Fig. 5 pH and temperature dependence of catalysis with free or immobilized GOX. (a) The relative catalytic activity of free or immobilized enzymes at pH 4-9. (b) The relative catalytic activity of free or immobilized GOX at 40-70 °C. (c) The relative catalytic activity of free GOX after heating at 40 °C, 50 °C, 60 °C, or 70 °C for 0-90 min. (d) The relative catalytic activity of immobilized GOX after heating at 40 °C, 50 °C, 60 °C, or 70 °C for 0-90 min.
Temperature is one of the key factors for the catalysis reaction and the stability of enzymes. Fig. 5b showed that the catalytic activity of both free and immobilized GOX decreased with temperature above 50 °C. For the immobilized GOX, the decrease of activity was much slower than that of free GOX, indicating better tolerance of immobilized enzymes to heating. The favorable ability of resistance to thermal inactivation for the immobilized enzymes may ascribed to the protection of the alginate carriers. The thermal stability of immobilized enzymes was further investigated by the catalysis assays of enzyme-loaded microfibers after heat treatments at different temperature for 0-90 min (Figs. 5cand d ). For the heat treatments at 40 °C and 50 °C, immobilized GOX exhibited moderate decrease in the relative catalytic activity with the increase of heating time, which is similar to that of free GOX. Meanwhile, heating at 70 °C readily resulted in the substantial inactivation of both free GOX and immobilized GOX. For the heat treatment at 60 °C, enzyme-immobilized microfibers retained about 60% of the catalytic activity after heating for 60 and 90 min whereas the relative catalytic activity of free GOX was less than 20% under the same conditions. The results demonstrated that the thermal stability of immobilized enzymes in alginate-based microfibers was enhanced due to the increased rigidity of enzyme caused by the firm attachment to microfibers and the protection of alginate matrix for enzymes (Bedade et al., 2019; Todea et al., 2021; Zhang et al., 2015).
3.5 HRP and GOX co-immobilized microfibers for the visual detection of glucose
Besides the excellent thermal stability and recyclability of the immobilized enzymes, microfluidic fabrication endowed alginate-based fibers with tunable composition and size as well as good mechanical properties for knitting, which suggests the potential applications in the construction of detection chips on demand. To validate this potential, microfibers loaded with HRP, GOX, and the chromogenic substrate TMB were prepared under the optimal conditions and wrapped on a glass slide for the visual detection of glucose (Fig. 6 ). By the catalysis with GOX, glucose could be oxidized to gluconic acid in aqueous solution while oxygen was simultaneously converted to H2O2. which oxidizes TMB to a green-blue product OxTMB in the presence of HRP (Scheme 1b ) (Dong et al., 2012; Lin et al., 2014; Ren et al., 2019).
First, the weight ratio of GOX-PAA and HRP-PAA was optimized to improve the catalytic efficiency of the multi-enzyme immobilized microfibers. When the total amount of GOX and HRP was constant, the conversion of TMB to OxTMB increased with GOX as HRP/GOX weight ratio decreased from 8:1 to 1:2. Because the molecular weight of GOX is about 4 times as that of HRP, the molar ratio of HRP to GOX was about 32:1 to 2:1, suggesting that the chromogenic reaction was dominated by the catalysis with GOX under these conditions. More GOX improved the oxidation of glucose and the subsequent conversion of TMB to OxTMB in the presence of abundant HRP. Further increase of GOX did not improve the chromogenic reaction due to the decrease of HRP when HRP/GOX weight ratio was lower than 1:2 (Fig. 6a ). In general, enzymes co-immobilized microfibers exhibited obvious absorption for the successive reactions at HRP/GOX weight ratio of 1:1 to 1:8. These microfibers showed great GOX activity at the ratio of 4:1 to 1:2 (Fig. S5 ). HRP and GOX with equivalent weight were selected for the preparation of enzyme-loaded microfibers for glucose detection.