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.

Microfluidic Magneto Immunosensor for Rapid, High Sensitivity Measurements of SARS-CoV-2 Nucleocapsid Protein in Serum.

The COVID-19 pandemic has highlighted the importance and urgent need for rapid and accurate diagnostic tests for COVID-19 detection and screening. The objective of this work was to develop a simple immunosensor for rapid and high sensitivity measurements of SARS-CoV-2 nucleocapsid protein in serum. This assay is based on a unique sensing scheme utilizing dually-labeled magnetic nanobeads for immunomagnetic enrichment and signal amplification. This immunosensor is integrated onto a microfluidic chip, which offers the advantages of minimal sample and reagent consumption, simplified sample handling, and enhanced detection sensitivity. The functionality of this immunosensor was validated by using it to detect SARS-CoV-2 nucleocapsid protein, which could be detected at concentrations as low as 50 pg/mL in whole serum and 10 pg/mL in 5× diluted serum. We also adapted this assay onto a handheld smartphone-based diagnostic device that could detect SARS-CoV-2 nucleocapsid protein at concentrations as low as 230 pg/mL in whole serum and 100 pg/mL in 5× diluted serum. Lastly, we assessed the capability of this immunosensor to diagnose COVID-19 infection by testing clinical serum specimens, which revealed its ability to accurately distinguish PCR-positive COVID-19 patients from healthy, uninfected individuals based on SARS-CoV-2 nucleocapsid protein serum levels. To the best of our knowledge, this work is the first demonstration of rapid (<1 h) SARS-CoV-2 antigen quantification in whole serum samples. The ability to rapidly detect SARS-CoV-2 protein biomarkers with high sensitivity in very small (<50 µL) serum samples makes this platform a promising tool for point-of-care COVID-19 testing.