Colorimetric and fluorimetric dual mode detection of Fe²⁺ in aqueous solution based on a carbon dots/phenanthroline system

2.1 Materials

CrCl₃, NiCl₂, Pb(NO₃)₂, CuCl₂, MnSO₄, KCl, AgNO₃, Cd(NO₃)₂, BaCl₂, Hg(Ac)₂, Zn(NO₃)₂, LiCl, NaCl, MgCl₂, CoCl₂, Fe(NO₃)₃, AlCl₃ and FeSO₄ were purchased from Damao Chemical Corp (Tianjin, China). Sulphuric acid, m-phenylenediamine, PEG 1500, sodium acetate trihydrate, acetic acid and phenanthroline were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All chemicals were used without any further purification. The ultrapure water applied throughout all the experiments was purified through Water Purifier Nanopure water system (18.3 MΩ cm).

2.2 Characterization

The morphology and size of mPD-CDs were analysed using a transmission electron microscopy (TEM, Philips Tecnai G2F20, operated at 200 kV). UV–vis absorption spectra were performed by the use of a U-3900 UV–vis spectrophotometer (Hitachi, Japan). Fluorescent spectra were collected by a fluorescence spectrometer F-4600 (Hitachi, Japan). The X-ray diffraction (XRD) of mPD-CDs was recorded using a D8 ADVANCE X-ray diffractometer (Bruker AXS, German) with Cu-Kα radiation (40 kV, 40 mA, λ = 1.5418 Å) at a scanning rate of 1° min⁻¹ in the range from 10° to 80°. The Fourier transform infrared spectroscopy (FTIR) spectrum of mPD-CDs was scanned using a FT-IR 200 spectrometer (Thermo, America) with KBr pellets technique, over the range of 500–4000 cm⁻¹. The quantum yield (QY) of mPD-CDs was determined using rhodamine 6G in ethanol (literature QY: 95%) as the standard sample by comparing the integrated fluorescence intensities (excitation at 440 nm) and absorbance values at 440 nm of the mPD-CDs aqueous solution with those of rhodamine 6G.

2.3 Synthesis of mPD-CDs

mPD-CDs was synthesized by a facile hydrothermal method (Lim et al., 2018; Pan et al., 2018). 0.2 g m-phenylenediamine, 0.1 g PEG 1500 and 25 mL sulphuric acid (0.75 M) were put into a Teflon-equipped stainless steel autoclave sequentially. Then, the mixture was hydrothermally treated for 10 h at 180^◦C. After cooling down to room temperature, the transparent product was subjected to centrifugation (10000 rpm, 10 min), passed through a 0.22 μm micron filter and dialysis against deionized water though a dialysis membrane (500 MWCO) for 8 h. The final solid product was collected by lyophilization and stored at 4 °C for further analysis.

2.4 Determination of quantum yield

The QY of the obtained mPD-CDs was determined by a relative method (Jiang et al., 2015; Jiang et al., 2015). Specially, rhodamine 6G (QY = 95% in ethanol) for the emission range of 480–560 nm was chosen for the determination. The QY of a sample was then calculated according to the following equation: $$\mathrm{\Phi }={\mathrm{\Phi }}^{\text{'}}\times \frac{A^{\text{'}}}{I^{\text{'}}}\times \frac{I}{A}\times \frac{n^2}{{n^{\text{'}}}^2}$$ where ϕ is the QY of the testing sample, I is the testing sample’s integrated emission intensity, n is the refractive index (1.33 for water and 1.36 for ethanol), and A is the optical density. The superscript “′” refers to the referenced fluorescence dye of known QYs. To obtain more reliable results, a series of solutions of mPD-CDs and referenced fluorescence dye were prepared with concentrations being adjusted so that the optical absorbance values were between 0.0 – 0.1 at 440 nm. The fluorescence spectra were measured and the fluorescence intensity was integrated. QYs were determined by comparison of the integrated fluorescence intensity vs absorbance curves (refractive index, n, had also to be considered).

2.5 Selectivity

In order to investigate the selectivity, the fluorescence spectra of mPD-CDs/phenanthroline system were collected in the presence of the potential interferences, including Al³⁺, Pb²⁺, Cr³⁺, Mn²⁺, K⁺, Cd²⁺, Ni²⁺, Ba²⁺, Zn²⁺, Li⁺, Na⁺, Mg²⁺, Co²⁺, Cu²⁺, Fe³⁺, Hg²⁺, Ag⁺, and Fe²⁺. All the determinations were repeated for three times.

2.6 Colorimetric and fluorimetric detection of Fe²⁺

For the detection of Fe²⁺, mPD-CDs (0.025 mg/mL) and phenanthroline (0.18 mM) were mixed in the HAc-NaAc buffer solution (50 mM, pH 6.0), thereafter, Fe²⁺ was added into the mPD-CDs/phenanthroline system at different concentrations. The mixture solutions were incubated at ambient temperature for 5 min, and then their absorption and fluorescence spectra were collected.

2.7 Real samples preparation

In this work, tap water, lake water and human blood samples were used to evaluate the feasibility of the proposed method for the detection of Fe²⁺ in practical samples. The tap water samples were taken from our lab without any pretreatment, and then spiked with Fe²⁺ at different concentrations. The lake water was sampled from the Hongzi Lake in the campus of Qingdao Agricultural University, in which the solid suspensions and other impurities were removed by qualitative filter paper and 0.22 μm filter membrane. And the human blood samples were centrifuged at 12000 rpm for 15 min, treated by 0.45 μm filter membrane, then diluted for 100 times. Then, the treated lake water and human blood samples were spiked with Fe²⁺ at different concentrations, respectively. Finally, their recoveries were performed by the proposed method.

3.1 Synthesis and characterization of mPD-CDs

mPD-CDs were synthesized by hydrothermal carbonization of mPD and PEG 1500 in sulfuric acid solution at 180 °C for 10 h. It emits green fluorescence with a high quantum yield of 74.13% using rhodamine 6G (literature QY: 95%) as a reference in ethanol solution under excitation at 440 nm ***(Table S1) (Jiang et al., 2015). TEM was used to characterize the morphology and size of mPD-CDs. As shown in Fig. 1a, mPD-CDs are near spherical and mono-dispersed with the average diameter of 3.45 ± 0.27 nm (Fig. 1b). Two sharp diffraction peaks centered at 20.2°, 26.8° are observed on the XRD pattern of mPD-CDs corresponding to interlayer spacing of 0.44 and 0.33 nm (Fig. 1c), which are ascribed to the (0 0 2) lattice of the graphitic carbon based materials (Feng et al., 2016; Feng et al., 2016). FTIR was used to identify the functional groups on the surface of mPD-CDs. As can be seen from Fig. 1d, the broad bands at 3363 and 3169 cm⁻¹ are ascribed to the stretching vibrations of –OH and N–H (Yan et al., 2019; Edison et al., 2016), while the absorption band at 2870.0 cm⁻¹ belongs to the stretching vibration of –CH₂ (Liu et al., 2017). The peaks located at 1598.3 and 1549.1 cm⁻¹ are assigned to the amide I and II bands (Li et al., 2019), and the absorption at 1028.9 cm⁻¹ is assigned to the C–O stretching vibration (Lesani et al., 2019).

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Fig. 1 (a) TEM image, (b) The particle size distribution, (c) XRD pattern, and (d) FT-IR spectrum of mPD-CDs.

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The optical properties of mPD-CDs in dilute aqueous solution were investigated by UV and fluorescence spectra (Fig. 2a). Obviously, there are a shoulder peak at 237 nm ascribed to the π-π* transition, a distinct peak centered at 290 nm attributed to the n-π* transition and a weak band at 440 nm corresponding to surface state in the absorption spectrum of mPD-CDs (Ren et al., 2018), overlapping with the excitation spectrum (Fig. 2a). On the other hand, as shown in Fig. 2b, the fluorescence emission of mPD-CDs is peaked at 516 nm with an excitation independent property.

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Fig. 2 (a) The absorption, excitation (monitoring wavelength at 516 nm) and fluorescence (excitation at 440 nm) spectra of mPD-CDs. (b) The fluorescence spectra of mPD-CDs at the excitation wavelengths as indicated with 20 nm increments.

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In addition, the effect of pH on the fluorescence intensity of mPD-CDs was investigated. As shown in Fig. 3a, the fluorescence intensity of mPD-CDs was strong and stable in acidic environment, and reached the highest at pH = 6. As pH increased from 6 to 8, the fluorescence emission decreased quickly and kept stable under strong alkaline condition. Furthermore, the influences of ions strength, temperature and time on the photostability of mPD-CDs were also explored. As shown in Fig. 3b–d, the fluorescence intensity of mPD-CDs was not sensitive to the variations of temperature, ionic strength and irradiation time under excitation at 440 nm with a 150 W Xe lamp.

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Fig. 3 The effects of (a) pH, (b) ionic strength, (c) temperature and (d) irradiation time on the fluorescence intensity of mPD-CDs at the emission wavelength of 516 nm in a fluorescence spectrophotometer under excitation at 440 nm with a 150 W Xe lamp.

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3.2 IFE between the Fe(II)-phenanthroline complex and mPD-CDs

It is interesting that, upon addition of Fe²⁺, the colourless solution of the mixture of mPD-CDs and phenanthroline gradually became rufous, and the colour was getting deeper and deeper, which even can be detected by naked eyes (Fig. 4a). Hence, Fe²⁺ formed a rufous complex with phenanthroline (Fe(II)-phenanthroline) without interference from mPD-CDs. A broad peak centered at 510 nm is observed in the absorption spectrum of the Fe(II)-phenanthroline complex, while no obvious peaks are found in the absorption spectra of individual Fe²⁺ or phenanthroline (Fig. 4b). Interestingly, the addition of mPD-CDs negligibly affected the absorption of the Fe(II)-phenanthroline complex (Fig. 4b). A colorimetric method based on the formation of the Fe(II)-phenoline complex would not be affected by the presence of mPD-CDs. On the other hand, the absorption spectrum of the Fe(II)-phenanthroline complex is overlapping with the excitation and emission spectra of mPD-CDs peaked at 440 and 516 nm, respectively, resulting in an IFE on the fluorescence of mPD-CDs. Thus, the fluorescence of mPD-CDs was significantly quenched with the addition of Fe²⁺ into the mPD-CDs/phenanthroline system. However, individual Fe²⁺ or phenanthroline did not affect the fluorescence of mPD-CDs, suggesting there was no interaction between Fe²⁺ (phenanthroline) and mPD-CDs (Fig. 4b).

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Fig. 4 (a) The photographs of the mPD-CDs/phenanthroline system up addition of Fe²⁺ at various concentrations in the range of 0.0–100.0 μM. (b) The absorption spectra of Fe²⁺, phenanthroline and the Fe(II) – phenanthroline complex in the absence and presence of mPD-CDs; The excitation spectrum of mPD-CDs; The fluorescence spectra of mPD-CDs in the absence and presence of Fe²⁺, phenanthroline and the Fe(II) – phenanthroline complex.

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Subsequently, fluorescence lifetimes were measured to confirm IFE of the Fe(II)-phenanthroline complex on the fluorescence of mPD-CDs. As shown in ***Fig. S1, phenanthroline nearly exerted no effect on the fluorescence lifetime of mPD-CDs, while the Fe(II)-phenanthroline complex influenced the fluorescence lifetime of mPD-CDs slightly since the lifetime decreased from 5.57 to 5.05 ns with the concentration of Fe²⁺ at 100 μM ***(Table S2). Therefore, the decrease in the fluorescence of mPD-CDs induced by the Fe(II)-phenanthroline complex is due to static quenching.

3.3 Colorimetric detection of Fe²⁺

Upon addition of Fe²⁺, the absorbance of the Fe(II)-phenanthroline complex at 516 nm gradually increased (see Fig. 5a). A linear relationship between the absorbance at 516 nm and the Fe²⁺ concentration was well calibrated in the range of 1.0–60.0 μM with a linear equation of A = 0.0122 + 0.0106c (R² = 0.9992, see Fig. 5b). The LOD was estimated to be 2.98 μM based on 3σ/s. Meanwhile, the solution colour changing from colourless to rufous and was getting deeper. When the concentration of Fe²⁺ reached 3.0 μM, an obviously rufous colour could be differentiated with naked eyes (see Fig. 4a).

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Fig. 5 (a) The absorption spectra of the mPD-CDs (0.025 mg/mL)/phenanthroline (0.18 mM) system upon addition of Fe²⁺ from 1.0 to100.0 μM, incubated at ambient temperature for 5 min. (b) The linear relationship between absorbance and the concentration of Fe²⁺ in the range of 1.0 to 60.0 μM.

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3.4 Fluorimetric detection of Fe²⁺

A fluorimetry built for the detection of Fe²⁺ is possible since there is an IFE of the Fe(II)-phenanthroline complex on the fluorescence of mPD-CDs. Its selectivity was surveyed by collecting the fluorescence spectra of mPD-CDs and the mPD-CDs/phenanthroline system in the presence of potential interferences, including Al³⁺, Ba²⁺, Ni²⁺, Mn²⁺, Ca²⁺, Cd²⁺, Cu²⁺, Li⁺, Na⁺, Co²⁺, Mg²⁺, Fe³⁺, K⁺, Cr³⁺, Zn²⁺, Ag⁺, Hg²⁺, Pb²⁺ and Fe²⁺. As shown in ***Figure S2, all the metal ions exerted negligible effect on the fluorescence intensity of the mPD-CDs. However, Fe²⁺ significantly quenched the fluorescence of the mPD-CDs/phenanthroline system while the other metal ions showed insensitivity to the mPD-CDs/phenanthroline system except that Fe³⁺ and Cd²⁺exhibited slight quenching effect (Fig. 6a). As shown in ***Figure S3, EDTA will not affect the absorption of the Fe(II)-phenanthroline complex. Hence, 150.0 μM of EDTA as masking agent was contained in the the mPD-CDs/phenanthroline system to improve the selectivity of the fluorimetric method to Fe²⁺.

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Fig. 6 (a) The responses of the mPD-CDs/phenanthroline system toward different metal ions (200.0 μM) in HAc-NaAc buffer solution (0.05 mol/L, pH 6.0). (b) The effect of the ratio of Fe²⁺ to phenanthroline on the fluorescence quenching of mPD-CDs. (c) The fluorescence spectra of mPD-CDs/phenanthroline system upon addition of Fe²⁺. (d) The linear relationship between (F₀-F)/F₀ and the concentration of Fe²⁺ in the range of 0.0–50.0 μM.

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Moreover, mPD-CDs exhibited the strongest emission at pH6.0 (Fig. 3a), the fluorescence of mPD-CDs did not suffer from temperature (Fig. 3c), the balance of the fluorescence quenching of mPD-CDs by the Fe(II)-phenanthroline complex was reached very soon ***(Figure S4), and the fluorescence of mPD-CDs were quenched to the maximum at the ratio (Fe²⁺ to phenanthroline) of 1:3 (Fig. 6b). Therefore, pH6.0, room temperature, 5 min and the ratio of Fe²⁺ to phenanthroline at 1:3 were selected as the optimal condition for the fluorometric method. Under this condition, the fluorescence intensity of mPD-CDs/phenanthroline was gradually quenched with an increase in the concentration of Fe²⁺ (Fig. 6c). Accordingly, two excellent linear relationships (R² = 0.9958) were built between (F₀ - F)/F₀ and the concentration of Fe²⁺ in the range from 0.0 to 25.0 and 25.0 to 50.0 μM under excitation at 440 nm. The linear regression equations were calibrated as (F₀ - F)/F₀ = 0.00850 + 0.01621c and (F₀ − F)/F₀ = 0.16805 + 0.00938c (c represents the concentration of Fe²⁺), and the LOD was estimated to be 0.59 μM (Fig. 6d). Therefore, a fluorimetric method for the detection of Fe²⁺ with higher sensitivity was developed.

A comparison between the colorimetric and fluorimetric dual mode method with the previous methods for the detection of Fe²⁺ was performed in ***Table S3. The LOD and linear range of the present dual mode method for the detection of Fe²⁺ are comparable or even better than those of the other methods previously reported on the basis of CDs.

3.5 Detection in real sample

Due to the higher sensitivity and selectivity, the fluorimetric method based on the mPD-CDs/phenanthroline system was directly applied for the detection of Fe²⁺ in real samples. In this work, lake water, tap water and human blood samples were applied as the real samples to assess the feasibility of the mPD-CDs/phenanthroline system for the detection of Fe²⁺. All the samples were pretreated by 0.45 μm Supor filters to remove any suspension, and then spiked with Fe²⁺, respectively. Finally, the spiked Fe²⁺ was recovered by the proposed method. As presented in Table 1, the recoveries for the selective detection of Fe²⁺ were in the range of 96.3–104.7% (Table 1). Although the components of the real samples are very complicated and they may interfere with the measurements, the spiked Fe²⁺ can be recovered from these samples with accuracy, which indicates that the proposed method is of high selectivity.