Silver Nanoparticle-Based Fluorescence-Quenching Lateral Flow Immunoassay for Sensitive Detection of Ochratoxin A in Grape Juice and Wine

Abstract

A silver nanoparticle (AgNP)-based fluorescence-quenching lateral flow immunoassay with competitive format (cLFIA) was developed for sensitive detection of ochratoxin A (OTA) in grape juice and wine samples in the present study. The Ru(phen) 3 2 + -doped silica nanoparticles (RuNPs) were sprayed on the test and control line zones as background fluorescence signals. The AgNPs were designed as the fluorescence quenchers of RuNPs because they can block the exciting light transferring to the RuNP molecules. The proposed method exhibited high sensitivity for OTA detection, with a detection limit of 0.06 µg/L under optimized conditions. The method also exhibited a good linear range for OTA quantitative analysis from 0.08 µg/L to 5.0 µg/L. The reliability of the fluorescence-quenching cLFIA method was evaluated through analysis of the OTA-spiked red grape wine and juice samples. The average recoveries ranged from 88.0% to 110.0% in red grape wine and from 92.0% to 110.0% in grape juice. Meanwhile, less than a 10% coefficient variation indicated an acceptable precision of the cLFIA method. In summary, the new AgNP-based fluorescence-quenching cLFIA is a simple, rapid, sensitive, and accurate method for quantitative detection of OTA in grape juice and wine or other foodstuffs.

Keywords: fluorescence quenching; lateral flow immunoassay; ochratoxin A; quantitative detection; silver nanoparticles.

Figure 1

Schematic of the AgNP–RuNP–cLFIA sensor: (a) fabrication; (b) detection of ochratoxin A (OTA)-free and OTA-positive samples.

Figure 2

Characterization of Ru(phen)${}_3^{2+}$-doped silica nanoparticles (RuNPs) and silver nanoparticles (AgNPs) used in AgNP–RuNP–cLFIA sensor: (a) Transmission electron microscopy (TEM) images of RuNPs; (b) TEM images of AgNPs; (c) Ultraviolet absorption spectrum of the AgNPs and fluorescence excitation and emission spectra of the RuNPs.

Figure 3

(a) Optimization of the amount of anti-OTA ascites labeled on the AgNPs. The vertical bars indicate the standard deviation (n = 3); (b) Ultraviolet absorption spectra of AgNPs before and after labeling anti-OTA ascites.

Figure 4

(a) Standard curve for OTA quantitative analysis in phosphate buffer solution (0.01 M, pH 7.0). The regression equation is: y = 0.272 lnx + 0.828 (0.08 ≤ x ≤ 5.0 µg/L, R² = 0.98); (b) Matrix-matched standard curves for OTA quantitative analysis in red grape wine and juice samples. The regression equations are: y = 0.245 lnx + 0.786 (0.10 ≤ x ≤ 5.0 µg/L, R² = 0.98) for grape juice sample and y = 0.202 lnx + 0.797 (0.10 ≤ x ≤ 5.0 µg/L, R² = 0.99) for red grape wine sample. The vertical bars indicate the standard deviation (n = 3).

Figure 5

Specificity experiments for OTA, aflatoxin B₁ (AFB₁), patulin (PAT), citrinin (CIT), zearalenone (ZEN), deoxynivalenol (DON), and fumonisin B₁ (FB₁) using the AgNP–RuNP–cLFIA sensor. 1: AFB₁ (10 µg/L), 2: PAT (10 µg/L), 3: CIT (10 µg/L), 4: ZEN (10 µg/L), 5: FB₁ (10 µg/L), 6: DON (10 µg/L), 7: OTA (0.5 µg/L), and 8: OTA (0.5 µg/L) containing 10 µg/L of AFB₁, PAT, CIT, ZEN, FB₁ and DON. The vertical bars indicate the standard deviation (n = 3).