A 3,5-dinitropyridin-2yl substituted naphthalimide-based fluorescent probe for the selective detection of biothiols and its application in cell-imaging

A naphthalimide-based fluorescent probe was developed for the sensitive and selective detection of biothiols. The fluorescence of the probe was quenched by the electron-withdrawing 3,5-dinitropyridin-2-yl group via the photoinduced electron transfer process, and turned on by biothiol-triggered nucleophilic aromatic substitution. The sensing mechanism was confirmed by HPLC analysis and theoretical calculations. The probe shows a satisfactory response time of 30 min with low detection limits (Cys: 0.32 μM; Hcy: 0.88 μM; GSH: 0.46 μM). Furthermore, the probe was successfully utilized to detect endogenous and exogenous biothiols in HeLa cells.

Electronic Supplementary Material (ESI) for RSC Advances. This journal is © The Royal Society of Chemistry 2021

Materials and instruments
1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance-III 400 MHz Spectrometer (at 400 and 100 MHz, respectively). High-resolution mass spectroscopy (HRMS) was performed with an ESI source and a TOF detector. Fluorescence emission spectra and UV-Vis spectra were collected on a SHIMAZU UV-2450 and an RF-5301 spectrometer, respectively. The melting point was determined with a X-5A melting point apparatus (uncorrected). The pH measurements were recorded on a Rex PHS-3G pH/mV/temperature benchtop meter equipped with an E-201-C pH combined electrode.
Double-distilled water was used throughout the experiments.

Spectroscopic measurements
The absorption and fluorescence experiments were conducted with DMSO-Tris buffer In the fluorescence spectral experiments, the excitation wavelength was set at 460 nm, and the excitation and emission slit widths were set at 5 and 3 nm, respectively.

Cytotoxicity assay
The cellular toxicity of probe NAP-DNP was performed using a Cell Counting . HeLa cells were seeded into 96-well plates at a density of 4000/well, cultured at 37 °C with 5% CO 2 for 24 hours, and then different concentrations of probe NAP-DNP (5, 10, 20, 50 μM) were added to the wells. Subsequently, 10 μL of CCK-8 was added to each well followed by incubation for an additional 4 hours at 37 °C under 5% CO 2 . The absorbance of each well was measured on a micro-plate reader (Tecan, Austria) at a detection wavelength of 450 nm.
The following formula was used to calculate the inhibition of cell growth: Cell viability (%) = (mean of absorbance value of treatment group/mean of absorbance value of control)×100%.

Cell imaging
HeLa cells were cultured in DMEM with 10% fetal bovine serum (FBS) at 37°C, 5% CO 2 and 95% humidity. All the cells were washed 3 times with phosphate-buffered saline (PBS) and then incubated with PBS free fresh media for subsequent cell imaging.