A Novel Immunosensing Method Based on the Capture and Enzymatic Release of Sandwich-Type Covalently Conjugated Thionine–Gold Nanoparticles as a New Fluorescence Label Used for Ultrasensitive Detection of Hepatitis B Virus Surface Antigen

[Image: see text] A novel ultrasensitive and simple amplified immunosensing strategy is designed based on a surface-enhanced fluorescence (SEF) nanohybrid made from covalently conjugated thionine–gold nanoparticles (GNP–Th), as a novel amplified fluorescence label, and magnetic nanoparticles (MNPs), as a biological carrier, used for hepatitis B virus surface antigen (HBsAg) detection. This immunosensing strategy operates on the basis of the capture and then release of the amplified fluorescence label. Capturing of the antiHBs-antibody (Ab)-modified GNP–thionine hybrid (GNP–Th-Ab) is carried out through the formation of a two-dimensional (sandwich) probe between this amplified label and antiHBs-antibody-modified magnetic nanoparticles (MNP-Ab), in the presence of a target antigen and using an external magnetic force. Afterward, releasing of the captured fluorescence label is performed using a protease enzyme (pepsin) by a digestion mechanism of grafted antibodies on the GNP–thionine hybrid. As a result of antibody digestion, the amplified fluorescent hybrids (labels) are released into the solution. To understand the mechanism of enhanced fluorescence, the nature of the interaction between thionine and gold nanoparticles is studied using the B3LYP density functional method. In such a methodology, several new mechanisms and structures are used simultaneously, including a SEF-based metal nanoparticle–organic dye hybrid, dual signal amplification in a two-dimensional probe between the GNP–thionine hybrid and MNPs, and a novel releasing method using protease enzymes. These factors improve the sensitivity and speed, along with the simplicity of the procedure. Under optimal conditions, the fluorescence signal increases with the increment of HBs antigen concentration in the linear dynamic range of 4.6 × 10(–9) to 0.012 ng/mL with a detection limit (LOD) of 4.6 × 10(–9) ng/mL. The proposed immunosensor has great potential in developing ultrasensitive and rapid diagnostic platforms.