Fig.
2 Preparation and the catalytic activity of microfibers based on
alginate and enzyme-PAA conjugates. (a) Synthesis of enzyme-PAA
conjugates. (b) FT-IR spectra of PAA, GOX, HRP, GOX-PAA conjugate, and
HRP-PAA conjugate. (c) Reusability of GOX-PAA immobilized alginate
microfibers.
To reduce the leakage from microfibers, enzymes were conjugated to PAA
prior to mix with alginate for microfluidic fabrications (Fig.
2a ). FTIR spectroscopy was utilized for the analysis of enzyme-PAA
conjugates (Nie et al., 2005). The FTIR spectra of PAA showed typical
absorption of COOH at 1702 cm-1 which corresponds to
the stretching vibration of C=O bonds while the spectra of enzymes
showed absorption of amide I and II at 1634 cm-1 and
1538 cm-1, respectively (Fig. 2b ). After
coupling reaction and subsequently dialysis, the IR spectra of resulting
mixture of PAA and enzymes demonstrated the coexistence of the
characteristic absorption of the starting components, indicating the
successful preparation of enzyme-PAA conjugates (Gejji & Fernando,
2018). It is worth noting that enzyme-alginate conjugates cannot be
prepared via EDC/NHS coupling strategy (Fig. S4 ). The enzyme
cannot covalently bind to alginate which may be ascribed to the steric
hindrance of the cyclic structure to the coupling reaction on the COOH
of alginate. Abundant carboxyl groups in the flexible PAA enables not
only the feasible conjugation with proteins but also the robust
chelation with Ca2+ which is beneficial for the
formation of microfibers and the immobilization of enzymes in the
fibers.
By the covalent attachment to Ca2+-crosslinkable PAA,
the leakage of enzymes was inhibited and the reusability of the
enzyme-immobilized microfibers was substantially improved (Fig.
2c ). The insert images showed that the color of solution after
catalysis reaction with GOX-immobilized microfibers did not change
significantly in the first to the seventh use, indicating the consistent
catalytic activity of enzymes in microfibers during these reactions. The
quantitative data showed that the immobilized GOX-PAA retained over 85%
of its initial activity after seven cycles of utilization. The highly
remained activity could be attributed to the firm attachment of enzymes
to PAA and the robust crosslinking of alginate and GOX-PAA with
Ca2+, which leads to efficacious prevention of enzyme
from leakage during the enzymatic reaction. These microfibers were
capable of ease collection and handling, as well as modulatable size and
component, thus, offering promising potential in enzyme immobilization
for multiple purposes.
3.2 Characterization of alginate-based microfibers
SEM images of alginate microfibers with diameter of 50 and 100 μm (the
size measured by optical microscope during microfluidic fabrication)
showed that the microfibers had smooth surfaces and the change of
preparation settings (e.g., the flow rate and the inner diameter of
capillary) did not remarkably affect the morphologies of microfibers
(Figs. 3a and 3b ). The rapid chelation of sodium
alginate with calcium ions at the orifice of tapered aperture, and the
high viscosity of PEG solution may suppress the diffusion of alginate
from the inner phase to the outer phase, leading to the formation of
smooth surfaces. Moreover, the size of dry microfibers observed by SEM
was consistent with that at swollen state measured by optical microscope
in the preparation of microfibers, indicating the robust crosslinking in
the microfibers and no significant shrinkage during drying for SEM
observation.