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.