Nanoflower Microreactor Based Versatile Enhancer for Recognition Cofactor-Dependent Enzyme Biocatalysis toward Saxitoxin Detection.

Investigating organic carriers' utilization efficiency and bioactivity within organic-inorganic hybrid nanoflowers is critical to constructing sensitive immunosensors. Nevertheless, the sensitivity of immunosensors is interactively regulated by different classes of biomolecules such as antibodies and enzymes. In this work, we introduced a new alkaline phosphatase-antibody-CaHPO4 hybrid nanoflowers (AAHNFs) microreactor based colorimetric immunoprobe. This system integrates a biometric unit (antibody) with a signal amplification element (enzyme) through the biomineralization process. Specifically, the critical factors affecting antibody recognition activity in the formation mechanism of AAHNFs are investigated. The designed AAHNFs retain antibody recognition ability with enhanced protection for encapsulated proteins against high temperature, organic solvents, and long-term storage, facilitating the selective construction of lock structures against antigens. Additionally, a colorimetric immunosensor based on AAHNFs was developed. After ascorbic acid 2-phosphate hydrolysis by alkaline phosphatase (ALP), the generated ascorbic acid decomposes I2 to I-, inducing the localized surface plasmon resonance in the silver nanoplate, which is effectively tuned through shape conversion to develop the sensor. Further, a 3D-printed portable device is fabricated, integrated with a smartphone sensing platform, and applied to the data of collection and analysis. Notably, the immunosensor exhibits improved analytical performance with a 0.1-6.25 ng·mL-1 detection range and a 0.06 ng·mL-1 detection limit for quantitative saxitoxin (STX) analysis. The average recoveries of STX in real samples ranged from 85.9% to 105.9%. This study presents a more in-depth investigation of the recognition element performance, providing insights for improved antibody performance in practical applications.

Multi-branched gold nanoflower-embedded iron porphyrin for colorimetric immunosensor.

Here, we for the first time used a thiolated amino-ligand modified multi-branched gold nanoflower as skeleton to encapsulate iron porphyrins (AuNF@FeTPPCl) as alternatives to horseradish peroxidase (HRP). After the FeTPPCl encapsulation, the HRP mimicking activity of FeTPPCl was effectively blocked by the steric hindrance of the hydrophobic layer on the AuNF surface. Upon the addition of ethanol, the loaded FeTPPCl was released into the solution in its free format and exposed the catalytic sites, resulting in the recovery of catalytic activity. The proposed encapsulation method effectively avoided the loss of catalytic activity that originated from the blocking of the catalytic active sites during immobilization. Additionally, the resultant AuNF@FeTPPCl nanocomposite exhibited a high loading level with 2.4 × 106 FeTPPCl molecules per AuNF, and showed considerably high catalytic activity for hydrogen peroxide and 3, 3' 5, 5'-tetramethylbenzidine, which is approximately 40- and 172-folds higher than native HRP. Through using the as-prepared AuNF@FeTPPCl as an alternative of HRP for trace labeling, we successfully developed colorimetric immunosensors for fumonisin B1 (FB1) and hepatitis B surface antigen (HBsAg) with competitive and sandwich-type formats, respectively. The developed AuNF@FeTPPCl-based colorimetric immunosensor exhibited higher detection sensitivity for FB1 and HBsAg than the corresponding HRP-based immunosensors. Thus, the proposed AuNF@FeTPPCl can be used as HRP mimicking analogs for developing highly sensitive colorimetric immunosensor and for loading other hydrophobic iron porphyrins or catalysts.