Dot-blot immunoassay based on antibody-nanocluster biohybrids as tags for naked-eye detection

Paper-based assays such as dot-blot show high promise to develop point-of-care testing devices fulfilling the ASSURED requirements suggested by the World Health Organization (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable). In this technique, natural enzymes are conventionally employed tags to provide bioreceptors such as antibodies with catalytic activity for quantitative assessment. Nonetheless, their inherent biomolecular limitations pose significant challenges, including cost and storage constraints. We propose an alternative conjugation for antibodies based on catalytic bimetallic nanoclusters and their integration in such simple colorimetric paper-based immunoassay. The nanoclusters are composed of gold and platinum and they are embedded in the structure of an anti-rabbit antibody, integrating in a single component the biorecognition and transduction elements required for biosensing. The detection is based on the catalytic properties of the NCs to oxidize an insoluble chromogenic substrate, generating a visible signal on the surface of the paper that can be further analysed for quantitative results. We demonstrate the detection of antibodies against the inflammation biomarker interleukin-6 with a limit of detection of 200 ng mL⁻¹. Experimental results reveal improvements in terms of stability compared to the natural enzyme horseradish peroxidase, retaining most of its activity after a storage equivalent to 6 months at 4 °C. Additionally, incorporating the NCs within the antibody structure instead of attaching them via a covalent bond provides an enhanced sensitivity of 69.7%. This assay could be transferred to other specific antibodies to detect and quantify other analytes of interest.