18β-Glycyrrhetinic acid derivative-based metallo-hydrogels with highly selective and sensitive for histidine detection

2.1 Materials

18β-Glycyrrhetinic acid (GA), 6-trifluoromethylpyridine-3-formaldehyde, and all amino acids were obtained from Sigma-Aldrich Ltd. (America). Europium nitrate, cerous nitrate, lanthanum nitrate, and terbium nitrate were procured from Promega Biotech Co., Ltd. (Beijing, China). All other chemicals used were of analytical grade.

2.2 Apparatus

The fluorescence images were recorded by the FS5 Fluorescence spectrometer (Edinburgh, UK). Fourier transforms infrared (FT-IR) spectra were conducted with an IRAffinity-1S FT-IR spectrometer (Shimadzu, Japan). Scanning electron microscopy (SEM) image was obtained using an FE-SEM 5000 electron microscope (Hitachi, Japan). The test of rheological properties was conducted with MCR 702 MultiDrive Rheometer (Anton Paar, Austria). Energy dispersive spectrometer (EDS) elemental mapping was obtained by using Bruker EDS QUANTAX (Bruker, Germany). The powder X-ray diffraction (PXRD) image was obtained with D8 VENTURE QUEST XRD (Bruker, Germany). Brunauer Emmett Teller (BET) data was measured by using BSD-660 Physical Adsorption Analyzer (BSD, China). The surface contact angel was measured by CA200 automatic type optical contact angle measuring instrument (Beidou, China).

2.3 Preparation of metallo-hydrogels

The method of synthesizing GA-O-09 (Scheme S1) has been described in detail in previous study (Guo et al., 2021b). Firstly, GA-O-09 (15.00 mg) was dissolved in ethanol (0.52 mL) to form a GA-O-09 ethanol solution. Then, the solution was heated at 50 °C for 1 min with ultrasonic simultaneously. Secondly, one equivalent of the europium nitrate (10.71 mg), cerous nitrate (10.42 mg), lanthanum nitrate (7.80 mg), and terbium nitrate (8.28 mg) were dissolved in ultrapure water (0.48 mL) respectively, to afford four kinds of aqueous solutions containing rare earth metal ions. Finally, the four kinds of aqueous solution were added dropwise into the GA-O-09-ethanol solution, respectively, and the metallo-hydrogels were obtained.

2.4 Preparation of amino acids-loaded metallo-hydrogels

lysine (Lys), Asparagine (Asn), histidine (His), cysteine (Cys), tryptophan (Trp), arginine (Arg), valine (Val), aspartic acid (Asp), tyrosine (Tyr), leucine (Leu), glutamic acid (Glu), methionine (Met), alanine (Ala), isoleucine (Ile), glutamine (Gln), threonine (Thr), proline (Pro), glycine (Gly), phenylalanine (Phe) or serine (Ser), of the same equivalents of GA-O-09 were dissolved in ultrapure water (0.48 mL), respectively, and formed different solutions containing amino acids and GA-O-09. The hydrogels were constructed by repeating the above steps of the metallo-hydrogels.

2.5 The pH stability test of the hydrogels

Firstly, one equivalent of the rare earth metal nitrates was dissolved in ultrapure water (0.48 mL) and the desired pH values (range from 1 to 12) were achieved by adding trace amounts of 0.01 M HCl and 0.01 M NaOH solutions. Then the aqueous solution (0.52 mL) was added dropwise into the GA-O-09-ethanol solution entirely to form the hydrogels, the pH stability of the hydrogel was determined by inverting the test tube.

2.6 The thermodynamic stability test of the hydrogels

A series of GA-O-09/M³⁺ (M³⁺ = Ce³⁺, Tb³⁺, Eu³⁺ and La³⁺) hydrogels were prepared in 3 mL sample bottles, respectively. Then, placed these hydrogels in a water bath heating device for heating gradually and stopped until the gel collapsed, and recorded the concentration of hydrogel and the sol–gel phase transition temperatures (T_gel).

2.7 His detection in human urine samples

Firstly, three healthy volunteers were randomly selected and the then fresh urine was collected from them (Wang and Fan, 2018). The urine was filtered through the membrane and the filtrate was mixed with acetonitrile (5:1, V/V) to precipitate the proteins, Then, the supernatant was collected after being centrifuged at 5000 r/min for 5 mins, and diluted 100 times with 0.01 M PBS buffer. Subsequently, the above steps for the preparation of metallo-hydrogels were repeated to prepare the GA-O-09/M³⁺ hydrogels with different diluted urine solutions instead of ultrapure water.

3.1 Preparation and characterization of the metallo-hydrogels

The GA-O-09/M³⁺ (M³⁺ = Ce³⁺, Tb³⁺, Eu³⁺ and La³⁺) hydrogels were prepared and characterized by the SEM, FT-IR, BET, and Surface contact angel test. As shown in the SEM images (Fig. 1a-1d and S1), the GA-O-09/M³⁺ hydrogels (Fig. 1a-1d) existed with more compact orderly rod structures than the GA-O-09 hydrogel (Fig. S1) after being freeze-dried, it was attributed to the strong coordination between metal ions and pyridine group in GA-O-09, and the metal ions served the hydrogel network more compact orderly rod structures (Guo et al., 2021b; Guo et al., 2022). The FT-IR image (Fig. 1e and S2) displayed the absorption peaks of the carbonyl group (C = O) in GA-O-09 moved from 1740 and 1650 cm⁻¹ to 1720 and 1640 cm⁻¹ after the self-assembled hydrogels formed. These results also indicated that hydrogen bonds might be formed between the carbonyl groups and the molecule of water in these assembly gels (Liu et al., 2019c; Guo et al., 2022). Moreover, the absorption peaks did not change between GA-O-09 and GA-O-09/M³⁺ hydrogels as shown in Fig. 1e, which also suggested that the hydrogen bonds were undistorted. BET test (Table S1) indicated that the BET surface area of the GA-O-09 hydrogel (14.57 m²/g) was lower than the GA-O-09/Eu³⁺ (17.68 m²/g) and GA-O-09/La³⁺ (17.81 m²/g) hydrogels and the average pore diameter the GA-O-09 hydrogel (16.08 nm) was higher than the GA-O-09/Eu³⁺ (15.21 nm) and GA-O-09/La³⁺ hydrogels (15.48 nm), which may due to the more compact orderly hydrogel network in GA-O-09/M³⁺ hydrogels than in GA-O-09 hydrogel (Maiti et al., 2004). Surface contact angel test (Fig. S3) displayed that there is no significant change in the water contact angle of GA-O-09 hydrogel and GA-O-09/M³⁺ hydrogels (M³⁺ = Eu³⁺ and La³⁺), all of them were around 130°, which also indicated that they were hydrophobic. (Yang et al., 2022).

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Fig. 1

The SEM images of (a) GA-O-09/Eu³⁺; (b) GA-O-09/La³⁺; (c) GA-O-09/Tb³⁺; (d) GA-O-09/Ce³⁺ hydrogels; (e) the FT-IR spectra of GA-O-09 powder, GA-O-09 and GA-O-09/Eu³⁺ hydrogels; (f) the EDS elemental mapping of GA-O-09/Eu³⁺ hydrogel.

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Besides, the GA-O-09/M³⁺ (M³⁺ = Eu³⁺, La³⁺) hydrogels were chosen to make the energy dispersive spectrometer (EDS) elemental mapping images and powder X-ray diffraction (PXRD) images. Fig. 1f and S4 showed that the main elementals (C, O, N, F, Eu and La) were distributed in hydrogels uniformly and obviously. Therefore, it was inferred that the lanthanide ions were well incorporated within the gel network. The powder XRD images (Fig. S5) showed the peaks of GA-O-09/Eu³⁺ at around 2θ = 14.98° and 17.81°, and the peaks of GA-O-09/La³⁺ at around 2θ = 14.95° and 17.81°, respectively (Calvo-Castro et al., 2016; Son et al., 2018), which was indicated that the π–π stackings that yield between pyridine groups in GA-O-09 were the main driving forces for the self-assembly of hydrogels. Furthermore, it was also implied that GA-O-09/M³⁺ (M³⁺ = Eu³⁺, La³⁺) hydrogels were formed with amorphous phase structures. (Liu et al., 2019c; Guo et al., 2021a; Guo et al., 2021b).

In addition, as shown in Fig. 2, the GA-O-09/M³⁺ (M³⁺ = Eu³⁺, La³⁺) hydrogels were chosen for the rheological experiment to investigate viscoelasticity. The hydrogels exhibited a typical gelatinous behavior since Fig. 2a showed G′ > G″ when the strain amplitude ranged from 0.1 to 100% (Fan et al., 2021; Zeng et al., 2022). When the G′ < G″ at strains that are higher than 100%, the network of hydrogels had been disrupted. The frequency scan (Fig. 2b) also showed the G′ > G″ at the frequency ranging from 0.1 to 100 rad/s which confirmed the gel-like behavior of the hydrogels. Besides, the complex viscosity (η ∗ ) scan (Fig. 2c) reflected that the negatively correlated relationship between complex viscosity and frequency, and the stress–strain curves (Fig. 2d) showed that the hydrogels can withstand at 16 kPa at the strain of 40% approximately.

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Fig. 2

The rheology test of the GA-O-09/M³⁺ (M³⁺ = Eu³⁺ and La³⁺) hydrogels: (a) amplitude scan; (b) frequency scan; (c) complex viscosity (η ∗ ) scan; (d) stress–strain curves.

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3.2 The stability of the GA-O-09/M³⁺ hydrogels

The pH stability of the GA-O-09/M³⁺ hydrogels has been studied in a range values of 1 to 12 (Su et al., 2022). Fig. 3 showed that the GA-O-09/M³⁺ (M³⁺ = Tb³⁺, Ce³⁺, Eu³⁺ and La³⁺) hydrogels exhibited pH stability during the range of 3–7, 4–10, 3–10, and 3–10, respectively, which indicated that the excellent pH stability of these metallo-hydrogels except the GA-O-09/Tb³⁺ hydrogel.

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Fig. 3

The stability of the GA-O-09/M³⁺ hydrogels at varied pH values.

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Subsequently, the sol–gel phase transition temperatures (T_gel) were obtained by the test tube inversion method (Liu et al., 2019c) to explore the thermodynamic stability of GA-O-09/M³⁺ hydrogels. As listed in Table 1, the values of T_gel were increased with increasing the concentration of GA-O-09/M³⁺ within a certain range (from 12.5 to 17.0 mg·mL⁻¹), and the T_gel of GA-O-09/M³⁺ (M³⁺ = Eu³⁺, La³⁺, Tb³⁺ and Ce³⁺) hydrogels were 66.2 ± 0.75 °C, 67.4 ± 0.49 °C, 62.4 ± 0.49 °C, and 62.6 ± 0.49 °C, respectively, in which the concentration of GA-O-09 was 17.0 mg·mL⁻¹. Interestingly, the T_gel tended to be more/less stable when the concentration of GA-O-09/M³⁺ higher than that of 17.0 mg·mL⁻¹. Besides, the minimum gel concentrations of these hydrogels were calculated and illustrated in the Table 1 ranging from 1.25 to 1.30 wt%, which were close to the reported super hydrogels (Liu et al., 2019c).

Moreover, Fig. S5 displayed the gel–sol phase change and Van’t Hoff plot diagram (Javed et al., 2022), the thermodynamic parameters were calculated in formulas Equations (1) and (2) that can be expressed as:

Where T = 298 K, R = 8.314 J·mol⁻¹·K⁻¹, $\mathrm{\Delta }$ H was obtained according to the slope of Van’t Hoff plot, $\mathrm{\Delta }$ S was obtained according to the intercept, $\mathrm{\Delta }$ G was obtained according to Gibbs function.

The comprehensive interpretation of Fig. S6, showed that the ΔG values of the GA-O-09/M³⁺ (M³⁺ = Eu³⁺, La³⁺, Tb³⁺ and Ce³⁺) hydrogels were 11.43, 12.27, 12.40, and 12.42 kJ·mol⁻¹, respectively. The comparison indicated that the GA-O-09/M³⁺ hydrogels had excellent thermodynamic stability with the positive values of the ΔG, and also the lowest ΔG values revealed the reason why the T_gel of GA-O-09/Eu³⁺ hydrogel (67.4 ± 0.49 °C) was higher than others at a concentration of 17.0 mg·mL⁻¹, which meant the GA-O-09/Eu³⁺ hydrogel displayed the best thermodynamics stability.

3.3 The fluorescence detection of amino acids-loaded GA-O-09/M³⁺ hydrogels

The GA-O-09 and GA-O-09/M³⁺ hydrogels were prepared and remained in an inverted test glass cuvette. Then their fluorescence intensity was recorded at 300 nm due to both GA-O-09 and GA-O-09/M³⁺ (M³⁺ = Eu³⁺, La³⁺, Tb³⁺ and Ce³⁺) hydrogels having the strongest UV absorption peaks around this wavelength (Fig. S7a). The fluorescence emission spectra of the GA-O-09 and GA-O-09/M³⁺ hydrogels exhibited a maximum peak of around 600 nm upon excitation at 300 nm.

As shown in Fig. 4a and 4b, a series of amino acids-loaded GA-O-09/M³⁺ hydrogels were constructed with then their changes in fluorescence intensity were verified during the selective recognition of amino acids. The remarkable fluorescence enhancements of the His-, Lys-, and Arg-loaded GA-O-09/Eu³⁺ hydrogels were observed. Interestingly, both the His-loaded GA-O-09/Eu³⁺ hydrogel and the His-loaded GA-O-09/Eu³⁺ hydrogel displayed the most remarkable fluorescence enhancement upon excitation at 300 nm. Besides, the specific selectivity of the GA-O-09/M³⁺ (M³⁺ = Eu³⁺, La³⁺) hydrogels for His was further verified by competitive experiments (Fig. 4c, 4d). 1.0 equiv. His and 1.0 equiv. competitive amino acids were loaded to the hydrogels, respectively. The corresponding fluorescence intensities of hydrogels indicated that there was no obvious interference to recognize the His selectively while other competitive amino acids were presented.

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Fig. 4

Photographs of various amino acids-loaded (a) GA-O-09/Eu³⁺ and (b) GA-O-09/La³⁺ hydrogels under 365 nm UV lamp irradiation and the corresponding fluorescence intensity of hydrogels upon excitation at 300 nm; Photographs of (c) GA-O-09/Eu³⁺ and (d) GA-O-09/La³⁺ hydrogels loading 1.0 equiv. His and 1.0 equiv. coexisting amino acids, respectively, under 365 nm UV lamp irradiation and the corresponding fluorescence intensity of hydrogels upon excitation at 300 nm.

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Meanwhile, 365 nm UV lamp was treated as the most common detection wavelength, which was attempted to irradiate the GA-O-09 and GA-O-09/M³⁺ hydrogels loaded with histidine in this study according to the previous study (Liu et al., 2019c). Interestingly, although 365 nm was not the optimal absorption wavelength for detection, the GA-O-09 and GA-O-09/M³⁺ hydrogels emitted green fluorescence (Fig. S7b) and the fluorescence emission color altered from faint green to pink, purple, and cyan while the GA-O-09/Eu³⁺ hydrogel loaded 1.0 equiv. His, Lys, and Arg, respectively. However, when His, Arg, and Lys were added to GA-O-09/La³⁺ hydrogel, the fluorescence emission color only altered from faint green to cyanine, but the His-loaded GA-O-09/La³⁺ hydrogel also displayed the most remarkable fluorescence enhancement than the others.

3.4 The mechanism of selective recognition for His

The possible mechanism of GA-O-09/M³⁺ hydrogels selective to recognize His (Fig. 5a) was explored. Fig. 5b showed that the hydroxyl group (–OH) of the GA-O-09/Eu³⁺ hydrogels shifted to 3421, 2956 cm⁻¹ from 3380, 2950 cm⁻¹, and the carboxyl group (C = O) of the GA-O-09/Eu³⁺ hydrogels shifted to 1726, 1647 cm⁻¹ from 1720, 1670 and 1640 cm⁻¹, respectively. Besides, it was worth noting that the hydroxyl group shifted to 3440, 2964 cm⁻¹ and the carboxyl group shifted to 1732, 1649 cm⁻¹ when the GA-O-09/Eu³⁺ hydrogels loaded His. Fig. 5c also suggested that the hydroxyl group and carboxyl group shifted to 3437 and 2958 cm⁻¹, 1728 and 1649 cm⁻¹ while the GA-O-09/La³⁺ hydrogels loaded Arg and Lys, and shifted to 3446, 2964 cm⁻¹, and 1728 cm⁻¹, 1649 cm⁻¹ while the GA-O-09/La³⁺ hydrogels loaded the His. In summary, the FT-IR characterization indicated that the hydrogen-bonding interactions and electrostatic interactions were generated between carboxylic acid groups and carbonyl groups on the GA-O-09 and the amino group on His, Arg, and Lys. Interestingly, His, Arg, and Lys were all the basic amino acids and the hydrogen-bonding interactions and electrostatic interactions were the major driving force that caused electron transfer between these basic amino acids and GA-O-09/M³⁺. Besides, the covalent affinity between the unique imidazole group on His and the pyridine group on the GA-O-09/M³⁺ caused further electron transfer and remarkable fluorescence changes, in which the His-loaded GA-O-09/M³⁺ (M³⁺ = Eu³⁺, La³⁺) hydrogels displayed more remarkable fluorescence enhancement than the others.

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Fig. 5

(a) The possible mechanism of the hydrogels’ selective recognition for His; Infrared spectra of (b) GA-O-09/Eu³⁺ and GA-O-09/La³⁺ hydrogels loaded His, Arg, and Lys, respectively; (c) GA-O-09/La³⁺ and GA-O-09/Eu³⁺ hydrogels loaded His, Arg and Lys, respectively; the SEMs of (d) the His-loaded GA-O-09/Eu³⁺ hydrogel; (e) the Arg-loaded GA-O-09/Eu³⁺ hydrogel; (f) the Lys-loaded GA-O-09/Eu³⁺ hydrogel; (g) the His-loaded GA-O-09/La³⁺ hydrogel; (h) the Arg-loaded GA-O-09/La³⁺ hydrogel; (i) the Lys-loaded GA-O-09/La³⁺ hydrogel.

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As displayed in Fig. 5d-5i, the amino acid particles were attached to the GA-O-09/M³⁺ (M³⁺ = Eu³⁺, La³⁺) xerogels, respectively, and the His-, Arg-, Lys-loaded GA-O-09/M³⁺ (M³⁺ = Eu³⁺, La³⁺) xerogels possessed layered and agminated structures, which were different from the nano rod-like structures of the GA-O-09/M³⁺ (M³⁺ = Eu³⁺, La³⁺) xerogels. Meanwhile, the coordination interactions between the GA-O-09/M³⁺ (M³⁺ = Eu³⁺, La³⁺) and amino acids were further supported by the SEM characterization.

3.5 The sensitivity study of His detection

To evaluate the detection sensitivity of the GA-O-09/M³⁺ (M³⁺ = Eu³⁺, La³⁺) hydrogels for amino acids, His was selected in this study for that more remarkable fluorescence enhancement was observed when the His was loaded by GA-O-09/La³⁺ and GA-O-09/Eu³⁺ hydrogels. Fig. 6 and S8 showed the fluorescence intensity was enhanced upon excitation at 300 nm, and 365 nm respectively, and the position of the emission peak shifted from short wavelength to long wavelength (red-shifted) while the hydrogels loaded 1.0 equiv. His. The insets in Fig. S8a and S8b displayed corresponding emission colors change of hydrogel under 365 nm UV lamp irradiation.

Furthermore, the fluorescence titration experiments were investigated to monitor the changes in the fluorescence intensity for different equiv. His (ranging from 0.0 to 1.0 equiv.) loaded by GA-O-09/M³⁺ (M³⁺ = Eu³⁺, La³⁺) hydrogels, respectively (Fig. 6c, 6d). The results indicated that the fluorescence was enhanced with the increasing of the His concentration, which further confirmed the existence of electron transfer between His and GA-O-09/M³⁺ and which caused the change of the emission colors of hydrogel to change. In addition, the LODs was calculated by using the 3 σ/m method, where σ is the standard deviation of the blank and m is the slope of the calibration plot. (Committee, 1987; Nafaâ et al., 2003; Hua et al., 2017; Lin et al., 2019b; Liu et al., 2019a), and the calculation formulas Equations S1 to S4 were represented in the supplementary material.

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Fig. 6

Comparison of fluorescence intensity between (a) GA-O-09/Eu³⁺ and (b) GA-O-09/La³⁺ hydrogels prepared with 1.0 equiv. of His or without His upon excitation at 300 nm, respectively. The fluorescence intensity of (c) GA-O-09/Eu³⁺ and (d) GA-O-09/La³⁺ hydrogels which were added with different equivalents of His upon excitation at 300 nm, respectively; the fluorescence titration linear range and the LODs of (e) GA-O-09/Eu³⁺ and (f) GA-O-09/La³⁺ hydrogels loading His.

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As shown in Fig. 6e and 6f, the LODs of GA-O-09/Eu³⁺ and GA-O-09/La³⁺ hydrogels for His were 1.14 × 10^-8 M and 1.07 × 10^-8 M, respectively. Compared to the calculated LODs reported (Table 2), the hydrogels displayed more remarkable selectivity and sensitivity than the others.

Furthermore, the pH and thermodynamic stability of the His-loaded GA-O-09/M³⁺ (M³⁺ = Eu³⁺, La³⁺) hydrogels were explored. The result indicated the His-loaded hydrogels still exhibited excellent pH stability over a range of 4–10 and 3–9, respectively (Fig. S9), and the thermodynamic stability were not significantly affected with the addition of His (Table S2).

3.6 The determination of His in human urine samples

To further evaluate the practical value of this study, His of human urine samples were detected and analysed (Gu et al., 2018; Gu and Cao, 2018). As shown in Table 3, the concentration of His in the tested urine sample was 834 μM (the concentrations of Founds in Table 3 multiplied by 100), which was within the normal range of His in human urine (the normal level of His was 130–2100 μM in urine) (Sun et al., 2012b). Meanwhile, a series of known concentrations (5, 10, 20, and 50 μM) of His were the spiked respectively to the samples. The result showed that the high recoveries of spiked His ranged from 99.7% to 103.8% and the low relative standard deviations (RSDs) were between 0.8% and 1.9%, which indicated that this detection method could be successfully applied for His determination in human urine sample.