A mitochondrial-targetable dual functional near-infrared fluorescent probe to monitor pH and H₂O₂ in living cells and mice

Abstract

A lower pH level and high hydrogen peroxide (H₂O₂) concentration in mitochondria is closely associated with a variety of diseases including cancer and inflammation. Thus, determination of changes in the level of acidic pH and H₂O₂ is of great importance and could provide new insights into the key functions under both physiological and pathological conditions. Herein, we present a novel mitochondria-targetable probe NIR-pH-H₂O₂, as the first near infrared (NIR) fluorescent small molecule, to monitor changes of endogenous pH (pk_a = 6.17) and H₂O₂ with high sensitivity, good compatibility and low cytotoxicity. Futhermore, it was successfully employed to monitor pH and H₂O₂ in a mouse acute inflammation model. These results demonstrate that NIR-pH-H₂O₂ is a novel bifunctional mitochondrial-targeted NIR probe to sense acidic pH and H₂O₂ in vitro and in vivo, indicating its huge potential for the diagnosis of pH and H₂O₂-related diseases.

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Introduction

Intracellular pH is an important parameter in mediating many physiological processes such as cell metabolism, proliferation, apoptosis, ion transport and homeostasis and so on.^1–4 However, abnormal intracellular pH is associated with cellular dysfunctions such as cancer and neurodegenerative disorders.^5,6 Hydrogen peroxide (H₂O₂), an important member of reactive oxygen species (ROS) mediates signal cascades involved in redox homeostasis.⁷ Substantial evidence has showed that excessive H₂O₂ is relevant to serious diseases including cardiovascular disorders, neurodegenerative diseases, cancer and diabetes.^8–13 Wang et al. monitored low pH and high H₂O₂ in cancer cells, using dual responsive supramolecular polymer by the host–guest interactions of β-CD-hydrazone-DOX and PEG-Fc.¹⁴ Colon cancer cells were extremely sensitive to pH reduction, which naturally cause metabolic stress in the colon lumen, as well as H₂O₂ and ionizing radiation.¹⁵ A trend for reduced pH and increased H₂O₂ in lower airway inflammation and chronic obstructive pulmonary disease was observed.^16,17 Mitochondria are cellular powerhouses that produce energy through the respiratory chain.¹⁸ Abnormal levels of pH and H₂O₂ in the mitochondria are associated with various diseases like cancer, Alzheimer's disease and cardiovascular diseases.^19–22 Thus, determination of mitochondrial changes in pH and level of H₂O₂ is of great importance and could provide new insights into the key functions of mitochondria under both physiological and pathological conditions.

Recently, Zhou et al. developed a type of two-photon dyes with D–π–A–π–D structure, which possesses near-infrared (NIR) properties, large two-photon absorption cross-section ∼160 G and high fluorescence quantum yield ∼0.15.²³ The effect of pH on this type of dyes had been studied, pH changes had significant effect on this kind of dyes fluorescence intensity, speculating that in a strong base condition the oxygen-bridge bond could be cut off and in acidic medium recovered easily. To the best of our knowledge, we firstly propose that oxygen-bridge group could be appropriate for the development of acidic pH fluorescent probes. In fluorescent detection field, boric acid group has widely been used as the recognition site toward H₂O₂.^24–26 The chemo-selective deprotection of boric acid to phenols provides a useful reaction-based method which detects H₂O₂ over other ROS.²⁷ Additionally, phenylboronic acid (pK_a = 8.70) with relatively weak Lewis acidity is more stability to different pH.²⁸ Moreover, carbonate as the self-immolative system was applied as a linker of probe which can release CO₂ through a 1,6-benzyl elimination.^29,30

In order to study the biological function or harmful effects of pH and H₂O₂ in mitochondria, we present the first mitochondria-immobilized NIR bifunctional fluorescent probe NIR-pH-H₂O₂ for monitoring pH and H₂O₂ (Table S1†). As illustrated in Scheme 1, NIR-pH-H₂O₂ consists of TP-NIR-OH fluorophore, phenylboronic acid and carbonate linker. The TP-NIR-OH fluorophore was predicted to display a NIR fluorescence “off–on” signal response to acidic pH and its positively charged was expected to display mitochondria-specific staining capability. Phenylboronic acid moiety was oxidized by H₂O₂ to release CO₂ through a 1,6-benzyl elimination, resulting in the recovery of fluorescence. It was successfully applied for image of pH and H₂O₂ in mitochondria of live cells and monitoring inflammation in living mice. The present results strongly demonstrated that NIR-pH-H₂O₂ would serve as an excellent bifunctional fluorescent probe to monitor mitochondrial pH and H₂O₂ fluctuations in living biological samples.

Scheme 1 Proposed reaction mechanism of NIR-pH-H₂O₂ towards pH and H₂O₂.

Experimental section

Materials and instruments

Absorption spectra measurements were performed on a Shimadzu UV-vis spectrophotometer, UV-2550. Fluorescent spectra measurements were performed on a Thermo Scientific Lumina fluorescence spectrophotometer. The pH was carried out by a PB 10 digital pH meter. ¹H NMR (400 MHz) and ¹³C NMR (125 MHz) spectra were measured on a Bruker ACF-300 spectrometer (Bruker Corp., Billerica, MA, USA) using TMS as internal reference. Mass spectra was carried on an Agilent 1100 Series (Agilent Technologies, Santa Clara, CA, USA) LC/MSD high performance ion trap mass spectrometer and a Mariner ESI-TOF spectrometer. All starting materials and reagents were purchased from commercial sources and used without further purification. Deionized water was prepared through a water purification system. Fluorescent microscopy imaging was carried out by a confocal laser scanning microscope (CLSM, LSM700, Zeiss, Germany). Fluorescent images of mice were taken by an IVIS Spectrum.

General procedure for fluorometric analysis

A stock solution of NIR-pH-H₂O₂ (1 mM) was prepared in DMSO. Stock solutions of H₂O₂ and other various analytes were prepared with distilled water freshly. A series of standard pH buffer solutions with different values were prepared by the standard procedures. Before absorption and fluorescence spectral measurements, all the resulting solutions were shaken well and incubated for 60 min at room temperature, all fluorescence measurements were executed with the excitation wavelength of 560 nm and the emission wavelength of 680 nm unless other noted.

The pK_a values of NIR-pH-H₂O₂

The pK_a values of NIR-pH-H₂O₂ were calculated using the Henderson–Hasselbalch equation: log[(I_max − I)/(I − I_min)] = pH − pK_a. Where I was the observed fluorescence intensity, I_max and I_min were the corresponding maximum and minimum fluorescence intensity, respectively.³¹

Cytotoxicity assay and fluorescence imaging toward pH and H₂O₂ in living cells

The cytotoxic effect of NIR-pH-H₂O₂ was assessed by the MTT assay. After cells attachment, MCF-7 and Hela cell lines pretreated in medium with NIR-pH-H₂O₂ at different concentrations. 200 μL DMSO was added to each well and shaken for 20 min. The absorbance was measured at 490 nm with a microplate reader (Tecan US, Inc. USA). Cell viability was calculated using the following equation:

Cell viability (%) = optical density value (treatment group)/optical density value (control group) × 100%.

Before imaging, cells were passed and seeded in glass dishes. For labeling, the cells were incubated with 10 μM of NIR-pH-H₂O₂ for the specific incubation time. Then, cells were washed three times with PBS (phosphate buffered saline). Cell images were then recorded using a confocal laser scanning microscope (CLSM, LSM700, Zeiss, Germany) and processed using the ZEN imaging software. Other information is available in the figure captions.

Animal models and in vivo animal studies

Athymic nude mice were purchased from Qinglongshan Animal Breeding Farm (Nanjing, China) for in vivo imaging studies. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of China Pharmaceutical University and the experiments were approved by the Animal Ethics Committee of China Pharmaceutical University.

Before animal imaging, nude mice were given an intraperitoneal injection of lipopolysaccharide (2 mg mL⁻¹) in 100 μL PBS for group A. NIR-pH-H₂O₂ (250 μM) in 200 μL buffered solution was injected in the same way for group B. Lipopolysaccharide (2 mg mL⁻¹) in 100 μL PBS buffer solution was injected intraperitoneally firstly. After 12 h, NIR-pH-H₂O₂ (250 μM) in 200 μL of buffered solution was injected at the same region for group C. All fluorescence images were acquired with auto exposure (IVIS Spectrum) after incubation for 30 min by using the NIR fluorescence imaging system (excitation wavelength = 560 nm, emission wavelength = 680 nm).³²

Synthesis of NIR-pH-H₂O₂

The probe NIR-pH-H₂O₂ was synthesized by the procedure shown in Fig. S1.† TP-NIR-OH and B1 was synthesized according to the reported methods.^23,30

TP-NIR-OH (62 mg, 0.2 mmol), compound B1 (80 mg, 0.2 mmol) and K₂CO₃ (65 mg, 0.6 mmol) were dissolved in 5 mL anhydrous DMF under nitrogen. The mixture was allowed to stir at room temperature for 2 h. The solvent was removed under reduced pressure. Then the residue was purified on a silica gel column with CH₂Cl₂ to CH₂Cl₂/methanol (v/v, 200/1 to 15/1) as eluent to afford NIR-pH-H₂O₂ as black solid (30 mg, 21% yield). ¹H NMR (500 MHz, DMSO-d₆) δ 8.55 (s, 1H), 8.06 (s, 2H), 8.04 (s, 1H), 7.90 (d, J = 9.5 Hz, 1H), 7.79 (d, J = 7.7 Hz, 2H), 7.67 (d, J = 8.6 Hz, 2H), 7.48–7.46 (m, 1H), 7.38–7.36 (m, 4H), 7.30 (d, J = 1.7 Hz, 1H), 5.25 (s, 2H), 3.68 (m, 4H), 2.90–2.83 (m, 4H), 1.81 (m, 2H), 1.21 (t, J = 6.8 Hz, 6H). ¹³C NMR (125 MHz, DMSO-d₆) δ 161.81, 159.16, 156.67, 153.13, 151.66, 149.34, 137.02, 134.82, 134.61, 133.65, 132.67, 132.45, 129.25, 127.66, 123.94, 122.15, 120.02, 119.37, 95.98, 70.51, 70.27, 55.40, 46.21, 40.52, 40.36, 40.19, 40.02, 39.86, 39.69, 39.52, 31.76, 30.88, 29.49, 27.19, 26.99, 22.57, 21.59, 14.42, 12.89. HRMS (ESI): calcd for C₃₂H₃₃BNO₆⁺ [M]⁺ m/z 538.2395, found 538.2416. Purity: 98.5% (HPLC).

Results and discussion

UV-vis and fluorescence responses to pH

Scheme 2 shows the synthetic route adopted for the preparation of NIR-pH-H₂O₂, whose structure was further characterized by ¹H NMR, ¹³C NMR and HRMS (Fig. S8–S10†). The fluorescence behaviors of NIR-pH-H₂O₂ (10 μM) towards pH were obtained containing 6% CH₃CN (v/v). As shown in Fig. 1, a significantly large stokes shift of 120 nm was noticed of NIR-pH-H₂O₂ with an absorption maximum (λ_abs) of 560 nm (Fig. 1A) and an emission maximum (λ_em) of 680 nm (Fig. 1B). Both intensity of absorption at 560 nm and fluorescence at 680 nm were dramatically reduced as the value of pH (2.96–9.08) gradually increased (Fig. 1C). Moreover, there is an excellent linear relationship of pH over the range of 4.93 to 7.47 (Fig. 1C inset, R² = 0.9937). By the nonlinear regression analysis, the pK_a value was calculated to be 6.17, indicating that NIR-pH-H₂O₂ is capable of assessing acidic media. Additionally, the probe shows satisfactory reversibility between pH 4.0 and pH 8.0 (Fig. 1D).

Scheme 2 Synthesis route for NIR-pH-H₂O₂.

Fig. 1 (A) Absorption and (B) fluorescence emission spectra of NIR-pH-H₂O₂ (10 μM) in the phosphate buffer (6% CH₃CN) with various pH values. (C) Plot of I 680 vs. pH in the range of 1.02 to 9.94. Inset: the linear fitting curve of I_680nm vs. pH from 4.93 to 7.47. (D) Reversible fluorescence changes of NIR-pH-H₂O₂ between pH 4.0 and 8.0 with λ_ex at 560 nm.

UV-vis and fluorescence responses to H₂O₂

To investigate the interaction between H₂O₂ and NIR-pH-H₂O₂, UV titration was performed (Fig. 2A). The main absorption peak at 560 nm obviously increased with H₂O₂ (200 μM). Subsequently, the fluorescence of NIR-pH-H₂O₂ were explored (Fig. 2B). The free probe exhibited a weak fluorescence emission at 680 nm in PBS buffer. However, its fluorescence immediately increased in the presence of H₂O₂ (200 μM). This result was consistent with its UV absorption spectrum to H₂O₂. As expected, NIR-pH-H₂O₂ is weak-fluorescent (Fig. 2B) in NIR area. The introduction of H₂O₂ could specifically convert the phenylboronic acid unit into an electron-donor-OH group with CO₂ released through a 1,6-benzyl elimination, which resulted a significant fluorescence enhancement at 680 nm. A fluorescence titration of NIR-pH-H₂O₂ was carried out to further investigate the quantitative relationship between NIR-pH-H₂O₂ and H₂O₂. With the increasing of H₂O₂, its fluorescence intensity at 680 nm gradually increased (Fig. 2C and D). When the concentration increased to 130 μM, the fluorescence almost kept stable. In addition, the fluorescence intensity of NIR-pH-H₂O₂ showed an excellent linear relationship with the H₂O₂ concentration (R² = 0.9968 Fig. 2D inset). The detection limit (utilizing the 3σ/k method) for H₂O₂ was determined 2.097 μM. which was enough for direct image of intracellular H₂O₂ as its concentrations are usually in sub-micromolar range.²⁵ To ensure whether NIR-pH-H₂O₂ could be applied for real-time detection, the kinetic experiments towards H₂O₂ were explored (Fig. S1†). It was displayed that the detection process balanced within 30 minutes, after adding H₂O₂ to the probe solution and a small quantity of enhancement was observed within the next 20 minutes.

Fig. 2 Absorption (A) and fluorescence (B) spectra of NIR-pH-H₂O₂ (10 μM) in PBS buffer (6% CH₃CN). (C) Fluorescence spectra of NIR-pH-H₂O₂ (10 μM) in the presence of various amounts of H₂O₂ (0–130 μM) in PBS buffer. (D) The curve was plotted with fluorescence intensity vs. H₂O₂ concentration (0–200 μM) inset: linear relationship between fluorescence intensity of NIR-pH-H₂O₂ and H₂O₂ concentration (0–130 μM) at 680 nm (λ_ex = 560 nm).

Sensitivity detection

Subsequently, the fluorescence specificity of NIR-pH-H₂O₂ was examined over various other species. As shown in Fig. 3, the fluorescence intensity at 680 nm of H⁺ and H₂O₂ are stronger than the other tested analytes in the phosphate buffer (6% CH₃CN) indicating that NIR-pH-H₂O₂ was sensitive to protonic acid (pH = 4) and H₂O₂. These unique properties enable it to be feasible to accurately measure intracellular pH and H₂O₂ with high selectivity.

Fig. 3 Fluorescence response of NIR-pH-H₂O₂ (10 μM) upon addition of various analytes (200 μM: CaCl₂; MgCl₂; KCl; NaS; NaBr; NaOAC; NaHCO₃; NaCl; NaF; Cys; Hcy; GSH; ¹O₂; ·OH; ROO·; TBHP; NaOCl; ONOO–; H₂O₂ and H⁺ (pH = 4) in PBS buffer (6% CH₃CN)).

The response between NIR-pH-H₂O₂ and H₂O₂ at different pH

It was of great significance to know the response between NIR-pH-H₂O₂ and H₂O₂ at different pH levels. The fluorescence intensity of NIR-pH-H₂O₂ and NIR-pH-H₂O₂ + H₂O₂ was then measured in PBS buffer from pH 1 to 10, respectively (Fig. S2†). It was found that fluorescence intensity of NIR-pH-H₂O₂ can increase upon the addition of H₂O₂ in the pH (1–10) range and the most obvious increase toward H₂O₂ could be observed from pH 6.0 to 8.0, indicating it can be applied in most living organisms (the physiological environment pH = 7.4).

Mechanism studies

The phenylboronic acid group in NIR-pH-H₂O₂ was the reaction site with H₂O₂ (Scheme 1). The starting material TP-NIR-OH was produced through a 1,6-benzyl elimination. The result of the electrospray ionization mass spectrometry (ESI-MS) confirmed the mechanism of this reaction. Addition of H₂O₂ in the NIR-pH-H₂O₂ solution resulted in a new peak at 360.19578 which correspond to TP-NIR-OH [M + H⁺] (Fig. S12†). The same results were also shown in the HPLC analysis (Fig. S13†). Thus, the results were in line with the proposed mechanism.

Cytotoxicity and cell mitochondria-targeting specificity of NIR-pH-H₂O₂

Before intracellular experiments, a standard MTT assay of MCF-7 and Hela cell lines were carried out to evaluate the biocompatibility of NIR-pH-H₂O₂. When the probe concentration reached 50 μM, NIR-pH-H₂O₂ displayed negligible cell toxicity since nearly 80% of MCF-7 cells (Fig. S3A†) and 85% of HeLa cells (Fig. S3B†) survived and could be applied to biological cells. To confirm the presumed mitochondria-targeting specificity of NIR-pH-H₂O₂, the colocalization experiments were performed in MCF-7 cells using Mito Tracker Green (a commercial mitochondria-targeting dye). As expected, the fluorescence image produced using NIR-pH-H₂O₂ (Fig. 4A) overlaps with that obtained using Mito Tracker Green (Fig. 4B), the fluorescence distribution of NIR-pH-H₂O₂ overlaps well with that of Mito Green (Pearson’ s coefficient, 0.90; Fig. S4, parts D and E†). In summary, these data proved that NIR-pH-H₂O₂ was a good mitochondria-targeted fluorescent probe.

Fig. 4 Colocalization experiments involving NIR-pH-H₂O₂ and Mito Tracker Green (MTG) in MCF-7 cells. The cells were incubated with NIR-pH-H₂O₂ (10 μM) and MTG (5 μM) for 30 min at 37 °C, followed by treatment with 200 μM H₂O₂ for 30 min at 37 °C. Images for NIR-pH-H₂O₂ (A) and MTG (B) were then recorded using excitation wavelengths of 555 nm and 485 nm, and band-path emission filters at 680 nm and 514 nm, respectively. Panels (C) shows a merged image of (A) and (B). (D) Intensity scatter plot of channels 1 and 2. (E) Intensity correlation plot of NIR-pH-H₂O₂ and MTG (Pearson's coefficient 0.90). Scale bar: 2 μm.

Cell imaging of NIR-pH-H₂O₂ for pH and H₂O₂

To explore capacity of NIR-pH-H₂O₂ for living cell fluorescence imaging of pH changes, MCF-7 cells and Hela cells were evaluated using the confocal fluorescence imaging. The fluorescence intensity of NIR-pH-H₂O₂ in MCF-7 cells (Fig. 5A) and Hela cells (Fig. S4A†) gradually decreased with increasing pH over the range of pH 5.08 to 7.84, indicated that NIR-pH-H₂O₂ could be applied for the detection of pH in living cells. We then employed NIR-pH-H₂O₂ to determine the pH change in mitochondria with a rapamycin model.³² Rapamycin, a widely used macrolide antibiotic, induces mitophagy in a variety of cell types and acidification of mitochondria has been observed during the mitophagy.^33–35 MCF-7 cells and HeLa cells were treated with NIR-pH-H₂O₂ and rapamycin for 1 h then fluorescence images were recorded. As expected, the merged images of the rapamycin-treated MCF-7 cells (Fig. 5B) and HeLa cells (Fig. S4B†) exhibit more obvious colour change, which is ascribed to the rapamycin-induced generation of mitophagy and decrease of the mitochondrial pH.

Fig. 5 (A) Fluorescent microscopic images of MCF-7 cells with NIR-pH-H₂O₂. Incubated cells with 10 μL of NIR-pH-H₂O₂ (10 μM) under the pH 5.08, 6.28, 7.4, 7.84 and (B) rapamycin (10 μM) of the culture medium for 30 min and observed under the microscope (λ_ex = 555 nm, λ_em = 680 nm for the red channel). Scale bar: 10 μm.

Next, we investigated the fluorescence imaging of NIR-pH-H₂O₂ for H₂O₂ in MCF-7 (Fig. 6a–d) cells and Hela cells (Fig. S5a–d†). After incubation with probe NIR-pH-H₂O₂ for 30 min at 37 °C, almost no fluorescence could be observed for non-H₂O₂. However, fluorescence intensities became increased after treated with increased exogenous H₂O₂. The results were consistent with our previous results of fluorescence spectra, showing that NIR-pH-H₂O₂ could be used for H₂O₂ detection in living cells. Next, application of NIR-pH-H₂O₂ to visualize endogenous H₂O₂ in MCF-7 cells and Hela cells by confocal fluorescence microscopy. To induce acute endogenous H₂O₂ production, the nonspecific protein kinase C activator phorbol myristate acetate (PMA; 200 ng mL⁻¹) was co-incubated with NIR-pH-H₂O₂ for 30 min, followed by confocal imaging.^36,37 Fluorescence intensity of NIR-pH-H₂O₂ sharply enhanced due to PMA treatment triggered an oxidative burst in for MCF-7 cells (Fig. 6e) and Hela cells (Fig. S5e†) compared with the control group. These results indicate the capability of NIR-pH-H₂O₂ in determining the endogenous changes of H₂O₂ in living cells. Dynamic fluorescence imaging for NIR-pH-H₂O₂ was further conducted. After incubated with exogenous H₂O₂ and probe NIR-pH-H₂O₂ in MCF-7 (Fig. S6†) and Hela cell lines (Fig. S7†), red fluorescence was enhanced gradually after incubated for 0 min, 10 min, 20 min and 30 min. The results were consistent with our previous results of fluorescence spectra.

Fig. 6 Fluorescence images of NIR-pH-H₂O₂ (10 μM) with various concentrations of H₂O₂ solution and PMA (λ_ex = 555 nm, λ_em = 680 nm for the red channel). The MCF-7 was treated with NIR-pH-H₂O₂ and different concentration of H₂O₂ and PMA for 30 min. Scale bar: 10 μm.

Fluorescence imaging of NIR-pH-H₂O₂ in mice

Having achieved successful cell imaging with NIR-pH-H₂O₂, a further exploratory effort was made to determine whether it would be competent of fluorescence imaging in vivo and reflect occurrence of biological processes induced by exogenous stimulation. Since abdominal inflammation induced by lipopolysaccharide (LPS) could cause changes of pH and overproduction of hydrogen peroxide,^38–40 we investigated the application of NIR-pH-H₂O₂ to visualize fluorescence changes in an abdominal inflammation mouse model induced by LPS. As shown in Fig. 7b, the mouse treated with saline and NIR-pH-H₂O₂ exhibited low fluorescence intensity. However, the fluorescence intensity in the mouse treated with LPS and NIR-pH-H₂O₂ changed evidently (Fig. 7c), which was attributed to that the inflammatory response could reduce the local pH and increased the level of H₂O₂. As the control, the mouse treated with LPS but no NIR-pH-H₂O₂ injection showed no fluorescence (Fig. 7a). Therefore, these results demonstrate that NIR-pH-H₂O₂ is a prominent fluorescent probe for imaging inflammation in vivo.

Fig. 7 In vivo fluorescence images in the peritoneal cavity of mice injected with NIR-pH-H₂O₂ during LPS-induced inflammatory response. (a) Only LPS was injected for control. (b) Saline was injected in the peritoneal cavity of mouse, followed by injection of NIR-pH-H₂O₂ (50 μM). (c) LPS was in-jected into the peritoneal cavity of the mouse, followed by injection with NIR-pH-H₂O₂ (50 μM). The mice were imaged with an excitation filter at 560 nm and emission filter at 680 nm.

Conclusions

In summary, we firstly reported a novel “off–on” dual functional NIR fluorescent probe NIR-pH-H₂O₂ to monitor pH and H₂O₂ in vitro and in vivo. The cellular assay confirmed that NIR-pH-H₂O₂ is co-localized in mitochondria with low cytotoxicity. Furthermore, the probe shows excellent pH sensitivity, good response to H₂O₂, and successful imaging of pH and H₂O₂ in mice inflammation model induced by LPS. Mitochondria acidification is seen during the mitophagy. Abnormal levels of mitophagy and H₂O₂ are associated with various pathological conditions such as cancer, inflammation and neurodegenerative diseases. On the basis of these results, we envision that NIR-pH-H₂O₂ could be employed to understand the roles of reduced pH and increased H₂O₂ in pathological conditions and further for the pH and H₂O₂ related diseases diagnosis and real-time monitoring.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81973187), “Six Talent Peaks” Project of Jiangsu Province (2016-YY-042), National Key Research and Development Program (2017YFC0107700), “Double First-Class” University project (CPU2018GY24, CPU2018GY38) and National Found for Fostering Talents of Basic Science (NFFTBS, No. J1310032).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0ra03905e

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