Enzyme immobilization is a core technique of enzymatic biochemical engineering because it can remarkably reduce the cost of enzymes and improve the enzyme recovery procedure. The most crucial issues for enzyme immobilization include (1) maintaining its activity, both in the immobilization process and in the batchwise catalyst course; (2) separating the immobilized enzyme from the reaction mixture; and (3) the readiness and cost of the immobilization process. Herein, we report a new strategy to immobilize xylanase within a hydrophilic and nonswelling polyethylene glycol (PEG) net-cloth grafted on a polypropylene nonwoven fabric (PPNWF) membrane by a visible light-induced surface graft cross-linking polymerization. The xylanase was in situ entrapped within the PEG net-cloth. The nonswelling PEG net-cloth can effectively maintain the xylanase without leakage in long-term operation. As for the hydrolysis of corncob hemicelluloses, the experimental results showed that the as-formed immobilized xylanase retained 80% of its original activity after being reused for 25 cycles and 60% after 50 cycles, which is far better than that of other immobilization methods by entrapment. Notably, this simple in situ entrapment of enzymes on routine polymeric matrix would lead to an easy industrial production at low cost, while the form of end-products as a sheet can be readily separated from the reaction mixture and reused for batchwise production. After immobilization, the xylanase showed no significant shift in pH or temperature optima as compared with its free form. These results suggest that the immobilization of xylanase within the PEG net-cloth grafted on PPNWF is promising for industrial applications because of its long-term operation stability and convenient recovery for reuse.

The use of the mixed catalytic system with several enzymes can provide multiple benefits in terms of the cost, simplification of a multistep reaction, and effectiveness of complex chemical reactions. Although study of different enzyme coimmobilization systems has attracted increasing attention in recent years, separately immobilizing enzymes which can not coexist on one support is still one of the great challenges. In this paper, a simple and effective strategy was introduced to separately encapsulate incompatible trypsin and transglutaminase (TGase) into different poly(ethylene glycol) (PEG) network layer grafted on low-density polyethylene (LDPE) film via visible light induced living photografting polymerization. As a proof of concept, this dual-enzyme separately loaded film was used to catalyze the synthesis of a new target antitumor drug LTV-azacytidine. The final results demonstrated that this strategy could maintain higher activities of both enzymes than the mixed coimmobilization method. And the mass spectra analysis results demonstrated that LTV-azacytidine was successfully synthesized. We believe that this facile and mild separately immobilizing incompatible enzyme strategy has great application potential in the field of biocatalysis.

A new visible light induced graft polymerization method was utilized to prepare pH-sensitive hydrogel layers covalently attached to polymer substrates for drug delivery. In our strategy, isopropyl thioxanthone semi-pinacol (ITXSP) dormant groups were firstly introduced on the surface of a polycaprolactone (PCL) film by a UV-induced abstracting hydrogen-coupling reaction. Then visible light induced graft cross-linking polymerization was performed to initiate polymerization of poly(ethylene glycol) diacrylate (PEGDA) and acrylic acid (AA), resulting in the formation of a hydrogel layer. The thickness of the hydrogel film can be controlled by varying the exposure time and monomer composition. The grafted hydrogel layers showed a flat morphology and dense structure, which is different from the traditional reported porous structure. The water contact angle of the hydrogel layer exhibited a reversible change from 38° to 18° when the film was alternatively treated in buffers of pH 2.0 and 7.4, respectively. Patterned hydrogel layers were prepared as a model to determine the change in the height of the grafted hydrogel layer as a function of pH. As the pH changed from 2.0 to 7.4, the hydrogel pattern showed an increase in height due to the swelling of the hydrogel network, and the hydrogel layer formed by 0.2 wt% PEGDA and 25 wt% AA showed the most increase (30%) in height. Bovine serum albumin (BSA) and lysozyme as models of protein drugs were incorporated in the hydrogel network, and their release also showed obvious pH-sensitivity. At pH 2.0, hydrogels present a faster initial burst release due to the squeezing mechanism. Tertiary structure analysis showed that encapsulation and release did not affect the protein conformation. These findings have improved our understanding of hydrogel thin films, which may be useful as potential vehicles of therapeutic proteins in drug delivery applications.

Protein microarrays have become vital tools for various applications in biomedicine and bio-analysis during the past decade. The intense requirements for a lower detection limit and industrialization in this area have resulted in a persistent pursuit to fabricate protein microarrays with a low background and high signal intensity via simple methods. Here, we report on an extremely simple strategy to create three-dimensional (3D) protein microarrays with an anti-fouling background and a high protein capacity by photo-induced surface sequential controlled/living graft polymerization developed in our lab. According to this strategy, “dormant” groups of isopropyl thioxanthone semipinacol (ITXSP) were first introduced to a polymeric substrate through ultraviolet (UV)-induced surface abstraction of hydrogen, followed by a coupling reaction. Under visible light irradiation, the ITXSP groups were photolyzed to initiate surface living graft polymerization of poly(ethylene glycol) methyl methacrylate (PEGMMA), thus introducing PEG brushes to the substrate to generate a full anti-fouling background. Due to the living nature of this graft polymerization, there were still ITXSP groups on the chain ends of the PEG brushes. Therefore, by in situ secondary living graft cross-linking copolymerization of glycidyl methacrylate (GMA) and polyethylene glycol diacrylate (PEGDA), we could finally plant height-controllable cylinder microarrays of a 3D PEG network containing reactive epoxy groups onto the PEG brushes. Through a commonly used reaction of amine and epoxy groups, the proteins could readily be covalently immobilized onto the microarrays. This delicate design aims to overcome two universal limitations in protein microarrays: a full anti-fouling background can effectively eliminate noise caused by non-specific absorption and a 3D reactive network provides a larger protein-loading capacity to improve signal intensity. The results of non-specific protein absorption tests demonstrated that the introduction of PEG brushes greatly improved the anti-fouling properties of the pristine low-density polyethylene (LDPE), for which the absorption to bovine serum albumin was reduced by 83.3%. Moreover, the 3D protein microarrays exhibited a higher protein capacity than the controls to which were attached the same protein on PGMA brushes and monolayer epoxy functional groups. The 3D protein microarrays were used to test the immunoglobulin G (IgG) concentration in human serum, suggesting that they could be used for biomedical diagnosis, which indicates that more potential bio-applications could be developed for these protein microarrays in the future.

During recent research on the fabrication of DNA microarrays, polymers have been intensively investigated as substrates for immobilizing oligonucleotides, due to their low cost, disposability and excellent processing flexibility. Among these, cyclic olefin copolymers (COCs) are of special interest because of their many favorable properties, including high glass transition temperature, low auto-fluorescence, optical clarity and resistance to organic solvents. In the present study a novel strategy has been developed by introducing epoxy groups on the COC surface, based on a confined photocatalytic oxidation (CPO) method. Firstly, a sulfate anion (–SO4−) was introduced on to the COC film by CPO, accompanied by brief (120 s) UV irradiation. The sulfate anions were then hydrolyzed to form hydroxyl groups (–OH), forming a glass-like surface, which readily reacts with a silane coupling agent. In the present study (3-glycidoxypropyl)trimethoxysilane was used as a model for introducing epoxy groups on to COC film, the result confirmed by X-ray photoelectron spectroscopy (XPS), water contact angle measurement and atomic force microscopy (AFM). DNA probes were subsequently spotted on the COC surface and immobilized by reaction between the epoxy groups and amino groups on strands of DNA. The immobilization efficiency of different concentrations of DNA probes on the COC surface ranged from 45% to 65%, comparable to a traditional epoxy-functionalized glass slide. Hybridization with complementary strands of this microarray was successfully achieved, and the fluorescence intensity after hybridization was readily tuned by adjusting the probe immobilization density, or the target DNA sequence concentration in a hybridization solution. This simple approach has considerable potential in the construction of low-cost polymer biochips.

Although the hydrogel network has been widely investigated as a carrier for enzyme immobilization, to in situ encapsulate enzymes into a hydrogel network in an efficient, practical, and active way is still one of the great challenges in the field of biochemical engineering. Here, we report a new protocol to address this issue by encapsulating enzyme into poly(ethylene glycol) (PEG) hydrogel network grafted on polymeric substrates. In our strategy, isopropyl thioxanthone semipinacol (ITXSP) dormant groups were first planted onto the surface of a plastic matrix with low density polyethylene (LDPE) film as a model by a UV-induced abstracting hydrogen-coupling reaction. As a proof of concept, lipase, which could catalyze esterification of glucose with palmitic acid, then was in situ net-immobilized into a PEG-based hydrogel network layer through a visible light-induced surface controlled/living graft cross-linking polymerization. This strategy demonstrates the following novel significant merits: (1) in comparison with the UV irradiation or high temperature, the visible light and room temperature used provide a friendly condition to maintain activity of enzyme during immobilization; (2) the uniqueness of controlled/living cross-linking polymerization not only makes it easy to form a uniform PEG hydrogel network, which is a benefit to avoid the leakage of net-immobilizing enzyme, but also to tune the net-thickness or capacity to accommodate enzyme; and (3) as compared to systems of nanoparticles and porous matrixes, the flexible/robust end-products of the surface net-immobilizing enzyme with polymer film are more suitable to be applied in a bioreactor due to their features of easier separation and reuse. We confirmed that this catalytic film could retain almost all of its initial activity after seven batches of 24 h esterifications. The proposed strategy provides an extremely simple, effective, and flexible method for enzyme immobilization.

A new visible light induced graft polymerization method was utilized to prepare pH-sensitive hydrogel layers covalently attached to polymer substrates for drug delivery. In our strategy, isopropyl thioxanthone semi-pinacol (ITXSP) dormant groups were firstly introduced on the surface of a polycaprolactone (PCL) film by a UV-induced abstracting hydrogen-coupling reaction. Then visible light induced graft cross-linking polymerization was performed to initiate polymerization of poly(ethylene glycol) diacrylate (PEGDA) and acrylic acid (AA), resulting in the formation of a hydrogel layer. The thickness of the hydrogel film can be controlled by varying the exposure time and monomer composition. The grafted hydrogel layers showed a flat morphology and dense structure, which is different from the traditional reported porous structure. The water contact angle of the hydrogel layer exhibited a reversible change from 38° to 18° when the film was alternatively treated in buffers of pH 2.0 and 7.4, respectively. Patterned hydrogel layers were prepared as a model to determine the change in the height of the grafted hydrogel layer as a function of pH. As the pH changed from 2.0 to 7.4, the hydrogel pattern showed an increase in height due to the swelling of the hydrogel network, and the hydrogel layer formed by 0.2 wt% PEGDA and 25 wt% AA showed the most increase (30%) in height. Bovine serum albumin (BSA) and lysozyme as models of protein drugs were incorporated in the hydrogel network, and their release also showed obvious pH-sensitivity. At pH 2.0, hydrogels present a faster initial burst release due to the squeezing mechanism. Tertiary structure analysis showed that encapsulation and release did not affect the protein conformation. These findings have improved our understanding of hydrogel thin films, which may be useful as potential vehicles of therapeutic proteins in drug delivery applications.