Analytical Sciences

Development of Metal Nanoparticle-immobilized Microplate for High-throughput and Highly Sensitive Fluorescence Analysis

Kyohei MATSUI,* So TANABE,* Shuyi SUN,* Dung NGUYEN,* Takamasa KINOSHITA,* Yojiro YAMAMOTO,*,** and Hiroshi SHIIGI*

*Department of Applied Chemistry, Osaka Prefecture University, 1-2 Gakuen, Naka, Sakai, Osaka 599-8570, Japan
**GreenChem. Inc., 19-19 Tsuruta, Nishi, Sakai, Osaka 593-8323, Japan

Enzyme-linked immunosorbent assay (ELISA) is a widespread analytical biochemistry assay. In this work, a direct ELISA method using a metallic nanoparticle (NP)-immobilized 96-well plate was developed for high-throughput, highly sensitive fluorescence analysis. Immobilization of metallic NPs on a 96-well plate effectively amplified fluorescence signals of the assay. The silver (Ag) NP-immobilized plate showed the best fluorescence enhancement effect of all the metal-immobilized plates tested. We used the Ag NP-immobilized plate to detect biomolecules and bacteria and found that both the fluorescence intensity and the limit of detection (LOD) were strongly enhanced by more than 100 times compared with those of the unmodified 96-well plates. Quantitative and qualitative considerations for target bacteria regarding the impact of autofluorescence on detection were successfully obtained for several strains. Our results demonstrate the potential of applying Ag NPs for enhancing the efficiency of direct and indirect ELISA assays.

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