Immobilization–Stabilization of Proteins on Nanofibrillated Cellulose Derivatives and Their Bioactive Film Formation

Abstract

In a number of different applications for enzymes and specific binding proteins a key technology is the immobilization of these proteins to different types of supports. In this work we describe a concept for protein immobilization that is based on nanofibrillated cellulose (NFC). NFC is a form of cellulose where fibers have been disintegrated into fibrils that are only a few nanometers in diameter and have a very large aspect ratio. Proteins were conjugated through three different strategies using amine, epoxy, and carboxylic acid functionalized NFC. The conjugation chemistries were chosen according to the reactive groups on the NFC derivatives; epoxy amination, heterobifunctional modification of amino groups, and EDC/s-NHS activation of carboxylic acid groups. The conjugation reactions were performed in solution and immobilization was performed by spin coating the protein-NCF conjugates. The structure of NFC was shown to be advantageous for both protein performance and stability. The use of NFC allows all covalent chemistry to be performed in solution, while the immobilization is achieved by a simple spin coating or spreading of the protein-NFC conjugates on a support. This allows more scalable methods and better control of conditions compared to the traditional methods that depend on surface reactions.

Effect of citric acid crosslinking cellulose‐based hydrogels on osteogenic differentiation

“…20 Materials surface properties influence the composition of the adsorbed protein compounds, which in turn regulates how cells respond to the material. [21][22][23][24] Understanding the relationship between material surface properties, adsorbed molecules, and cellular responses is essential to designing optimal material surfaces for implantation and tissue engineering. In fact, it is recognized that the behavior of the adhesion and proliferation of different cell lines on polymeric materials depend on the surface characteristic such as hydrophilicity/ hydrophobicity, chemistry, charge, roughness, and so forth.…”

Nanocellulose and Proteins: Exploiting Their Interactions for Production, Immobilization, and Synthesis of Biocompatible Materials

“…Immobilization can be carried out by different mechanisms, involving covalent or noncovalent attachment, biochemical affinity, and physical adsorption (van de Waals forces, hydrogen bonds, electrostatic and hydrophobic interactions). The immobilization of enzymes onto a material can help to increase their thermal and pH stability and provide relative longevity and reusability [15]. This could also allow substrates to be modified for biosensors, industrial applications, and continuous catalytic processes [15][16][17], as discussed in the next sections.…”

“…This technique was shown to extend enzyme activity (although decreased to 42% by immobilization) and helped to stabilize it against thermal and pH fluctuations [14]. Since the undertakings of Ong et al [14], other successful studies utilizing covalent attachment have also been conducted [15,18,19]. Arola et al [15] used CNF to covalently immobilize two types of proteins (alkaline phosphatase and anti-hydrocortisone antibody).…”

“…Since the undertakings of Ong et al [14], other successful studies utilizing covalent attachment have also been conducted [15,18,19]. Arola et al [15] used CNF to covalently immobilize two types of proteins (alkaline phosphatase and anti-hydrocortisone antibody). Specialized techniques were utilized to conjugate the proteins to three CNF-derived substrates based on their prominent functional groups (epoxy, amine, and carboxylic acid) [15].…”

Regenerated Cellulose Fiber and Film Immobilized with Lysozyme

“…Xyloglucan, xyloglucan endotransglycosylase, and chemically modified xyloglucan oligosaccharides were used to produce aminated xyloglucan adsorbed to cellulose surfaces [7]. Arola and his co-workers conjugated alkaline phosphatase to nano fibrillated cellulose activated by amine, epoxy, and carboxylic acid [8]. A fusion protein containing cellulose-binding-domain (CBD) was found effective and stable when adsorbed to cellulose [9][10][11][12][13][14][15].…”