Bifunctional Au@Pt/Au nanoparticles as electrochemiluminescence signaling probes for SARS-CoV-2 detection

Abstract

A novel immunosensor based on electrochemiluminescence resonance energy transfer (ECL-RET) for the sensitive determination of N protein of the SARS-CoV-2 coronavirus is described. For this purpose, bifunctional core@shell nanoparticles composed of a Pt-coated Au core and finally decorated with small Au inlays (Au@Pt/Au NPs) have been synthesized to act as ECL acceptor, using [Ru (bpy)₃]²⁺ as ECL donor. These nanoparticles are efficient signaling probes in the immunosensor developed. The proposed ECL-RET immunosensor has a wide linear response to the concentration of N protein of the SARS-CoV-2 coronavirus with a detection limit of 1.27 pg/mL. Moreover, it has a high stability and shows no response to other proteins related to different virus. The immunosensor has achieved the quantification of N protein of the SARS-CoV-2 coronavirus in saliva samples. Results are consistent with those provided by a commercial colorimetric ELISA kit. Therefore, the developed immunosensor provides a feasible and reliable tool for early and effective detection of the virus to protect the population.

Keywords: COVID-19; Electrochemiluminescence resonance energy transfer; Immunosensor; Metal nanoparticles; N protein; SARS-CoV-2.

Graphical abstract

Scheme 1

Steps followed for the preparation of the immunosensor (Au@Pt/Au NPs/DAb/Nprotein/CAb-MBs/SPCE) for N protein detection.

Fig. 1

(A) TEM image of Au NPs. (B) HR-TEM image of Au@Pt/Au NPs with a magnification of a single Au@Pt/Au NP. Au protuberances are labeled with a red circle. (C) AFM topographic image onto HOPG, and topographic profile across the line drawn as an inset. (D) Bright field STEM micrograph of a single nanoparticle, together with Energy dispersive X-ray (EDX) mapping of Au (E) and Pt (F). (G) Normalized UV–vis absorption spectra of 4.500 × 10¹⁴ NPs/mL of Au NPs (a), Pt–Au NPs (b) and Au@Pt/Au NPs (c). Normalized ECL emission spectrum (d) of 2.0 mM [Ru (bpy)₃]²⁺ in 0.1 M phosphate buffer, pH 8.0, containing 0.25 mM TPrA obtained under a cyclic potential scan from 0.0 to +1.1 V at 0.03 V/s.

Fig. 2

(A) EDX analysis and SEM image of Au@Pt/Au NPs/DAb/Nprotein/CAb-MBs/SPCE. (B) ECL responses in presence of 2.0 mM [Ru (bpy)₃]²⁺ in 0.1 M phosphate buffer, pH 8.0, at 0.03 V/s and (C) Nyquist diagrams obtained in 0.1 M phosphate buffer, pH 8.0, with 25 mM KCl in the presence of 1.0 × 10⁻² mol/L K₃Fe(CN)₆/1.0 × 10⁻² mol/L K₄Fe(CN)₆ for a bare SPCE (a), MBs/SPCE (b), CAb-MBs/SPCE (c), Au@Pt/Au NPs/DAb/Nprotein/CAb-MBs/SPCE (d). The N protein concentration was 20 pg/mL.

Fig. 3

Optimization of different experimental variables: (A) amount of MBs, (B) concentration of Au@Pt–Au NPs, (C) concentration of CAb, (D) incubation time of MBs with CAb, (E) concentration of DAb and (F) incubation time of CAb-MBs with the mixture of N protein and Au@Pt–Au NPs/Dab. Normalized ECL responses (left) measured in the absence (light grey bars) or in the presence (dark grey bars) of 20.0 pg/mL N protein, and the resulting S₀/S ratio (right). All the ECL responses were obtained in 0.1 M phosphate buffer, pH 8.0, in presence of 2.0 mM [Ru (bpy)₃]²⁺.

Fig. 4

(A) Normalized ECL immunosensor response to various concentrations N protein. (B) Calibration curve of normalized ECL intensity vs N protein concentrations (n = 3). (C) Linear plot of the normalized ECL intensity vs logarithmic concentration of N protein (n = 3). (D) Immunosensor response (n = 3) to 20.0 pg/mL N protein obtained in the absence and in presence of 20.0 pg/mL different potential interfering proteins. (E) ECL signal-time curve of 20.0 pg/mL N protein obtained under continuous cycles. (F) Storage stability of the immunosensor measured for 45 days. All measurements were carried out in 0.1 M phosphate buffer, pH 8.0 aqueous solution containing 2.0 mM [Ru (bpy)₃]²⁺.