Stable, small, specific, low-valency quantum dots for single-molecule imaging

Abstract

We have developed a strategy for synthesizing immediately activable, water-soluble, compact (∼10-12 nm hydrodynamic diameter) quantum dots with a small number of stable and controllable conjugation handles for long distance delivery and subsequent biomolecule conjugation. Upon covalent conjugation with engineered monovalent streptavidin, the sample results in a population consisting of low-valency quantum dots. Alternatively, we have synthesized quantum dots with a small number of biotin molecules that can self-assemble with engineered divalent streptavidin via high-affinity biotin-streptavidin interactions. Being compact, stable and highly specific against biotinylated proteins of interest, these low-valency quantum dots are ideal for labeling and tracking single molecules on the cell surface with high spatiotemporal resolution for different biological systems and applications.

Figure 1

Schematic of QD construct synthesis. (a) To make 5% SPDP-QDs, poly-imidazole ligands (PILs) containing methoxy-terminated poly(ethylene)₁₁glycol (mPEG₁₁), imidazole, and amine-terminated poly(ethylene)₈glycol (AminoPEG₈) groups were conjugated to SPDP prior to ligand exchange with native hydrophobic ligands on the QD surface. (b) To make 5% biotin-QDs, AminoPEG₈-PILs were reacted with NHS-biotin, purified, and exchanged with the native ligands on the QD surface, allowing self-assembly with streptavidin molecules for immediate labeling and detection.

Figure 2

Characterization of SPDP-QD. (a) The broad absorbance and narrow emission bands of QD570 that has been ligand exchanged with PILs containing SPDP. (b) Size of SPDP-QDs after various hours of incubation at room temperature as measured by gel filtration chromatography (GFC). Symmetric GFC elution peaks at ~8.2 min correspond to an unaggregated, monodisperse QD solution of 10-12 nm in hydrodynamic diameter. (c) Verification of the average number of SPDP per QD by saturating with incremental equivalents of maleimide-Alexa594. (Alexa594 coefficient at 588 nm = 96,000 M⁻¹cm⁻¹, QD absorption coefficient at 350 nm = 1.53 × 10⁶ M⁻¹cm⁻¹). The maximum number of conjugated dyes is 6 per QD. (d) Stability of thiol reactivity on SPDP-QDs in Dulbecco’s Modified Eagle’s Medium at room temperature, as measured by the number of maleimide-dyes that can be conjugated. SPDP-QDs were stirred on a stir plate at 25°C and aliquots were taken at 2, 48, and 120 hrs. Each aliquot was reacted first with dithiothreitol (DTT) to deprotect pyridylthiol groups, and then with an excess of maleimide-dye. Dye per QD ratio was calculated by measuring the absorbance at 575 nm (Rox absorbance peak, absorption coefficient at 575 nm = 82,000 M⁻¹cm⁻¹) and at 350 nm (QD absorbance, absorption coefficient at 350 nm = 1.53 × 10 M⁻¹cm⁻¹).

Figure 3. Specific cell staining by QDs

(a) Schematic showing the mSA and QD conjugation strategy. Thiols on the QD were exposed by reducing pyridyldithiol groups with DTT and were reacted with maleimide-conjugated mSA. (b) Specific staining of biotinylated RBCs with mSA-QDs validated by flow cytometry. The orange histogram represents specific staining, and the red and blue dotted histograms represent unstained RBCs and the blockage of mSA-QD staining by incubating mSA-QDs with excess biotin beforehand, respectively. (c) Schematic showing the dSA and biotin-QD conjugation strategy. A biotin-QD binds to a dSA and forms a dSA-QD. (d) Specific staining of biotinylated RBCs by dSA-QDs validated by flow cytometry. The blue and orange histograms represent the specific staining of 3% and 5% biotin-QD constructs conjugated with dSA. The red dotted histogram represents the control staining by biotin-QDs alone.

Figure 4. Single-molecule imaging of homemade specific, compact, low-valency QDs

(a) Imaging single QDs on glass coverslip. Single QDs were excited with a 488-nm laser and imaged with a 10 ms exposure. The “on” and “off” blinking property of single QDs on a glass coverslip proves single particle detection (Supplementary Video S1). Result is from one representative experiment out of 23 experiments. The arrow indicates one representative single QD. (b) Single QD detection on glass surface shown by interactive 3D surface plot using ImageJ. (c) Imaging single QDs on live cell membrane. Single QDs at the membrane of a CH27 cell were excited with a 488-nm laser and imaged with 50 ms exposure. Reduced blinking of single QDs was observed at the cell membrane (Supplementary Video S2). Result is from one representative experiment out of 20 experiments. The arrow indicates one representative single QD; the yellow dashed line indicates the cell boundary. (d) Single QD detection at cell membrane shown by interactive 3D surface plot using ImageJ.