A membrane scaffold for oriented-controlled biosensing in complex samples

Abstract Detecting target molecules in complex samples with both high sensitivity and selectivity remains a central challenge in biosensing, owing to low analyte abundance and interference from coexisting substances. Here, we present a membrane scaffold based on a ZZ-tag-displaying hepatitis B virus-derived L protein (ZZ-L membrane) that enables the oriented immobilization of sensing molecules. On quartz crystal microbalance sensor chips, the ZZ-L membrane enables controlled orientation of immunoglobulin G (IgG), leading to up to ~200-fold improvements in detection sensitivity and markedly enhanced binding capacity for purified food allergens, including ovomucoid, lectin, and tropomyosin, compared with direct immobilization. Whereas conventional methods failed to detect targets in complex matrices, the ZZ-L membrane enabled sensitive and selective detection of gliadin in wheat gluten and tropomyosin in heated shrimp extracts. Oriented immobilization of anti- hemagglutinin IgG further reduced the detectable amount of UV-inactivated influenza A virus by approximately 12-fold. The approach also extends beyond antibodies: Fc-fused leptin receptors were similarly immobilized, resulting in an approximately 15-fold increase in detection sensitivity. Across all targets, the number of bound analyte molecules per sensing molecule was consistently increased, indicating an increased active fraction at the molecular recognition interface. Together, these results demonstrate that membrane-mediated orientation control improves molecular accessibility and increases the active fraction, establishing the ZZ-L membrane as a robust platform for biosensing in complex samples with broad applicability in food safety, viral diagnostics, and biomarker detection.