Surface-Anchored Poly(2-vinyl-4,4-dimethyl azlactone) Brushes as Templates for Enzyme Immobilization

Abstract

We explored surface-anchored poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA) brushes as potential templates for protein immobilization. The brushes were grown using atom transfer radical polymerization from surface-anchored initiators and characterized by a combination of ellipsometry, atomic force microscopy, and X-ray photoelectron spectroscopy. RNase A was immobilized as a model enzyme through the nucleophilic attack of azlactone by the amine groups in the lysines located in the protein. The surface density of RNase A increased linearly from 5 to 50 nm. For 50 nm thick poly(2-vinyl-4,4-dimethyl azlactone) brushes, 7.5 microg/cm2 of RNase A was bound. The kinetics and thermodynamics of RNase A immobilization, the activity relative to surface density, and the pH and temperature dependence were examined. A Langmuir-like model for binding kinetics indicates that the kinetics are controlled by the rate of adsorption of RNase A and has an adsorption rate constant, k(ads), of 2.8 x 10(-8) microg(-1) s(-1) cm3. A maximum relative activity of approximately 0.95, which is near the activity of free RNase A, was reached at 1.2 microg/cm2 (approximately 3.0 monolayers) of immobilized RNase A. The immobilized RNase A had a similar temperature and pH dependence as free RNase A, indicating no significant change in conformation. The PVDMA template was extended to other biotechnologically relevant enzymes, such as deoxyribonuclease I, glucose oxidase, glucoamylase, and trypsin, with relative activities higher than or comparable to those of enzymes immobilized by other means. PVDMA brushes offer an efficient route to immobilize proteins via the ring opening of azlactone without the need for activation or pretreatment while retaining high relative activities of the bound enzymes.

From Self-Assembled Monolayers to Coatings: Advances in the Synthesis and Nanobio Applications of Polymer Brushes

“…Cullen et al used a poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA) brush, which was covalently bonded to the enzyme RNase A to create a type of biosensor ( Figure 13) [174]. The bound RNase A retained up to 95% of the activity of the same concentration of free enzyme.…”

“…Detection methods for biosensors include, micro-ring resonators [176], voltammetry [177][178][179], electrochemical detection [89,92,180], interferometric detection of scattering (iSCAT) [181], as well as secondary label methods such as ELISA [182] and fluorescence [174]. Methods such as fluorescence and ELISA can have a high degree of specificity and are amenable to microarray applications allowing detection of multiple components at once (Figure 14) [183,184].…”

Immobilization of α-amylase onto poly(glycidyl methacrylate) grafted electrospun fibers by ATRP

“…Compared to many substrates such as nanoparticles, nanotubes and nanofiber templates have many advantages, including good mechanical and thermal properties, large surface area, easy handling and controllable porosity [9]. Three fundamental methods used for immobilization of proteins are epoxide ring opening, amidation and nitrilotriacetate complexation [10]. Epoxy groups are attractive among various chemical groups for biomolecule immobilization.…”

Post‐polymerization Modification of Polymer Brushes

“…The Rühe group [56] has polymerized n-methacryloyl-β-alanine succinimide ester from a surface-bound free radical initiator and demonstrated the functionalization of the activated ester with small molecules and oligomers with 80 nm polymer brushes. Cullen et al [57] used ATRP to grow polymer brushes of 2-vinyl-4,4-dimethyl azlactone on a surface to immobilize RNase A. They showed that the enzyme maintained activity while covalently attached to the polymer matrix.…”

“…They showed that the enzyme maintained activity while covalently attached to the polymer matrix. Polymer brushes bearing an azlactone group can also be quantitatively functionalized in aqueous environments without competitive hydrolysis [57][58][59].…”