Citation: WEI Chun-Hao, YANG Guang-Xin, CHI Hai, TAO Le-Ren, KONG Cong, CAI You-Qiong. Inner Filter Effect-based Fluorescence Immunoassay with GoldNanoclusters for Malachite Green Detection[J]. Chinese Journal of Analytical Chemistry, ;2022, 50(1): 73-81. doi: 10.19756/j.issn.0253-3820.201750 shu

Inner Filter Effect-based Fluorescence Immunoassay with GoldNanoclusters for Malachite Green Detection

WEI Chun-Hao ^1,2 ,
YANG Guang-Xin ^1,, ,
CHI Hai ^1,2,, ,
TAO Le-Ren ² ,
KONG Cong ¹ ,
CAI You-Qiong ¹

1.
East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, China;
2.
Schoolof Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China

Corresponding author: YANG Guang-Xin, CHI Hai,
Received Date: 11 December 2020
Revised Date: 9 June 2021

Fund Project: Supported by Central Public-interest Scientific Institution Based Research Fund (No.2016T09)

Malachite green (MG), an effective insecticide and preservative, is commonly used in the aquaculture industry. However, MG has acute toxicity to terrestrial and aquatic animals, which can lead to mutagenicity, carcinogenicity, and teratogenicity on animals and human bodies. Therefore, it is necessary to explore a reliable and sensitive method for the rapid detection of MG. Herein, a novel and sensitive fluorescence-linked immunosorbent assay (FLISA) was constructed based on glod nanoclusters (BSA-AuNCs) for MG detection. The assay signal was originated from inner filter effect (IFE) between BSA-AuNCs and p-nitrophenol (PNP). Firstly, a double emission peak fluorescent gold nanocluster with good light stability and ultra-small size was quickly synthesized in 7 minutes, which was used as fluorescence probe in this immunoassay. Secondary, alkaline phosphatase labeled secondary antibody (IgG-ALP) was used to replace IgG-HRP in the immune reaction, which could generate PNP with the substrate of p-nitrophenyl phosphate (PNPP). As the maximum excitation wavelength of BSA-AuNCs (390 nm) overlapped with the absorption peak of PNP (400 nm), the fluorescence of BSA-AuNCs could be strongly quenched by PNP. Thus, with the absence of MG in the indirect immunoassay, much more IgG-ALP would be adsorbed in the 96-well plates, more PNP would be produced, then with the fluorescence quenched more strongly. The fluorescence signal could be used for MG detection indirectly. In this immunoassay, several factors including coating antigen, antibody concentration, secondary antibody concentration, substrate concentration, reaction temperature and reaction time were optimized. Under the optimized conditions, the linear range of this method was 0.1-100 ng/mL, linear equation was y=181.53lgC_MG+241.3 (R²=0.995), and the limit of detection (LOD) was 0.089 ng/mL. The sensitivity of FLISA was 30 times that of ic-ELISA. Furthermore, the proposed method was successfully applied to determination of MG in spiked aquaculture water and tap water. The recoveries of MG were in the range of 84.0%-110.2%, and the coefficient of variation was 4.6%-11.2%. The new immunoassay could be used as a promising tool for rapid detection of MG in real environmental samples.
- Malachite green,
- Fluorescence,
- Inner filter effect,
- Gold nanoclusters,
- Enzyme-linked immunosorbent assay
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