On-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry for the determination of SARS-CoV-2 nucleocapsid protein

• An aptamer affinity sorbent was prepared against the SARS-CoV-2 N protein. • AA-SPE-CE-MS allowed the unequivocal and accurate determination of the N protein. • The method required important adaptations compared to previous studies. • Up to 500 times sensitivity enhancement compared to CE-MS was achieved. • The method was applied to saliva samples. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19), which has sparked a significant global health crisis in recent years. Among its structural proteins, the nucleocapsid protein (N protein) stands out as one of the most abundant. Despite being well-recognized as an immunodominant antigen in host immune responses and a promising diagnostic biomarker, further insight into this protein with novel analytical methods is crucial for understanding the disease mechanisms. This study focuses on the development of an aptamer affinity sorbent for the purification, preconcentration, separation, characterization, and quantification of the N protein using on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS). Microcartridges packed with a sorbent composed of magnetic bead (MB) particles modified with an aptamer against the N protein were utilized. A rigorous optimization of several method parameters resulted in the use of a lab-made hydroxypropyl cellulose (HPC)-coated capillary to prevent protein adsorption and a neutral background electrolyte (BGE) of 10 mM ammonium acetate (pH 7.0) for the separation. The sample was loaded in the BGE, and the retained protein was subsequently eluted with 1 M acetic acid (pH 2.3). The developed method demonstrated repeatability in terms of migration times and peak areas, exhibited linearity between 2.5 and 25 µg mL −1 , and achieved a limit of detection (LOD) of 0.5 µg mL −1 , providing a sensitivity enhancement of 500 times compared to CE-MS. It was finally applied to the analysis of the N protein in human saliva, pointing out its potential for establishing accurate SARS-CoV-2 complementary analytical methods.