A novel mimetic enzymatic fluorescence immunoassay for hepatitis B surface antigen by using a thermal phase separating polymer
Iron tetrasulfonatophthalocyanine (FeTSPc), a peroxidase mimic, was used as a labeling reagent and poly(N-isopropylacrylamide) (PNIP) as the separation support of the immune complex for the mimetic-enzymatic immunoassay of hepatitis B surface antigen (HBsAg). PNIP was precipitated from aqueous solution when the ambient temperature was higher than its lower critical solution temperature of 31 °C. In a sandwich immunoassay, the antigen (HBsAg) first reacted with mouse anti-human HBsAg antibody immobilized on PNIP (PNIP–antibody) and then further reacted with FeTSPc-labeled mouse anti-HBsAg antibody (antibody–FeTSPc) at room temperature in a homogeneous format. After changing the temperature to separate the PNIP–antibody–HBsAg–antibody–FeTSPc conjugate moiety, it was re-dissolved and determined by coupling with the fluorogenic reaction of hydrogen peroxide and p-hydroxyphenylpropionic acid. The sensitivity of this method (3 ng mL−1) was close to that of the traditional ELISA using the same reactants. However, the assay was much faster (the assay time decreased from 100–120 to 45 min). This method was applied to determine HBsAg in human serum with satisfactory results.