Poly(benzoxazine) as an Immobilization Matrix for Miniaturized ATP and Glucose Biosensors

Abstract

The reproducible immobilization of enzymes represents a key requirement in developing sensitive and fast‐responding amperometric microbiosensors. A microbiosensor for the respective detection of glucose and adenosine‐5′‐triphosphate (ATP) is presented by using a poly(benzoxazine) derivative for the entrapment of the enzymes glucose oxidase (GOD) and hexokinase (HEX) at platinum (Pt) microelectrodes (MEs). For glucose, a sensitivity of 123.05±10.78 pA/mM (n=5) was obtained, which shows twice as high sensitivity compared to microbiosensors that use electrophoretic paints as the immobilization matrix for the same size ME (radius: 25 μm). For the determination of ATP, a sensitivity of 48.47±5.12 pA/μM and a signal‐to‐noise ratio of 40 at physiological pH values were obtained. Apart from their enhanced sensitivity, a significant improvement of these sensors is related to their improved mechanical stability. The applicability of these poly(benzoxine)‐based microbiosensors for ATP detection was demonstrated with measurements at receptor protein tyrosine phosphatase zeta (PTPRζ) osteoblastic cells during mechanical stimulation.

Mechanical stretch activates piezo1 in caveolae of alveolar type I cells to trigger ATP release and paracrine stimulation of surfactant secretion from alveolar type II cells

“…Subsequent measurements of stretch‐induced ATP release using ATP microbiosensors 54 (Figure 5A) confirmed that ATP release from hAELVi cells is dependent on the expression of caveolin‐1 and Ca 2+ ‐entry via piezo1. ATP release from hAELVi wt and hAELVi cav−/− cells increased with increasing stretch amplitudes, however, release from hAELVi cav−/− was significantly lower.…”

“…ATP measurements were performed using an electrochemical biosensor based on surface immobilized glucose oxidase/hexokinase as recently described 54,55 …”

“…ATP measurements were performed using an electrochemical biosensor based on surface immobilized glucose oxidase/ hexokinase as recently described. 54,55 ATP microbiosensors were prepared by immobilizing glucose oxidase (from Aspergillus niger, 1500.000 U/g, Sigma-Aldrich Chemie GmbH) and hexokinase (from yeast, 2500 IU solid, Calbiochem) in a poly(benzoxazine) layer. 54 Pt microelectrodes with a radius of 25 µm were modified by electrodeposition using mixtures of BA-TEPA and GOD/HEX in a ratio of 1:2.5 w/w; the activity ratio for the two enzymes was 1:1.…”

ATP is stored in lamellar bodies to activate vesicular P2X4 in an autocrine fashion upon exocytosis

“…ATP microbiosensors were prepared by immobilizing glucose oxidase (from Aspergillus niger ; 1,500,000 U/g; Sigma-Aldrich Chemie) and hexokinase (from yeast; 2,500 IU solid; Calbiochem, Merck) in a poly(benzoxazine) layer ( Ziller et al, 2017 ) Pt microelectrodes with a radius of 25 µm were modified by electrodeposition using mixtures of BA-TEPA and GOD/HEX in a ratio of 1:2.5 wt/wt; the activity ratio for the two enzymes was 1:1. The synthesis of the precursor was prepared as previously described ( Andronescu et al, 2014 ).…”

Electrochemical Biosensors Based on Membrane-Bound Enzymes in Biomimetic Configurations

“…However, a very interesting transmembrane enzyme for biosensing is ATP-synthase or ATPase. The measurement of adenosin triphosphate (ATP) concentration is of great interest for studying cell metabolism [92] and for the detection of microbial contamination on surfaces [93]. In order to develop a sensitive and versatile biosensor for ATP detection, the ATPase from Escherichia coli was reconstituted on a floating phospholipid bilayer over a gold electrode modified with a 4-APh SAM ( Figure 5).…”