Reversible Two‐Enzyme Coimmobilization on pH‐Responsive Imprinted Monolith for Glucose Detection

Abstract

A new enzymatic glucose biosensor based on reversible co-immobilization of horseradish peroxidase (HRP) and glucose oxidase (GOx) on a pH-responsive imprinted monolith is prepared. The poly(4-vinylphenylboronic acid)-grafted imprinted polymer using HRP as a template is formed via surface initiated atom transfer radical polymerization within the pores of brominated poly(glycidyl methacrylate-co-ethylene dimethacrylate) macroporous monolith contained in a 100 μm I.D. capillary column. The two enzymes conjugate is formed via the strong affinity interaction between biotin-labeled GOx and streptavidin-labeled HRP. The modulation of the external pH value enables reusability of the biosensor simply using stripping of the inactive enzymes at a low pH value and subsequent immobilization of fresh enzymes at a high pH value. Under the optimized conditions, the enzymatic biosensor features excellent performance in detection of glucose with a linear range of its concentration from 0.11 to 38.85 mmol/L and a limit of detection of 0.03 mmol/L. A relative standard deviation of 3.7% is calculated from determination of twenty glucose samples. This novel enzymatic sensing system is successfully applied for determination of glucose in human serum, and confirms an enhancement both in selectivity and specificity compared to the more traditionally clinical methods.

Immobilizing Enzyme Biocatalysts onto Porous Polymer Monoliths Prepared from Cocontinuous Polymer Blends

“…Melt-processing is industrially scalable and allows control over polymer scaffold size and shape, which, combined with the robust mechanical properties, facilitates their integration into continuous flow systems, as demonstrated in this work with their insertion in silicone tubing using tie-wrapsfeatures that are also interesting for the development of sensing devices. 24,25 In addition, fluid circulation within the scaffolds only requires the application of low pressures (realized manually in this work), preventing the necessity of using high pressure and/or costly pumping systems. Finally, a variety of enzymes can be immobilized and reused, with the possibility of tuning the grafted enzymes' surface density and the total grafted amount on the dextran layer, by controlling the specific area of the PCL scaffolds via the quiescent annealing time and content in silica microparticles.…”

Molecularly imprinted monoliths: Recent advances in the selective recognition of biomacromolecules related biomarkers

“…Surface-initiated atom transfer radical polymerization is a robust and versatile strategy for the preparation of various MIP monoliths with controlled molecular characteristics of imprinted polymer chains [36][37][38]. On the other hand, smart materials that can respond in their structure and polarity by altering external stimuli (temperature, pH, ionic strength, etc.)…”

Flow‐through enzymatic reactors using polymer monoliths: From motivation to application

“…The preparation and application of glucose oxidase (GOx)‐bearing reactors based on polymer monoliths were described in several works [82,83]. In a recent work [84], a new enzymatic glucose biosensor with a detection range from 0.11 to 38.85 mmol/L was developed, where horseradish peroxidase (HRP) and GOx were reversibly coimmobilized on a pH‐responsive imprinted monolith. The poly(4‐vinylphenylboronic acid)‐grafted imprinted polymer using HRP as a template was formed within the pores of the brominated poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) macroporous monolith.…”