1. Introduction
Hearing loss is a serious and widespread disease affecting more than 1.5 billion people worldwide, more than 400 million of whom have moderate or severe hearing loss [
1]. This disease not only reduces the quality of life but also increases the psychological burden on patients; therefore, it is crucial to prevent and treat hearing loss [
2], [
3], [
4], [
5]. Among the various treatments, a feasible strategy is to reduce the intake of heavy metal ions, such as lead, cadmium, and copper. Copper agents are widely used in agriculture and industry [
6], [
7], [
8], [
9], [
10]. Although copper is an important microelement in the human body, the excess uptake of copper ions (Cu
2+) in the environment and foods often causes severe health problems [
11], [
12], [
13]. Excess Cu
2+ generates hydroxyl radicals and induces apoptosis in hair cells (HCs), resulting in hearing loss [
14], [
15], [
16], [
17], [
18]. Therefore, it is necessary to control Cu
2+ uptake. Monitoring Cu
2+ levels in foods and drinking water is a possible method. Many technologies, including fluorescence [
19], [
20], [
21], spectrophotometry [
22], inductively coupled plasma-atomic emission spectrometry [
23], inductively coupled plasma mass spectrometry [
24], atomic absorption spectroscopy [
25], and electrochemistry [
26], [
27]. Despite their high accuracy and specificity, these techniques are limited by the complex pretreatment of samples and expensive equipment. Therefore, a novel facile, low-cost, and high-specificity platform for Cu
2+ detection is still lacking.
We developed chitosan (CS)-based Cu
2+ ionic imprinted hydrogel bead (IIHB) with an inverse opal structure for the label-free detection of Cu
2+ (
Fig. 1). CS is a natural-derived polymer with abundant functional groups that can form coordination bonds with various cationic ions. Thus, it is widely used in many fields [
28], [
29], [
30], [
31], [
32], [
33], [
34], [
35], [
36]. CS-based materials have been developed for the binding and removal of heavy metal ions [
37], [
38], [
39]. However, their poor selectivity makes them unsuitable for the detection of heavy metal ions. By contrast, imprinted polymers are materials with imprinted sites that can specifically bind to imprinted molecules and ions specifically [
40], [
41], [
42]. Therefore, ionically imprinted polymers (IIPs) are widely used for ion recognition and removal [
43], [
44]. Inverse opals, a type of material with an ordered microporous structure that is generally fabricated by replicating colloidal crystals [
45], [
46], [
47], [
48], [
49], [
50], [
51], [
52], [
53], have shown great potential in sensing, catalysis, and tissue engineering [
54], [
55], [
56], [
57], [
58], [
59], [
60], [
61]. Owing to their unique microstructures, inverse opals exhibit vivid structural colors. In particular, when coupled with responsive hydrogels, the response of the hydrogels to the stimuli may cause a color change, which allows inverse opal hydrogels to be ideal sensors for the label-free detection of target stimuli.
We integrated a CS-based ionic imprinted hydrogel with structural color beads derived from the silica colloidal crystal beads (SCCBs) template to present a label-free detection platform for Cu2+. A mixture of CS, polyethylene glycol diacrylate (PEGDA), and Cu2+ was used as a pregel to replicate the SCCBs. After polymerization of the pregel and removal of the Cu2+ and SCCB templates, IIHBs with inverse opal structures were obtained. The IIHBs exhibited a good degree of sphericity and monodispersity, and were coupled with magnetic nanoparticles to obtain mobility. When the IIHBs were immersed in a solution containing Cu2+, they could recognize and rebind with the ions with high specificity and satisfactory repeatability and showed a shift in the reflective peak related to the concentration. Notably, we demonstrated that excess Cu2+ negatively influenced House Ear Institute-Organ of Corti 1 (HEI-OC1) cells’ activity, revealing the positive significance of Cu2+ detection in hearing loss prevention. Subsequently, an assay of Cu2+ in tap water by spiking confirmed its potential for practical applications. These results suggest that the presented IIHBs are feasible as a facile, low-cost, and high-specificity detection platform for Cu2+; therefore, they are promising indicators for foods and drinking water and prevent hearing loss.
2. Experimental section
2.1. Materials
CS (deacetylation degree 80%–95%, 50–800 mPa·s), ethylenediamine tetraacetic acid disodium (EDTA·2Na), Cu(NO3)2, NaNO3, KNO3, Pb(NO3)2, Mg(NO3)2, Zn(NO3)2, Ca(NO3)2, and Al(NO3)3 were bought from Shanghai Sinopharm Co., Ltd. (China). 2-hydroxy-2-methylpropiophenone photoinitiator (HMPP) and PEGDA were obtained from Sigma-Aldrich (USA). Glutaraldehyde (GA), hydrofluoric acid (HF; 40%, v/v), silicone oil, sodium diethyldithiocarbamate (DDTC·Na), and acetic acid were obtained from Macklin (China). All reagents were of analytical grade or higher and used as received. Water was purified and with a resistivity higher than 18 MΩ·cm.
2.2. Preparation of SCCBs
SCCBs were prepared according to a previously reported method. First, silica nanoparticles were dispersed in water to form homogeneous solutions (20%, w/v). The solution was then pumped into a single-emulsion microfluidic chip and cut into droplets using silicone oil. The droplets were collected in a container containing silicon oil and placed in an oven at 75 °C overnight. Subsequently, n-hexane was used to remove the silicon oil. Finally, the beads were collected in a crucible and calcined in a muffle furnace at 800 °C for 4 h.
2.3. Fabrication of CS-based IIHBs
A solution of CS (2%, w/v), Cu(NO3)2, and PEGDA (15%, w/v), was used as the pregel (20 μL) to infiltrate the SCCBs (number: approximately 200) for 6 h. The system was then polymerized under ultraviolet (UV) light for 10 s and treated with GA for 4 h. Thereafter, the beads were separated from the bulk hydrogel and incubated with EDTA·2Na solution (2%, w/v) for 3 h at a shaker. Finally, the beads were treated with HF (2%, w/v) for 2 h. The NIHBs were fabricated using the same process but without Cu(NO3)2.
2.4. Detection of Cu2+ using IIHBs
The reflection wavelengths of the IIHBs and NIHBs were measured before detection. They were then immersed in 3 mL of Cu(NO3)2 solution (0, 1, 10, 102, 103, 104, 105, and 106 nmol·L−1) for 2 h. Finally, the IIHBs and NIHBs were washed gently, and their reflective wavelengths were measured again. The concentrations of IIHBs and NIHBs used in each group were 5.
2.5. Selectivity of IIHBs
The reflection wavelengths of the IIHBs were measured before detection. They were then incubated in a 3 mL solution containing different types of ions (105 nmol·L−1) for 2 h. Finally, the IIHBs were washed gently and the reflective wavelength was measured again. The IIHBs used in each concentration groups were 5.
2.6. Quantitative analysis of IIHBs
The standard curve of Cu2+ was obtained by mixing the DDTC·Na solution with Cu(NO3)2 solutions at different concentrations for 10 min, and the absorbance of the solution at 452 nm was detected first. Thereafter, 300 μL IIHBs or NIHBs were incubated in 5 mL Cu(NO3)2 solution (200 μmol·L−1) for 3 h, respectively. Subsequently, 50 μL of the supernatant was added to the mixture of 50 μL DDTC·Na solution (400 μmol·L−1) and 100 μL ammonia solution (pH 9.0–9.2) for 10 min. The absorbance of the solution was measured at 452 nm. Each experiment was repeated five times.
2.7. Cytotoxicity of Cu2+ on HEI-OC1 cells
HEI-OC1 cells were co-cultured with the culture medium containing 0, 20, 50, 100, 200, and 300 μmol·L−1 Cu2+ in a 12-well plate with glass coverslips. After incubation for 1 and 6 h, the cells were strained by adding 1 μL·mL−1 Calcein-AM and propidium iodide (PI) into the culture medium and incubated for 30 min at 37 °C. HEI-OC1 cells were added to 96-well plates and cultured in a medium containing 0, 20, 50, 100, 200, and 300 μmol·L−1 Cu2+. After incubation for 1 and 6 h, the cells were treated with the cell counting kit-8 (CCK-8) in accordance with the manufacturer’s instructions, and the absorbance was read using a microplate reader at 450 nm.
2.8. Detection of Cu2+ in tap water
The Cu2+ concentration in tap water was determined using the spiking method. The reflection wavelengths of the IIHBs were measured before detection. Thereafter, they were incubated in a 3 mL solution with different concentrations of Cu2+ (1, 5, 10, 50, and 100 µmol·L−1, respectively) for 2 h. Finally, the IIHBs were washed gently and the reflective wavelength was measured again. The IIHBs used in each concentration group were 5.
3. Results and discussion
In a typical experiment, template SCCBs were prepared by microfluidics [
62], [
63], [
64], [
65], [
66], [
67], [
68], [
69], which is a reliable technology that can generate microparticles and fibers with uniform size [
70], [
71], [
72]. In brief, silica nanoparticles were dispersed in water to form a colloidal solution and used as the inner phase of a single-emulsion microfluidic chip, whereas silicon oil was employed as the outer phase. When the microfluidic system was operated, the inner phase was cut into droplets and collected in a container with silicone oil. After drying and calcination, the nanoparticles self-assembled into the SCCBs. Owing to the precise control of the microfluidics, the SCCBs exhibited good monodispersity (Fig. S1 in Appendix A). To obtain the IIHBs, a pregel solution containing CS, Cu
2+ and PEGDA was used to replicate the microstructure of the SCCBs. It has been demonstrated that CS can bind with Cu
2+ to form a complex structure through coordination bonds and electrostatic interactions, and then crosslink with glutaraldehyde to form a CS hydrogel. However, pure CS hydrogels often suffer from fragility and poor elasticity, as well as sightless color of the resultant IIHBs. Therefore, PEGDA was added to the pregel solution, which formed a soft hydrogel network after polymerization and improved the optical properties of the resulting hydrogel beads. When the SCCBs were incubated in the pregel solution, their nanovoids were filled with the solution, and silica/hydrogel composite beads were obtained after polymerization. Following treatment with EDTA·2Na and HF, the Cu
2+ and silica templates were removed to acquire IIHBs [
73], [
74].
The microstructures of the beads were through scanning electron microscopy (SEM), as shown in
Fig. 2. It could be found that the silica nanoparticles assembled into the close-packed arrangement on the surface of an SCCB (
Fig. 2(a)), and it extended to the inside (
Fig. 2(b)). The packing of the nanoparticles formed many nanovoids within the SCCB, which the hydrogel filled to form silica/hydrogel composite beads (Fig. S2(a) in Appendix A). After removing the templates, the hydrogel-based IIHBs were obtained. However, the poor mechanical strength of the hydrogel often leads to the collapse of the microstructure (Fig. S2(b) in Appendix A). Thus, we used a high-crosslinking agent to form an inverse opal structure. As shown in
Fig. 2(c) and (d), the beads displayed an ordered microporous structure on the surface and inside, indicating successful replication and inheritance of the microstructure of the SCCB.
The unique microstructure of the beads results in a photonic bandgap that inhibits the spread of light at a specific frequency and reflects it to exhibit the corresponding structural colors. In general, the peak position λ of the reflective wavelength can be estimated using the Bragg–Snell law:
where
d refers to the nearest center-to-center distance of the nanoparticles or nanopores, and
naverage refers to the average refractive index of the entire bead. Therefore, by changing
d and
naverage, the structural color of the beads can be tuned. In this study, the components of the beads and the ambient solution environment exhibited negligible changes; thus,
naverage was relatively stable, and the structural color was mainly dependent on
d. As shown in
Fig. 3(a) and Figs. S3 and S4 in Appendix A, silica nanoparticles of different sizes formed SCCBs with different structural colors. Moreover, the composite beads and IIHBs derived from the SCCBs displayed colors corresponding to those of the SCCBs (
Fig. 3(b) and (c)). Generally, the composite beads had the same
d as the SCCBs but a slightly higher
naverage, which led to a slight red shift of the color. In contrast, IIHBs had a lower
naverage and thus displayed an apparent blue shift (
Fig. 3; Figs. S3 and S4).
Notably, the IIHBs had a similar density to that of water. Therefore, to realize fast separation of IIHBs from the solution, IIHBs were functionalized with magnetic nanoparticles to obtain magnetic field-regulated mobility. As shown in
Fig. 4 and Movie S1 in Appendix A, the functionalized IIHBs were initially placed on one side of a dish. When a magnetic field was applied to the other side, the IIHBs displayed excellent responsiveness and moved quickly in the direction of the magnetic field. This result indicates that the IIHBs could be easily enriched and separated from the solution using a magnet, which made it possible to save time in collecting the IIHBs.
When IIHBs were incubated in a solution containing Cu
2+, they bound to the ions and showed changes in the reflective wavelength peak. As shown in
Fig. 5, the IIHBs displayed a red shift in the Cu
2+ solution, and the shift increased with the concentration of Cu
2+. In addition, the shift values of the IIHBs were significantly different from those of non-imprinted hydrogel beads (NIHBs), indicating that the shift was caused by the interaction between the imprinted sites and Cu
2+. Therefore, by simply measuring the reflective wavelength of the IIHBs before and after incubation in the solution, the assay of Cu
2+ concentration could be realized without any labels. Notably, although a higher concentration of Cu
2+ resulted in a larger shift value of the IIHBs, the shift value of the NIHBs also increased in this case, indicating that more non-specific adsorption occurred. To ensure detection accuracy, IIHBs should be used in solutions containing Cu
2+ at concentrations lower than 1 mmol·L
−1.The different binding capabilities of IIHBs and NIHBs to Cu
2+ were further studied by quantitative analysis. DDTC·Na is a sensitive reagent for Cu detection. It can form a brown–yellow complex with Cu
2+ in the ammonia solution with pH 9.0–9.2, and the complex has an apparent peak of absorbance at 452 nm (Fig. S5(a) in Appendix A). The absorbance exhibited a linear relationship with the concentration of Cu
2+ (Fig. S5(b) in Appendix A). Based on this mechanism, we incubated IIHBs and NIHBs in the Cu
2+ solution (100 μmol·L
−1) for 3 h and analyzed the residual concentration of Cu
2+ after the reaction. As shown in
Fig. 6(a) and Table S1 in Appendix A, the IIHBs and NIHBs displayed a wide difference in Cu
2+ adsorption, with an imprinting factor (IF) of 19.14, indicating the good specificity of the developed IIHBs.
In addition, the selectivity of the IIHBs was studied. For this purpose, the IIHBs were treated 100 μmol·L
−1 solution with different cations. The IIHBs showed a large shift value (15.67 nm) in the Cu
2+ solution, whereas the value in the solution of other types of ions was smaller than 4 nm, confirming the accurate selectivity of the IIHBs for Cu
2+ detection (
Fig. 6(b)).
According to previous studies, excess Cu
2+ generates hydroxyl radicals and induce HC apoptosis to lead to hearing loss. Therefore, it is important to monitor Cu
2+ levels in drinking water to control Cu
2+ uptake. Before being applied in practical application, the influence of Cu
2+ on HEI-OC1 cells was studied. As shown in
Fig. 7(a) and (b), HEI-OC1 cells displayed normal growth in the culture medium without Cu
2+. However, when Cu
2+ was added to the medium, cell viability reduced, and this reduction was closely related to the concentration of added Cu
2+. The results of the cell viability assay agreed with those of the live/dead staining (
Fig. 7(c)). These results confirmed that Cu
2+ severely damaged HEI-OC1 cells, indicating that it is necessary to avoid high Cu
2+ intake to prevent hearing loss. To achieve this goal, we used the developed IIHBs to monitor Cu
2+ in food and drinking water. For example, they have been used to assay Cu
2+ in tap water using a spiking strategy. As shown in
Table 1 and Fig. S6 in Appendix A, the IIHBs in the solution displayed a shift in value with the added amount, and the recoveries were acceptable when compared with the standard curve, revealing little Cu
2+ in the tap water. These results indicated that the prepared IIHBs are promising for the Cu
2+ detection of actual samples.
4. Conclusion
In summary, we developed novel CS-based IIHBs with a spherical shape, uniform size, and magnetic responsiveness for the label-free detection of Cu2+ in water to prevent excessive Cu2+ uptake and hearing loss. The CS component could bind with Cu2+ to form imprinting sites, whereas PEGDA improved the mechanical properties of the hydrogels. When IIHBs were incubated in a solution containing ions, they could specifically bind to Cu2+ and showed a redshift in the reflective wavelength peak, which could sense the concentration that could cause damage to HEI-OC1 cells. The applicability of IIHBs was also evaluated by spiking them with tap water. These features indicate that the developed IIHBs are feasible for detecting Cu2+ in water and are promising indicators for food and drinking water to prevent hearing loss.
Acknowledgments
This work was supported by grants from the National Key Research and Development Program of China (2021YFA1101300, 2021YFA1101800, and 2020YFA0112503); the National Natural Science Foundation of China (82030029, 81970882, 92149304, and 22302231); the Science and Technology Department of Sichuan Province (2021YFS0371); the Guangdong Basic and Applied Basic Research Foundation (2023A1515011986); the Shenzhen Fundamental Research Program (JCYJ20190814093401920, JCYJ20210324125608022, JCYJ20190813152616459, and JCYJ20190808120405672); the Futian Healthcare Research Project (FTWS2022013 and FTWS2023080); the Open Research Fund of State Key Laboratory of Genetic Engineering, Fudan University (SKLGE-2104); and the Fundamental Research Funds for the Central Universities, Sun Yat-sen University (23qnpy153).
Authors’ contribution
Huan Wang and Renjie Chai provided the idea and designed the experiment. Huan Wang carried out experiments and data analysis. Huan Wang and Hui Zhang wrote the manuscript. Xiaoli Zhang, Hong Chen, and Ling Lu contributed to the scientific discussion.
Compliance with ethics guidelines
Huan Wang, Hui Zhang, Xiaoli Zhang, Hong Chen, Ling Lu, and Renjie Chai declare that they have no conflict of interest or financial conflicts to disclose.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1016/j.eng.2023.09.001.
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