A Novel Fluorescence Immunoassay Based on Two-time Amplified Fluorescence Signal by Hemin and Magnetic Nanoparticles for the Detection of Antigen

Abstract

A novel, selective, and sensitive magnetic-mimetic enzyme fluorescence immunoassay method for antigen detection has been developed by taking advantage of a magnetic separation process and the amplification feature of the hemin label. This method is based on a twice amplified fluorescence signal. The signal is first amplified due to the ultrasmall size and the high surface-to-volume ratio of the silica-coated magnetite nanoparticles, which enable the nanoparticles to carry much more antibodies. Second, the mimetic enzyme (hemin) as a labeling reagent catalyzes the reaction of p-hydroxyphenyl acetic acid and H₂O₂ can further amplify the fluorescence signal. This protocol was also evaluated for a sandwich-type immunoassay of human IgG, and the calibration graph for human IgG was linear over the range of 0–100 ng mL⁻¹ with a detection limit of 9.8 ng mL⁻¹. This method can easily separate magnetic nanoparticles from the solution, which simplified the process and played a promising role for various applications in immunoassay.

Keywords:

• Fluorescence detection
• human IgG
• mimetic enzyme immunoassay
• Silica-coated magnetite nanoparticles

This work was supported by the Priority Specialty Construction Project of Xiamen Medical Center (Xiamen financial construction, 2007, No. 46).

Notes

^a Mean of six determinations.