Introduction

Metal ion fluorescent probes have received extensive attention due to their wide application in environmental and biochemical fields [1]. Fe3+ ion is one of the essential ions for life. Many cell-level biochemical processes require the participation of iron ions [2, 3]. Therefore, the research on the detection of iron ions is of great significance in the environment and life sciences. The lack or excess of Fe3+ can induce many diseases, such as diabetes, hepatitis, anemia and Parkinson’s disease [4,5,6], so it is very important to detect Fe3+. Commonly used detection methods are colorimetry [7], atomic absorption spectrometry [8, 9], electrothermal atomic absorption spectrometry [10], cold vapor atomic absorption spectrometry [11], inductively coupled plasma emission spectrometry [12, 13], spectroscopy [14,15,16] and anodic stripping voltammetry [17], among them, fluorescence spectroscopy has the advantages of high sensitivity and simple operation, so it has attracted widespread attention, and many have been designed and synthesized probe [18].

Pyrazoline is an important nitrogen-containing five-membered heterocyclic compound. There are many methods to synthesize this compound [19,20,21] with many biological activities [22,23,24,25,26,27,28]. Pyrazoline has strong fluorescence properties, so it is also widely used in the field of optoelectronics. It is used as an optical brightener for textiles and papermaking materials. The pyrazoline fluorescent compound also has excellent stability, so it can be used in laser dyes, fluorescent dyes, for biological analysis, tracking detection, drug tracking and sun traps. Pyrazole is a colorless or white crystalline substance with high fluorescence quantum yield, high light stability, non-toxic and other optical properties, it is usually used as stabilizer, chelate, organic synthesis intermediate, etc [29,30,31]

Due to the excellent properties of pyrazole and pyrazoline, we designed and synthesized compound M that combines the advantages of both. This compound contains two fluorophores, a pyrazole unit and a pyrazoline unit. It has good selectivity and high-sensitivity fluorescence response to Fe3+. In order to clarify the complex relationship between the probe and Fe3+, theoretical calculations of density functional theory (DFT) were carried out. These results illustrate the reasonable design of fluorescent probes for Fe3+ detection. At the same time, the probe M has good yellow fluorescence, its emission wavelength is about 600 nm, the fluorescence quantum yield is high, the hole transport efficiency is high, it is easy to detect, and the color change can be directly observed with the naked eye. And it is also a good photoluminescent material. In addition, the resulting M-Fe3+ group reacts specifically with pyrophosphate (PPI) anion through fluorescence quenching.

Experimental Part

Materials and Characterization

FA1004 Electronic Balance (Changzhou Nuoji Instrument Co., Ltd.); SGWX-4 Micro-melting Point Instrument (Shanghai Precision Scientific Instrument Co., Ltd.); Bruker-AV400, 400 MHz Nuclear Magnetic Resonance Instrument; HPLC Q-Tof HR-MS Mass Spectrometer (Waters Micromass); Bruker DTX-400 carbon spectrum analyzer; UV-1800PC spectrometer; HITACHIF-4500 fluorescence spectrometer.

During the experiment, various metal ions correspond to chloride salts (Na+, K+, Ca2+, Cu2+, Zn2+, Ni2+, Pb2+, Mg2+, Al3+, Ba2+, Fe3+, Co2+, Mn2+) and nitrate (Ag+), Other reagents are commercially available analytical grade, and all samples are performed at room temperature.

Synthesis Route of Probe M

Scheme 1 shows the synthetic route of compound M.

Scheme 1
scheme 1

Synthesis route of probe M

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The synthesis of probe M is as follows: o-hydroxyacetophenone was purchased from a commercial supplier. Compound a was prepared according to literature method [32] m.p.140–143 °C(lit [32]. m.p.144–146 °C), in 50 ml round-bottom flasks, add 0.210 g (1 mmol) of compound a, 0.136 g (1.0 mmol) of o-hydroxyacetophenone, 10 ml of ethanol and 1 ml of 10% NaOH at 75 °C was heated to reflux for 3 h. After the reaction was completed, it was cooled to room temperature to produce a bright yellow powder. The crude product was obtained by suction filtration. Recrystallized with V ethanol: V ethyl acetate (1:1) to obtain compound b. The rate is 88%. Dissolve 0.366 g (1.0 mmol) of compound b in 2 ml of acetic acid, completely dissolve it and transfer to an oil bath (120 °C), then add 1.0 ml of 80% hydrazine hydrate dropwise, heat for 4 h, and detect the completion of the reaction by TCL. After cooling to room temperature, white crystal M was produced with a yield of 78%, with a yield of 78%, mp: 230–233 °C The analysis data are as follows: 1H NMR (CDCl3, 400 MHz) δppm 2.39(s, 3H),3.21(dd, J = 4.1, 4.0 Hz, 1H),3.71(d, J = 4.0 Hz,2H),5.84(m, 1H),6.87(t, J = 4.0 Hz, 1H),7.01(s, 2H),7.25(s, 1H),7.33(d, J = 3.1 Hz, 2H),7.41(dd, J = 5.1, 1.2 Hz, 3H),7.69(dd, J = 4.0, 2.3 Hz, 4H),7.81(s, 1H), 10.18(s, 1H).13C NMR (DMSO-d6, 100 MHz):167.93,156.59,156.79,154.00,150.46,146.86,139.70,136.81,132.93,131.40,128.59,126.99,125.84,119.69,119.14,117.45,116.99,115.73,114.99,112.44,65.43,50.86,47.31,42.41,22.64,22.13. calculated for [M + H]+ 423.2411, found423.2497.

Spectral Test

The M mixed stock solution was prepared 1.0 × 10−3 M. The configuration of the cation stock solution is prepared from the corresponding chloride or nitrate. Make volume with soft water, the concentration is 5.0 × 10−3 M. Add 10 μL, 20 μL of Compound M solution (1 × 10−3 M) to the cell, with an optical path of 1 cm, which contains 2 mL of THF/water (1:1 (v/v) and HEPES buffer (0.02 mM pH = 7.4), then add a certain amount of cation, and measure its UV-visible absorption, fluorescence spectrum and infrared spectrum.

Results and Discussion

Identification of Metal Ions by Probes

In the THT/water system, under the excitation wavelength of 280 nm, the probe showed strong fluorescence emission at 410 nm. The experimental results show that metal ions other than iron ions have little effect on the fluorescence of the system or have a very small fluorescence quenching effect (Fig. 1). This shows that probe M has a better response to iron ions.

Fig. 1
figure 1

Fluorescence emission spectrum of M in THF / water mixed solvent (1: 1, v / v) containing HEPES buffer (0.02 mM, pH = 7.4) at room temperature

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Fluorometric Titration Test of Iron Ion by Probe

Add a different multiple of iron ions to the probe in a THF/water (1:1: v/v) solution containing HEPES buffer (0.02 mM, pH = 7.4), as shown in Fig. 2a, as the iron ion concentration as the factor increases, the fluorescence intensity of probe M decreases continuously until it reaches saturation. The quantitative response of probe M to Fe3+ has a good linear relationship. As shown in Fig. 2b, the linear regression equation is y = 640.19–350.28x, R2 = 0.9902. The Benesi-Hildebrand nonlinear curve fits a straight line, showing a linear fit, and confirms that the stoichiometric relationship between Fe3+ and probe M is 1:1. From Fig. 2c, we can conclude that the binding constant of the M-Fe3+ complex is 2.6 × 105 M−1. The detection limit calculated from the multiple of the fluorescence intensity of the probe M relative to the standard deviation is 3.9 × 10−10 M (when the probe concentration is 1.0 × 10−5 M). Table 1 lists several probes for Fe3+ detection. Compared with the LOD of other probes, M probe has a lower detection line and a higher Fe3+ detection sensitivity.

Fig. 2
figure 2

a Fluorescence emission intensity curve of adding different concentrations of Fe3+ to probe M. b The fluorescence spectrum of the emission spectrum of probe M and the change in Fe3+ concentration. c Complexation constant curve of Fe3+ to probe M titration

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Table 1 Detection limits of Fe3+ by other fluorescent sensors and fluorescent probe M
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Competitive Testing

In order to further detect the actual application of the probe M for the recognition of Fe3+, an anti-interference experiment was performed on the probe M (Fig. 3a). Only adding iron ions to the probe will significantly reduce the fluorescence intensity, and then add other equal amounts of metal ions. The fluorescence intensity remains basically unchanged. The results prove that the probe M does not interfere with other ions during the recognition process of Fe3+, and has good anti-interference ability. In addition, in order to further explore the influence of anions on the identification process of iron ions in probe M, we configured iron sulfate ion, sulfate ion and chloride ion solutions in HEPES buffer (0.02 mM, pH = 7.4) THF /water Fluorescence spectroscopy tests were carried out in water (1:1: v/v) solution, as shown in Fig. 3b, the fluorescence response of the probe did not change significantly. The results show that in the process of identifying metal salt ions, anions will not affect the identification of metal ions.

Fig. 3
figure 3

a In the presence of Fe3+ and various other metal ions in a mixed solvent of THF/ water (1:1, v/v) containing HEPES buffer (0.02 mM, pH = 7.4) at room temperature Next, the fluorescence response of probe M. The black bar indicates the probe M plus other metal ion solutions. The gray bar indicates that the same amount of Fe3+ is subsequently added to the mixture solution. b represents the fluorescence spectrum of probe M in a mixed solvent THF/water (1:1, v/v) containing HEPES buffer (0.02 mM, pH = 7.4) and different iron salts at room temperature

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Selective Features

In order to explore the practical application of probe M in a biological environment at room temperature, the absorption spectrum characteristics of probe L in a mixed solvent containing THF/water (1:1, v/v) for various metal ions were studied. As shown in Fig. 4a, without adding Fe3+ ions, the probe M showed a maximum absorption peak at 250 nm. Then, Fe3+ ions were added to the probe M. We found that it did cause a significant change in absorption, the absorbance at 310 nm increased, and a new band appeared at 310 nm. It can be clearly seen from Fig. 4b that when adding 1 eq Fe3+ and other metal ions (Ba2+, Ca2+, Mg2+, Na+, Al3+, Mn2+, Ni+, Pb2+, Cu2+, Co2+, K+, Zn2+, Ag+, M), only Fe3+ The absorption peak has obvious changes.

Fig. 4
figure 4

a UV-Vis spectra of probes M and M + Fe3+ in THF/water (1:1, v/v) containing HEPES buffer (0.02 mM, pH = 7.4). b After adding various metal ions, the UV-visible spectrum of probe M changes

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UV Absorption Spectrum of Fe3+ Recognized by Probe

In THF/water (1:1, v/v) buffer containing HEPES (0.02 mM, pH = 7.4), as shown in Fig. 5a, adding different concentrations of Fe3+ ions, the probe M starts to absorb at 350 nm. It shows that a new complex is formed between compound M and Fe3+. The UV-vis titration in Fig. 5b shows a good linear relationship between probe M and Fe3+. The linear regression equation is y = 0.022 + 0.8343x, R2 = 0.9946. The spectral fit of the absorption data obtained with Fe3+ titration probe M shows that the stoichiometric ratio between probe M and Fe3+ is 1:1.

Fig. 5a
figure 5

UV absorption spectrum of fluorescent probe M and addition of different equivalents of Fe3+ metal ions. b Linear ultraviolet absorption spectrum of fluorescent probe M and iron ion concentration

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Combination Ratio

In order to further explore the binding ratio of probe M to Fe3+, the molar ratio of probe M to Fe3+ (9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9), to detect its fluorescence properties, and draw the curve shown in Fig. 6, the maximum molar fraction of probe M appears at 0.5, which supports the above 1:1 (M:M + Fe3+) In combination with stoichiometry.

Fig. 6
figure 6

Used to determine the stoichiometry of probes M and Fe3+ in THF/water (1:1, v/v) with HEPES buffer (0.02 mM, pH = 7.4), total concentration 1.0 × 10−5 M Working graph

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Detection Limit

The detection limit is calculated by LOD = 3ơ/k, where & is the standard deviation of the relative fluorescence intensity of the 15 sets of probes at the position of their emission peaks. After probe M is combined with different concentrations of Fe3+, the emission peaks are relative to each other. The intensity is plotted against the corresponding concentration to obtain the slope k. Through calculation, the LOD of the probe M to Fe3+ is 3.9 × 10−10M. Compared with the fluorescent probe for detecting Fe3+ listed in Table 1, the fluorescent probe M made by us has a lower detection limit.

Optimized Calculation

To have a better understanding about the electronic structures of M and M + Fe3+, DFT calculations were performed at B3LYP/def2-SVP [39,40,41]. For the composite M+ Fe3+, HOMO and LUMO are different from M. Due to the addition of Fe3+ in pyrazoline, the HOMO of M + Fe3+ is limited to this fragment, and the CT from the electron donor to the electron acceptor group is interrupted in M + Fe3+, resulting in a decrease in fluorescence. Compared with M, the LUMO-HOMO orbital energy of M + Fe3+ is reduced (Fig. 7), and the acceptor system of the compound is destroyed. This is due to the hydrogen bonding interaction between C=N and C=O.

Fig. 7
figure 7figure 7

Geometric optimization of Gaussian

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Infrared Spectrum Test

Figure 8 shows the FT-IR spectra of probes M and M + Fe3+. The characteristic bands of -OH, pyrazoline C=O and C=N extend at 3430 cm−1, 1664 cm−1 and 1101 cm−1, respectively. After adding probe M, these three bands dropped from 3466 cm−1, 1652 cm−1 and 1287 cm−1 to 3428 cm−1, 1643 cm−1 and 1258 cm−1, which may be due to -OH, C=O and C=N The density of the group and Fe3+ coordination electron cloud decreases [41].

Fig. 8
figure 8

Infrared spectra of probes M and M + Fe3+

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PPI Test Fluorescence Probe M Detection Fe3+ Reversibility Experiment

By adding the chelating agent PPI to the fluorescent probe M to recognize the fluorescence emission peak of Fe3+, we can gain a deeper understanding of the response of the fluorescent probe M to Fe3+, as shown in Fig. 9, when adding PPi to the mixed solution of M and Fe3+ At this time, the fluorescence intensity of the complex almost quenches to the intensity of the fluorescent probe M itself, which shows that the complexation between the fluorescent probe M and Fe3+ is reversible.

Fig. 9
figure 9

Fluorescence spectrum of M combined with Fe3+ when PPI was added to THF/water (1:1, v/v) containing HEPES buffer (0.02 mM, pH = 7.4)

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Recognition Mechanism of Fluorescent Probe M and Fe3+

The possible binding mechanism of M with Fe3+ induced the fluorescence changes is shown in Scheme 2. Based on the previously reported pyrazoline-based probes [42,43,44], we can speculate that Fe3+ may interact with C = N, C = O and -OH on the fluorescent probe M to undergo photoinduced electron transfer and chelation. The cooperation thus weakens the binding of fluorescence [45], but the chelation of probe M with Fe3+ makes the complex more rigid and limits the C = N isomerization [46, 47]. And because the binding ratio of probe M and Fe3+ ions is 1:1, the phenomenon of fluorescence quenching is further confirmed and their binding mechanism.

Scheme 2
scheme 2

The complexation mechanism between probe and ion is speculated

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Conclusion

In summary, a novel fluorescent probe based on pyrazole-pyrazoline for Fe (III) ions recognition was designed and synthesized. By observing the ultraviolet absorption spectrum and fluorescence spectrum, it was found that tetrahydrofuran: water = 1:1 (v: v) in the presence of other metal ions, the probe M in the mixed solution has high selectivity, sensitivity and anti-interference ability for the detection of Fe3+, and the recognition process is reversible. The combination of probe M and iron ions is 1:1, and the detection limit is 3.9 × 10−10 M. The binding mode and sensing mechanism of M with Fe3+ was verified by theoretical calculations using Gaussian 09 based on B3LYP/6-31G(d) basis.