A two-dimensional molecular beacon for mRNA-activated intelligent cancer theranostics† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4sc03894k

A two-dimensional quantum dot molecular beacon with interconnected imaging and therapy modalities is developed for intelligent cancer theranostics.

was purified with a Milli-Q water purification system. All other reagents and solvents were of analytical grade. All the DNA molecules were synthesized and HPLC purified by Takara Biotechnology Co., Ltd (Dalian, China).

Synthesis of CdTe/CdS/ZnS core/shell/shell QDs.
Synthesis of CdTe QDs: First, a NaHTe solution was prepared by mixing Te powder (0.5 mmol) with NaBH 4 (2 mmol) in 1 mL of degassed deionized water in an eppendorf tube. A needle was inserted into the capped tube to release the pressure, and the solution was maintained for 40 min at 60 °C. The Cd 2+ precursor solution was prepared by dissolving GSH (0.125 mmol) and CdCl 2 (0.05 mmol) in 20 mL of deionized water. The solution pH was adjusted to 8.5 with 1 M NaOH solution. The NaHTe solution (25 µL) was injected into N 2 saturated Cd 2+ precursor solution with a molar ratio of 4:1:10 for Cd 2+ :NaHTe:GSH. After injection, the color of the solution immediately changed from colorless to yellow. The reaction mixture was heated at 100°C, and aliquots of the reaction mixture (0.2 mL) were collected and characterized every 10 minutes until the QD emission maximum reached 535 nm, after which the reaction was quenched quickly by cooling down to 0 °C in an ice-bath. Synthesis of CdTe/CdS core/shell QDs: First, the CdTe core nanocrystals were purified and resuspended in 25 mL of degassed water, and the pH of the solution was adjusted to 10.5. The Cd 2+ precursor solution was prepared to include 30 mM CdCl 2 , 75 mM GSH, and 25 mM thiourea.
Assuming that one CdS monolayer adds 0.335 nm to the radius of the core, precise amounts of the Cd 2+ and thiourea solutions were injected to form the first monolayer, with a separate addition step for the second monolayer. The temperature of each reaction mixture was held at 90 °C for 60 min before samples were collected for subsequent purification and characterization.
Synthesis of CdTe/CdS/ZnS core/shell/shell QDs: First, Zn 2+ precursor solution was prepared by dissolving GSH (0.2 mmol) and Zn(OAc) 2 (0.1 mmol) in 25 mL of deionized water, with subsequent adjustment of the solution pH to ~7. Next, 0.025 µmol purified CdTe/CdS nanocrystals and 0.1 mmol thiourea were added to Zn 2+ precursor solution, and the pH was adjusted to 11. The molar ratio of Zn 2+ /thiourea/GSH in the reaction mixture was 1:1:2. The reaction mixture was heated at 90 °C, and aliquots of the mixture (0.2 mL) were collected at various time points for characterization. The reaction was subsequently quenched in an ice bath. The resulting QDs were purified by 2-propanol precipitation and were resuspended in deionized water (2 mL) for characterization.
The concentration of CdTe QDs was calculated according to a method reported in Chem. Mater. 2003Mater. , 15, 2854Mater. -2860. The concentrations of CdTe/CdS QDs and CdTe/CdS/ZnS QDs are directly derived from the CdTe core concentration.

Optical characterization.
Photoluminescence spectra were recorded on a fiber fluorescence spectrophotometer (AvaSpec-ULS2048-USB2) equipped with a 405 nm laser (110 mW) as excitation light source. Absorption spectra were recorded on a UV-Vis spectrophotometer (Agilent 8453). Excitation spectrum of Ce6 was recorded on an Edinburgh FLS920 steady state & time-resolved fluorescence spectrometer.

TEM characterization.
A few drops of each QD sample were dispersed onto a 3 mm copper grid covered with a continuous carbon film and were dried at room temperature. Low magnification and high resolution TEM characterization was performed on a FEI Tecnai G20 and F20 transmission electron microscope operating at 185 kV and 200 kV, respectively.

Dynamic light scattering.
Dynamic light scattering (DLS) measurements were performed on a Zetasizer Nano ZS90 (Malvern) with 90° scattering angle and a He-Ne laser.

Quantum yield determination.
The QY is calculated according to the equation below: Where Φ is the quantum yield, I is the measured integrated emission intensity, η is the refractive index of the solvent, and A is the optical density. The subscript "st" refers to standard with known quantum yield and "x" refers to the QD sample. Rhodamine 6G (QY = 95% in ethanol) was chosen as the standard.
CdTe/CdS/ZnS QDs, CdTe QDs, and Ce6 solutions were continuously excited with a 405 nm laser (110 mW) and the fluorescence spectra were record at different time points.

Stability of CdTe/CdS/ZnS QDs in buffer solutions with different pH values
Three aliquots of as-prepared CdTe/CdS/ZnS QDs (1.45 µM) were purified using a Microsep TM Advance Centrifugal Device (YM-30, Pall Corporation) by centrifugation at 12000 rpm for 10 min.

Conjugation of QDs with dual-thiol-modified hairpin DNA.
Before conjugation, the dual-thiol-modified hairpin DNA (50 µM) were activated by 75 µM TCEP in 50 mM PBS (pH 7.4) for 2 h at room temperature in a glove box to reduce the disulfide bond.
The as-prepared CdTe/CdS/ZnS QDs were purified by 2-propanol precipitation (1:3 v/v) and redispersed in water. The purified QDs were mixed with activated dual-thiol-DNA with a molar ratio of 1:10. The mixture was then incubated at room temperature for 16 h, aged for another 12 h in the presence of 0.2 M NaCl, and finally purified using a Microsep TM Advance Centrifugal Device (YM-30, Pall Corporation) by centrifugation at 12 000 rpm for 10 min.

FRET Analysis.
The donor emission spectrum and the acceptor absorption spectrum were used to calculate the spectral overlap integral: where F D is the wavelength dependent donor emission spectrum normalized to an area of 1, ε A is the extinction coefficient spectrum of the acceptor in units of M -1 cm -1 , and λ is the wavelength in cm. The overlap integral is used to calculate the Förster distance, R 0 , using the equation: where κ 2 is the dipole orientation factor, assumed to be 2/3, Q D is the quantum yield of the donor, and η is the refractive index of the medium (η(H 2 O)=1.333).
The average number of acceptors per donor, n, as determined by absorbance spectroscopy, was taken into account. In the case where one donor species can interact with several acceptors the energy transfer efficiency can be expressed as: where r is the separation distance between the donor and the acceptor. Denaturing PAGE was performed to further confirm that the DNA attached to QDs remains intact after nuclease treatment. 20 µM MB and 2 µM TMB were treated with 5 µL of DNase I in reaction buffer (1 U/µL) in a total volume of 10 µL and incubated for 12 h at 37 ºC. Afterwards the samples was mixed with 5 µL HCl (pH=3) to digest QDs and the pH was adjusted to 7.4 with Tris-HCl (20 mM). MB without nuclese treatment was used as a negative control.

Cell culture.
Breast cancer cell line MCF-7 was cultured in 25 cm 2 cell culture flasks with vent caps (Corning) in DMEM supplemented with 10% FBS, 0.01 mg/mL insulin, and 1% antibiotics penicillin/streptomycin (100 U/ml). Normal immortalized human mammary epithelial cell line Hs578Bst was cultured in DMEM/F12 supplemented with 10% FBS and 30 ng/ml EGF. MDA-MB-humidified incubator at 37 °C containing CO 2 (5%). Cells that had been grown to subconfluence were dissociated from the surface with a solution of 0.25% trypsin/EDTA. Then aliquots of cells were seeded into an 8-well chamber slide (Lab-Tek) and grown overnight in FBS-containing cell media before experiments.
Confocal laser scanning microscopy. Tumor growth curves of each group were recorded by measuring tumor sizes in two dimensions using a vernier caliper. Tumor was sized once a day, and the tumor volume in cubic mm was approximated by the formula V(tumor) = (width) 2 × length/2.       . In order to track the QD signal, the BHQ3 quencher was not conjugated to the probe. The QD fluorescence was shown in red and the lysotracker fluorescence was shown in green. Figure S9. Whole body fluorescence images of MCF-7 tumor-bearing mouse injected with TMB and Ce6 (same Ce6 concentration). The images were recorded pre-injection (0 hour) and post-injection at different time points (1, 2, 3, 4 hours). Images were acquired with 455 nm excitation and the emission signals were collected between 610 and 750 nm. Figure S10. Quantitative analysis of biodistribution of QD-DNA nanoprobes using ICP-AES. Five nude mice were injected with QD-DNA nanoprobes via tail vein and were sacrificed after 4 hours. The biodistribution of QDs in each organ is calculated to be: liver (43.5%ID/g), spleen (25.2%ID/g), lung (18.6%ID/g), kidney (27.3%ID/g), heart (7.2%ID/g), intestine (5.8%ID/g), and tumor (68.1%ID/g) Figure S11. Representative organ histology H&E staining images of the mouse injected with QD-DNA nanoprobes and untreated healthy control mouse. The QD-DNA nanoprobes are not destructive to all the examined organs.