Automatic label-free immunoassay with high sensitivity for rapid detection of SARS-CoV-2 nucleocapsid protein based on chemiluminescent magnetic beads

Accurate and efficient early diagnosis is crucial for the control of COVID-19 pandemic. However, methods that can balance sensitivity, high throughput, detection speed and automation simultaneously are still scarce. Here, we report an automatic label-free chemiluminescence immunoassay (CLIA) for rapid SARS-CoV-2 nucleocapsid protein (NP) detection with high sensitivity and throughput. N-(4-aminobutyl)-N-ethylisoluminol and Co2+ dualfunctionalized chemiluminescent magnetic beads (dfCL-MB) were first applied to the detection of protein by a novel and simple strategy. Sulphydryl polyethylene glycol was coated on the surface of dfCL-MB so as to assemble dfCL-MB and antibody conjugated gold nanoparticles through Au-S bond. Considering the high-risk application scenarios, the immunosensor was integrated with an automatic chemiluminescence analyzer so that the whole testing procedure could be carried out automatically without manual operation. A linear correlation between CL intensities and the logarithm of NP concentration was obtained in the range of 0.1-10,000 pg/ mL with a detection limit of 21 fg/mL. The whole process cost 25 min and the sample compartment can bear 24 samples simultaneously. The spiked human serum samples and serum samples from COVID-19 patients were determined with satisfactory recoveries of 91.1-109.4%, suggesting that the proposed label-free CLIA is of great potential for SARS-CoV-2 NP detection in practice.