Development of a self-assembled heptameric nanobody/streptavidin-binding peptide fusion for ultrasensitive detection of serum biomarkers

[Display omitted] •Self-assembled nanobody/streptavidin-binding peptide heptamer (Nb-C4bpα-SBP) for AFP.•An NSH-EIA based on Nb-C4bpα-SBP and SA-polyHRP for trace analysis of AFP in serum.•High affinity of Nb-C4bpα-SBP for AFP and large enzyme load of SA-polyHRP.•The NSH-EIA is 70.5-fold more sensitive than the method based on Nb-SBP and SA-HRP.•The bifunctional Nb-C4bpα-SBP shows great potential in immunodiagnosis of diseases. Since serum biomarkers are commonly used as the indicators of human health, sensitive detection methods for biomarkers can facilitate the diagnosis of related diseases. Herein, to improve sensitivity of the traditional enzyme immunoassay for the model macromolecule alpha fetoprotein (AFP), a novel signal amplification strategy using nanobody/streptavidin-binding peptide (Nb/SBP) heptamer and streptavidin poly-horseradish peroxide conjugate (SA-polyHRP) was proposed to develop an Nb/SBP heptamer-based enzyme immunoassay (NSH-EIA), which integrates the heptamerization of antibody and enzyme. The Nb/SBP heptamer was generated by prokaryotic expression, during which the heptamerization was completed by self-assembly through the α-chains of C4-binding protein (C4bpα). Owing to the strong affinity of Nb/SBP heptamer and high enzyme load of SA-polyHRP, the NSH-EIA shows a limit of detection of 0.065 ng/mL with the linear range of 0.125–8 ng/mL. The NSH-EIA is highly selective for AFP and shows satisfactory accuracy and precision for the analysis of spiked serum samples. Moreover, the NSH-EIA was used to test clinical serum samples and confirmed by the automated chemiluminescence immunoassay analyzer with a good correlation. Thus, this study demonstrated that the Nb/SBP heptamer is a valuable immunoreagent and the NSH-EIA could be a useful diagnostic tool for ultrasensitive detection of AFP and other serum biomarkers.