Boron doped diamond thin films for the electrochemical detection of SARS-CoV-2 S1 protein

Abstract

Amidst a global pandemic, a precise and widely accessible rapid detection method is needed for accurate diagnosis and contact tracing. The lack of this technology was exposed through the outbreak of SARS-CoV-2 beginning in 2019. This study sets the foundation for the development of a boron doped diamond (BDD)-based impedimetric sensor. While specifically developed for use in the detection of SARS-CoV-2, this technology uses principles that could be adapted to detect other viruses in the future. Boron doped polycrystalline diamond electrodes were functionalized with a biotin-streptavidin linker complex and biotinylated anti-SARS-CoV-2 S1 antibodies. Electrodes were then incubated with the S1 subunit of the SARS-CoV-2 spike surface protein, and an electrical response was recorded using the changes to the electrode's charge transfer resistance (R_ct), measured through electrochemical impedance spectroscopy (EIS). Detectable changes in the R_ct were observed after 5-min incubation periods with S1 subunit concentrations as low as 1 fg/mL. Incubation with Influenza-B Hemagglutinin protein resulted in minimal change to the R_ct, indicating specificity of the BDD electrode for the S1 subunit of SARS-CoV-2. Detection of the S1 subunit in a complex (cell culture) medium was also demonstrated by modifying the EIS protocol to minimize the effects of sample matrix binding. BDD films of varying surface morphologies were investigated, and material characterization was used to give insight into the microstructure-performance relationship of the BDD sensing surface.

Keywords: Biosensor; Boron doped diamond; Impedimetric sensor; SARS-CoV-2.

Graphical abstract

Fig. 1

Raman spectra of the 3.6 μm film (black), 8.0 μm film (red) and 0.7 μm film (green). The peak at 520 cm⁻¹ for the 0.7 μm film corresponds to the silicon substrate, which is visible due to the much lower film thickness of that sample. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Representative AFM images of the samples before (left) and after (right) surface functionalization with SARS-CoV-2 (2019-nCoV) Spike S1 Antibody, Rabbit Mab, where A1 and A2 correspond to the 3.6 μm film, B1 and B2 correspond to the 8.0 μm film, and C1 and C2 correspond to the 0.7 μm film.

Fig. 3

Representative EIS spectra (Nyquist plots) of the 3.6 μm film after exposure to increasing concentrations of SARS-CoV-2 spike S1 subunit in PBS. The cell conditions were as follows: 3 mm diameter BDD film working electrode, graphite rod counter electrode, Pt wire reference electrode, and 1 mM K₃Fe(CN)₆/PBS electrolyte solution. Inset: equivalent circuit model for data fitting.

Fig. 4

Baseline subtracted ΔR_ct values as a function of SARS-CoV-2 spike S1 subunit concentration for the 3.6 μm film (green) and the 8.0 μm film (red). The 3.6 μm film was also tested for binding of Influenza B Hemagglutinin protein (purple). The plotted data represents n = 2 total sensors ± 1 standard deviation. Lines are best linear fit. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Normalized ΔR_ctvs antigen concentration for sensors comprised of 3.6 μm (A) and 0.7 μm (B) BDD films tested against SARS-CoV-2 spike S1 subunit (red) and Influenza B Hemagglutinin protein (blue). The plotted data represents n = 3 total sensors ± 1 standard deviation. Lines are linear best fit to guide the eye. Equations are logarithmic best fit with 0 fg/mL excluded. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)