Fluorescein Derivative Immobilized Optical Hydrogels: Fabrication and Its Application for Detection of H2O2

A novel fluorescein-based probe FLA-Boe was developed for detecting H2O2. Modified by 2-Bromomethylphenylboronic acid pinacol ester, FLA-Boe is a Fluorescein derivative with eminent photostability and remarkable H2O2 sensitivity and selectivity. FLA-Boe was utilized to synthesize hydrogel sensors in the manner of guest–host interaction by taking advantage of its aforementioned features. The hydrogel sensor can be used to detect H2O2 effectively in both flowing and static water environments with satisfactory performance. It is expected that this application may open a new page to develop a neoteric fluorescent property analysis method aiming at H2O2 detection.


Introduction
Hydrogen peroxide (H 2 O 2 ), a typical reactive oxygen species (ROS), functions as a key throughout cell growth, proliferation, host defense, and signal transmission pathways under a physiological environment [1,2]. However, the chemical and pharmaceutical industries inevitably use hydrogen peroxide as an oxidizer for related production activities, resulting in the discharge of excessive hydrogen peroxide in wastewater, which severely impacts people's living environment and the water treatment system [3]. Excessive H 2 O 2 in the human body triggers the pathogenesis of many disorders, such as inflammation, Alzheimer's disease, cardiovascular disease and, more seriously, cancer [4]. Consequently, it is vital to monitor H 2 O 2 in the inner condition and outer environment of the human body [5]. Undoubtedly, the development of H 2 O 2 fluorescent sensors has been arousing people's interest. Thus, the design and fabrication of sensing systems with H 2 O 2 responsive characteristics are key for human health and environmental protection.
Macroscopic supramolecular hydrogels are excellent candidate materials for intelligent sensing activity. In comparison with naturally existing hydrogels, such as polysaccharides and proteins, assorted synthetic hydrogels have been applied as smart materials on account of their functionalization during synthesis. Furthermore, most hydrogels are economical and environmentally friendly with transparent and stretchable features [6,7]. Nowadays, composite materials based on macroscopic supramolecular hydrogels have been investigated for optical detection. Using hydrogel sensors to detect test objects in environmental samples is a handy, easily operated, low-cost, analytical method with remarkable sensitivity [8]. Through research, it is possible to conduct naked-eye observation and direct analysis without spectroscopic devices and apparatus. Hitherto, diverse synthetic strategies using hydrogel sensors have been extensively explored and studied, accompanied by their functionalization procedures, performance control, and potential applications [9][10][11][12][13]. According to experience, device-free sensor systems aiming at colorimetric and fluorometric detection by the naked eye are of the most extensive and applicable utility ad hoc; the aforementioned hydrogel sensors are easily manipulated, as well as featuring eminent sensitivity and high signal-to-noise ratio [14,15]. Furthermore, these materials can be easily and cheaply prepared, and are highly adaptive to various conditions. As mentioned above, cyclodextrin moieties can be incorporated into pre-existing polymeric materials via grafting reactions. Interpenetrated networks can also be used to develop new selective and synergistic sorption capacities for specific purposes, such as the fabrication of a hydrogel sensor. Surmounting the shortcomings existing among traditional fluorescence detection methods, such as monitoring limited, complicated detection processes and man-made mistakes requires the application of hydrogel immobilized sensors with response signals that are adaptive to the living environment and that are able to simplify the detection operation and acquire more convincing and urgently needed detection results [16][17][18][19]. Thus, the development of hydrogel sensors for detecting H 2 O 2 with dual colorimetric and fluorescence signals is of profound significance. Crosslinking and grafting are applied to prepare covalently-attached cyclodextrins [20,21]. The application of a supramolecular sensing system has a promising application prospect [22]. With regard to complexity, functionality and capabilities, the evolution of cyclodextrin polymers matches the modern requirements of macromolecular materials.
In this study, fluorescein-based derivatives are selected as the fluorophores by structural modifications and the phenylboronic acid ester acts as the recognition site for H 2 O 2 . The collected results underpinned the development of a responsive fluorescence probe, in an aqueous medium, that has the potential for discovering hydrogen peroxide in the inner condition and outer environment of the human body. Hence a novel fluorescence probe FLA-Boe was devised and synthesized, which exhibited ideal properties for H 2 O 2 detection. By utilizing this probe, a transparent optical hydrogel sensor with a fluorescence feature for H 2 O 2 sensing was prepared and synthesized; the result remained effective based on this hydrogel sensor after a series of experiments were conducted. After being exposed to H 2 O 2 , the hydrogels were colored purplish red; the intensity was associated with H 2 O 2 concentration. Thus, the hydrogel sensor features selective and integrated detection and monitoring of H 2 O 2 within one system. This effective approach to developing a novel macroscopic hydrogel sensor provides a convenient and intelligent sensing system that is adaptive to complex aqueous environments.

Materials and Instrumentation
Fluorescein and 2-Bromomethylphenylboronic acid pinacol ester were purchased from Aladdin Co., Ltd. (Jinan, China) All chemical reagents reached analytical grade or obtained the highest purity availability, and were free of further purification. The preparation in terms of metal ions solutions was conducted in deionized water. 1 H and 13 C NMR spectra were recorded on a Bruker 400 MHz spectrometer ( 1 H 400 MHz; 13 C 100 MHz, Jinan, China) by using DMSO-d 6 as solvents. FT-IR spectra were obtained on a Thermo fisher Nicolet 6700 FT-IR spectrophotometer (Jinan, China). Q-TOF LC/MS mass spectrometry acted as the recorder for high-resolution mass spectra (HRMS). Shimadzu UV-2600 spectrophotometer was utilized to perform UV-Vis absorption spectra (Jinan, China). Fluorescence spectra were measured on Hitachi F-4600 (Jinan, China) which functions as the gauge of fluorescent intensity.

Synthesis of FLA-Boe
As shown in Scheme 1, Fluorescein (3.3230 g, 1.00 mmol) and hydrazine monohydrate (50%, 3 mL) was dissolved in 100 mL of methanol, after which the mixture was heated to reflux for 4 h. Then the solvent was evaporated, and the resultant residue was subjected to column chromatography (dichloromethane/ethanol = 20/1, v/v) to obtain the intermediate N-(Fluorescein) lactam (2.9611 g, 0.86 mmol). N-(Fluorescein)lactam (3.4634 g, 1.00 mmol) and acetic acid (0.5 mL) were dissolved in 150 mL of acetone and the mixture was heated to 50 • C and stirred for 5 h. The mixture was filtered and the filter cake was washed with acetone and then purified by column chromatography (dichloromethane: ethanol = 30/1, v/v) to obtain the N-(Fluorescein) lactam-N -methylethylidene (2.7814 g, 0.75 mmol). N-(Fluorescein) lactam-N -methylethylidene (3.8640 g, 1.00 mmol) was added into a flask containing the mixture of 4-Bromomethylphenylboronic acid pinacol ester (2.3760 g, 0.8 mmol), K 2 CO 3 (3.0 g), and 20 mL of DMF at 40 • C for 6 h, and the mixture was poured into H 2 O and extracted with ethyl acetate. The organic phase was separated and dried by MgSO 4 , meanwhile, the solvent was removed by vacuum distillation. FLA-Boe was obtained as the white solid after being purified by column chromatography with dichloromethane/ethanol (40/1, v/v) as eluent (2.5295 g, yield = 42%). 1 13

Synthesis of FLA-Boe
As shown in Scheme 1, Fluorescein (3.3230 g, 1.00 mmol) and hydrazine monohydrate (50%, 3 mL) was dissolved in 100 mL of methanol, after which the mixture was heated to reflux for 4 h. Then the solvent was evaporated, and the resultant residue was subjected to column chromatography (dichloromethane/ethanol = 20/1, v/v) to obtain the intermediate N-(Fluorescein) lactam (2.9611 g, 0.86 mmol). N-(Fluorescein)lactam (3.4634 g, 1.00 mmol) and acetic acid (0.5 mL) were dissolved in 150 mL of acetone and the mixture was heated to 50 °C and stirred for 5 h. The mixture was filtered and the filter cake was washed with acetone and then purified by column chromatography (dichloromethane: ethanol = 30/1, v/v) to obtain the N-(Fluorescein) lactam-N′-methylethylidene (2.7814 g, 0.75 mmol). N-(Fluorescein) lactam-N′-methylethylidene (3.8640 g, 1.00 mmol) was added into a flask containing the mixture of 4-Bromomethylphenylboronic acid pinacol ester (2.3760 g, 0.8 mmol), K2CO3 (3.0 g), and 20 mL of DMF at 40 °C for 6 h, and the mixture was poured into H2O and extracted with ethyl acetate. The organic phase was separated and dried by MgSO4, meanwhile, the solvent was removed by vacuum distillation. FLA-Boe was obtained as the white solid after being purified by column chromatography with dichloromethane/ethanol (40/1, v/v) as eluent (2.5295 g, yield = 42%). 1

Synthesis Process of Hydrogel Sensor
The hydrogel sensors were prepared and synthesized by guest-host interaction as shown in Figure 1. At first, Acrylamide (AAm), β-cyclodextrin (β-CD) and

Synthesis Process of Hydrogel Sensor
The hydrogel sensors were prepared and synthesized by guest-host interaction as shown in Figure 1. At first, Acrylamide (AAm), β-cyclodextrin (β-CD) and N,N -methylene bisacrylamide (MBA) (0.3% of monomer content) were mingled together and dissolved in DMSO solution. Then the mixture was stirred under a vacuum at 5 • C until a homogeneous solution was formed. In the next step, 100 µL of TEMED and initiator (APS, 2% of monomer content) were put into the mixture. The mixture was stirred constantly until a homogeneous solution was witnessed during the reaction, which was subsequently poured into a cylindrical mold and maintained at 50 • C for 12 h. In the following step, the hydrogel was extracted and collected from the mold and then rinsed with DMSO for 36 h. AAm-co-β-CD hydrogel was steeped in 10 −2 mol/L of FLA-Boe DMSO solution for 10 h before being dialyzed in H 2 O for 2 h to fabricate the hydrogel sensor in accordance with the host-guest interaction.
Polymers 2022, 14, x FOR PEER REVIEW 4 of 11 N,N′-methylene bisacrylamide (MBA) (0.3% of monomer content) were mingled together and dissolved in DMSO solution. Then the mixture was stirred under a vacuum at 5 °C until a homogeneous solution was formed. In the next step, 100 μL of TEMED and initiator (APS, 2% of monomer content) were put into the mixture. The mixture was stirred constantly until a homogeneous solution was witnessed during the reaction, which was subsequently poured into a cylindrical mold and maintained at 50 °C for 12 h. In the following step, the hydrogel was extracted and collected from the mold and then rinsed with DMSO for 36 h. AAm-co-β-CD hydrogel was steeped in 10 −2 mol/L of FLA-Boe DMSO solution for 10 h before being dialyzed in H2O for 2 h to fabricate the hydrogel sensor in accordance with the host-guest interaction.

Optical Properties of FLA-Boe
The The fluorescence emission property of FLA-Boe toward different test objects (H2O2, NaCl, Na2CO3, NaNO3, NaNO2, Na2S, NaClO, glutathione (GSH), glutamic acid (Glu), histidine (His), levodopa (L-DOPA), lactate (Lac), fructose (Fru), glucose (Glc), urea, alanine (Ala), choline oxidase (ChOx), and glucose oxidase (GOD)) was analyzed and investigated in DMSO and H2O solution (1/1, v/v), as shown in Figure 2b. From the standard solution of FLA-Boe in DMSO and H2O (1/1, v/v), no distinct fluorescence emission was performed. However, as long as H2O2 was added, significant fluorescence emission was displayed, whose emission level was maximized at 526 nm, which could indirectly affect the π-electron push-pull function and resulted in the intramolecular charge transfer (ICT). Additionally, the color change of the solution from transparent to red clearly appeared (Figure 2c). For experiment comparison, other metal ions were added in the solution, and the fluorescence spectra remained unchanged. Hence the result demonstrated eminent fluorescent selectivity of FLA-Boe towards H2O2.  The fluorescence emission property of FLA-Boe toward different test objects (H 2 O 2 , NaCl, Na 2 CO 3 , NaNO 3 , NaNO 2 , Na 2 S, NaClO, glutathione (GSH), glutamic acid (Glu), histidine (His), levodopa (L-DOPA), lactate (Lac), fructose (Fru), glucose (Glc), urea, alanine (Ala), choline oxidase (ChOx), and glucose oxidase (GOD)) was analyzed and investigated in DMSO and H 2 O solution (1/1, v/v), as shown in Figure 2b. From the standard solution of FLA-Boe in DMSO and H 2 O (1/1, v/v), no distinct fluorescence emission was performed. However, as long as H 2 O 2 was added, significant fluorescence emission was displayed, whose emission level was maximized at 526 nm, which could indirectly affect the π-electron push-pull function and resulted in the intramolecular charge transfer (ICT). Additionally, the color change of the solution from transparent to red clearly appeared (Figure 2c

Sensitivity and Selectivity
As shown in Figure 3a, the fluorescence titration experiments were conducted. FLA-Boe solution was incubated by changing the amounts of H2O2 in pH 7.4 HEPES buffer solution. On the basis of the aforementioned investigation, the emission of FLA-Boe is dominated by the intense excimer emission (DMSO/H2O = 1/1, v/v). When the probe was excited at 417 nm, the fluorescence intensity evidently increased at 526 nm after the addition of H2O2. FLA-Boe rapidly responded to H2O2 and the fluorescence intensity was distinctly boosted, which could be caused by the intramolecular charge transfer (ICT) process. Phenylboronic ester in FLA-Boe was cleaved through an oxidative reaction when contacting with H2O2, which resulted in the enhancement of fluorescence intensity. As shown in Figure 3b, the detection limit (DL) was investigated and obtained from the data, reaching 0.22 μM.

Sensitivity and Selectivity
As shown in Figure 3a

Sensitivity and Selectivity
As shown in Figure 3a, the fluorescence titration experiments were conducted. FLA-Boe solution was incubated by changing the amounts of H2O2 in pH 7.4 HEPES buffer solution. On the basis of the aforementioned investigation, the emission of FLA-Boe is dominated by the intense excimer emission (DMSO/H2O = 1/1, v/v). When the probe was excited at 417 nm, the fluorescence intensity evidently increased at 526 nm after the addition of H2O2. FLA-Boe rapidly responded to H2O2 and the fluorescence intensity was distinctly boosted, which could be caused by the intramolecular charge transfer (ICT) process. Phenylboronic ester in FLA-Boe was cleaved through an oxidative reaction when contacting with H2O2, which resulted in the enhancement of fluorescence intensity. As shown in Figure 3b, the detection limit (DL) was investigated and obtained from the data, reaching 0.22 μM.

Competition Experiments
To further check the practical applicability of receptor FLA-Boe as the selective fluorescent sensor for H 2 O 2 , competitive experiments were carried out with 2.0 equiv of H 2 O 2 in the presence of other test objects in an aqueous solution, as shown in Figure 4. It was interesting to note that only H 2 O 2 rendered significant fluorescence turn-on responses among all the tested objects, whereas all the coexistent test objects had no noteworthy variance with regard to fluorescence intensity. These investigated results clearly demonstrated that the sensor FLA-Boe for detecting H 2 O 2 had no interference and could be a good sensor for H 2 O 2 detection in aqueous media.

Competition Experiments
To further check the practical applicability of receptor FLA-Boe as the selective fluorescent sensor for H2O2, competitive experiments were carried out with 2.0 equiv of H2O2 in the presence of other test objects in an aqueous solution, as shown in Figure 4. It was interesting to note that only H2O2 rendered significant fluorescence turn-on responses among all the tested objects, whereas all the coexistent test objects had no noteworthy variance with regard to fluorescence intensity. These investigated results clearly demonstrated that the sensor FLA-Boe for detecting H2O2 had no interference and could be a good sensor for H2O2 detection in aqueous media.

Detection Mechanism of FLA-Boe
The presupposition of the FLA-Boe probe design aims to block the possible ICT process by protecting the hydroxyl group with boronate ester. Hence, Fluorescein was selected as the fluorophore and phenylboronic acid pinacol ester acted as the receptor unit. The inferred detection mechanism of the probe FLA-Boe was recommended in Scheme 2. Due to the protection mechanism of the boronate ester on the hydroxyl group, the ICT process of FLA-Boe was hindered, which demonstrates no fluorescence emission of FLA-Boe was triggered. While FLA-Boe was exposed to H2O2, the decomposition of the boronate ester group was taken and the hydroxyl group was released; then the ICT process of Fluorescein was recovered, through which the strong fluorescence emission from Fluorescein was inevitably triggered. The FT-IR and HRMS tests were investigated to deduce the rational mechanism. In order to verify the response mechanism in detail, the generation of Compound 2-the product of FLA-Boe and H2O2 reaction-was monitored by means of HRMS. As shown in Figure 5a, FT-IR spectrum results confirmed the reasonableness of the aforementioned response mechanism hypothesis; after the addition of the H2O2, a notable hydroxyl peak appeared at around 3250 cm −1 . Moreover, the HRMS result proved that Compound 2 was the product of the FLA-Boe and H2O2 reaction (Figure 5b). The inference concerning the fluorescence response mechanism of FLA-Boe to H2O2 was preliminarily confirmed from these results of the experiments.

Detection Mechanism of FLA-Boe
The presupposition of the FLA-Boe probe design aims to block the possible ICT process by protecting the hydroxyl group with boronate ester. Hence, Fluorescein was selected as the fluorophore and phenylboronic acid pinacol ester acted as the receptor unit. The inferred detection mechanism of the probe FLA-Boe was recommended in Scheme 2. Due to the protection mechanism of the boronate ester on the hydroxyl group, the ICT process of FLA-Boe was hindered, which demonstrates no fluorescence emission of FLA-Boe was triggered. While FLA-Boe was exposed to H 2 O 2 , the decomposition of the boronate ester group was taken and the hydroxyl group was released; then the ICT process of Fluorescein was recovered, through which the strong fluorescence emission from Fluorescein was inevitably triggered. The FT-IR and HRMS tests were investigated to deduce the rational mechanism. In order to verify the response mechanism in detail, the generation of Compound 2-the product of FLA-Boe and H 2 O 2 reaction-was monitored by means of HRMS. As shown in Figure 5a, FT-IR spectrum results confirmed the reasonableness of the aforementioned response mechanism hypothesis; after the addition of the H 2 O 2 , a notable hydroxyl peak appeared at around 3250 cm −1 . Moreover, the HRMS result proved that Compound 2 was the product of the FLA-Boe and H 2 O 2 reaction (Figure 5b). The inference concerning the fluorescence response mechanism of FLA-Boe to H 2 O 2 was preliminarily confirmed from these results of the experiments.

The pH Effect
The influence and phenomenon of the probe FLA-Boe to H2O2 were analyzed in different pH conditions. As shown in Figure 6, within the scale of pH 3.0-4.0, the probe failed to respond notably to H2O2. While FLA-Boe was exposed to H2O2 within the scope of pH 5.0-8.0, the decomposition of the boronate ester group was taken, then the ICT process of Fluorescein was recovered, through which the strong fluorescence emission from Fluorescein was triggered. As a result, the FLA-Boe could respond efficiently to H2O2 in the pH range of 5.0-8.0. The fluorescence intensity reached 581 nm and the response of the probe to H2O2 gradually receded after the pH value was improved to

The pH Effect
The influence and phenomenon of the probe FLA-Boe to H2O2 were analyzed in different pH conditions. As shown in Figure 6, within the scale of pH 3.0-4.0, the probe failed to respond notably to H2O2. While FLA-Boe was exposed to H2O2 within the scope of pH 5.0-8.0, the decomposition of the boronate ester group was taken, then the ICT process of Fluorescein was recovered, through which the strong fluorescence emission from Fluorescein was triggered. As a result, the FLA-Boe could respond efficiently to H2O2 in the pH range of 5.0-8.0. The fluorescence intensity reached 581 nm and the response of the probe to H2O2 gradually receded after the pH value was improved to

The pH Effect
The influence and phenomenon of the probe FLA-Boe to H 2 O 2 were analyzed in different pH conditions. As shown in Figure 6, within the scale of pH 3.0-4.0, the probe failed to respond notably to H 2 O 2 . While FLA-Boe was exposed to H 2 O 2 within the scope of pH 5.0-8.0, the decomposition of the boronate ester group was taken, then the ICT process of Fluorescein was recovered, through which the strong fluorescence emission from Fluorescein was triggered. As a result, the FLA-Boe could respond efficiently to H 2 O 2 in the pH range of 5.0-8.0. The fluorescence intensity reached 581 nm and the response of the probe to H 2 O 2 gradually receded after the pH value was improved to 8.0-9.0. Thus, the phenomenon illustrated that the probe FLA-Boe had eminent fluorescent responses to H 2 O 2 from weakly acidic to weakly alkaline conditions. 8.0-9.0. Thus, the phenomenon illustrated that the probe FLA-Boe had eminent fluorescent responses to H2O2 from weakly acidic to weakly alkaline conditions.

The Fabrication of Hydrogel Sensor
The work noted that the formation of the β-CD-FLA-Boe inclusion complex was distinctly performed by 1 H NMR; meanwhile, the inclusion procedure was demonstrated preliminarily. The 1 H NMR spectrum of β-CD during the appearance and disappearance of FLA-Boe was shown in Figure 7. The integral signal strengths in the 1 H NMR spectrum were investigated. The inclusion of β-CD with FLA-Boe exhibited small changes, and a conspicuous influence was shown on the chemical shifts of protons of β-CD-FLA-Boe, which varied notably ( Table 1), revealing that these protons primarily contributed to the inclusion with FLA-Boe. More supporting evidence regarding the inclusion of FLA-Boe in the central cavity of β-CD was acquired from 1 H NMR studies. In comparisons of the free β-CD and FLA-Boe, the chemical shifts of the inclusion complexes were investigated, demonstrating that FLA-Boe had mutually interacted with β-CD.

The Fabrication of Hydrogel Sensor
The work noted that the formation of the β-CD-FLA-Boe inclusion complex was distinctly performed by 1 H NMR; meanwhile, the inclusion procedure was demonstrated preliminarily. The 1 H NMR spectrum of β-CD during the appearance and disappearance of FLA-Boe was shown in Figure 7. The integral signal strengths in the 1 H NMR spectrum were investigated. The inclusion of β-CD with FLA-Boe exhibited small changes, and a conspicuous influence was shown on the chemical shifts of protons of β-CD-FLA-Boe, which varied notably ( Table 1), revealing that these protons primarily contributed to the inclusion with FLA-Boe. More supporting evidence regarding the inclusion of FLA-Boe in the central cavity of β-CD was acquired from 1 H NMR studies. In comparisons of the free β-CD and FLA-Boe, the chemical shifts of the inclusion complexes were investigated, demonstrating that FLA-Boe had mutually interacted with β-CD. 8.0-9.0. Thus, the phenomenon illustrated that the probe FLA-Boe had eminent fluorescent responses to H2O2 from weakly acidic to weakly alkaline conditions.

The Fabrication of Hydrogel Sensor
The work noted that the formation of the β-CD-FLA-Boe inclusion complex was distinctly performed by 1 H NMR; meanwhile, the inclusion procedure was demonstrated preliminarily. The 1 H NMR spectrum of β-CD during the appearance and disappearance of FLA-Boe was shown in Figure 7. The integral signal strengths in the 1 H NMR spectrum were investigated. The inclusion of β-CD with FLA-Boe exhibited small changes, and a conspicuous influence was shown on the chemical shifts of protons of β-CD-FLA-Boe, which varied notably ( Table 1), revealing that these protons primarily contributed to the inclusion with FLA-Boe. More supporting evidence regarding the inclusion of FLA-Boe in the central cavity of β-CD was acquired from 1 H NMR studies. In comparisons of the free β-CD and FLA-Boe, the chemical shifts of the inclusion complexes were investigated, demonstrating that FLA-Boe had mutually interacted with β-CD.

Detection Performance of the Hydrogel Sensor
The hydrogel sensor responded conspicuously to H 2 O 2 with prominent selectivity and sensitivity due to the role of ICT. To ascertain the capability and effectiveness of the hydrogel sensor, hydrogel detection limits were adjusted and confirmed among varied concentrations of H 2 O 2 solutions, from 10 −4 to 10 −7 mol/L. By observation, the color of the hydrogel sensor could still be notably changed when H 2 O 2 concentration reached 10 −6 mol/L. The color of the hydrogel sensor changed when H 2 O 2 concentration was 10 -4 , 10 −5 , and 10 −6 mol/L for 50 s, 4 min, and 20 min, respectively. In Figure 8, the hydrogel sensor performed visible red light in the aqueous solution environment, which was the same as FLA-Boe's; under 365 nm UV light, the color of the hydrogel sensor in the aqueous solution also conspicuously changed.

Detection Performance of the Hydrogel Sensor
The hydrogel sensor responded conspicuously to H2O2 with prominent selectivity and sensitivity due to the role of ICT. To ascertain the capability and effectiveness of the hydrogel sensor, hydrogel detection limits were adjusted and confirmed among varied concentrations of H2O2 solutions, from 10 −4 to 10 −7 mol/L. By observation, the color of the hydrogel sensor could still be notably changed when H2O2 concentration reached 10 −6 mol/L. The color of the hydrogel sensor changed when H2O2 concentration was 10 -4 , 10 −5 , and 10 −6 mol/L for 50 s, 4 min, and 20 min, respectively. In Figure 8, the hydrogel sensor performed visible red light in the aqueous solution environment, which was the same as FLA-Boe's; under 365 nm UV light, the color of the hydrogel sensor in the aqueous solution also conspicuously changed. A comparison of the results indicates that the Fluorescein derivative immobilized system in this work exhibits better ability for H2O2 detection compared to the other previously reported systems (Table 2). In addition, many of the reported sensors involve poorer reuse performance. On the other hand, the present sensing system is cost-effective, selective, exhibits simple naked-eye sensing, and is suggested to be utilized to detect H2O2 in a real water environment.

Universality of the Hydrogel Sensor
Inspired by the remarkable function of the hydrogel sensor in solution for H2O2, the response of the hydrogel sensor for H2O2 in flowing and static water environments was further investigated. The hydrogel sensor was colored from transparent to light aubergine A comparison of the results indicates that the Fluorescein derivative immobilized system in this work exhibits better ability for H 2 O 2 detection compared to the other previously reported systems ( Table 2). In addition, many of the reported sensors involve poorer reuse performance. On the other hand, the present sensing system is cost-effective, selective, exhibits simple naked-eye sensing, and is suggested to be utilized to detect H 2 O 2 in a real water environment.

Universality of the Hydrogel Sensor
Inspired by the remarkable function of the hydrogel sensor in solution for H 2 O 2 , the response of the hydrogel sensor for H 2 O 2 in flowing and static water environments was further investigated. The hydrogel sensor was colored from transparent to light aubergine (about 6 min) after the sensor was exposed to the flowing H 2 O 2 solution (10 −4 mol/L). The color changed to sharp aubergine after 10 min. In a large container insulated from the atmosphere, the hydrogel sensor was exposed to a static H 2 O 2 solution. An interesting fact was that when the gel was steeped in 10 −7 mol/L H 2 O 2 solution for 3 h, the hydrogel sensor changed color. During the experiment for detection performance, the hydrogel sensor had no response when contacting a small quantity of H 2 O 2 solution whose concentration was no more than 10 −7 mol/L. In accordance with the experimental phenomenon, it was summarized that a cumulative effect may appear in the hydrogel sensor that could result in the reduction of the detection limit to some extent. Thus, analyzing the sensing performance and process of the hydrogel sensor can open a new avenue for developing significant, long continuous, and fast-responding test strips for precisely detecting H 2 O 2 by convenient naked-eye observation, which is of particular importance in the immediate monitoring of H 2 O 2 in both natural aqueous environments and liquid environments around chemical industrial regions. Meanwhile, observing color changes by the naked eye simplifies and speeds up the detection of H 2 O 2 . Therefore, this technological proposal will be a reliable method and a suitable selection for the detection of H 2 O 2 in aqueous environments.

Conclusions
In summary, based on the H 2 O 2 probe FLA-Boe, we developed an easily operated and commonly applicable strategy to synthesize a H 2 O 2 responsive hydrogel sensor with on-off switching and color-changing fluorescence features. Hydrogel sensors were utilized smoothly for the determination of trace amounts of H 2 O 2 in water samples, which indicates that hydrogel sensors are available for use by common people and scientists depending on practical requirements. Additionally, this method has potential applications in establishing novel monitoring and detection mechanisms and systems that cover detection systems for flowing and static water environments, etc. It is expected that this method may provide diverse and novel strategies to exploit smarter systems that include functions, such as synergistic visual detection and efficient sensing.