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.
Feeding of drugs and chemicals to cattle can leave residues in milk and meat. For example, aflatoxin M1 (AFM1), the hydroxylated metabolite of aflatoxin B1, is a carcinogenic substance detected in milk and dairy products [
Currently, several methods have been developed for the detection of AFM1 and CAP, such as enzyme linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC), and gold immune chromatographic assay (GICA) [
Recently, a quantitative assay named background fluorescence quenching immune chromatographic assay has been developed based on the fluorescence quenching and nitrocellulose membrane background signals. The method has been used for the alpha fetoprotein in clinical practice. Unlike the conventional GICA assay, in the background fluorescence quenching immunochromatographic assay (bFQICA), the fluorescence donors are fluorescein that are precoated on the entire nitrocellulose membrane and quenching occurs between the gold particles and nitrocellulose membrane [
The bFQICA analyzer was provided by the Simp Bio-Science Co., Ltd. (Shanghai, China). The reference substance and the monoclonal antibody of AFM1 were purchased from Rohi Biotech Co., Ltd. (Shanghai, China). The AFM1-bovine serum albumin (BSA) conjugate, reference substance and the monoclonal antibody of CAP, CAP-BSA conjugate, and the goat anti-mouse IgG were purchased from Rohi Biotech Co., Ltd. (Shanghai, China).
GNPs were made through trisodium citrate reduction of hydrogen tetrachloroaurate (iii) hydrate (HAuCl4·3H2O) as previously described [
The test strip consisted of sample pad, background fluorescence (
Diagram of test strip used in the background fluorescence quenching immunochromatographic assay (bFQICA).
For the preparation of GNP-labeled AFM1 antibody, AFM1 antibody was labeled by GNP solutions (pH 7.0) with a concentration of 0.30
For the preparation of AFM1 or CAP coupled antibody, the AFM1-BSA was diluted using PBS buffer into a concentration of 0.30 mg/mL, 0.50 mg/mL, and 0.70 mg/mL, respectively, while that of CAP-BSA was 0.20 mg/mL, 0.40 mg/mL, and 0.60 mg/mL, respectively. Subsequently, the solution was coated onto the test line of the nitrocellulose membrane and kept at room temperature for 8 hrs. On this basis, three test strips coated with AFM1-BSA (0.30 mg/mL, 0.50 mg/mL, and 0.70 mg/mL) and CAP-BSA (0.20 mg/mL, 0.40 mg/mL, and 0.60 mg/mL) on the test line were obtained.
The reference substance of AFM1 (0.25 ng/mL, 0.50 ng/mL, 1.00 ng/mL, and 2.00 ng/mL) or CAP (0.10 ng/mL, 0.30 ng/mL, 0.50 ng/mL, and 1.00 ng/mL) diluted by PBS was add to each test tube. After that, 100
The bFQICA reader consisted of several core parts including an optical sensor, a scanning platform, and the stepping system, as well as signal processing system (Figure
Fabrication of the bFQICA system.
Using serial dilutions of the standard solution, we established a standard curve for the analysis of known samples with AFM1 or CAP in the range of 0–2.0 ng/mL. In brief, 100
Upon preparation of the test strips, AFM1 or CAP standards at various concentrations were used to assess the performance of bFQICA, including assay limit of blank and variability, test repeatability, and concentration recovery, as well as test stability. For the detection of AFM1 and CAP, 100
LC-MS detection was performed according to the conventional description. The samples were extracted using the methyl cyanide-Mcilvaine buffer and then subjected to the Eclipse XDB-C18 column (150 mm × 2.1 mm, 3.5
In this study, the optimal density of GNP-labeled AFM1 antibody was 1.2
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
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
The formula of AFM1 was as follows:
To test the limit of blank, a blank sample of AFM1 or CAP (0 ng/mL) was analyzed with the bFQICA system. The test was repeated 20 times, and a detection limit of 0.0009 ng/mL was obtained for AFM1, while that for the CAP was 0.0008 ng/mL. The assay variability was determined with 3 AFM1 standards at 0.25 ng/mL, 0.5 ng/mL, and 1.0 ng/mL, and the actual detection value was 0.2497 (RSD = 0.21%), 0.5329 (RSD = 0.13%), and 1.0941 (RSD = 0.15%), respectively. For the assay variability of 3 CAP standards at 0.10 ng/mL, 0.30 ng/mL, and 0.50 ng/mL, the actual detection value was 0.0996 (RSD = 1.76%), 0.3096 (RSD = 1.03%), and 0.4905 (RSD = 0.26%), respectively.
To assess the test repeatability of the system, the concentration of AFM1 and CAP in the milk obtained in a local supermarket was determined. The concentration of AFM1 was 0.008 ng/mL (RSD = 0.54%), while that for the CAP was 0.0011 ng/mL (RSD = 0.45%).
The milk samples (1 mL) were add to an Eppendorf tube, followed by addition of 10 ng/mL AFM1 or CAP of a volume of 50
Recovery rate of AFM1 and CAP.
Sample | Actual value (ng) | Measured value (ng) | Recovery rate (%) | RSD |
---|---|---|---|---|
AFM1 | 0.25 | 0.2503 | 100.1 | 0.20 |
0.50 | 0.4994 | 99.7 | 0.35 | |
1.00 | 1.0167 | 101.7 | 0.87 | |
|
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CAP | 0.10 | 0.0953 | 95.3 | 0.60 |
0.30 | 0.2933 | 97.7 | 1.18 | |
0.50 | 0.4883 | 97.6 | 0.59 |
AFM1, aflatoxin M1; CAP, chloramphenicol; RSD, relative standard deviation.
In this section, we determined the test stability by AFM1 or CAP standard and the standard of the same batch at months 1, 2, 3, 4, and 5, respectively. Both AFM1 and CAP showed satisfactory test stability even at month 5 (Table
Test stability of the strip for AFM1 and CAP.
Concentration |
|
|
|
|
|
RSD (%) | |
---|---|---|---|---|---|---|---|
AFM1 | 0.00 | 0.5996 | 0.5892 | 0.5930 | 0.6004 | 0.5915 | 0.84 |
0.25 | 0.6594 | 0.6588 | 0.6479 | 0.6581 | 0.6482 | 0.89 | |
0.50 | 0.7184 | 0.7091 | 0.7122 | 0.7093 | 0.7159 | 0.57 | |
1.00 | 0.8458 | 0.8513 | 0.8475 | 0.8434 | 0.8522 | 0.43 | |
2.00 | 0.9416 | 0.9425 | 0.9368 | 0.9570 | 0.9485 | 0.82 | |
|
|||||||
CAP | 0.0 | 0.6531 | 0.6508 | 0.6549 | 0.6528 | 0.6545 | 0.25 |
0.1 | 0.7063 | 0.7122 | 0.7051 | 0.7066 | 0.7081 | 0.39 | |
0.3 | 0.8054 | 0.8031 | 0.8024 | 0.8105 | 0.8062 | 0.40 | |
0.5 | 0.8509 | 0.8473 | 0.8516 | 0.8526 | 0.8479 | 0.27 | |
1.0 | 0.9406 | 0.9380 | 0.9415 | 0.9402 | 0.9411 | 0.14 |
AFM1, aflatoxin M1; CAP, chloramphenicol; RSD, relative standard deviation.
The test milk containing AFM1 or CAP was subject to bFQICA detection and LC-MS detection. The detected concentration of AFM1 and CAP was listed in Table
Comparison of test efficiency between LC-MS and bFQICA.
Sample | bFQICA ( |
LC-MS ( |
---|---|---|
AFM1 in sample 01 | 0.1151 | 0.0988 |
AFM1 in sample 02 | 0.1492 | 0.151 |
CAP in sample 03 | 0.1768 | 0.160 |
CAP in sample 04 | 0.4925 | 0.500 |
bFQICA, background fluorescence quenching immunochromatographic assay; LC-MS, liquid chromatography-mass spectrometry. Two samples were used for the AFM1 (samples 01 and 02) and CAP (samples 03 and 04), respectively. No statistical difference was noticed between the efficiency of bFQICA and LC-MS.
Food safety has been a great concern worldwide. Harsh demanding has been exposed on the concentration of AFM1 and CAP in milk. To date, several methods have been developed for the detection of these drugs such as ELISA and LC-MS. In this study, we developed a new method for the detection of AFM1 and CAP in milk which was more convenient with high specificity and sensitivity.
The presence of AFM1 and CAP in milk has been a concern in some countries, which promotes the emergence of determination of these chemicals using various methods, such as ELISA, HPLC, and immunochromatographic assay. For example, in a previous study [
Using this method, we confirmed that the AFM1 antigen coating concentration was 0.5 mg/mL in the test line, and the corresponding GNP-labeled AFM1 antibody concentration in the test tube was 1.2
As is known to all, false positive samples were noticed which may be related to the application of such technique and enzyme labeling [
In this study, we developed a new method for the detection of AFM1 and CAP in milk which was more convenient with high specificity and sensitivity. The detection limit for AFM1 was 0.0009 ng/mL, while that for the CAP was 0.0008 ng/mL. Besides, the method showed satisfactory stability and test efficiency.
All the authors declare no competing interests.
Xiaoxia Wu did the experiment; Xiaofeng Tian and Lihua Xu did the data collection and analysis; Yuwen Wang and Xinxia Li revised the manuscript and approved the submission.
This study was supported by the Science and Technology Program of Xinjiang Uygur Autonomous Region (no. 2016E02036) and program by Science and Technology Committee of Shanghai (no. 15CT24D7300).