- Open Access
Blue shift of CdSe/ZnS nanocrystal-labels upon DNA-hybridization
© Riegler et al; licensee BioMed Central Ltd. 2008
Received: 06 December 2007
Accepted: 19 May 2008
Published: 19 May 2008
Luminescence color multiplexing is one of the most intriguing benefits, which might occur by using semiconductor Quantum Dots (QDs) as labels for biomolecules. It was found, that the luminescence of QDs can be quenched, and replaced by a luminescence peak at approximately 460 nm on hybridization with certain regions of Arabidopsis thaliana tissue. This effect is site selective, and it is unclear whether it occurs due to an energy transfer process, or due to quenching and scattering of the excitation light. The article describes methods for phase-transfer of differently coloured, hydrophobically ligated QDs, coupling of DNA strands to the QD's surface, and hybridization of the labelled DNA to different cell types of Arabidopsis thaliana. The reason for the luminescence blue-shift was studied systematically, and narrowed down to the above mentioned causes.
Fluorescence is a widely used tool in biology to study the complexity and dynamics of biological processes. Compared to conventional organic dye molecules, fluorescent semiconductor nanocrystals (QDs) have several promising advantages. They can be excited by a broad range of wavelengths from UV up to their individual absorption edge, and they have narrow, tuneable emission spectra, which can be well resolved over the same spectral range. Moreover, in contrast to most organic fluorophores they are highly resistant to chemical and metabolic degradation and have a higher photobleaching threshold [1–5]. The challenges for using QDs in biological studies include designing hydrophilic QDs with surface chemistry well adapted to different biological applications. Surface modified QDs should be luminescent with optical properties not differing from the unmodified QDs [6–10].
Here we report the preparation of water-soluble CdSe/ZnS QDs, which have been surface modified for versatile and selective coupling of biological probes and subsequent specific labeling of cells. Because of the wide emission range, narrow spectral linewidth, brightness, and the adjustable, size dependent emission wavelengths of these QDs, they are expected to be a good choice for multiplex-imaging. Theoretically our CdSe/ZnS QDs should allow labeling of several different probes and imaging of up to eight different biological molecules in the visible range of the spectrum [11, 12]. We demonstrate that synthetic oligonucleotides can be efficiently covalently linked to these QDs. We further show that they can be used for subsequent analysis of expressed genes by in situ hybridisation experiments. The technique was successfully applied to detect transcripts in the plant Arabidopsis thaliana, a fully sequenced model organism .
Materials and methods
Preparation of QDs
CdSe/ZnS core/shell QDs were prepared according to a method published previously . Briefly, cadmium stearate and trioctylphosohine-selenid (TOP-Se) were reacted at temperatures above 200°C by fast injection of TOP-Se into a mixture of trioctylphosphine-oxide (TOPO) and cadmium stearate. The CdSe-cores were passivated and annealed by growing a shell of two additional monolayers of ZnS on their surface. Diethylzinc and hexamethyldisilathian were reacted for 12 hours with the CdSe-cores at 160°C in the presence of TOPO and TOP, again. The core/shell particles obtained were repeatedly washed with methanol, and re-dispersed in chloroform. Finally the particles were stored in 50 ml chloroform as stock solution.
Phase transfer and conjugation of DNA
Phase transfer of the CdSe-QDs to water phase and conjugation to oligonucleotides was carried out by double ligand exchange by modifying the procedure of Mirkin et al. . In a first step the TOPO on the surface of the particles was exchanged with mercaptopropionic acid (MPA). 10 ml (0.1 molar) of a solution of MPA in dimethylformamide (DMF) were added to solid QDs, precipitated out of 5 ml of each stock solution by addition of 10 ml methanol and subsequent centrifugation. To complete the ligand exchange, the QD solutions were incubated for 12 hours at 80°C. To precipitate MPA-QDs, 100 μg of dimethylaminopyridine (DMAP) were added to each sample, followed by centrifugation at 10,000 g. The supernatants with unincorporated MPA and DMPA were discarded and the pellets containing QDs were dispersed in 1 ml of specific thionylated oligonucleotides in water. The slightly colored solutions were incubated for additional 24 hours at room temperature to partially exchange MPA against the thionylated oligonucleotides. Finally the QD-oligonucleotide conjugates were yielded by adding 3 M NaCl and dialysis the solution against water for 72 hours while the receiver was three times renewed. Five different antisense specific QD-oligonucleotide conjugates were prepared with emission-wavelengths at 543 nm (At5g05600-5'gcatgcatgaaggcaaatcatcctttgaaaattcaaaatataaatgattgtacacatatacaagtcagacgtaatatc3'), 563 nm (At1g73590-5'agaaagattagaggctctaggggttaagcacaaggagggggacataa3'), 598 nm (At5g47910-5'cagagatctatacaaataaacacccgtaaggttactgtattagttgatagagaaaaaataaccgctctc3'), 610 nm (At5g50960 5'cgtcgacttgagacttctcgaagggaatttttcgtttatatgtgaaactctctgcttatggcggcg'), and 653 nm (At2g24200-5' ctgcacgactaaaacaaagtaccactttattcaacttttgacgattttacttttcataac) respectively. Same oligonucleotides labeled with fluorescein (Genedetect-New Zealand) were used as controls.
In situ hybridization
Arabidopsis thaliana floral meristems of 24 days old plants were fixed with 4% paraformaldehyde in PBS (pH 7.3) and embedded in paraffin. 7 μm tissue sections mounted on SuperFrost® slides (Carl Roth, Germany) were used for in situ hybridization. In situ hybridization was performed as follows. SuperFrost® slides holding sectioned paraffin embedded Arabidopsis thaliana inflorescence were de-waxed by placing them in 3 changes of histoclear (PLANO GmbH) for 3 minutes each, followed by 2 changes of histoclear/ethanol (2:1 and then 1:2) followed by 3 changes of 100% ethanol for 3 minutes each. Tissues were re-hydrated in 95%, 70%, 50% and 30% ethanol, for 2 minutes each. Slides were incubated in 0.2 M HCl for 20 min at room temperature. Slides were washed 2 × 5 min in PBS and tissue were then permeabilized for 20 min at 37°C with TE buffer (20 mM Tris-HCl pH 7.5, 2 mM CaCl2) containing 20 μg/ml of RNAse free Proteinase K (Roche Diagnostics, Basel, Switzerland). The enzymatic reaction was stopped by incubating slides in 0.2 mg/ml glycine. After two rinses in PBS, tissues were carefully overlaid with prehybridization buffer [50% formamide deionised, 2× SSC (from 20× SSC, sodium chloride and 300 mM trisodium citrate, pH 7.0), 10 mg/ml yeast tRNA, 2% dextransulfate, 10 mg/ml Poly A, 10 mg/ml ssDNA, 100 mM DTT, 50× Denhardts] and incubated in a humid sealed chamber at 37°C for 2 hours. After 5 minute rinse in 2× SSC, slides were overlaid with prehybridization buffer supplemented with labelled oligonucleotides (25 ng/ml; 1/400 dilution) and incubated overnight at 37°C. Post hybridization washes were done as follows. A quick wash in 1× SSC (10 mM DTT) at RT, 2 × 15 min in 1× SSC (10 mM DTT) at 55°C, 2 × 15 min in 0.5× SSC (10 mM DTT) at 55°C and 1× in 0.5× SSC (10 mM DTT) at RT. For sample processing the In SituPro robot (Intavis AG) was used.
Plant sections were imaged using a laser scanning microscope (LSM) (Zeiss LSM 510 META). QD-labeled oligonucleotides were excited with a laser beam in the UV range (405 nm excitation wavelength) and the full visible emission spectrum was recorded. This allowed to precisely differentiate expected emission wavelength from background emission. This method also allowed detection of any shift in QD emission wave length.
Photoluminescence (PL) and absorption spectra were recorded on a J&M TIDAS diode array spectrometer using standard quartz cuvettes. QD spectra were recorded in aqueous buffer solution.
Results and Discussion
Generalized approach for QD labeling of oligonucleotides
In situ hybridization using QD-oligonucleotide conjugates
Generalized approach for multiparametric labeling
Simultaneous detection of several different biomolecules in the cellular context is essential for addressing many biological questions. In order to establish multiparametric analysis of mRNAs, five different oligonucleotides were covalently linked to QDs with distinguishable emission wavelengths. The spectral properties of these QD-oligonucleotide conjugates were as expected.
This blue emission does not correspond to any known fluorophore present in the experimental setup. Figure 3b shows the corresponding spectral analysis. After blue-shifting, the luminescence maximum was found at about 460 nm independent of the initial luminescence of the QD-label (not shown in Figure 3). It was observed, that the initial luminescence of the QDs decreased, while the luminescence peak at around 460 nm increased. In other words: the luminescence did not shift gradually to the blue, but "leaped" to the blue. The shapes of the luminescence spectra around 460 nm are similar to those of the original QDs.
The observations reported above rule out the obvious explanation for blue-shift of QD-emission, namely photo corrosion. Therefore the blue shift seems to be caused by an energy-transfer process that is related to hybridization or by selective quenching of the QD luminescence and light scattering . So far, it was not possible to explain this phenomenon fully. Nevertheless, it is an interesting observation which may pave the way for potential QD-based in-vivo sensors.
In this paper we report the specific labeling and imaging of Arabidopsis tissue sections by the use of distinguishable QD-oligonucleotide conjugates. After hybridization, regions within the Arabidopsis flower like pistil, leaves or pollen could be depicted respectively on a cellular level by LSM. Surprisingly, a strong blue-shift together with a reduction of luminescence intensity of the initial QD-fluorescence was observed. This remarkable blue-shift does not originate from the oxidative corrosion of QDs as it appears only after hybridization of coupled QDs. Finally, the origin of the blue shift could not be clarified within the presented work. Most likely, the blue-shift is caused by selective luminescence quenching and light scattering at hybridized QDs. The discussed blue-shift was not observed with organic fluorophores. Even though this effect is not fully understood yet, it might be potentially interesting for in-vivo molecular imaging, because of its sensitivity against the biological microenvironment.
The work was supported by grants from the Alexander von Humbold Foundation, the FCI, the Landestiftung ("Neue informatische, bildgebende und mikrotechnische Werkzeuge zur hochauflösenden Exploration komplexer biologischer Strukturen") and the European Union (STREP project LSHB-CT-2006-037639). We also thank Dr Roland Nitschke at the Freiburg University Life Imaging Centre for advice.
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