Near-infrared fluorescence activation probes based on disassembly-induced emission cyanine dye

In the presence of target analyte, bright fluorescence in the near-IR region is emitted through the recognition-induced disassembly of the probe aggregate.


Fluorescence imaging of transfected HeLa Cells
The cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. 1.5 × 10 4 cells were seeded in 8-well chamber slides and cultured overnight at 37 °C in air with 5% CO 2 . Cells were then transfected by using X-treme GENE HP DNA transfection reagent (Roche Applied Science) according to the manufacturer's protocol.
Thirty hours after transfection, the cells were washed with DMEM supplemented with 10% FBS twice and cultured overnight. The cells were washed twice with Opti-MEM and stained with 0.5 μM Hoechst 34580. After thirty minutes incubation, excess Hoechst 34580 was removed. The cells were treated with 0.5 μM of probe prepared in Opti-MEM (1.0 % DMSO (v/v)). The fluorescence imaging was carried out by using Laser Scanning Confocal Microscope (LSM 700, Zeiss, Germany). For Cy5 channel, the images were taken by using 639 nm laser and LP640 emission filter. For Hoechst, we used 405 nm laser and SP490 emission filter. For CFP, we used 405 nm laser and SP555 emission filter.

Fluorescence imaging of HeLa cells under hypoxia condition
The cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. 2 × 10 4 cells were seeded in 8-well chamber slides and cultured 24 hours at 37 °C in air with 5% CO 2 . The cells were cultured for 24 hours at 37°C in Opti-MEM under hypoxic conditions (<0.1% O 2 ) generated with an Anaero Pack (Mitsubishi Gas Chemical Company Inc.). For hypoxia-mimetic conditions, the cells were cultured in Opti-MEM with 200 μM DFO for 24 hours at 37 °C in air with 5% CO 2 . The cells were imaged in Opti-MEM with the addition of 0.5 μM probe1 (1% DMSO (v/v)) in the absence or presence of 100 μM EZA (1% DMSO (v/v)). The images were taken after staining without any washing operations.

Transmission electron microscopy (TEM)
Sample (5 µM 1 or with 10 µM hCAII) were prepared by drop casting onto 200 mesh carbon-coated copper grids and air-dried overnight. The samples were washed with water before measurement. TEM imaging was carried out with JEOL-2100 microscope with an accelerating voltage of 160 kV.

Dynamic light scattering (DLS)
DLS analyses were performed on Brookhaven 90Plus at 25℃ in PBS buffer using a plastic cuvette (3 mL volume). At least six measurements were taken. All samples were incubated for 15 minutes before measurement.

Determination of probe 1 quantum yields
The quantum yields of 1 in the absence or presence of hCAII were determined by comparing the integrated area of the corrected emission spectrum of Probe 1 with a Cy5 reference (φ =0.28 in PBS buffer). The quantum yield can be calculated using the following equation:

Synthesis of compound 7
POCl 3 (2.1 mL, 22.5 mmol) was added dropwise to dry DMF (10 mL) at 0 °C. The mixture was stirred for one hour at room temperature. Subsequently, phenylacetic acid (1.02 g 7.5 mmol) was S26 added to the solution mixture. The clear solution formed was heated at 95 °C for 4 hours and then at room temperature overnight. The dark mixture was poured onto 20 g crushed ice followed by saturated NaClO 4 solution. The resulting white crystalline was filtered and washed two times with 5 mL water to afford compound 6 in 70 % yield (1.07 g). Compound 6 was used in the next step without further purification. Compound 6 (1.07 g, 5.9 mmol) was added to 6 mL warm NaOH (1.15 g, 28.6 mmol) solution, and the mixture was heated with stirring at 90 °C until compound 6 was dissolved. The reaction mixture was cooled to room temperature and diluted with 10 mL water. The solution was acidified to pH 5 with 10% HCl solution resulting in the precipitation of the compound 7 (quantitative yield). 1 H NMR (400 MHz, d 6 -DMSO) δ 8.52