Keywords
Immobilized enzyme, microfluidics, microfiber, glucose oxidase, sodium alginate
Introduction
The increasing importance of industrial biocatalysis has led to the extensive development of heterogeneous biocatalysts employing immobilization technologies (Rudroff et al., 2018). The rational design of stable, active, and reusable immobilized enzymes is an urgent need for the broad application of industrial enzymes. So far, the immobilization of enzymes is a facile and economically feasible strategy to improve their stability and reusability through a variety of methods such as entrapment, encapsulation, adsorption, crosslinking, and covalent attachment to water-insoluble matrices (Basso & Serban, 2019; Bayramoglu & Arica, 2008; Bilal et al., 2019; Cantone et al., 2013; Liese & Hilterhaus, 2013; Liu et al., 2018). Substantial progresses have been made in the development of enzyme immobilization in recent years. Nevertheless, the practical application of a large number of immobilized biocatalysts still suffers from varying problems, e.g., enzyme leaching, low stability, limited diffusion, deactivation, sophisticated recycling, which reduces the catalytic efficiency and hinders their industrial application (Bilal & Iqbal, 2019; Liang et al., 2020; Secundo, 2013; Zhou & Hartmann, 2013). The exploration of appropriate supporting matrix and novel technologies for enzyme immobilization has attracted intensive attentions (Grant et al., 2018; He et al., 2020; Henderson et al., 2019; Ho et al., 2019; Jannat & Yang, 2020; Ko et al., 2019; Liang et al., 2020; Liu & Nidetzky, 2021; Ren et al., 2019; Teepakorn et al., 2021; Yang et al., 2020; Zanker et al., 2021; Zhu et al., 2019b).
Microfluidic platform is a promising technique for manufacturing customizable structures integrating with rapid crosslinking strategies (Hu et al., 2020; Huang et al., 2020; Jeong et al., 2004; Jun et al., 2014; Ren et al., 2019; Shao et al., 2019; Teepakorn et al., 2021). The convergence of the emerging microfluidic technology and materials science enables novel applications in varying fields including enzyme immobilization (Ho et al., 2019; Kabernick et al., 2022; Teepakorn et al., 2021; Zhu et al., 2019b). Alginate is a natural biomaterial that widely used in immobilization of protein/enzyme, drug, and cell because of the rapid crosslinking by multivalent cations (e.g., Ca2+ or Ba2+) (Bedade et al., 2019; Cheng et al., 2014; Jeon et al., 2009; Kahya & Erim, 2019; Pawar & Edgar, 2012; Qin, 2008; Yu et al., 2017). Alginate-based microfibers, microspheres, and microparticles can be feasibly fabricated via microfluidics technique which are widely used in fundamental researches and practical applications (Cheng et al., 2014; Jeong et al., 2004; Jun et al., 2014; Pawar & Edgar, 2012; Qin, 2008; Shin et al., 2007; Yu et al., 2017). They are capable of modifying or blending with other organic or inorganic materials for the immobilization of protein/enzyme (Bedade et al., 2019; Coppi et al., 2002; Lee & Lee, 2016; Wang et al., 2011; Wu et al., 2015), controlled release of protein drug (Kahya & Erim, 2019), or the detection of alkaline phosphatase, hydrofluoric acid, lactate, glucose (Gunatilake et al., 2021; Lee & Lee, 2016; Li et al., 2019). It has been reported that the calcium-crosslinked alginate hydrogel possesses a microporous structure, which benefits the rapid diffusion of substrates and products during catalysis while leads to the leakage of physically adsorbed enzymes from the matrix as well (Shao et al., 2018; Zdarta et al., 2018). To address this challenge, covalent binding or crosslinking was introduced to enzyme immobilization to improve the stability and suppress enzyme leaching (Jannat & Yang, 2020; Sheldon & van Pelt, 2013). In addition, the microfluidic fabrication endows alginate microfibers with flexible manipulation on the diameter, composition, and pore size of microfibers, which plays a substantial role in the development of supports for enzymes with specific physicochemical features (Cheng et al., 2014; Jun et al., 2014; Yu et al., 2017; Zhu et al., 2019b).
In this study, we developed co-flow microfluidic devices for the fabrication of alginate-based microfibers in coaxial glass capillaries. This approach is capable of fine tuning the diameter and crosslinking degree of microfibers. Moreover, alginate microfibers with covalently bound enzymes were readily constructed and easily collected using this microfluidic platform, showing excellent reusability and improved thermal stability. After loading with glucose oxidase and horseradish peroxidase, the alginate microfibers are able to sense glucose in a wide range of concentrations (Scheme 1 ). Due to the feasible modulation of the physicochemical properties of microfibers, the microfluidic platform may have great potential in the construction of microfiber carriers for enzyme immobilization and multitarget detection.