Performance assessment of disposable carbon-based immunosensors for the detection of SARS-CoV-2 infections.

We designed, developed, and clinically tested two rapid antigen-based immunosensors for SARS-CoV-2 detection, enabling diagnosis and viral load quantification for under USD $2. In a first clinical study, a screen-printed disposable carbon-based (SPC) sensor was assessed on prospectively recruited adult participants classified into three study groups: healthy donors (n = 46); SARS-CoV-2-infected symptomatic patients (n = 58); and co-habitants of patients without prior testing (n = 38). Nasopharyngeal aspirates (NA), oropharyngeal swabs (OS), and saliva (SA) samples were obtained from all participants. Performance was measured in terms of clinical sensitivity and specificity against a reference diagnostic RT-qPCR kit and analytical sensitivity (limit of detection, LoD) and specificity using recombinant material in lab tests. A second study was performed using the same sensor design, albeit with laser-induced graphene (LIG) electrodes, using nasopharyngeal swabs (NS) on 224 patient samples obtained at different stages of the pandemic, of which 110 tested negative and 114 positive via RT-qPCR. We find OS was the most informative sample, when compared to NA and SA. The SPC-based sensors had a 93.8% sensitivity and 61.5% specificity with OS samples, while the LIG-based sensors with NS had a lower sensitivity of 68.93%, albeit a significantly higher specificity of 86.17%. We believe specificity values for the SPC sensors were driven by positive results from co-habitants and healthy donors and were affected by the low sensitivity (75.5%) and high LoD (> 20,000 viral copies/mL) of the reference RT-qPCR kit used, and the lower sensitivity of the LIG-based was due to a reduced set of effective antigen-binding sites caused by the non-covalent LIG-mAb ligands used. The immunosensor's LoD to spike protein in phosphate-buffered saline (PBS) for both types of sensors was near 1 fg/mL and showed no cross-reactivity to recombinant structural proteins of Epstein-Barr and Influenza. Performance metrics and time-to-result (5 < 12 min) provide proof-of-principle of the immunosensor's applicability as a low-cost, rapid technology for determining SARS-CoV-2 infections. Changing the working electrode material to LIG, instead of SPC, improved specificity even in the presence of pathogen variants. Discordant results between our two immunosensor versions and RT-qPCR tests are attributed not only to limited antibody effectiveness in the former but also to the quality of RT-qPCR probes used at the height of the pandemic.