Systematic\nStudy of Various Functionalization Steps\nfor Ultrasensitive Detection of SARS-CoV‑2 with Direct Laser-Functionalized\nAu-LIG Electrochemical Sensors

The\n2019 coronavirus (COVID-19) pandemic impaired global health,\ndisrupted society, and slowed the economy. Early detection of the\ninfection using highly sensitive diagnostics is crucial in preventing\nthe disease’s spread. In this paper, we demonstrate electrochemical\nsensors based on laser induced graphene (LIG) functionalized directly\nwith gold (Au) nanostructures for the detection of SARS-CoV-2 with\nan outstanding limit of detection (LOD) of ∼1.2 ag·mL<sup>–1</sup>. To achieve the optimum performance, we explored\nvarious functionalization parameters to elucidate their impact on\nthe LOD, sensitivity, and linearity. Specifically, we investigated\nthe effect of (i) gold precursor concentration, (ii) cross-linker\nchemistry, (iii) cross-linker and antibody incubation conditions,\nand (iv) antigen–sensor interaction (diffusion-dominated incubation\nvs pipette-mixing), as there is a lack of a systematic study of these\nparameters. Our benchmarking analysis highlights the critical role\nof the antigen–sensor interaction and cross-linker chemistry.\nWe showed that pipette-mixing enhances sensitivity and LOD by more\nthan 1.6- and 5.5-fold, respectively, and also enables multimodal\nreadout compared to diffusion-dominated incubation. Moreover, the\nPBA/Sulfo-NHS: EDC cross-linker improves the sensitivity and LOD compared\nto PBASE. The sensors demonstrate excellent selectivity against other\nviruses, including HCoV-229E, HCoV-OC43, HCoV-NL63, and influenza\nH5N1. Beyond the ability to detect antigen fragments, our sensors\nenable the detection of antigen-coated virion mimics (which are a\nbetter representative of the real infection) down to an ultralow concentration\nof ∼5 particles·mL<sup>–1</sup>.