3.1 Characterization of ZrPR1R2
The ZrPR1R2 is prepared by the covalent linking of aminoethoxy and carboxypropyl to the inorganic framework of zirconium phosphate. As shown in curve d of Fig. 1, The FTIR spectra of the zirconium phosphonate has the characteristic absorbance around 1080 cm− 1 similar to α-ZrP (curve a), attributing to the stretching vibration of P–O in both them. Compared with the carboxypropyl-functionalized zirconium phosphonate (ZrPR1, curve b), the stretching vibration of O-H around 3450cm− 1 and the stretching vibration of C = O around 1700cm− 1 decreased significantly, because of the decrease of the carboxypropyl group and the increase of the aminoethoxy group. The characteristic bands around 3362cm− 1 and 1644cm− 1 were primarily attributed to the stretching vibration of the N-H and C-N respectively, similar to aminoethoxy-functionalized zirconium phosphonate (ZrPR2, curve c).
3.2 The activity of POD/ZrPR1R2 composite
The characteristic absorbance band of native POD is located 400nm, as shown in curve a of Fig. 2A. After POD combined with zirconium phosphonate nanosheets, the characteristic wavelength (curve b) of the POD/ZrPR1R2 composite is similar to the native POD, and the absorbance intensity enhanced slightly. The results indicated the protein structure remained well after the combination of POD and ZrPR1R2 nanosheets.
The activity of POD/ZrPR1R2 composite was detected as follow, glucose oxidase reacts with glucose to generate gluconic acid and hydrogen peroxide. The generated hydrogen peroxide is then catalyzed by peroxidase to generate oxygen. Oxygen oxidizes the reduced 2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid Ammonium Salt), abbreviated as ABTS, into oxidized product, and its absorbance is measured at 425nm.
As shown in curve a of Fig. 2B, no absorbance around 425nm can be observed of the reduced ABTS. After the addition of the native POD (curve b) or POD/ZrPR1R2 composite (curve c), a new intensive absorbance at 425nm displayed, attributed to the oxidized product. Compared to the native POD (curve b), the absorbance intensity at 425nm caused by the POD/ZrPR1R2 composite enhanced slightly, indicated the higher activity after POD combined with the ZrPR1R2 nanosheets.
3.3 Direct electrochemistry of the POD/ZrPR1R2 composite modified electrode
Figure3 shows the cyclic voltammograms (CVs) of the POD/ZrPR1R2 composite modified electrodein a 0.1mol L− 1 PBS buffer solution at the pH of 7.0. A couple of reversible and well-defined redox peaks at -320 and − 370 mV were observed, with an apparent formal peak potential (Ep) of -345 mV and a peak-to-peak separation (△Ep) of 50 mV at the scan rate of 100mV s− 1. The apparent formal peak potential (Ep) of this nafion-POD/ZrPR1R2-GCE was close to the value of the HRP–TiO2-GCE(Zhang et al. 2004), demonstrated clearly that the direct electrochemistry of the POD on the glassy carbon electrodewas achieved by immobilizing the POD into the matrixs of the ZrPR1R2 nanosheets.
The effect of the scan rate on the response of the POD/ZrPR1R2 composite electrode is also exhibited in Fig. 3. With the increase of the scan rate from 50 to 300mV s− 1, both the reduction peak currents (ip,c)and the oxidation peak currents (ip,a) increased linearly, suggesting a surface-controlled process. The linear regression equations of the peak currents with the scan rate werey = 15.00x + 0.16µA (n = 6, R = 0.9998) and y = -12.92x + 0.20µA (n = 6, R = 0.9999) respectively. The electron transfer rate constant (ks) can be estimated according to the formula ks= mnFv/RT if the peak-to-peak separation is less than 200mV, where m is a parameter related to the peak-to-peak separation, n isthe number of transferred electron, F is the Faraday’s constant, v is the scan rate, T is the temperature and R is the universal gas constant. The peak-to-peak separationswere48, 50, 51, 52, 53 and 55 mV at 50, 100, 150, 200, 250 and 300 mV s− 1, respectively, and then the ks was calculated to be 4.11 s− 1according to the Laviron’s theory. The electron transfer rate constant (ks) of this POD/ZrPR1R2 composite electrode was faster than the value reported previously(Xiao et al. 2014; Zhao et al. 2011; Zhao et al. 2008; Kong et al. 2003), suggested the electron transfer between POD and glassy carbon electrode was enhanced remarkably by the ZrPR1R2 nanosheets.
3.4 Electrocatalytic properties of the POD/ZrPR1R2 composite modified electrode
The CVs in Fig. 4A displayed the sensitive response of the POD/ZrPR1R2 modified electrode to hydrogen peroxide.The cathodic peak (~-0.27V) was increased remarkably after the addition of H2O2 into 0.1 mol L− 1 PBS (pH 7.0), and the anodic peak decreased correspondingly. Furthermore, the reduction peak increased along with the increase of H2O2 concentration, indicating a typical electrocatalytic reduction process.
The reduction currents exhibited a linear response to H2O2 in the range of 8.82×10− 8 ~ 8.82×10− 7mol L− 1 and 8.82×10− 7 ~ 8.82×10− 6mol L− 1 respectively, with the detection limit of 3.31×10− 8mol L− 1. The linear regression equations are y = 0.67x + 1.1µA (n = 10, R = 0.9973) and y = 0.037x + 6.89µA (n = 10, R = 0.9908) respectively. The detection limit of this POD/ZrPR1R2 composite modified electrode is better than the value reported previously(Xiao et al. 2014; Zhao et al. 2011; Zhao et al. 2008; Zhang et al. 2007c), indicated the higher sensitivity of the immobilized POD to H2O2.
The POD/ZrPR1R2 composite modified electrode also displayed an excellent response to dissolved oxygen. As showed in Fig. 4B, without the presence of nitrogen stream, a rapid increase of the cathodic peak around − 0.29V was observed, and the anodic peak decreased and even disappeared. The reduction current increased with the increase of dissolved oxygen concentration. After stopping the nitrogen stream by 30 minutes, the reduction current was unchanged almost, indicating a saturated concentration of dissolved oxygen. The highest catalytic current of 22.34 µA suggested the higher sensitivity of the immobilized POD to dissolved oxygen, facilitating the detection of antioxidant capacity in the real samples, such as tea water.
3.5 Detection of the total antioxidant capacity of the tea water
Figure 5 displayed the amperometric response of the POD/ZrPR1R2 composite modified electrode to the successive additions of deionized water at room temperature and tea water prepared at different temperature for soaking time of 0.5 hours, indicated the higher dissolved oxygen concentration in deionized water, as shown in Fig. 5A. However, the electrocatalytic currents (~-0.27V) after the additions of tea water were lower than the corresponding values of deionized water, as indicated in Fig. 6. The results suggested the dissolved oxygen concentration in tea water was lower than deionized water, in other words, the antioxidant capacity of tea water was higher than deionized water at the same prepared temperature, according with the common sense of life.
In order to investigate further the detection ability of the total antioxidant capacity in real tea water, the dissolved oxygen concentrations of tea water prepared at different temperature were compared. As shown in Fig. 5B to Fig. 5D, the amperometric response of the POD/ZrPR1R2 composite modified electrode to the successive additions of tea water prepared at room temperature, 60 oC and 80 oC, indicated the prepared temperature of tea water was the key factor of antioxidant capacity.
As shown in Fig. 6, the electrocatalytic currents (~-0.27V) after the additions of tea water was decreased along with the increasing of the prepared temperature, attributing to the more antioxidants dissolved from the tea, such as tea polyphenols. When the prepared temperature higher than 60oC, the antioxidant capacity of tea water was not increased along the soaking temperature, the reason may be the more antioxidants dissolved but the more antioxidants oxidized at the higher temperature. So, the optimum temperature to soak tea may be from 60oC to 80oC, according to the antioxidant capacity.
We also explored the influence of soaking time on the antioxidant capacity of tea water by soaking the tea in the room temperature water from 0.5 to 2.5 hours. As shown in Fig. 7 and Fig. 8, the electrocatalytic currents (~-0.27V) after the additions of tea water was decreased along with the increase of soaking time from 0.5 hours to 1.0 hours, but increased along with the increase of soaking time from 1.0 hours to 2.5 hours. The reason maybe attributing to the more antioxidants dissolved from the tea along with the increased time before 1.0 hours. When the soaking time longer than 1.0 hours, the more antioxidants dissolved but the more antioxidants oxidized along with the increased time, so the antioxidant capacity was not increased along with the soaking time. The results suggested the optimum time to soak tea maybe from 1.0 hours to 1.5 hours, according to the antioxidant capacity.