Immunoassay for sepsis

Nearly 50m cases of sepsis were reported in 2017 worldwide, with some 20m cases occurring in children under five years old, according to a recent report in The Lancet (doi: 10.1016/S0140-6736(19)32989-7). Millions of people die as a result every year.

‘Sepsis diagnosis is a difficult task, but talking about neonates, sepsis is especially challenging,’ says Alberto Escarpa from the University of Alcala in Madrid. Blood cultures remain the gold standard, however, the necessary blood samples may not be available particularly for low-birth-weight neonates.

Instead, Escarpa and his colleagues have now reported a proof-of-concept immunoassay using micromotors (Anal. Chem., doi: 10.1021/acs.analchem.9b05384). As micromotors can propel themselves, they could swim around, promote mixing and find target biomarkers for sepsis, such as C-reactive protein, present in very small volume samples. Microfluidic platforms help to automate the diagnostic process.

The micromotors were made with the conical pores of a polycarbonate membrane as a template. An outer layer of reduced graphene oxide was deposited, followed by a middle layer of magnetic nickel, and finally a core of platinum nanoparticles. Dissolving the membrane released the tubular micromotors, which were incubated with streptavidin and subsequently with an antibody of C-reactive protein to functionalise their surfaces.

The micromotors were placed in the microfluidic system’s external reservoir with a sample, hydrogen peroxide fuel and a second antibody for C-reactive protein, this time tagged with an enzyme. The micromotors’ platinum cores catalyse the decomposition of hydrogen peroxide, generating oxygen bubbles that propel them around. Once C-reactive protein is found, a micromotor attaches to it on one side and the enzyme-tagged antibody on the other side, forming a sandwich.

After five minutes, the mixture enters the microfluidic platform. The micromotors are retained in a channel with a magnet and rinsed. The level of C-reactive protein is then measured indirectly, by allowing the enzyme to mediate the oxidation of hydroquinone to benzoquinone. The benzoquinone flows to a gold working electrode, where it is reduced back to hydroquinone. The electrical signal produced is then measured and calibrated against standard reference solutions of C-reactive protein.

When tested with 10 microlitre blood samples from neonates in a hospital setting, the immunoassay produced results in 8 minutes. The results were in good agreement with those determined by the hospital with standard methods.

For sepsis diagnosis, clinicians want results as fast as possible, ideally within half an hour, says Hakho Lee, a biomedical engineer at Harvard University. Current blood cultures require a few hours to fully capture the target proteins, he says, so producing results in 8 mins is ‘very impressive’. However, Lee notes that sepsis is a very complex problem, which requires considering multiple biomarkers, and he hopes to see the technique extended to detect biomarkers in a multiplexed way.