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.