Immobilization of glucose oxidase to nanostructured films of polystyrene-block-poly(2-vinylpyridine)

Abstract

A critical step for the development of biosensors is the immobilization of the biorecognition element to the surface of a substrate. Among other materials that can be used as substrates, block copolymers have the untapped potential to provide significant advantages for the immobilization of proteins. To explore such possibility, this manuscript describes the fabrication and characterization of thin-films of polystyrene-block-poly(2-vinylpyridine) (PS-b-P2VP). These films were then used to investigate the immobilization of glucose oxidase, a model enzyme for the development of biosensors. According to the results presented, the nanoporous films can provide significant increases in surface area of the substrate and the immobilization of larger amounts of active enzyme. The characterization of the substrate-enzyme interface discussed in the manuscript aims to provide critical information about relationship between the surface (material, geometry, and density of pores), the protein structure, and the immobilization conditions (pH, ionic strength, and protein concentration) required to improve the catalytic activity and stability of the enzymes. A maximum normalized activity of 3300 ± 700 U m−2 was achieved for the nanoporous film of PS-b-P2VP.

In-situ Neutron Reflectometry Study on Adsorption of Glucose Oxidase at Mesoporous Aluminum Oxide Film

“…The protein distribution on and in nanoporous materials is thus an important subject to be clarified, and in-situ spectroscopic techniques for protein adsorption have been developed. [4][5][6][7][8][9][10][11][12][13][14][15][16][17] The distribution of adsorbate on a single nanoporous particle has been often observed by microspectroscopy techniques. 4,5 For a nanoporous film on a solid substrate, spectroscopic methods utilizing optical interference [6][7][8][9][10][11][12][13][14] and waveguide mode [15][16][17] are available for the in-situ observation of the molecular adsorption.…”

Protein adsorption onto nanomaterials for the development of biosensors and analytical devices: A review

“…Surface features and topography can be determined using atomic force microscopy (AFM) [89, 92-94], scanning electron microscopy (SEM), transmission electron microscopy (TEM) [95], and scanning tunneling microscopy (STM)[6]. AFM can render 3D images of surface topography but can be affected by a series of artifacts [96].…”

“…Since it measures the ratio of two values originated by the same signal, the measurements are highly accurate and reproducible. The measurements are complementary to the information obtained by QCM and can be used to determine the optical properties of asubstrate and the thickness of multiple layers on the surface.The technique is simple, nondestructive, has angstrom resolution, and has the capability of allowing the observation of the adsorption process inreal time[94, 134, 135]. The use of imaging ellipsometry can also provide spatial resolution of protein binding [136].…”

“…The latter is particularly interesting due to its high IEP(about 9.5) which serves as a positively charged substrate for the immobilization of GOx (and other negatively-charged proteins) via electrostatic interactions. With the intention of preserving the native structure of the enzyme, Bhakta et al recently demonstrated the potential advantages of nanoporous substrates fabricated using block co-polymers of polystyrene-block-poly(2-vinylpyridine) for the immobilization of GOx [94]. …”

Quantum dot-modified paper-based assay for glucose screening

“…Glucose was selected as a model system because it is important for several fields including biology [20,21], biochemistry [22], and food science [23]. …”