Silk cocoon membrane-based immunosensing assay for red blood cell antigen typing

•The application of natural free-standing silk cocoon membrane in blood typing by immunosensing assay was proposed.•The cocoon membranes were treated with heat and detergent to increase wettability.•The cocoon membranes were functionalized by affinity binding with specific silk monoclonal antibodies and crosslinking antibodies.•Blood grouping antibodies were coupled to functional cocoon membranes to form the immunosensor for blood typing.•This affinity biosensor yielded accurate visualization results for ABO and RhD blood grouping within 30 s. Accurate and fast identification of blood groups is a critical step in the blood transfusion procedure. In this study, we reported a rapid and reliable immunosensing assay for ABO and RhD blood group typing based on natural free-standing silk cocoon membranes. Natural silk cocoon membranes were punched into discs (SMDs) and were treated with heat and detergent to increase wettability. Monoclonal antibodies (mAbs) against silk proteins were successfully prepared through animal immunization. The SMDs were functionalized by affinity binding with specific silk mAbs and crosslinking antibodies. Blood grouping antibodies were coupled with functional SMDs to form the immunosensor for blood typing. Only three pipetting steps were required to perform the assay, without the need for any external analytical equipment. Results could be obtained and distinguished by the naked eye within 30 s, making this immunosensor suitable for point-of-care testing. A positive result was obtained when the SMD showed a visualization red spot after washing, no red spot was observed if there was no reaction. We compared our sensing results with those from the gold-standard tube test on 112 real blood samples. The accuracy was 100 %. Due to the availability of the cocoon membranes-based immunosensor and the facile fabrication process, the SMART can be scaled up easily for mass production.