Abstract
BACKGROUND: Tropomyosin (TM) is a major and highly stable allergen in crustaceans such as shrimp. The high stability of TM during food processing or gastrointestinal digestion impels the substantial allergenic risk to sensitive individuals, which strengthen the urgent requirement of rapid and sensitive detection to guarantee the avoidance of relevant food allergens. RESULTS: In this study, we report the development of a nanobody (Nb)-based "colorimetric-photothermal" dual-readout immunosensing system for ultrasensitive TM detection based on the utilization of a nanozyme of manganese-gold nanoflowers (MnAuNPs). TM-specific Nbs with high affinity and good specificity were isolated from an immune library using phage display technology. To enhance the detection signal, MnAuNPs with a core-shell structure were prepared by a hydrothermal method. The Mn-rich shell and gold core confer intrinsic oxidase-like activity and strong near-infrared absorption capacity to MnAuNPs, enabling simultaneous catalysis of 3,3',5,5'-tetramethylbenzidine oxidation and photothermal conversion of the oxidized product. By decorating TM-specific Nbs with MnAuNPs, a dual-mode immunoassay combining enzymatic and photothermal signal amplification was established. The colorimetric detection limit was 6.17 ng/mL, while the sensitivity of the photothermal mode was further enhanced to 0.25 ng/mL, approximately 48 times higher than that of the conventional ELISA. The immunosensing system exhibited high specificity, reproducibility, and accuracy in real food matrices. The recovery rates of the dual-signal immunosensing system ranged from 79.1% to 108.8%, with a coefficient of variation of 0.6% to 12.3%. CONCLUSIONS: We have generated a highly sensitive detection platform based on TM specific Nbs and nanozyme-mediated enhanced signaling. The versatile Nb-nanozyme detection platform integrates electron-transfer catalysis and photothermal transduction for dual-mode readout. The established method could offer a promising foundation and inspire further investigation on portable, low-cost point-of-care testing of food allergens and other biomolecular targets.