title: 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.
authors.affiliation: Biosensor Development Division, DRDE, Jhansi Rd., Gwalior-474002, India.
authors.forename: Mukesh K
authors.initials: MK
authors.lastname: Sharma
authors.forename: Vepa K
authors.initials: VK
authors.lastname: Rao
authors.forename: Gauri S
authors.initials: GS
authors.lastname: Agarwal
authors.forename: Ganga P
authors.initials: GP
authors.lastname: Rai
authors.forename: N
authors.initials: N
authors.lastname: Gopalan
authors.forename: Shri
authors.initials: S
authors.lastname: Prakash
authors.forename: S K
authors.initials: SK
authors.lastname: Sharma
authors.forename: R
authors.initials: R
authors.lastname: Vijayaraghavan
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan / Q000097:blood*; D015374:Biosensing Techniques / Q000379:methods*; D006801:Humans; D007159:Immunologic Tests / Q000379:methods*; D016778:Malaria, Falciparum / Q000175:diagnosis*; D051379:Mice; D011237:Predictive Value of Tests; D015800:Protozoan Proteins / Q000097:blood*; D011817:Rabbits; D012680:Sensitivity and Specificity; D044967:Serum / Q000737:chemistry*
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; C514457:HRP-2 antigen, Plasmodium falciparum; D015800:Protozoan Proteins
references.citation: Annu Rev Biochem. 1984;53:537-72
references.pmid: 6383199
references.citation: J Am Chem Soc. 2005 Apr 20;127(15):5689-94
references.pmid: 15826209
references.citation: Am J Trop Med Hyg. 2006 Dec;75(6):1209-15
references.pmid: 17172395
references.citation: Analyst. 2001 Nov;126(11):1919-22
references.pmid: 11763067
references.citation: Parasitol Res. 2003 Mar;89(5):371-4
references.pmid: 12632149
references.citation: J Clin Microbiol. 1999 Sep;37(9):2992-6
references.pmid: 10449488
references.citation: Trans R Soc Trop Med Hyg. 1993 Nov-Dec;87(6):646-8
references.pmid: 8296363
references.citation: Proc Natl Acad Sci U S A. 1986 Aug;83(16):6065-9
references.pmid: 3016741
references.citation: Anal Chem. 2002 Jul 15;74(14):3262-6
references.pmid: 12139027
references.citation: Scand J Infect Dis. 2001;33(10):752-4
references.pmid: 11728042
references.citation: Anal Chem. 2001 Mar 1;73(5):915-20
references.pmid: 11289436
references.citation: Anal Biochem. 2002 Aug 1;307(1):110-6
references.pmid: 12137787
references.citation: Biosens Bioelectron. 2004 Jan 15;19(6):575-82
references.pmid: 14683641
references.citation: Am Fam Physician. 2003 Aug 1;68(3):509-14
references.pmid: 12924833
references.citation: Mol Biochem Parasitol. 2005 Dec;144(2):177-86
references.pmid: 16174539
references.citation: J Clin Microbiol. 1991 Aug;29(8):1629-34
references.pmid: 1761684
references.citation: Anal Biochem. 2002 Sep 15;308(2):405-8
references.pmid: 12419358
references.citation: Anal Chim Acta. 2007 Jul 23;596(2):222-30
references.pmid: 17631100
references.citation: Diagn Microbiol Infect Dis. 2004 May;49(1):25-9
references.pmid: 15135496
references.citation: Anal Sci. 2006 Sep;22(9):1207-11
references.pmid: 16966811