Sensitive electrochemical detection of horseradish peroxidase at disposable screen-printed carbon electrode

Abstract

A rapid, simple and sensitive electrochemical assay of horseradish peroxidase (HRP) performed on disposable screen-printed carbon electrode was developed. HRP activities were monitored by square-wave voltammetric (SWV) measuring the electroactive enzymatic product in the presence of o-aminophenol and hydrogen peroxide substrate solution. SWV analysis demonstrated a greater sensitivity and shorter analysis time than the widely used amperometric and differential-pulsed voltammetric methods. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were optimized. Under optimized conditions, a linear response for HRP from 0.003 - 0.1 U/mL and a detection limit of 0.002 U/mL (1.25×10(-15) mol in 25 μL) were obtained with a good precision (RSD = 8%; n = 6). This rapid and sensitive HRP assay with microliters-assay volume could be readily integrated to portable devices and point-of-care (POC) diagnosis applications.

Scheme 1

Scheme 2

Fig. 1

Cyclic voltammograms of o-AP in the absence and presence of HRP using the SPCE in a pH5.7 BR buffer solution containing 0.04 M each of H₃PO₄, HOAc and H₃BO₃: (a). 1.2×10⁻⁵ M o-AP + 2×10⁻⁴ M H₂0₂; (b). as in (a) + 0.25 U/mL HRP; Potential scanning rate: 0.1 V/s; scanning potential range: +0.50 V to -0.60 V. Curve b was recorded after the enzyme catalytic reaction preceded for 20 minutes.

Fig. 2

Responses at the SPCE to o-AP enzymatic product catalyzed by HRP in BR buffer recorded using (a) SWV, (b) DPV and (c) amperometric techniques. Equal concentration of product solution was used. SWV and DPV scanning were performed from 0.00 to −0.60 V with a step potential of 0.004 V, an amplitude of 0.025 V, and a frequency of 15 Hz for SWV and a pulse width of 0.05 seconds for DPV. Potential was applied at −0.4 V for amperometric analysis. All curves were shown with o-AP background current subtraction.

Fig. 3

Effect of initial scanning potential on SWV responses of enzymatic product (grey bars) and blank substrate (white bars) solution. Signal-to-noise ratio (line graph) represents the ratio of response of product to that of blank substrate. Conditions of SWV measurement were the same as in Figure 2.

Fig. 4

Optimization of substrate concentration. Signal-to-noise ratio represents the ratio of response of product to that of blank substrate. Product solution was produced by 5 minutes of 0.1 U/mL HRP enzymatic reaction in the o-AP substrate solution containing 2×10⁻⁴ M H₂O₂. Conditions of SWV measurement were the same as in Figure 2.

Fig. 5

Typical SWV responses of increasing HRP concentration in PBS buffer. From bottom to top, the concentration of HRP in PBS buffer is 0.1, 0.05, 0.025, 0.012, 0.006, 0.003 and 0 U/mL, respectively. Insert shows the calibration curve of HRP. Enzymatic reaction preceded 5 minutes at room temperature in the substrate solution containing 0.06×10⁻³ M o-AP and 2×10⁻⁴ M H₂O₂ in BR buffer (pH 5.7).