Quantum dot – Deoxyribonucleic Acid – Gold nanoparticle composite - Part I

December 12, 2018

by Abhilasha "Abby" Dehankar

Goal: Our goal was to test the Quantum dot – Deoxyribonucleic Acid – Gold nanoparticle (QD-DNA-AuNP) composites formed by hybridization of complementary single-stranded DNA (ssDNA) modified QDs and AuNPs and with ssDNA that did not contain azobenzene modification. Formation of QD-DNA-AuNP will yield quenching of QD fluorescence via a Forster Resonance Energy Transfer mechanism. This study will help us quantify the QD fluorescence quenching observed on formation of stable QD-DNA-AuNP composites and allow us to tune the DNA sequence for maximizing the fluorescence quench while maintaining room temperature stability of the QD-DNA-AuNP composites. The ultimate goal is to employ azobenzene modified ssDNA to form QD-azoDNA-AuNP composites that would undergo photoactivated reversible QD fluorescence quenching (Fig 1). When UV light is applied the azobenzene molecules switch from a trans to cis conformation, promoting dissociation of the dsDNA complexes. Thus, QDs are detached from AuNPs, restoring QD fluorescence. These photoswitchable QD-DNA-AuNP conjugates will allow us to employ QDs for STORM imaging because of their predicted and controllable stochastic on/off behavior.

Figure 1: QD-DNA-AuNP conjugate reversible photoswitching mechanism. QD fluorescence quenches in proximity of AuNPs through a mechanism called as “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 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 dibenzocyclooctyne (DBCO) to -NH2 terminations on the surface of PC3-QDs to form QD-DBCO conjugates using commonly-employed carbodiimide chemistry. Carbodiimide chemistry forms an amide (-CONH2-) bond between –COOH groups and -NHgroups. This allowed us to employ “click” chemistry to successfully conjugate ssDNA to PC3-QDs through the attached DBCO group. To implement this approach, we mixed the QD-DBCO conjugates with azide (-N3) terminated ssDNA to permit strain promoted alkyne - azide cycloaddition via a click reaction, generating QD-ssDNA conjugates.

3. We used a DNA origami platform to test the functionality of the ssDNA-QD and ssDNA-AuNP conjugates. For this, we mixed the nanoparticle (NP) – ssDNA conjugates with DNA origami hinges containing corresponding complementary ssDNA sequences only at a specific location on the hinge arm. Successful NP binding is observed only at a specific location on the hinge arms, indicating functional ssDNA-NP conjugates.

4. In the previous report, we briefly studied switchability of the photoresponsive, hybridized azobenzene-modified-deoxyribonucleic acid (azoDNA) without nanoparticles on our current optical setup as a control experiment. One of the ssDNA employed was modified with a fluorophore while the complementary pair was modified with azobenzene and a FRET based quencher, such that the fluorophore and quencher were separated by the length of the DNA on dsDNA formation. We saw significant quenching of the fluorophore on hybridization, however, the photoswitching did not work. Further investigation to enable the photo-switch is in progress.

Monthly Update:

QD-ssDNA purification (Successful):

As mentioned in the previous report, I had been working on investigating different purification strategies for separation of ssDNA-QD conjugates from excess, unconjugated ssDNA/QD. As mentioned earlier, QD-DNA-AuNP composites form via hybridization of complementary ssDNA-QD and ssDNA-AuNP conjugates. In practice, the synthesis involves mixing of colloidal solutions of ssDNA-QD conjugates with that of complementary ssDNA-AuNP conjugates at known concentrations (higher concentrations are desired) and proportions (preferably ssDNA-QD: AuNP-ssDNA = 1: 1). Presence of excess, unconjugated ssDNA in ssDNA-NP colloidal solutions results in competition between the unconjugated ssDNA and ssDNA-NP conjugates for hybridization with corresponding complementary ssDNA-NP conjugates. This could significantly decrease or even eliminate the formation of QD-DNA-AuNP composites. Thus, a high excess, unconjugated ssDNA separation efficiency is extremely important. Further, a high yield is desirable for precise determination of concentration. Therefore, a high yield and ssDNA purification efficiency of ssDNA-QD conjugates is crucial for optimal formation of QD-DNA-AuNP composites.

I worked on three different purification strategies: column separation, gel electrophoresis and centrifugal filtration. Different separation efficiencies and yields were observed for each of these techniques, based on their operating principle and implementation (Table 1). Since centrifugal filtration satisfied the purification criteria mentioned above, I decided to use centrifugal filtration for future experiments.

Table 1: Efficiency and yield of different purification strategies

Parameters

Size exclusion column

Gel electrophoresis

Centrifugal filtration

Separation of excess and unconjugated ssDNA

~100%

~100%

~95%

Separation of excess and unconjugated QDs

High

Low

No

QD recovery (yield)

Low

Low

High

 

QD-DNA-AuNP composite (Unsuccessful):

Synthesis: For initial experiments, we decided to start with 16 base-pairs T sequence for the ssDNA to form ssDNA-QD (16T-QD) conjugates because of low non-specific interactions of the nucleotide T with nanoparticle surfaces as suggested in the literature. Next, AuNPs of 15 nm size were conjugated to 20 base-pairs A sequence to form complementary ssDNA-AuNP (20A-AuNP) conjugates. The 16T-QD and 20A-AuNP conjugates were mixed together at equimolar ratio with 10 nM individual NP concentration and allowed to hybridize overnight to form QD-16(AT)-AuNP composite.

Detection Strategy: The formation of QD-DNA-AuNP composite was analyzed using gel electrophoretic separation.  As mentioned in a previous blog (“Testing quantum dot – single stranded DNA conjugates”), Gel electrophoresis is a charge driven separation technique used for separating mixtures of charged components (e.g., ssDNAs (as they have negative phosphate backbone) from negatively charged NPs) based on their charge and/or size. In this technique, components to be separated are pushed by an applied electric field through a gel consisting of small pores. Smaller components migrate faster through the pores compared to larger components, resulting in size-based separation. Since QD-DNA-AuNP composite will have significantly higher size as compared to ssDNA-QD and ssDNA-AuNP conjugates alone, we expect much slower migration of QD-DNA-AuNP composites through the gel. Therefore, on gel electrophoresis purification of the unpurified QD-16(AT)-AuNP composite solution, the stable QD-16(AT)-AuNP composites should be present closer to the sample addition well of the gel in comparison to excess, individual 16T-QD and 20A-AuNP conjugates.

Detection technique: Since QD fluorescence quenches in close proximity of AuNPs, the formation of QD-DNA-AuNP composites was detected by imaging AuNPs. AuNPs in the gel were imaged using visible light as the excitation source. As AuNPs hinder visible light transmission through the gel, a dark band is observed at locations where AuNPs are present in the gel (Fig 2). Based on the QD-DNA-AuNP conjugate formation efficiency, this should result in appearance of atleast two well separated dark bands of AuNPs with QD-16(AT)-AuNP conjugates closer to the sample addition well, while 20A-AuNP conjugates farther away from the well.

Result: Fig 2 shows three lanes where both lane 1 and 2 contains replicas of unpurified QD-16(AT)-AuNP conjugates while lane 3 contains only 20A-ssDNA conjugates. Only one dark band appeared at equivalent distance from the sample addition well in all the lanes that could only correspond to 20A-AuNP conjugates. This indicated that the yield of QD-16(AT)-AuNP composite was either negligible or zero. Therefore, the initial experiment of QD-16(AT)-AuNP composite synthesis was unsuccessful.

Figure 2: Gel electrophoresis separation AuNP imaging. Lane 1 and Lane 2 contain unpurified QD-16(AT)-AuNP composite sample,
Lane 3 contains only 20A-AuNP conjugates. The black band denotes presence of AuNPs. Presence of all three bands at the same location indicate that they are 20A-AuNP conjugate bands

Future steps:

Based on the understanding of our system, the failure of QD-16(AT)-AuNP composite synthesis could result from the following:

1. Increase in the temperature of the system above DNA dehybridization/melting temperature.

2. Unavailability of sufficient amount of complementary ssDNA for conjugation.

For the current QD-16(AT)-AuNP composite, the DNA melting temperature is 30 C. Electrophoretic separation often results in heat dissipation during the process. Ice water cooling was performed during gel electrophoresis to avoid overheating of the sample. However, repeating the above synthesis at lower temperature did not solve the problem. This clearly indicated that failure of QD-16(AT)-AuNP composites resulted from insufficient complementary ssDNA for conjugation.

Current 16T-QD conjugates have extremely low conjugation efficiency (0-1 ssDNA/QD). However, our previous tests of the functionality of 16T-QD conjugates on DNA origami platform (“Testing quantum dot – single stranded DNA conjugates”) clearly indicate that the low conjugation efficiency of 16T-QD conjugates did not affect its hybridization efficiency with complementary ssDNA. Thus, the insufficiency and unavailability of ssDNA could result from 20A-AuNP conjugates. Although, ssDNA-AuNP conjugation has been thoroughly investigated in the past, the conjugation efficiency of the procedure is extremely dependent on NP size, ssDNA length and ssDNA sequence. Preliminary test suggests low ssDNA/AuNP in current 20A-AuNP conjugates. Although low ssDNA/NP does not affect the hybridization functionality of ssDNA-NP conjugates individually, insufficiency of ssDNA on both the particles can significantly decrease the composite formation efficiency. Therefore, future experiments would focus on optimizing ssDNA conjugation of AuNPs to permit QD-DNA-AuNP composite formation.