A Label and Probe-Free Zika Virus Immunosensor Prussian Blue@carbon Nanotube-Based for Amperometric Detection of the NS2B Protein

Abstract

Zika virus (ZIKV) is a mosquito-borne infection, predominant in tropical and subtropical regions causing international concern due to the ZIKV disease having been associated with congenital disabilities, especially microcephaly and other congenital abnormalities in the fetus and newborns. Development of strategies that minimize the devastating impact by monitoring and preventing ZIKV transmission through sexual intercourse, especially in pregnant women, since no vaccine is yet available for the prevention or treatment, is critically important. ZIKV infection is generally asymptomatic and cross-reactivity with dengue virus (DENV) is a global concern. An innovative screen-printed electrode (SPE) was developed for amperometric detection of the non-structural protein (NS2B) of ZIKV by exploring the intrinsic redox catalytic activity of Prussian blue (PB), incorporated into a carbon nanotube-polypyrrole composite. Thus, this immunosensor has the advantage of electrochemical detection without adding any redox-probe solution (probe-less detection), allowing a point-of-care diagnosis. It was responsive to serum samples of only ZIKV positive patients and non-responsive to negative ZIKV patients, even if the sample was DENV positive, indicating a possible differential diagnosis between them by NS2B. All samples used here were confirmed by CDC protocols, and immunosensor responses were also checked in the supernatant of C6/36 and in Vero cell cultures infected with ZIKV.

Keywords: NS2B; Prussian blue; Zika virus; point-of-care testing; probeless; reagentless.

Figure 1

Schematic diagram of ZIKV immunosensor. (a) Preparation steps of the electrode and (b) analytical measurement principle.

Figure 2

(a) Cyclic voltammograms at 0.1 V s⁻¹ scan rate: (I) bare SPE; (II) PPy/SPE; (III) PPy-CNT and (IV) PB@CNT-PPy. (b) Effect of the scan rate on the current response of the PB@CNT-PPy/SPE (from 0.01 to 0.12 V s⁻¹) (inset: relationship between Ipa and Ipc vs. scan rate). (c) Plot of log Ipa and Ipc vs. log of scan rate. All measurements were performed in KCl (0.1 mol L⁻¹) as support electrolyte.

Figure 3

FTIR spectrum of the (a) bare SPE, (b) PPy/CNT/SPE and (c) PB@CNT-PPy/SPE.

Figure 4

Cyclic voltammograms of the stepwise preparation of the immunosensor: (I) bare SPE, (II) PB@CNT-PPy/SPE, (III) anti-NS2B/PB@CNT-PPy/SPE and (IV) Gly/anti-NS2B/PB@CNT-PPy/SPE. Measurements were performed in the presence of KCl (0.1 mol L⁻¹) at a 0.05 V s⁻¹ scan rate.

Figure 5

Optimization of anti-NS2B concentrations (0.5; 0.1; 2; 3 and 4 mg L⁻¹) immobilized (inset: CVs of the immunosensor in the different anti-NS2B concentrations). Measurements were performed in KCl (0.1 mol L⁻¹) solution at a 0.05 V s⁻¹ scan rate.

Figure 6

(a) Chronoamperograms obtained after successive incubations with supernatants of the infected ZIKV cell-culture and PBS washes before measurements obtained in presence 0.1 mol L⁻¹ KCl solution at 0.4 V fixed potential. Inset: linear fit adjusted for successive cell-culture incubations. (b) Analytical curve of the immunosensor in response to successive incubations with serum samples 1:100 PBS dilution of the ZIKV (positive)/DENV (negative) and ZIKV (negative)/DENV (positive), measured by chronoamperograms at 0.4 V vs. Ag/AgCl. (c) Bar diagram of current (with error bar of three replicates) as function of incubation number of ZIKV (positive)/DENV (negative) and ZIKV (negative)/DENV (positive).

Figure 6

(a) Chronoamperograms obtained after successive incubations with supernatants of the infected ZIKV cell-culture and PBS washes before measurements obtained in presence 0.1 mol L⁻¹ KCl solution at 0.4 V fixed potential. Inset: linear fit adjusted for successive cell-culture incubations. (b) Analytical curve of the immunosensor in response to successive incubations with serum samples 1:100 PBS dilution of the ZIKV (positive)/DENV (negative) and ZIKV (negative)/DENV (positive), measured by chronoamperograms at 0.4 V vs. Ag/AgCl. (c) Bar diagram of current (with error bar of three replicates) as function of incubation number of ZIKV (positive)/DENV (negative) and ZIKV (negative)/DENV (positive).