Amperometric immunosensor based on gold nanoparticles/alumina sol-gel modified screen-printed electrodes for antibodies to Plasmodium falciparum histidine rich protein-2.

We report herein the amperometric immunosensor for antibodies to Plasmodium falciparum histidine rich protein-2 (PfHRP-2). Screen-printed electrodes (SPEs) were modified with alumina sol-gel (Al(2)O(3) sol-gel) derived film and gold nanoparticles i.e. AuNPs/Al(2)O(3)sol-gel/SPE. A thin film was formed by dripping Al(2)O(3) sol on SPE followed by electrochemical deposition of gold nanoparticles (AuNPs). The modified SPEs were characterized by scanning electron microscopy/energy dispersive X-ray analysis (SEM-EDAX), Raman spectra and voltammetric experiments. Antibodies in rabbit serum sample were allowed to react with the PfHRP-2 protein that was immobilized on the modified SPE to form antigen-antibody immune complex (PfHRP-2/anti-PfHRP-2). The bound antibodies were quantified by alkaline phosphatase (AP) enzyme labeled secondary antibodies (anti-rabbit immunoglobulins-AP conjugate). Enzymatic substrate, 1-naphthyl phosphate was converted to 1-naphthol by AP and an electroactive product was quantified using amperometry. The performances of the developed immunosensor and Dot-ELISA were tested against different dilutions of hyper immune serum (rabbit anti-PfHRP-2). Dot ELISA and the developed immunosensor (AuNPs/Al(2)O(3)sol-gel/SPE) results for the hyper immune serum containing anti-PfHRP-2 were distinctly positive when diluted upto 8 times (1 : 12800 dilution) and 11 times (1 : 102400 dilution), respectively. The developed immunosensor was applied for antibodies to PfHRP-2 in human clinical samples.

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

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.