Enhanced colorimetric detection of norovirus using in-situ growth of Ag shell on Au NPs

Indra Memdi Khoris; Kenshin Takemura; Jaewook Lee; Toshimi Hara; Fuyuki Abe; Tetsuro Suzuki; Enoch Y Park

doi:10.1016/j.bios.2018.10.067

Enhanced colorimetric detection of norovirus using in-situ growth of Ag shell on Au NPs

Biosens Bioelectron. 2019 Feb 1:126:425-432. doi: 10.1016/j.bios.2018.10.067. Epub 2018 Nov 1.

Authors

Indra Memdi Khoris¹, Kenshin Takemura², Jaewook Lee³, Toshimi Hara⁴, Fuyuki Abe⁵, Tetsuro Suzuki⁶, Enoch Y Park⁷

Affiliations

¹ Department of Applied Biological Chemistry, College of Agriculture, Graduate School of Integrated Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan. Electronic address: indra.memdi.khoris.17@shizuoka.ac.jp.
² Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan. Electronic address: takemura.kenshin.16@shizuoka.ac.jp.
³ Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan. Electronic address: nanojaewook@outlook.com.
⁴ Department of Microbiology, Shizuoka Institute of Environment and Hygiene, 4-27-2, Kita-ando, Aoi-ku, Shizuoka 420-8637, Japan. Electronic address: toshimi1_hara@pref.shizuoka.lg.jp.
⁵ Department of Microbiology, Shizuoka Institute of Environment and Hygiene, 4-27-2, Kita-ando, Aoi-ku, Shizuoka 420-8637, Japan. Electronic address: fuyuki1_abe@pref.shizuoka.lg.jp.
⁶ Department of Infectious Disease, Hamamatsu University School of Medicine, 1-20-1 Higashi-ku, Handa-yama, Hamamatsu 431-3192, Japan. Electronic address: tesuzuki@hama-med.ac.jp.
⁷ Department of Applied Biological Chemistry, College of Agriculture, Graduate School of Integrated Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan; Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan; Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan. Electronic address: park.enoch@shizuoka.ac.jp.

PMID: 30471568
DOI: 10.1016/j.bios.2018.10.067

Abstract

Norovirus (NoV) is a leading cause of acute gastroenteritis. The low infectious dose and environmental stability of NoV facilitate its effective transmission through a variety of modes such as food, water and person-to-person. The available enzyme-linked immunosorbent assay (ELISA) for NoV detection has low sensitivity due to the low catalytic activity of the peroxidase used, and thus, a reliable ultrasensitive bioassay is needed. In this study, we apply the enhanced peroxidase-like activity of silver ion-incorporated gold nanoparticles (Au/Ag NPs) in a colorimetric bioassay for NoV detection. NoV was captured by anti-NoV genogroup II antibodies, which were immobilized on the surface of a 96-well microtiter plate and formed a sandwich structure among anti-NoV Ab, NoV and Ab-Au NP. Then, Ag ion-containing hydroquinone was added to form Au/Ag core/shell NPs. When H₂O₂/3,3',5,5'-tetramethylbenzidine (TMB) solution was added to the wells, Ag ions were liberated from the surface of Au/Ag NPs and enhanced the oxidation of TMB. These reactions enhanced the oxidation of TMB and developed an intense blue color. The Au/Ag NPs were shown to exhibit higher affinity and catalytic efficiency for H₂O₂ and higher catalytic velocity based on the k_cat of up to 7-fold compared with Au NPs. The bioassay was then optimized to detect clinically isolated NoV using NoV-like particles (NoV-LPs). NoV-LPs were detected with a limit of detection of 10.8 pg/mL, corresponding to 1000- and 100-fold higher sensitivity compared to the gold-immunoassay and horseradish peroxidase-based ELISA, respectively. Clinically isolated NoV GII.4 and NoV GII.3 were detected in the range of 10²-10⁶ copies of viral RNA/mL fecal solution with a detection limit of 13.2 copies/mL fecal solution for NoV GII.4, equivalent to 132 copies of viral RNA/g feces and indicating significantly higher sensitivity compared to commercial immunoassay kits. This bioassay represents a workable detection assay for low concentrations of NoV that is applicable for early-stage diagnosis for public hygiene.

Keywords: Colorimetric bioassay; Gold/silver nanohybrid; Norovirus; Norovirus-like particles; Peroxidase-like activity.

MeSH terms

Biosensing Techniques*
Catalysis
Colorimetry*
Gold / chemistry
Horseradish Peroxidase / chemistry
Hydrogen Peroxide / chemistry
Immunoassay*
Limit of Detection
Metal Nanoparticles / chemistry
Norovirus / isolation & purification*
Norovirus / pathogenicity
Silver / chemistry

Substances

Silver
Gold
Hydrogen Peroxide
Horseradish Peroxidase