A New Approach for Interferent-Free Amperometric Biosensor Production Based on All-Electrochemically Assisted Procedures

Abstract

A new approach in amperometric enzyme electrodes production based on all-electrochemically assisted procedures will be described. Enzyme (glucose oxidase) immobilization was performed by in situ co-crosslinking of enzyme molecules through electrophoretic protein deposition, assuring enzyme immobilization exclusively onto the transducer surface (Pt electrode). Analogously, the poor selectivity of the transducer was dramatically improved by the electrosynthesis of non-conducting polymers with built-in permselectivity, permitting the formation of a thin permselective film onto the transducer surface, able to reject common interferents usually found in real samples. Since both approaches required a proper and distinct electrochemical perturbation (a pulsed current sequence for electrophoretic protein deposition and cyclic voltammetry for the electrosynthesis of non-conducting polymers), an appropriate coupling of the two all-electrochemical approaches was assured by a thorough study of the likely combinations of the electrosynthesis of permselective polymers with enzyme immobilization by electrophoretic protein deposition and by the use of several electrosynthesized polymers. For each investigated combination and for each polymer, the analytical performances and the rejection capabilities of the resulting biosensor were acquired so to gain information about their sensing abilities eventually in real sample analysis. This study shows that the proper coupling of the two all-electrochemical approaches and the appropriate choice of the electrosynthesized, permselective polymer permits the easy fabrication of novel glucose oxidase biosensors with good analytical performance and low bias in glucose measurement from typical interferent in serum. This novel approach, resembling classical electroplating procedures, is expected to allow all the advantages expected from such procedures like an easy preparation biosensor, a bi-dimensional control of enzyme immobilization and thickness, interferent- and fouling-free transduction of the electrodic sensor and, last but not the least, possibility of miniaturization of the biosensing device.

Keywords: biosensor; electroanalytical chemistry; electrophoretic enzyme immobilization; electropolymerization; glucose biosensor; permselective polymers.

Scheme 1

Schematization of the EPD process referring to the electrophoretic protein deposition assisted in situ co-crosslinking enzyme immobilization used. The large rectangle couples in each left panel represent the electrodes, each with its proper charge. Circles and ellipses between electrodes for each left panel represent the albumin protein and the enzyme molecules, respectively, while each right panel shows their concentrations near the electrode surface when the electrical field is applied (lower panel) or not (upper panel) between electrodes.

Figure 1

Typical cyclic voltammograms of potassium ferricyanide 5 mM in phosphate buffer (pH 7, I 0.1 M) on bare Pt electrode (a) and Pt-modified electrodes with poly-2-naphtol (P2NAP) before (b) and after (c) the current pulse application for EPD enzyme immobilization. The scan rate was 50 mV/s and the electrode diameter 3 mm; other conditions as described in the Materials and Methods section.

Figure 2

Typical cyclic voltammograms of potassium ferricyanide 5 mM in phosphate buffer (pH 7, I 0.1 M) on bare Pt electrode (a) and Pt-modified electrodes with poly-o-aminophenol (PoAP) before (b) and after (c) the current pulse application for EPD enzyme immobilization. The scan rate was 50 mV/s and the electrode diameter 3 mm; other conditions as described in the Materials and Methods section.

Figure 3

(a) Left panels—Typical cyclic voltammograms relevant to the electrosynthesis of poly-2-naphtol (P2NAP) from a solution of 2-naphtol 5 mM in phosphate buffer (pH 7, I 0.1 M) on bare Pt electrode (upper panel) and Pt-modified electrode by in situ co-crosslinking of GOD and BSA by EPD (lower panel). (b) Right panels—Typical cyclic voltammograms relevant to the electrosynthesis of poly-o-phenylenediamine (PoPD) from a solution of o-phenylenediamine 5 mM in phosphate buffer (pH 7, I 0.1 M) on bare Pt electrode (upper panel) and Pt-modified electrode by in situ co-crosslinking of GOD and BSA by EPD (lower panel). In all panels, the arrows show the time course of scan cycles. The scan rate was 50 mV/s and the electrode diameter 3 mm; other conditions as described in the Materials and Methods section.

Figure 4

Typical current–time responses at a rotating disk Pt/PoAP/GOD electrode for sequential additions of glucose to an air-saturated phosphate buffer (pH 7, I 0.1 M). Glucose adding in figure refers to total glucose concentrations of 1.25, 2.49, 3.72, 4.95, and 7.39 mM. The disk rotation rate was 1000 rpm and the electrode diameter 2 mm; other conditions as described in the Materials and Methods section.

Figure 5

Typical current–time responses at a rotating disk Pt/PoAP/GOD electrode for additions of glucose (G) 5 mM, ascorbic acid (AA) 0.1 mM, uric acid (UA) 0.5 mM, paracetamol (PA) 0.2 mM, and cysteine (CYS) 0.1 mM to an air-saturated phosphate buffer (pH 7, I 0.1 M). The disk rotation rate was 1000 rpm and the electrode diameter 2 mm; other conditions as described in the Materials and Methods section.

Figure 6

Typical current–time responses at a rotating disk Pt/GOD/PoAP electrode for successive additions of glucose to an air-saturated phosphate buffer (pH 7, I 0.1 M). Each response in the figure refers to total glucose concentrations of 1.25, 2.49, 3.72, 4.95, and 7.39 mM and more. The disk rotation rate was 1000 rpm and the electrode diameter 2 mm; other conditions as described in the Materials and Methods section.

Figure 7

Typical current–time responses at a rotating disk Pt/GOD/PoAP electrode for additions of glucose (G) 5 mM, ascorbic acid (AA) 0.1 mM, uric acid (UA) 0.5 mM, paracetamol (PA) 0.2 mM, and cysteine (CYS) 0.1 mM to an air-saturated phosphate buffer (pH 7, I 0.1 M). The disk rotation rate was 1000 rpm and the electrode diameter 2 mm; other conditions as described in the Materials and Methods section.

Figure 8

Typical current–time responses at a rotating disk Pt/GOD/P2NAP electrode for successive additions of glucose to an air-saturated phosphate buffer (pH 7, I 0.1 M). Glucose addition in the figure refers to total glucose concentrations of 1.25, 2.49, 3.72, 4.95, and 7.39 mM, and more. The disk rotation rate was 1000 rpm and the electrode diameter 2 mm; other conditions as described in the Materials and Methods section.

Figure 9

Typical current–time responses at a rotating disk Pt/GOD/P2NAP electrode for additions of glucose (G) 5 mM, ascorbic acid (AA) 0.1 mM, uric acid (UA) 0.5 mM, paracetamol (PA) 0.2 mM, and cysteine (CYS) 0.1 mM to an air-saturated phosphate buffer (pH 7, I 0.1 M). The disk rotation rate was 1000 rpm and the electrode diameter 2 mm; other conditions as described in the Materials and Methods section.