Electrochemical detection of interleukin-8 in human saliva using a polyenzyme label based on diaphorase and neutravidin

Abstract

Polyenzymes have been commonly employed as catalytic labels to obtain high signal amplification in immunoassays, but they are mainly constructed using horseradish peroxidase that requires the use of H2O2, whose storage is difficult. Here, we present a new polyenzyme label based on diaphorase (DI), a dehydrogenase redox enzyme with high catalytic activity and long-term stability. The polyenzyme label is constructed using a complex of a biotinylated DI and an avidin analog. Among the five commercially available DIs, the one that shows the highest signal amplification using electrochemical-enzymatic redox cycling involving an electron mediator, a DI, and NADH is chosen. Among the three avidin analogs (avidin, neutravidin, and streptavidin), neutravidin provides the lowest nonspecific binding of the polyenzyme label. The polyenzyme label shows higher signal amplification and lower nonspecific binding than a single enzyme label of DI. Comparative studies reveal that the detection limit for the detection of interleukin-8 (IL-8) in saliva using the polyenzyme label (∼1 pg/mL) is one order of magnitude lower than that using a single enzyme label (∼10 pg/mL). IL-8 concentrations measured in clinical saliva samples agree well with those measured using a conventional microplate immunoassay. Thus, the polyenzyme label can be applied for the noninvasive detection of other clinically relevant proteins in saliva.