A Methylene Blue-Enhanced Nanostructured Electrochemical Immunosensor for H-FABP Myocardial Injury Biomarker

Abstract

A sensitive electrochemical immunosensor for the detection of the heart-type fatty acid binding protein (HFABP), an earlier biomarker for acute myocardial infarction than Troponins, is described. The sensing platform was enhanced with methylene blue (MB) redox coupled to carbon nanotubes (CNT) assembled on a polymer film of polythionine (PTh). For this strategy, monomers of thionine rich in amine groups were electrosynthesized by cyclic voltammetry on the immunosensor's gold surface, forming an electroactive film with excellent electron transfer capacity. Stepwise sensor surface preparation was electrochemically characterized at each step and scanning electronic microscopy was carried out showing all the preparation steps. The assembled sensor platform combines MB and PTh in a synergism, allowing sensitive detection of the H-FABP in a linear response from 3.0 to 25.0 ng∙mL^-1 with a limit of detection of 1.47 ng∙mL^-1 HFABP that is similar to the clinical level range for diagnostics. H-FABP is a newer powerful biomarker for distinguishing between unstable angina and acute myocardial infarction.

Keywords: H-FABP; acute myocardial infarction; immunosensor; methylene blue; polythionine.

Figure 1

Stepwise step modifications of the electrode surface for immunosensor assembling, and immunoassay for analytical responses.

Figure 2

(a) Cyclic voltammograms of PTh electropolymerization process for 30 successive cycles in the presence of PBS (7.4) as support electrolyte. (b) Bar diagram indicating standardized electroactive areas to the clean electrode in the respective assembly steps (I) Bare GE, (II) PTh/GE, (III) CNT@MB/PTh/GE. The experiment was carried out in the presence of 0.1 M KCl as an electrolyte.

Figure 2

(a) Cyclic voltammograms of PTh electropolymerization process for 30 successive cycles in the presence of PBS (7.4) as support electrolyte. (b) Bar diagram indicating standardized electroactive areas to the clean electrode in the respective assembly steps (I) Bare GE, (II) PTh/GE, (III) CNT@MB/PTh/GE. The experiment was carried out in the presence of 0.1 M KCl as an electrolyte.

Figure 3

Voltammetric profiles performed in the CNT@MB/PTh/GE. (a) Effect of PSS on CNT. (b) Effect of CNT-PSS@MB on the assembling of the electrode.

Figure 3

Voltammetric profiles performed in the CNT@MB/PTh/GE. (a) Effect of PSS on CNT. (b) Effect of CNT-PSS@MB on the assembling of the electrode.

Figure 4

(a) Voltammetric profiles of the EAu/PTh/CNT@MB surface obtained under different scan rates. (b) Plot of scan rate vs. anodic and cathodic peaks. (c) Log of scan rate vs. log of the modulus of the anodic and cathodic peaks. Experiments were performed in the presence of PBS (pH 7.4).

Figure 4

(a) Voltammetric profiles of the EAu/PTh/CNT@MB surface obtained under different scan rates. (b) Plot of scan rate vs. anodic and cathodic peaks. (c) Log of scan rate vs. log of the modulus of the anodic and cathodic peaks. Experiments were performed in the presence of PBS (pH 7.4).

Figure 4

(a) Voltammetric profiles of the EAu/PTh/CNT@MB surface obtained under different scan rates. (b) Plot of scan rate vs. anodic and cathodic peaks. (c) Log of scan rate vs. log of the modulus of the anodic and cathodic peaks. Experiments were performed in the presence of PBS (pH 7.4).

Figure 5

SEM images (a) CNT/GE (b) PSS-CNT/GE; (c) PSS-CNT@MB/GE; (d) PTh/GE; (e) PSS-CNT@MB/PTh/GE. Mag 16.10 KX.

Figure 6

(a) Calibration curves of different concentrations of H-FABP (2.5; 5; 7.5; 10; 12.5; 15; 17.7; 20; 22.5; 25 ng∙mL⁻¹). Measurements were obtained in the presence of 5 mM of (K₃[Fe(CN)₆])/(K₄[Fe(CN)₆]), at 30 mV∙s⁻¹ scan rate. (b) Linear adjustment of the calibration curve. (c) Negative and positive H-FABP concentrations in standard calibrator serum samples, in the presence of 5 mM of (K₃[Fe(CN)₆])/(K₄[Fe(CN)₆]) at mV∙s⁻¹ scan rate. In (a) the mean and standard deviation were obtained from testing three samples.

Figure 6

(a) Calibration curves of different concentrations of H-FABP (2.5; 5; 7.5; 10; 12.5; 15; 17.7; 20; 22.5; 25 ng∙mL⁻¹). Measurements were obtained in the presence of 5 mM of (K₃[Fe(CN)₆])/(K₄[Fe(CN)₆]), at 30 mV∙s⁻¹ scan rate. (b) Linear adjustment of the calibration curve. (c) Negative and positive H-FABP concentrations in standard calibrator serum samples, in the presence of 5 mM of (K₃[Fe(CN)₆])/(K₄[Fe(CN)₆]) at mV∙s⁻¹ scan rate. In (a) the mean and standard deviation were obtained from testing three samples.