Magnetic Nanoparticle-based Immunoassays for Rapid, High-Sensitivity Detection of Protein Biomarkers

The detection of protein biomarkers in human biofluids is a standard clinical practice for diagnosis, and monitoring of disease progression and therapeutic response. Current gold standard techniques for protein measurement are based on immunoassays, in which antibodies specific to the analyte of interest are used to bind and isolate the target protein. The most commonly used immunoassay is the enzyme-linked immunosorbent assay (ELISA), which produces a color change proportional to the concentration of the target analyte. A point-of-care friendly alternative to ELISA is an electrochemical amperometric immunosensor, which measures a current change. These techniques are highly sensitive, often detecting target analyte levels in the pg’s/mL, and specific due to their use of antibodies. However, they require long (3-4 hr) incubation times, skilled laboratory personnel, single-use substrates, and may not achieve the sensitivity necessary to detect ultralow levels of protein biomarkers. To address these challenges, we have employed magnetic nanoparticles to amplify the detection signal produced by immunoassays and enhance binding kinetics in both ELISA and electrochemical sensing. We have developed a magneto-ELISA employing dually-labelled magnetic nanoparticles (DMPs), which are bound to an excess of detection antibody (dAb) and enzymatic reporter, in order to increase the binding of target analytes, enhance signal amplification, and reduce incubation times within the assay. By using an external magnetic field, DMP-immunocomplexes are concentrated at the bottom of each well, facilitating binding with the capture antibody (cAb). Using Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a marker for malaria, as a proof-of-concept biomarker, we found that this assay can detect proteins in the 10’s pg/mL range within 30 minutes, maintaining the sensitivity of a standard ELISA and producing results up to 4-fold faster. The magneto-ELISA was further adapted for serological detection of antibodies against Trypanosoma cruzi (T. cruzi), as would be found in chronic Chagas disease. Using DMPs conjugated to both Tc24, a protein specific to T. cruzi, and an enzymatic reporter, we show that anti-Tc24 antibodies can be identified in 6400x diluted clinical serum samples with an equivalent accuracy to a standard ELISA. We have also leveraged the capability of magnetic nanoparticles to be positioned using an external magnetic field to develop a reusable electrochemical sensor. Standard electrochemical immunoassay techniques render the sensors to be single-use consumables, increasing waste and costs associated with point-of-care quantitative protein biomarker detection. By using cAb-labelled magnetic nanoparticles (MNPs), we are able to temporarily immobilize immunocomplexes onto the working electrode, which can subsequently be washed away using a mild detergent. Furthermore, the addition of dually-labelled gold nanoparticles, bound to both dAb and a reporter molecule, allows for signal amplification. Together, these modifications to standard electrochemical sensing allow the sensor to be reused up to 100 times with a minimal reduction in analytical performance, while allowing for the detection of pg’s/mL of protein biomarkers in approximately one hour using point-of-care friendly instrumentation. Overall, we demonstrate the benefits of dually-labelled nanoparticles and MNPs in enhancing the sensitivity and reducing incubation times required for protein biomarker detection. The techniques described here are done with proof-of-concept biomarkers and biofluids, but can be easily adapted for the detection of other biomarkers. Furthermore, these techniques do not require additional laboratory equipment for protein detection, thus facilitating their adoption into clinical practice.