Water-soluble semiconducting nanocrystals or quantum dots (QDs) have attracted much interest in recent years due to their tuneable emission and potential applications in photonics and biological imaging. Fluorescence resonance energy transfer (FRET) processes are very important for elucidating biochemical mechanisms
Quantum dots (QDs) are semiconductor nanoparticles where the electrons and holes are quantum-confined, which translates into strong photoluminescence (PL) in the visible range. The emission wavelength is directly related to the particle size as larger particles will absorb lower energy, that is, longer wavelength photons [
The aqueous synthesis of thiol-stabilised CdTe QDs has been reported and optimised over the last years and adapted for different types of ligands [
The oligonucleotides with the sequences (5′ NH2-A10 TAG GAA TAG TTA TCA (T6) 3′ and 5′ NH2-A10 TGA TAA CTA TTC CTA (T6) 3′) were prepared according to standard on-support oligonucleotide synthesis. T6 phosphorothioate tail was prepared by using Beaucage reagent during oxidation steps and 5-amino group was introduced at the last step of DNA synthesis by using the corresponding phosphoramidite C6-amino linker from Eurogentec. Al2Te3 was purchased from Cerac Inc. All other chemicals for QD synthesis were purchased from Sigma-Aldrich.
We used a modification of the method developed by Gaponik et al. [
HT1080 cells were cultured in a medium (500 mL Minimum Essential Medium (MEM) supplemented with 0.055 g of sodium pyruvate, 5 mL of a solution of penicillin (2 mM) and streptomycin (2 mM), 5 mL of 1 mM gentamicin, and 100 mL of fetal bovine serum (FBS) at 37°C and in a 5% CO2 atmosphere. Cells were plated in 35 cm2 glass-bottomed petri dishes (from iBidi) at a final concentration of 105 cells per dish and left to adhere overnight. Half of the cell medium was then removed from all dishes and replaced by serum-free medium. Cells were incubated for another 4 hours. QDs were added to a final concentrations of 10−7 mol/L in Dulbecco’s modified phosphate buffered saline (DPBS) with sodium chloride and magnesium chloride, and cells were incubated for another 4 hours. The QD-containing medium was aspirated out of the dishes and the cells were washed three times with DPBS. The cell cultures were finally imaged using an Olympus FV-1000 confocal microscope.
Complementary sequences of oligonucleotides bearing a T6 phosphorothioate tail at their 3′end and an amino function at their 5′-end (5′ NH2-A10 TAG GAA TAG TTA TCA (T6) 3′ and 5′ NH2-A10 TGA TAA CTA TTC CTA (T6) 3′) were used to synthesise three series of modified QDs. The procedure was adapted from the work of Tikhomirov et al. [
The presence of oligonucleotides on the surface of QDs and their ability to hybridise with the complementary sequence immobilised on other QDs were confirmed by several instrumental techniques. First, circular dichroism (CD) spectra were recorded for QDs modified with complementary oligonucleotides as well as a one-to-one mixture of those. They exhibited a weak signal in the 500–800 nm range, which corresponds to the position of the exciton peak. The corresponding spectra are shown in Figure
CD spectra of QDs modified with complementary oligonucleotide sequences and their mixture in 1 : 1 ratio.
For further assessment of the binding effect of oligonucleotides, a 1 : 1 mixture of QD-oligo 1 and QD-oligo 2 was scanned by dynamic light scattering (DLS) in order to measure the changes of the hydrodynamic diameter over time. Figure
DLS measurements of a 1 : 1 mixture of QDs modified with complementary oligonucleotide sequences and incubated for 0 to 55 min.
The initial mixing of both types of QDs resulted in an average diameter of around 10 nm with a polydispersity index (PDI) of 0.431. As incubation time was prolonged, the PDI did not significantly vary; the average diameter, however, slightly decreased to around 7 nm after 20 min and remained in that range afterwards. This indicated that QDs in initial clusters which were formed in the first 20 min interacted more stronger than and packed more tightly with time. The cluster size of 7 nm was consistent with dimeric structures. Subsequently, the role of oligonucleotide hybridisation in the process was ascertained by measuring the hydrodynamic diameter of the same one-to-one mixture with increasing temperature. Results are displayed on Figure
DLS measurements of a 1 : 1 mixture of QDs modified with complementary sequences of oligonucleotides at increasing temperatures.
Having ensured the presence and hybridisation ability of complementary oligonucleotides on QD surfaces, we investigated the energy transfer processes in the nanocomposites. Three sets of donor-acceptor pairs were considered. Their full characterisation data are summarised in Table
Characterisation of oligonucleotide-modified QDs.
Sample | Oligonucleotide | Absorption (nm) | Emission (nm) | Quantum yield | Diameter (nm) | Zeta potential (mV) |
---|---|---|---|---|---|---|
Donor (a) | Oligo-2 | 525 | 558 | 27% | 2.7 | −44 |
Acceptor (a) | Oligo-1 | 556 | 592 | 25% | 3.7 | −29 |
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Donor (b) | Oligo-1 | 535 | 560 | 50% | 2.9 | −35 |
Acceptor (b) | Oligo-2 | 601 | 644 | 51% | 5.2 | −40 |
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Donor (c) | Oligo-2 | 531 | 567 | 6% | 2.9 | −47 |
Acceptor (c) | Oligo-1 | 556 | 592 | 25% | 3.7 | −29 |
In the first pair, the donor emission and acceptor absorption perfectly overlapped which was expected to produce very efficient energy transfer. In the second case, the PL emissions were well separated which enabled good discrimination between them. In the third pair, there was near complete overlap between both absorption and emission spectra, allowing for mutual energy transfer. In each pair, the donor and acceptor were functionalised with oligonucleotide sequences complementary to each other.
Energy transfer in each donor-acceptor pair was investigated by measuring the PL emission of various donor-acceptor solutions of different compositions. The donor quenching and acceptor enhancement were plotted against the donor-to-acceptor ratio (Figures
(a): Donor (a) and acceptor (a) UV-visible absorption and PL emission spectra. (b): Donor (a) quenching and acceptor (a) enhancement versus donor-to-acceptor ratio.
(a): Donor (b) and acceptor (b) UV-visible absorption and PL emission spectra. (b): Donor (b) quenching and acceptor (b) enhancement versus donor-to-acceptor ratio.
(a): Donor (c) and acceptor (c) UV-visible absorption and PL emission spectra. (b): Donor quenching (c) and acceptor enhancement (c) versus donor-to-acceptor ratio.
Considering the donor-acceptor pair (b) with well-separated absorptions and emissions, the interaction pattern appeared slightly different as presented in Figure
Figure
In all cases, energy transfer was observed between QDs modified with complementary oligonucleotide sequences, and there was always a range of ratio for which one was quenched and the other was enhanced. Both donor quenching and acceptor enhancement plateaued between 3 and 4 donors per acceptor, consistently with the calculated valency of the acceptors.
To be suitable for
Confocal microscope images of HT-1080 cells exhibiting internalised QDs. First row: negative control; second row: donor (b); third row: acceptor (b); fourth row: donor (b) and acceptor (b) added simultaneously; fifth row: donor (b) and acceptor (b) with preincubation. First column: excitation 543 nm, emission 603 nm; second column: excitation 633 nm, emission 668 nm; third column: overlay of green and red channels with bright field. Arrows indicate green QDs.
Prehybridisation of QDs also resulted in a higher degree of aggregation and accumulation in individual cells as illustrated in Figure
Confocal microscope close-up images of HT-1080 cells exhibiting internalised green and red QDs. (a) Without preincubation. (b) With preincubation. Overlay of green and red channels with bright field. Excitation 543 nm, emission 603 nm; excitation 633 nm, emission 668 nm.
Histograms representing the number (b) and size (a) of QD aggregates internalised by cells depending on QD type: donor, acceptor, and 1 : 1 mixture with or without preincubation.
This observation opened to another possible type of application, where DNA-hybridisation processes could be monitored through QD aggregation using a system where FRET does not occur. Similar cellular testing of the donor-acceptor pair (c) provided an example of such system. As predicted by the fluorescence titration described above, neither species was quenched at 1 : 1 ratio, thus allowing visualising them simultaneously in cells. The presence in cytoplasms of large aggregates containing both red and green QDs confirmed the occurrence of oligonucleotide binding after preincubation. Confocal microscopy images of cells treated with these QDs are displayed in Figure
Confocal microscope images of HT-1080 cells exhibiting internalised QDs. (a) donor (c), (b) acceptor (c), (c) donor (c) + acceptor (c) added simultaneously, (D) donor (c) + acceptor (c) after a one-hour preincubation. Overlay of bright field with green and red channels.
Thus, the results above demonstrated that our composites were suitable for studying energy transfer processes or DNA binding/digesting processes in cell cultures
In this work, a series of new oligonucleotide-modified CdTe QDs have been prepared, characterized, and tested
The authors acknowledge financial support from Science Foundation Ireland (Grants SFI 07/IN.1/I1862 and SFI 12/IA/1300) and the Ministry of Education and Science of the Russian Federation (Grant no. 14.B25.31.0002). E. Defrancq thanks the NanoBio program for the facilities of the synthesis platform.