Determination of Aflatoxin M1 and Chloramphenicol in Milk Based on Background Fluorescence Quenching Immunochromatographic Assay

Abstract

Harsh demanding has been exposed on the concentration of aflatoxin M1 (AFM1) and chloramphenicol (CAP) in milk. In this study, we developed a new method based on background fluorescence quenching immunochromatographic assay (bFQICA) to detect AFM1 and CAP in milk. The detection limit for AFM1 was 0.0009 ng/mL, while that for the CAP was 0.0008 ng/mL. The assay variability was determined with 3 AFM1 standards (i.e., 0.25 ng/mL, 0.5 ng/mL, and 1.0 ng/mL), and the actual detection value was 0.2497, 0.5329, and 1.0941, respectively. For the assay variability of 3 CAP standards (i.e., 0.10 ng/mL, 0.30 ng/mL, and 0.50 ng/mL), the actual detection value was 0.0996, 0.3096, and 0.4905, respectively. The recovery rate of AFM1 was 99.7%-101.7%, while that for CAP was 95.3%-97.6%. For the test stability, AFM1 and CAP showed satisfactory test stability even at month 5. Compared with the sensitivity of liquid chromatography-mass spectrometry (LC-MS) method, no statistical difference was noticed in results of the bFQICA. Our method is convenient for the detection of AFM1 and CAP in milk with a test duration of about 8 minutes. Additionally, an internal WiFi facility is provided in the system allowing for quick connection and storage in the intelligent cell phone.

Figure 1

Diagram of test strip used in the background fluorescence quenching immunochromatographic assay (bFQICA).

Figure 2

Fabrication of the bFQICA system.

Figure 3

Determination of optimal density of GNP-labeled antibody and concentration of coupled antibody of AFM1. AFM1 antibody was labeled by GNP solutions (pH 7.0) with a concentration of 0.30 μg/mL, 0.60 μg/mL, 0.90 μg/mL, and 1.20 μg/mL. The antibody with a concentration of 0.3 mg/mL (a), 0.5 mg/mL (b), and 1.0 mg/mL (c) was used to select the optimal density of GNP-labeled AFM1 antibody and concentration of AFM1 coupled antibody, defined as presence of significant difference between F₁/F₀ at four concentrations.

Figure 4

Determination of optimal density of GNP-labeled antibody and concentration of coupled antibody of CAP. CAP antibody was labeled by GNP solutions (pH 7.0) with a concentration of 0.30 μg/mL, 0.60 μg/mL, 0.90 μg/mL, and 1.20 μg/mL. The antibody with a concentration of 0.2 mg/mL (a), 0.4 mg/mL (b), and 0.6 mg/mL (c) was used to select the optimal density of GNP-labeled CAP antibody and concentration of coupled antibody, defined as presence of significant difference between F₁/F₀ at four concentrations.