Mn, B, N co-doped graphene quantum dots for fluorescence sensing and biological imaging

3.1 Characterization analysis of Mn-BN-GQDs

The synthesis strategy of Mn-BN-GQDs is shown in Fig. 1a (The molecular structural formulas of citric acid, urea, borax, BN-GQDs and Mn-BN-GQDs are shown in Figure S2). Three synthetic materials and MnCl₂ are dissolved in distilled water and then the obtained solution is transferred to Teflon-lined autoclave and heated at 180 °C for 8 h to obtain a kind of GQDs with photoluminescence properties. On the basis of this, the transparent electronic microscopy (TEM) was used to display the morphology and microscopic size of the synthesized doped GQDs. Compared with the TEM images of BN-GQDs in Fig. 1b, it can be illustrated from Fig. 1c that the synthesized Mn-BN-GQDs have better dispersion and more uniform particle size. By randomly sampling the sizes of 150 particles, the corresponding size square distribution map was obtained, as shown in Figure S3, through square distribution mathematical calculation, the particle size distribution of Mn-BN-GQDs is within the range of 1.5 ∼ 6.5 nm., with an average size of 3.7 nm. It is obvious that the doping of Mn²⁺ ions effectively change size of distribution of BN-GQDs. According to HRTEM image, as shown in Fig. 1 d and e, it can be seen that the synthesized BN-GQDs and Mn-BN-GQDs have clear lattice fringes, indicating that they have high crystallinity. Their lattice spacing is 0.232 nm and 0.272 nm respectively corresponding to the (1 0 0) and (0 2 0) planes of GQDs (Li et al., 2012, Ju and Chen 2014) this indicates that they have a good crystal structure, which can also be investigated by XRD pattern.

Close

Fig. 1

Preparation and characterization of Mn-BN-GQDs and BN-GQD. a. Schematic illustration of the synthesis of Mn-BN-GQDs as idealized model. b, d. TEM (scale bar: 50 nm) and HRTEM (scale bar: 2 nm) images of BN-GQDs. c, e. TEM (scale bar: 20 nm) and HRTEM (scale bar: 2 nm) images of Mn-BN-GQD. f. XRD pattern of the as-prepared BN-GQDs and Mn-BN-GQDs. g. FTIR spectra of BN-GQDs and Mn-BN-GQDs.

Export to PPT

As shown in the figure S4a, b and c, we analyzed the X-ray Scattering (XRD) image of citric acid, urea and borax. By comparing with the synthesized BN-GQDs and Mn-BN-GQDs, a clear difference in the (0 0 2) of peak from the XRD image of BN-GQDs and Mn-BN-GQDs integrated the formation of new substance (Fig. 1f). Moreover, the XRD pattern of the synthesized BN-GQDs and Mn-BN-GQDs have two peaks at 8.4° (2θ) and 24.8° (2θ), corresponding to the plane of (0 0 1) and (0 0 2) of GQDs, respectively (Bao et al., 2015). According to the XRD image, the intensities of synthesized Mn-BN-GQDs at 8.4° (2θ) and 24.8° (2θ) are lower than those at BN-GQDs, indicating that manganese ions bind together with the functional groups at the edges of BN-GQDs, making the graphene sheets divided into smaller sheets.

In order for further analysis on chemical bonds and structure information of the synthesized BN-GQDs and Mn-BN-GQDs, the Fourier Transform Infrared Spectrometer (FTIR) have been applied on the as-prepared samples. As shown in Fig. 1g, the BN-GQDs and Mn-BN-GQDs show the vibrational absorption of 3182 cm⁻¹ and 2990 cm⁻¹ are related to O-H, N-H, C-H (unsaturated carbon) equivalent electron vibrations, indicating the presence of amino and hydroxyl groups on the GQDs. Besides, the characteristic absorption band from 1496 cm⁻¹ to 1753 cm⁻¹, and the peak at the 1558 cm⁻¹ were due to the stretching vibration of the C = O and C = C double bonds in the aromatic ring, respectively. Meanwhile, the relatively small peaks at 1311 cm⁻¹ and 1179 cm⁻¹ proved the hydrogen bonds of B-O and B-O-H structure, indicating boron was present in GQDs. Notably, recent works in graphene-based ORR catalysis have revealed a strong coordination effect between the N_x molecular and Mn²⁺ (Zhou et al., 2014). Therefore, the detection of abnormal abundance of nitrogen bonding structure in the as-prepared GQDs is able to determine the existence of Mn ions in the structure reversely. Similarly, we compared the N 1 s of synthesized Mn-BN-GQDs with BN-GQDs, and found that the peak position was significantly shifted as shown in Figure S5, so there is a strong coordination effect between N_x molecule and Mn²⁺. According to the FTIR image, the C-N stretching vibration of Mn-BN-GQDs at 1396 cm⁻¹ increases significantly as a result of the higher density of nitrogen elements on Mn-BN-GQDs in comparison to BN-GQDs, which provides evidence some element doping in BN-GQDs has an effect on the bond vibration. On the other hand, the reaction of Mn²⁺ with the carboxyl group on the residual citric acid would prevent the condensation reaction. Thereby, the suppression of carboxyl group from further condensation can be demonstrated by the significantly enhance of the peak value of Mn-BN-GQDs at 3000 cm⁻¹, which led to more O-H bonds retained on Mn-BN-GQDs and proved Mn²⁺ has an effect on the bond vibration. We calculate the density of states of them later and combine the analysis of doping level with its properties.

Furthermore, we made X-ray Photoelectron Spectroscopy (XPS) for further analysis of the synthesized Mn-BN-GQDs, and get the proportion of each element and functional groups (Table S1). From the full spectrum in Fig. 2a, we can observe that the synthesized BN-GQDs and Mn-BN-GQDs all contain C, N, O, and B. As shown in Table S1, the contents of C, N, O, B and Mn in the synthesized Mn-BN-GQDs were 48.75%, 7.64%, 39.28%, 3.87% and 0.47%, respectively. The contents of C, N, O and B in the synthesized BN-GQDs were 37.73%, 9.63%, 46.62% and 6.03%, respectively. Fig. 2b shows the XPS spectra of C 1 s in Mn-BN-GQDs, with four adjacent peaks of 284.6 eV, 285.2 eV, 285.7 eV and 289.1 eV respectively corresponding to C-C/C = C, C-O, C-N and O-C = O bonds. Fig. 2c shows the XPS spectra of N 1 s in Mn-BN-GQDs, with two peaks of 400.9 eV and 401.9 eV respectively corresponding to N-(C)₃ and C-N-C groups. The groups indicate that the N element has been successfully doped into the skeleton of GQDs. Fig. 2d shows the spectrum of O 1 s, which contains two peaks of 531.6 eV, 532.6 eV corresponding to C − OH/C − O − C and C = O bonds, respectively. Fig. 2e shows that Mn 2p has a dominant peak at 643.1 eV and a weak peak at 654.5 eV, corresponding to Mn²⁺ and Mn³⁺, respectively. This indicates that Mn exists on the surface of GQDs in the form of a mixture of Mn²⁺ and Mn³⁺, and the content of Mn²⁺ is higher. This indicates that Mn²⁺ ions have been successfully doped into the skeleton of GQDS (Ju and Chen 2014). Fig. 2f shows a spectrograph of B 1 s, with two peaks of 187.9 eV and 191.8 eV corresponding to B-O and B-C bonds, respectively. It shows that the B element is doped into the skeleton of GQDs. The above characterization analysis indicates the successful synthesis of Mn-BN-GQDs.

Close

Fig. 2

a. XPS survey spectra of Mn-BN-GQDs and BN-GQDs. b. C 1 s, c. N 1 s and d. O 1 s region of Mn-BN-GQDs. XPS high resolution of e. Mn 2p and f. B 1 s.

Export to PPT

3.2 Characterization of optical properties of Mn-BN-GQDs

The optical properties of the synthesized Mn-BN-GQDs were analyzed. The Mn-BN-GQDs showed pale yellow solution under the sunlight and a bright blue light under the irradiation of the ultraviolet lamp, as shown in Fig. 3a, which indicated that the Mn-BN-GQDs has the characteristics of photoluminescence in ultraviolet range. The synthesized Mn-BN-GQDs luminance spectrum is clearly represented in the Figure S6. Mn-BN-GQDs were dissolved in distilled water and the excitation wavelength, emission wavelength, UV-Vis spectrum of the synthesized Mn-BN-GQDs were measured at pH = 7. As can be observed from the UV-Vis spectrum in the Fig. 3a, two obvious absorption peaks are 216 nm and 326 nm respectively. The peak value at 216 nm corresponds to the π-π* transition on the sp² structure of the aromatic ring, the peak at 326 nm corresponds to the absorption band of n-π* (Jing et al., 2015). The excitation and emission peak wavelengths of the synthesized Mn-BN-GQDs are 345 nm and 434 nm, respectively. Compared with the UV-Vis spectrum, the absorption band is 326 nm, and the maximum excitation wavelength of Mn-BN-GQDs is 345 nm, which may be caused by the different distribution of GQDs energy traps in different signals (Liu et al., 2012, Ju and Chen 2014, Miao et al., 2020). As shown in Fig. 3b, we detected the change of emission wavelength under different excitation wavelength from 325 nm to 365 nm. When the excitation wavelength changes, the emission wavelength does not change at 434 nm, which indicates that the synthesized Mn-BN-GQDs have independent-excitation characteristics (Zhu et al., 2016, Li et al., 2019, Kaewprom et al., 2020). This is due to the uniform size of the synthesized Mn-BN-GQDs and the single photoluminescence process of the N element from n-p* in the aromatic hydrocarbon of Mn-BN-GQDs. This characteristic is beneficial to the practical application of Mn-BN-GQDs as fluorescence sensors, and avoids the phenomenon of automatic discoloration. From Fig. 3c, d, we compare the effects of doping of different ions on the fluorescence intensity and quantum yield of BN-GQDs. It can be clearly seen from the figure that Mn²⁺ enhances the fluorescence intensity of BN-GQDs better than other metal ions, and the QY of Mn-BN-GQDs is 30.52% (BN-GQDs = 20.12%). At the same time, we analyzed the fluorescence lifetime of Mn-BN-GQDs and BN-GQDs, As shown in Fig. 3e, its fluorescence intensity attenuation is fitted as a single exponential attenuation function and the results showed that the fluorescence lifetime of Mn-BN-GQDs was 7.444 ns, while that of BN-GQDs was 7.005 ns (Figure S7), and the fluorescence lifetime was improved. Thus, it can be further confirmed that Mn²⁺ ion is doped into BN-GQDs, and the optical properties of BN-GQDs are improved.

Close

Fig. 3

Optical properties of Mn-B, N-GQD and influence of pH. a. The fluorescence excitation (red), emission (blue) and UV-Vis (green) absorption spectra of Mn-BN-GQDs. Inset: photographs of the solution of the Mn-BN-GQDs taken under visible light (left) and 365 nm UV light (right). b. Fluorescence emission spectra of the Mn-BN-GQDs with different excitation wavelengths. c. The fluorescence intensity of BN-GQDs doped with different metal ions and the fluorescence intensity of BN-GQDs. d. The QY of different metal ions doped with BN-GQDs and the QY of BN-GQDs. e. Fitting curves of time-resolved fluorescence decay curve of Mn-BN-GQDs. f. Fluorescence intensities of synthesized Mn-BN-GQDs at different pH values at 434 nm (black) and in the presence of Fe³⁺ (red). [Fe³⁺] = 3.7 × 10^-4 mol/L [Mn-BN-GQDs] = 0.054 mg/mL.

Export to PPT

The principle that manganese ions can enhance the fluorescence and QY of BN-GQDs is analyzed. During the synthesis of BN-GQDs, some carboxyl groups of citric acid will remain on the surface of BN-GQDs without being carbonized. These carboxyl groups can promote further condensation carbonization reaction of BN-GQDs, which leads to large irregular agglomerations of BN-GQDs samples in TEM. However, manganese ions can combine with the carboxyl group and other functional groups on the surface of BN-GQDs, thus preventing further condensation reaction of BN-GQDs, and thus preventing the growth of BN-GQDs particles. Thus, the synthesized Mn-BN-GQDs particles are smaller and more evenly distributed. Because of the quantum confining effect, the synthesized Mn-BN-GQDs have better optical properties (Li et al., 2011, Ananthanarayanan et al., 2014).

3.3 Effect of pH on fluorescence Influence

Fig. 3f shows the fluorescence intensity of Mn-BN-GQDs synthesized in the pH range of 2–12 (black) and the quenched fluorescence intensity in the presence of Fe³⁺ (red). From the difference between the two lines, we can see the fluorescence quenching effect of Fe³⁺ on Mn-BN-GQDs at different pH values. On the whole in the figure, we can see that Fe³⁺ has a good quenching effect in the pH range of 2–12. However, when the pH is 2–4, the fluorescence intensity is obviously the worst, so the synthesized Mn-BN-GQDs as fluorescence sensors are not suitable for application under acidic conditions. Secondly, when the pH is 9–12, the fluorescence quenching effect is good, but the quenching effect is not stable. Therefore, it is not suitable for the practical use of Mn-BN-GQDs as fluorescence sensors. Finally, the quenching efficiency is the highest and relatively stable when the pH is 6–8, which is suitable for the use of Mn-BN-GQDs, and the quenching effect is the best when the pH is 7. Therefore, the subsequent tests are carried out at pH = 7.

3.4 Mechanism of Mn-BN-GQDs and Fe³⁺ quenching

Then we studied and analyzed the quenching mechanism of Mn-BN-GQDs as fluorescence sensors. Generally speaking, there are three main forms of fluorescence quenching: (1) internal filtration effect (IFE), (2) electron transfer, and (3) conversion of fluorescent materials to non-fluorescent materials (Lin et al., 2015, Shi et al., 2020). To know whether the fluorescence quenching process is dynamic quenching process or static quenching process, we have carried out the characterization analysis of fluorescence decay. The fluorescence intensity attenuation fitting of synthesized Mn-BN-GQDs in the presence and absence of Fe³⁺ is a single exponential decay function, and the fluorescence lifetime was shortened with the increasing of Fe³⁺ ions. The average fluorescence lifetime of Mn-BN-GQDs is 7.444 ns. The average fluorescence lifetime of Mn-BN-GQDs (in the presence of 10 μM Fe³⁺) and Mn-BN-GQDs (in the presence of 100 μM Fe³⁺) are 7.355 ns in Fig. 4a, and 7.267 ns in Figure S8, as shown in the table S3, which indicates that the fluorescence quenching between Fe³⁺ and Mn-BN-GQDs is a dynamic quenching process. It is also indicated that the fluorescence resonance electron transfer occurs during the process, because the fluorescence lifetime of the fluorescent material does not change during the process of IFE. This may be due to the fact that Fe³⁺ is a paramagnetic ion with empty d shells, which can quench the fluorescence of the fluorophore by electron transfer, as shown in Figure S9. At the same time, since Fe³⁺ ion has three positive charges, the free amino group can provide electrons to iron ion, so it can have better coordination ability with the free amino group (Xu et al., 2015). In order to further explore the quenching mechanism of Mn-BN-GQDs as fluorescence sensors, we tested the ultraviolet absorption spectra of Mn-BN-GQDs in the absence and presence of Fe³⁺ ions. As shown in Fig. 4b, the peak value of absorption spectra decreased in the presence of Fe³⁺ ions. In addition, the absorption peak redshifted from 326 nm to 329 nm, indicating that new chelating substances may be generated to change the absorption peak. Meanwhile, according to previous literature reports, oxygen-containing functional groups such as hydroxyl (–OH) and carboxyl (–COOH) have a good affinity with Fe³⁺ ions and can chelate and form new complexes (Zhang et al., 2015, Liu et al., 2017, Zhu et al., 2017). Then, as shown in Figure S10, we tested the FTIR of Mn-BN-GQDs in the presence and absence of Fe³⁺ and it can be seen from the figure that the O-H vibration and O-H/C-O changes around 3000 cm⁻¹ and 1000 cm⁻¹, but other parts are basically unchanged, indicating that Fe³⁺ has a good affinity with hydroxyl carboxyl group. This indicates that a new non-fluorescent substance may be formed on the surface of Mn-BN-GQDs to quench the fluorescence. Finally, as shown in Figure S11 1 mM-1 M EDTA was added to the Mn-BN-GQDs solution of Fe³⁺, and the fluorescence intensity recovered was 47.2%. It can be inferred that Fe³⁺ chelates with the above functional groups to produce quenching effect. In summary, Mn-BN-GQDs, as the quenching mechanism of fluorescence sensing, takes place the electron transfer and conversion process from fluorescent materials to non-fluorescent materials.

Close

Fig. 4

Mechanism of Mn-BN-GQDs and Fe³⁺ quenching. a. Fitting curves of time-resolved fluorescence decay curve of the Mn-BN-GQDs for the 10 μM Fe³⁺ analysis. b. The UV absorption spectra of Mn-BN-GQDs without Fe³⁺ and in the presence of Fe³⁺. c. Density of states for the Mn-BN-GQDs in different doping level.

Export to PPT

In order to investigate the relation between GQDs electronical properties with the doping level density functional theory calculations were performed by Vienna ab Initio Simulation Package with Perdew − Burke − Ernzerhof (PBE) (Fig. 4c). The graphene quantum dot structure is simplified to 16 aromatic rings, which reduce the computational burden without losing generality to provide qualitative insights and comparisons (Jin et al., 2013, Yu et al., 2021). Then we add the doped nitrogen to the rings, boron and Mn²⁺ as functional groups, according to the FTIR we obtained above, as shown from top to bottom (Yan et al., 2020). The number of functional groups indicates the expansion of doping level. Compared with the Ⅳ structure, III structure has only one functional group with N doped, and the second structure has four functional groups with N doped, the Ⅰ structure has four functional groups with N, Mn doped, respectively. The graphene quantum dot benchmark shows a bandgap near 2.50 eV which in accordance with the previous reports (Jin et al., 2013). Meanwhile the location of Fermi energy from the density of states shows that GQD is a p-type semiconductor. With the increase of doping level, structure Ⅱ and structure III show a tendency of metallization, and Fermi energy level moves to the negative energy region. Continuous doping with N and B gives a narrower band and larger carrier density, which reflected in the good electronic properties. However, after doping with Mn, the structure has larger carrier density and lower barrier compared with the Ⅱ structure and the III structure. It donates electrons that works like a n-type semiconductor which is easily react with Fe³⁺ than undoped and unsaturated doped GQD, which is also proved in our experiment.

3.5 Detection of Fe³⁺ ions

In order to explore the sensitivity of Mn-BN-GQDs to detection of Fe³⁺, we designed Mn-BN-GQDs to detect their emission wavelengths at different concentrations of Fe³⁺ (0.1–800 μM) under the condition of excitation wavelength of 345 nm, as shown in Fig. 5a. With the increase of Fe³⁺ concentration, the fluorescence intensity of Mn-BN-GQDs decreased gradually, which indicated that Mn-BN-GQDs had very good sensitivity to Fe³⁺ ions, and the CIE chromaticity coordinates of the synthesized Mn-BN-GQDs correspond to the color filtered PL spectrum as shown in the Figure S12. And Fig. 5b shows the curve of quenching efficiency (F/F₀) and Fe³⁺ concentration. It can be clearly seen from the figure that the quenching of Fe³⁺ by Mn-BN-GQDs shows a bilinear relationship and a good linear relationship. As shown in the illustration in Fig. 5b, the linear equation at 10–100 μM is $\mathrm{F}/\mathrm{F}\text{0}=0.78598-0.00466\mathrm{C}$ (R² = 0.99757, where F₀ and F correspond to the fluorescence intensities of Mn-BN-GQDs at 434 nm, with the 345 nm excitation wavelength, in the absence and presence of Fe³⁺ ions, respectively, and C is the concentration of Fe³⁺). Under this experimental condition, the limit of detection (LOD) is 0.78 μM according to the calculation formula $\mathrm{L}\mathrm{O}\mathrm{D}=3\mathrm{\delta }/\mathrm{K}$ is the standard deviation of 11 blank samples and K is the slope of the linear coordinate curve). Similarly, as shown in the illustration in Fig. 5b, the linear equation in the range of 100–800 μM is $\mathrm{F}/\mathrm{F}\text{0}=0.36169-3.9633\times 10\text{-4}\mathrm{C}$ (R² = 0.98256), and the LOD is 9.08 μM. As shown in the table S2, we compared the detection of Fe³⁺ by different fluorescence sensors.

Close

Fig. 5

Fluorescence quenching of Fe³⁺ by Mn-BN-GQDs and its application in biological imaging. a. Emission spectra (λ_ex = 345 nm) of in the presence of different concentrations of Fe³⁺ (from top to bottom:0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800 μM). b. The relationship between F/F₀ and Fe³⁺ ions concentrations range from 0 to 800 μM. (Insert: Linear relationship between F/F₀ and Fe³⁺ concentrations 10 ∼ 100 μM. Linear relationship between F/F₀ and Fe³⁺ concentrations 100 ∼ 800 μM.) c. Fluorescence responses of Mn-BN-GQDs to the different metal ions (pH = 7). The concentration of each metal ion is 800 μM. F₀ and F correspond to the fluorescence intensities of Mn-BN-GQDs at 434 nm with the 345 nm excitation wavelength in the absence and presence of metal ions, respectively. d. Relative cell viability of HepG2 cells after incubation with Mn-BN-GQDs (0–200 mg/mL) for 24 h. e. CLSM images bright field of HepG2 cells incubated with Mn-BN-GQDs (40 mg/mL) for 3 h by excitation at 405 nm. (scale bar: 20 μM magnified 400 times by CLSM). f. Morphology of Mn-BN-GQDs and HepG2 cells cultured together (scale bar: 20 μM magnified 400 times by CLSM).

Export to PPT

In order to further explore the selection characteristics of the synthesized Mn-BN-GQDs, we added the same concentration of metal ions to the synthesized Mn-BN-GQDs fluorescence sensor to observe the fluorescence quenching effect of different ions on Mn-BN-GQDs. As shown in Fig. 5c, Cd²⁺, Li⁺, Mg²⁺, Na⁺, Zn²⁺, Fe³⁺, Al³⁺, Ca²⁺, CO²⁺, Cu²⁺, K⁺ and Fe²⁺ ions were added into the fluorescence sensors of Mn-BN-GQDs, and it was found that besides Fe³⁺ had obvious quenching effect on the synthesized Mn-BN-GQDs. After the addition of other ions, the fluorescence intensity is basically unchanged, which fully demonstrates that the Mn-BN-GQDs synthesized by us have the characteristic of specific selection as fluorescence sensors, reflecting that Mn-BN-GQDs can detect Fe³⁺ in practical application as fluorescence sensors, so as to effectively control the pollution of Fe³⁺.

3.6 Cellular toxicity

In order to evaluate the application of Mn-BN-GQDs in bioimaging, we conducted cytotoxicity tests for its biocompatibility. HepG2 cells were cultured with different concentrations of Mn-BN-GQDs. After 24 h, the survival of HepG2 cells was calculated by using MTT cell relative viability method. As shown in the Fig. 5d, after cultured HepG2 cells for 24 h, the cell viability basically did not change when the concentration of Mn-BN-GQDs was 0–40 μg/mL, and the cell viability slightly decreased when the concentration was 60–200 μg/mL. By comparing the cytotoxicity test of Jiang et al. (Kai et al., 2015) at 0–50 μg/mL and Li et al. (Li et al., 2015) at 0–200 μg/mL, which indicates that the synthetic Mn-BN-GQDs have low cytotoxicity.

3.7 Cell fluorescent imaging

Confocal laser scanning microscope (CLSM) was used to observe the application of Mn-BN-GQDs imaging. Under the light confocal scanning microscope, we can see the blue fluorescence in Mn-BN-GQDs co-cultured with HepG2 cells at a concentration of 40 μg/mL, as shown in Fig. 5e, which indicates that Mn-BN-GQDs can enter the HepG2 cells and conduct cell imaging. Moreover, as shown in Fig. 5f, Mn-BN-GQDs and HepG2 cells were cultured together and HepG2 cells maintained their normal morphology and adhered to cell plates, indicating that Mn-BN-GQDs had good biocompatibility. Therefore, as a kind of low toxicity, Mn-BN-GQDs also has a good application prospect in the field of fluorescent biological imaging probes (Li et al., 2021a, 2021b).