Fibroblast growth factor inducible-14 (Fn14) is a receptor protein that plays an important role in the progression of cancer and some other diseases. Here, an ultrasensitive assay was developed for the detection of Fn14 based on a digital sandwich enzyme-linked immunosorbent bead-based assay (dELISA). Beads containing a single immunocomplex are loaded into microwells (~46 fL) and produce fluorescence through enzyme-catalyzed reactions in extremely small volumes. By measuring the number of fluorescent microwells in arrays arranged on a circular Disc, the concentration of Fn14 was determined. To obtain better performance for Fn14 detection, assay conditions including reagent concentrations and measurement parameters were optimized and 44 different antibody pairs were screened. The detection range of Fn14 is 1.26 pg/mL to 3683 pg/mL with a lower detection limit of 0.32 pg/mL, which is much lower than that of conventional ELISAs . In addition, the total operation of this assay is automated and only takes approximately an hour to accomplish. Furthermore, this assay was successfully applied to the determination of spiked Fn14 in serum samples with satisfactory performance.
Prostate-Specific Antigen (PSA) is a crucial biomarker for screening prostate cancer, but a sensitive and selective immunosensor for rapid quantification of serum PSA remains to be developed. In this study, a sandwich pair of nanobodies (Nbs) (i.e., Nb2 and Nb40) against PSA surface antigen was obtained from an alpaca-derived immune phage display library. A sandwich-type immunosensor for the sensitive and selective detection of PSA in serum samples was ingeniously designed based on the pair of Nbs. The small size of Nb40 allowed high capture densities on the surface of reduced graphene oxide (rGO) nanocomposed with massive Au nanoparticles ( [email protected] ), which significantly improved the conductivity and provided a large area to anchor many primary antibodies. The secondary antibody Nb2 fused with streptavidin -binding peptide (SBP) cooperated with Nb40 for PSA sandwiching. Accompanying introduction of horseradish peroxidase-streptavidin (HRP-SA) coupled with Nb2-SBP, the faradaic current was linearly correlated with the logarithm of PSA concentration in a range of 0.1–100 ng mL −1 . More importantly, this immunosensor exhibited excellent selectivity, stability, and reproducibility due to the sandwich pair Nbs. The proposed immunosensor was successfully applied in determining PSA in serum samples and could be used for the sensitive and specific detection of PSA.
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