Highly sensitive amperometric immunosensor for detection of Plasmodium falciparum histidine-rich protein 2 in serum of humans with malaria: comparison with a commercial kit

Abstract

A disposable amperometric immunosensor was developed for the detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP-2) in the sera of humans with P. falciparum malaria. For this purpose, disposable screen-printed electrodes (SPEs) were modified with multiwall carbon nanotubes (MWCNTs) and Au nanoparticles. The electrodes were characterized by cyclic voltammetry, scanning electron microscopy, and Raman spectroscopy. In order to study the immunosensing performances of modified electrodes, a rabbit anti-PfHRP-2 antibody (as the capturing antibody) was first immobilized on an electrode. Further, the electrode was exposed to a mouse anti-PfHRP-2 antibody from a serum sample (as the revealing antibody), followed by a rabbit anti-mouse immunoglobulin G-alkaline phosphatase conjugate. The immunosensing experiments were performed on bare SPEs, MWCNT-modified SPEs, and Au nanoparticle- and MWCNT-modified SPEs (Nano-Au/MWCNT/SPEs) for the amperometric detection of PfHRP-2 in a solution of 0.1 M diethanolamine buffer, pH 9.8, by applying a potential of 450 mV at the working electrode. Nano-Au/MWCNT/SPEs yielded the highest-level immunosensing performance among the electrodes, with a detection limit of 8 ng/ml. The analytical results of immunosensing experiments with human serum samples were compared with the results of a commercial Paracheck Pf test, as well as the results of microscopy. The specificities, sensitivities, and positive and negative predictive values of the Paracheck Pf and amperometric immunosensors were calculated by taking the microscopy results as the "gold standard." The Paracheck Pf kit exhibited a sensitivity of 79% (detecting 34 of 43 positive samples; 95% confidence interval [CI], 75 to 86%) and a specificity of 81% (correctly identifying 57 of 70 negative samples; 95% CI, 76 to 92%), whereas the developed amperometric immunosensor showed a sensitivity of 96% (detecting 41 of 43 positive samples; 95% CI, 93 to 98%) and a specificity of 94% (correctly identifying 66 of 70 negative samples; 95% CI, 92 to 99%). The developed method is more sensitive and specific than the Paracheck Pf kit.

FIG. 1.

Steps involved in the preparation of a modified SPE and steps involved in immunosensor detection. Red diamonds represent PfHRP-2 antigen. BSA, bovine serum albumin.

FIG. 2.

CVs measured with an SPE modified with 0.1% Nafion (a), 0.5% Nafion (b), and 1% Nafion (c) in an aqueous solution containing 1 mM K₃Fe(CN)₆-K₄Fe(CN)₆ (1:1) and 0.1 M KCl as the supporting electrolyte at a scan rate of 50 mV/s. I/A, current in amperes; E/V, electrode potential in volts.

FIG. 3.

CVs measured with an SPE modified with Au nanoparticles and MWCNTs (a), Au nanoparticles (b), and MWCNTs (c) and with a bare SPE (d) in a solution containing 1 mM K₃Fe(CN)₆-K₄Fe(CN)₆ (1:1) and 0.1 M KCl as the supporting electrolyte at a scan rate of 50 mV/s. I/A, current in amperes; E/V, electrode potential in volts.

FIG. 4.

CVs measured with a bare SPE (a) and with an SPE modified with MWCNTs (b) and Au nanoparticles and MWCNTs (c) in 0.2 mM 1-naphthol in DEA buffer, pH 9.8, at a scan rate of 50 mV/s. I/A, current in amperes; E/V, electrode potential in volts.

FIG. 5.

SEM images of the electrode surfaces of the bare SPE (a), the MWCNT/SPE (b), the Nano-Au/SPE (c), and the Nano-Au/MWCNT/SPE (d). Acceleration voltage, 10 kV.

FIG. 6.

Raman spectra of the Nano-Au/MWCNT/SPE. Laser source, 514 nm; exposure time, 10 s.

FIG. 7.

Calibration plot for the amperometric current with different concentrations of PfHRP-2 under optimized conditions for the bare SPE, the MWCNT/SPE, and the Nano-Au/MWCNT/SPE in DEA buffer, pH 9.8. Applied potential, 450 mV versus the Ag/AgCl/sat-KCl reference electrode, with a stirring speed of 700 rpm (n = 6). I/nA, current in nanoamperes.

FIG. 8.

Amperometric responses on the Nano-Au/MWCNT/SPE immunosensor for P. falciparum-positive serum samples (a) and pooled healthy human serum samples (b) in a DEA buffer solution of pH 9.8. Applied potential, 450 mV versus the Ag/AgCl/sat-KCl reference electrode, with a stirring speed of 700 rpm. I/A, current in amperes.

FIG. 9.

Amperometric responses for P. falciparum-negative PUO serum samples in DEA buffer, pH 9.8. Applied potential, 450 mV versus the Ag/AgCl/sat-KCl reference electrode, with a stirring speed of 700 rpm (n = 6). I/A, current in amperes.