Received on August 15, 2006
Accepted on May 9, 2007

Automation and Analytical Techniques

Multiple Fluorescent Labeling of Silica Nanoparticles with Lanthanide Chelates for Highly Sensitive Time-Resolved Immunofluorometric Assays

¹ Molecular Diagnostics Laboratory, Department of Biomedical Sciences and the Key Laboratory of the Ministration of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China
² Molecular Diagnostics Laboratory, Department of Biomedical Sciences and the Key Laboratory of the Ministration of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China and The Key Laboratory of Chemical Biology of Fujian, Xiamen University, Xiamen, Fujian 361005, China

^* To whom correspondence should be addressed. E-mail: qgli{at}xmu.edu.cn.

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Background: Time-resolved immunofluorometric assays (TrIFA) using lanthanide-labeled nanoparticles have greatly increased the sensitivity of immunoassays. Current labeling strategies, however, use either physical doping of lanthanide chelates into preformed nanoparticles or covalent linking of lanthanide chelates to precursors used for making nanoparticles; both these strategies have drawbacks.

Methods: Luminescent Eu(III) and Tb(III) chelates were covalently coated on the surface of preformed silica nanoparticles to which detection antibodies or bridging proteins for antibody binding were conjugated. We used the resulting conjugates in TrIFA for detection of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg), both individually and simultaneously. We compared the results of the newly established method with results of an ELISA for serum samples. Positive samples identified by TrIFA but not by ELISA were confirmed by additional assays, including real-time PCR detection of viral DNA.

Results: The prepared nanoparticle conjugates were homogeneous in size, at 55 (5) nm in diameter [mean (SD)], were stable for long-time storage (>2 years), and contained more chelates (6.86 x 10⁵ for Eu(III), 4.73 x 10⁴ for Tb(III)) per nanoparticle than particles made as previously reported. The TrIFA established for HBsAg had a comparable or lower detection limit (0.0092 µg/L) than existing nanoparticle-based TrIFA or ELISA. The TrIFA for HBeAg had a much lower detection limit [10.0 National Centre Unit (NCU)/L] than ELISA and detected HBeAg in 5 samples missed by the ELISA method. Simultaneous TrIFA for both HBsAg and HBeAg was achieved with detection limits (0.033 µg/L for HBsAg and 27.0 NCU/L for HBeAg) close to those of the individual assays.

Conclusions: Covalent surface labeling of silica nanoparticles with lanthanide chelates provides good fluorescent labels that can be used in TrIFA for highly sensitive and robust detection of clinical targets.