A Crosstalk- and Interferent-Free Dual Electrode Amperometric Biosensor for the Simultaneous Determination of Choline and Phosphocholine

Abstract

Choline (Ch) and phosphocholine (PCh) levels in tissues are associated to tissue growth and so to carcinogenesis. Till now, only highly sophisticated and expensive techniques like those based on NMR spectroscopy or GC/LC- high resolution mass spectrometry permitted Ch and PCh analysis but very few of them were capable of a simultaneous determination of these analytes. Thus, a never reported before amperometric biosensor for PCh analysis based on choline oxidase and alkaline phosphatase co-immobilized onto a Pt electrode by co-crosslinking has been developed. Coupling the developed biosensor with a parallel sensor but specific to Ch, a crosstalk-free dual electrode biosensor was also developed, permitting the simultaneous determination of Ch and PCh in flow injection analysis. This novel sensing device performed remarkably in terms of sensitivity, linear range, and limit of detection so to exceed in most cases the more complex analytical instrumentations. Further, electrode modification by overoxidized polypyrrole permitted the development of a fouling- and interferent-free dual electrode biosensor which appeared promising for the simultaneous determination of Ch and PCh in a real sample.

Keywords: alkaline phosphatase; choline analysis; choline oxidase; dual electrode biosensor; electropolymerized non-conducting polymer; enzyme immobilization; flow injection analysis; overoxidized polypyrrole; phosphocholine analysis; simultaneous determination.

Scheme 1

The approach for the simultaneous determination by the dual electrode (parallel configuration) amperometric biosensor thin-layer cell (as shown in Scheme S1 in Supplementary Materials) used in the flow injection experiments for Ch and PCh analysis (a) and the example of a crosstalk effect in measurements (b). ChO and ALP/ChO refer to ChO and ALP/ChO biosensors, respectively, the black curved arrows (left side in both sketches) represent the enzymatic detection, the blue ones, the carrier flow direction while i_Ch and i_PCh the currents for Ch and PCh measurements, respectively, as measured at their respective electrical connections. The larger black curved arrow (right side in right sketch) represents the potential crosstalk effect due to hydrogen peroxide detection from the side electrode.

Figure 1

Typical flow injection responses at the dual electrode biosensor for replicate injections of Ch 0.1 mM (Ch1), 0.05 mM (Ch2), 0.025 mM (Ch3), 0.01 mM (Ch4) and PCh 0.5 mM (PCh1), 0.25 mM (PCh2), 0.1 mM (PCh3), 0.05 mM (PCh4). Upper and lower traces refer to responses at Pt/ChO and Pt/Cho-ALP biosensors, respectively. Carrier solution: borate buffer (pH 9, I 0.1 M); flow rate 1 mL/min; injection volume 20 µL. Other experimental conditions as described in Section 2.

Figure 2

Calibration curves at the dual electrode biosensor for replicate injections of Ch and PCh. Continuous lines refer to Michaelis-Menten fitting of data. Carrier solution: borate buffer (pH 9, I 0.1 M); flow rate 1 mL/min; injection volume 20 µL. Other experimental conditions as described in the Section 2.

Figure 3

Typical flow injection responses at the dual electrode biosensor for replicate injections of Ch 2.5 µM (Ch1), 1.25 µM (Ch2), 0.625 µM (Ch3) and PCh 25 µM (PCh1), 12.5 µM (PCh2), 6.25 µM (PCh3); B refers to responses due to replicate injections of the carrier solution, i.e., the buffer used for preparing sample solutions. Upper and lower traces refer to responses at Pt/ChO and Pt/ChO-ALP biosensors, respectively. Carrier solution: borate buffer (pH 9, I 0.1 M); flow rate 1 mL/min; injection volume 20 µL. Other experimental conditions as described in Section 2.

Figure 4

Calibration 3d plot at the Pt/ChO electrode of the dual electrode biosensor for replicate injections of mixtures of Ch and PCh. Carrier solution: borate buffer (pH 9, I 0.1 M); flow rate 1 mL/min; injection volume 20 µL. Other experimental conditions as described in Section 2.

Figure 5

Calibration lines at the Pt/ChO electrode of the dual electrode biosensor for replicate injections of mixtures of Ch and PCh at several concentration levels of PCh (upper plot) and Ch (lower plot). Continuous lines refer to linear fitting of data. Carrier solution: borate buffer (pH 9, I 0.1 M); flow rate 1 mL/min; injection volume 20 µL. Other experimental conditions as described Section 2.

Figure 6

Calibration 3d plot at the Pt/ChO-ALP electrode of the dual electrode biosensor for replicate injections of mixtures of Ch and PCh. Carrier solution: borate buffer (pH 9, I 0.1 M); flow rate 1 mL/min; injection volume 20 µL. Other experimental conditions as described in Section 2.

Figure 7

Calibration lines at the Pt/ChO-ALP electrode of the dual electrode biosensor for replicate injections of mixtures of Ch and PCh at several concentration levels of PCh (upper plot) and Ch (lower plot). Continuous lines refer to linear fitting of data. Carrier solution: borate buffer (pH 9, I 0.1 M); flow rate 1 mL/min; injection volume 20 µL. Other experimental conditions as described in Section 2.

Figure 8

Typical flow injection responses at the oPPy modified dual electrode biosensor for replicate injections of Ch 2.5 µM (Ch1), 1.25 µM (Ch2), 0.625 µM (Ch3), PCh 25 µM (PCh1), 12.5 µM (PCh2), 6.25 µM (PCh3) compared to responses due to AA 0.1 mM, AU 0.5 mM, and CYS 0.2 mM; B refers to responses due to replicate injections of the carrier solution, i.e., the buffer used for preparing sample solutions. Upper and lower traces refer to responses at Pt/oPPy/ChO and Pt/oPPy/ChO-ALP biosensors, respectively. Carrier solution: borate buffer (pH 9, I 0.1 M); flow rate 1 mL/min; injection volume 20 µL. Other experimental conditions as described in Section 2.