Construction of Prussian Blue Fluorescent Nanoprobe for Specific Detection of HClO in Cancer Cells

doi:10.15541/jim20220119

Abstract

Abstract:

Hypochlorous acid (HClO) is one of the reactive oxygen species (ROS), taking crucial parts in many physiological and pathological processes. However, excessive HClO causes tissue injuries, atherosclerosis, neurodegeneration diseases, and even cancers. Therefore, real-time detection of HClO in cancer cells is of importance for exploring the effect of HClO in tumor progression or immunotherapy. Quite different from present organic molecular probes, a novel inorganic-based hydrophilic fluorescent nanoprobe was developed by simply integrating fluorescein isothiocyanate (FITC) into hollow mesoporous Prussian Blue nanoparticles (HMPB) in this work. Owing to inner filter effect, fluorescence of FITC within HMPB quenches to some extent, which can be restored via the Fe²⁺-ClO^- redox reaction. A typical fluorescence increase of FITC at emission peak of 520 nm can be clearly observed in the presence of HClO in vitro, which exhibits a good linear relationship in the range of 5×10^-6-50×10^-6 mol/L in HClO detection and its detection limit is calculated to be 2.01×10^-6 mol/L. Furthermore, the cellular experiment demonstrates the specific detection capability of HClO in cancer cells with high sensitivity by the obtained nanoprobe.

Key words: Prussian Blue, fluorescent probe, detection of HClO

CLC Number:

TQ138

DU Qiujing, LIU Tianzhi, CHEN Jufeng, CHEN Hangrong. Construction of Prussian Blue Fluorescent Nanoprobe for Specific Detection of HClO in Cancer Cells[J]. Journal of Inorganic Materials, 2023, 38(1): 55-61.

Figures/Tables 10

Fig. 1 Illustration of activation mechanism of fluorescence signal when F@H presenting in the HClO

Fig. 2 Characterization of the prepared HMPB and F@H (a) Typical TEM image, (b) XRD pattern, and (c) pore-size distribution curve of HMPB with inset showing N2 adsorption-desorption isotherm; (d) UV-Vis spectra of FITC, HMPB and F@H; (e) Fluorescence spectra of free FITC and F@H with inset showing corresponding fluorescence intensity ratio at 520 nm; (f) Fluorescence life of FITC and F@H

Fig. S1 Dynamic light scattering (DLS) result of HMPB with insets showing digital photographs of HMPB suspension standing still for 0 and 12 h.

Fig. S2 Standard curves (a) and the corresponding calibration curve (b) of FITC, UV-Vis spectrum of F@H supernatent (c) ppm: μg/mL

Table S1 Calculated encapsulation efficiency and loading efficiency by the standard curve and UV-Vis spectrum of F@H supernatant

Encapsulation Efficiency	83.9%
Loading Efficiency	14.4%

Fig. 3 In vitro detection of HClO and mechanism (a, b) Fluorescence (FL) spectra (a) and the corresponding calibration curve (b) of F@H (50 μg/mL) with the addition of NaClO (0-50 μmol/L) in Tris-HCl (10 mmol/L, pH 5.5). λex=488 nm, λem=520 nm; (c) Absorbance of F@H varied with time before and after addition of NaClO; (d) XPS profiles of F@H without/with addition of NaClO

Fig. S3 Time-dependent fluorescence intensity of F@H (50 μg/mL) upon the addition of 50 μmol/L NaClO in Tris-HCl buffer (10 mmol/L, pH=5.5). λex = 488 nm, λem = 520 nm.

Fig. 4 Fluorescence of F@H in the presence of other ROS (a) Fluorescence spectra (inset of (a)) and the corresponding fluorescence (FL) intensity (a) of F@H (50 μg/mL) with the addition of different interfering substances (500 μmol/L, 1-blank, 2-TBHP, 3-ROO, 4-NO, 5-H2O2, 6- · ·OH, 7-ONOO-, 8-ClO-) in Tris-HCl (10 mmol/L, pH 5.5) λex=488 nm, λem=520 nm; (b) Absorbance change of F@H with addition of different interfering substances (500 μmol/L) Colorful figures are available on website

Fig. S4 Cytotoxicity of F@H on 4T1 cells. Cells were incubated with 0-100 μmol/L F@H in DMEM medium containing 10% fetal bovine serum (FBS) for 24 h. ppm: μg/mL

Fig. 5 Detecting HClO in living cancer cells (a-d) Confocal fluorescence and (e-h) bright field images for detecting exogenous or endogenous HClO in 4T1 cells Blank: without any treatments; Control: 50 μg/mL of F@H and 0 μmol/L NaClO; NaClO: 50 μg/mL of F@H and 100 μmol/L NaClO; Elesclomol: 50 μg/mL of F@H and 50 μmol/L elesclomol. λex=488 nm, λem=520 nm; (i) Statistical analyses of the confocal images

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