Graphene oxide based ultrasensitive flow-through chemiluminescent immunoassay for sub-picogram level detection of chicken interferon-γ

• A new method for chemiluminescent immunoassay of ChIFN-γ is constructed.
• Graphene oxide is used as immobilization matrix of chemiluminescent immunoassay.
• The immunoassay system shows ultrahigh sensitivity for detection of ChIFN-γ.
• This method provides a very promising tool for quantifying ChIFN-γ.

The quantitative detection of chicken interferon-γ (ChIFN-γ) released by T cells after in vitro stimulation is a good evaluation of cell-mediated immunity in chickens after infection or vaccination. This work reports a new flow-through the chemiluminescent (CL) immunoassay method for rapid and specific determination of ChIFN-γ at a level of sub-picogram per milliliter. The biocompatible graphene oxide nanosheet is introduced into CL immunoassay for highly efficient immobilization of capture antibody. The detection limit of the proposed method at a signal to noise ratio of 3 is 0.36 pg ml−1, which is 138-fold lower than the current lowest value of 50 pg ml−1 for ChIFN-γ. Coupling with a flow-through system, the whole immunoassay process can be completed within 25 min. The resulting ChIFN-γ immunosensor shows excellent detection and fabrication reproducibility, good specificity and stability. The assay results of nature ChIFN-γ samples with the proposed method are in an acceptable agreement with the reference values. Compared to the present assay methods, this method is more flexible, simple, rapid and sensitive. The aim of this work is to demonstrate that the ultrasensitive, specific and rapid CL immunoassay format can become a very potential application for quantifying ChIFN-γ and further studying its role in immune response in poultry.