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A new time-resolved fluorometric microarray detection system using core–shell-type fluorescent nanosphere and its application to allergen microarray

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Abstract

We have developed a new time-resolved fluorometric (TRF) microarray detection system consisting of fluorescent NH2 nanosphere, TRF microarray detector and gamma-irradiated polystyrene chip. Using the TRF microarray detector, we detected 500 particles of the fluorescent nanosphere in one channel. Cross-talk fluorescence from the adjacent channels was little observed in the TRF microarray detector (<0.0004 %). The TRF microarray detection system was further applied for serum allergen-specific immunoglobulin E (IgE) multi-analyses. As a labeled tag antibody, an anti-human IgE Fab’ fragment-conjugated fluorescent nanosphere (Fab’ nanosphere) was prepared as described previously. As a chip surface appropriate for allergen immobilization, the polystyrene chip surface was modified by gamma irradiation. The immunoassay reactivity using the gamma-irradiated polystyrene chip was approximately 2.5-times improved compared with that of the non-treated polystyrene chip. Non-specific adsorption of the Fab’ nanosphere onto the gamma-irradiated polystyrene chip surface was very low level (<0.0009 %). In only 20 μl of serum, six allergen-specific IgEs could be simultaneously determined in one reaction well in fewer than 90 min. Good correlation curves were obtained between the microarray immunoassay and the CAP RAST fluoro-enzyme immunoassay (CAP/RAST FEIA) method (r>0.961). Reproducibility (CVs) of the microarray immunoassay was 8.6 % to 19.0 % (n=5).

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Acknowledgments

The authors wish to thank Mr. I. Shindo, for his advice on the construction of the TRF microarray detector, and the Corona Electric Co., Ltd., for construction of the TRF microarray detector. The present work was financially supported by the New Energy and Industrial Technology Development Organization (NEDO).

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Authors and Affiliations

  1. Research & Development Division, Mitsubishi Kagaku Iatron, Inc., 1144 Ohwadashinden, Yachiyo-shi, Chiba, 276-0046, Japan

    Takeshi Matsuya, Kazuhiko Otake, Shigeru Tashiro & Nobuhiro Hoshino

  2. Graduate School of Science, Tokyo Metropolitan University, Hachioji, Tokyo, Japan

    Motomi Katada

  3. Protein Technos Institute, Atsugi-shi, Kanagawa, 243-0206, Japan

    Tsuneo Okuyama

Authors
  1. Takeshi Matsuya
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  2. Kazuhiko Otake
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  3. Shigeru Tashiro
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  4. Nobuhiro Hoshino
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  5. Motomi Katada
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  6. Tsuneo Okuyama
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Correspondence to Takeshi Matsuya.

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Matsuya, T., Otake, K., Tashiro, S. et al. A new time-resolved fluorometric microarray detection system using core–shell-type fluorescent nanosphere and its application to allergen microarray. Anal Bioanal Chem 385, 797–806 (2006). https://doi.org/10.1007/s00216-006-0455-9

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  • DOI: https://doi.org/10.1007/s00216-006-0455-9

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