Construction and Characterization of a Chitosan-Immobilized-Enzyme and β-Cyclodextrin-Included-Ferrocene-Based Electrochemical Biosensor for H2O2 Detection

An electrochemical detection biosensor was prepared with the chitosan-immobilized-enzyme (CTS-CAT) and β-cyclodextrin-included-ferrocene (β-CD-FE) complex for the determination of H2O2. Ferrocene (FE) was included in β-cyclodextrin (β-CD) to increase its stability. The structure of the β-CD-FE was characterized. The inclusion amount, inclusion rate, and electrochemical properties of inclusion complexes were determined to optimize the reaction conditions for the inclusion. CTS-CAT was prepared by a step-by-step immobilization method, which overcame the disadvantages of the conventional preparation methods. The immobilization conditions were optimized to obtain the desired enzyme activity. CTS-CAT/β-CD-FE composite electrodes were prepared by compositing the CTS-CAT with the β-CD-FE complex on a glassy carbon electrode and used for the electrochemical detection of H2O2. It was found that the CTS-CAT could produce a strong reduction peak current in response to H2O2 and the β-CD-FE could amplify the current signal. The peak current exhibited a linear relationship with the H2O2 concentration in the range of 1.0 × 10−7–6.0 × 10−3 mol/L. Our work provided a novel method for the construction of electrochemical biosensors with a fast response, good stability, high sensitivity, and a wide linear response range based on the composite of chitosan and cyclodextrin.


Introduction
Reactive oxygen species (ROS) play an important role in cell signal transduction as a cell signalling molecule, and participate in the initiation of biological effects of various factors. Hydrogen peroxide (H 2 O 2 ) is a representative of ROS. Its concentration is closely related to other ROS [1][2][3][4], such as superoxide anions, hydroxyl radicals, and so on. H 2 O 2 can regulate the cell metabolism of the life system. Low concentrations of H 2 O 2 can act as the second messenger of signal transduction and expansion, while high concentrations of H 2 O 2 may cause damage to cell compositions and organisms, lead to oxidative stress, a variety of diseases, and physiological system disorders [5][6][7]. Therefore, H 2 O 2 is considered as a key factor regulating the apoptosis process. Reliable and sensitive detection of H 2 O 2 in cells is of great significance in physiology and pathophysiology [8,9]. However, the H 2 O 2 detection at the cellular level is limited by many factors, such as small cell size, short half-life of intracellular superoxide radicals, low steady state concentrations, lack of efficient H 2 O 2 capture probes, and so on [10]. electron-mediator-doped chitosan membrane. A novel method for the construction of electrochemical biosensors with fast response, good stability, high sensitivity, and wide linear response range based on a composite of chitosan and cyclodextrin was explored. The experimental structure diagram was shown in Figure 1. electrode that was used to detect H2O2. The above method could overcome the disadvantages of the electron-mediator-doped chitosan membrane. A novel method for the construction of electrochemical biosensors with fast response, good stability, high sensitivity, and wide linear response range based on a composite of chitosan and cyclodextrin was explored. The experimental structure diagram was shown in Figure 1.

Spectral Characterization of β-CD-FE
Ferrocene (FE), β-CD, the β-CD/FE mixture, and the β-CD-FE complex were respectively characterized with infrared spectroscopy and the results are shown in Figure 2a. FE exhibited three strong absorption peaks at 817 cm −1 (δπC-H), 1000 cm −1 (cyclopentadienyl ring δC-H), and 1100 cm −1 (cyclopentadienyl ring νC-C) [37]. The peaks at 1156 cm −1 and 1028 cm −1 in the spectrum of β-CD were attributed to the characteristic absorption of C-O-C [38]. The characteristic absorption peaks of both FE and β-CD were observed in the mixture of FE and β-CD. However, no characteristic absorption peaks of FE were observed in the spectrum of the ferrocene-included complex formed by the solution method, indicating that FE molecules successfully entered the cavities of β-cyclodextrin.

Spectral Characterization of β-CD-FE
Ferrocene (FE), β-CD, the β-CD/FE mixture, and the β-CD-FE complex were respectively characterized with infrared spectroscopy and the results are shown in Figure 2a. FE exhibited three strong absorption peaks at 817 cm −1 (δπ C-H ), 1000 cm −1 (cyclopentadienyl ring δ C-H ), and 1100 cm −1 (cyclopentadienyl ring ν C-C ) [37]. The peaks at 1156 cm −1 and 1028 cm −1 in the spectrum of β-CD were attributed to the characteristic absorption of C-O-C [38]. The characteristic absorption peaks of both FE and β-CD were observed in the mixture of FE and β-CD. However, no characteristic absorption peaks of FE were observed in the spectrum of the ferrocene-included complex formed by the solution method, indicating that FE molecules successfully entered the cavities of β-cyclodextrin.
The UV spectroscopy analysis indicated that neither the ferrocene solution nor Fe 3+ solution could absorb visible light at 619 nm. However, ferrocene in the presence of Fe 3+ exhibited a strong absorption peak at 619 nm. The coexistence of β-CD-FE and Fe 3+ also resulted in a strong absorption peak at 619 nm (Figure 2b), indicating that ferrocene was included in β-CD. These results, along with the results of IR analysis, indicate that the β-CD-FE complex was successfully synthesized. The UV spectroscopy analysis indicated that neither the ferrocene solution nor Fe 3+ solution could absorb visible light at 619 nm. However, ferrocene in the presence of Fe 3+ exhibited a strong absorption peak at 619 nm. The coexistence of β-CD-FE and Fe 3+ also resulted in a strong absorption peak at 619 nm (Figure 2b), indicating that ferrocene was included in β-CD. These results, along with the results of IR analysis, indicate that the β-CD-FE complex was successfully synthesized.

Thermal Stability of β-CD-FE
The thermogravimetric analyses of FE, β-CD, the β-CD/FE mixture, and the β-CD-FE complex were carried out. Their TG and DTG curves are shown in Figure 2c,d, respectively. The mixture of β-CD and FE was decomposed in two steps. The weight loss of 55.3% in the temperature range of 87.7-161.9 °C was caused by the decomposition of FE and the weight loss of 38.0% in the temperature range of 288.7-348.3 °C was attributed to the decomposition of β-CD. β-CD-FE was only subjected to a rapid weight loss in the temperature range of 279.2 °C to 348 °C. The temperature at which the decomposition of β-CD-FE started was higher than the temperature (259.2 °C) required to start the decomposition of β-CD. The decomposition of β-CD-FE complex was finished at a temperature lower than that to finish the decomposition of β-CD (259.2 °C) and the weight loss of the complex was 94.0%, higher than that of β-CD (86.8%). These results indicate that the thermal stability of FE was significantly improved due to the inclusion of FE in β-CD. FE started to break down as β-CD was decomposed, which further accelerated the thermal decomposition of β-CD. These observations further confirmed that the ferrocene-included complex was successfully prepared 2.1.3. Determination of Inclusion Constant, Inclusion Amount, and Inclusion Rate of β-CD-FE As shown in Figure 2e for the standard curve of the inclusion constant, 1/(F − F0) is linearly related to 1/C0, indicating that the inclusion ratio was 1:1 [39]. The inclusion constant (K) was calculated to be 7.48 × 10 2 , suggesting that the inclusion reaction could occur spontaneously at room temperature [40].
The inclusion amounts and inclusion rates at different feeding ratios were calculated from the standard curve. The inclusion rate increased with the increase of mFE/mβ-CD, peaked at the mFE/mβ-CD ratio of 3:1, and decreased as mFE/mβ-CD further increased. It can be explained that the chance for

Thermal Stability of β-CD-FE
The thermogravimetric analyses of FE, β-CD, the β-CD/FE mixture, and the β-CD-FE complex were carried out. Their TG and DTG curves are shown in Figure 2c,d, respectively. The mixture of β-CD and FE was decomposed in two steps. The weight loss of 55.3% in the temperature range of 87.7-161.9 • C was caused by the decomposition of FE and the weight loss of 38.0% in the temperature range of 288.7-348.3 • C was attributed to the decomposition of β-CD. β-CD-FE was only subjected to a rapid weight loss in the temperature range of 279.2 • C to 348 • C. The temperature at which the decomposition of β-CD-FE started was higher than the temperature (259.2 • C) required to start the decomposition of β-CD. The decomposition of β-CD-FE complex was finished at a temperature lower than that to finish the decomposition of β-CD (259.2 • C) and the weight loss of the complex was 94.0%, higher than that of β-CD (86.8%). These results indicate that the thermal stability of FE was significantly improved due to the inclusion of FE in β-CD. FE started to break down as β-CD was decomposed, which further accelerated the thermal decomposition of β-CD. These observations further confirmed that the ferrocene-included complex was successfully prepared 2.1.3. Determination of Inclusion Constant, Inclusion Amount, and Inclusion Rate of β-CD-FE As shown in Figure 2e for the standard curve of the inclusion constant, 1/(F − F 0 ) is linearly related to 1/C 0 , indicating that the inclusion ratio was 1:1 [39]. The inclusion constant (K) was calculated to be 7.48 × 10 2 , suggesting that the inclusion reaction could occur spontaneously at room temperature [40].
The inclusion amounts and inclusion rates at different feeding ratios were calculated from the standard curve. The inclusion rate increased with the increase of m FE /m β-CD , peaked at the m FE /m β-CD ratio of 3:1, and decreased as m FE /m β-CD further increased. It can be explained that the chance for β-CD molecules contacting ferrocene molecules increased with the increase of m FE /m β-CD , resulting in more ferrocene molecules in the cavities of β-CD. Meanwhile, the increased volume fraction of ethanol in the reaction system as m FE /m β-CD increased led to a decrease in the solubility of β-CD in the system and significantly reduced the contact between ferrocene molecules and β-CD molecules, which was unfavorable to the inclusion reaction. Therefore, β-CD-FE was prepared at m FE /m β-CD = 3:1 in the following experiments.
Half a gram of β-CD-FE prepared at m FE /m β-CD = 3:1 was dispersed in 20 mL of 0.1 mol/L PBS (pH = 7.0) and let stand for 3 h. Then it was precipitated and dried. It was found that the inclusion amount of FE was 98.73% to the original amount. The above results indicate that the stability of β-CD-FE in 0.1 mol/L PBS is enough to ensure the stability requirement of the composite electrode for H 2 O 2 detection. β-CD molecules contacting ferrocene molecules increased with the increase of mFE/mβ-CD, resulting in more ferrocene molecules in the cavities of β-CD. Meanwhile, the increased volume fraction of ethanol in the reaction system as mFE/mβ-CD increased led to a decrease in the solubility of β-CD in the system and significantly reduced the contact between ferrocene molecules and β-CD molecules, which was unfavorable to the inclusion reaction. Therefore, β-CD-FE was prepared at mFE/mβ-CD = 3:1 in the following experiments. Half a gram of β-CD-FE prepared at mFE/mβ-CD = 3:1 was dispersed in 20 mL of 0.1 mol/L PBS (pH = 7.0) and let stand for 3 h. Then it was precipitated and dried. It was found that the inclusion amount of FE was 98.73% to the original amount. The above results indicate that the stability of β-CD-FE in 0.1 mol/L PBS is enough to ensure the stability requirement of the composite electrode for H2O2 detection.  Based on the cyclic voltammogram curves of the complex at different scan rates (Figure 3a), a linear relationship between the peak current and square root of scan rate was established, as shown in Figure 3b. These results indicate that the redox process of β-CD-FE is a diffusion process, rather than the dissociation of the FE from the cavities of the β-CD, to the surface of the electrode.  Figure 3d). It is clear that the redox peak potential remained unchanged, but the peak current increased, with the increases of β-CD-FE concentration and the inclusion amount. Based on the cyclic voltammogram curves of the complex at different scan rates (Figure 3a), a linear relationship between the peak current and square root of scan rate was established, as shown in Figure 3b. These results indicate that the redox process of β-CD-FE is a diffusion process, rather than the dissociation of the FE from the cavities of the β-CD, to the surface of the electrode.  (Figure 3d). It is clear that the redox peak potential remained unchanged, but the peak current increased, with the increases of β-CD-FE concentration and the inclusion amount.

Optimization of Preparation Conditions for CTS-CAT
Adsorption, coating, and cross-linking are the most widely-used traditional chitosan-based enzyme immobilization methods [17]. However, the enzymes immobilized by adsorption can easily fall off due to the weak electrostatic interactions, which is not conducive to the stability and performance of the modified electrode. The carrier-binding method immobilizes an enzyme on a carrier via chemical bonding using a cross-linking agent, which forms a modified electrode with stable electrochemical properties. In general, chitosan, glutaraldehyde (GD), and enzyme are mixed together to prepare a modification solution. However, chitosan is insoluble in water and can only be dissolved in dilute acid. The cross-linking reaction is time consuming and the enzyme tends to become inactive during the reaction. In the present work, a step-by-step method was used to cross-link chitosan with GD and the cross-linked product used to immobilize CAT.
The activity of immobilized enzyme is an important factor to evaluate the performance of the chitosan biosensor. Therefore, the preparation conditions for the enzyme immobilization were optimized by monitoring the activity of CTS-CAT. Figure 4a shows the relative enzyme activity of the CTS-CAT prepared with different amounts of GD, where the highest activity is set to 100%. The activity of the immobilized enzyme increased with the increase of m GD /m CTS , peaked at m GD /m CTS = 5%, and decreased as the m GD /m CTS ratio further increased. For the enzyme immobilization, one of the aldehyde groups of GD reacts with the amino group of chitosan and the other one reacts with the amino group, phenol group, or the mercapto group of CAT. At low m GD /m CTS ratios, only a limited amount of aldehyde can react with chitosan, which provided low amounts of active sites on the carrier. Only a small amount of enzyme was immobilized on the carrier. More GD reacted with chitosan as m GD /m CTS increased, resulting in more enzyme on the carriers and, thus, higher enzyme activity. However, the high amount of cross-linked product formed at extremely high m GD /m CTS ratios tended to undergo intramolecular or intermolecular cross-linking, which reduced the active site (aldehyde) on the carrier, increased the steric hindrance and, thus, decreased the enzyme loading. The enzyme activity was then decreased. In addition, the high amount of GD on the carrier might react with the key sites of the enzyme to form multiple bindings, which also reduced the enzyme activity. Therefore, the amount of GD was optimized as m GD /m CTS = 5%.

Effects of the Chitosan Modification Temperature on Enzyme Activity
The effects of the temperature for chitosan modification were determined and the results are shown in Figure 4b. The activity of catalase immobilized on chitosan increased with the increase of the modification temperature, peaked at 25 • C, and decreased as the temperature was further increased. Therefore, the temperature for the chitosan modification with GD was optimized as 25 • C, at which point the modification produced a modified chitosan that was suitable for enzyme immobilization with the highest enzyme activity.

Effects of the Chitosan Modification Time on Enzyme Activity
As shown in Figure 4c, the activity of CTS-CAT increased with the increase of the reaction time for chitosan modification with GD, reached the highest value after a 60 min reaction, and decreased as the modification was prolonged further. It can be explained that the reactive sites for the enzyme immobilization gradually increased with the modification time, which increased the enzyme loading and, thus, the enzyme activity. As the modification time was prolonged greater than 60 min, the cross-linking degree gradually increased with the modification time and the cross-linked molecules tended to undergo intramolecular or intermolecular crosslinking. The steric hindrance was then increased which, as well as the high degree of cross-linking, led to a decrease in the adsorption efficiency of the enzyme. Therefore, the modification time of chitosan for the preparation of CTS-CAT was optimized as 60 min.
group of CAT. At low mGD/mCTS ratios, only a limited amount of aldehyde can react with chitosan, which provided low amounts of active sites on the carrier. Only a small amount of enzyme was immobilized on the carrier. More GD reacted with chitosan as mGD/mCTS increased, resulting in more enzyme on the carriers and, thus, higher enzyme activity. However, the high amount of cross-linked product formed at extremely high mGD/mCTS ratios tended to undergo intramolecular or intermolecular cross-linking, which reduced the active site (aldehyde) on the carrier, increased the steric hindrance and, thus, decreased the enzyme loading. The enzyme activity was then decreased. In addition, the high amount of GD on the carrier might react with the key sites of the enzyme to form multiple bindings, which also reduced the enzyme activity. Therefore, the amount of GD was optimized as mGD/mCTS = 5%.

Effects of the Enzyme Amount on Enzyme Activity
The amount of CAT for the immobilization can also affect the enzyme activity of CTS-CAT. As shown in Figure 4d, the overall enzyme activity increased with the increase of enzyme amount, reached the maximum at m CAT /m CTS = 0.315, and remained unchanged as the enzyme amount was further increased. It can be explained that the enzyme loading increased with the increase of the enzyme amount, which increased the overall enzyme activity, until the active sites (aldehyde group) on the carrier were saturated at m CAT /m CTS = 0.315. Further increasing the enzyme amount caused no effect on the enzyme loading or the activity of CTS-CAT. Excessive enzyme reduces the utilization efficiency of the enzyme. Therefore, the optimal amount of CAT was found to be m CAT /m CTS = 0.315.

Effects of the Immobilization Temperature on Enzyme Activity
The effects of immobilization temperature were determined by immobilizing CAT on the GD modified chitosan at different temperatures. The relationship between enzyme activity and immobilization temperature is shown in Figure 4e. The reaction rate for immobilization was low at low temperature, resulting in a low enzyme loading per unit time. However, low temperature is favorable to maintain the enzyme activity. Increasing the immobilization temperature can promote the thermal motion of the enzyme molecules and, thus, increase enzyme loading. However, high temperature could deactivate the enzyme and the deactivation is irreversible. Therefore, the immobilization temperature was set to 25 • C, at which point the enzyme could be completely immobilized while its activity was maintained at a reasonable value.

Effects of the Immobilization Time on Enzyme Activity
The effects of immobilization time were investigated by immobilizing CAT on the GD-modified chitosan for different reaction times. The relationship between the enzyme activity and immobilization time is shown in Figure 4f. The extension of the immobilization time facilitates the contact and reaction between the enzyme and the aldehyde group. The activity sites (aldehydes) on the carrier were gradually saturated and the activity of the CTS-CAT became constant as the reaction time was prolonged to 180 min. The enzyme molecules on the carrier tended to block each other as the reaction time increased further, which reduced the enzyme activity. Therefore, the immobilization time of CAT on GD-modified chitosan was optimized as 180 min.
The pH of the reaction system can also affect the enzyme activity. The CTS-CAT obtained in the neutral solution exhibited the highest enzyme activity. It can be explained that free catalase has the highest activity under the conditions similar to the biological environment. Due to prolonged exposure to acidic or basic immobilization conditions may deactivate the catalase, the pH for the immobilization was set to 7 in the following experiments.
Based on these results, the preparation conditions of CTS-CAT were optimized as follows: chitosan was modified with 5% (wt %) GD at 25 • C for 60 min and the GD-modified chitosan was reacted with CAT at m CAT /m CTS = 0.315 and pH = 7 and cured at 25 • C for 180 min. The control electrode A coated with CTS-CAT and the CTS-CAT/β-CD-FE composite electrode exhibited strong peak current signals in response to H 2 O 2 , indicating that they were very sensitive to H 2 O 2 . It can be explained that CAT obtained electrons from the electrode to turn into a reduced state that was oxidized back to the oxidation state by H 2 O 2 . The oxidation of CAT significantly increased the reduction peak current. The signal amplification intensity of the CTS-CAT/β-CD-FE composite electrode was significantly higher than that of electrode A, indicating that β-CD-FE played a significant role in amplifying the electrical signal and CTS-CAT/β-CD-FE electrode was more sensitive to H 2 O 2 . These results indicated that the CAT and FE included in β-CD played a major role in the response of the composite electrode to H 2 O 2 . The pH of the reaction system can also affect the enzyme activity. The CTS-CAT obtained in the neutral solution exhibited the highest enzyme activity. It can be explained that free catalase has the highest activity under the conditions similar to the biological environment. Due to prolonged exposure to acidic or basic immobilization conditions may deactivate the catalase, the pH for the immobilization was set to 7 in the following experiments.
Based on these results, the preparation conditions of CTS-CAT were optimized as follows: chitosan was modified with 5% (wt %) GD at 25 °C for 60 min and the GD-modified chitosan was reacted with CAT at mCAT/mCTS = 0.315 and pH = 7 and cured at 25 °C for 180 min. Figure 5 shows the cyclic voltammetry curves of the control electrodes (bare electrode, electrode A which was coated with CTS-CAT, and electrode B coated with CTS) and the CTS-CAT/β-CD-FE composite electrode in a H2O2 solution. The procedure for preparation of different electrodes is described in Section 3.7. It could be found that neither the bare electrode without any coating nor the control electrode B coated with chitosan exhibited any obvious redox activity in 0.1 mol/L PBS (pH = 7.0) of H2O2 in the range of −0.80-0.80 V. The control electrode A coated with CTS-CAT and the CTS-CAT/β-CD-FE composite electrode exhibited strong peak current signals in response to H2O2, indicating that they were very sensitive to H2O2. It can be explained that CAT obtained electrons from the electrode to turn into a reduced state that was oxidized back to the oxidation state by H2O2. The oxidation of CAT significantly increased the reduction peak current. The signal amplification intensity of the CTS-CAT/β-CD-FE composite electrode was significantly higher than that of electrode A, indicating that β-CD-FE played a significant role in amplifying the electrical signal and CTS-CAT/β-CD-FE electrode was more sensitive to H2O2. These results indicated that the CAT and FE included in β-CD played a major role The pH of the reaction system can also affect the enzyme activity. The CTS-CAT obtained in the neutral solution exhibited the highest enzyme activity. It can be explained that free catalase has the highest activity under the conditions similar to the biological environment. Due to prolonged exposure to acidic or basic immobilization conditions may deactivate the catalase, the pH for the immobilization was set to 7 in the following experiments.

Oxidation-Reduction Properties of CTS-CAT/β-CD-FE Composite Electrodes
Based on these results, the preparation conditions of CTS-CAT were optimized as follows: chitosan was modified with 5% (wt %) GD at 25 °C for 60 min and the GD-modified chitosan was reacted with CAT at mCAT/mCTS = 0.315 and pH = 7 and cured at 25 °C for 180 min.  The control electrode A coated with CTS-CAT and the CTS-CAT/β-CD-FE composite electrode exhibited strong peak current signals in response to H2O2, indicating that they were very sensitive to H2O2. It can be explained that CAT obtained electrons from the electrode to turn into a reduced state that was oxidized back to the oxidation state by H2O2. The oxidation of CAT significantly increased the reduction peak current. The signal amplification intensity of the CTS-CAT/β-CD-FE composite electrode was significantly higher than that of electrode A, indicating that β-CD-FE played a significant role in amplifying the electrical signal and CTS-CAT/β-CD-FE electrode was more The pH of the reaction system can also affect the enzyme activity. The CTS-CAT obtained in the neutral solution exhibited the highest enzyme activity. It can be explained that free catalase has the highest activity under the conditions similar to the biological environment. Due to prolonged exposure to acidic or basic immobilization conditions may deactivate the catalase, the pH for the immobilization was set to 7 in the following experiments.

Oxidation-Reduction Properties of CTS-CAT/β-CD-FE Composite Electrodes
Based on these results, the preparation conditions of CTS-CAT were optimized as follows: chitosan was modified with 5% (wt %) GD at 25 °C for 60 min and the GD-modified chitosan was reacted with CAT at mCAT/mCTS = 0.315 and pH = 7 and cured at 25 °C for 180 min.  The control electrode A coated with CTS-CAT and the CTS-CAT/β-CD-FE composite electrode exhibited strong peak current signals in response to H2O2, indicating that they were very sensitive to H2O2. It can be explained that CAT obtained electrons from the electrode to turn into a reduced state that was oxidized back to the oxidation state by H2O2. The oxidation of CAT significantly increased the reduction peak current. The signal amplification intensity of the CTS-CAT/β-CD-FE composite electrode was significantly higher than that of electrode A, indicating that β-CD-FE played a significant role in amplifying the electrical signal and CTS-CAT/β-CD-FE electrode was more  Figure 6 shows the results of the peak current in the range of −0.80-0.80 V of the CTS-CAT/β-CD-FE composite electrode in the solutions of different pH conditions. The peak current increased with the increase of pH. Therefore, the optimum working pH of the CTS-CAT/β-CD-FE composite electrode was determined to be 7.0. As discussed above, the enzyme played a major role in the response of the CTS-CAT/β-CD-FE composite electrode to H 2 O 2 . Therefore, the peak current is closely related to the activity of the enzyme and the effects of working pH on peak current were similar to those of the immobilization pH on the enzyme activity.  Figure 6 shows the results of the peak current in the range of −0.80-0.80 V of the CTS-CAT/β-CD-FE composite electrode in the solutions of different pH conditions. The peak current increased with the increase of pH. Therefore, the optimum working pH of the CTS-CAT/β-CD-FE composite electrode was determined to be 7.0. As discussed above, the enzyme played a major role in the response of the CTS-CAT/β-CD-FE composite electrode to H2O2. Therefore, the peak current is closely related to the activity of the enzyme and the effects of working pH on peak current were similar to those of the immobilization pH on the enzyme activity.

Sensitivity of the CTS-CAT/β-CD-FE Composite Electrodes
The concentrations and volumes of H2O2 used in the experiments are shown in Table 1. Figure 7 shows the time-current plot of the CTS-CAT/β-CD-FE composite electrode. The composite electrode responded to H2O2 rapidly and the reaction current reached the saturated state in less than 5 s, indicating that the activity of CAT on the composite electrode was well maintained. In addition, the peak current exhibited a linear relationship with the H2O2 concentration ( Figure 8) in the range of 1.0 × 10 −7 -6.0 × 10 −3 mol/L with the fitting equation as follows: I = 6.239 × 10 C + 0.016 where I is the peak current and C is the concentration of H2O2. Therefore, the CTS-CAT/β-CD-FE composite electrode can be used to quantitatively detect H2O2 in the range of 1.0 × 10 −7 -6.0 × 10 −3 mol/L. The relative standard deviation of the test was 1.7-11.2% and the detection limit was 5 × 10 −8 mol/L calculated at a signal-to-noise ratio of 3. The above composite electrode was stored in PBS in a fridge at 4 °C for 10 days, and then the properties of it were tested again. It was found that the decrease of the current response was 9.6%, which indicates that the stability of the composite electrode is ideal.   Figure 6. The effect of solution pH on the peak current.

Sensitivity of the CTS-CAT/β-CD-FE Composite Electrodes
The concentrations and volumes of H 2 O 2 used in the experiments are shown in Table 1. Figure 7 shows the time-current plot of the CTS-CAT/β-CD-FE composite electrode. The composite electrode responded to H 2 O 2 rapidly and the reaction current reached the saturated state in less than 5 s, indicating that the activity of CAT on the composite electrode was well maintained. In addition, the peak current exhibited a linear relationship with the H 2 O 2 concentration (Figure 8) in the range of 1.0 × 10 −7 -6.0 × 10 −3 mol/L with the fitting equation as follows: where I is the peak current and C is the concentration of H 2 O 2 . Therefore, the CTS-CAT/ β-CD-FE composite electrode can be used to quantitatively detect H 2 O 2 in the range of 1.0 × 10 −7 -6.0 × 10 −3 mol/L. The relative standard deviation of the test was 1.7-11.2% and the detection limit was 5 × 10 −8 mol/L calculated at a signal-to-noise ratio of 3. The above composite electrode was stored in PBS in a fridge at 4 • C for 10 days, and then the properties of it were tested again. It was found that the decrease of the current response was 9.6%, which indicates that the stability of the composite electrode is ideal.

Inclusion of Ferrocene in β-CD
One gram of β-CD was added to 6.0 g of deionized water. Ferrocene was suspended in anhydrous ethanol at the mass ratio of 1:10. These two suspensions were, respectively, heated to micro-boiling, mixed at mass ratios of 1:1, 2:1, 3:1, or 4:1, stirred in an oil bath at 60 °C for 6 h, cooled to room temperature and filtered by suction filtration. The solid residue was washed with tetrahydrofuran to remove the unreacted ferrocene and dried to afford the ferrocene-included product, β-CD-FE.

Inclusion of Ferrocene in β-CD
One gram of β-CD was added to 6.0 g of deionized water. Ferrocene was suspended in anhydrous ethanol at the mass ratio of 1:10. These two suspensions were, respectively, heated to micro-boiling, mixed at mass ratios of 1:1, 2:1, 3:1, or 4:1, stirred in an oil bath at 60 °C for 6 h, cooled to room temperature and filtered by suction filtration. The solid residue was washed with tetrahydrofuran to remove the unreacted ferrocene and dried to afford the ferrocene-included product, β-CD-FE.

Inclusion of Ferrocene in β-CD
One gram of β-CD was added to 6.0 g of deionized water. Ferrocene was suspended in anhydrous ethanol at the mass ratio of 1:10. These two suspensions were, respectively, heated to micro-boiling, mixed at mass ratios of 1:1, 2:1, 3:1, or 4:1, stirred in an oil bath at 60 • C for 6 h, cooled to room temperature and filtered by suction filtration. The solid residue was washed with tetrahydrofuran to remove the unreacted ferrocene and dried to afford the ferrocene-included product, β-CD-FE.

Determination of Ferrocene Inclusion Constant, Inclusion Amount, and Inclusion Ratio
The reaction between β-CD and ferrocene can be expressed as: The equilibrium constant is: The stoichiometric proportion and binding constants K, from spectrophotometric data, could be calculated using Scott's Equation [41]: where C FE0 is the initial concentration of ferrocene, C β0 is the initial concentration of β-CD, F − F 0 is the absorbance difference before and after β-CD is added, and α is the difference of molar absorptivity for free and included ferrocene. If the resulting curve of C β0 /F − F 0 against C β0 is a straight line, the 1:1 complexing system is expected and the binding constant K (M −1 ) can be calculated as: To obtain a standard curve of UV absorption vs. ferrocene concentration, 20 mL of anhydrous ethanol, 10 mL of HCl (37 wt %), and 10 mL of Fe 3+ solution (1.00 mg/mL) were added to a 50 mL volumetric flask. Then different amounts of ferrocene standard solutions (5.00 mg/mL) were respectively added to the volumetric flasks containing the Fe 3+ . The mixtures were allowed to stand for 30 min and their absorption at 619 nm was measured. The standard curve of UV absorption vs. ferrocene concentration can be expressed as: To determine the inclusion amount and ratio, 0.5 g β-CD-FE was suspended in 30.00 mL anhydrous ethanol and allowed to stand for 24 h. The supernatant was collected and 20.00 mL of anhydrous ethanol was added to the β-CD-FE suspension. The suspension was allowed to stand for another 24 h and filtered. The filtrate was combined with the supernatant collected above and measured for the UV absorption at 619 nm. The ferrocene concentration was calculated from the standard curve of UV absorption vs. ferrocene concentration to determine the inclusion amount and ratio.

Electrochemical Properties of β-CD-FE
The cyclic voltammogram curves of β-CD-FE were measured in the potential range of 0-0.60 V using the CHI-660 electrochemical workstation of Chunghwa Instrument Co., Ltd. (Dongguan, China), by which the relationship between the electrochemical properties of β-CD-FE and FE concentration or inclusion amount were determined.

Preparation of CTS-CAT
CAT was immobilized on chitosan using a step-by-step method. A 2.0% chitosan solution was prepared in 1% acetic acid aqueous solution. Two milliliters of 5% glutaraldehyde (GD) solution was mixed with 1 mL chitosan solution, reacted for a certain period of time at a certain temperature, and centrifuged for 20 min at 3500 rad/min. The suspension was further filtered by suction filtration and the solid residue was washed with water to remove free GD to afford GD modified chitosan. The GD modified chitosan was added to a 3% CAT solution and reacted for a certain period of time. The suspension was then suction filtered and the unreacted CAT was removed with 0.1 mol/L PBS buffer solution to afford CTS-CAT.

Determination of the Enzyme Activity of CTS-CAT
The activity of CAT was determined by measuring the UV absorption spectra of a H 2 O 2 solution at 240 nm before and after CAT was added. Enzyme activity was calculated as follows: where P is the specific activity of the enzyme, A is the difference between the absorptions of the test solution and blank solution, V is the solution volume, ε is the extinction coefficient of H 2 O 2 at 240 nm, l is the thickness of cuvette, t is the reaction time, and m is the mass of CTS-CAT.

Preparation of the CTS-CAT/β-CD-FE Composite Electrode
A glassy carbon electrode was polished to obtain a mirror surface with 0.3 µm Al 2 O 3 , sequentially followed by the ultra-sonication in deionized water, anhydrous ethanol, and deionized water for 1 min. The electrode was placed vertically and air-dried at 25 • C. A solution of 5.00 mmol/L (determined by ferrocene content) of β-CD-FE was prepared in dimethyl sulfoxide and mixed with 2 × 10 −3 g CTS-CAT. The mixture was shaken for 30 min and 6 µL of the mixture was added dropwise to the glassy carbon electrode. The electrode was placed vertically and air-dried at 25 • C to form a modified CTS-CAT/β-CD-FE composite electrode.
Two milligrams of CTS-CAT was suspended in 2 mL of dimethyl sulfoxide to prepare control electrode A using the same procedure. Control electrode B was prepared with 6 µL of chitosan solution in acetic acid using the same procedure described above.

Electrochemical Properties of the CTS-CAT/β-CD-FE Composite Electrode
The cyclic voltammetry curves of different electrodes were measured at potential of 0.80-0.80 V in 0.1 mol/L PBS (pH = 7.0) and H 2 O 2 solution, respectively, using a CHI-660 electrochemical workstation (Chunghwa Instrument Co., Ltd., Dongguan, China) to determine the electrochemical properties of control electrode A and B and the CTS-CAT/β-CD-FE composite electrode.

Sensitivity of the CTS-CAT/β-CD-FE Composite Electrode
The chrono-current curve of the CTS-CAT/β-CD-FE composite electrode was measured at the potential of −0.30 V in 5.00 mL 0.1 mol/L PBS (pH = 7.0) with a certain amount of H 2 O 2 solution added at regular intervals (Table 1) using the CHI-660 electrochemical workstation. The relationship between the peak current and H 2 O 2 concentration was established to determine the effective analysis range of H 2 O 2 on the CTS-CAT/β-CD-FE composite electrode.

Conclusions
In the present work, ferrocene was included in the cavity of β-CD to improve its stability. The inclusion constant and thermal stability of the β-CD-FE complex were determined. The effects of the preparation conditions on the inclusion amount, inclusion rate, and electrochemical properties of the complex were evaluated. At the same time, enzyme-immobilized chitosan derivatives were prepared by a stepwise cross-linking method. The immobilization conditions were optimized by monitoring the enzyme activity as follows: Chitosan was reacted with 5% (mass fraction) GD at 25 • C for 60 min and the GD modified chitosan and CAT reacted under the condition of m CAT /m CTS = 0.315 and pH = 7 and cured at 25 • C for 180 min. Then a glassy electrode was modified with CTS-CAT and β-CD-FE for the electrochemical detection of H 2 O 2 . The immobilized enzyme responded to H 2 O 2 as a strong reduction peak current and β-CD-FE amplified the current signal to improve the sensitivity of the electrode. The optimal working pH for the modified electrode was 7.0. The peak current of the electrode exhibited a linear relationship with the concentration of H 2 O 2 in the range of 1.0 × 10 −7 -6.0 × 10 −3 mol/L. The above work constructed an electrochemical biosensor model based on enzyme-immobilized chitosan and an electron mediator-included cyclodextrin derivative to overcome the issues of a traditional chitosan-encapsulated electron mediator electrochemical sensor for the detection of H 2 O 2 .