Development and Application of an SPR Nanobiosensor Based on AuNPs for the Detection of SARS-CoV-2 on Food Surfaces

Abstract

A new transmission route of SARS-CoV-2 through food was recently considered by the World Health Organization (WHO), and, given the pandemic scenario, the search for fast, sensitive, and low-cost methods is necessary. Biosensors have become a viable alternative for large-scale testing because they overcome the limitations of standard techniques. Herein, we investigated the ability of gold spherical nanoparticles (AuNPs) functionalized with oligonucleotides to detect SARS-CoV-2 and demonstrated their potential to be used as plasmonic nanobiosensors. The loop-mediated isothermal amplification (LAMP) technique was used to amplify the viral genetic material from the raw virus-containing solution without any preparation. The detection of virus presence or absence was performed by ultraviolet-visible (UV-Vis) absorption spectroscopy, by monitoring the absorption band of the surface plasmonic resonance (SPR) of the AuNPs. The displacement of the peak by 525 nm from the functionalized AuNPs indicated the absence of the virus (particular region of gold). On the other hand, the region ~300 nm indicated the presence of the virus when RNA bound to the functionalized AuNPs. The nanobiosensor system was designed to detect a region of the N gene in a dynamic concentration range from 0.1 to 50 × 10³ ng·mL^-1 with a limit of detection (LOD) of 1 ng·mL^-1 (2.7 × 10³ copy per µL), indicating excellent sensitivity. The nanobiosensor was applied to detect the SARS-CoV-2 virus on the surfaces of vegetables and showed 100% accuracy compared to the standard quantitative reverse transcription polymerase chain reaction (RT-qPCR) technique. Therefore, the nanobiosensor is sensitive, selective, and simple, providing a viable alternative for the rapid detection of SARS-CoV-2 in ready-to-eat vegetables.

Keywords: COVID-19; LAMP; SPR; UV–Vis; optical biosensor.

Figure 1

Schematic representation of the main stages of development and application of a nanobiosensor based on AuNPs functionalized with oligonucleotides for the detection of SARS-CoV-2 in food surfaces.

Figure 2

Design of N-gene-specific primer sets for SARS-CoV-2 (Table 1), designed to selectively amplify the 28,530–28,740 nucleotide segment. Their corresponding positions are demonstrated in the magnification of the image.

Figure 3

Key design steps of AuNP-based nanobiosensor for detection of SARS-CoV-2 on food surfaces: (A) RT-LAMP reaction and virus sample addition; (B) AuNPs containing thiol groups were functionalized with oligonucleotides; (C) separate amplicons bind oligonucleotide groups to the target for further detection in UV–Vis; (D) in natura samples of vegetables (cabbage, spinach, and lettuce) decontaminated with UV light were infected with SARS-CoV-2 and recovered for RT-LAMP and RT-qPCR reactions.

Figure 4

Response surface obtained from 11 experiments carried out according to the rotational central compound design (RCCD).

Figure 5

(A) TEM image of AuNPs indicating distribution with scale bar magnification: 200 nm and 50 nm. (B) Particle size distribution histogram determined from the TEM image. (C) UV–Vis spectrum indicates the absorbance changes between the AuNPs (solid line) and the functionalized AuNP-Oligos (dashed line). (D) UV–Vis spectrum indicates the displacement of the SPR band after hybridization of the target amplicon with AuNP-Oligos, evidencing the detection of SARS-CoV-2.

Figure 6

(A) UV–Vis spectra for dynamic concentrations ranging from 0.1 to 50 × 10³ ng mL⁻¹ of RNA of SARS-CoV-2 virus, negative control, and betacoronavirus SA44. (B) Electrophoresis with 1% agarose gel confirming distinct concentration of RNA amplification and identification of amplified fragment in the target range of 100 to 200 bp.

Figure 7

(A,B) The UV–Vis spectra after the application of the biosensor to detect SARS-CoV-2 at high (Ct:19) and low viral concentrations (Ct:31) in vegetables (lettuce, cabbage, and spinach), respectively. (C) Electrophoresis with 1% agarose gel to confirm RNA amplification in vegetable samples. (D) Image generated by RT-qPCR, showing that there was no detection in the samples subjected to the RNA extraction.

Figure 8

Absorbance values for food samples infected (positive) or not infected (negative) were recorded using a plate reader. A threshold line (red line) was set that maximized the assay’s discrimination performance.