Turn-on quinoline probe for selective sensing of hypochlorite in live cells

Abstract

Hypochlorite is a highly reactive oxygen species (ROS) implicated in diverse physiological and pathological processes. Elevated hypochlorite levels cause oxidative stress, leading to structural and functional disruptions in biomacromolecules and contributing to the onset and progression of disease. Despite significant progress, many reported hypochlorite probes suffer from relatively high molecular weights and often depend on emission shifts, which complicate sensitivity and quantitative analysis. To overcome these limitations, we developed a low-molecular-weight quinoline-derived probe 5 that, to our knowledge, represents the first example of a quinoline-phenothiazine-based hypochlorite sensor that is non-fluorescent in its basal state yet undergoes a selective oxidative transformation to yield a robust emission at 523 nm. The probe exhibits exceptional temporal resolution, with fluorescence activation observed within 10 s in cell-free assays and generates a visible signal that scales directly with ROS concentration. Importantly, the probe maintains performance in live-cell studies, where activation occurs in a concentration-dependent manner. Altogether, these results establish 5 as a rapid, sensitive, and biologically compatible tool for monitoring hypochlorite dynamics in real time, advancing the design of next-generation ROS probes by combining low molecular weight, rapid response time, and straightforward readout.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-45327-1.

Introduction

Reactive oxygen species (ROS) are oxidative derivatives generated through complete or incomplete oxidation processes¹ and are recognized for their diverse roles in biological processes, functioning as antimicrobial agents within the immune system², redox signaling agents³, and regulators of hematopoietic stem cell quiescence and self-renewal^4,5. However, the overproduction of these species causes oxidative stress, which can lead to a variety of negative cellular consequences due to alterations in the structure and function of biomacromolecules^6–8. These changes can lead to disease and tissue damage^9,10. Important advances have been made to elucidate the roles and significance of ROS. Technically, accurate quantification and analysis of ROS are crucial, as these species naturally occur at low concentrations¹¹.

Hypochlorite is a ROS that has been extensively studied because of its stability in live cells¹². Techniques for sensing and quantifying hypochlorite include electrochemical methods, spectrophotometry, chromatography, electron spin resonance, and chemiluminescent probes, among others^13–15. However, fluorescence analysis has become increasingly favored due to probe availability, high sensitivity, and low cytotoxicity¹⁶. The physical properties of activity-based probes, such as color, quantum yield, solubility, molar absorption coefficient, Stokes’ shift, photostability, specificity, and selectivity, are all critical factors that influence their applicability^17–21. Quinoline and its derivatives have been important motifs in organic chemistry since its discovery in the 1800s²². Because quinoline and its derivatives contribute to binding through pi-stacking or hydrophobic interactions through the aromatic ring systems, they can intercalate DNA, disrupt biological redox systems, or chelate metal ions²³, which leads to their application as antimalarials^24,25, antioxidants^26,27, and fluorescent dyes amongst others. The quinoline scaffold has been studied extensively as fluorescent probes for the detection and measurement of metals^28–31, hydrogen sulfide³², Tau aggregates³³, and reactive oxidative species³⁴. But due to the weak fluorescence of these quinoline compounds, studies on the scaffold for ROS have mostly employed addition of a fluorophore to the structure, focusing on positions 2, 3, and 4 of the motif²⁹. In these reported approaches, quinoline probes either achieve emission wavelengths higher than 500 nm at the expense of a higher molecular weight or maintain low molecular weights but with shorter emission wavelengths^35–37. Generally, achieving both low molecular weight and longer emission wavelength at the same time is uncommon (Supplementary Table S1). There has been little research on the benzylic motif. Here, we studied the 7-position of the ring and synthesized a quinoline-phenothiazine structure for hypochlorite detection. We synthesized a low molecular weight probe that maintains an emission wavelength at 523 nm upon hypochlorite interaction. This turn-on probe is not fluorescent by itself but responds selectively with high sensitivity to hypochlorite while remaining unresponsive to other analytes. Furthermore, the probe can detect hypochlorite in a concentration-dependent manner in live cells.

Results

Design and synthesis of probe 5

Synthesis of the precursor, 4, was carried out in three simple steps from easily available starting materials (Fig. 1), and all intermediates and the target molecule were characterized using well established methods of ¹H NMR, ¹³C NMR, mass spectrometry, and UV–Vis spectroscopy (Supplementary Figs. S1–S16).

Fig. 1.

Synthesis of probe precursor 4 from commercially available starting materials.

Very little solvatochromic shift was seen in absorbance of 4 (Supplementary Fig. S17a). Precursor 4 exhibited limited fluorescence in various solvents except for phosphate buffered saline (PBS) (Supplementary Fig. S17b). Thus, a sulfur ring closure reaction was carried out with 4 to make target molecule 5 (Fig. 2a). Vedamalai et al. have determined the phenothiazine ring to be very susceptible to hypochlorite oxidation via the intramolecular charge transfer (ICT) process³⁸. Their molecule exhibited a blue shift upon addition of the ROS. We hypothesized that the mechanism of action of hypochlorite on probe 5 is through the oxidation of the sulfur atom in 5 to generate the corresponding sulfoxide, 6. Thus, we treated 5 with sodium hypochlorite and isolated compound 6 (Fig. 2b).

Fig. 2.

Synthesis of 5 and 6 (a) Synthetic route to probe 5 (b) Reaction of probe with hypochlorite and synthesis of 6.

The ¹H NMR spectra of 5 and 6 were stacked (Fig. 3). An obvious change in the chemical shift of most of the protons down field showed that a strong electron-withdrawing group was added to the molecule. Further proof of both structures was obtained from mass spectrometry and fluorescence analysis of both compounds (See Supplementary Figs. S14, S15, S18, S19). Thus, 5 was further characterized for use in detection of hypochlorite.

Fig. 3.

Stacked ¹H NMR of 5 (below) and 6 (above) in CDCl₃ showing downfield shift of aromatic protons numbered 1–7.

Response rate and selectivity of the probe

Probe 5 showed no fluorescence in various solvents, polar or non-polar at 50 µM (Supplementary Fig. S19). Specificity of the probe toward different ions and ROS was tested using the following analytes: OH^•, SO₃^2-, ONOO^-, H₂O₂, Cys, Hcy, GSH, OH^-, Fe²⁺, Zn²⁺, Sn²⁺, Ca²⁺, Na⁺, Cl^-, Br^-, F^-, NO₂^-, and ClO^- (Fig. 4a). Upon addition of hypochlorite, an intense fluorescence peak was observed at 523 nm. Probe 5 only showed fluorescence response toward ClO^-, with no fluorescence detected towards all other analytes tested. The sensitivity of probe 5 towards hypochlorite was determined by titration of ClO^- against the probe (Fig. 4b). Hypochlorite concentrations between 20 and 90 µM were tested with probe constant at 5 µM (Fig. 4b). An increase in intensity of the peak at 523 nm proportional to the increase in molarity of hypochlorite was observed, with a steady increase in fluorescence intensity up to 70 µM of hypochlorite (Fig. 4c). At high concentrations of hypochlorite, the florescence intensity began to decrease, although still well above that detected at lower hypochlorite concentrations. This is likely due to the over-oxidation of the probe to the sulfone at higher hypochlorite concentrations. Production of sulfone at high hypochlorite concentrations was confirmed by isolating it as compound 7 and performing NMR, mass spectrometry analyses and spectrophotometer analyses (Supplementary Figs. S13, S16 and S22). The sulfone had lower fluorescence intensity compared to the sulfoxide at similar concentrations. The high intensity of the sulfone was only realized at 0.18 mM concentration of the sample (Supplementary Fig. S22). Since a dependable straight-line curve is possible up to 70 µM of analyte, the limit of detection of 5 was measured by the calibration curve method as 0.19 μM (see Supplementary information). Furthermore, the fluorescence of probe 5 treated with ClO^- could be visualized by the naked eye (Supplementary Fig. S20).

Fig. 4.

Analysis of probe showing interaction with hypochlorite (a) Probe 5 (50 µM in 3% DMSO:PBS) interaction with 100 µM analytes; OH^•, SO₃^2-, ONOO^-, H₂O₂, Cys, Hcy, GSH, OH^-, Fe²⁺, Zn²⁺, Mg²⁺, Sn²⁺, Ca²⁺, K⁺, Na⁺, Cl^-, Br^-, F^-, NO₂^- and 20 µM ClO. (λ^Exc= 400 nm, excitation and emission slits = 5, scan rate = 600 nm/min and data interval = 1) (b) Sensitivity of 5 (5 µM in 3% DMSO:PBS) towards hypochlorite at 523 nm (c) Calibration curve showing concentration relationship with intensity (correlation coefficient = 0.80) (d) Competing reaction showing fluorescence intensity ratio change of probe interaction with hypochlorite alone and hypochlorite with other common analytes.

To determine how other biologically present ions and analytes could affect probe interaction with hypochlorite, we recorded fluorescence intensity changes of the probe with hypochlorite both with and without the presence of common interfering species (Fig. 4d). The following cations, anions, bio-thiols and ROS were chosen because of their high concentrations in cells; Na⁺, K⁺, Mg²⁺, Zn²⁺, Ca²⁺, NO₂^-, ^.OH, Cys, Hcy, GSH, H₂O₂, ONOO^- and ClO^-. Interaction assays revealed that cations like K⁺, Mg²⁺, Zn²⁺ and Ca²⁺ increase the fluorescence up to 20%. This is likely due to possible sulfoxide binding interaction with cations slightly resulting in enhanced fluorescence. Biologically present thiols like cysteine attenuated the fluorescence signal by 36%. However, none of these analytes ultimately caused major disruptions in hypochlorite interaction with probe. Assessing these competing pathways is critical to confirm probe selectivity, minimize off-target oxidation, and ensure that observed fluorescence changes arise specifically from the intended hypochlorite-triggered mechanism.

To determine the time sensitivity of the probe, a time-dependent fluorescence analysis was carried out on 5. Probe and analyte were kept at 5 and 50 µM, respectively, and probe fluorescence was investigated at various times between 10 s and 20 min. A change in intensity could be seen by 10 s (Supplementary Fig. S21). A linear relationship between intensity and time, up to 20 min, was observed with a correlation coefficient of 0.93.

Detection of hypochlorite in live cells

To determine if 5 could detect hypochlorite in live cells, mouse bone marrow cells were freshly isolated and incubated with 5 at 5 µM, a concentration with limited cytotoxicity to live cells (Supplementary Fig. S23). Cells were washed and then treated with various concentrations of exogenous ClO^- for 30 min. The reaction of cellular probe 5 with exogenous hypochlorite was detected using flow cytometry, violet laser excitation at 405 nm and emission at 512/25 nm. Compared to cells with no treatment (Mean Fluorescence Intensity (MFI): 27.6), cells incubated with probe 5 exhibited a minimal fluorescence shift (MFI: 37.7) when no hypochlorite was added (Fig. 5a), suggesting that endogenous hypochlorite levels in untreated, freshly isolated cells are likely low, although detectable by the slight increase in 5 fluorescence intensity. In contrast, upon addition of 50 µM ClO^-, a clear shift in the cells toward high probe 5 fluorescence (MFI: 2323) was detected. About 80% of single cells showed fluorescence greater than probe 5 only upon addition of ClO^-. The change in probe 5 fluorescence upon exogenous ClO^- was verified via fluorescence microscopy (Fig. 5b). To determine the sensitivity of probe 5, live bone marrow cells were treated with various concentrations of ClO^- within a range tolerated in vivo^39,40. The hypochlorite concentrations used were validated to induce limited cytotoxicity during the course of the experiment (Supplementary Fig. S24). The fluorescence intensity of cells treated with 5 increased with increasing concentration of ClO^-, suggesting that probe 5 detects ClO^- in live cells in a concentration-dependent manner (Fig. 5c). Thus, probe 5 enters into live bone marrow cells and can detect exogenously added ClO^-.

Fig. 5.

Detection of hypochlorite by probe 5 in live mouse bone marrow cells. (a) Histogram showing probe 5 fluorescence in mouse bone marrow cells at 405 nm excitation and 512/25 nm emission, measured by flow cytometry. (b) Representative fluorescence images of fixed cells under the indicated treatment conditions. Pyronin Y was used to identify cells. (c) Detection of probe 5 fluorescence in live bone marrow cells by flow cytometry after exogenous treatment with the indicated concentrations of ClO^-. Each dot represents a single sample from 6 independent experiments, where each experiment was performed on cells from a single mouse. p < **0.01 using one-way ANOVA with Dunnett’s multiple comparison test.

Physical properties

The physical properties of isolated compound 6 were recorded in Table 1. The excitation and emission spectra of 6 show significant dependence on solvent, with intensities highest in polar protic solvent ethanol and PBS, implying a possibility of electron transfer suppression via hydrogen bonding (Supplementary Fig. S18). The Stokes’ shift, defined as the difference between the absorbance and emission wavelengths, was highest in polar solvents DMSO and PBS. The efficiency of the compound to absorb light was measured by calculating its molar absorptivity (ϵ) according to the Beer-Lambert equation (See Supplementary Methods) with the results indicating best absorptivity in PBS. The quantum yield in each solvent was calculated according to the comparative method with Coumarin-30 (Φ = 0.67 in acetonitrile) as a standard (See Supplementary Methods). Quantum yield values showed moderate fluorescence indicating that compound 6 is an easily detectable fluorophore.

Table 1.

Physical properties of compound 6 in a range of organic solvents at 20 µM with respect to a reference standard (Coumarin-30).

Solvent	Abs	λEmission (nm)	λExcitation (nm)	ϵ ()	Stokes shift (cm-1)	Quantum yield
ACN	0.37	529	405	18,675	5787.8	0.43
EtOH	0.36	501	394	18,153	5420.6	0.56
Toluene	0.46	516	406	22,920	5250.7	0.27
PBS	0.50	521	395	25,655	6122.6	0.39
DMSO	0.48	543	404	23,825	6336.3	0.34

Photobleaching is a common problem in fluorophore design and can be affected by the cellular environment. A good probe should have sufficient stability under continuous irradiation⁴¹. The photostability of compound 6 was studied both in solution and in live cells (Supplementary Figs. S25 and S26). The stability of compound 6 in solution was determined by focused and continuous excitation over a 1800s period. Compound 6 showed minimal photobleaching over this time period, maintaining more than 80% fluorescence. The rate constant of decay was calculated according to first order principles and averaged at 8.30 × 10⁻⁵ s⁻¹ while the half-life was found to be approximately 2.41 h, indicating that it is quite stable under typical solution excitation (Supplementary Fig. 25). . Next, photostability was determined by constant fluorescent irradiation of live cells and time-lapse imaging over 300 s (Supplementary Fig. S26). Photostability was determined for cells treated with probe 5 and hypochlorite, along with two commercial fluorescent probes, Cell ROX Deep Red and Mitotracker Green. Compared with commercial probes, probe 5 was more stable, with a rate constant of decay of 6.34 × 10^-4 s^-1 and half life of 1093 s as determined using a single phase exponential decay. Together, the data suggest that probe 5, upon reaction, is stable in solution and in cells.

Discussion

A simple turn-on quinoline-based fluorescent probe was successfully synthesized, characterized, and studied for its sensitivity towards hypochlorite. The probe combines high fluorescence efficiency by emitting under standard green filter sets (~ 500–550 nm) with a compact molecular structure, ensuring brightness without compromising cell permeability or diffusion compared to other recently known quinoline probes that either compromise molecular weight for high emission or vice versa. Probe 5 exhibits selectivity for hypochlorite compared to other reactive oxygen species, metals, and anions with its limit of detection towards hypochlorite at 0.19 μM and time sensitivity as low as 10 s. We showed that probe 5 can detect exogenous hypochlorite in cells by flow cytometry and microscopy. Probe product is photostable in solution and in cells, and the probe product possesses physical properties that are highly favored in polar solvents. Thus, probe 5 has high potential for being able to selectively identify cells with high concentrations of hypochlorite experienced under stress or disease conditions.

Methods

¹H, ¹³C, and ¹⁹F NMR were recorded in CDCl₃ with TMS as internal standard on a Bruker 500 MHz spectrometer at 500, 125, and 471 MHz, respectively, unless otherwise specified. UV–Vis Spectroscopy was carried out on a Carey Eclipse UV–vis Spectrophotometer with a 1 cm cuvette. Fluorescence was recorded on the Carey Eclipse Spectrometer with excitation and emission slits set at 5, and scan rate 600 nm /min with a 1 cm cuvette.

Ethics declaration

Male and female C57BL/6 mice at 7–12 weeks of age were used to acquire cells for live cell studies. Animals were generated from breeding colonies, maintained on C57BL/6N or C57BL/6 J backgrounds, housed in the Southern Illinois University animal facilities. Animal procedures involving live animals were conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory animals. All procedures were approved by the Southern Illinois University Institutional Animal Care and Use Committee (IACUC) under protocols 23–021, 22–015 and 2023–125. The study is reported following the recommendations of the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

Synthesis of 2 (2,4-bis(trifluoromethyl)quinolin-7-amine):

To a flask containing 40 ml chloroform, was added m-phenylenediamine (5.00 g, 46 mmol) and 1,1,1,5,5,5-hexafluoropentane-2,4-dione (9.60 g, 46 mmol) and the resulting solution was refluxed at 90 °C for 7 h. After completion, the solution was concentrated and 2 was recrystallized from ethanol–water as a fluffy yellow solid (12.8 g, 100%). ¹H NMR (DMSO-d₆) δ 7.81 (dd, J = 9.3, 2.3 Hz, 1H), 7.58 (s, 1H), 7.33 (dd, J = 9.2, 2.4 Hz, 1H), 7.10 (d, J = 2.3 Hz, 1H), 6.46 (s, 2H). ¹³C NMR (DMSO-d₆) δ 152.8, 150.7, 146.6 (q, J = 34.4 Hz), 134.9 (q, J = 31.7 Hz), 124.9 – 124.2 (m), 123.9, 122.6 (d, J = 33.7 Hz), 120.4 (d, J = 33.5 Hz), 115.7, 108.4, 106.8. ¹⁹F NMR (DMSO-d₆) δ -60.53 (d, J = 2.2 Hz), -66.68. HRMS (EI-TOF) m/z: [M]⁺ Calcd for C₁₁H₆F₆N₂ 280.04352; Found 280.04428.

Synthesis of 3 (N-methyl-2,4-bis(trifluoromethyl)quinolin-7-amine):

To a solution containing 2 (500 mg, 1.79 mmol) and NaOCH₃ (482 mg, 8.92 mmol) in 30 ml methanol, was added paraformaldehyde (71.4 mg, 2.23 mmol) in 5 ml methanol dropwise. The mixture was refluxed and monitored with TLC. NaBH₄ (67.5 mg, 1.79 mmol) was then added, and solution stirred for additional 2 h at reflux temperature. The crude solution was concentrated and washed with water twice. It was then extracted with dichloromethane and dried with anhydrous sodium sulfate. The filtrate was concentrated and subject to column chromatography using 30% dichloromethane in hexane to yield 3 as a bright yellow solid (178 mg, 52%). ¹H NMR (DMSO-d₆) δ 7.84 (dq, J = 9.3, 2.2 Hz, 1H), 7.69 (s, 1H), 7.34 (dd, J = 9.3, 2.4 Hz, 1H), 7.07 (q, J = 4.9 Hz, 1H), 6.92 (d, J = 2.4 Hz, 1H), 2.81 (d, J = 4.9 Hz, 3H). ¹³C NMR (DMSO-d₆) δ 152.8, 151.2, 147.7 – 145.8 (m), 134.8 (d, J = 31.7 Hz), 124.7, 124.2 (d, J = 17.2 Hz), 122.7 (d, J = 35.9 Hz), 120.6, 116.1, 108.7, 102.8, 29.7. ¹⁹F NMR (DMSO-d₆) δ -60.11 (d, J = 2.2 Hz), -66.32. HRMS (EI-TOF) m/z: [M]⁺ Calcd for C₁₂H₈F₆N₂ 294.05917; Found 294.05995.

Synthesis of 4 (N-methyl-2,4-bis(trifluoromethyl)quinolin-7-amine-N-methyl-N-phenyl-2,4-bis(trifluoromethyl)quinolin-7-amine):

20 ml toluene was degassed for 15 min by continuous bubbling of argon. 3 (N-methyl-2,4-bis(trifluoromethyl) quinolin-7-amine) (300 mg, 1.02 mmol), bromobenzene (320 mg, 2.04 mmol), palladium acetate (84.0 mg, 0.37 mmol), BINAP (120 mg, 0.19 mmol) and NaO-t-Bu (1.57 g, 16.3 mmol) was added to the solvent in a glove box, the solution was stirred at 110 °C and the reaction was monitored with TLC until completion. After completion, the solution was filtered and the filtrate concentrated. The filtrate was then subject to column chromatography to yield product as a deep yellow solid (304.8 mg, 80.7%).¹H NMR (CDCl₃) δ 7.88 (dt, J = 9.0, 1.7 Hz, 1H), 7.69 (s, 1H), 7.47 (d, J = 2.6 Hz, 1H), 7.46 – 7.41 (m, 2H), 7.30 (dd, J = 2.4, 1.1 Hz, 1H), 7.26 (ddt), J = 7.5, 6.4, 1.2 Hz, 3H), 3.46 (s, 3H). ¹⁹F NMR (CDCl₃) δ -61.32 (d, J = 2.0 Hz), -67.70 (d, J = 1.1 Hz). HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₁₈H₁₃N₂F₆ 371.09830; Found 371.09840.

Synthesis of 5 (11-methyl-2,4-bis(trifluoromethyl)-11H-pyrido[2,3-b] phenothiazine):

Sulfur (26.9 mg, 0.84 mmol) and Iodine (6.72 mg, 0.03 mmol) were added to a solution of 4 (142 mg, 0.38 mmol) in 1,2-dichlorobenzene and the solution was stirred at 190 °C for 48 h. The crude sample was purified using silica gel chromatography with 30% DCM in hexanes to yield 5 as a reddish-purple solid. ¹H NMR (400 MHz, CDCl₃) δ 7.96 (dd, J = 9.2, 2.1 Hz, 1H), 7.80 (s, 1H), 7.33 (d, J = 9.3 Hz, 1H), 7.22 – 7.13 (m, 2H), 6.97 (t, J = 7.5 Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H), 3.48 (s, 3H). ¹⁹F NMR (CDCl₃) δ -60.38, -67.48 (d, J = 2.3 Hz). HRMS (EI-TOF) m/z: [M + H]⁺ Calcd for C₁₈H₁₁F₆N₂S 401.05471; Found: 401.05510.

Flow cytometry analysis of live mouse bone marrow cells

Mice were euthanized using carbon dioxide at a 30–70% chamber volume displacement rate per minute controlled by an adjustable flow meter followed by cervical dislocation. Mouse bone marrow cells were isolated by flushing the long bones (femurs and tibias) with a needle and syringe using staining media (Ca²⁺- and Mg²⁺-free Hank’s buffered salt solution (HBSS; Gibco) supplemented with 2% heat-inactivated bovine serum (Gibco)). Cells were gently triturated and filtered through 40-μm nylon mesh to obtain a single cell suspension. Cell counts were determined using a hemocytometer and trypan blue exclusion. For experiments, 2 × 10⁶ cells were incubated with 200 µl of probe 5 in 1X PBS at 37 °C for 30 min. Cells were washed with 1X PBS and treated with exogenous hypochlorite for 30 min at 37 °C. Cells were then washed in 1X PBS, resuspended in 500 µl 1 × PBS, and samples analyzed by flow cytometry using an Attune NxT flow cytometer and the 512/25 emission with excitation by the violet, 405 nm laser (VL2 channel). Flow cytometry data was analyzed using FlowJo software. Graphs and statistical analysis were generated with GraphPad Prism.

Imaging

5 × 10⁶ freshly isolated bone marrow cells were incubated with 5 µM of 5 and 1 mg/ml of Pyronin Y for nuclear staining for 30 min at 37 °C. Cells were then washed in 1X PBS. Next, cells were fixed with 1% Paraformaldehyde in 1X PBS for 10 min at room temperature, washed and then treated with 40 µM ClO^- in 1X PBS for 5 min. Cells were washed with 1X PBS and then resuspended in 1X PBS and fluorescent mounting medium (VECTASHIELD vibrance antifade mounting medium) for imaging on a Leica Thunder Fluorescence Imager with DM6 B upright microscope. Probe 5 fluorescence was detected with a 405 nm excitation and 535 nm emission.

Author contributions

Synthesis and Chemistry—F.F.O. and Y.H. Biological Analysis- S.Y.S.K and R.J.B. F.F.O wrote the main manuscript text. F.F.O and S.Y.S.K prepared all the figures. All authors reviewed the manuscript.

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.