Rapid magneto-enzyme-linked immunosorbent assay for ultrasensitive protein detection

Protein-based diagnostics are the standard of care for screening and diagnosing a broad range of diseases and medical conditions. The current gold standard method for quantifying proteins in clinical specimens is the enzyme-linked immunosorbent assay (ELISA), which offers high analytical sensitivity, can process many samples at once, and is widely available in many diagnostic laboratories worldwide. However, running an ELISA is cumbersome, requiring multiple liquid handling and washing steps, and time-intensive (∼2 − 4 h per test). Here, we demonstrate a unique magneto-ELISA that utilizes dually labeled magnetic nanoparticles (DMPs) coated with horseradish peroxidase (HRP) and an HRP-conjugated detection antibody, enabling rapid immunomagnetic enrichment and signal amplification. For proof of concept, this assay was used to detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a malaria parasite biomarker, which exhibited a lower limit of detection of 2 pg mL(−1) (33 fM) in human serum. Measurements of PfHRP2 in clinical blood samples from individuals with and without P. falciparum infection revealed that this magneto-ELISA offers a superior diagnostic accuracy compared to a commercial PfHRP2 ELISA kit. We also demonstrate the versatility of this platform by adapting it for the detection of SARS-CoV-2 nucleocapsid protein, which could be detected at concentrations as low as 8 pg mL(−1) (174 fM) in human serum. In addition to its high analytical performance, this assay can be completed in 30 min, requires no specialized equipment, and is compatible with standard microplate readers and ELISA protocols, allowing it to integrate readily into current clinical practice.