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.