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.