDNA conjugates: orange) and C (QD-ssDNA conjugates + complementary AuNP-ssDNA conjugates: grey)

The observed gap between the fluorescence of Q and NC can be attributed to the Inner-filter effect (IFE). IFE is a known concern in fluorescence spectroscopy that results in an apparent decrease of sample fluorescence because of the attenuation of excitation or emission beam, especially in the presence of a highly absorbing component. IFE effect is generally negligible at low concentrations (absorbance of the sample < 0.1). At 3 nM, QDs do display IFE-affected fluorescence (e.g., sample Q). However, IFE becomes strongly dominant on the addition of highly absorbing AuNPs (e.g., sample NC and C). Therefore, it is essential to compensate the IFE effect to accurately determine the FRET dependent drop in the fluorescence of QDs in sample C. The observed drop in QD fluorescence of C is cumulative because of the IFE and the FRET effect while that in NC is only because of the IFE. As a result, the actual drop in QD fluorescence in C can be determined by matching the QD fluorescence in Q and NC. Based on this correction, Fig 3b displays the IFE-corrected fluorescence of C and NC, demonstrating an overall ~75% drop in sample C exclusively because of the FRET effect. Further, we also determined that ~85% of the QDs in sample C were actually hybridized with AuNPs. By taking into account the fluorescence because of free QD-ssDNA conjugates in sample, we calculated an effective decrease of ~90% in QD fluorescence in a QD-DNA-AuNP composite as compared to a free a QD, meaning, QD On/Off ratio of 10, bringing very close to the goal of 20 for optimal QSTORM imaging.

Figure 3 (a) Comparative fluorescence of Q (QD-ssDNA conjugates), NC (QD-ssDNA conjugates + non-complementary AuNP-ssDNA conjugates), and C (QD-ssDNA conjugates + complementary AuNP-ssDNA conjugates) before IFE-correction; (b) Comparative fluorescence of Q, NC and C after IFE correction.

Finally, we also tried to actuate the NC and C sample thermally by heating them above the melting point of DNA (30 oC) to 50 oC, followed by cooling them below the melting point to 10 oC. In sample C, the heating should induce dehybridization and consequent increase in the QD fluorescence, while cooling should result in re-hybridization and reduced fluorescence. In contrast, the heating and cooling should not induce any change in fluorescence for sample NC because of the absence of a hybridized DNA (Fig 4). This successful actuation of QD from its off-state to the on-state and back to the off-state confirmed the functionality of our composite, bringing us one-step close to our goal. In the future, we will move towards photo-actuation of QD-DNA-AuNP composites by employing azo-benzene modified DNA for the experiments.

Figure 4: Temperature dependent fluorescence intensity of sample C (QD-ssDNA conjugates + complementary AuNP-ssDNA conjugates) and sample NC (QD-ssDNA conjugates + non-complementary AuNP-ssDNA conjugates).