Highly Sensitive Detection of Iron Ions in Aqueous Solutions Using Fluorescent Chitosan Nanoparticles Functionalized by Rhodamine B.

Detection of iron ions in aqueous solutions is of significant importance because of their important role in the environment and the human body. Herein, a fluorescent rhodamine B-functionalized chitosan nanoparticles probe is reported for the efficient detection of iron ions. The chitosan nanospheres-rhodamine B (CREN) was prepared by grafting rhodamine B onto the surface of chitosan nanospheres through an amidation reaction. The as-prepared CREN fluorescent probes exhibit high fluorescence intensity under ultraviolet light. When iron ions are added to the CREN solution, they can be coordinated with weak-field ligands such as N and O on the surface of chitosan nanoparticles (CSNP) by a high-spin method. The self-assembly of Fe3+ on the surface of the CREN led to the generation of single electrons and the presence of high paramagnetism, resulting in fluorescence quenching. The quenching effect of Fe3+ on the CREN fluorescent probe can achieve the efficient detection of Fe3+, and the detection limit reaches 10-5 mol/mL. Moreover, this fluorescence quenching effect of Fe3+ on the CREN fluorescent probe is specific, which could not be disturbed by other metal ions and counteranions.

Introduction

Water resources, as an indispensable portion for human survival, occupy an extremely important position in human life and processes.[1] However, with the rapid development of industry and economic society, the generation of plenty of industrial, agricultural, and domestic wastewater has caused serious pollution on water sources. In particular, contamination of water with heavy-metal ions poses significant threats to ecosystems, destroying biological diversity and threatening human processes and life.[2] Moreover, the pollution of surface water has gradually changed from single heavy metals to multiple heavy metals.[3−7] When wastewater containing heavy metals is discharged into the environment, the self-purification ability of soil and water is not enough to treat these heavy metals. Throughout the food chain, these heavy metals will cause potential health hazards due to their migration and accumulation in the environmental water system.[8−10]

The iron ion, as an important heavy-metal ion in human processes and life, plays an important role in the environment and the human body.[11] For example, Fe3+ plays a key role in metabolic processes and constitutes an important element of proteins in organisms. Moreover, the lack and imbalance of Fe3+ in the body will cause anemia, leading to heart diseases and cancer. So, when people drink contaminated water sources containing Fe3+, abnormal levels of Fe3+ will damage the normal operation of the human body. Moreover, iron ions also play a crucial role in environmental processes such as limiting the phytoplankton primary productivity in water environments. Therefore, establishing simple and effective methods for the detection of Fe3+ in aqueous solutions is essential due to its significant role in the environment and human health. Recently, various methods including spectrophotometry, voltammetry, inductively coupled plasma mass spectrometry, atomic absorption spectrometry, etc. have been used to detect iron ions.[12−22] However, these detection methods are easily interfered by other metal ions, limiting their applications in the detection of Fe3+. Fluorescence analysis is the use of fluorescence signals to record, store, and transmit information to identify analytes by fluorescence.[13−26] The sensitivity of a fluorescence-based technology is mainly reflected in the difference in fluorescence intensity before and after analyte binding, while the selectivity is realized by the interaction between recognition units and receptors.[27] Lately, fluorescence approaches have gained huge attention for metal ion detection due to their rapid response and high sensitivity and selectivity. Various small molecular fluorescence probes have already been used to detect Fe3+ sensitively, among which rhodamine B exhibited great promising potential due to high fluorescence quantum yield and good light stability. For example, Sun et al. prepared a rhodamine–pyridine-conjugated fluorescent probe for Fe3+, exhibiting obvious “turn-on” fluorescence response.[14] However, the application of rhodamine B for the detection of Fe3+ in aqueous solutions is limited by its low sensitivity and poor solubility.

Chitosan is the only natural alkaline polysaccharide obtained from partial deacetylation of chitin, which is antibacterial, biocompatible, and renewable.[28,29] Chitosan is hydrophilic and has a large number of hydroxyl and amino groups, allowing it to strongly interact with metal ions while providing the sites for numerous attractive chemical modifications.[30,31] Especially, there are plenty of N and O in chitosan chains, which are hard bases, and iron ions are hard acids. According to the soft and hard acid–base theory,[32,33] iron ions are easily combined with O and N. Therefore, the introduction of more N and O elements can greatly improve the ability of fluorescent probes to identify and detect iron ions when preparing fluorescent probes.[34−38] Thus, the combination of chitosan and fluorescent small molecules may be an effective method for preparing fluorescent probes for the detection of Fe3+ in aqueous solutions with high sensitivity and selectivity.

In this work, we presented a new sensitive chitosan nanospheres-rhodamine B (CREN) fluorescent probe by grafting rhodamine B (RB) fluorophores to the surfaces of chitosan nanoparticles (CSNP). The prepared CREN was uniformly spherical with a diameter of about 180 nm, which was able to detect Fe3+ sensitively in an aqueous medium. In this sensing platform, iron ions can be coordinated with weak-field ligands such as N and O on the surface of CSNP by a high-spin method. The chelation of Fe3+ on the surface of the CREN led to the generation of single electrons and the presence of high paramagnetism, which resulted in fluorescence quenching, allowing the CREN to detect Fe3+ more sensitively. The CREN fluorescent probe was simple and accurate, providing a good method for the detection of Fe3+ based on the fluorescence quenching effect.

Results and Discussion

Synthesis and Characterization of Chitosan Nanospheres-Rhodamine B

Figure a illustrates the synthesis of a chitosan nanospheres-rhodamine B (CREN) fluorescent probe and the mechanism of Fe3+ detection with the as-prepared CREN. The chitosan nanospheres-rhodamine B was prepared by grafting rhodamine B onto the surface of chitosan nanospheres through an amidation reaction with the assistance of the activator EDC/NHS (Figure b). The CREN solution exhibited strong fluorescence intensity under the excitation of ultraviolet light. According to the soft and hard acid–base theory,[27] iron ions can easily combine with nitrogen and oxygen. When iron ions are added to the CREN solution, these weak-field ligands of CREN such as nitrogen and oxygen can be coordinated with iron ions by a high-spin method, resulting in the generation of single electrons and at the same time the presence of high paramagnetism, leading to fluorescence quenching. Thus, the CREN fluorescent probe can realize the detection of Fe3+ with high sensitivity and selectivity based on the fluorescence quenching effect.

Figure 1

Schematic diagram of the chitosan nanospheres-rhodamine B (CREN) synthesis and detection mechanism. (a) Schematic diagram of the process of CREN synthesis and the detection of Fe3+. (b) Synthetic route of rhodamine B-functionalized chitosan.

First, the chitosan nanospheres (CSNP) were fabricated by oxidative degradation of chitosan and subsequent cross-linking via sodium tripolyphosphate (TPP). The Fourier transform infrared (FT-IR) spectra (Figure S1) of chitosan under different degradation times indicate that the degradation procedure did not affect the chemical components of chitosan, and a low-molecular-weight chitosan with the same chemical components as the original chitosan can be obtained. Subsequently, the obtained low-molecular-weight chitosan under different degradation times was used to prepare chitosan nanospheres by TPP cross-linking, and TEM images (Figure S2) of these chitosan nanospheres show that the prepared chitosan nanospheres were in good shape and uniform in size when using low-molecular-weight chitosan degraded for 9 h, which was used in the following experiments. Micrographs of the dried CSNP characterized by SEM are shown in Figure a. It could be seen that CSNP had relatively uniform sizes and exhibited an aggregation tendency. Moreover, the corresponding mappings of C, N, and O elements (Figure b–e) exhibited even distribution, indicating that the CSNP were evenly cross-linked by TPP. The TEM image in Figure f clearly shows that the prepared chitosan nanospheres were uniformly spherical, and the diameter of the nanospheres was about 180 nm. Chitosan has two different crystal morphologies of the monoclinic system: form-I (2θ = 10°) and form-II (2θ = 20°).[35] The crystal structures of the original chitosan and CSNP were characterized by XRD, as shown in Figure g. It was found that the original chitosan had only one obvious diffraction peak at 20.18°. After oxidative degradation, the diffraction peak of CSNP at 20.18° decreased, and a new diffraction peak appeared at 10.10°, indicating that oxidative degradation led to the formation of new crystals. Furthermore, the particle size distribution of CSNP is shown in Figure h. Under experimental conditions, the diameter of chitosan nanoparticles was about 180 nm with a small dispersion coefficient and uniform particle size distribution.

Figure 2

(a,b) SEM images of chitosan nanospheres; corresponding EDX elemental mapping images of (c) O, (d) C, and (e) N in chitosan nanospheres; (f) TEM image, (g) XRD patterns, and (h) size distribution of chitosan nanospheres.

Based on the successful preparation of chitosan nanoparticles above, the CREN was synthesized by an amidation reaction. The FT-IR spectra of CSNP, Rh B, and CREN are shown in Figure . Compared with CSNP, the bending vibration of N–H at 1650 cm–1 was weakened, and the bending vibration of −OH at 950 cm–1 disappeared in CREN compared with Rh B, all of which indicated that rhodamine B was successfully grafted to the surface of chitosan nanospheres by amido bonds. The morphology of CREN was analyzed and characterized by SEM and TEM tests. From the SEM image in Figure a and the corresponding elemental distribution in Figure b,–e it could be seen that the CREN was agglomerated, and the block shape consisted of plenty of nanoparticles. Figure f shows the TEM image of the CREN. The prepared CREN was almost spherical and monodisperse with a particle size of about 180 nm.

Figure 3

FT-IR spectra of CSNP, Rh B, and CREN.

Figure 4

(a) SEM image of CREN; corresponding EDX elemental mapping images of (b) C, (c) O, (d) N, and (e) P in CREN; (f) TEM image of CREN.

Recognition of Metal Ions by the CREN Fluorescent Probe

First, the CREN solution was tested to see if there was multiexcitation and multiemission. The excitation wavelengths were set to be 365, 390, 415, 440, 445, 465, 490, 505, 515, and 530 nm, and the corresponding spectrum was tested. As shown in Figure a, the CREN fluorescence probe exhibited a single emission under multiple excitations, and the fluorescence emission peaks of different excitation wavelengths were all around 590 nm. The excitation wavelength was selected to be 505 nm for the following experiments due to the strongest fluorescence emission intensity at this wavelength. Subsequently, different concentrations (10–9–10–1 mol/L) of Fe(NO3)3 solutions were prepared and mixed with the CREN solution to study the fluorescence quenching. From the UV–vis absorption spectrum in Figure b, it could be seen that the mixed solution has a wide absorption peak at 500–575 nm and the absorption intensity gradually decreased when the concentrations of the Fe(NO3)3 solution increased from 10–5 to 10–2 mol/L. According to the UV–vis absorption spectrum, the fluorescence intensity of the solution was also tested, as shown in Figure c. Due to paramagnetism, the fluorescence intensity decreased with the increase in the Fe3+ concentration. The relationship between the fluorescence intensity and Fe3+ concentration is shown in Figure d. When the concentration of Fe(NO3)3 was 10–5 mol/L, the fluorescence intensity began to decrease, and the fluorescence intensity was almost zero when the concentration of Fe(NO3)3 reached 10–2 mol/L. The detection limit and the linear range of the CREN fluorescent probe are 10–5 mol/L 10–4–10–2 mol/L, respectively. These results demonstrated that the CREN could be selected as a fluorescent probe for the sensitive detection of Fe3+ based on the fluorescence quenching effect.

Figure 5

(a) Fluorescence spectra of CREN under the different excitation wavelengths. (b) Ultraviolet spectra and (c) fluorescence spectra of the CREN solution mixed with the Fe(NO3)3 solution with different concentrations. (d) Fluorescence intensity changes (590 nm) as a function of the Fe(NO3)3 concentration.

In order to study the antianion interference ability of CREN for the detection of Fe3+, different iron salts such as FeCl3 and Fe2(SO4)3 were prepared, and the fluorescence measurements were performed. When the Fe(NO3)3 solution was changed to the FeCl3 solution with the same concentration gradients, UV–vis absorption spectroscopy and fluorescence spectroscopy were carried out under the same test conditions, as shown in Figure S3a,b. It was found that the absorption intensity of the mixed solution at 500–575 nm also decreased with the increase in the FeCl3 concentration. Moreover, the fluorescence intensity of the mixed solution gradually decreased with the concentrations of FeCl3 increasing. According to the relationship of the fluorescence intensity and the concentration of the FeCl3 solution in Figure S3c, it could be observed that the fluorescence intensity exhibited high responsiveness when the concentration of the FeCl3 solution ranged from 10–5 to 10–2 mol/L, which was similar to the responsiveness of the fluorescence intensity to the Fe(NO3)3 solution concentration. By comparison, it was speculated that the change in the fluorescence intensity of CREN was related to the chelation of Fe3+ with CREN. In order to further explore whether the fluorescence quenching of CREN was related to Fe3+ or anions, the UV–vis absorption spectrum and the fluorescence spectrum of the mixture solution of Fe2(SO4)3 and CREN were also tested (Figure S4). The changes in the fluorescence intensity with the Fe2(SO4)3 concentration exhibited a similar change trend to that of Fe(NO3)3 and FeCl3. The three solutions of Fe(NO3)3, FeCl3, and Fe2(SO4)3 have similar fluorescence quenching effects on CREN. The above results confirmed that the anions in the Fe3+ salt solutions had almost no effect on the quenching effect, and the CREN fluorescent probe could achieve the highly efficient detection of iron ions with a wide detection range of 10–5 to 10–2 mol/L. The possible fluorescence quenching mechanism could be ascribed to that there was plenty of weak-field ligands such as nitrogen and oxygen on the surface of chitosan nanoparticles, and the iron ions can be easily coordinated with these weak-field ligands by a high-spin method, which resulted in the generation of single electrons and high paramagnetism, leading to fluorescence quenching of CREN.

Anti-interference Performance of the CREN Fluorescent Probe

The fluorescence quenching effect of Fe(NO3)3, FeCl3, and Fe2(SO4)3 on the CREN fluorescent probe was similar, and the anions in the Fe3+ salt solution had little effect on the quenching effect. Furthermore, we selected iron salts with different valences to explore the selective detection of the CREN fluorescent probe for Fe3+. For this purpose, a ferrous sulfate solution with different concentrations ranging from 10–9 to 10–1 mol/L was prepared and was mixed with the CREN solution to react. As shown in Figure a, there was a wide absorption peak at 500–575 nm for Fe2+, which was similar to that of Fe3+. According to the ultraviolet absorption spectrum, the fluorescence spectrum of the solution was tested, as shown in Figure b. The fluorescence intensity decreased with the increase in the Fe2+ concentration due to paramagnetism, but this change was inconspicuous. Moreover, the fluorescence intensity was still very strong when the Fe2+ concentration reached 10–1 mol/L. Therefore, the CREN fluorescence probe was more suitable for identifying and detecting Fe3+.

Figure 6

(a) Ultraviolet spectra and (b) fluorescence spectra of the CREN solution mixed with the FeSO4 solution with different concentrations.

In order to explore the interference effect of other metal ions on Fe3+ detection, various typical metal ions such as Mg2+, Na+, Ni2+, Cu2+, Ag+, Al3+, Hg2+, K+, and Mn2+ with the same concentration were selected to react with the CREN fluorescence probe. As shown in Figure a, although the ultraviolet–visible absorption spectra of mixed solutions of these metal ions and CREN were similar to that of Fe3+, they exhibited a wide absorption peak at 500–575 nm. However, the fluorescence intensity of the mixed solutions of these metal ions and CREN under UV light was nearly the same with that of the original CREN solution, and the fluorescence intensity exhibited a large decrease only when Fe3+ was mixed with the CREN solution. Figure c shows the optical photo of solutions in which different metal ions were uniformly mixed with CREN under visible light, and the solution marked with a yellow arrow in the figure was the solution containing ferric ions. Under visible light, the color of the solution containing Fe3+ was slightly darker. When these solutions were observed under ultraviolet light, other solutions had obvious fluorescence, while the solution containing Fe3+ had no obvious fluorescence (Figure d), which proved that Fe3+ had an obvious fluorescence quenching effect on CREN. Figure e shows the images of the solutions in the fluorescence gel imaging system. The solution marked by the yellow arrow was the Fe3+ solution. In the solution containing 12 kinds of metal ions, only the solution containing Fe3+ had no obvious fluorescence. Similarly, imaging of the fluorescence gel system also proved that the fluorescence quenching effect of Fe3+ on CREN was unique. To sum up, the CREN fluorescent probe had a strong anti-interference ability and could specifically identify Fe3+.[39−44]

Figure 7

Anti-interference test. (a) Ultraviolet spectra and (b) fluorescence spectra of various cations mixed with CREN. Macrographs of mixture solutions of CREN and different metal ions (the yellow arrow in the figure indicates the solution containing ferric ions) under (c) visible light and (d) ultraviolet light and (e) in the fluorescence gel imaging system. Photograph courtesy of Zhiwei Liu. Copyright 2021.

Conclusions

In summary, we have presented the successful synthesis of chitosan nanospheres-rhodamine B (CREN) by grafting rhodamine B (RB) fluorophores to the surfaces of chitosan nanoparticles (CSNP) as a fluorescence quenching sensor for the detection of Fe3+. The as-prepared CREN was uniformly spherical with a diameter of about 180 nm. Plenty of N and O elemental groups in the CREN were found to be the key to the adsorption of Fe3+. The adsorbed Fe3+ led to the generation of single electrons and the presence of high paramagnetism, causing the decrease in fluorescence intensity. The quenching effect of Fe3+ on the CREN fluorescent probe can achieve the sensitive detection of Fe3+ in the concentration range of 10–5–10–2 mol/L. Moreover, the detection of the CREN fluorescent probe for iron ions is specific without being affected by the addition of various metal ions and counteranions. The CREN fluorescence probe designed in this work was simple to prepare and easy to operate, which could be applied to the identification and detection of iron ions in aqueous solutions.

Materials and Methods

Materials

Chitosan (CS, deacetylation degree of ≥95%) and rhodamine B were purchased from Aladdin Co., Ltd. (Shanghai, China). Hydrochloric acid (HCl), sodium hydroxide (NaOH), and metal ion salts such as iron trichloride hexahydrate (FeCl3·6H2O), nickel nitrate hexahydrate (Ni(NO3)2·6H2O), sodium nitrate (NaNO3), manganese nitrate tetrahydrate (Mn(NO3)2·4H2O), aluminum nitrate nonahydrate (Al(NO3)3·9H2O), iron nitrate nonahydrate (Fe(NO3)3·9H2O), iron sulfate (Fe2(SO4)3), potassium chloride (KCl), mercury nitrate (Hg(NO3)2), zinc chloride (ZnCl2), and ferrous sulfate heptahydrate (FeSO4·7H2O) were provided by the North Tianjin Chemical Reagent Factory (Tianjin, China). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxy-succinimide (NHS) were purchased from Sigma-Aldrich Trading Co., Ltd. Deionized water used in all the experiments was obtained using a Milli-Q ultrapure water purification system.

Preparation of Chitosan Nanospheres

Oxidative degradation was first used to obtain low-molecular-weight chitosan. Briefly, 3 g of chitosan was dissolved in 90 mL of a 3% acetic acid solution followed by addition of 30 mL of a 1.5% H2O2 solution. The mixture solution was heated to 40 °C under mechanical stirring for 9 h. At the end of the reaction, the mixture solution was poured into a 5 mol/L sodium hydroxide solution to precipitate the degraded chitosan. The obtained low-molecular-weight chitosan was centrifugally washed with ultrapure water to neutrality and dried in vacuum for later use. The chitosan nanospheres were prepared according to the method of a previous report.[34] Low-molecular-weight chitosan (0.05 g) was dissolved in 10 mL of a 1% acetic acid solution. Then, the pH value of the solution was adjusted to about 4.6 using 5 mol/L NaOH. The chitosan nanoparticles (CSNP) can be formed by adding dropwise 3 mL of 0.25% sodium tripolyphosphate (TPP) into the low-molecular-weight chitosan solution under magnetic stirring and then centrifuging and freeze-drying.

Preparation of Chitosan Nanoparticles-Rhodamine B

First, the rhodamine B (Rh B) solution was prepared by adding 0.01 g of Rh B into 10 mL of ultrapure water under magnetic stirring at room temperature without light. Then, 0.04 g of N-hydroxy-succinimide (NHS) and 0.05 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added to the Rh B solution. After magnetic stirring for 30 min, 0.2 g of chitosan nanoparticles and 30 mL of ultrapure water were added to the above solution, and the reaction mixture was stirred at room temperature in the dark for 48 h. Centrifugal washing was carried out to remove unreacted Rh B and other impurities, and the chitosan nanoparticles-rhodamine B was obtained by freeze-drying, which was named as CREN.

Characterization

Field-emission scanning electron microscopy (FE-SEM) (S-4800II, Hitachi, Japan) was used to observe the surface morphology of all samples. The morphology structures of the samples were studied by transmission electron microscopy (TEM) (HT7700, Hitachi, Japan). The infrared spectra measured by Fourier infrared spectroscopy (Nicolet Corporation, America) through the KBr sheet method were used to analyze the structure and composition of the samples. The UV–vis spectra and fluorescence spectra of the liquid samples were obtained using a Shimadzu UV-2550 system (Shimadzu Corporation, Japan).

Fluorescence Determination Procedures

The detection of Fe3+ based on the fluorescence quenching effect was carried out in an aqueous solution at room temperature. In the test, different metal salt solutions were mixed with a small amount of CREN and left to react. For the fluorescence test, the excitation wavelengths were set to be 365, 390, 415, 440, 445, 465, 490, 505, 515, and 530 nm, and the intensity of fluorescence was measured at the scanning range of 575–620 nm in a 1 cm quartz cell with a slit width of 5 nm. Different concentrations of iron ions were detected and analyzed using the same method.