Mn, N co-doped CDs as a fluorescent nanosensing platform for the detection of tannic acid and hafnium ion and in vitro fluorescence imaging of U2OS osteosarcoma cells

Abstract

Multi-wavelength emission fluorescent manganese-nitrogen co-doped carbon dots (Mn, N co-doped CDs) were synthesized by solvothermal method using β-cyclodextrin, O-phenylenediamine, and manganese chloride as raw materials. The prepared Mn, N co-doped CDs were used as fluorescent nanosensing platforms for the detection of metal ions and biomolecules and were found to be capable of fluorescence detection of tannic acid (TA) and hafnium (Hf) ion at 320, 380, and 480 nm excitation wavelengths with multi-response linear ranges of 0.7 ~ 1.2 µM and 6.35 ~ 13 µM and detection limits of 0.45 µM and 6.3 µM, respectively. The wide linear ranges and low detection limits may be due to the fluorescence resonance energy transfer effect between the platform and TA and Hf ions. In addition, it was found that Mn, N co-doped CDs had good photostability, biocompatibility, and low cytotoxicity, which could be used for in vitro fluorescence imaging of exogenous TA and Hf ion imaging in U2OS osteosarcoma cells. Thus, the probe has a promising application in biomedical fields as a new multi-responsive fluorescence nanosensing platform member.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s00604-024-06848-6.

Introduction

Tannic acid (TA) is a polyphenol found primarily in plants that act as natural barriers against pathogens or herbivores. Due to their ability to interact with proteins and stabilize their structure, allowing the skin to be tanned into leather, TA has long been used to treat animal skins to create leather and prevent rotting [1, 2]. In addition, when TA is released into the environment, it causes water pollution and leads to diseases such as sclerosis of the mucous membranes of the human gastrointestinal tract [1, 3, 4]. Hafnium (Hf), as a high atomic number element with chemical properties very similar to zirconium, has good corrosion resistance and is not susceptible to erosion by acidic and alkaline aqueous solutions [5]. Complexes formed by chelation of the element Hf and its oxides with different substances are suitable for magnetic resonance imaging (MRI) and computed tomography (CT) imaging contrast agents. They have promising applications in fluorescence nanosensing platforms and biomedicine [5–7]. Therefore, a simple and effective method for detecting TA and Hf ions is needed.

Traditional metal ion and biomolecule detection methods, such as electrochemical, plasma spectrometry, and high-performance liquid chromatography, are complex and time-consuming [8, 9]. In contrast, fluorescence detection is widely used because of its intuition, simplicity, and high selectivity and sensitivity [8, 9]. In recent years, fluorescence detection has received extensive attention in the field of analysis and bioimaging, and a variety of nanomaterials based on fluorescence detection have been developed, such as fluorescent carbon dots (CDs), metal nanoclusters, upconversion nanoparticles, and organic small molecules [3, 9, 10]. Among these fluorescent nanomaterials, fluorescent CDs, as a kind of zero-dimensional carbon nanomaterials, are characterized by good biocompatibility, photostability, and accessible surface functionalization. Due to its easy and environmentally friendly synthesis method and the abundance and low price of synthetic raw materials, researchers have emphasized it in various fields [11–13], such as Yang et al. [11]. Successfully realized the preparation of red fluorescent CDs with high quantum yield by a solvothermal method using 1, 3-dihydroxynaphthalene and potassium periodate as raw materials used in the research and exploration of warm-white light-emitting diodes. Zhou et al. [12]. Synthesized fluorescent CDs with red emission by one-pot hydrothermal method, and the CDs could be used as fluorescent probes for ex vivo detection and imaging tracking of particular noble metal ions. Yan et al. [13]. Prepared green fluorescent CDs with natural vitamin riboflavin as a photosensitizer and used them for the photodynamic treatment of tumors through endocytosis. However, to the best of our knowledge from researching the literature, there is no fluorescence method for simultaneous detection of TA and Hf ion. Therefore, developing a selective, susceptible, and reproducible fluorescence detection method for the ex vivo detection and analysis of TA and Hf ion is very necessary.

On this basis, we successfully synthesized multi-wavelength emission fluorescent Mn, N co-doped CDs with fluorescence that can be transformed from purple to red at room temperature using a simple and environmentally friendly one-step solvothermal method. This unique property of Mn, N co-doped CDs was utilized to successfully construct a multi-response fluorescent nanosensing platform for the detection of TA and Hf ions (Scheme 1), which exhibited a wide multi-response linear range of 0.7 ~ 1.2 µM and 6.35 ~ 13 µM, respectively. In addition, we evaluated the sensitivity and anti-interference of Mn, N co-doped CDs for detecting TA and Hf ions and found that they exhibited better anti-interference and optical stability. Finally, we successfully realized the imaging analysis of exogenous TA and Hf ions in U2OS osteosarcoma cells by using Mn, N co-doped CDs as a fluorescent probe, which lays a preliminary foundation for the construction and application of fluorescence nanosensing platforms for the detection of TA and Hf ions.

Scheme 1.

Schematic structure of Mn, N co-doped CDs synthesized and their multi-response fluorescent nanosensing platform constructed for TA and Hf ions detection

Experimental

Synthesis of Mn, N co-doped CDs

Based on previous studies, the classical solvothermal method synthesized Mn, N co-doped CDs [12]. Firstly, 0.3 g of O-phenylenediamine, 1.2 g of β-cyclodextrin, and 0.1 g of manganese chloride solid were sequentially added to 7 ml of N, N-dimethylformamide solution, and 1 mL (1%) of acetic acid solution was added to the above solution during magnetic stirring. After magnetic stirring for 10 min, the mixed solution was transferred to the polytetrafluoroethylene reactor and heated at 200 ℃ for 12 h. Then, the supernatant was collected by cooling, centrifugation, and solidification treatment to obtain Mn, N co-doped CDs.

Results and discussion

Morphological and structural analysis

Under the optimal conditions, we successfully synthesized multi-wavelength emission fluorescent Mn, N co-doped CDs using a simple and green solvothermal method. We analyzed the synthesized materials’ morphology size and particle height with the help of relevant instruments. The morphology and particle size distribution of the synthesized materials are shown in Fig. 1a and b, from which we found that the synthesized material has good mono-dispersity with a particle size of about 8.45 nm. In addition, as shown in Fig. 1c, we also performed X-ray diffraction (XRD) spectroscopy on the Mn, N co-doped CDs. It was found that two diffraction peaks were displayed at 18.4^o and 19.4^o, corresponding to amorphous and graphitized carbon, respectively, which were consistent with the lattice spacing illustrated in Fig. 1a, indicating that the surface of the material was enriched with oxygen-containing functional groups and graphitized carbon, resulting in better water solubility and stability [12]. In addition, we further analyzed the particle height of the synthesized materials using AFM. As shown in Fig. 1d, the average particle height of the CDs is about 6.3 nm (Fig. 1e and f), which is smaller than the average particle size of the TEM, indicating that the morphology of the CDs is a spherical-like structure.

Fig. 1.

Transmission electron microscopy (TEM) (a), particle size distribution (b), and XRD (c) spectra of Mn, N co-doped CDs. (d-f) Atomic force microscopy (AFM), particle size height and 3D height distribution of Mn, N co-doped CDs

We analyzed the Mn, N co-doped CDs’ surface structure and valence bond forms. The Fourier transform infrared (FT-IR) spectra are displayed in Fig. 2a, and the characteristic peaks of the synthesized material at 3256, 2927, and 1405 cm⁻¹ are attributed to the stretching vibrations of the O/N-H and C-H alkyl groups, respectively. The characteristic peaks near 1657 cm⁻¹ are related to the tensile vibration of C = O. In addition, the Mn, N co-doped CDs also exhibit metal-sensitive bonds in the range of 870 ~ 1100 cm⁻¹, which may correspond to N-Mn-N [9, 13, 14]; these results mentioned above indicate that O/Mn-related groups are formed on the surface and backbone of the synthesized materials, which endow the Mn, N co-doped CDs with unique optical properties. In addition, the elemental content, composition, and valence bond structure of the synthetic materials were analyzed by X-ray photoelectron spectroscopy (XPS). The total XPS spectrum of Mn, N co-doped CDs is displayed in Fig. 2b, which is mainly composed of four elements, C, N, O, and Mn, with percentage contents of 43.41%, 2.97%, 53.52% and 0.1%, respectively, indicating that Mn and N are successfully doped on the surface of the synthetic material. In the high-resolution spectra (c-f), the C 1s XPS spectra (Fig. 2c) can be deconvoluted into three peaks at 284.81, 286.31, and 287.61 eV, which correspond to C = C/C-C, C = N, and C = O bonds, respectively [13, 15]. The N 1s XPS spectra, shown in Fig. 2d, have binding energies at 399.61 and 401.71 eV that can be attributed to the pyridine N and amino N. The O 1s XPS spectra (Fig. 2e) can be deconvoluted into two peaks at 532.51 and 533.01 eV, corresponding to the C = C and C-O and C-O/C-O-C bonds, respectively [13, 16]. As displayed in Fig. 2f, there are four significant peaks at 641.91, 644.71 and 653.11, 655.01 eV, which can be attributed to the Mn 2p_3/2 and Mn 2p_1/2 spin-orbit peaks [17]. FT-IR and XPS data indicate that the hydrophilic groups such as -OH, -COOH, and -NH₂ in the Mn, N co-doped CDs improve the mono-dispersity of the material in aqueous solution, providing feasibility for the detection of metal ions and biomolecules.

Fig. 2.

(a) FT-IR spectra of Mn, N co-doped CDs. (b) XPS total spectra of Mn, N co-doped CDs. (c-f) C 1s, N 1s, O 1s and Mn 2p XPS spectra of Mn, N co-doped CDs

Optical properties

The UV-vis absorption spectra and fluorescence emission spectra of the multi-wavelength emission fluorescent Mn, N co-doped CDs are shown in Fig. 3. The CDs show significant absorption bands at 270, 376, and 498 nm, which originate from the π-π* transition of C = C and the n-π* transition of C = O/N, respectively (Fig. 3a) [18, 19]. In addition, the fluorescence emission peaks of the Mn, N co-doped CDs were gradually red-shifted when the excitation wavelength was changed from 300 nm to 500 nm (Fig. 3b). This photoluminescence property indicated that their sizes and surface states were inhomogeneous [20]. Therefore, we chose the excitation wavelengths of 320, 380, and 480 nm for exploring the fluorescence lifetime changes of Mn, N co-doped CDs. As shown in Fig. 3c, the fluorescence lifetimes of Mn, N co-doped CDs at 320, 380, and 480 nm were 2.1, 4.3, and 3.1 ns, respectively. We also analyzed the fluorescence intensity changes of Mn, N co-doped CDs under different pH values, sodium chloride concentrations, and UV irradiation. As shown in Fig. S1, the fluorescence intensities of Mn, N co-doped CDs at 320, 380, and 480 nm excitation wavelengths remained almost unchanged after 60 min continuous irradiation. Moreover, the fluorescence intensity of Mn, N co-doped CDs at different excitation wavelengths also maintained relatively good optical stability with increased NaCl content (Fig. S2). In addition, in studying the effect of pH on the optical properties of the CDs, we found that the Mn, N co-doped CDs had the most vigorous fluorescence intensity at pH 5 (Fig. S3). Therefore, we chose the above optimal conditions for selective detection of metal ions and biomolecules.

Fig. 3.

UV-vis absorption spectra (a) and fluorescence emission spectra (b) of Mn, N co-doped CDs. (c) Fluorescence lifetimes of Mn, N co-doped CDs at 320, 380 and 480 nm excitation wavelengths

Detection of Hf ions

The doping of Mn and N is an important reason to improve the ability of CDs detection. Mn and N can introduce a positive charge and enhance the interaction between the CDs and the analyte [13, 21]. In addition, Mn is easy to chelate with most small molecule compounds, which improves the sensitivity and selectivity of CDs to analytes.

We successfully established a fluorescent nanosensing platform for Hf ion detection using the above-optimized conditions. Briefly, under the optimal conditions, we found that other metal ions and biomolecules could not induce significant changes in the fluorescence intensity of the CDs except for Hf ion and TA, which suggests that Mn, N co-doped CDs have high selectivity for Hf ion and TA. With the gradual addition of Hf ion (0 ~ 15 µM), the fluorescence emission peak intensities of Mn, N co-doped CDs at the excitation wavelengths of 320, 380, and 480 nm all underwent regular decreases (Fig. 4a, b, c). The fluorescence intensity ratios of the CDs showed an excellent linear relationship with the content of Hf ion in the range of 6.35 ~ 13 µM (Fig. 4d, e, f), with the detection limits of 6.3, 3.1, and 4.1 µM, respectively, and the signal-to-noise ratio of 3. In summary, the Mn, N co-doped CDs can perform multi-response fluorescence detection of Hf ion in the range of 6.35 ~ 13 µM with a detection limit of 6.3 µM, showing good sensitivity and selectivity.

Fig. 4.

Fluorescence emission spectra of Mn, N co-doped CDs at the excitation wavelengths of 320 (a), 380 (b), and 480 nm (c) under the effect of different content of Hf ion. Linear plots of fluorescence intensity ratios of Mn, N co-doped CDs with varying contents of Hf ions at 320 (d), 380 (e), and 480 nm (f) excitation wavelengths

Detection of TA

Under the best conditions, we observed the fluorescence intensity changes of Mn, N co-doped CDs at the TA content of 0 ~ 1.5 µM and explored the feasibility of this probe for detecting TA. Therefore, TAs were added to the Mn, N co-doped CDs solution, respectively, to observe further the fluorescence intensity changes of the CDs at 320, 380, and 480 nm excitation wavelengths. As shown in Fig. 5a, b, c, TA effectively suppressed the fluorescence intensity of Mn, N co-doped CDs. Moreover, the fluorescence emission peaks of the CDs were gradually quenched with the increase of TA content, indicating that the accumulation of interaction with TA caused the Mn, N co-doped CDs, as can be seen in Fig. 5d, e, f, there is an excellent linear relationship between the TA content and the fluorescence intensity changes of the Mn, N co-doped CDs at different excitation wavelengths in the range of 0.7 ~ 1.2 µM (R² = 0.9981, 0.987 and 0.991), and the corresponding linear regression equations are y=−0.76x + 0.1288, y=−0.294x + 0.791 and y=−0.296x + 0.7873, with detection limits of 0.32, 0.14 and 0.45 µM, respectively, and the signal-to-noise ratio of 3. Compared with Table S1, this nanofluorescence sensor can perform ultra-sensitive multi-response fluorescence detection of TA in the range of 0.7 ~ 1.2 µM, with a detection limit of 0.45 µM, providing the possibility for monitoring TA in drinking water and industrial water.

Fig. 5.

Fluorescence emission spectra of Mn, N co-doped CDs at 320 (a), 380 (b), and 480 nm (c) excitation wavelengths with different TA contents. The linear relationship between the change of fluorescence intensity and TA content at the excitation wavelengths of 320 (d), 380 (e), and 480 nm (f)

Selectivity

The anti-interference of the probes was investigated by exposing the optimized Mn, N co-doped CDs to different metal ions and biomolecules. As shown in Figs. S4, S5, and S6, the fluorescence intensity ratios of Mn, N co-doped CDs at 320, 380, and 480 nm excitation wavelengths were varied by the addition of Ca²⁺, Al³⁺, Lys, Glu, Cu²⁺, Ala, Cys, Zn²⁺, TA, Hf²⁺, Na⁺, Gly, GSH, AA, Mel, Met, Ba²⁺ and DA. Among them, Al³⁺, Lys, Ca²⁺, Cys, and Zn²⁺ had less than 5% influence on the changes of fluorescence intensity ratios of Mn, N co-doped CDs at different excitation wavelengths. The impact of Ala, Na⁺, Cu²⁺, Glu, GSH, AA, and DA on the fluorescence intensity ratios of Mn, N co-doped CDs at 380 nm excitation wavelength was relatively significant, about 5 ~ 18%, and the impact of fluorescence intensity ratios at 320 and 480 nm excitation wavelengths was relatively small. The effects of Mel and Gly on the fluorescence intensity ratios of Mn, N co-doped CDs at different excitation wavelengths were relatively significant, about 10 ~ 15%. In summary, the Mn, N co-doped CDs have specific selectivity and anti-interference for the selective detection of TA and Hf ion.

To explore the utility of multi-wavelength emission fluorescent Mn, N co-doped CDs as a fluorescent nanosensing platform for detecting TA and Hf ions, we evaluated the variation of TA and Hf ions concentrations in water samples. First, we treated actual water samples with a simple pretreatment and obtained three concentration points at linear ranges of 0.7 ~ 1.2 µM and 6.35 ~ 13 µM, respectively. Then, we added TA and Hf ions separately to the aqueous solution of Mn, N co-doped CDs and analyzed the fluorescence intensity at excitation wavelengths of 320, 380, and 480 nm. Using the linear relationship curves shown in Figs. 4 and 5, we calculated the contents of TA and Hf ions in water samples based on the linear function. As shown in Table 1, the recoveries of TA and Hf ion samples ranged from about 95 to 104% and 96.89 to 104.64%, respectively, indicating that the nanosensing platform has good reproducibility and reliability in detecting TA and Hf ion in natural water samples.

Table 1.

Multi-wavelength emission fluorescent Mn, N co-doped CDs prepared in this study as a fluorescent nanosensing platform for the analysis of TA and Hf ions in real water samples

Samples	Added (µM)	Measurement			RSD (%, n = 3)
		(320 nm, µM)	(380 nm, µM)	(480 nm, µM)
TA	0.75	0.78	0.73	0.75	2.83
	0.80	0.76	0.83	0.81	4.52
	0.85	0.82	0.87	0.84	1.35
Hf ions	7.00	6.84	7.36	6.95	5.26
	9.00	8.72	9.37	9.13	2.62
	11.00	10.78	11.05	11.51	3.41

Detection of exogenous TA and hf ions in U2OS osteosarcoma cells

Due to the tight binding between Mn, N co-doped CDs and the above analytes and exhibiting quenching effects on aqueous solutions of TA and Hf ion under optimal conditions, we further explored whether Mn, N co-doped CDs could penetrate living cells and detect exogenous intracellular TA and Hf ions by fluorescence quenching changes. Therefore, we chose exogenous Hf ion and TA in U2OS osteosarcoma cells as a model system for in vitro monitoring by confocal laser scanning microscopy (CLSM). For the first time, we chose the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide)) method for evaluating the biocompatibility of Mn, N co-doped CDs on U2OS cells. As shown in Fig. S7, U2OS osteosarcoma cells showed more than 80% cell survival even with the concentration of Mn, N co-doped CDs of 1.0 mg/mL, indicating that the CDs had better biocompatibility and lower cytotoxicity [22, 23].

To confirm that the significant decrease in fluorescence intensity was caused by the binding between Mn, N co-doped CDs and analytes, we co-mixed the medium containing Mn, N co-doped CDs with TA and Hf ion and then co-cultured the U2OS cells respectively to observe the changes in fluorescence intensity of different groups of U2OS cells. As shown in Fig. 6a, Mn, N co-doped CDs exhibited bright green fluorescence in U2OS cells after co-culture with U2OS cells. In contrast, the fluorescence intensity of U2OS cells co-cultured with Mn, N co-doped CDs/TA (Fig. 6b) and Mn, N co-doped CDs/Hf ion (Fig. 6c) was weaker, suggesting that Mn, N co-doped CDs could be used as intracellular fluorescent probes to detect the changes in the content of exogenous Hf ion and TA.

Fig. 6.

Confocal fluorescence images of TA and Hf ions in U2OS osteosarcoma cells. (a) U2OS cells with only Mn, N co-doped CDs were added as a negative control. (b) Staining image of Mn, N co-doped CDs co-incubated with TA in U2OS cells. (c) Staining image of Mn, N co-doped CDs in U2OS cells co-incubated with Hf ion. (i) Bright-field images of U2OS cells. (ii) Fluorescence images of Mn, N co-doped CDs were obtained using a 488 nm laser emission filter. (iii) Superimposed final rendered images of (i) and (ii)

Conclusions

In summary, the synthesis and characterization of Mn, N co-doped CDs, as well as a new method for the detection of exogenous TA and Hf ions in U2OS osteosarcoma cells, are discussed in detail, demonstrating the mechanism of photoluminescence action of the synthesized materials. In addition, we also analyzed the Mn, N co-doped CDs with relevant instruments. Their surfaces contain abundant amino, carboxyl, and Mn doping sites. The linear ranges of TA and Hf ions analyzed at the excitation wavelengths of 320, 380, and 480 nm were 0.7 ~ 1.2 µM and 6.35 ~ 13 µM, which provide a broad application prospect for detecting TA and Hf ions in actual samples. Due to their excellent biocompatibility and low cytotoxicity, we have successfully realized in vitro fluorescence imaging of exogenous TA and Hf ions in U2OS osteosarcoma cells. This study provides a new multi-response fluorescence method for accurately monitoring TA and Hf ions and a new detection tool for early diagnosis of related diseases in the future.

Supplementary information

Below is the link to the electronic supplementary material.

Author contributions

Zhao X.L., Wang S.Z., Zhang L.H., Wang Z. wrote the main manuscript text. Huang J.Y., Liao S, Lu M., Yang Z. Zhao X.J., Zhao Z.Y., and Guo Z.X. prepared Figs. 1, 2, 3, 4, 5 and 6. Zhang L.N., Zhu PD. and Xu M. as the co-corresponding authors participated in the design of this study and revision of the manuscript. All authors reviewed the manuscript.

Funding

This work was supported by the National Key Research and Development Program of China (2023YFB4706305), Doctoral Start-up Funds for Xinxiang Medical University (505554) and the National Defense Science and Technology Excellence Youth Science Fund Program (2022-JCJQ-ZQ-018).

Data availability

No datasets were generated or analysed during the current study.

Declarations

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical approval

This research did not involve human or animal samples.

Competing interests

The authors declare no competing interests.