A Redox-Probe-Free Immunosensor Based on Electrocatalytic Prussian Blue Nanostructured Film One-Step-Prepared for Zika Virus Diagnosis

Abstract

The Zika virus (ZIKV) is a great concern for global health due to its high transmission, including disseminating through blood, saliva, urine, semen and vertical transmission. In some cases, ZIKV has been associated with microcephaly, neurological disorders, and Guillain−Barré syndrome. There is no vaccine, and controlling the disease is a challenge, especially with the co-circulation of the Dengue virus, which causes a severe cross-reaction due to the similarity between the two arboviruses. Considering that electrochemical immunosensors are well-established, sensitive, and practical tools for diagnosis, in this study we developed a sensor platform with intrinsic redox activity that facilitates measurement readouts. Prussian blue (PB) has a great ability to form electrocatalytic surfaces, dispensing redox probe solutions in voltammetric measurements. Herein, PB was incorporated into a chitosan−carbon nanotube hybrid, forming a nanocomposite that was drop-casted on a screen-printed electrode (SPE). The immunosensor detected the envelope protein of ZIKV in a linear range of 0.25 to 1.75 µg/mL (n = 8, p < 0.01), with a 0.20 µg/mL limit of detection. The developed immunosensor represents a new method for electrochemical measurements without additional redox probe solutions, and it is feasible for application in point-of-care diagnosis.

Keywords: Prussian blue; Zika virus; envelope protein; immunosensor; point-of-care diagnosis.

Figure 1

Diagram of the ZIKV immunosensor: (a) steps performed in the synthesis of the nanostructured electrode and (b) process of immobilization of the anti-ZIKV E protein and immunoassay.

Figure 2

Cyclic voltammograms at 0.1 V·s⁻¹ scan rate with different film compositions: (a) CHI–CNT, (b) CHI–PB, (c) CNT–PB, and (d) CHI–CNT@PB. Measurements were performed in the presence of KCl (100 mmol·L⁻¹) at a 0.05 V·s⁻¹ scan rate.

Figure 3

CV profiles according to the PB concentration (inset: electroactive area as function of different PB concentrations). Measurements performed in 100 mmol·L⁻¹ KCl at 0.05 V·s⁻¹ scan rate.

Figure 4

SEM micrographs of the following: (a) CHI–CNT, (b) CHI–CNT@PB, (c) CNT–PB, (d) CHI–PB.

Figure 5

(a) CVs obtained from the CHI–CNT@PB film at 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 mV·s⁻¹ scan rates (inner to outer); (b) plot of anodic and cathodic peak currents from CVs vs. square root of scan rate; (c) plot of log Ipa and Ipc vs. log scan rate. All measurements were performed in KCl (100 mmol·L⁻¹) as the supporting electrolyte.

Figure 5

(a) CVs obtained from the CHI–CNT@PB film at 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 mV·s⁻¹ scan rates (inner to outer); (b) plot of anodic and cathodic peak currents from CVs vs. square root of scan rate; (c) plot of log Ipa and Ipc vs. log scan rate. All measurements were performed in KCl (100 mmol·L⁻¹) as the supporting electrolyte.

Figure 6

CVs of the stepwise preparation of the immunosensor: (a) bare SPE, (b) CHI–CNT@PB, (c) Anti-ZIKV E, (d) glycine (100 mmol·L⁻¹). Measurements were performed in the presence of KCl (100 mmol·L⁻¹) for a 0.05 V·s⁻¹ scan rate.

Figure 7

Analytical curve of the CV anodic peaks for immunosensor response to the serum pool spiked with ZIKV E protein diluted in PBS 100 mmol·L⁻¹ (pH 7.4) and the unspiked serum pool diluted under the same conditions. Measurements were performed in the presence of KCl (100 mmol·L⁻¹) for a 0.05 V·s⁻¹ scan rate.

Figure 8

Immunosensor response to successive incubations with serum samples spiked and unspiked with ZIKV supernatant culture.