O-phenylenediamine, ascorbic acid (AA), glycine (Gly), glutathione (GSH), dopamine (DA), iron chloride, bismuth chloride, hafnium chloride, hydrochloric acid, indium chloride, and lysine (Lys) were obtained from Aladdin Reagent Co., Ltd. Sulfamic acid (SA), manganese acetate, NaCl, copper chloride, manganese chloride, N, N-dimethylformamide (DMF), and NaOH were gained from Tianjin Guangfu Fine Chemicals Co., Ltd. The ultrapure water in the whole experiment was prepared by the BK-10B system.

The particle size of Mn³⁺/Mn⁴⁺ ion-doped CDs was recorded by a JEM-2100 transmission electron microscope (TEM). The height distribution of Mn³⁺/Mn⁴⁺ ion-doped CD was detected by atomic force microscopy (AFM). The UV-vis absorption and fluorescence spectra of Mn³⁺/Mn⁴⁺ ion-doped CDs were analyzed by an FS5 spectrophotometer. Fourier transform infrared (FT-IR) spectra of Mn³⁺/Mn⁴⁺ ion-doped CDs were measured by Bruker spectrometer. The structure of Mn³⁺/Mn⁴⁺ ion-doped CDs was recorded by X-ray diffraction (XRD) using Cu Kα radiation at a voltage of 40 kV and current of 40 mA. X-ray photoelectron spectroscopy (XPS) of Mn³⁺/Mn⁴⁺ ion-doped CDs was obtained by ESCALAB 250 Xi electron spectrometer.

During the stirring process, 0.31 g O-phenylenediamine and 0.331 g manganese acetate were dissolved in 8 ml DMF solution (containing 1 ml (1%) acetic acid) to form a mixed solution. The above-mixed solution was transferred to a 25 ml reaction vessel and heated at 200°C for 12 h. Subsequently, the solution was centrifuged to remove the solid precipitates at room temperature. Finally, solid Mn³⁺/Mn⁴⁺ ion-doped CDs were obtained by rotation.

Add different contents of DA (0–0.55 μM) aqueous solution to 3 ml of Mn³⁺/Mn⁴⁺ ion-doped CDs. The fluorescence spectra were then measured at 380 nm excitation. Selectivity was achieved by adding other biomolecules (including SA, Gly, GSH, AA, Bi³⁺, Cu²⁺, Hf⁴⁺, Fe³⁺, In³⁺, Mn²⁺, Na⁺, and Lys) instead of DA. The competitive experiments were carried out by adding DA to the Mn³⁺/Mn⁴⁺ ion-doped CDs solution.

The structure, morphology, and optical properties of Mn³⁺/Mn⁴⁺ ion-doped CDs were studied under heating at 200°C for 12 h. The TEM image of Mn³⁺/Mn⁴⁺ ion-doped CDs was shown in Figures 1A,B, and the as-prepared CDs exhibited good dispersion and uniform size of quasi-spherical shape with an average particle size of 3.5 nm (Figure 1B). Furthermore, the HRTEM in the inset of Figure 1A showed that the lattice spacing of the Mn³⁺/Mn⁴⁺ ion-doped CDs was 0.21 nm, corresponding to the (002) crystal plane of graphite carbon (Tang et al., 2018). In addition, as shown in Figure 1C, Supplementary Figures S1, S2, the height and morphology of Mn³⁺/Mn⁴⁺ ion-doped CDs were further studied by AFM. The AFM image showed that the height of Mn³⁺/Mn⁴⁺ ion-doped CDs was about 2.0 nm (Supplementary Figures S1, S2), similar to the TEM results. The X-ray diffraction (XRD) pattern of Mn³⁺/Mn⁴⁺ ion-doped CDs was shown in Figure 1D. A new wide diffraction peak appears in the XRD pattern of the CDs, located at 26.8°C, which is related to the (002) crystal plane of the graphitic carbon material (Tian et al., 2017; Gao et al., 2018). The aqueous solution of Mn³⁺/Mn⁴⁺ ion-doped CDs showed two UV-vis absorption peaks at 270 and 434 nm, as shown in Figure 1E. The absorption peak at 270 nm was attributed to the π-π conjugation of the benzene ring, which exhibited a lower energy absorption band at approximately 434 nm (Shi et al., 2020). The fluorescence emission spectra of Mn³⁺/Mn⁴⁺ ion-doped CDs are shown in Figure 1F. It can be seen that the fluorescence emission peak moved in the direction of a long wavelength, and the fluorescence intensity changed with the increase of the excitation wavelength. The illustration in Figure 1F shows the fluorescent color of Mn³⁺/Mn⁴⁺ ion-doped CDs. These fluorescence characteristics may be related to the functional groups on the surface of Mn³⁺/Mn⁴⁺ ion-doped CDs, and various functional groups can introduce different defects into the surface of Mn³⁺/Mn⁴⁺ ion-doped CDs as excitation energy traps, resulting in different fluorescence properties (Zhao et al., 2018). In addition, photostability is the premise of Mn³⁺/Mn⁴⁺ ion-doped CDs for biomolecular or metal ion detection and imaging. We studied the changes in fluorescence intensity of Mn³⁺/Mn⁴⁺ ion-doped CDs under different salt solution concentrations, time, and pH values. As shown in Supplementary Figures S3, S4, the fluorescence intensity of Mn³⁺/Mn⁴⁺ ion-doped CDs remain almost unchanged with the increase in salt solution concentration and time. Based on this, we chose to carry out the related experiments of DA detection in the later stage without sodium chloride solution. As shown in Supplementary Figure S5, the fluorescence intensity of Mn³⁺/Mn⁴⁺ ion-doped CDs is relatively low under strong acid or alkali conditions. Therefore, we choose pH 7 as the best condition for the anti-interference experiment of DA and related metal ions or biomolecules. Under the optimum conditions, the highest quantum yield of Mn³⁺/Mn⁴⁺ ion-doped CDs is 4.29%.

FT-IR and XPS evaluated the chemical composition and functional groups of Mn³⁺/Mn⁴⁺ ion-doped CDs. FT-IR spectra were shown in Figure 2A, the peak of Mn³⁺/Mn⁴⁺ ion-doped CDs were 3,367.8, 1,680.9, 1,600, 1,400, and 610 cm⁻¹, respectively, which were attributed to -NH₂/-OH, C=O, benzene ring, and Mn-O functional groups (Zhu et al., 2020). These results indicated abundant functional groups on the surface of Mn³⁺/Mn⁴⁺ ion-doped CDs, which makes Mn³⁺/Mn⁴⁺ ion-doped CDs have better dispersibility in an aqueous solution. In addition, XPS also further confirmed the presence of C=O, C=C, -NH₂/-OH and Mn-O chemical structures and elemental compositions on the surface of Mn³⁺/Mn⁴⁺ ion-doped CDs. The total spectrum of XPS shown in Figure 2B was mainly composed of C 1s, N 1s, O 1s, and Mn 2p, with the proportion of elements being 69.96, 8.55, 19.13, and 2.37% (Supplementary Table S1), respectively. As shown in Figure 2C, the high-resolution spectrum of C 1s showed three prominent peaks, and the binding energies of 284.7, 286, and 288 eV correspond to C=C, C-N, and C=O functional groups, respectively. The high-resolution spectrum of O 1s (Figure 2D) could be decomposed into two binding energies of 531.3 and 532.1 eV, which could be attributed to Mn-O and C-O, respectively. There were two binding energies of 399.1 and 400 eV in the high-resolution spectrum of N 1s, which could be attributed to C-N and N-H functional groups (Figure 2E). Moreover, Figure 2F shows the XPS spectrum of Mn 2p in the Mn³⁺/Mn⁴⁺ ion-doped CDs with four prominent peaks at 640.9, 645.8 eV, and 642.5, 653.3 eV, respectively, indicating that Mn³⁺ and Mn⁴⁺ ions exist in the surface of Mn³⁺/Mn⁴⁺ ion-doped CDs, and the ratio of Mn³⁺/Mn⁴⁺ on the surface was 0.82 (Wang J et al., 2019). FT-IR and XPS spectra showed a large number of hydrophilic functional groups on the surface of Mn³⁺/Mn⁴⁺ ion-doped CDs, which not only provided interaction sites for specific ions or biomolecules; but also improved the biocompatibility of the material itself.

Due to Mn³⁺ and Mn⁴⁺ions on the surface of CDs, the material had Fenton-like catalytic properties. The effect of DA on the fluorescence intensity of Mn³⁺/Mn⁴⁺ ion-doped CDs at 390 and 478 nm was studied under optimal conditions. As shown in Figure 3A, with the gradual increase of DA content, the fluorescence intensity of Mn³⁺/Mn⁴⁺ ion-doped CDs at 478 nm showed a continuous decrease, and the fluorescence intensity at 390 nm showed a continuously increasing trend. Figures 3B,C illustrate the linear relationship between ΔF/F₀ (ΔF = F-F₀, where F₀ and F are the fluorescence intensities of Mn³⁺/Mn⁴⁺ ion-doped CDs in the absence and presence of DA, respectively) and DA content. The linear range of Figure 3C was 100–275 nM with a correlation coefficient of 0.9966, and the detection limit was as low as 3 nM (n = 3); the linear range of Figure 3D was 325–525 nM with a correlation coefficient of 0.9918, and the detection limit was as low as 12 nM (n = 3). This value is equivalent to or even better than the previous fluorescence detection of DA (Table 1), indicating that this platform is suitable for the determination of DA.

To study the mechanism of Mn³⁺/Mn⁴⁺ ion-doped CDs as a fluorescent probe for detecting DA, the valence states of Mn ion in Mn³⁺/Mn⁴⁺ ion-doped CDs without and containing DA were analyzed, respectively. As shown in Figure 2F and Supplementary Figure S6, compared with Mn³⁺/Mn⁴⁺ ion-doped CDs without DA, the Mn³⁺/Mn⁴⁺ ratio of DA-containing Mn³⁺/Mn⁴⁺ ion-doped CDs increased from 0.82 to 1.04. These results suggest that the mechanism of Mn³⁺/Mn⁴⁺ ion-doped CDs and DA was a redox property. As shown in Figure 3D, when DA was added to the aqueous solution containing Mn³⁺/Mn⁴⁺ ion-doped CDs, the DA reacted with Mn⁴⁺ ion on the surface of CDs, resulting in the conversion of phenolic hydroxyl functional groups on DA into O-quinone structure (Shi et al., 2020). At the same time, Mn⁴⁺ ions on the surface of CDs act as oxidants to obtain electrons, which changes the Mn³⁺/Mn⁴⁺ ratio on the surface of CDs, resulting in an increase or decrease in the fluorescence intensity of emission peak at 390 and 478 nm of Mn³⁺/Mn⁴⁺ ion-doped CDs with the rise of DA content. In addition, we also carried out the time-dependent single-photon counting spectra of Mn³⁺/Mn⁴⁺ ion-doped CDs under different conditions. As shown in Figure 3E, the average fluorescence lifetime of single Mn³⁺/Mn⁴⁺ ion-doped CDs was 4.82 ns. The average fluorescence lifetime of Mn³⁺/Mn⁴⁺ ion-doped CDs decreased to 4.62 ns after adding DA. The decreased average fluorescence lifetime indicates that the electron transfer between Mn³⁺/Mn⁴⁺ ion-doped CDs and DA was a dynamic fluorescence quenching induced process. Between them, Mn³⁺/Mn⁴⁺ ion-doped CDs and DA were used as energy receptors and energy donors, respectively. To induce Fenton-like catalysis between catechol functional groups on DA molecule and Mn³⁺/Mn⁴⁺ ion pairs on CDs surface, jointly constructing donor/receptor pairs of FRET.

Mn³⁺/Mn⁴⁺ ion pairs with Fenton-like catalytic properties exist on the surface of CDs, which have the potential to be used as fluorescent probes for oxidation/reduction of biomolecules or metal ions. DA, a suitable electron donor, will quench and enhance the fluorescence intensity at 478 and 390 nm respectively after mixing with Mn³⁺/Mn⁴⁺ ion-doped CDs. Firstly, we studied the fluorescence intensity changes of Mn³⁺/Mn⁴⁺ ion-doped CDs in NaCl solutions with different concentrations. It can be seen that the fluorescence intensity of Mn³⁺/Mn⁴⁺ ion-doped CDs was not affected by the NaCl solution. Secondly, the effects of biomolecules or metal ions on the fluorescence intensity of Mn³⁺/Mn⁴⁺ ion-doped CDs were also investigated. As shown in Figure 3F and Supplementary Figure S7, biomolecules or metal ions such as Lys, Gly, Bi³⁺, Hf⁴⁺, In³⁺, Mn²⁺, and Na⁺ had almost no influence on the fluorescence intensity ratio F/F₀ of Mn³⁺/Mn⁴⁺ ion-doped CDs. SA had a weak fluorescence quenching effect on the fluorescence intensity ratio F/F₀ of Mn³⁺/Mn⁴⁺ ion-doped CDs, indicating that Mn³⁺/Mn⁴⁺ ion-doped CDs had relatively good selectivity for the above biomolecules or metal ions. Finally, to verify the Fenton-like catalytic properties of Mn³⁺/Mn⁴⁺ ion-doped CDs, we further studied the effect of oxidation/reduction biomolecules or metal ions on the fluorescence intensity ratio F/F₀ of Mn³⁺/Mn⁴⁺ ion-doped CDs. As shown in Figure 3F and Supplementary Figure S7, after adding oxidation/reduction biomolecules or metal ions such as AA, GSH, Cu²⁺, and Fe³⁺, the fluorescence intensity ratio F/F₀ of Mn³⁺/Mn⁴⁺ ion-doped CDs at 478 nm was relatively lower than that of the blank control group. At the same time, the fluorescence intensity ratio F/F₀ of Mn³⁺/Mn⁴⁺ ion-doped CDs at 390 nm was higher than that of the blank control group. The results indicate that Mn³⁺/Mn⁴⁺ ion-doped CDs have the potential as fluorescence probes for the detection of oxidation/reduction biomolecules or metal ions. However, the interference of other oxidation/reduction biomolecules or metal ions should be avoided during the detection of a certain oxidation/reduction biomolecule or metal ions, to enhance the accuracy and reliability of Mn³⁺/Mn⁴⁺ ion-doped CDs.